INFORMATION TO USERS

Thi s manu script has been reprod uced from the microfilm master. UMI films the

text directl y from lhe origi nal or copy submitted. Thus , some thesis and

dissertation copies are in typewriter face, while others may be from any type of

computer printer .

The quality of this reproduction Is dependent upon the quality of the copy

submitted. Broken or indi stinct print. co lor ed or poor qual ity illus trat ions and

photographs, print bleedthrough, substandard margins, and improper alignment

can adversely affect reproduction.

In the unlikely event that the author did not send UMI a complete man uscript and

there are missing pages, these will be noted. Also. if unau thorized copyright

material had to be removed, a note will indi cat e the deletion .

Oversize mater ials (e.g., maps , drawings, charts) are repro duced by sectioning

th e orig inal , beg inning at the upper left ·hand comer and cont inui ng from left to

right in equal sect ions with small overlaps. Each original is also pho tographed in

one exposure and is included in reduced form at the back of the boo k.

Photographs induded in the original manuscript hav e been reproduced

xerographically in this copy. Higher qual ity 6 " x 9" black and whit e photographic

prints are available for any photographs or illustrations appearing in this copy for

an additional charg e . Contact UMI directly to order.

UMfBell & Howellintormation and Leaming

300 North zeee Road, Ann Areer, Ml 481Q6..1346 USA800-521-0600

.+.Acquisitions and AcquisitionsetBbIiographicServices servCes bibl~

3llSW~~ 3llS• .-w-...=.ON K1A.0N( =00 K''''OtW

Th e auth or bas granted a non­exclusive licence allowin g theNa tional Library ofCanada toreproduce. loan, distribute or se llcopies of this thesis in microform .paper or electronic form ats .

The autho r retains ownership oftheco pyright in this thes is. Neither thethes is nc r substan tial extrac ts from itmay be printed or otherwiserep roduced with out the author'spermis sion.

L ' auteur a accord e une licence nonexclusi ve permettant alaBibliotheque nationale du Cana da dereproduce, prefer. distribuer ouvendr e des copies de cett e these SOllS

la forme de microfich e/film, dereproduction sur papier au sur form atelectr onique .

L "auteur conserve la prop riete dudroi t d 'auteur qui protege cetre these .Ni la these ni des extraits substan tielsde ce lle-ci DC doivent etre imprimesau autrement reproduits sans sonaut orisation .

0-612-55 504-6

Canada

Th e iun uence of phos phod iaterase inhibitor, rolip ram , on plasma tumor e eeee stsfaclor -a tevejs a nd baemodyn am ics in lipopolysaccharid e-Ireat ed rals

by

Pras a Dn aj it Dutt a

A th a i' sub mitt ed 10 th e Schoo l of Grad ua le Siu dies in partial fulfillment of Ihedegree of Master e r Sc teeee

Division of Basic Med ica l Sciences, Facul ty of Med icin eMemori al Uoiversily of Newfound land

Se ptem ber 2000SI. John's, Newfoundland

ABSTRACT

Septic shock is the thirteenth most common cause of death in the United States and the

leading cause ofdeath of individuaJs in intensive care uni ts once it progresses to multiple

organ dysfunction syndrome (MO DS) (Parri llo et al.• 1993). Morti lity ranges from 20%

to 95% (E idleman er el., 1995; Panillo et al., 1993; Wie ssne ret al ., 1995). Septic shock

is caused most often by gram-nega tive bacteria and it has increased dram atically in the

past 10 years . Even when pro per ly treated with avai lable therapi es. it carries a 60%

mortality (Wiessner et al ., 1995).

The administration of lipopo lysaccharide (LPS) bas been repo ned to produce

hypotension and reduced card iac outpu t. The aim a f the present investigation was to (a)

exami ne the influence of type IV phosphodiesterase inhib ito r rolipram on

baemcdynarnics, plasma levels of twnor necrosis factor-a (INF-a) levels, and

productio n of inducib le nitric oxide synthase CiNOS) in the lungs, ex vivo, in LPS-trea ted

rats, and (b) dete nnine the cardio vascular effects of a selec tive a t-adrenoceplor agonist,

methoxami ne. in the absence or presence of rolipram in rats trea ted with LPS.

Blood pressure, cardiac index. heart rate and arterial resistance were assessed in Long­

Evans rats anaesthetized with thiobutabarbital. Cardiac outp ut was measured using

radioactive labeled micmspheres and arterial blood pressure was measured via an intra­

arterial cathe ter. Plasma levels of TNF- a were measured by an imm unoass ay technique,

-ii-

and nitric oxide synthase (inducible & constitutive) activity in lung homogenate was

assessed by measurin g the conversion of eHlL-arginine to e HlL-eitruiline .

Administrat ion o f LPS (0 .8 mglkg i.v.) to animal s resulted in a significant reduction in

cardiac index ove r time. Changes in arterial resistance, heart rate and blood pressure

were insignificant over time in LPS-treated animals. The administration of LPS to rats

resulted in a substantial rise in the plasma levels of lNF-a.. Furtherm ore , the injection of

LPS resulted in a significant increase in the iNOS activity in lungs. Pre-treatment with

rolipram (10 mglkg) or dexamethasone (5 mglkg) prevented the decline in cardiac index

in animals that received LPS. Infusion of methoxamine into animals injected with

rolipnun and pre-treated with LPS did not result in significant changes in cardiac index .

In contrast, in animals treated with dexamethaso ne and subseque ntly LPS, infusion of

methoxamine significantly reduced cardi ac index and increased blood pressure and

arterial resistanc e. Pre-treatment with rolipram ( 10 mglkg) or dexam ethasone in animal s

injected with LPS significantly prevented the rise in lNF-a. when co mpared to respective

values in vehicle treated animal s. Howe ver, pre-treatment wi th dex amethasone but not

rolipram was found to significantly reduce iNOS activity in the lungs ofanimals injected

with LPS.

The present observations suppo rt the view that cardiac index can be maintained in

animals treated with LPS indepen de nt of LNOS inhib ition. Furthermore. our findings

seem to support the idea that induct ion of NOS may occur indepen dentl y of TNF-a. in

LPS-treated rats .

-iii-

ACKNOWLEDGMENTS

I would like to thank Dr. R. Tabrizc hi for his excellent supervis ion, gui dance and

cooperati on. Many thanks to Dr. J. Smeda, Dr. B. Van Vliet. Dr. D. Bieger, Dr. S.

Vasdev and Dr. P. Mood y-Corben.

Spec ial thanks to my Mum, Dad, Brother and wife Nee ladri for the ir he lp,

TAB LE or CONTENTS

Abstra ct Pg. i

List of Tables Pg. vi

List of Figures Pg. vii

I . Introduction Pg. I1.1. Nitric oxide production Pg. 21.2. Regul ation of NO synthes is Pg. 41.3. Gram negative endotoxin and NOS Pg. 51.4 . Septic shock and lNF-a. Pg. 71.5. The role o f cGMP/cAMPin TNF -aproduction Pg. 1I1.6 . The relati onship between TNF-a and phosphod iesterase Pg. 121.7 . Nature o f tile problem and Experime ntal Objectives Pg. 14

2. Methods Pg. 162.1. Surgical preparation of animal s Pg. 162.2 . Measurem ent of cardiac output Pg. 172.3. Experimental protocols Pg. 172.4 . TNF-a assay in plasma Pg. 192.5. Nitric oxide synthase assay in lungs Pg. 192.6 . Chemicals Pg. 202.7 . AnaJysis of data Pg. 20

3. Results Pg. 2 13.1. Effects of rclipram and dexamethasone on haemoclynamics in LPS·treated rats Pg. 2932 . Effects of a.l-adrenoceptor stimulation on haemodynamics in LPS-treated rats Pg. 293.3. Effects o f rolipram and dexamethasone on plasma levels of lNF-a Pg. 323.4. Effects of rotipram and dexamethasone on NOS activity in lungs in LPS- Pg. 34

treated animals

4. Discuss ion Pg. 35

5. References Pg. 44

LlST OF T ABLES

Tab le I Values ofenzymatic activity of inducible nitric oxide synthase Pg. 28and constitutive forms of nitric oxide synthase (pmoVmgprotein/min) in lungs of various gro ups of animals treated withsaline. LPS. and vehicle. rolipram and dexamethasone prior totreatme nt with LPS .

Ta ble 2 Haemodynami c changes in vario us gro ups of animalstreated Pg. 30with LPS in the absence or pres ence of vehicle. rolipram ordexamethasone.

Tab le 3 Haemodyn amic change s in various gro ups of animals treated Pg. 31with LPS in the absence or presence of vehicle. rolipram ordexamethasone.

Table 4 Plasma TNF-a. values in various gro ups of animals treated with Pg. 33LPS in the absence or presence of vehicle, rolipram ordexamethasone.

LIST OF FIGURES

Figure I The effects of treatment of anaesthetized rats with saline or LPS Pg- 22on cardiac index:and heart rate over time.

Figure 2 The effects of treatment of anaesthetized rats with saline or LPS Pg. 24on blood pressure and arterial res istance over time.

Figure 3 The effects of treatment of anaes thetized rats with saline or LPS Pg. 26on plasma concentration ofTNF-a. over time.

-vii-

1.0 INTRODUcnON

The cardi ovascular sys tem serves the body by supplying blood to the tissues in

sufficient volume to meet metabolic needs. Blood carried in the vascul ature provides

oxygen and nutrients to di fferent parts of the body and remove s carbon dioxide and

metabolic waste products . These transport functions arc mad e possible by the

cardiovascul ar system (Guyto n et al., 1973; Sagawa, 1973; Levy, 1979; Greenway,

1982).

It is recognised that the main dctcmrinants of cardiac output are heart rate, cardiac

contractil ity, blood volume. preload and afterload (Greenway, 1982). Under certain

pathoph ysiological conditions , alteratio n in the functio n of the cardio vasc ular system play

a critical role in the demi se of circulation (Forfi a et al., 1998). For instances, in

endotoxaernia, hypotension and reduction of cardiac output are evident. Recently it has

been suggeste d that changes that resul t from endotox:aemia are du e to an over-production

of nitric oxide (NO) (Forfia et al., 1998). Clearly, there is evide nce in the current

literature to indicate that the administration of lipopolysaccharide (LPS ) results in the

inductio n of inducible nitric oxide synthase (iNOS) (Salter et al ., 1991; Thie mermann et

al., 1993) . Evidently, the admi nistration of l PS has been reported to result in reduction

in card iac output and assoc iated with this is a fall in blood press ure (Poon et al., 1997;

Chen g et al ., 1998) . The prevai ling view seems to support the idea that l PS mediated

-1-

reduction in cardiac outp ut is the result of an over-prod uction of NO. which is to be the

key mediato r responsible for the eollapse of the cardiovasc ular sys tem.

t.t Nitric oxid e production

The production of NO in mammal ian cells is dependent on the: enzym e: nitric o xide

synthase: (NOS) which produces NO from the: amino acid Lcarginin e (p almer e t al ..

1988). NO is generat ed by oxidatio n from the terminal guanidinium. nitrogen o f L-

argini ne: and the reaction is both oxygen and nieotinamide adenine: dinucleotid e

phosphate (NADP) dependent, and produces L-citrulline in additio n to NO (Ta yeh. 1989;

Bush et al., 1992). To date, three differen t iscfc rms of NOS have been identified by

protein purification and molecular clo ning approaches. These iso forms are : ( I) a

eonstitu tive form (NOS-lor nNOS). which is depc:ndent on CaJ' and calmodulin fo r its

enzymatic action and is mainl y present in neural tissue, both cen tral as we ll as perip heral

(Mayer et al., 1990; Fcrstermann et al., 1991; Schmidt et al., 1991). (2) A constitu tive

(NOS-III or eNOS) form which also requires CaJ' and calmodul in for its enzym atic

action and is present to a major extent in vasc ular endothelial cells. (po llock et et., 1'991).

(3) A Ca1- independent form CiNOS or NOS-lO which was original ly iso lated from

macrophag es (He vel er aL, 199 1), and subseque ntly found to be present in vasc ular

smooth muscle cells and hepatic cel ls and can be induced by the action of cvtcktne s

(Stuehr et al., 199 1; Evans et al., 1992; Wood et al., 1990) .

-2-

NOS activity requires a parti cular co factor , NADPH, as a so urce of electrons for

oxyg en activation and substrate oxidation (Watanabe et aI., 1992 ). The heme moiety of

NOS resembles cytochro me P-450. Due to this similarity, carbon mo noxide (CO) and

othe r heme bindin g agents inhibit NOS activi ty . It is believed that the heme co mponent

of NOS represen ts the catalytic cen ter, responsib le for bindin g and red ucing molecular

oxygen and subsequent oxidation (Havel et al.. 199 1). Calmodulin is also imponarn: for

the regulatio n of NOS activity but different quantities of calmodulin are required by

different isoenzymes (Abu -Soud et al ., 1993).

There are two react ions by which NO is produced from L-arginine. The initial

reactio n involves N-hydroxy lation of the guanidium nitro gen 10 form N-hydroxy-L­

arginine. This reactio n utilizes one equivalent of NAD PH and oxygen to conduct a

simple two-electron oxidation of nitrogen (Marlena er al., 1988). However, the

subsequent steps in the conversion of Nehydrcx y-Learginine to NO and L-citrul line

rema in unclear . Recent studies suppo rted the view that there are two mechan isms

respons ible for the produc tion of NO . Firs t, nitroxyl (RN O) has been shown [0 possess

biological activity indistinguis hable from NO, which seems attributabl e to the rapid

conversion of HNO to NO, by a variety of physiological ly relevant oxidants including

superoxide dismutase (SOD), oxygen and hemoproteins (Fukuto er aI., 1992, 1993).

Seco nd, SOD has been demons trated to directl y enhance the fonoation of free NO from

L-argi nine by NOS (Hobbs et al., 1994 ). Thus , SOD appears to acce lerate the conversio n

of an intermediate in the L-arginine and NO pathway. In a similar mann er to cytoc hrome

-3-

P-450. NOS also appears to be able to uncouple from its substrate . L-.arginine. and

generate superoxi de anion and hydrogen peroxide via the NADPH-dependent reduction

of molecular oxygen (Klatt et aI.• 1992; Heinzel er aI.• 1992; Pou et aI.• 1992). Therefore.

it can be concluded that oxygen and NADPH are essential for the production of NO from

L-arginine and SOD plays an interme diate role.

1.2 Regu la tioD oCNO syDthe sis

Regulation of NOS activity and NO synthesis is different for constitutive and

inducible isoforms. For both nNOS and eNOS . the main mechanism of regulation is

provided by the Ca! >' calmodul in system. At resting intrace llular free Ca » concentrations

([Ca!-l -100 nM ), eNOS does not interact with calmod ulin and therefore it is inactive.

But when [Cal> l, increases, calmod ulin binds to NOS and stimulates NO formation

(Schmidt et al., 1991). In endothelial cells, the presence of specific receptors for

bradykinin. throm bin, or adenosine-SO-tri phosphate or simply shear stress of blood flow

increases [Ca» l and therefore activa te eNOS activity (Buga et al., 199 1). Thus, due to

cellular regulation of [Ca'l, the production of NO by eNOS can be controlled. It has

been demonstra ted that calmodulin antagonists such as triflu perazine and calmidazolium

block [Ca»l depe ndent activation of eNOS (Mayer et al., 1992; Schini et aI.• 1992).

Inducible fonn of NOS contains highly bound calmoduli n and therefore it is not

controlled by Cal' (Xi e er aI., 1994). For this isofonn , NO synthesis is regulated by

expression of prote in that are not constitutively expressed in tissues, but that require

-4-

induction by spec ific cytokines such as twnour necrosis factor al pha (TNf-a),

iater leukia- I, and interfc:ron-gama (Hevel et al.. 1991; Stuc:hr er al ., 1991). The: actions

of these: cytokinc:s ca use: an increase: in the transcription of appropriate NOS gene thus

resulting in produ ction ofiNOS (Stuc:hr et al ., 1991). Cytokines no t only induce NOS but

also increase the availability o f co -factors. to increase:NO synthesis (Hattori et al., 1993).

Evans and assoc iates (1992) have report ed that cytokines such as TNF-<:t can regulate the

syn thesis of iNO S fro m immune ce lls (Evans et al ., 1992).

NOS activity also appears to be regul ated by a negative feedback mechanism that is

media ted by NO. Recent studies have shown that NO generated by nNOS and iNOS is

capable of inhibiting subsequent enzymatic activity (Griscavage et al., 1993). It appears

that the enzymatic activity of nNOS and eNOS may also be regu lated by phosphorylation

(Bred t et al., 199 1). In addi tion, it seems that cycl ic adeno sine S'-mo nopnosphate

(cAMP)-dependent protein kinase C and Cal' /calmodulin -dependent protein kinase have

been found to modulate the activity of NOS (Bredt et al., 199 1; Nakane e t al., 199 1) .

These: reports ind icate tha t the activity of NOS is under the regulation of a number of

inuaeellular mediators.

1.3 Gra m negati ve end otoxin a nd NOS

It hasbeen found tha t during septic shock induced by endotoxin from gram -negative

bacteri a, the left ventricular ejection fraction is decreased (p arker et al., 1990). This

reducti on of ejection fract ion indicates that there is a decrease in left ventricular

-5-

contraction due to reduction of tbe left ventric ular end-systolic pressure to vo lume ratio

(parker er al ., 1990). A number of studies suggest that NO may play a pivotal role in

decreasi ng myocan1ial contracti lity . Ir bas been suggested that TNF-a. interleukm-Z (ll.­

2), and interleuldn-6 (IL-6), are mainly responsi ble for the induction of NO (Finkel et al.,

1992; Walley et al., 1994). As mentioned earlier these cytokin es can ind uce a Cal'

independent fonn of NOS. iNOS (Gross et al ., 1990). As result of induction of NOS,

there is enhanced production of NO within myocytes, and nearby endo lhelial and

macrophages which can cause a reduction of myocardial contractil iry (Finkel et aI..

1992). A number of potential pathways have been implicated by which NO may produce

depression of the myocardium. Fiest, there could be an increase in tum-over of cyclic

guanosine monophosphate (cG MP) which can result in a decrease in cytoso lic availability

of Ca!' leading to depression oflhe myocardium (Stuehr et al., 1991). Seco nd. NO may

decrease myocardial contractility by forming tox ic peroxynitrites in the presence of

oxygen free radicals (Schutz et al., 1992). Finally, NO binds to heme related proteins

thereby inhib iting the cytoso lic and mitochondrial proteins (Estrada et al ., 1992).

Furthermore , NOS inhibition during endotexaemla bas been fowxl. to increase capillary

leak. and this may cause edema and thus "indirectly" impair ventricular systolic and

diastol ic function (Hu tcheson er al., 1990).

Previously, it has been demonstrat ed that NOS inhib itor, ~-nitro-L-arginine (L­

NNA), prevents the decrease in left ventricular contractility during endcto xaemia in intact

animals (S tuehr et al., 1991). Moreover, based on the measurem ent of the end-systolic

-6-

pres sure vo lume relationship, it has been demonstrated that L-NNA partially prevents the

dep ress ion in left. ventricular ccmractility in ancsthctiscd pig with end otoxaemia (Kaszaki

et al ., 1996). Therefore , it would appe ar that NO plays a critical ro le in the depress ion of

the m yocard ium in the latter stages of sepsis.

Although NO has been suggested to be an important mediator in reducin g

myocardial contractility in vitro (Finkel et al., 1992). it would appear that it bas a minor

pro tecti ve role in the early stage of myocardi al depression in endotoxaemia in vivo

(Kaszak.i et aI.• 19%). TIlls is a parad ox ical effect of NO whic h appears to occur at the

initial stages of sepsis. Th is difference between the in vitro and in vivo effects of NO

initial ly could be attri buted 10 vario us prot ective effects o f NO in the intact animal.

Certainly, NO may playa direct role in the maintenance and. control of microvascular

blood flow by virtue of it dilato r effect (parker et al., 1990). In addition. NO may have an

indirect effect on microvascular blood flow by inhibiting platelet aggregatio n and

leuk ocyt e adhesion (Radom ski et al., 1990 ; Kubes et al., 199 1). It is evident tha t a

co mparis on between the impact of inhib iting NOS in vivo and in vitro is quite complex as

diffe rent varia bles play different roles in each paradigm.

1.4 ~pric sh()('k a nd TNF-a.

There is evidence in the current literature which indicates that administration of LPS

results in the re lease of cytokines such as lNF-a: (Thiemennann et aI., 1993; Liebeman er

al., 1989) . In addit ion, it is believed that TNF -a. is one of the factors that activates the

-7-

processrespo nsib le for iNO S. ultimately leading to an over -production of NO in the body

(Ruette n er at.. 1997) . Multi ple organ and system failure is the main pathology associated

with septic shock which occurs afte r a serious bacterial, vira l.or parasitic infection initiate

a series of immunological , metabolic, and haemodynamic reacti ons in the host (Solorzano

er aI., 1981). Over the past ten years, it has been recognized that one class of endogenous

host mediators, the cytokines (TNF-a. , and inter leukin- l) , contributes significantly to the

pathophysiology of septic shock (Lieberman et al., 1989) . Cytc kines are capable of

media ting a wide range of biological effec ts.

TNF-a. was first purifi ed and charac terised by Aggarwal and co lleague s (1985).

They established that this polypeptid e has a molecular weight of 17 kDa. Subse quently,

Beutler and associa tes (19 81) repo rted tha t it co ntains 157 amino acids. It is secreted by

a variety of myeloid cells. such as monocytes, lymphocyte, Kupfer cells (Hesse et al.,

1988), and perit oneal mac rophages (Halme er al., 1989). Mast cells and endothelial cells

also synthes ize TNF-a.. Expressio n of TNF-a. is very stric tly controlle d on a

transcriptional as welt as translationa l level (Beutler e r al., 1988). Unstimulated

mcnocytes express low levels of TNF-a. messenge r ribonu cleic acid (mRNA) , and

stimula tion causes both increased translation and transcri ption of the mature protein

within minutes (Be utler et aI., 19 88). Numerous infections and inflammatory stimuli

el icit TNF -a. syn thesis, including bact erial ce ll wall-derived tPS bacteri al exotoxins,

protozoa, fungi and viral particles (Wong et aI., 1986). Bacterial infections afte r injecting

bacterial endotoxin in rats, rabb its, and baboons cause an incre ase in circulating levels of

-$ -

lNF~ which reaches a peak within 90 to 120 minutes post injection (Beutler et al.,

1985; Hesse et al., 1988). Furth er studi es demonstra ted that bolus infusion of endo toxin

in anim als and humans induces a similar monophasic peak 1.5 hours after infus ion

(Michi e et al.. 1988). Experim ental studies have further demonstrated a causal

relationshi p between TNF-a: and sepsis. Beutler and assoc iates (1985) have report ed that

circulating lNF-a: levels appear in rabbits within IS minutes after a sublethal intravenous

dose of endotoxin. where TNF-a levels peaked within 2 hours, and returned to base line

within 5 hours (Beutlerer al., 1985). Hesse and cowork ers (1988) demonstrated the same

findings in hwnans . They have shown that TNF-a is detectabl e in the hwnan plasma

within 30 minutes and reaches the peak level at 1.5 hours after infus ion of endot oxin. and

these respo nses occ urred temporall y correlated with the appearan ce of symptoms (M ichie

et al., 1988; Ding et al .• 1989). Similar observations have also been reported in rabbits

(Ulevitch et al., 1989) and rats (He et al., 1992).

More specific effects oflNF-a were identified following admini stration of TNF -a..

For example. high doses of TNF-a in animals have been reported to precip itate a

syndro me similar to that seen in human septic shock (T I1lCey et al ., 1987). Further , acute

infusion of TNF -a in rats has been shown to produce hypotens ion , lactic acidosis, and

subseq uently death (Tracey ee al ., 1986). Pathological findings followin g a lethal dose of

TNF-a have shown adrenal necro sis, pulmonary congestion, and intestinal congestion

and intestinal necrosis consistent with those found in sept ic shock (Tracey et aI., 1987).

Furthermore, admini stration of lNF-a 10 humans elicits simi lar metabo lic and

-9-

hemod ynamic chan ges, including an increase in glucose and free fatty acid turnover ,

amino acid efflux and energy expendi ture (Warren er al ., 1987; Michie et al., 1988; Van

der Poll et al ., 1991). Also TNF-a. stimu lated expression of a cell surface tiss ue factor

initiates coagulation via generat ion of thrombin (Van der Poll et aI., 1990).

It has been repon ed that prophylactic administra tion of a polyclonal anti TNF -a.

antiserum protected. mice from the lethal effects of endotoxin (Beutler et al., 1985).

Tracey and associates (1981) also demonstrated that the septic shock due to lethal

Escherich ia coli infusion in primates can be prevented with a monoclonal murin e anti­

human TNF-a. antibody. These results clearly identified the proximal role ofTNF-a. in

the inflammatory cascade of sepsis. Although TNF-a. has the capacity to elicit

deleterious responses in the host, it has also beenfound tha t TNF~ possesses significant

beneficial properties, including the capac ity to elicit an endogenous anti-viral and anti ­

bacterial response . It serves as an endogenous pyrogen wi th immuno-stimulatory activity

(Dinarello et al., 1986). lNF-a. promot es the release of neutrophil from the bone

marrow, as well as enhance neutro phi l function. It initiates neutrophils margination,

transendo the lial passage (Mose r et al., 1989) and activa tion (Ulich et al., 1981), inclu ding

degran ulation., producti on of super oxide radical s, and release of Iysozymes (Shalaby et

al., 1985), which enhance antibody dependent cellular cytotoxic and neutrophil mediated

inhibition of functional growth (Dj eu et aI., 1986). Furthenn ore, it promotes

differentiation of myelogenous cells to monocytes and macropha ges. as well as activation

- 10-

of these cells . TNF'.-a. aIso particip ates in inhibition of intrace llular replicatio n of viral

and parasitic or ganisms (Beutler et al., 1985).

1.5 The ro le or c:GMP/cAMP in TN F-a. production

Most of the effects oCNO are mediated through a unique cOMP signal ing pathway .

NO activates the enzyme guanyl ate cyclase, and thereby elevating intracellular cG MP

concentration (lgnarro et al., 198 2; Crave n et al., 197 8; Ignarro , 1990). This increase in

cGMP subsequently activates certai n protein kinase , which phospho rylate targ et proteins

involv ed in regulation of ee l! function (lgnarro , 1990; Stewart et al ., 1994 ; Kuo et al.,

1995). Alth ough the role of cGMP as a NO seco nd messe nger is undisputed. some

findin gs have led to the speculation regarding the existence of cGMP .independed signal

transd uction pathways for NO. Studies have shown tha t some effects oCNO cannot be

reproduced with cell permeable cGMP analo gs. For example, the synthesis of TNF-a. is

increased in hwnan peripheral blood mononuc lear cells (Van Dervort et el., 1994 ), as

well as LPS · stimulated neutrophil preparations by exogenous NO (lander et at.. 1995).

Although NO increases cGMP co ncentration s in these cells. cGMP analogs ha ve no

effect on TNF -a: production (Van Dervcn et aL, 1994 ; Lander er al., 1995). Co llecti vely ,

these in vesti gations suggest that NO might use cGMP.indepe ndent signaling pathways

for some of its ce llular functions.

Studies have found that enzyme adenylate cyclase can be modified by NO (Duhe et

al., 1994). Treatment of cel l memb ranes with NO decreases cAMP prod uction by

-II·

inhibiting calmodulin activation of typc I adenyl cyclase. presumabl y through thiol

nitrosylation at the calmod ulin-binding site (Duhe et al., 1994 ; Vorherr et al., 1993).

Notab ly, Incre ase in cAMP in leukocyte activate cAMP.d epended protein kinase. This

cAM P.depended protein kinas e phosphorylate transcription factors that bind to the

cAMP-responsc: element on the TNF-a. promoter, thereby inhibiting TNF-a. mRN A

transcri ption (Zho ng et al. , 1995). The effect of NO on type [ adenyl cyclase suggest that

NO might up-regu late lNF-a. systhes is in human monocytes by decreas ing cAMP

conce ntrations .

1.6 T he rel ation shi p between TNF -a. and phcsph cdiesterase

A pan of the physiological responses to both cG MP and cAMP are gove rned by a

family o f phosphodiesterase (POE) lhat specifical ly hydro lyse the cyclic nucleotide to

biologically inert S·-nucleotide. There are five sub-types of PO E that have been isolated

and charact erised . The Cal~/calmodulin dependent or type I POEs catalyze both cGMP

and cAMP hydrolysis and there are at least six lsoforms of this type (Beavo et al., 1990;

Wu et al., 1992). This form of POE is found in high concentrations in the brain, heart.

lung and testis, and to a lesser extent in the kidney, adrenal glands, pancreas, and thyro id.

One of its isoforms , a 63-kDa protein. catalyses cOMP hydrolysis several times more

efficientl y thancAMP hydro lysis (Sharma et 3.1., 1986). Furthermore, a 75-kDa form that

is expressed in the central nervous system appears to specifically degrad e cGMP

(Yamamoto et 81., 1983). The Types II and III are both cAMP selective POEs, but they

· 12·

have significant relevance to the: NO-cGMP transduction system since the rate of

hydro lysis by these two etazymes is stimulated and inhibited respectively, by cGMP

(Manins et al., 1982; Yamarnotc et al., 1983). Type IV is a cG rvlP specific POE, but its

activity is not affect ed by cGJMP (Li et al ., 1990; Charbo nnea u e t al., 1990). Type V POE

is an important regulato r cef cGMP function . This enzym e was first identified and

partiall y purified from rat hm g and platelets (francis et al., 1980 ; Coquil et aI., 1983). It

has been reponed that the imtraee llular concentration of cAMP plays an important role

during inflamma tion (Brand-t et aI., 1992). TNF-a. prod uction inside the monocyte and

mac rophages increases durimg inflamma tion (Semm ler et al ., 1993; Evan et al ., 1992;

Torphy et al., 1992; Troplay et al., 1993). cAMP analogs such as dibutyryl cAMP

(dbcAMP) can also increase the production of TNF-a.. POE inhibitors, attenuate the

catabo lism of cAMP and cGMP and regula tion of inflammatory function in many cells,

inc luding monocytes, mast celts, basophil s and neutrophils (forph y et al ., 1992;

Giembycz et al., 1992; Tor-phy et al., 1993). Evidently a spec ific putative se lective

inhib itor of type IV POE ro Iipram, (Nemoz et al., 1985) reportedly was ab le to reduce

TNF-a. production in vitro (Navarro et al., 1998; Navikas er al. , 1998) and in vivo

(Griswold et aI., 1998 ). It has been suggested from in vitro studies that POE inhi bitors

can suppress LPS induced THF-a. production, and the type-lV PO E is mainl y invo lved in

this process . This type of agent which increases the intracellular concentration of cAMP

can inhibit the TNF-a. prod uc:tion inside the macrophages.

1.7 N8tu n of prob lem 8nd Es.perimenbl Obj ectins

Tbe administration of LPS has bee n reported to produce hypo tension (Ibiemennann.

1994). It has also been repo rted tha t a reduction in cardiac output is assoc iated with the

fall in blood pressure (p oon er al., 1997; Cheng et al ., 199 8). It has also bee n su ggested

that changes in haemod ynam ics resulting from the adminis tration of LPS are the result of

an over-prod uctio n of NO (Forfi a et aI., 1998). There is ev idence in the literature that

indicates tha t the adm inistra tion of LPS does result in the induction of inducible iNOS

(Salter et aI., 1991; Thieme rmann et al., 1993). It has been repo rted tha t LPS is

responsible for the over-productio n of NO within the system (Thiemennann. 1994) . The

Increase in NO production has been suggested to result in vascular hyporeectivity

ultimately producing loss of vascul ar tone and cardiovascular co llapse (F leming et al.,

1991; Gray et al ., 199 1; Kengatharan et al ., 1996).

There is evide nce in the curre nt literature which indicates that admi nistra tion of LPS

results in the release of'cytokln es such as lNF-o. (Thiemermann et al., 1993; Liebe rman

et at.. 1989). In add ition, it is believed that TNF-o. is one of the factors tha t acti vates the

process responsible for iNOS induc tion, ultimately leadin g to an over-production of NO

in the body (Ruetten et aI.. 1997), and this over prod uctio n of NO can be inhibi ted by

rolipram. a puta tive selecti ve inhibitor of PDE type IV (Ne moz er al., 1985) .

Collectively, published data in the literature seem 10 support the view that in se ptic shock

cardiovascu lar coll apse results from an increase in plasma TNF -a which eventual ly

results in from an increas e in over-productio n of NO (Kengatharan et aI., l996; Avontuur

-14-

et al., 1990). The over-production of NO has been implicated in the development of

vascul ar hypo-reactivity treated vasoco nstrictor such as noadrena.Iine . Thus , the purpose

of the present investigation was twofo ld: (a) to exam ine the influence of rolipram on

hemod ynam ics, plasma TNF.a levels and production of iNU S in the lungs, ex vivo, in

LPS-treated rats, and (b) to determ ine the effects of admi nistration of a vasoconstrictor

selective a.1-adrenorecepl or ago nist, methoxamine to observe the vascul ar hypo-reactivity

and baemodynamics in LPS-trea ted rats in the absence or presence of rolipram .

Furthermore, in an additional serie s of expe riments a paral lel co mparison was made

betwe en the effects of de xamethasone, an inhibitor of iNOS, and rolipram on the

card iovasc ular system in rats treated wi th LPS.

-15-

1. METHO DS

1.1. Surgical prepa ra ti on of ani mals

Male Long-Evans rats (330 - 360 g) were anaesthetized wi th thiobutabarbital (100

mglkg) i.p.. Ca theters (poly ethylene tubin g; LO. 0.58 mID, 0 .0 . 0.965 mm) were

inserted into the left and right iliac arteries and veins. The catheters inserted into left iliac

artery and vein were used for the measurement of blood press ure , and drug/vehicle

administrations, respectively, while the cathe ters inserted into right iliac artery and vein

were used for blood withdrawal of radio labe led microspheres and return of blood sampl es

after each cardiac ou tput measure ments, respectively. An additional catheter was inserted

into the left ventri cle via the right caroti d artery for injection of radiolabeled

microsphercs . The animal s were tracheotomized and allowed to sta bilize for a period of I

b while arte rial pressure and heart rate were monito red continuous ly.

All cathe ters were filled with heparini zed saline (25 iulm l). Bod y tempe rature was

maintained at 37 ± I"C using a heat ing lamp and monitored using a rectal thermometer.

Arterial blood pressure was recorded with a pressure transducer (Gou ld Statham, USA ;

Model P02JB) co nnected to a Gou ld Universal amplifier and recorder (Go uld, France.

Model 8188.2202-00) . Heart rate was calculated from the blood pressure tracing .

-16-

2.2 . Measu rement o r cardia c:: ou tp u t

This technique has been describe d in detai l elsewhere (Pang, 1983). Briefly,

suspensions of microspberes (Mandel Canada ; 15 urn diameter) labe led with 11CO

(20.000-22,000 in 150 I-I-L) were injected into the left ventricle over a period of LOs.

Blood was withdrawn from the right femoral artery at the rate of 0.35 mUntinstarting 15

s. before microsphere injection using an infusionlwilhdrawal pwn p (Kd Scientific USA;

Model 120) for I min . The blood sample and syringes used for injection of microspheres

or wi thdra wal of blood were counted for radioac tivity at 80-160 Kev using a dual channel

automatic gamma counter (LKB Wallac, Cl inic Gamma Counter , Canada; Model 12TI ).

The withdrawn blood samp le was slowly injected back into The animal s immediately after

counting of radioactivity.

2.3 . Ell:perimcolal Pro toc::o l

& riu I: Animals were randomly assigned to two groups (n = 5): saline-treated (0.8

mlIkg bolus; Group I) and LPS (0.8 mglkg; Group 0) . After the com pletion of surgery ,

blood pressure and heart rate were continuously monitored for 60 min, after which each

animal received either saline or LPS. Five blood samples (120 J.11 each) were collected

into a pre-chil led syringe containin g EDTA to yield a final concentrati on of I mglml.

After centrifugation., the plasma was frozen and stored at ·SO"C unti l it was assayed for

TNF-a . The first blood sam ple was taken just before the adminis tration of saline or LPS

and the other four samples were collected at 30, 60, t 20 and ISO min post saline or LPS

·1 7·

administration.. Cardi ac outp ut was also measured five times in these grou ps ofanimals.

the first measurem ent being just before the administration of saline o r lPS and four other

measure ments thereafter every hr. At the end of each experime nt, the lungs were quickly

excised, placed in liquid nitrogen and stored at -80QC.

Series l/: Animals were randoml y assigned to four groups (n - 5): vehicle-treated

(2· hydroxypropyl-jkyclode xtrin; 2.0 mlIkg; Group Ill), rolipram-treated (3 mgtq;

Group IV and 10 mglkg; Group V), and dexamethasone-trea ted (5 mglkg ; Group VI) .

After the stabilization period each animal was treated with vehicle or drug s. 15·20 min

post treatment with vehicle/drugs a blood sample (120 Vol) was collected for plasma TNF -

a measuremen ts as previous ly described. and cardiac output was measured. Each animal

(Gro ups [u NI) was then trea ted with a bolus dose of l PS (0.8 mglkg). Two more blood

samp les (120 ....1each time) were subsequentl y collected for plasma TNF..a. assessment at

60 and [20 min post· l PS treatm ent. Four hr afte r the admini stration of LPS a second

card iac output measurement was made. Subsequently, methoxamine (100 or 300

....g!kg/mi n) was infused and cardiac output was measur ed 14- [6 min after the start of

infusion. In each animal, repea ted cardiac output measurements we re mad e durin g the

infusion of each dose of methoxamine . The time allowed between each dose of

methoxamine was 15- 16 min . At the end of each experiment, the lungs were quickly

excised, placed in liquid nitrog en and stored at -8O"C.

2.4. TNF~ anay iD plasm a

The totallNF~ in plasma was determined by a commercial ly available colorimetric

enzyme linked immunosorbent assay kit (R&D Systems . MN. USA) for rat lNF.-a. The

sensitivity ofthe assay was 6 pg/ml .

2.5. i'liitri c oxide synthase assa y ia luo gs

Nitric oxide synthase was assessed by measurin g the conversion of CH]L-argini ne to

CH]L-c itrul line as described by Thie mermann et al., (199 3), with slight modifications.

Frozen lungs were homo genized on ice in buffer composed of (in mM) : Tris-H CI. SO;

EDTA. 0.1; EGTA. 0. 1; z-mercaptoetbanot, 12; and phenylmethylsulfonyl fluorid e. I

(pH 7.4). 50 1!1 of homcgenat es were incubated in the prese nce o f [lH]L_arginineIL·

arginine (10~. NADPH (1.0 mM) . calmodul in (10 l.J.g/ ml ). tetrahydro biopterin (5.0

p.M) and Ca l+ (2.0 mM) (total volume of 200 ,.d ) at 37"'C for 30 min. The reactio n was

stopped using stop buffer (LO ml) of the followin g composition (in mM): HEPES . 20;

EDTA. 2.0; and EGTA. 2.0 (pH 5.5). Each samp le was applied to a 2-ml co lumn of

Dowex AG 50W-X8 (sod ium form ) (Bio-Rad Laboratory. Canada ) and eluted four times

with 1.0 mI of stop buffer . Radioactivity in each sample was measured wing a

scinti llation counter (Beckman., USA; Model LS 380 1). Assays were performed in

dup licate in the prese nce of NADPH to determine constiruti ve NOS activity. the absence

of NADP H to determine the exten t of CHJL-citrul line formation independent of NOS.

and in a Cal +_free buffer containing NADPH and EGTA (5 mM) to determine Ca",

indepen dent iNO S activi ty. Protein concentration was measured using Bradfo rd 's

method (B radford. 1976).

2.6. Chemicals

Rolipram , thiobutabarbital. Lcarg inlne and 2-hydroxypropyl-~-cyclodextrin were

purchased from Research Biochemical International (Natick, MA, USA). All other fine

chemic als were purchased from Sigma Chemical Company (Ontario, Canada). Rolipram

was disso lved in 2. hydroxypropyl-tl-cyclodextrin and this was the vehicle used in the

experiments .

2.7. ADalysis or d ata

Mean arte ria l blood pressure (mmHg) is reported as diasto lic blood pressure plus one

third of the differe nce between systo lic and diasto lic blood pressures. Cardiac output

(mllmi n) was calc ulated as the rate of with drawal of blood multiplied by inject ed

c.p.m. divided by c.p. m. in withdra wn blood . Cardiac index is cardiac outp ut divided by

body weigh t. Arterial resistance (mmH g minfmllkg) was obtained by dividing mean

blood pressure by cardiac index.

The data were analyzed by one-way anal ysis of variance with repea ted measures for

comparison. Duncans mul tiple range test was used for comparison between means. In all

cases, a probab ility of error of less than 0.0 5 was selected as the criterion for statistical

significance.

-21>-

3. RESULTS

There were no sign ificant changes in haemodynamic values (cardiac index. mean

blood pressure, arterial resistance and heart rate) over time after the administra tion of

saline (Figure I & 2). In addition, plasma levels ofTNF-a did not change following the

administratio n of saline (Figure 3).

The administra tion of LPS to animals resulted in a significant reduction in cardiac

index over time (Figure IA). At 4 hr post-LPS treatment, cardi ac index was reduced by

over 27% when compared to cardiac index measured prior to the admini stration of LPS.

ALtho ugh arterial res istance and heart rate did increase over time in Lj' Svtreated animals,

these changes were found to be insignificant (Figure IB & 28). Ther e were no

appreciab le changes in mean blood pressure in animals treated with LPS over time

(Figure 2A). The administration of LPS to rats resulted in a substant ial rise in the plasma

levels of TNF-a (Figure 3). The time course for the peak and subseq uent decline in

plasma concentrations of TNF -a was within the 180 min time frame afte r the injection of

LPg (Figure 3). In the present investigation, the peak concentra tion ofTNF-<l detected in

the plasma at 120 min post- LPS injectio n was 190 times that of control levels prior to

LPS administration . Furthermore, the injection of LPg resulted in a significant increase

in the iNOS activity in the lungs of animals ex vivo (Ta ble I) .

l 300

~si~ 200

<:l

TIme c.)

200

100 '-_--;,.-__;-__-;-__-:-__-..;-__-;

Time (h)

-22-

Figure 1. The effects of treatment of anaesthetized rats with saline (0.8 m1Ikg) (do sedcircles) or LPS (0.8 mglkg) (opened circles) on (A) card iac index and (B) heartrate over time. Data represents the mean of five experiments ± SEM.'S ignificantly different from respective values in saline-treated animals, P<0.05.

-23-

10

Tim e (h)

o

T ime (h)

-24-

Figure 2. The effects of treatment of anaesth etized rats with salin e (0 .8 mlIk g) (d osedcircles) or LPS (0 .8 mglkg) (opened circ les) on (A) blood pressure and (B)arte rial resistan ce over time . Data represents the mean of five experiments ±SEM.

-25-

20

15

T

as 1.0

T ime (b)

-26-

Figure 3. The effects of treatment of anaesthetized rats with saline (0.8 mllkg) (closedbars) or LPS (0.8 mglkg) (cross-hatched bars) on plasma concentration ofTNF-a over time. Data represents the mean of five experiments t SEM.'Significantly different from respective values in saline-treated animals, P <O.OS.

T ab le 1. Values of enzymatic activity o f inducible nitric oxide synthase (iN OS) andco nsti tutive (eNOS) fonns of nitric oxide syn thase (pmollmg pro tein/min) in lungs ofvarious group s of animals treated with salin e (0. 8 mIIkg; Group I) . LPS (0 .8 mg/kg;Group II), and vehicle (2 ml/kg ; Group lIT), rolipram (3 mg/kg; Group IV or 10 mg/kg;Group V) and dexamethasone (S mglkg; Group VI) prior to treatment with LPS (0.8mg/kg ). Each value represents mean of five experime nts ± S.E.M .

Group'

Saline (I)LPS(lI)Vehicle+ LPS (III)Rolipram3 +LPS (IV)Rolipram 100L PS (V)Dexam ethasone (VI )

iNOS

0.3 ± O.04L5.0± 3.o-"L3.2± 1.6·19.0±3.~

17.0±4.oat'4.30±0.7"

eNO S

O.4 ± 0.12.8±0.8"1.8± 0 .6"1.8 ±O .6"2.6 ± 0 .S·0.80 ± O.2

-Significan tly di fferent from group I; P -c0.0 5"Signi ficantly different from group VI; p < 0.05

-28-

3.1. Effects of roli pram and dexa metbas one on haem odynam ics in LP8- lrea led ra ts

Prior to the admini stration of LPS, pre- treatm ent of animals with rolipram (3 & 10

mglkg) or dexam eth asone (S mglkg) did not result in significant chan ges to cardiac index,

mean blood pressure, arterial resistance or heart rate when compared to animals that were

treated with vehicl e (Table 2 & 3). There were no significant chang es detected in mean

blood press ure, arte rial resistance or heart rate in animals that were pretreated with

vehicle, rolipram or dexamethasone after LPS injection (Table 2 & 3). In animals that

were pre-treated with vehicle, lPS-treaDDent reduced cardiac index by over 25% when

compared to cardiac index measured prior to the adminiscration of LPS (Table 2). In

contrast. pre-trea tment with rolipram (10 mglkg) and dexamethaso ne prevented the

decline in cardia c index in animals that received LPS (Table 2). Card iac index was

significan tly higher in rolipram (10 mglkg) and dexamethasone treated animals when

compared to vehicle-treated animal s injected with l PS (Table 2).

3.2. Effcels of a,-adnnoce p tor slimulatio n o n ha emod yn am ics in LPS-tnatcd rats

Infusion of methoxamin e (100 & 300 IJglkglmio) did not appear to have any

significant effects on cardiac index in either vehicle-treated or rolipram -treated cats when

compared to the respecti ve values within each group prior to infus ion of the a ,­

adrenoce ptor agonist 4 hr po5t-LPS treatment (Ta ble 2). In contrast, the administra tion

of methoxam ine ( 100 & 300 lJg!kglmin) to dexam ethason e-treated rats produced a

Table 2. Hae mod ynamic changes in various groups of animal s treated with LPS (0.8mg/k g) in the absence or presence of vehicle (2 mlIkg ), rotipram (3 or 10 mg/k g) ordexamethasone (Den; 5 mglkg). The values represe nts the mean of five experiments ±S.E.M.

Pre-LPS Post·LPS Methoxam ine Methoxamine(+4M)

(Groups) Cardiac Index (lOOlJ.g/kglmin) (300 /1g!kgfmin)(mVminIkg)

Vehi cle(lll) 295 ± 22.0 220 ± io.o- 236± 11.0 200 ± 8.0Ro lipram (IV) 334 ± 17.0 258 ± 12.0 258 ± 20.0 226 ±7.0Ro lipramM 342 ± 15.0 280 ± 21.0" 288 ± 29J)" 290 ± 27.ff'"Dexa (VI) 302±27.0 318 ±36.0" 192± to .nt' 2t6±20.0b

Mean BloodPressure(mmHg )

Vebi c1e(lll) to2 ± 2.0 89 ±6.0 99 ± 4.0" to7 ±3JtRo lipram(IV) 96±3.0 81 ±6.0 S4±6.0" 85± 6.~

Ro lipram (V) I03 ± 5.0 83 ± 6.0 88 ± 5.0" 92 ±4.~

De xa (VI) 1l 7± 5.0 107 ± 7.0 1l 4 ± 7.nt' 1l6± 6.ot'

"Signi f icantly diffe rent from Pre·L PS within the same group ; P < 0.05"Signific antly different from Post- LPS-treatm ent (+4 he) withi n the same grou p; P < 0.05"Significan tly different from respect ive values in vehicle-treated group; P < 0.05dSigni fican tly different from respective values in dexamethasone-treated group; P < 0.05

-30-

Table 3. Haemodynarnic changes in various groups of animals treated wi th LPS (0.8mglkg) in the absence or presen ce of vehicle (2 mlIk g), rolipram (3 or 10 mglkg ) ordexamethasone (Den; 5 mglkg). The values represen ts the mean of five experim ents ±S.E.M.

(Groups) AR(mmHgminlmUkg)

Post-LP S(+4"')

Methoxamin e Methoxamine

(100 ""glk glmin) (300 }lglkglm in)

Vehicle (Ill )Rolipram (IV)Rolipram MDexa (VI )

Vehicle (l ll )Rotipram (IV)RoLipram MDexa(Vl)

0.38 ±0.030.29 ± 0.010.30 ± 0.020.40:10.03

Heart Rate(bealsfmi n)

344 ± 11424 ± l3398± 19332 :1 14

0.39 ± 0.030.31 ± 0.020.30 ± 0.030 .35 ± 0.02

430 ± 28462 ± 13445 ± 16380± 12

0.42 ±O.024

0.33 ±0.034

0 .31 t 0.024

0.61 t o.OT'

392± 23460 ± 12428 ± 16388 ± 24

0.51 ± 0.0211cl

0.38 :10.04·0.33 :10.02 ·0.56 ±0.0s'

382± 19456± 17430 ± 24372:1 20

"significantly different from Pest- LPS-lreatDlent (+4 hr) within the same group; P < 0.05"Significan tly different from respec tive values in dexamethasone-treated group; P < 0.05

significant reduc tion in cardiac index whe n compared to respective values prior to

methoxamine infusion in dexamethasone-treated rau 4 hr post-LPS treatment (Table 2).

Administra tion of methoxamine did not significantly affect mean blood pressure and

arterial resistance in rolipram treated animals when com pared to respective values prior to

infusion of methoxamine 4 hr pcs t-Lf'S treatment. Howev er. in vehicle -treated animals.

infusion of methox am ine sign ificantly increased blood. pressure and arteria l resistance at

the higher bu t not lower dose when compare d to respective values prior to the

admini stra tion of C11-ad.renocep tor agonist 4 hr pcst- Ll'S treatment (Ta ble 2 & 3).

Moreover. administration of methoxamine also signifi cantly increased mean blood.

pressure and arteri al resistance at both dose levels in dexamethasone-treated rats when

compared to res pective values prior to the infusion of methoxami ne 4 hr pln-LPS

treatment (Table 2 & 3). Stimulation of C1 1-adrenoceptors did not resu lt in an y significan t

changes in heart rate in any group of anim als (Table 3).

3.3. Erreds of rolipram and dn a mdhasoo e on pla sma levels of TN F-a

Plasma co nce ntrations of the cyto lcine TNF-a were significantly elevated in all

groups subsequent to injection of LPS (fable 4). In animalsthat had received rolipram

(10 mg/kg) or dexamethaso ne. significantly lower plasma levels oflNF-a were detected

at both 60 and 120 min post LPS-trea tme nt when co mpared to respective values in

vehicle-treated animals that had also recei ved LPS (Table 4). However, even though

plasma levels of TNF -a were lower in LPS-treated rats admin istered the lower dose of

Ta ble 4. Plasma TNF-a: values in various groups or animals treat ed with LPS (0.8mglkg) in the absence or presence o r vehicle (2 mlIkg), rol ipram (3 or 10 mgtkg) ordexamethasone (Dexa; 5 mglkg) . The values represents the mean of five experiments ±S.E .M.

TNF-a.(nglml)

(Groups) ~LPSTreatm.ent t -hr PoSl-LPS 2-hr Post- LPS

Vehicle(lJl) 0.004 ± O.OOt 6.0±2.1· 14.8 ± 3.S"Rolipram (IV) 0.00 8 ± 0.004 3.4 ± 0.9" 9.6±3.S"RolipramM 0.003 ± 0.00 1 3.1 ± 1.0"" 4.0± 1.2'"Dcx:a(VI) O.OOS ± 0.005 0.8±0.2ob O.9±O.3ob

' Significantly di fferent from respectiv e values pre-Lf'S treatm ent ; P < 0.05"Slgnlflcan tly di fferent from respec tive values in group III; P < 0.05

·33-

rolipram (3 mglkg) when compared to vehicle-treated animals. differences in plasma

levels oflNF-a were co t found to be significant (Tab le 4).

3.4. Eff~c:ts of ro lipra m an d d~um~thason~ on NOS activi ty in lun gs in LPS-treated

anima ls

The activity of NOS was elevated significantly following the treatment of animal s

with LPS (Table I) . Pre -treatment of animals with roliprnm or vehicle did not affect

NOS activity in animalsthat had received LPS. However . dexamethasone pre-treatment

significantly reduced iNOS activity in the: lungs of animals that had received LPS when

compared to vehicle-treated animals that had also received LPS (fable I).

-34-

4.0. DIS CUSSIO N

Cytokines, such as TNF-a and interleuki n· 1p (lL.I P) play an im po rtan t role in the

cardiovascular sequelae of endotoxaemia (Cavaillon et aI., 1992; Blackwell and

Chritsman, 1996). In a number of patho pys iological states such as septic shock (Kumar

et aL , 1996), acute viral myocar ditis (Smith et al., 1992), card iac allograft rejection

(Arbustini e t al., 1991), myocardial infarction (Maury et aI., 1990), and conges tive heart

fai lure (Levine er aI., 1990 ) the conce ntration ofTNF-a in blood increases . Recently, it

has been reported that the administration ofTNF-a. alone or in combination with a low

dose of endotoxin causes several cardiovascular effects including peripheral

vasodilatation, hypotension , circu latory shock and organ damag e (BiUiau and

Vand erkerckhove, 1991). It has been established that during endotoxaernia, there is a rise

in TNF -a concentration in blood (Beutler et al., 1985). However, it is not known if co­

infusi on of TNF-a and IL-Ill produces cardiovascular changes similar to those that have

obse rved with LPS administration. Like LPS. TNF--a also induces the Calo independent

isofonn of NOS in vitro (Drapier et 81., 1988; K.i1bowneand Be llonl , 1990). Systemic

administration oflNF-a increases NO production (Kosaka et al ., 1992). An increase in

NO prod uction resul ts in systemic vasodi latation (Kilbourne et aI., 1990 ) and vascular

hypo ree ctivity to vasoconstric tors (Vicaul, 1992).

Gardin er and associates (1998) have reported that TNF-a evokes significant

hypo tension accompanied by tachycardia, and dilatation in the renal and hindquart ers

-35-

vascular beds . But this effect is not seen in mesenteric vessels due to endothe lin

producti on by the lNF-a. (Go to et et., 1996; Gard iner et aI.,I 996). It has been aIso

reported that TNF-a. induces rap id (minutes) as well as slow (hours) effects on heart

muscl e (Tracy er al ., 1993). It has been established that TNF-a. is responsible for many

of tbe cardiovascular seque lae ofseptic shock (Tracey and Cerami 1994).

Current evidence in the litera ture suggests that phosphodiesterase type-Iv inhibito rs

arc potential too ls that can inhibi t the producti on of TNF-a. as a result of the action in

elevatin g the intracellular cAMP level (Strieter et al ., 1988; Endres et al., 199 1).

Rolipnun is a specifi c type (V PO E inhibitor, the type IV POE being the predominant

isoenzyme present in mooocytes and it is the enzyme responsible for controlling the

cellular prod uction of TNF-<l (Beavo and Reifsnyder. 1990; Nicholson et aI., 1991;

Torph y et aI., 1991). Semml er and associa tes (1993) have found that PO E inhibitors like

rolipram , methylxanthine and pentcxifyl line, markedly suppress the TNF-a production in

human mo nonuclear cells. They also have established that the inhibitory action of

rolipram is very selective for decreasing TNF -a. level rather than lL-l13 level. The

substantial effectiveness of rolipram in suppressi ng TNF-a. synthesis may be explained

by its effec t in inhib iting type rv POE (forphy et al., 1991 ).

Evidence from the present study indicates that treatment wi th LPg results in a

progres sive decli ne in cardiac index over time . There was also an increase in circula ting

levels of TNF-a. in plasma, as well as an induction of NOS activity in lungs ex vivo.

Pretreatment of ani mals with the putativ e selective phosphodiesterase type lV inhibitor .

-36-

rolipram , or syn thetic glucocorti co id. dexam ethasone. prevented the dec line in cardiac

output due to LPS administrati on . Rolipram and dexameth asone also signi ficantly

reduced the rise in plasma levels of TNF,,- that had resulted from injectio n of LPS.

However, only dexamethasone but not rolipram was able to significantly inhib it iNOS

activity in lungs of animal s that had rece ived LPS.

Administration of LPS to rats leads to hypotension (Thiem ermann. 1994 ). More

recently, it was reported that a single bolus injection of LPS to the rat resulted in a

progressive reduction of cardiac output over time. Associated with this reduction in

cardiac output there was a reduction in mean circulatory filling pressure, an index of total

venous ton e (Poon et aI., 1997). Treatment of rats with LPS did not appear to

significantly affect vascular resistance to venous return (poo n et al., 1997). However.

endotoxic shock has been reported to lead to an impairment o f portal venous flow with

portal venous resistance causing an increase in splanchnic blood pooling and subsequent

decrease in venous return and thus cardiac output. in anaesthetized pigs ( Ayuse et aI.•

1995). Taken together , the evidence would suggest that the reduction in cardiac output in

endotoxic shock is, in part, due to a reduction in venomoto r tone. In the present

investigatio n, da ta indicate that pretre atmen t with LPS resulted in a reduction in cardiac

output without any significant change in arterial resistance. In additio n there were no

significan t changes in heart rate. Thus it is possible that the reduction in cardiac output

observed in animal s treated with LPS in the present investigation was the result of a

reduction in venous return (poon et at.• 1997; Ayus et at.• 1995). It is possi ble that

-37-

treatment with rolipram and dexamethasone augmented venous return thereby resulting in

the maintenance of cardiac output in LPS-treated rats.

However , it is also possib le that reduction of cardiac output may be due to reduction

of cardiac contrac tility. Finkel and assoc iates (1992) have reported that TNF-a. causes a

reduction in the contractile force in the isolated hamster papill ary muscle and this effect

can be blunted by NOS inhibitor (Finkel et al ., (992). A simil ar NO dependent effect of

TNF-a. described in isolated rabbit ventricular tissues (Goldhabe r et aI.• 1996). In

contrast, Yoko yama and associate s (199 3) showed that the reductio n of contractile force

of feline myocytes induced. by TNF-a. was not affected by pretreatment with either W·

uitro-Lcarginine or~ monomethy l· L-arginin e (Yokoyama er al .• 1993). The impact of

TNF-a. on isolated hamster papillary musc le. was immediate , and this effect of TNF -a. on

the myocardiu m is unlikely to be due to over-prod uction ofNO by the activation of (NOS

(Finkel et a]•. 1992; Goldhaber et al., 1996; Yokoyama et al., 1993). In the present

investigation, the reduction of cardiac output in LPS treated rats may have been due to a

reduction in contracti le function of the myocardium .

It is evident from the present inves tigatio n that pre-treatment with rolipram in LPS·

treated rats did not result in inhibition of (NOS but that cardiac index in these animal s

was maintained . This may imply that the reduction in cardiac index following the

administration of LPS is not necessarily due to an over-production o f NO. There is

evidence to indicate that treatment of patients in a slate of septi c shock with a non

selective inhibitor of nitric oxide synthase, ~·nitro·L-arginine methyl ester, results in

-38-

increase mean arteri al press ure and anerial resistance with conco mi tan t reduction in

cardiac output (Avo ntuur et al., 1998). Furthermore, unde r expe rimental conditions,

treatment of rats with methy lene blue in endotoxaemia results in increased arterial

resistance and reduction in cardiac outp ut (Cheng et al., 1998 ). The evidence in the

literature seems to suggest that inhibition of nitri c oxide synthase and/ or NO pathways

does not improve cardiac output in endotoxaemia, and that cardiac output can be

main tained indepe nde ntly of iNOS inhibi tion in endotoxaemia.

In the present investigation, we have found thai pretrea tment of animal s with

dexamethasone attenuated the rise in TNF -a concentration in plasma following treatment

with LPS . This finding is consistent with the evidence in the litera ture which also

indicates that glucocorti co ids are capable of suppressing the rise in TNF -a plasma

concen tration (Srosic-Gru jicic et al., 1982; Smi th et al. , 1980; Oppenhe im et al ., 1982). It

is also apparen t from the prese nt investig ation that pretrea tmen t with de xamethasone also

inhibited the activity of iNOS . Such a finding is consistent with reports presented in the

literature (Beu tler et al. , 1990) . However, in our view it is unlikely that the inhibitory

effect of this substance in mainlaining the cardiac output is the result of inhibition of

NOS. It is perhap s most likely that the effects of dexamethasone in maintaining cardiac

outp ut in LPS treate d rats wasin pan, due to its effec t in reducing levels of plasma TNF­

a. It is evid ent from the literature that inhibition of NOS does not resul t in improvrnent

in cardiac output during endoto xaemia (Avontuur et at, 1998; Cheng et al ., 1998).

-39-

NO is an important medi ator for regulation of vascular tone as well as blood pressure

(Huang et aI., 1995; Takahashi et aI., 1995; Kassab et aI., 1998). How ever evide nce

sugges t that there is an ev er-productio n of NO in endotoxi c and hemo rrhagic shoc k

(Szabo and Thiemermann, 1994 ). It has also been esta blished that the acute

vasodilatation., sustained hypo tension., and hypo reactivity to adrenergic agcn ists, which

characterise the circulatory failure in endotoxic shoc k in vivo, are mediated by increased

release of NO (Thiemermann and Vane. 1990; Kilbourn et al., 1990; Wright et al., 1992).

Unde r physiological co nditions , prod uction of NO from Lcarginine by the constitutive

NO synthase (NOS ) pres ent in vascular cells keeps the vascu lature in a state of active

vasodilatation (Rees et al ., 1990 ). An enhanced formation of NO in response to LPg is an

im portant mediator of hypot ens ion., periphe ral vasod ilatation and vascular hyporeacti vity

to vasoconstrictor agents in endotoxaemia. In additi on., LPS and a numbe r of cytoltines

induce NOS in phagocytic cell s (Stuehr et aI.• 1989).

Th e current result indica te that infusion of metho xamine into animals injec ted with

rolipram and pre-treated with LPS did not result in a signi fican t increase or decrease in

either card iac index or heart rate. However, in animals treat ed with dexamethasone and

pre-treated with LPS, infusion of methoxamine signi fican tly reduced cardiac index . This

was probably due to a substantial increase in arterial resistance. Since, dexamethasone

did inhibit iNOS, vasc ular reactivity to methoxamine was not reduced and thus an

increas e in arterial resistance occurred followin g infus ion with methoxam ine.

-40 -

It appears that the reducti on in cardiac output in dexamethaso ne treated rats was not

the result of signi fican t chang es in the heart rate. Similar pattern was ob served with

infusion of noradr enaline in animal s that were hemorr haged but were pre-trea ted with the

same dose of dexam ethasone (Tabrizchi , 1998). Under norma l circumstances an increase

in arterial res istance (aft erload) can result in a reduction in cardiac ou tput which occurs

due to increased impedance to flow (Nekooeian et al., 1996) . Moreover, a reduction in

arterial resistance can result in the opposite effect and thus increase cardi ac output under

normal , as well as patho physiological conditions (Ta brizchi , 1991 ; Nekooeian et al.,

1998) .

Treatment of animals with LPS resulting in endo toxaemia leads to an induction of

iNOS secondary to an elevatio n of circula ting levels ofTNF-a. However, the decline in

cardiac output followi ng administration of LPS does not co rrelate well with induct ion of

iNOS. At this point, it is evide nt that cardiac index is reduc ed within an hour following

injection of LPS. Sim ilar observa tion were made by other investig ators (poon et al.,

1991; Forfla et al., 1998 ). It seems tha t iNOS begins to have a substan tial impact on the

cardiovascular system within approximately ) hr post-LPS injection (Thi emerm ann et al.,

1994). It is also evident from the present investigat ion tha t the peak co ncentra tion of

TNF-a in plasma does not occur until at least 120 min post -LPS injections. Such an

observatio n is supported by other reports (Michie et al ., 1998 ; Ruenen et el ., 199 7).

However , it is also apparent tha t the concentration of TN F-a is significantly elevated one

hr post-LPS, and certainly this co uld trigger other processes that may have an imm ediate

-4,-

impact on cardiac output and circula tory system. It has been demonstrated that anti -TNF­

a antibodies preven t endotoxi n-induced depress ion of myocardiac contractility (Trac ey et

al., 1981) . Tracey and associates also hypothesized that myocard.iac depression occurs in

response to TNF -a. beca use th~ found tha t infusion of 1lIlF-a. produces hypo tens ion and

decreased card iac output whereas pre treatment with TNF-a. monod onal antibod y

attenua tes th ese effects (1987) . Myocardial contractil ity however, was not measured in

their studies . Collectively, evide nce in the literature and prese nt findings indicate that the

initial negativ e impact of l PS on the cardi ovascular system, and espec ially on cardiac

output., may be independent of iNOS acti vity .

The possibility that lPS can produce inductio n of NOS independent of TNF-a. also

needs to co ns idered . Recently, it was reponed that in vivo expo sure of hwn an colon

epithel ial ce lls to Escherichia coli results in induction of NOS (W inhoft et el ., 1998). In

addition, there is evidence in the literatur e which sugges ts that LPS is able to induce NOS

in cytc kine rece ptor-defic ient mice (le Roy et al., 1998). Moreover , in a recent report, it

was demonstrated that endogenous lNF-a. is not requ ired for LPS-mediated induction of

NO in rats (Xi e et aI.• 1997) . However, in contrast to these observations. earli er studi es

had ind icated that pre-treatment with TNF-a. monoclonal antibodies prevented symptoms

associated with shock in anaesthetized baboons (Tracey et al., 1987). Recently ,

Thiemermann and associa tes (1993 ) had reponed tha t pretreatm ent of rats wi th

monoclonal antibody for TNF-a. prevented the induction of NOS activity in the lung of

anim als that were subsequently treated with LPS (Thiemermann et al., 1993). However,

-42·

in lhe present investigation. the data indicate that a significant reduction in the circulating

levels ofTNF-a in animals treated with rolipram did not prevent induction of NOS in the

lungs of animals treated with LPS. The present findings suppon the vi ew that perhaps

mediatoes oerer than TNF-a may contribute to induction o{NOS in animal s treated with

LPS (Ruetten et at.. 1997). However, it is possible that the effect of LPS may directly

activat e pathways that result in induction of NOS .

To summarise, the present findings indicat e that treatment of rats with LPS results in

a reduction in cardiac index, elevated plasma levels ofTNF-a, as we ll as, induction of

NOS in lungs. Pre-treatment with rolipram and dexamethasone prevents the decline in

cardiac index and signi ficantly reduces the rise in plasma levels of TNF-a as a result of

LPS. Our findings show that dexamethasone inhibits induction of NOS where as

rolipram does not. The results suggests that the induction of NOS may occur , in pan.

independ ent of TNF-a. Furthermore, circ ulating TNf-a may affect cardiac output

independe nt of iNOS.

-43-

5.0 REF EREN CES

Abu-Soud HM. Stuehr OJ. Nitric oxide synthases reveal a role for calmodulin in

controlling electron transfer. Proc Nat.!Acad Sci U S A 1993;90 :10769·12 .

Adams MA. Hirst M. Metopro lol suppresses the development of ethan ol-induced cardiac

hypertrophy in the rat. Can I Physiol Pharmac ol 1990;68 :562-7 .

Aggarwal BB, Kohr WI . Hass PE, Moffat B. Spencer SA, Henze l WJ. Bringman TS.

Ncdwin GE, Goedde! DY, Harkins RN. Hwnan tumo r necrosis factor production,

purifica tion, and characterization. J Bioi Chern 1985;260:2345· 54.

Arbustini E, Grasso M. Diego li M, Bramerio M, Foglieni AS, Albertario M, Martinelli L,

Gavazzi A, Gogg i C. Campana C. et aI. Expression of twnoc necro sis factor in human

acute cardiac rejection. An immunohistochemical and immuno blotting study. Am I

PathoI 1991;139: 709-15.

Avontuur I A. Biewenga M, Buijk SL, Kanhai Kl , Bruinin g HA. Pulm onary

hypcnc nsio n and reduced cardiac output during inhibition of nitric oxide synthes is in

hwnan septic shock.. Shock.199 8;9 : 451-4.

Ayuse T, Brienza N. Revelly JP, O'Donnell CP , Boitnott IK.,Robotham lL . Alterations

in liver hemodynamics in an intac t porcine mode l of endotoxi n shock. Am I Physiol

1995;268: H I 106-14 .

-44-

Beasley 0 , Sc hwartz JH, Brenner 8M. lnter leukin 1 induces prolo nged Li-arginine­

dependen t cycli c guan osine moeophosphate and nitrite production in raa vascular

smoo th muscle ce lls. JClin lnvest 1991;87:602-8 .

Scavo JA, Reifsnyder DH. Primary sequence of cycl ic nucleotide phosphoxti estetase

isozymes and the des ign of selective inhibitors . Trends Pharmacol Sci 1990;.11:150-5.

Beutler B. Cerami A. The endoge nous mediator of endc toxic shock . Clio Res

1987;35:192· 7.

Beutler B, Milsatk [W, Cerami AC. Passive imm unization against cechecctin'tumo r

necrosi s factor protects mice from lethal effect of endotoxin.. Science 19850;229 :869­

71.

Beutler B, 1bompson P, Keyes J, Hageny K. Crawford D. Assay of a ribo nuclease that

preferentially hydro lyses mRNAs containing cyto kine-derived UA-rich rinstab ihry

sequences. Bio chem Biophys Res Cornmun 1988;152:973·80.

Beutler BA, Milsark IW, Cerami A. Cachectinltum or necros is factor: pe odu ction,

distri bution. and metabolic fate in vivo. J Immun oI 198S;135:39n-7.

Billiau A, Vandekerckhove F Cytcki res and their interactio ns with other inflammatory

mediato rs in the pathogenesis of sepsis and septic shock. Eur J Clin Invest 1 99 1~ 1 :

559-73.

Blackwell TS, Christm an IW . Sepsis and cyto kines : current status. Br J" Anaesth

1996;77: 110- 7.

.....

Bradford MM. A rap id and sensitive method for the quantitatio n of micro gram quantities

of protein util izin g the principle of protein -dye binding . Anal Biochem 1976;72:248­

54 .

Brand t E, Peterse n F, Flad HD. Recom binant tumor necrosis factor -alpha potentiates

neutrop hil degran ulat ion in response to host defense cytckines neutrophi l-activating

peptide 2 and IL-8 by modulating intracell ular cyclic AMP levels . J Immuno l

1992; 149:1356-64.

Bredt OS, Hwang PM. Glan CEo Lowenstein C, Reed RR., Snyder SH. Cloned and

expressed nitric oxide synthase structura lly rese mbles cytochrome P450 reductase .

Nature 1991;351 :714-8 .

Buga GM , Gold ME , Fukut o 1M, Ignarro U . Shear stress- induced release o f nitric oxide

from endothelial ce lls grown on beads. Hypertens ion 1991;17: 187-93 .

Bush PA, Gonza lez NE, lgnarro U . Biosynth esis of nitric oxide and citrulline from L­

arginine by cons titutive nitric oxide synthase present in rabb it corpus cavernosum.

Bioc hem Biophys Res Commun 1992;186:308-14 .

Busse R. Mulsch A . Inducti on of nitric oxid e synthase by cytc kinee in vascular smooth

muscle cells. FEBS Lett 1990;275: 87-90 .

Cavaill cn 1M, Munoz C. Fining C, Misset B. Cadet J. Circulating eytckines : the tip of

the iceberg. Circ Shock 1992;38: 145-52.

-46-

Charbonnea u H. Be ier N, Walsh KA. Beavo IA. Identification of a conserved domain

among cyclic nucleotide phosphodiesterases from diverse species . Proc Nat! Acad Sci

U S A 1986;83:9308.12.

Charbonn eau H. Prusti RK, LeTrcug H, Sonn enburg WK, Mullane y PI, Walsh KA,

Beavc IA . Identification of a noncatalytic cGMP-binding domain conserved in both

the cGMP-stimulated and photoreceptor cyc lic nucleotide phosphodie sterases . Proc

Nat! Acad Sci U SA 1990;8 7:288-92.

Cheng X. Pang CCY . Pressor and vasoconstrictor effects of methylene blue in

endotoxaemic rots. Naunyn Schmiedebergs Arch Pharmaco l l998 ;3S7: 648·53 .

Coquil JF. Properties oflhe cyclic GMP phosphod iesterase from rot lung. Inhib ition by

unsaturated fatty acids. Biochim Bioph ys Acta 1983;743:359-69.

Craven PA, DeRuberti s FR. Restoration of the respon siveness of purified guany late

cyclase to nitrosoguanidine, nitric oxide, and related activators by heme and

hemeproteins. Eviden ce for involvement of the param agnetic nitrosyl-heme complex

in enzyme acti vation. J Blol Chern 1978;253 (23) :8433-43 .

Dinaxello CA, Cannon JG. Wolff SM, Bernheim HA. Beutler B. Cerami A, Figari IS,

Palladino MA lr , O'Connor N . Tumor necrosis factor (cachectin) is an endogenous

pyrogen and induces production ofi nterleukin I . J Exp Med 1986;163:1433-50.

Ding AH, Sanchez E, Srimal S, Nathan CF . Macroph ages rapidly internal ize their tumo r

necrosis factor receptors in response to bacterial lipopolysaccharide. 1 Bioi Chern

1989;264:3924-9.

-47-

Ojeu IV. Blanchard OK. Halkias 0 , Friedman H. Growth inhibition of Candida albicans

by human polymorphonuclear neutrophils: activa tion by interferon-gamma and tumor

necrosis factor. J Immun oI1 986;137:2980-4 .

Drapier I e , Wle tzerbln J. Hibbs 18 Jr Interferon-gamm a and tumor necrosis factor induce

the L-argini ne-dependent cytotoxic effector mechanism in murine macrophage s. Eur I

Immunel 1988;18: 1587-92 .

Duhe RI . Nielsen MD. Dittman AH, Villacres EC. Choi EJ. Storm DR. Oxida tion of

critical cysteine residues of type I adenylyl cyclase by o-iod osobenzoa te or nitric oxide

revers ibly inhibits stimulation by calcium and calmodulin. J Bioi Chern

1 994~69( 1 0) :7290-6 .

Eidlernan LA, Pizov R. Sprung CL. New therapeutic approa ches in seps is: a critical

review .lnteVSiveCateMed21 :S269 .1995.

Endres S, Fulle HI. Sinha B, Stoll D. Dinarello CA, Gerzer R, Weber PC. Cyclic

nucleotid es d ifferent ially regulate the synthesis of tumour necrosis factor-alpha and

interleuki n-I beta by human mononuclear cells. Immun ology 1991;72: 56-60 .

Estrada C, Gom ez C. Manin C. Moncada S. Gonzalez C. Nitric oxide media tes rumo r

necrosis factor -alpha cytotoxicity in endothel ial cells. Biochem Bicph ys Res

Comm un 1992;186 :475-82 .

Evans T. Carpenter A, Cohen J. Purificatio n of a distinctive form of endcrc xin-induced

nitric oxide synthase from rat liver. Proc Nat! Acad Sci USA 1992;89:536 1.5.

-48-

Evans T, Carpenter A. Silva A, Cohen J. Differenti al effects of monoclo nal an tibod ies to

tumo r necro sis facto r alpha and gamma interferon on induc tion of hepatic nitric oxide

synthase in ex perime ntal gram- negative sepsis. Infect Immun 1992;60:4133-9.

Fahey TJ 3rd, Yoshioka T, Shires QT, Fanti ni GA . The role of tumor necrosis factor and

nitric oxide in the acute card iovascul ar response 10 endotoxin. Ann Surg 1996;223:63

9.

Finkel MS. Oddi s CV, Jaco b TO, Watkins SC. Hanler BG, Simmons RL. Negative

inotro pic effec ts of cytokiaes on the heart mediated by nitric oxide . Science 1992;257:

387-9.

Fleming I, Gra y GA., lul ou-Schaeffer G. Parratt JR Incubati on with endotoxin activ ates

the L-argin ine pathway in vascular tissue . Biochem Biophys Res Comm un

1990 ;171:562-8.

Fleming I. Julc u-Schaeffer G. Gray GA., Parrett JR. Stod ct IC. Evidence that an L.

arginine/nitric ox ide dependent elevation of tissue cyclic G MP content is involved in

de pression of vascular reactivi ty by endotoxin . Br I Pbarm acol 1991;103:1047-52 .

Fa ng Y. Trace y KJ. Moldawer l l , Hesse 00. Manogue KB. Kenney IS, l ee AT. Kuo

GC . Alliso n AC, lowry SF. et at Antibodies to cachc:ctinfWmor necro sis factor

reduce interie ukin 1 beta and interleukin 6 appearance during lethal bacterem ia. I Exp

Med 1989;170 :1627-33.

Forfia PRo Zhan g X. Ocho a F. Ochoa M. Xu X. Bernstein R, Sehgal PB, Ferreri

NR,Hintze TH . Relationship betwe en plasm a NOx and cardiac and vascular

....-

dysfuncti on afte r LPS injec tion in anesth etized dogs. Am 1 Physi ol 1998 ;274:HI93­

20 t.

Forstennann U, Pollock 1S, Sd unidt HH, Heller M, Murad F. Calmodulin-dependent

endothe lium-derived relaxing factor/nitri c oxide synthase activity is presen t in the

particulate and cyt oso lic fiactions of bovi ne aortic endothelial cell s. Proc Natl Acad

Sci U S A 199 1;88: 1788-92.

Francis SH, Lincoln TM, Corbin ID . Characterization of a novel cGMP bind ing protein

from rat lung. 1 Bioi Chern 1980;255:620-6.

Fukuto 1M, Wallace GC, Hszieh R.. Chaudhuri G. Chemical oxidation of N­

bydroxyguanidine compounds Release of nitric oxide, nitrox yl and possib le

relationship to the mechanism of biological nitric oxide genera tion. Biochem

Pharmacc l 1992;43:607-13.

Fukuto 1M, Hobbs N , fgnarro U . Conversio n of nitroxyl (HNO) [0 ni tric oxi de (NO) in

biological systems: the role of pbysiological oxidants and relevance to the biological

activity of HN'O. Biochem Biophys Res Co nunun 1993;196:707-13.

Gardiner SM, Kemp PA, March IE, Woolley J , Bennett T. The influence of antibodies to

TNF-a1pha and IL- l beta on haemodynamic responses to the cyto kines, and to

lipopol ysaccharide, in consc ious rats. Br J Phannacol 1998;125: 1543 -50 .

Gardin er SM, March JE, Kemp PA, Bennett T. Regional haemodynam ic effects of

antagonists of angiotensin Il , endothelia and adrenoceptors in cons cious, vasopressin­

deficient, genetically hypertensive rats. Br J Pharmacol 1996;118: 325 -34 .

-so-

Giembycz MA. Dent G. Prospects for selective cyc lic nucleotide phosphodiesterase

inhibitors in the treatmen t ofbronc:hial asthma. Clin Exp Allergy 1992a2:337-44 .

Goldhabc r Jl , Kim KH, Nanerso n PO, Lawrence T, Yang P, Weiss IN . Effects ofTNF­

alpha on [Ca2+]i and contractili ty in iso lated adult rabbit ventricular myocytes. Am J

PhysioI1 996;271: HI 449-SS.

Goto K, Hama H. Kasuya Y. Molecular pharmaco logy and pathophys iological

signi ficance of endothe lin. Jpn J Pharma col l996;n: 261·90.

Gra y GA. Schon C, Julcu-Schaeffer G, Fleming I, Parratt JR..,Stoclet JC. The effect of

inhibit ors of the L·arginin clnitric oxide pathwa y on endotoxin-induced loss of vascular

respons iveness in anaesthetized rats . Br J Pharmacoll99 I;103 :1218-24 .

Greenway SM . Renal transplantation in the USA. Nun Times 1982 Dec IS·

21;78(SO):2131-4.

Griscavage JM, Rogers NE, Sherman MP, Ignarro U . Inducible nitric oxide synthase

from a rat alveolar macrophage cell line is inhibited by nitric oxi de. J Immuno!

1993 ;ISI :6329-37.

Griswold DE, Webb EF. Badger AM, Gorycki PO, Levandos ki PA, Barne tte MA. Grous

M. Chris tensen S. Torphy TJ. 58 207499 (Aritlo), a second generation

phosphodiesterase 4 inhibitor, reduces tumor necrosis factor alpha and interleukin-4

production in vivo. J Pharmaco l Exp Ther 1998;287:70S· 11.

Gross 55, Stuehr OJ, Aisaka K, Jaffe EA, Levi R, Griffith OW Macrophag e and

endothe lial cell nitric oxide synthesis: cell-type selectiv e inhibition by NO-

·51-

aminoargininc , NG-nitroarginine and NG-methylarginine . Biocbem Biophys Res

Commun 1990;170: 96-103.

Guyton AC, Jones CEo Central venous pressure: physiolog ical significance and clinical

implications. Am Hean J I 973;86;43 1 ~7 ,

Halme J. Release of tumo r necrosis factor-alpha by bwnan peritoneal macropha gcs in

vivo and in vitro . Am J ObstctGynecoI1989;16 1:1 718~25 .

Hattori R.., Kosuga K., Eizawa H, Sese K. Inoue R, Sunamoto M, Ichimori Y, Sate K.

Mori. Stabi lization of inducible nitric oxide synthase by monoclonal antibodies.

Hybridoma 1 993 ;1 2:763~70 ,

Haynes RC Jr and Murad F 1985 in Goodman and Gilman's The Phanna cologica1 Basis

of The:rapeutics .,. Ed. Pp 1459-1489.

He W, Fong Y, Marano MA, Gershenwald JE. Yurt RW, Moldawer Ll, Lowry SF.

Tolerance to endot oxin preve nts mortal ity in infected thermal injury: assoc iation with

attenuated cytokine responses , J Infect Dis 1992;165:859-64 .

Heinzel B, John M, Klatt P, Bohme E, Mayer B. Ca1+lcalmodulin-d.ependent formation

of hydrogen peroxide by brain nitric oxide synthase. Biochem J 1992;281:627-30.

Hesse DO, Tracey KJ, Fong Y, Manogue KR, Palladino MA Jr, Cerami A, Shires QT,

Lowry SF. Cytokine appcanance: in human endotoxemia and primate bacterem ia.

Surg Gynecol Obste t 1988;166:147-53.

-52·

Hevel 1M, White K.A. Marletta MA . Purification of the inducible murine macrophage

nitric oxide synthase. Identification as a flavoprotein. J BioI Chern 1991;266:22789·

91.

Hintze TH Relationship betwee n plasma NOx and cardi ac and vascul ar dysfunc tion after

LPS injection in anestheti zed dogs. Am J Physiol 1998 ;274: H I93·201.

Hintze TH . Relationship between plasma NOx and card iac and vascular dysfunctio n after

LPS injection in anesthetized dogs. Aml Physioll998; 274: H I93 .201.

Hobbs AJ, Fukuto 1M, Ignarro U . · Forma tion of free nitric oxide from l-arginine by

nitric oxide synthase : direct enhancement of generation by superoxi de dismutase.

Proc Natl Acad Sci USA 1994;91:10992-6.

Huang PL, Huang Z. Mashimo H, Bloch reo , Moskowitz MA., Bev an lA , Fishman Me .

Hypert ension in mice lackin g the gene for endothel ial nitric oxide synthase. Nature

1995;377: 239-42.

Hutcheson IR.., Whittle Bl , Boughton-Smith NK. Role of nitric oxide in maintaining

vascular integrity in endotoxin -induced acute intestinal dama ge in the rat. Br 1

Pharma co l l 990; I01:815-20.

Ignarro U , Degnan lN , Baricos WI{. Kadowitz Pl , Wolin MS . Activa tion of purified

guan yla te cyclase by nitric oxid e requ ires heme. Comparison of heme-deflcien t, heme.

reconsti tuted and heme-containing forms of soluble enzyme from bovine lung.

Biochim Bioph ys Acta 1982,17;7 18(1):49-59.

-53-

Ignarro U . Haem-depcndent activation of guan ylate cyclase and cyc lic GMP formati on

by endogenous nitri c oxide: a unique transduct ion mechani sm for tran scellular

signaling. Pharma col Toxicot 1990 ;67(1):1. 7.

Kass DA,. Yamazaki T. Burkhoff 0 , Maughan WL, Sagawa K. Detennination of left

ventricular end-systolic pressure -vo lume relationships by the co nduc tance (volume)

catheter technique. Circulation 1986;73:586-95.

Kassab S, Miller MT, Hester R, Nov ak J, Granger JP Sys temic hemod ynamics and

regional blood flow during chro nic nitric oxide synthesi s inhibition in pregnant rats .

Hypertension 1998 ;31:315·20.

Kaszaki J, Wolfard A,. Sari F, Boro s M, Parran IR., Nagy S. Effec t of nitric oxide

synthase inhibition on myocard ial contracti lity in anesthe tized norm al and

endoroxemic dogs. Shock 1996;6 :279· 85.

Kengatharan KM, De Kimpc SJ, Thie mermann C. Role of nitric oxid e in the circulatory

failure and organ injury in a rodent model of gram-positive shock . Br I Pharmac ol

1996;119:14 11-21.

Kilboum RG, Belloni P. Endothelial cell production of nitrogen oxid es in response to

interferon gamma in combination with tumor necrosi s facto r, interle ukin- I, or

endotoxin. I Natl CancerInst 1990 ;82:772-6.

Kilbourn RG, Jubran A, Gross S8 , Griffi th OW, Levi It. Adams J, Lodato RF. Reversal

of endotoxin-media ted shock by NG-memyl·L-arginine, an inhib itor of nitric oxide

synthesis. Biochern Biophy s Res Commun 1990;172: t t 32·8 .

-54 -

Klan P, Heinze l B, John M, Kastner M, Bohme E, Mayer B. Ca2+/calmodulin-dependent

cytochrome c reductase acti vity of brain nitric oxide synthase. J Bioi Chern

1992;267 :11374-8 .

Klatt P, Schm idt K, Mayer B. Brain nitric oxide synthase is a haemo protei n. Biocbem J

1992;288:15· 7.

Know les RO, Merre tt M, Salter M, Moncada S. Differentia l induc tion of brain, lung and

liver nitric oxide synthase by endotoxin in the rat. Bioc hem J 1990:270:833-6.

Knowl es RO, Salter M, Brooks st, Moncada S. Anti-inflammatory glucocortico ids

inhibit the induction by endotoxin of nitric oxide syn thase in the lung, liver and aorta

of the rat. Biochem Biophys Res Commun 1990; l n : 1042-44.

Kosaka H. Harada N, Watana be M. Yoshihara H, Katsuki Y, Shiga T. Synergistic

stimu lation of nitric oxide hemoglobin production in rats by recombi nant interleukin 1

and tumo r necrosis factor. Bioc hem Biophys Res Commun 1992; 189: 392·7.

Kubes P, Suzuki M. Grange r ON. Nitric oxide: an endoge nous modu lator of leukocyte

adhesion. Proc NatlAcad Sci U 5 A 1991;88:465 1·5 .

Kumar A, Theta V, Dee L, Olson J, Uretz E, Parrill o IE. Tumor necros is factor alpha

and interleukin l beta are responsible for in vitro myocardial cell dep ression induced

by hwnan septic shock serum. J Exp Moo 1996;183 : 949-58.

Kuo PC . Schroeder RA. The emerging multifaceted roles of nitric oxide . Ann Surg

1995;221(3):220-35 .

-55-

Lander HM. Sehajpal P, Levin e OM , Novogrodsky A. Activation of human peripheral

blood mononuclear cells by nitric oxide-generating compounds. J Imm unol 1993 Feb

15;150(4 ):1509- 16.

Le Roy 0 , Heumann D, Glauser MP, Maud J, Smith I , Betz Corrad in S. Nitric oxide

production in experimen tal gram-negative infection: studie s with cytckine recep tor-

deficient mice . Shock 199 8;10: 37-42.

Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated circu lating levels of tumor

necros is factor in severe chronic heart failure. N Engl I Med 1990;323: 236-41 .

Levy B. Fixed -dose co mbina tion diure tics in congestive heart failure: an eval uation. J

C lio PbarmacoI1979;19:743--6.

Li TS, Vo lpp K, App lebury ML . Bovine cone photorecep tor cO MP phosphodi esterase

structure deduc ed from a cD NA clone. Proc Natl Acad Sci U S A 1990;87:293-7.

Lieberman AP , Pitha PM, Shin HS, Shin ML . Productio n of twnor necros is factor and

other cytokines by astroc ytes stimulated with lipopolysaccharide or a neurotrop ic

virus. Proc Nat! Acad Sci US A 1989;86:6348-52.

Marlena MA , Yoon PS, Iyengar R.Leaf CD, Wishnok IS . Mac rophage oxidation o f L-

arginine to nitrite and ni trate : nitric oxide is an intermed iate. Biochemistry

1988~7:87()6.. 1 1.

Marlena MA. Nitric oxide : biosynthes is and biological signi fican ce . Trends Biochem

Sci 1989; 14: 488-92 .

-56 -

Martins TJ, Mumb y Me, Beavo JA. Purifica tion and characteriza tion o f a cyclic GMP­

stimulated cyclic nucleotide phosp hodiesterase from bovine tissues . Bioi Chem

1982;257:1973-9.

Mayer B, John M, Bohm e E. Purificatio n of a Ca2+/calmoduIin-dependent nitric oxide

synthase from porcine cerebellum. Cofactor-role of tetrahydro bio pterin. FEBS Lett

1990;277:2 15-9 .

Mayer B. Klan P, Bohme E.Schmidt K. Regulation of neuronal nitric oxide and cyclic

GMP formation by Ca2+. J Neuroc hem 1 992~9 :2024-9.

McDonald U . Moss J. Stimulation by nitric oxide of an NAD linkage to glyce raldehyde­

3-phosphate dehydro genase Proc Nat! Acad Sci US A 1993;90(13):623 8-4 1.

Michie HR..,Man ogue KR.. Spriggs DR, Revhaug A. O'Dwyer S, Dinarello CA, Cerami

A, Wolff SM, Wilmore DW. Detec tion of circulating rumor necro sis factor after

endotoxin admini stration . N Eng1J Med 1988;318:1481-6.

Moser R, Schle iffenbaum B, Grcsc urth P, Fehr J. Imerleukin I and tum or necrosis factor

stimulate human vascular endothel ial ce lls to promote ttansen doth elial neutrophil

passage. J Cli n Invest 1989;83:444-5 5.

Nakane M. Mitche ll J. Fomermann U. Murad. F. Phosphorylation by calcium

calmodulin-dependent protein kinaseITand protein kinase C modul ates the activity of

nitric oxide synthase. Biochem Biophys Res Commun 1991;180:1396-402.

Navarro J, Punzo n C. Jimenez IL. Fernandez-Cruz E, Pizarro A. Fresno M, MWlOZ-

Fernandez MA. Inhibi tion of phosph odiesterase type IV suppresses human

-57-

immunodeficiency virus type 1 replication and cytokine production in primary T cells:

involvement ofNF-kappaB and NFAT. J Virol 1998;72:4712-20.

Navikas V. Matusevicius D. Soderstrom M, Pirskanen R, Fredrikson S. Link H. The

phosphodiesterase i v, inhibitor rolipram in vitro reduces the numbers of MBP­

reactive IFN-garruna and TNF-alpha mRNA expressing blood mononuclear cells in

patients with mul tiple sclerosis. Clin Neuropharmacol 1998;21:236-44 .

Nedwin GE, Goedde! DV, Harkins RN. Human tumor necrosis factor. Production,

purification, and characte rization. J Bio i Chem 1 985~60 : 2345-54 .

Nek.ooeian AA, Pang CCY . Estrogen restores role of basal nitric oxide in control of

vascular tone in rats with chronic heart fai lure. Am 1 Physiol 1998; 274: H2094-9

Nekooeian AA, Tab rizchi R. Effects of adenosine Al A receptor ago nist. CGS 2 1680, on

blood pressure, cardiac index and arterial conductance in anaesthetized rats. Eur 1

Pbannaco I 1996;307: 163-9.

Nemoz G, Prigent AF. Moueqqit M. Fougier S. Macovschi O. Pacheco H. Selective

inhibition of one of the cyclic AMP phosphodiesterases from rat brain by the

neurotropic compoun d rolipram. Biochem Pharmacol 1985;15;34:2997-3000.

Nicholson D. White TO. Sawynok 1. Forskolin and phosphodieste rase inhibitors release

adenosine but inhibit morphine-evoked release of adenosine from sp inal cord

synaptosomes. Can 1 Physiol Pharmacal 1991;69: 877-85.

Pang CCY. Effect of vasopressin antagon ist and saralasin on regional blood flow

following hemorrhage. Am 1 Physiol 1993;245:H749-5S.

-58-

Parker JL. Keller RS, Behm Lt. Adams HR. Left ventricular dysfunction in early E-coli

endotoxemia: effects of naloxone. Am 1 Physiol 1 990~9:H504-11 .

Parrillo IE : Pathog eneti c mechanisms of septic shock. N Engl J Med 328 (20): 1471,

1993.

Petit JF. Schott C. Lepoivre M. Stodet JC. Production of an arginine-derived relaxing

factor induced by IFN-gamma plus endotoxin in murine adenocarc inoma EMT 6 cells .

Biochem Biophys Res Commun 1990;173:1-5.

Pollock JS. Forstermann U. Mitchell JA, Warner ro. Schmid t HH. Nakane M. Murad F.

Purification and charac terization of particulate endothelium-derived relaxing factor

synthase from cultur ed and native bovine aort ic endo thelial cells. Proc Narl Acad Sci

USA 1991;88 :10480 -4.

Poon CS. Merri ll CK. Decrease of card iac chaos in congestive heart failure. Nature

1997;389:492-5.

Pou S. Pou WS. Bredt OS, Snyder SH. Rosen GM. Generation of superoxide by purified

brain nitric oxide synthase . J Bioi Chern 1992;267:24 173-6 .

Radomski MW, Palmer RM, Moncada S. Characteriza tion of the L-arginine:nitric oxide

pathway in human platelets. Br J Pharm acol 1990;101:325-8.

Radomski MW. Palmer RM. Moncada S. Glucoco rticoid s inhibi t the expression o f an

inducible. but not the constitutive, nitric oxide synthase in vascu lar endothelial cells.

Prcc Natl Acad Sci USA 1990;87: 10043-7.

Rees DD, Ce llek S, Palmer R.M. Moncada S. Dexamethasone pre vents the inductio n by

endotoxi n of a nitric oxide syn thase and the associated effec ts on vascular tone: an

insight into endotoxin shock . Biochem Bioph ys Res Commun 1990;173: 54 1-7.

Ruetten H, Thiemermann C. Interleuk in-13 is a more potent inhibito r of the expression

of induci ble nitric oxide synthase in smooth muscle cells than in macrophages: a

comparison with interleukin-4 and interieukin-IO . Shock 1997; 8: 409-14.

Rutten AJ, Bersten AD, Whit ing MI . Endotoxin alters the systemi c dispos ition of nitric

oxide synthase inhibitors in the awake sheep. Clin Exp Pharma col Physiol 1997;24 :

391-7.

Sagawa K, Kwnada M, Shoukas A. A systems approach to baro recep tor reflex control o f

the circ ulation. Aust J Exp Bio i Med Sci 1973;5 I :33 ~52.

Salter M, Knowles RG, Moncada S. Widespread tissue distributi on, species distribution

and changes in activity of Ca(2+).dependent and Ca(2+)-ind epend ent nitric oxide

synthases. FEBS Lett 1991;29 1:145-9.

Salter M, Knowles RG, Moncada S. Widespread tissue distribution. species distribution

and changes in activity of Ca(2 +)-dependent and Ca(2 +)-independent nitric oxide

synthases . FEBS Lett 1991;29 1: 145-9.

Sharma RIC.Shalaby MR. Aggarwal BB, Rinderknecht E, Svedersk y LP. Finkle BS,

Palladino MA Jr, , Wang lli. Purificati on and charac terizati on of bovine lung

calmodulin-dependent cyclic nucl eotide phosphodie sterase. An enzyme containing

calmodul in as a subunit. J Bioi Chern 1986;26 1:14160-6.

....

Schini VB. Vanhoutte PM. Inhib itors of calmod ulin impair the constitutive but DOt the

inducible nitric oxide synthase activity in the rat aorta. J Pharmaco l Exp Tber

1992;261:55J-9.

Schm idt HH. Murad F. Purification and characterization of a human NO synthase.

Biochcm Biophys Res Commun 1991:181: lJn·7 .

Schulz R.., Nave E. Moncada S. Inductio n and potential biological relevance of a Ca(2+}­

independent nitric oxide synthase in the myocardium. Br J Pharmacal 1992;105 :575·

80 .

Schulz R, Panas DL. Catena R, Moncada S, Olley PM. Lopaschuk GO. The role of nitric

oxi de in cardiac dep ression induced by interleukin- I beta and tumour necrosis factor-

alpha. Br J Pharm aca l 1995;114:27-34.

Semmler J. Wachtel H. Endres S. The specific type IV phosphodiesterase inhibitor

rolipram suppresses tumor necrosis factor-alpha production by hwnan mononuclear

cell s. lnt J Immunophann acoI 199J ;15:409- I3.

Smith KA. T-ce ll growth factor . Immuno! Rev 1980;5 l :J J 7·57.

Sm ith SC. Allen PM. Neutralization of endogenous tumo r necrosis factor ameliorates the

severity of myosin. induced myocarditis. Ctre Res 1992 ;70: 856-6J .

Solo rzano ce. Ksontini R..,Pruitt 1H, Auffcnberg T. Tannahill C. Galardy R.E. Schultz

GP. MacKa y SL. Copeland EM Jrd, Moldawer LL. A matrix metal loproteinase

inhibitor prevents processing of tumo r necrosis factor alpha (TNF alpha) and abrogates

endc tcxin-induced lethality . Shock 1997;7:427· J 1.

-6,.

Stewart AG, Phan LH. Grigoriadis G. Physio logical and pathophysiological roles of

nitric oxide. Microsurgery 1994;1S(10);693-70 2.

Stosic-Grujicic S, Simic MM. Modulation of interleukln I production by activated

macrophages: in vitro action of hydrocorti sone, colchicine, and cytochal asin B. Ccll

ImmunoI 1982;69;235-47.

Strieter RM, Remick 00. Ward PA., Spengler RN. Lynch JP 3d., Larrick J, Kunkel SL.

Cellular and molecul ar regulation of tumo r necrosis factor-alpha production by

pentoxifylline. Biochem Biophys Res Commun 1988;155: 1230-6.

Stuehr OJ, Cho HJ, Kwon NS, Weise MF, Nathan CF. Purification and characterization

of the cytokine-induced macrophage nitric oxide synthase : an FAD and FMN­

containing flavoprote in, Proc Natl Acad Sci U SA 1991;88:7773-7.

Stuebr OJ, Fasehun OA., Kwcn NS. Gross SS, Gonzalez JA., Levi R.. Nathan CF.

Inhibition of macrop hage and endothelial cell nitric oxide synthase by

diphenyleneiodoniwn and its analogs . Biochem Bioph ys Res Commun

1991;18 1:1372-7.

Stuehr OJ, Gross SS. Sakwna I, Levi R.. Nathan CF. Activated murine macrophages

secrete a metabolite of arginine with the bioactivity of endotheli um-derived relaxing

factor and the chemical reactivity of nitric oxide . J Exp Moo1989;169: 1011-20.

Stuehr 0 1. Kwon NS, Gross 55 . Thiel BA, Levi R.. Nathan CF. Synthes is of nitrogen

oxides from L-arginine by macrophage cytoso l: requirement for inducible and

constituti ve components. Biochem Biophys Res Commun 1989;161:420-6.

Szabo C. Thiemermann C. Invited opinion: role of nitric oxide in hemo rrhagic,

traumatic, and anaphy lactic shock and. thermal injlllJ' . Shock 1994;2: 1 45~55 .

Tabrizchi R. Cardiovascular effects of noradrenali ne in hypo volemic haemorrhag e: role

of inducib le nitric oxide synth ase. Eur J Pharmacal 1998;361: 227-34.

Tabrizchi R. Effects of adenosine and adenosine analog ues on mean circulatory filling

pressure and cardiac output in anesthetized rats . Naunyn Schm iedebergs Arch

PhannacoI1 997;3S6: 69-75.

Takahashi H. Han K, Kom iyama Y, Masuda M, Murakami T, Nishimura M. Nambu A..

Yoshimura M. Mechani sm of hypertension induced by chronic inhibition of runic

oxide in rats . Hypertens Res 1995;18: 319-24.

Takahashi H, Nakanishi T, Nishimura M. Tanaka H. Yoshimura M. Effects of

endothe lia- 1 and inhibition of nitric oxide production with NO-monomethyl·L ·

argini ne on arterial pressure and regional blood flow in anes thetized rats. J Cardiovasc

Pharmacol 1992;20 Suppl 12: SI76-8.

Tayeh MA, Marlena MA. Macrophage oxidatio n of L-arg inine to nitric oxide. nitrite.

and nitrate , Telrahydrob iopterin is required as a cofacto r. J Bioi Chern

1989;264 :1%5 4. 8.

Thiemennann C, Vane J. Inhibi tion of nitric oxi de synthes is reduces the hypotens ion

induced by bacterial lipopo lysaccharides in the rat in vivo. Eur J Pharmacal 1990; 182:

591-5.

"3-

Thiemennann C, Wu Cc. Szabo C. Perretti M, Vane JR. Role of tumour necrosis factor

in the induction of nitric oxide synthase in a rat model of endotoxin shock. Br J

Pharmaco I 1993;l lO:177-82.

Thiemennann C. The role of the L-arginine: nitric oxide pathway in circulatory shock.

Adv PharmacoI 1994;28:45-79.

Torphy TJ, Undem BJ, Cieslinski LB, Luttrnann MA, Reeves ML. Hay OW.

Identification, characterization and functional role of phosphodiesterase isozymes in

hwnan airway smooth muscle . J Pharmacol Exp Tber 199];26 5:1213-23.

Tocphy TI, Zhou HL, Burman M, Huang LB. Role of cycl ic nucleotide

phosphodiesterase isozymes in intact canine ttae healis. Mol Phannaco l 1991;39: 376­

84 .

Torphy TJ, Zhou HL. Cieslinski LB. Stimulation of beta adrenoceptors in a human

monocyte cell line (U937) up-regulates cyclic AMP-specific phosphodiesterase

activity. J Pharmacol Exp Ther 1992;263:1195-205.

Tracey KJ, Beutler B, Lowry SF, Merryweather J, Wolpe S. Milsark IW, Hariri RJ,

Fahey TJ Jd, amelia A. Albert ro, et al. Shock and tissue injwy induced by

recombinan t human cachect.in. Science 1986;234:47Q..4.

Tracey KJ, Cerami A. Tumor necrosis factor, other cytokines and disease . Annu Rev

Cell Bioi 1993;9: 317-43 .

Tracey KJ. Cerami A. Tumor necrosis factor: a pleiotropic cytukine and therapeutic

target. Annu Rev Med 1994;45:491-503.

-64-

Tracey KJ, Cerami A. Twnor necrosis factor: an updated revie w of its biology. Crit Care

Med 1 993~1 :S415-22.

' Tracey KJ, Fong Y, Hesse DG, Manog ue KR, Lee AT, Kuo GC, Lowry SF, Ce ram i A.

Anti-ca chectinfTNF monoclonal antibo dies prevent septic shock during lethal

becte raemia. Nature 1987;330 :66 2-4.

Tracey KJ, Lowry SF, Fahey TJ 3d.. Albert ro, Fong Y, Hesse 0 , Beutler B, Manogue

KR. C alvano S, Wei H, et al . Cac hectinftwnor necrosis factor induces lethal shoc k:

and stress horm one responses in the dog. Surg Gyneco l Obstet 1987; 164:415-22.

Ulevitch RJ, Wolfson N, Virca G O, Kim S, Kline L, Mathison JC. Macrophages regulate

the host respo nse to bacterial lipopolysaccharides . Prog Clin Bioi Res 1989;299 :193-

202.

Ulich TR., del Casti llo J, Keys M. Grange r GA., Ni RX. Kinetics and mechanisms o f

reco mbinan t human interteukin 1 and tumor necro sis factor-alpha-induced chan ges in

circulating numbers of neutrophils and lymphocytes . J Imm unol 1987;139:3406- 15.

Van der Poll T, Buller HR, ten Care H, Wortel CH, Bauer KA. van Devenrer 5J, Hack:

CE, Sauerwein HP. Rosenbe rg RD. len Cate JW . Activa tion of coaguJation afte r

administration of tumor necros is factor to normal subj ects. N Engl J Med

1990;322 :1622 .7 .

Van der Poll T, Romijn JA, EM ert E. Borm JJ. Buller HIt. Sauerwe in HP. T umor

necrosis factor mimics the metabolic response to acute infection in healthy human s.

Am J PhysioI1 991;261:E457-65.

..S-

Van Dcrvo rt AL, Van L. Madara PJ. Cobb JP. Wes ley RA, Corrivea u CC . Tro pea MM,

Danner RL. Nitric oxid e regula tes endo to xin-induced TNF -alpha production by

human neutrophi ls. J Imrn unol 1994 ;152(8 ):4 102-9.

Vieaut E. Hype rtension and the microcirculation: a brief overv iew of experimental

studi es. J Hyperte ns SuppI 1992; l O: S59-68.

Vorhe rr T. Kncp fel L. Ho fmann F, Mollner S. Pfeuffer T. Carafo li E. The calmo dul in

bindin g do main of nitric oxide synthase and ade nylyl cyclase Bioche mistry

1993;32(23):6081-8

Wall ey KR, Hebert PC. Wakai V. Wilcox PG. Road m. Cooper OJ Decrease in left

ventricular co ntractility after tumor necro sis facto r-alpha infusi on in dogs . J Appl

Physiol l 994 ;76:106o-7.

Warr en RS, Starnes HF Jr, Gabrilove IL. Oett gen HF. Brennan MF . The acute meta bolic

effects of tumo r necro sis factor administra tion in human s. Arch Surg 1987;122 :l396·

400.

Watanabe F. Nakan o V. Said o H. Tamura Y. Yamanaka H. NAD PH-<:ytochrome c (P.

450) reductase has the activity ofNADPH-linked aquacobalamin red uctase in rat liver

microsomes. Biochim Biophys Acta 1992 ;1119 :175-7.

Wiessne r WH. Case y LC. Zbil ut JP. Trea tment of sepsis and septic shock: a review,

Heart Lung 24 :380. 1995 .

Witthoft T. Eckmann L, Kim 1M. Kagnoff MF gnt eroia vasive bacteria directly activate

expression of iNOS and NO prod uctio n in human colo n epithelial cells . Am I Physio!

1 998~75: 0 564-7 1.

Wong G H, Goedde ! DV. Twn our necrosis factors alph a and beta inhibit virus replication

and synergize with interferons. Nature 1986;323:819-22.

Wood KS, Buga OM. Byrns RE. Ignarro U . Vascular smooth muscle-de rive d relaxin g

factor (MDRF) and its close simi larity to nitric oxide. Biochem Biophy s Res Commun

1990 ;170 :80-8 .

Wright CEo Rees DO. Moncada S. Protecti ve and pathological roles of nitric oxide in

endotoxin shoc k. Card iovasc Res 1992 ;26: 48· 57.

Wu Z. Sharma RK.. Wang JH. [The mec hanis m of inhibiti on of calmod ul in-depen de nt

cycl ic nucl eot ide phosphodiesterase by dih ydrop yrid ine calcium antago nists] {Arti cle

in Chin ese]. HUBHsi rkc Ta Hsueh Hsueh Pao 1992;23:17.20 .

Xie I . Ioseph KO . Bagby 01. Giles ro . Greenberg SS. Dissocia tion ofTNF-alpha from

endotoxi n- induced nitric oxide and ac ute-phase hypo tension . Am I Physio l 1997;273:

H I64-74 .

Xie Q W. Coo H. Kashiwabara Y. Baum M, Weidne r IR. Elliston K, Mumford R. Nathan

C. Carboxyl terminus of inducibl e nitric oxide synthase. Contributio n to NAD PH

bindin g and enzymatic activity. I Bioi Chern 1994 ;269:28500- 5.

-67-

Yamamoto T, Manganie llo VC. Vaughan M. Purification and characterization of cyclic

GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of

divalent cations on activity. J Bioi Chern 1 98J~8 : 1 2526-J3 .

Yokoyama T. Vaca L. Rossen RD, Durante W, Hazarika P, Mann DL. CeUular basis for

the negative inotrop ic effects of tumor necros is factor-alpha in the adu lt mammalian

heart. J Cl in Invest 1993;92: 2303· 12.

Yoshimura T. Kurita C. Nagao T, Usami E. Nakao T, Watanabe S. Kobayashi J.

Yamazaki F. Tanaka H.. Nagai H. Effects of cAMP-phosphodiesterase isozyme

inhibitor on cytokine production by lipopol ysaccharide-stimulated human peripheral

blood mononucl ear cells. Gen PharmacoJ 1997;29:633-8.

Zhong WW. Burke PA. Drotar ME, Cbavali SR. Forse RA. Effects of prostag landin E2.

cholera toxin and 8-bromo-cyclic AMP on lipopolysaccharide-induced gene

expression ofcytokines in human macrophages. lnunun ology 1995;84(3):446-52.

.. s.