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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-
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