Biotransformation of glyceryl trinitrate and isosorbide dinitrate in vascular smooth muscle made tolerant to organic nitrates

CHRISTOPHER J . SLACK, BRIAN E. MCLAUGMLIN, JAMES F. BRIEN, GERALD %. MARKS, AND KANJI NAKATSU" Department sf Bharmcskogy and Toxicsksgy, hcukty of Medicine, Queen's University, Kingston, Ont., Canada K7E 3N6

Received April 82, 1989

SLACK, C. J., MCLAWGHL~N, B. E., BRIEN, 9. F., MARKS, G. S., and NAKATSU, M. 1989. Biotransfomation of glyceryl trinitrate and isosorbide dinitrate in vascular smooth muscle made tolerant to organic nitrates. Can. J. Physiol . Phmacol . 67: 1381-1385.

It has been proposed that organic nitrates are prodrugs and biotransfomation to a phmacologica1Iy active metabolite (i.e., nitric oxide) must occur before the onset of vasodilation. Hf this postulated mechanism is correct, tolerance to organic nitrate-induced vasodilation might involve decreased biotransfomation of organic nitrates by vascular smooth muscle. In this study, biotransformation of isosorbide dinitrate (HSDN) and glycergvl trinitrate (GTN) was estimated by measuring isosorbide mononitrate (ISMN) and glyceryl dinitrate (GDN), respectively, rather than ehe nitrate mion, because of a more sensitive method for measurement of ISMN and GDN. To test this hypothesis, isolated rabbit aortic strips (WAS) were made tolerant in vidro by incubation with 500 FM GTN or ISDN for 1 h. After a washoutyiod and submaximal contraction with phenylephrine, the tissues were incubated witheither 2.0 pM [a4C]~SDN or 0.5 pM [I CIGTN for 2 min. ISDN- or GTN-induced relaxation of RAS was monitored and tissue parent drug and metabolite contents were determined by thin-layer chromatography and liquid scintillation spectrometry. ISDN- and GTN-induced relaxation of RAS and the metabolite concentrations were significantly less for both GTN- and ISDN-tolerant tissue compared with nontolerant tissue. 'These results are consistent with the hypothesis that organic nitrate biotransfomation is required for organic nitrate-induced vasodilation.

Key words: organic nitrates, glyceaqul trinitrate, isosorbide dinitrate, biotransfomation, p r o h g , tolerance.

SLACK, C. J., MCLAWGHLIN, B. E., BRIEN, J . F., MARKS, G. S., et NAKATSU, K. 8989. Biotransfomation of glyceryl trinitrate and isosorbide dinitrate in vascular smooth muscle made tolerant to organic nitrates. Can. 9. Physiol . Phmacol . 67 : 1381-1385.

On a tmis 19hypth&se que les nitrates organiques sont des bioprecurseurs et qu'ils doivent subir une biotransfomation en un mktabolite phmacologiquement actif (par ex., oxyde nitrique) avant le dCbut de la vasodilatation. Si ce mecanisme est correct, la tolkrance la vasodilatation induite par des nitrates organiques impliquerait une biotransfomation reduite de nitrates mganiques par les muscles lisses vasculaires. Dans cetbe ttude, on a estimC la biotransfomation de B'isosorbide dinitrate (ISBN) et du glycbryl trinitmte (GTN) en dkteminant l'isosorbide mononitrate (ISMN) et le glyc6ryl dinitrate (GDN) plutbt que l'mion nitrite, la mkthode de mesure de I'PSMN et du GDN ttant plus sensible. Pour vtrifier cette hypothi?se, des dmdes aortiques isolCes de lapin (BAL) ont Ct6 rendues toltrantes in vitro par une incubation avec 580 p M de GTN ou d'ISDN pendant 1 h. Apr&s une @ride de lavage et une contraction sous-maximale avec de la phCnyltphrine (PE), les tissus ont Ctt incuMs avec 2,0 de [14C]IS~N ou 0,5 FM de [ ' 4 ~ ] ~ ~ pendant 2 rmin. On a observt la relaxation des BAL induite par I'ISDN ou le GTN et dCtepmin6 les concentrations de m~tabslites et de composts mkres des tissus p a chromatographie ern couche mince et par spectrom&ie de scintillation liquide. La relaxation des BAL induite par Be GTN ou 1'ISDN et Bes concentrations de mktabolites ont Ctk significativement plus faibies dans le tissu toltrant B I'ISDN et au GTN que dans le tissu non tolerant. Ces rksultats sont en accord avec I'hypoth&se que Bes nitrates organiques doivent subip une biotxansfomation pour induire une vasodilatation.

[Traduit par la revue]

Introduction There have been a number of hypotheses proposed to explain

the mechanism of action s f glyceryl trinitrate (GTN) and the other organic nitrates as vasodilators. Much of the recent information is consistent with the idea that GTN biotransfoma- tion is an integral component of GTN-induced relaxation ice., the "pro&ug hypothesis." According to this hypothesis, GTN and other organic nitrates act as prodrugs to release a p h m a - cologically active ultimate drug, which some investigators have suggested is nitric oxide (NO) (Schroder and Noack 1987; Feelisch and Noack 1987). Previous work from our laboratory has demonstrated that GTN biotransfomation preceded the onset of relaxation, as would be predicted by this hypothesis (Brien et d. 1988). Furthermore, it was found that induction of in vidro tolerance to GTN resulted in inhibition of conversion of G a d to glyceryl dinitrate (GBN) in rabbit aorta (Brien et al. 1986). We interpreted this to mean that the rate-limiting step in the action of GTN was its metabolic activation and that this was the step which was compromised in tissues made tolerant to

'~u tho r to whom correspndence should be addressed.

GTN. If the prodrug hypothesis is extended to include other organic nitrates, it predicts that (a) when two organic nitrates are compared, equivalent amounts of relaxation will be accom- panied by equivalent amounts of metabolism of the parent organic nitrates; (b) induction of tolerance to GTN will result in reduction of denitration of GTN and other organic nitrates; (c) induction of tolerance to an organic nitrate other than GTN will result in reduction of denitration of the organic nitrate and other organic nitrates including GTN .

In this paper, we describe the results of studies in which the biotrmsfomation of radiolabelled GTN and isosorbide dinitrate (ISDN) was evduated in nontolerant rabbit aorta, and in aorta made tolerant to the two organic nitrates.

Methods and materials Drugs and solutions

Krebs' solueion contained the following (&): NaCI, 120; KCI, 5.6; MgS04, 1.2; NaH2P04, 1.2; CaCB2, 2.5; N&C03, 25; and dextrose, 10 (Perry 1968). The solution was aerated with 95% O2 - 5% C02, and maintained at 37'C. Phenyleghrine hydrochloride (PE) (Sigma Chemi- c d Co., St. Louis, MO) was dissolved in Khebs' solution. Glycepyl trinitrate (GTN) was obtained as the commercial fornulation, Tridil@

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

MC

GIL

L U

NIV

ER

SIT

Y o

n 11

/14/

14Fo

r pe

rson

al u

se o

nly.

1382 CAN. J. PHYSIBL. PHARMACQL. VOL. 67, I989

from AHS Medical Specialties (Mississauga, Ont., Canada). Isosor- bide dinitrate (ISDN) (25% wlw in lactose powder) was kindly supplied as a gift from Wyeth Ltd. (Toronto, ant., Canada). The GTN metabo- lites, 1,2-GDN and 1,3-GDN, and the ISDN metabolites, 5-isosorbide mononitrate (5-ISMN) and 2-HSMN, were prepared by alkaline hydrolysis of GTN and ISDN, respectively, followed by thin-layer chromatography (TLC) (Crew and Dicalo 1968). ['"]GTN was a gift from G.D. Sea~le and Co. (Chicago, HL). [I4c] ISBN and liquid scintillation counting fluid, Aquasol@, were obtained from New England Nuclear-Dupont Hnc. (Boston, MA). Dietkyl ether was distilled over KOH pellets before use. All other chemicals were at least reagent grade and were obtained from Fisher Scientific Ltd- or BDM Chemicals (Toronto, Qnt., Canada).

Purijication of [14c]@~fV a d ['"]ISDN 1 ,3-[14c]GTN (47.5 mCitmo1) (1 Ci = 37 GBq) was purified by

TLC as described by Brien et d. (1984, 1988). 2 , 5 - [ ' 4 ~ ] ~ ~ ~ ~ was purified in a similar manner using a solvent system of toluene and ethyl acetate (1 : 1, vlv) (modified from Dietz, 1963). The chemical purities were assessed by TLC -liquid scintillation spectrometry (LSS) and were >99.9% for both GTN and ISDN. Dilutions of the stock solutions were made with 0.9% saline and the concentsations sf the working solutions were checked by LSS. The purity of the stock solution was checked at least every 2 weeks by TLC-LSS.

Rabbit aortic strip preparation Mde New Zedand white rabbits (2.5 - 3.5 kg) were obtained from a

local supplier, and were housed individually with free access to laboratory rabbit chow 5321 (Wdston hr ina Canada Inc., Toronto, Ont., Canada) and water for at least I week before experimentation. Animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. On the day of experimentation the animal was killed by cervical dislocation and exsmguinatisn. The descending thoracic aorta was removed quickly and placed in ice-cold Krebs' solution. After removal sf adipose and connective tissue, the aorta was cut spirally into a single strip (160-200 m long and 4-5 rn wide) and then cut into four smaller strips, each 40 wuna in length. The four strips were trimmed with less than a 10-mg difference in weight among the strips for a given animal. The rabbit aortic strips (RAS) were suspended in individual tissue baths contain- ing 10 d of Krebs' solution at 3Y0C, and isometric responses of the tissues were recorded using force-displacement transducers (Grass R03D) coupled to a Grass model 7-B polygraph. The WAS were maintained at the optima1 resting tension of 2.0 g during the I-h equilibration period, during which the Krebs' solution was changed every 20 min.

After equilibration, the RAS were contracted maximally with 1W pM PE; following a 1-h washout period, 0.2 pM PE was added and the dose was increased until the tone achieved was 60-80% of the maximal tone initidly obtained with 100 pM PE. A 15-fin equilibration period was required to allow the submaximal tone of the RAS to stabilize before exposure to organic nitrates.

Biotran~orrraati~n of [l4c]CZ~ or [ 1 4 ~ ] ~ ~ ~ F 4 by tokmnt and non- to~erant rabbit aortic strips

Bioehmsfomation of [ ' 4 ~ ] ~ S ~ ~ by RAS dwing incubation was determined as outlined in Fig. 1. The four WAS were contracted submaximally with PE and incubated with 2.0 p M ISDN (nomadioac- tive) for 2 f i n ; the time of onset and percent organic nitrate-induced relaxation were monitored. Each tissue then was washed with &ebs' solution for 8.5 h, with buffer changes every 15 min. Tissues were subsequently put into one of f o u treatment goups: ISDN, GTN, vehicle (propylene glycol - ethanol - water, 3:3:4, v/v/v), and work- up. To these groups 0.1 rraL of 50 mhl ISBN, 0.1 waL of 50 KIM GTN, 0.2 mL vehicle, and 0.1 mL of 0.9% s h e was added, respectively. The find concentration of BSDN and GTN in the appropriate baths was 500 FM. After a 1-h incubation period, each tissue was then washed with Kiebs' solution for 1 h except for the ISDN group, which was washed for 15 nain. After this, each tissue was contracted submaximally (60-80%) w i h PE. Three RAS (ISDN, GTN, vehicle) were incubated

2. 2.0 pM ISDN 3. wash for 0.5 h

Vehicle 5 0 0 DM 500 BJM W 0 ~ k - u ~ GTN lSDN I

FIG. 1. Schematic representation of experimental protocol.

with 2.0 pM ['4C]IS~N for 2 min (n = 5) , and the time of onset and extent of ISDN-induced relaxation was monitored. At the end of the incubation period, all four RAS were removed from the tissue bath, and freeze-clamped with liquid nitrogen-cooled tongs. [ ' 4 ~ ] ~ ~ ~ ~ was added to the work-up sample. Biotransfomation of [ ' 4 ~ ] ~ ~ ~ by RAS was assessed in the same manner except that 0.5 pM GTN (nomadioac- five) and 0.5 pM [['"c]GTN replaced the nonradioactive ISDN and the [ 1 4 ~ ] ~ S ~ ~ .

Amkysis of [I4 C]GTN and [14C+]1~~N Quantitative determination of the content of [14C]~TN and two of its

metabolites, 1 , 2 - [ B 4 ~ ] ~ ~ ~ and 1 ,3-[14C]~DN or [14C~~DPJ and two of its metabolites 5 - [ 1 4 c ] I ~ ~ N and 2-[I4c]1s~N in GTN-tolerant, ISDN-tolerant, and wontolerant WAS was conducted as follows, Each frozen experimental WAS was placed in a 50-mL round-bottom tube containing 5 mL of ice-cold diethyl ether. The tissue was extracted for 10 min at 4"C, the organic extract was removed, and the 5-rPnL diethyl ether extraction was repeated once. Two 100-pL aliquots of the combined organic extract were added to individual 7 - d MinaivialsQ (Fisher Scientific) containing 5 mL of Aquas010 (New England Nuclear-Dupont), and the radioactivity was determined by LSS using a Bechan model LS-3800 instrument. The extracted RAS was digested in 1 d of 1 M NaOH for48 h. The digested tissue was placed in a 20-Hnk polyethylene vial (Fisher Scientific) to which was added two 5 - d Aquasola washes of the tubes used for the tissue digestion; 0.7 m.L of glacial acetic acid was added to the vid to prevent chemiluminescence, and the sample analyzed by LSS. The total radioactivity in the RAS was considered to be equal to the sum of the radioactivity in the combined diethy] ether extract and in the extracted RAS. The diethyl ether extract of the BAS was stored overnight at -20°C and then dried with anhydrous MgS04 for 20 min at 4°C. Each extract was concentrated to about 1 0 0 pL with a stream sf nitrogen gas at room temperature and then analyzed by TLC-LSS for [ B 4 ~ ] ~ ~ ~ , 1,2-["C]GDN, and 1,3-[14C]G~N or [ ' 4 ~ ] ~ ~ ~ ~ , 5-['v]ISMN, and 2 - [14~]1S~N, as described previously (Brien et all. 2986). The work-up control RAS was analyzed by the procedure described for experimental RAS .

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

MC

GIL

L U

NIV

ER

SIT

Y o

n 11

/14/

14Fo

r pe

rson

al u

se o

nly.

SLACK ET AE.

TABLE 1 . ISDN-induced and G'FN-induced relaxation of, and JSDN and GTN biotransfor- mation by, tolerant and nontolerant rabbit aortic strips (WAS)

Nontolerant ISBN-tolerant GTN-tolerant

ISDN Onset of relaxation (s)

% relaxation at 2 min

pmol ISDN -k ISMN/g RAS

5-ISMN/2-ISMN ratio

GTN Onset of relaxation (s)

% relaxation at 2 min

pmol GTN + GDN/g RAS

B,2-GDN/ 1,3-GDN ratio -

NOTE: The exprinnentd metlad for WAS treatment is described in Fig. 2. The data are expressed as the mean 2 SB ( a = 5) .

* p < 0.05, cornpami with nonmlerarst WAS.

Data anaiysis The degree of inhibition of PE-induced tone produced by the

vasodilator was expressed as the percent organic nitrate-induced relaxation in RAS. The radioactivity of the individual TLC band scrapings was corrected for background. The lower limit of sensitivity of the TLC-LSS assay was taken as twice background. Percentage recovery of radioactivity for each extracted, TEC-analyzed tissue was calculated as

total darn on TLC plate

dpm in diethyl ether extract + dpm in digested tissue ) x 1 m

Tissue GTN and GDN concentrations or ISDN and HSMN concentm- tions were cdculated from the radioactivity on the TLC plate at the respective Rf values, the specific activity of ['"C]GTN (47.5 mCi/msl) or [14G]ISDN (154.0 mCi/mol), and the weight of the WAS sample. The concentrations were corrected for percentage recovery and expressed as picomoles of GTN or %SPIN per gram WAS and picomoles of 1,2-GDN and 1,3-GDN or 5-ISMN and 2-HSMN per g m of U S , respectively. The data are presented a the mean & SE) of five animals unless otherwise indicated. Statistical analysis of the data was conducted by Student's E-test for paired data (two-tailed), or by repeated measures, one-way analysis of variance followed by a Newman-Keuls' test for a significant F-ratio ( p < Q.05), depending on which test was statistically appropriate (Zivin and Bartko 1976). Two groups of data were considered statistically different when p < 0.05.

responses ts concentrations of GTN md ISDN that caused relaxations of about 45% in nontslerant M S .

Bistransfomtion @['"]IsDN aper GTN sa ISDRr treatment The biotransfomation of ISDN by GTN-tolerant and ISDN-

tolerant RAS as well as by nontolerant WAS was measured during ISDN-induced relaxation. Biotransfomation of a single concentration of [ 1 4 ~ ] 1 s D ~ (2.0 yM) was affected as follows. The ISMN concentration in BAS was significantly less (p < 8.85) for both GTN-tolerant and ISDN-tolerant tissues com- pared with nontolerant tissue (Fig. 2a), but no significant difference existed between GTN- tolerant and ISDN-tolerant tissue. The total organic nitrate concentration (pmsl ISDNIg RAS + pmol ISMN/g RAS) was not significantly different for GTN-tolerant and ISDN-tolerant tissue compared with non- tolerant tissue (Table 1). Therefore the uptake of ISDN into tolerant and nontolerant RAS was similar and the lower I S W concentration in tolerant tissue was due primarily to decreased biotrawsfomation of ISDN. As the analytical method used to assess the ISMN concentration was able to distinguish isomers of ISMN, the 5-HSMN and the 2-ISMN could be considered separately. In nontolerant RAS , there was preferential bistrans- fomation of ISDN to 5-ISMN (Fig. 3a). For GTN-tolerant and

Results ISDN-tolerant tissues, there was still preferential fomation of 5-ISMN but the ratios of 5-ISMNI2-ISMN were significantly

GTN-induced andlSDN-induced relaration after GTN or ISDN less than in nontolerant (Table treatment

GTN was more potent than ISDN in relaxing vascular tissues. We also found that GTN was more effective than ISDN in producing tolerance to organic nitrates in the isolated rabbit aortic strip. To conduct the present experiments, it was necessary to prepare the RAS such that an equivalent amount of tolerance was induced in the tissues when using either GTN or ISDN. The following treatment schedule produced an equiva- lent amount of tolerance and cross-tolerance to GTN and ISDN. After equilibration, the RAS were incubated in the presence of 500 pM GTN or 500 pM ISDN for B h. The GTN-treated M S were washed for 1 hour and the ISDN-treated RAS were washed for 15 min. Tissues treated in this way displayed the same mount of tolerance to both GTN and ISDN as shown in Table 1. The conditions used were just sufficient to eliminate

Bistransfsrmartdon of [ " ~ G T N after GTN or ISDN treatment The biotransfomation sf GTN by GTN-tolerant and ISDN-

tolerant WAS was assessed relative to nontolerant M S during GTN-induced relaxation. For a single concentration of ['"CIGTN (0.5 pM), the GDN concentration in both GTN- tolerant and ISDN-tolerant WAS was significantly less (p < 0.85) than in nontolerant RAS (Fig. 2b). A significant differ- ence existed between the GDN concentrations in GTN-tolerant and ISDN-tolerant RAS. The total organic nitrate concentration (pmol GTNIg RAS + pmol GDNIg RAS) was not significantly different for G'PN-tolerant and ISDN-tolerant tissue compared with nontolerant tissue (Table 1). These data indicate that the uptake of GTN into tolerant and nontolerant RAS was similar and that the lower GDN concentration was due primarily to

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

MC

GIL

L U

NIV

ER

SIT

Y o

n 11

/14/

14Fo

r pe

rson

al u

se o

nly.

CAN. J. PHYSIOL. PHARMACQL. VOL. 67, 1989

pmsl ISMklBg R A S pmoD GDNBg RAS

NON-f Ob Gf N-TOk ISDN-TOL NOM-TOL GTN-TOL ISBN-TQL

FIG. 2. Reduction of metabolite fornation from GTN and ISDN in rabbit aortic strips (RAS) made tolerant to GTN or HSDN. The WAS from each of five m h l s were incubated with 500 pM GTN (GTN-TOIL), 500 pM ISDN (HSDN-TOL), or vehicle (NON-TOL) for I h before study. Vehicle control md GTN-exposed tissues were washed for I h, ISDN-exposed tissue was washed for 15 min. Each WAS was contracted submaximally with PE and then incubated with either 2.8 pM [ I 4 ~ ] 1 S ~ ~ or 0.5 pM [14c]GTN for 2 min. The tissues were freeze-clamped for determination of parent h g atad metabolite content by TEC-LSS. (a) Accumulation of ["CIISMN after 2 min (expressed as pmol BSMNIg M S ) ; (b) accumulation of ["CIGDN after 2 min (expressed as pmol GDNig WAS). AII values represent the mean + SD (n = 5). * p < 0.05, compaed with nsntolerant RAS; tp < 0.05, compared with GTN-tolerant RAS.

garnol BSMNPg RAS EL pmo! BDNlg RAS b

FIG. 3. Relative metabolite fomdion from GTN atad ISDN in nontolerant (NON-TOE), GTN-tolerant (GTN-TOE), md ISDN-tolerant (ISDN-TOL) rabbit aortic strips (RAS). The experimental method for RAS treatment is described in Fig. 2. (a) Accumulation of 2 - [ 1 4 ~ ] 1 ~ ~ ~ and 5 - [ 1 4 C ] ~ ~ ~ ~ in RAS (expressed as pmol ISMNIg RAS); (b) accumulation of 1 ,2-["CIGDN and 1 C]GDN in RAS (expressed as pmol GDNIg U S ) . All values Hepresent the m m & SD (n = 5). *p < 0.05, compared with nontderant RAS; ?a< 0.05, compared with GTN-tolerant RAS.

decreased biotrmsformation of GTN. Also, there was a per- Df scussion ferentid biotrmsfomation of GTN to 1,2-GDN compared with The aim of the present study was to further test the prodrug 1,3-GDN in nontolerant RAS (Fig. 3b). Furthermore, the 1,2- hypothesis by exmining the biotrmsformation of GTN and GDN/1,3-GDN ratio for GTN-tolerant md HSDN-tolerant RAS HSDN in naive and organic nitrate-tolerant rabbit aortic strips. In was significantly less than that for nontoleribnt RAS (Table I). earlier studies, we had demonstrated that GTN was biotransfor-

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

MC

GIL

L U

NIV

ER

SIT

Y o

n 11

/14/

14Fo

r pe

rson

al u

se o

nly.

SLACK ET AE. 1385

med to GDN in a time-dependent manner and the degree of relaxation sf the isolated rabbit aorta was related to the extent of biotransformation (Brien et al. 1986, 1988). One of the predictions made on the basis of the prodrug hypothesis was that the amounts of different organic nitrates metabolized by the naive tissues would be equivalent for equivalent degrees of relaxation. Figure 2 shows that naive RAS produced in the order of 90- B8Q pmol denitrated metabliteig WAS when either GTN or ISDN was the agonist. The concentrations of GTN and ISDN used produced virtually identical extents of relaxation in the BE-contracted RAS .

Other observations made earlier were that in rabbit aorta made tolerant to GTN, there was less GTN biotransformation

mean that the mechanism which releases the witrate group from the 2-position of ISDN is important in the same way as the mechanism which releases the nitrate group from the 3-position of GTN.

In s u m q , we have found that different, but equieffective, concentrations of GTN and ISDN were associated with equiva- lent molar mounts of dmg biotransformation. Furthemore, it has been demonstrated that GTN-induced and ISDN-induced tolerance inhibited relaxation to both GTN and ISDN, inhibiting biotrmsformation of both drugs as well. These results are consistent with the hypothesis that organic nitrates are prodmgs requiring biotransformation to an active vasodilating metabolite and that acute tolerance occurs due to inhibition of metabolic

and less GTN-induced relaxation compared with nontolerant activation. rabbit aorta (Baien et d. 1986). If one presumes that the organic nitrates act through a common mechanism sf action, that is, Acknowledgements acting as prodrugs, then treatments that interfere with the action This study was supported by the Hem and Skoke Foundation of one organic nitrate would be predicted to compromise the of Ontario (grant ~ - 6 9 1). The authors wish to thank E. actions of other organic nitrates. Results from the present Duffe for her assistance in the preparation of this manuscript. studies are consistent with these predictions; tolerance to GTN resulted in decreased biotransfo&ation of both GTN and ISDN, and tolerance to %SDN also resulted in decreased biotransforma- tion of both GTN and ISDN. Thus, the present data clearly support the idea that a metabolite of GTN and ISDN is responsible for their relaxant capability in vascular tissue and that acute tolerance is a manifestation sf inhibiting production of the active metabolite. A similar conclusion has been made by Henry et d. (1989) for their work on bovine coronary arteries. Based on cross-tolerance experiments in which a number of relaxants were employed, these workers suggested that GTN- induced tolerance affected the site discussed herein as well as a second site.

It was clew from the present study that there was preferential biotrmsformation of GTN to 1,2-GDN in nontolerant RAS as reported earlier by Brien et al. (1988). When tolerance to GTN was induced, inhibition of fomation of 1,2-GDN was more marked than inhibition of formation of 1,3-GDN. We interpret this to mem that the nitro metabolite released from the 3- position carhn atom of the GTN molecule is of prime impart- mce to the relaxation process. An analogous situation appears to hold for the biotransformation of ISDN by tolerant and nontolerant M S . In the case of nontolermt RAS, there was preferential biotransformation of ISDN to 5-ISMN relative to 2-ISMN. For GTN-tolerant and ISDN-tolerant tissues, the inhibition of 5-ISMN production was greater than inhibition of 2-%SMN production. If the analogy with GTN holds, this m y

BRIEN, J. I;., MCLAUGHLIN, B. E., B R E E ~ N , T. M., BENNETT, B. M., NAKATSU, K., and MARKS, G. S. 1986. Biotransfomation of glyceryl thinitrate occurs concurrently with relaxation of rabbit aorta. J. Phmacol . Exp. Ther. 237: 608-614.

BMEN, J. F., MCLAUGHLIN, B. E., KOBUS, S. M., KAWAMOTO, J. H., NAKATSU, K., and MARKS, G . S. 1988. Mechanism of glyceryI trinitrate-induced vasodilation. I. Relationship between drug bio- trmsfomation, tissue cyclic GMP elevation md relaxation of rabbit aorta. J . Phsumacol. Exp. Ther. 24%: 322-327.

Cmw, A%. C., and DICARLO, F. J. 1968. Identification and assay of isomeric B4~-glyceryl nitrates. J . Chromatog. 35: 506-5 12.

DIETZ, A. J. 1967. The biotransfomation of isosorbide dinitrate in dogs and humans. Biockem. Phmacol . 16: 2447-2448.

FEELPSCH, M., and NOACK, E. A. 1987, Conelation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. Eur. J . Phmaeol . 139: 19-30.

HENRY, B. J., HOROWITZ, J. D., and hvms, W. J. 1989. NitmgIycmin- induced tolerance affects multiple sites in the organic nitrate bioconversion cascade. J. Phrumacol . Exp. Ther . 248: 762-768.

Rzm~, W. L. M. (Editor). 1968. PhmacoBogical experiments on isolated preparations. E. & S. Livingstone Ltd., Edinburgh, Scot- land.

SCHRODER, HI., and NOAGK, E. A. 1987. Conelation between nitric oxide formation by cleavage of organic nitrates and activation of guatnylate cyclase. J . MOB. Cell. Cardiol. 17: 93 1-934.

ZIVIN, J. A . , and BARTKO, J. J. 1976. Statistics for disinterested scientists. Life Sci. 18: 15-26.

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

MC

GIL

L U

NIV

ER

SIT

Y o

n 11

/14/

14Fo

r pe

rson

al u

se o

nly.