Dissociation of sympathetic compensation from glyceryl trinitrate tolerance

EUROPEAN JOURNAL OF PHARMACOLOGY 21 (1973) 189-194. NORTH-HOLLAND PUBLISHING COMPANY

DISSOCIATION

OF SYMPATHETIC

FROM GLYCERYL

TRINITRATE

COMPENSATION TOLERANCE*

Eugene M. JOHNSON, Jr., Stanley LANG, and Philip NEEDLEMAN Department of Pharmacology and Department o f Physiology and Biophysics, Washington University School o f Medicine, St. Louis, Missouri, 6311 O, U.S.A.

Accepted 10 November 1972

Received 12 June 1972

E.M. JOHNSON, Jr., S. LANG and P. NEEDLEMAN, Dissociation o f sympathetic" compensation from glyceryl trinitrate tolerance, European J. Pharmacol. 21 (1973) 189-194, The hypothesis that changes in sympathetic function play an integral part in the development of tolerance to the vasodepressor effects of organic nitrates was tested. Glyceryl trinitrate (GTN) tolerant rats showed increased norepinephrine turnover but no changes in myocardial norepinephrine levels or in uptake of exogenous norepinephrine. GTN tolerant animals did not have altered sensitivities to sympathomimetics. Marked changes in sympathetic tone did not change the animals'sensitivity to glyceryl trinitrate (GTN) and did not alter the development of tolerance to GTN. No changes in sympathetic compensation to direct administration of a vasoconstrictor to the carotid sinus were observed. It is concluded that changes in sympathetic function play no role in the development of tolerance to GTN. Tolerance Vasodilators

Sympathetic compensation Norepinephrine turnover

1. Introduction The use of glyceryl trinitrate (GTN; nitroglycerin) and other organic nitrates in the treatment o f angina pectoris is hindered by the development o f tolerance to the vasodilator activity o f these agents. The mechanism by which this tolerance develops is not known. Tolerance to organic nitrates is not the result of their enhanced biotransformation (Needleman, 1970; Needleman et al., 1971; Lang et al., 1972; Clark and Litchfield, 1969). Clark and Litchfield (1969) suggested that tolerance develops because of an alteration o f sympathetic compensatory mechanisms to the vasodilation caused by chronic administration o f organic nitrates. Clark (1970) observed a supersensitivity to epinephrine (blood pressure and induction of arrythmias) 24 hr after cessation o f treatment o f rats with ethyl* A preliminary report of some of this work has appeared in Federation Proc., 13 (1971) 428.

Glyceryl trinitrate

ene glycol dinitrate (EGDN). He observed no changes in response to tyramine. On the other hand, Vigliani et al. (1968) observed that the tachycardia caused by tyramine was 'much greater and longer' in EGDN tolerant mice. This increase in response to tyramine was associated with an increase in cardiac norepinephrine levels which reached a maximum 24 hr after cessation o f treatment with EGDN. The purpose of the work presented here is to determine if changes in sympathetic function occur in rats made tolerant to GTN and what, if any, role these changes play in the development of tolerance.

2. Materials and Methods 2.1. Production o f tolerance to G T N Rats were administered 100 mg/kg GTN subcutaneously 3 times a day for 3 or 4 days utilizing the method described by Needleman (1970).

190

E.M. Johnson, Jr. et al., Glyceryl trinitrate tolerance

2.2. Labeled norepinephrine studies 3H-norepinephrine (3H-NE, 1 #Ci; New England Nuclear) was administered to conscious restrained female Sprague-Dawley rats (Zivic-Miller 175-225 g). The 3H-NE was injected in 0.05 ml saline from a 0.05 ml siliconized microsyringe via PE 50 jugular vein cannulae which were implanted and exteriorized 2 4 - 4 8 hr earlier. The effect of drugs on 3H-NE release was determined by their i.v. injection 1 hr after injection of the isotope. The animals were sacrificed by decapitation 4 hr after administration of the 3HNE. The hearts were immediately excised, rinsed in ice-cold saline, blotted dry, and frozen in liquid nitrogen. 3H-NE in the hearts was determined by dissolving the tissue in 5 ml of Soluene (Packard) Tissue Solubilizer and counting a 1-ml aliquot of the tissue solution. The counting efficiency was determined by internal standards. The assumption is made that essentially all of the radioactivity is in the form of 3HNE. It has previously been demonstrated that after injection of 3H-NE the radioactivity in the heart in the form of NE is 90% (Palaid, 1971) or greater (Kopin et al., 1962). 3H-NE uptake was determined by sacrificing the animals 3 min after administration of label. Hearts were prepared for counting as described above. The NE content of the myocardium was determined by the trihydroxyindole method utilizing the extraction procedure of Maickel et al. (1968). 2.3. Other blood pressure experiments Other blood pressure measurements were carried out on female Sprague-Dawley rats (175-255 g) or genetically hypertensive rats ( 1 6 0 - 2 3 0 g ) of either sex anesthetized with pentobarbital (30 mg/kg, i.p.). Recordings were made via PE 50 cannulas in the carotid artery (Physiograph, E & M Instrument Company, Houston, Texas, P-1000 linear core pressure transducer). Challenge doses of drug were administered i.v. via a PE cannula in a jugular vein. 2.4. Cold exposure studies Animals undergoing cold exposure were housed individually in a cold room at 4°C for periods up to 11

days. Blood pressure dose-response curves were run at room temperature immediately upon removal from the 4°C environment. 2.5. Direct application o f drugs to carotM sinus Sprague-Dawley rats of various weights and either sex were anesthetized with pentobarbital, 30 mg/kg, i.p. The femoral artery was cannulated (PE 10) and connected to a PG 23 Statham strain gauge and pressures were recorded on a Sanborn Recorder. A tracheal cannula was inserted and the left carotid bifurcation exposed. A cotton pledget (0.25 mm diameter or less) was placed in the bifurcation. After a control blood pressure reading, 10 btl of a solution of Angiotensin 11 (1 og/ml) or GTN (1 mg/ml) was pipetted onto the cotton pledget. After a 3-5-rain observation period, the pledger was removed and the area irrigated with 1 ml of saline, aspirated and the saline wash repeated twice. The area was then gently swabbed with small pieces of paper tissue and another dry pledget placed in the bifurcation.

3. Results 3.1. N E turnover after induction o f G T N tolerance The level of NE and the uptake of exogenous 3HNE by myocardium was unchanged (p> 0.5) in GTN tolerant rats (table 1). 4 hr after the administration of 3H-NE, control animals retained 185 dpm/mg. If control animals were treated with a high dose of GTN (100 mg/kg, s.c.) 1 hr after injection of 3H-NE there was an increase in sympathetic reflex activity as indicated by the decrease in cardiac radioactivity to 114 dpm/mg 3 hr after the GTN pulse (4 hr after injection of 3HNE). Of primary interest is the observation that the GTN tolerant animals show significantly lower (118 dpm/mg) 3H-NE remaining in their hearts 4 hr after injection of label. Since the endogenous cardiac NE content and SH-NE uptake are the same in both groups, this indicates that the turnover rate of heart NE is increased in tolerant animals. Tolerant animals, which do not exhibit a vasodepression following a GTN pulse show no further decrease in heart NE.

E.M. Johnson, Jr. et al. Glyceryl trinitrate tolerance Table 1 Effect of GTN tolerance on heart norepinephrine. Rats were made tolerant by administration of GTN (100 mg/ kg, s.c. rid) for 3 days. The uptake and release of 3H-NE and the level of NE in myocardium were determined as described in Materials and methods. In this and the remaining tables and figs., the numbers in parentheses indicate the number of animals used. A. Cardiac NE Content Control (7) GTN tolerant (7)

NE (ug/g ± S.E.M.)

191

EPINEPHRINE

[

* Normotel~s/ve (3) 0 Hypertensive [5)

TYRAMINE • IVon~v~/ve-GTN

|

ToleroM(4)

& HypertenMve-GTN To~erotiC(3)

cc 50

,L

;

3( ec u~ 2( t3

/

0.65 -+ 0.12 0.69 ± 0 . I t

20

e, B. 3H-NE Uptake Control (7) GTN tolerant (8) C. 3H-NE Release Control (11) Control +GTN Pulse (10) GTN tolerant (6) GTN tolerant + GTN pulse (I0)

3H-NE (dpm/mg ± S.E.M.) 281 -+ 14 269 ± 8

185 ± 12 114 -+ 15"

.1

1

I0

10

j~,'kg

I00 I000 .y@/kg

Fig. 1. Pressor responses to the i.v. injection of epinephrine and tyramine in groups of rats (as indicated). The single line drawn for each drug represents the mean response from the 4 groups of animals. Animals were made tolerant and curves generated as described in Materials and Methods.

118 ± 8* 129 ± 13

DEVELOPEMENT OF GTN TOLERANCE IN NORMAL AND GENETICALLY HYPERTENSIVE RATS

* p < 0.005.

3. 2. Sensitivity o f tolerant animals to epinephrine and tyramine

Untreated //]

Hy~rteas,v*f3)

Pressor d o s e - r e s p o n s e curves were g e n e r a t e d to e p i n e p h r i n e a n d t y r a m i n e o n G T N t o l e r a n t rats w h i c h e x h i b i t increased N E t u r n o v e r a n d o n g e n e t i c a l l y hyp e r t e n s i v e rats w h i c h r e p o r t e d l y ( L o u i s et al., 1 9 6 9 ) have d e c r e a s e d h e a r t N E t u r n o v e r rates. Fig. 1 s h o w s t h a t t h e r e is n o o b s e r v a b l e d i f f e r e n c e in r e s p o n s e s to e p i n e p h r i n e a n d t y r a m i n e in c o n t r o l a n d G T N tolera n t n o r m o t e n s i v e or genetically h y p e r t e n s i v e rats. N o d i f f e r e n c e was o b s e r v e d in the f r e q u e n c y o f d e a t h at t h e h i g h e s t dose b e t w e e n n o n - t o l e r a n t a n d t o l e r a n t animals. T h e s e e x p e r i m e n t s i n d i c a t e t h a t G T N tolerance p r o d u c e d in the m a n n e r d e s c r i b e d is associated w i t h an increased t u r n o v e r o f NE, b u t n o t w i t h an i n c r e a s e d sensitivity t o d i r e c t l y or i n d i r e c t l y a c t i n g sympathomimetics.

41

~

' /V. . . . . .

?/ o

Nro.

--

I

0.01

0.1

&l'-~-~

" ---"

1

~-~

//

f

.

ive{3}

,

.....

I

10 100 .,u.g/kg GTN

I

1000

Fig. 2. Vasodepressor responses to the i.v. injection of' GTN in GTN tolerant normotensive and genetically hypertensive rats. Vasodepressor responses expressed as a % decrease in mean arterial blood pressure. Mean resting blood pressures were 138 ± 8 (normotensive), 166 ± 4 (hypertensive), 118 -+ 7 (GTN tolerant normotensive), and 158 +-9 (GTN tolerant hypertensive). Animals were made tolerant as described in Materials and Methods.

3.3. G T N sensitivity and tolerance in normal and genetically hypertensive rats S e n s i t i v i t y t o the v a s o d e p r e s s o r activity o f G T N a n d the d e v e l o p m e n t o f G T N t o l e r a n c e is similar in

genetically hypertensive animals and control animals even t h o u g h the t u r n o v e r rate o f NE in the h y p e r t e n sive a n i m a l s is p r e s u m a b l y l o w e r (Fig. 2).

E.M. Johnson, Jr. et al., Glyceryl trinitrate tolerance

192

3.4. Effect o f marked changes in sympathetic tone on G T N sensitivity and the development o f G T N tolerance

T r e a t m e n t w i t h an i n h i b i t o r o f N E b i o s y n t h e s i s , a - m e t h y l t y r o s i n e (aMT), h a d n o e f f e c t ( p > 0.5) on the r e s p o n s e to GTN or papaverine (table 2). Conc o m i t a n t a d m i n i s t r a t i o n o f a M T and GTN had n o

E x p o s u r e t o c o l d m a r k e d l y increases the peripher-

e f f f e c t on the d e v e l o p m e n t o f a decreased sensitivity

al t u r n o v e r o f N E ( S p e c t o r , 1966). We also o b s e r v e d

to GTN. The degree o f i n h i b i t i o n o f s y m p a t h e t i c ac-

increases in 3H-NE m u c h greater t h a n t h a t seen in GTN t o l e r a n t animals ( d a t a n o t s h o w n ) . A n i m a l s

tivity using this or a similar regimen o f a M T p r o v e d

h o u s e d at 4 ° C s h o w n o significant d i f f e r e n c e in their

n o r m a l l y tolerable cold stress (4°C). Hence the devel-

r e s p o n s e t o GTN and h i s t a m i n e w h e n c o m p a r e d to c o n t r o l animals (table 2). H e n c e a m a r k e d increase in

o p m e n t o f tolerance does n o t appear d e p e n d e n t u p o n

sufficient t o kill 75% o f animals w h e n s u b j e c t e d to a

an increase in s y m p a t h e t i c t o n e .

s y m p a t h e t i c t o n e , in and o f itself, does n o t c o n f e r t o l e r a n c e to GTN or h i s t a m i n e .

Data (table

2) for the various vasodilators are

s h o w n at the a p p r o x i m a t e E D s o o f the agent. Full

Table 2 Effect of marked changes in sympathetic tone on GTN sensitivity and the development of GTN tolerance. Falls in blood pressure (B.P.) are given for approximated EDso of vasodilators. Cold stress was induced by housing rats at 4°C for 3-11 days. aMT was administered at a dose of 200 mg/kg bid × 3-0.5 days with or without simultaneous administration to produce tolerance (see Materials and methods). None of the pretreatment schedules described altered normal resting blood pressure. Numbers of animals used in parentheses. %Fallmeanarterial B.P.

Treatment

Control Cold stress c~MT treatment GTN tolerance aMT treatment + GTN tolerance

GTN (10 ug/kg)

Histamine (10 tag/kg)

Papaverine (1 mg/kg)

31 29 36 12 12

22 -+ 2 (11) 25 -+ 3 (9) 15 -+ 4 (8) -

36-+ 4 (2) 39 -+ 3 (3) 37 -+ 2 (3) -

-+ 4 -+ 2 -* 5 +- 2 -+ 4

(12) (10) (6) (3) (4)

Table 3 Change in mean blood pressure after direct administration of GTN or All to the carotid sinus region of GTN tolerant and nontolerant rats. Data shown as changes in mean blood pressure at various initial blood pressures. Carotid sinus exposed and drug applied as described in Materials and methods. Starting blood pressure

75 85 95 105 115 125 145

Pressure fall after GTN -+S.E.M.

Pressure rise after All -+ S.E.M.

Non-tolerant

Tolerant (n=5)

Non-tolerant

Tolerant (n=3)

6 +- 2 (3) 12-+ 2(5) 17 -+ 3 (7) 24 -+ 3 (11) 34 -+ 1 (6) 24 -* 6 (4) 15 -+ 3 (8)

0 0 0 0 0 0 0

24-+4(5) 30 +- 5 (6) 33 -+ 3 (6) 29 -+ 4 (3)

18-+ 1 28 +- 3 -

E.M. Johnson, Jr. et al., Glyceryl trinitrate folerance

d o s e - r e s p o n s e curves showed none of the pretreatment schedules described altered the response to histamine or papaverine. 3.5. Direct measurement o f compensatory reflexes

Application of angiotensin !1 ( A l l ) directly to the carotid sinus o f tolerant and non-tolerant rats produces similar increases in blood pressure in response to vasoconstriction produced locally (table 3), thus indicating that compensatory mechanisms which elevate blood pressure are unchanged. The response to local GTN application is quite different. Normal animals respond to the dilatation of the carotid sinus with the expected fall in blood pressure. Tolerant animals, however, are unresponsive. These observations are consistent with the observation (Needleman, 1970) that blood vessels (aorta) removed from tolerant animals are extremely resistant to the relaxant effects o f GTN in vitro (up to 1000-fold shift in in vitro d o s e - r e s p o n s e curve).

4. Discussion The results of this study demonstrate that the induction of tolerance to organic nitrates by the injection of GTN at high doses for 3 - 4 days is accompanied by an increase in sympathetic tone (table 1). This increase probably represents an adaptation to the chronic vasodepression caused by frequent administration of GTN. Although the turnover of heart NE is increased in tolerant animals, this change does not appear to play an integral role in the development of the tolerant state. No changes were observed in pressot response or lethality to epinephrine or the indirect acting adrenergic agent tyramine. The conclusion that changes in sympathetic function do not play a role in GTN tolerance is based on the experiments reported here which show no change in sensitivity to GTN caused by sustained increase in sympathetic tone induced by cold exposure, no changes in sensitivity to direct or indirect acting sympathomimetics, no alteration of tolerance induction when sympathetic compensation is severely impaired by inhibition of NE biosynthesis, and no change in response to local administration o f a vasoconstrictor (AII) to the carotid sinus. In support of this conclu-

193

sion it was observed (Rush et al., 1971) that impairment of sympathetic function by guanethidine or anesthesia during pretreatment with GTN had no effect on tolerance development. These authors suggest that central mechanisms are involved in tolerance. Our observations that spinal rats remain tolerant to GTN (unpublished data) and that vascular smooth muscle removed from a tolerant animal is less sensitive to GTN in vitro (Needleman, 1970) are not consistent with the involvement o f central mechanisms. These observations and previous studies (Clark, 1970; Needleman, 1970; Needleman et al., 1971) which demonstrate that enhanced biotransformation is not a factor in the development of tolerance lead to the conclusion that the decreased sensitivity to organic nitrates is due to some quantitative or qualitative change in the reaction of this class o f chemical compounds with their specific vascular receptors.

Acknowledgements The authors wish to thank Mrs. Anne H. Kauffman and Miss Jennifer M. Cady for their skillful assistance. This work was supported by USPHS grants HE 11771, HE 14397,GM 0096 and American Heart Association grants 68-115 and 69-722.

References Clark, D.G., 1970, The supersensitivity of the rat cardiovascular system to epinephrine after repeated injections of ethylene glycol dinitrate, To×icol. Appl. Pharmacol. 17, 433. Clark, D.G. and M.H. Litchfield, 1969, Metabolism ofethy!ene glycol dinitrate (ethylene dinitrate) in the rat following repeated administration, Brit. J. Industr. Med. 26, 150. Kopin, I.J., G. Hertting and E.K. Gordon, 1962, Fate of norepinephrine-H 3 in the isolated perfused rat heart, J. Pharmacol. 138, 34. Lang, S., E.M. Johnson, Jr. and P. Needleman, 1972, Metabolism and vascular responses of glyceryl trinitrate in the eviscerated rat, Biochem. Pharmacol. 21,422. Louis, W.J., S. Spector, R. Tabei and A. Sjoerdsma, 1969, Synthesis and turnover of norepinephrine in the heart of the spontaneously hypertensive rat, Circulation Res. 24, 85. Maickel, R.P., R.H. Cox, J. Saillant and F.P. Miller, 1968, A method for the determination of serotonin and norepinephrine in discrete areas of rat brain, Intern. J. Neuropharm. 7,275.

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ILM. Johnson, Jr. et al., Glyceryl trinitrate tolerance

Needleman, P., 1970, Tolerance to the vascular effects of glyceryl trinitrate, J. Pharmacol. Exptl. Therap. 171, 98. Needleman, P., D.J. Blehm, A.B. Harkey, E.M. Johnson, Jr. and S. Lang, 1971, The metabolic pathway in the degradation of glyceryl trinitrate, J. Pharmacol. Exptl. Therap. 179, 347. Palai6, D., 1971, Effect of angiotensin on noradrenalin-3H accumulation and synthesis in vivo, Can. J. Phys. Pharmacol. 49,495.

Rush, M.L., W.J. Lang and M.J. Rand, 1971, Studies on compensatory reflexes and tolerance to glyceryl trinitrate (GTN), European J. Pharmacol. 16, 148. Spector, S., 1966, Inhibitors of endogenous catecholamine biosynthesis, Pharm. Rev. 18,599. Vigliani, E.C., G. Cavagna, G. Locati and V. Foa, 1968, Biological effects of nitroglycol on the metabolism of catecholamines, Arch. Environ. Health 16,477.