Responses of dog large coronary arteries to constrictor and dilator substances: Implications for the...

Responses of Dog Large Coronary Arteries to Constrictor and Dilator Substances: Implications for the Cause and

Treatment of Variant Angina Pectoris

JAMES A. ANGUS, PhD, ROBERT M. BRAZENOR, PhD, and MATTHEW A. Le DUC

Canine circumflex coronary artery ring segments ceptor antagonists. In a blood-perfused left anterior were mounted in organ baths and contracted by descending coronary artery preparation, external ergonovine (ergometrine), serotonin, phenylephrine, diameter was measured by sonomicrometry under norepinephrine (in the presence of propranolol), conditions of controlled flow and resistance. Intra- potassium (K+) and a thromboxane A2 mimetic, arterial serotonin, U46619 and ergometrine infusions U46619. Ergometrine was classified as a serotonin decreased the diameter by up to 16% without agonist since concentration-response curves were causing spasm (0 lumen diameter). Lowering the competitively inhibited by methysergide but not by perfusion pressure from 90 to 60 mm Hg increased alpha-adrenoceptor antagonists. Arteries precon- the decrease in diameter during serotonin infusions. tracted to 60% of maximum were relaxed by Verapamil (50 to 150 ng/ml) and nifedipine (15 to verapamil, nifedipine and glyceryl trinitrate. How- 45 ng/ml) inhibited, to a similar degree, the de- ever, nifedipine was lOO-fold more potent against K+ than either serotonin- or phenylephrine-con-

crease in coronary artery diameter in response to serotonin infusions. However, verapamil was more

tracted vessels. Verapamil showed some selectivity for K+ and serotonin contractions compared with

effective than nifedipine in depressing the sinoatrial node, abolishing reflex tachycardia and lengthening

phenylephrine and was almost ineffective against the atrioventricular conduction time. These studies U46619. Glyceryl trinitrate was without selectivity. suggest that verapamil and nifedipine are only se- Electrical field stimulation (2 and 20 Hz, lo-second) lective antagonists of constrictor agents in the dog caused a weak contraction or relaxation or biphasic coronary artery, that both low perfusion pressure response in the isolated coronary ring. Contraction and beta-adrenoceptor blockade may amplify was amplified by propranolol (up to 1 PM) or me- constrictor responses, and that nifedipine has fewer toprolol(50 ng/ml) and inhibited by alpha-adreno- cardiotoxic effects than verapamil.

Large coronary arteries decrease in luminal diameter by a maximum of about 20% during intravenous ergonovine (ergometrine) administration in man,l or methoxamine2 or stellate ganglion stimulation in dogs.” This weak vasoconstriction of the large conduit vessels would not normally be sufficient to reduce coronary blood flow and induce angina. However, in some patients with Prinzmetal’s variant angina,4 the ergonovine provocative test may induce complete coronary artery occlusion (vasospasm), often at the site of a minimal organic lesion.5*6 Although this clinical test is useful in demonstrating that spasm can occur, it does not nec- essarily help to identify the cause of vasospasm that occur spontaneously. At least 2 factors may be involved in vasospasm. First, a constrictor substance such as

From the Baker Medical Research Institute, Commercial Road, Prahran, Victoria, Australia. This study was supported in part by grants from the National Heart Foundation of Australia and the National Health and Research Council.

Address for reprints: J. Angus, PhD, Baker Medical Research Institute, Commercial Road, Prahran, 3181, Victoria, Australia.

norepinephrine released from sympathetic nerve ter- minals, or serotonin or thromboxane As released from platelets could initiate focal vasoconstriction. Second, this constrictor stimulus would require an amplifier before vasospasm occurs.

We investigated the reactivity of dog large coronary artery segments to sympathetic nerve stimulation and to a range of vasoconstrictor substances. Interactions of verapamil, nifedipine and glyceryl trinitrate with various vasoconstrictors were also assessed. Experi- ments were performed in isolated coronary artery ring segments in which changes in circumferential force were measured, and in a blood-perfused left anterior descending coronary artery (LAD) in which external diameter was measured by sonomicrometry. These 2 ap- proaches have provided an insight into the variation of coronary constrictor agents in their requirement for extracellular calcium, suggested 2 factors other than fixed lesion that may amplify constrictor responses, and highlighted some differences in the cardiovascular properties of verapamil and nifedipine.

July 20, 1983 THE AMERICAN JOURNAL OF CARDIOLOGY Volume 52 53A

Methods

Isolated coronary artery ring segments: Greyhound dogs (20 to 25 kg) of either sex ‘were anesthetized with sodium pentobarbital (40 mg/kg, intravenously). The chest was opened and the heart removemd. Approximately 4 cm of the proximal circumflex coronary artery was cleared of connective tissue and cut into 3-mm-long ring segments. The segments were suspended in a jacketed organ bath by 2 stainless-steel hooks; the lower hook connected directly to an isometric force displacement transducer (Grass FT03C). The 30-ml organ baths contained a solution of a composition (in mM) of Na+ 144, K+ 5.9, Ca++ 2.5, Mg++ I .2, Cl- 128.7, HCO; 25, SO;- 1.2, HzPO; 1.2, and glucose 11 at 37’C, aerated with a gas mixture of 95% oxygen and 5% carbon dioxide. An initial force of 4 g was applied and readjusted to this level 30 minutes later if necessary. As many as 12 ring segments were cut from each circumflex artery and 6 rings ‘were used concurrently.

After 60 minutes of equilibration, concentration-response curves were constructed by the cumulative addition (0.5 log unit increments) of constrictor substance until the maximal response was achieved. Alternatively, concentrations of verapamil or serotonin antagonists were left in contact with the tissue during the 60-minute equilibration period. In all experiments, only a single concentration-response curve with or without antagonist was obtained in each segment. The advantages of this design have been discussed previously.7 In a second series of experiments, ring segments were constricted by a single concentration of agonist chosen to cause approximately 80% of the maximal response to that drug. When the constriction had reached a pl.steau, the ring segments were relaxed toward the preconstricted level by the cumulative addition of verapamil, nifedipine or glyceryl trinitrate.

Responses to sympathetic nerve stimulation were measured after square-wave monophasic electrical field pulses (2 or 20 Hz, 2 ms duration, 10 V/cm) (Grass SD9) for 10 seconds de- livered from platinum field electrodes placed either side of the coronary ring segment. The field pulses were monitored on an oscilloscope. Only 2 periods of 2- and 20-Hz field stimulation were given to any 1 ring segment, the second period after 30-minute equilibration with propranolol, metoprolol or phentolamine. Some segments were pretreated with an irreversible alpha-adrenoceptor antagonist (benextramine) or propranolol before the first period of field stimulation.

Measurement of coronary artery diameter: A 5-cm length of LAD together with a 3-cm-wide portion of the surrounding myocardium was separated from a dog heart im- mediately after its removal from an anesthetized greyhound. The tissue was immersed in cold oxygenated Krebs’ solution (see coronary ring method). The LAD was cannulated near its origin and again 3 to 4 cm distal. A small catheter was placed in a side branch of the LAD to monitor intraluminal perfusion pressure. Hepariniz,ed femoral arterial blood from an anesthetized support dog (pentobarbitone, 30 mg/kg intravenously and 6 mg/kg/h) was pumped (Watson-Marlow) through the coronary artery segment at 50 ml/min and then through a pneumatic Starling resistance before being returned to the femoral vein of the support dog (Fig. 1).

Continuous phasic external coronary artery diameter was measured by the transit-time ultrasound technique.8 With minimal dissection, a small, lightweight piezoelectric receiver crystal (1.5 mm X 3 mm, 5.4 mg) was placed beneath the LAD. A second emitter crystal (9 MHz) of similar dimensions was glued to the upper surface of the LAD (directly above the receiver crystal) with a small amount of tissue adhesive (cy- anoacrylate, Eastman Kodak 910). The accuracy, linearity and stability of the entire system :resolves changes in diameter of <O.Ol mm and does not drift over long periods.

SUPPORT DOG

SONOMICROMETER

PRESSURE : MSTAL SIDE BRANCH PROXIMAL

FIGURE 1. The in vivo experiments in which the arterial blood from a support dog was used to perfuse the left anterior descending coronary artery from another heart under conditions of controlled flow and resistance. BP = blood pressure.

The surrounding myocardium was clamped to limit the perfusion of the coronary resistance vessels as much as possible. When this perfusion did occur, the venous blood was collected in a polyethylene tray and returned to the supporting dog. Serotonin (or saline solution) was infused (at 1 ml/min by roller pump) directly into the coronary artery by a side-hole catheter in the proximal cannula. This procedure allowed cumulative concentration-response curves to be readily obtained by changing the concentration of the serotonin infusion in 10 ml vials. The proximal and distal perfusion pressures were monitored from side ports (Fig. 1). Mean and phasic LAD external diameters were continuously measured. Hemody- namic measurements from the support dog included aortic blood pressure from a catheter advanced from the femoral artery and heart rate and the PR interval from the lead II electrocardiogram run at 100 mm/s chart speed. During verapamil experiments, a bipolar pacing catheter (USCI-No. 6F) was inserted into the right ventricle through the external jugular vein. The effect of verapamil and nifedipine as vaso- dilators in the large coronary artery was evaluated by the degree of blockade of serotonin concentration-response curves. After the intracoronary infusion of serotonin, (&) verapamil was given by bolus and infusion intravenously to the support dog to increase the plasma level to 50,150 and 500 mg/ml. The plasma levels were not measured, but were calculated from the first-order kinetics of verapamil in dogs.g The actual doses were 0.67, 1.56 and 4.47 mg/kg given slowly over 5 minutes, followed by infusions of 9.3,31, and 93 rg/kg/min for 50,150, and 500 ng/ml levels, respectively. After lo-minute infusion of each level of verapamil, the serotonin concentration-response curve was started. A similar protocol was followed for nifedipine except that only intravenous bolus injections were given to the support dog, since this drug has a long half-life compared with verapamil (150 to 180 versus 15 to 30 minutes).lO Doses of nifedipine were 15,45 and 150 pg/kg.

At the end of each experiment, a double-bladed scalpel was used to remove a 4.33-mm length of LAD taken from the area of the sonomicrometer crystals. From the weight of this segment (W) and the tissue density (1.06), the wall volume (VW)

54A REACTIVITY OF CORONARY ARTERIES

was calculated as VW = W/1.06. The internal radius (Ri) was pro&adz 13E-dienoic acid) and glycerol trinitrate; nifedipine calculated from Ri2 = Re2 - (Vw/II.L), where Re is the ex- was dissolved in polyethylene glycol300 and protected from ternal radius. light.

Drugs were freshly prepared daily and diluted in distilled water. Drugs used were benextramine tetrahydrochloride, methysergide bimaleate, and pizotifen malate, (-) noradrenaline bitartrate dissolved in ascorbic acid, 0.1 nM, phenylephrine hydrochloride, cyproheptadine hydrochloride, (*) propranolol hydrochloride, prazosin hydrochloride, ergometrine maleate, serotonin creatinine sulphate, (*) verapamil, U46619 (15 S-hydroxy-9a,-lla-(epoxymethano)

Results

Isolated coronary ring segments: Constrictor substances: The most potent constrictor substance found on the circumflex coronary ring segment was ergonovine (ergometrine) (Fig. 2). However, the magnitude of the maximal response was substantially less than either serotonin (5HT) or a thromboxane AS mimetic drug, U44619. Norepinephrine in the presence of propranolol was similar (although less potent) to ergometrine in terms of magnitude. Phenylephrine, a more selective alpha-adrenoceptor agonist, was similar to norepinephrine.

Coronary Artery

U46_6 19

6 m

-* NA

. . . *

-10 -6 -6 -4

AGONIST log M

FIGURE 2. Comparison of the potency and magnitude of 4 constrictor substances in isolated coronary artery rings. Ordinate, change in isometric force (g); abscissa, concentration of constrictor (log units). EM = ergometrine; U46619 = thromboxane AZ mimetic: 5-HT = serotonin; NA = norepinephrine in the presence of propranolol(1 PM). Error bars are f 1 standard error at maximal response or at ECsO (horizontal).

” 0 -10 -6 -6

Ergometrine (log mol I I)

0 -9 -7 -5 -3

5-HT k-amolll)

ERGOMETRINE

FIGURE 3. Concentration-response curves to ergometrine and serotonin (5HT) in isolated coronary ring segments. Top, methysergide (1 /.MJ was a competitive antagonist of both ergometrine and 5-HT. Bottom, pizotifen (pizot) and cyproheptadine (cypro) (1 PM) were noncompetitive antagonists. Prazosin (pra), 10 nM, failed to antagonize ergometrine concentration-response curves. Error bars are f 1 standard error for 5 to 7 rings. ctrl = control.

The receptor mediating the response to ergometrine was classified as a serotonin receptor and not an alpha-adrenoceptor from the following observations.

a HZ r

METOPROLOL “k?hl

PROPRANOLOL 1jJI.I

PROXIMAL

DISTAL

20 HI

--L

FlGURE 4. Traces from isolated coronary ring segments during electrical field stimulation (2 or 20 Hz, 10 seconds). Top, metoprolol experiments. Two rings from 1 circumflex coronary artery before (left) and in the presence (right) of metoprolol, 50 ng/ml (top ring) and 100 ng/ml (bottom ring). Bottom, traces from 4 rings from 1 circumflex coronary artery (15 mm long) arranged in order from proximal to distal, showing different responses to ZO-Hz field stimulation. Left, control responses; right, in the presence of propranolol, 1 pM.

July 20,1983 THE AMERICAN JOURNAL OF CARDIOLOGY Volume 52 55A

In guinea pig aorta, a tissue responsive to alpha-adrenoceptor stimulation, both ergometrine and serotonin were inactive (up to 1 PM). In dog coronary artery, the specific alpha-adrenocepto:r antagonist prazosin, or the irreversible alpha-adrenoceptor antagonist benextramine, failed to block the response to ergometrine (Fig. 3). More precision was achieved with serotonin antagonists. Methysergide was the only antagonist that has been claimed to block serotonin receptors that displaced the ergometrine and serotonin concentration-response curves in parallel without reduction in maximum. The estimated dissociation constant (KB) for methysergide was similar for ergometrine and serotonin as agonists (13 and 10.2 nM), indicating that ergometrine was a serotonin receptor agonist i:n this tissue.ll Methysergide was also a constrictor substance itself in these experiments (partial agonist), as shown by the increase in baseline before the commencement of the agonist curves (Fig. 3). In contrast, other :serotonin antagonists, pizotifen and cyproheptadine (I p&f), were noncompetitive antagonists of ergometrine without causing a constrictor response on their own (Fig. 3).

Sympathetic nerve stimulation: Ring segments cut from a single 3-cm length of circumflex coronary artery often responded differently to electrical field stimulation. Some segments contracted and others relaxed, but most showed a biphasic response pattern of early constriction followed by more prolonged relaxation (Fig. 4). The responses were mediated by norepinephrine released from sympathetic nerves, since a combination of propranolol(1 pLM) and phentolamine (1 pA4) com- pletely abolished all respo:nses as did the Na+ channel blocker tetrodotoxin (0.1 to 1 PM). In segments pretreated with the irreversi’ble alpha-adrenoceptor antagonist benextramine, only marked relaxation was observed to field stimulation, a response that was sub- sequently blocked by propranolol (Fig. 5). In arteries pretreated with propranolol(1 PM) or metoprolol (50 to 100 ng/ml, a concentration observed in the plasma of patients in treatment for exercise-induced angina), the constrictor response was en.hanced (Fig. 4 and 5). These findings emphasize that large coronary arteries are neurally innervated and that responses may be mediated by alpha- or beta-adrenoceptors or both.

Vasodilator substances: In the isolated ring segments, the specificity of the dilator action of verapamil,

FIGURE 6. Relaxant effects of nifedi-

pine, verapamil, and glyceryl trinitrate (GTN) in isolated coronary artery rings contracted by K+ (25 m/M), phenyl-

z 100

ephrine (PE, 1 PM), serotonin (5HT., 0.3 •-:~huq..,,

PM), or thromboxane A2 mimetic s ? -\ (U46619, 0.03 PM). Ordinate, chmge in force, percentage of maximal response to constrictor agent; abscissa, concentration of dilator agent (log scale). Horizontal error bars are f 1 standard error at lCSO (concentration giving 50% inhibition of contraction) for 16 rings in each curve. Only 1 curve was obtained in each ring. NIFEDIPINE

CIRCUMFLEX CORONARY ARTERY

2 Hz 20 Hz 200 pulses

2

0

2

1

E 0

Q

-1

-2

-3

FIGURE 5. Group results for circumflex coronary artery ring segments to electrical field stimulation at 2 and 20 Hz for 10 seconds. Only 1 treatment was performed on each ring segment. Control (alpha- + beta-adrenoceptors), n = 7; benextramine 10 p M (beta-adrenoceptors only), n = 8; propranolol 1 /.LM (a-adrenoceptors only), n = 4. Error bars are f 1 standard error.

nifedipine and glyceryl trinitrate was compared in arteries contracted submaximally with K+, (25 mM), serotonin (0.3 PM), phenylephrine (1 ~n/r), and the thromboxane AZ mimetic U44619 (0.03 puM). Glyceryl trinitrate relaxed the arteries with a similar IC50 value (concentration giving 50% inhibition of contraction) for each of the 4 constrictor agents (Fig. 6). In contrast, the slow calcium-channel blockers nifedipine and verapamil showed marked differences in their ability to relax arteries contracted by the 4 constrictor substances. Ni- fedipine was very potent against K+ depolarized arteries (IQ,0 = 8.5 X lo-9M) but was relatively weak against both serotonin (I&,, = 1.8 X 10m6M) and phenylephrine (I&o = 1.7 X 10m6M) contracted arteries. Verapamil was much weaker than nifedipine against K+ (IQ,0 = 1.1 X lO_sM), but was similar against serotonin (ICSO = 1.5 X 10m6M). Verapamil was 4-fold more potent against serotonin-contracted tissues than against phenylephrine. However, in contrast to glyceryl trinitrate, verapamil was extremely ineffective against coronary rings contracted by U46619. This difference could not be accounted for by different absolute starting levels of constriction, since glyceryl trinitrate was equally ef-

Coronary Artery

6 -7 -5 -3

VERAPAMIL GTN log M


Verapamil

0 -9 -7 -5 0 -a

5-HT (log mol / I) PE

INTRACORONARY INFUSION

gJ_ - - - - -

s*b5op[- - - - - -

m’g; [ _-_--_ 4.3 I-_ - -

D%. 3.8 1

- -

-

Diam. II-

-- -

(pm01 / I)

r

-6 -4 0 -10 -a -6

(log mol / I) U-466 19 (log mol ! 0

IrJuLl

--

--

--

coronary artery was curvilinear, the artery showing greater passive changes in diameter in pressures from 50 to 100 mm Hg than from 100 to 150 mm Hg. In each preparation, the pressure/diameter type curve was de- termined by varying the pressure in the Starling resis- tor. Intracoronary serotonin infusions at a side branch pressure of 90 mm Hg lowered the external diameter, calculated internal diameter and area in a concentration-dependent manner (Fig. 8 and 9). The decrease in diameter was clearly an active response to the serotonin and was not due to a decrease in luminal pressure, em- phasizing an advantage of this preparation in defining the magnitude of diameter change in large coronary vessels. The diameter returned rapidly to preinfusion levels when the serotonin was replaced by 0.9% sodium chloride. Serotonin infusions did not alter the hemo- dynamics in the support dog, presumably because the metabolism of serotonin through the lungs and dilution on entering the femoral vein. Intracoronary infusions of U46619 up to 1 PM caused a decrease in diameter similar to serotonin, but was approximately 3-fold more potent. At 0.3 and 1 PM, U46619 caused marked cardiovascular changes in the support dog including hy- pertension, tachycardia and an increase in central venous pressure. Ergometrine (1 to 1000 nM) caused a maximal decrease in luminal diameter of only 12%, about half that of serotonin. The recovery of diameter after stopping U46619 was approximately 30 minutes, and >l hour after ergometrine. We never observed a decrease in luminal diameter of >40% for any of these constrictor substances.

z-

L 3.3 t t I’0 Id0 360 lb00 ha rot ’ LHT (nM) Recov8ry

FIGURE 6. Hemodynamic measurements during intraccronary infusions of serotonk(5-HT). From top, the records are mean proximal perfusion pressure (PPean side branch pressure (m), mean distal pressure (DP), mean (DIAM) and phasic (DIAM) external coronary artery diameter. All units of pressure are in millimeters of mercury and diameter is in millimeters. Recovery traces are shown at right.

fective against the same concentration of constrictor agents.

In a different experimental design in which verapamil was equilibrated with the artery before the addition of the constrictor agent, a similar pattern was noted (Fig. 7); that is, verapamil (1, 10 PM) was more effective against serotonin concentration-response curves than against phenylephrine. The poor inhibition by verapamil of the response curve to U46619 may suggest that this constrictor does not require extracellular Ca++ for contraction. In 6 coronary rings equilibrated for 45 minutes in Ca++-free Krebs’ solution, serotonin and phenylephrine failed to elicit a contraction, while U46619 was still most effective (80% of normal).

Diameter measurements in situ: Pressure-diameter relationship, serotonin, U46619, ergometrine: The relationship of intraluminal pressure (measured from side branch in the LAD) to luminal diameter of the

FIGURE 7. Effects of verapamil (0, 1 and 10 I.cM, 60-minute contact) on vasoconstrictor responses to serotonin (5-HT), phenylephrine (PE) and the thromboxane A2 mimetic U46619 in isolated coronary ring segments. Only 1 concentration-response curve was obtained in each ring. Values are mean f 1 standard error, with at least 5 tissues per verapamil concentration.

Amplification of constriction: The effect of perfusion pressure on the magnitude of the decrease in diameter to serotonin infusions was examined. In each artery complete concentration-response curves to serotonin were obtained at 60 and 90 mm Hg intraluminal pressure. These pressures were set before the infusion of serotonin by adjustment to the Starling resistance. The diameter decreased to a lower level during serotonin infusions with 60 mm Hg perfusion pressure, principally because the starting diameter was lower than at 90 mm Hg (Fig. 9). No significant change was observed in the potency of serotonin.

Simple geometric considerations of the effect of luminal encroachment by 20 to 40% (as in atheromatous


37 27 15 6 lun?an

0% snnoochmn1

X DECREASE OUTSIDE DIAMETER

l Ext. Diameter

A Lumen Dlometsf

7o- l Lumm Coo

.I - 0 I IO loo 1000

60 mmHg

*- 0 I IO loo 1000

-- 50 100 150 mlllig

SIDE BRANCH PRESSURE

5-HT (nmol/l)

FIGURE 9. Left top, theoretical relation between percent changes in outside diameter and percent change in luminal diameter of a coronary artery when the ratio of wall thickness tcl lumen radius varies from 0.1 to 0.75. If the ratio of 0.1 is taken as a normal artery, the percent luminal encroachment at rest is calculated for the higher ratio. Adapted from MacAlpin. 33 Left bottom, effect of altering perfusion pressure on the luminal diameter and

luminal area. Measurements were calculated as percentage of values at 90 mm Hg. Bight, effect of perfusion pressure (90 mm Hg left, 60 mm Hg right) on the magnitude of decrease in coronary artery diameter during intracoronary serotonin (5HT) infusions. External and internal diameter and luminal area were measured as oercentage of value at 90 mm Ha before the start of the serotonin infusion. Values are mean f standard error in 10 arteries. Both durves were completed in each artery.

lesions or by wall thickening) suggest that relatively small decreases in diameter (20%) should be sufficient to reduce the luminal diameter to almost O.(Fig. 9).

Comparison of verapamil and nifedipine in vivo: Full serotonin concentration-response curves could be readily repeated up to 4 times in the blood-perfused LAD preparation. The maximal decrease in luminal area from preinfusion level was 20 to 25%. As the concentration was increased further, the diameter started to return toward control (Fig. 10). If the maximal decrease in luminal area for the first concentration-response curve to serotonin was considered to be lOO%, the second and third curves repeated in the same artery were virtually identical in sensitivity and magnitude (Fig. 11). In support dclgs without calcium-channel blocker, systemic blood pressure decreased gradually (by 19 mm Hg) over 5 hours, the period in which 4 serotonin-response curves were completed. The heart rate and PR interval remained steady throughout this period (Fig. 12). In contrast, both nifedipine and verapamil lowered blood pressure in a concentration-dependent fashion. Heart rate was elevated by nifedipine administration but was reduced substantially by verapamil (Fig. 12). Atrioventricular conduction was unaffected by the lower concentrations (15,45 ng/ml) of nifedipine, whereas verapamil caused marked increase in PR interval even with the first concentration (50 ng/ml). With

CORONARY ARTERY T T

80

oqmo

SEROTONIN prnol/l

FfGURE 10. Effect of verapamil on constrictor responses to serotonin in blood-perfused left anterior descending coronary artery. Verapamil was given by intravenous bolus and infusion to the support dog to give plasma levels calculated to be 50, 150, and 500 ng/ml. Each artery (n = 6) had 0 and 50 ng/ml verapamil, but only 7 and 5 of these, respectively, had 150 and 500 ng/ml. Responses were calculated as luminat area, taking control values at the start of each serotonin curve as 100%.


i . ’ I

0 0.03 0.1 0.3 1 3 10 0 0.030.1 0.3 1 3 10 0 0.03 0.1 0.3 t 3 10

5-I-IT ph4 5-HT P,M 5HT ~JM

FIGURE 11. Effect of verapamil ano’ nifedipine on constrictor responses to serotonin (6HT) infusions in the perfused left anterior descending coronary artery. Ordinate, decrease in luminal area as a percentage of the maximal decrease to serotonin observed in the first curve in each artery. Subsequent curves (2, 3. and 4) were obtained without antagonist (lefi), or in the presence of verapamil(50 to 500 ng/ml, middle) or nifedipine (15 to 150 ng/ml, right). Both antagonists were administered to the support dog. Error bars are f 1 standard error. Controls, n = 5: verapamil, n = 5 to 8; nifedipine, n = 6.

MAP

SUPPORT DOG

150 r,

50 I& -+., l . \ %.

500s 1 2 3 4

FIGURE 12. Mean arterial pressure (MAP) and heart rate (HR) values for the support dogs during the 4 serotonin infusions (numbers 1 to 4 on abscissa). There were 3 groups of dogs; control, circles; nifedipine, triangles; and verapamil, squares. The concentrations of calcium antagonists are shown. Error bars are f 1 standard error.

150 ng/ml of verapamil, 5 of 7 dogs had atrioventricular block (Fig. 13). The range of concentrations of both verapamil and nifedipine were closely matched in their ability to reduce the coronary constrictor response to serotonin (Fig. 11). With the lower concentrations of

verapamil (curve 2 and 3), the decrease in diameter during serotonin infusions was less than that during the control curve. Nifedipine also lowered the sensitivity of the coronary artery to serotonin and reduced the maximal constrictor response, a response compatible with the concentration-response curves in the isolated coronary ring (Fig. 7).

Discussion

In these experiments we have qualified the potency and magnitude of a variety of constrictor substances on the coronary artery in the controlled environment of the organ bath where neural, hormonal and metabolic changes can be eliminated. In the blood-perfused LAD preparation, we could determine direct effects of constrictors and antagonists on coronary diameter without the influences of blood flow and pressure changes. Therefore, these 2 preparations have allowed a more rigorous analysis of the responses of the large coronary artery than is possible in man or in the intact myocardium in experimental animals2

Vasospasm: Vasospasm, defined as 0 luminal diameter, was not observed in the experimental preparations of the LAD during infusion of high concentrations of constrictor agents. The maximal decrease in luminal diameter was about 20 to 30%, a reduction that would be unlikely to influence flow, especially if the constriction was confined to a few isolated sites. How- ever, these dog coronary arteries were essentially normal, without atheromatous lesions, and were denervated by removal from the donor dog. Under appropriate conditions, one may be able to observe vasospasm ini- tiated by a relatively small decrease in luminal diameter. Such an amplified response could occur at sites of relatively small atheromatous lesions,e12 where luminal encroachment or medial hypertrophy would lead to a greater decrease in luminal diameter for a given constrictor stimulus.

Another factor that may amplify the response to constrictor stimuli is low coronary pressure. Our experiments show that the diameter of a large coronary artery decreases to a significantly lower level in the


presence of a constrictor substance when the perfusion pressure was reduced from 90 to 60 mm Hg. Therefore, 1 explanation of recurrent nocturnal pain that13 wakes patients from sleep (13 of 1.7 patients, all with rest pain) could be that lower arteri.al pressure during sleep am- plifies the constrictor response to some stimuli.

Possible mediators of coronary spasm: Experi- ments on isolated coronqy rings suggest that ergometrine is a potent coronary artery constrictor substance acting as a serotonin agonist.” Similar conclusions have been reported previously.14J5 The specificity of ergometrine as a diagnostic agent in variant angina may indicate a role for serotonin, perhaps released from platelets at the site of a minimal lesion. Platelets also produce thromboxane Az(TxAz), a product of arachi- donic acid metabolism that is a powerful vasoconstrictor agent and an inducer of both platelet aggregation and the platelet release reaction.ls TxAz is very unstable, at 37’ at pH 7.5 with a half-life of 30 seconds, making it very difficult to define responses quantitatively. Therefore, we used the stable analog of TxA2, U46619, which has a potent const:rictor action similar to TxAz in human and dog coronary artery.17J8 Although TxAz is a candidate for initiating vasospasm, clinical trials with synthesis inhibitors have been disappointing.lg

Norepinephrine may also be a trigger stimulus for vasospasm. Coronary artery ring segments, however, did not contract consistently to this catecholamine added to the organ bath unless beta-adrenoceptor blockade was present. In the perfu.sed coronary artery, norepinephrine infused into the lumen caused a small increase in diameter, which was also blocked by propranolol. These experiments do not support a role for circulating catecholamines in initiating coronary constriction. However, sympathetic nerve stimulation experiments may provide a clue to neurogenic vasospasm. Adjacent

3-mm segments from the circumflex coronary artery react very differently both in direction and magnitude of response to lo-second periods of stimulation. Therefore, it may be possible for a small segment to respond with marked constriction, although adjacent segments are less responsive. The receptors stimulated by the released norepinephrine are clearly alpha- and beta-adrenoceptors, as seen from the effects of the antagonists phentolamine and propranolol. Moreover, the density and type of adrenoceptor may vary along a large artery.

Because beta-adrenoceptors are apparently innervated in large coronary arteries, the use of beta-adrenoceptor antagonists could raise the incidence of episodes of variant angina by reducing neurogenic dilator tone. Indeed, this has been reported for propranolol in a small number of patients in a recent trial.20 Theoretically, if beta-adrenoceptors in coronary arteries were of the beta2 subclass, then a selective betal-adrenoceptor antagonist may still be useful in the treatment of variant angina.21 This does not appear to be the case for large coronary arteries, at least from the dog, where agonist and antagonist studies showed some evidence for a betal-adrenoceptor classification.22 Our studies showed that the so-called selective betal-adrenoceptor antagonist metropololz3 amplified the constrictor response to sympathetic stimulation. We deliberately chose a concentration of metoprolol(50 ng/ml) that was within the range of peak plasma levels (20 to 198 ng/ml) observed in patients receiving metoprolol therapy for typical angina pectoris.24J5 Therefore, beta-adrenoceptor blockade with nonselective or selective (betal) adrenoceptor antagonists may also act as an amplifier of focal neurogenic vasoconstriction.

Nifedipine, verapamil, and glyceryl trinitrate: Constrictor substances may vary in their requirement

SUPPORT DOG

VERAPAMIL no/ml

, I Cl CZ C3 ‘24

CONTROLS NIFEDIPINE Wml

fKiClRE 13. Effect of verapamil and nifedipine on the atrioventricular conduction (P-R interval in milliseconds) in the support dog. Each line represents the value for 1 experiment. Readings are the average of 3 to 6 intervals measured from lead II electrocardiogram, recorded just before the start of each of 4 serotonin curves.


for extracellular and intracellular calcium for activating contraction. Therefore, if dilator agents act by blocking the slow calcium channel without affecting the calcium release from intracellular stores, then only constrictors dependent on extracellular calcium should be sensitive to these antagonists. Both verapamil and nifedipine are classed as slow calcium channel blockers,10~2sJ7 so they should have similar relative potencies against a range of constrictor agents. This is clearly not the case in the present studies, since nifedipine was lOO-fold more potent against potassium-depolarized arteries than against serotonin or phenylephrine, whereas verapamil was similar in potency against serotonin and potassium but 3-fold less potent against phenylephrine. Similar selectivity by verapamil or methoxy verapamil (D600) has been noted in dog coronary28 and in rat aorta.2g Nifedipine has also been shown to be more potent against potassium than phenylephrine or serotonin in dog femoral arteries.30 Therefore, verapamil and nifedipine have very different constrictor selectivities and the present classification of these drugs into a single class appears to be inadequate. In contrast with the calcium antagonists, the dilator action of glyceryl trinitrate was relatively nonselective of the constrictor agents consistent with an intracellular mechanism at- tributed to the formation of active, unstable, interme- diate s-nitrosothiols.“l

These studies emphasize that the success of using slow calcium-channel blockers to treat coronary vasospasm will depend to some extent on the nature of the constrictor stimulus. For example, verapamil would appear most useful against serotonin-induced constriction but would be much less effective against noradrenaline and almost inactive, perhaps, against thromboxane AZ. However, the studies in vivo suggest that there are other important differences between nifedipine and verapamil. We chose to administer both calcium antagonists at a dose to bring the plasma levels into a range that would be found therapeutically. For verapamil, this is 15 to 100 ng/m1.10,32 Verapamil (50 and 150 ng/ml) was comparable to nifedipine (15 and 45 ng/ml) with regard to the antagonism of the serotonin constrictor response in the perfused coronary artery. However, verapamil appeared to be much more effective than nifedipine in depressing the sinoatrial node, abolishing reflex tachycardia, and lengthening the atrioventricular conduction time. These experiments, performed under barbiturate anesthesia, may have potentiated the effects of verapamil on nodal tissue. Nevertheless, a marked difference between the 2 calcium antagonists was observed. It is difficult to predict what degree of large coronary artery constriction would be required to trigger an episode of vasospasm in patients. It is equally hazardous to suggest from these experiments what degree of blockade is necessary for the calcium antagonists to be effective in vasospasm. What is clear, however, is that the actions of calcium antagonists are not confined to the large coronary artery at concentrations that are effective in reducing serotonin-induced constriction. Perhaps other actions of the drugs related to a decrease in myocardial oxygen con- sumption are just as important in the treatment of variant angina as in angina of effort.

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