Cardiac α-adrenoceptors: postjunctional and prejunctional

J Mol Cell Cardiol 18, (Supplement 5) 17-32 (1986)

Cardiac ~-adrenoceptors: postjunctional and prejunctional Michael J.Rand, Lun H.Tung, William J.Louis and David F.Story Department of Pharmacology University of Melbourne, PBrkville and

Departmentof Clinical Pharmacology,Austin Hospital, Heidelberg, Victoria, Australia

Postjunctional ~-adrenoceptors subserve positive inotropic and chronotropic responses. When B-adrenoceptorsare blocked, agonists that act on ~1-adrenoceptors evoke positive chronotropic responses in the pithed rat and rat isolated atria.

The

rank order of potency for this effect is adrenaline > noradrenaline > phenylephrine > methoxamine. The order of potency for antagonists to block the responses is prazosin > phentolamine > yohimbine. Thus, the postjunctional e-adrenoceptors are of the ~1-subtype. The positive chronotropic responses elicited by activating eladrenoceptors have a slower time course than those elicited by activation of Badrenoceptors. WhenB-adrenoceptors are blocked by propranolol, the positive chronotropic response to phenylephrine is enhanced by increasing the calcium concentration or by the calcium channel activator Bay K 8644 (0.1 uM), whereas the response is decreased by lowering the calcium concentration or by calcium antagonists (verapamil, nifedipine, nicardipine and diltiazem). Therefore, the positive chronotropic response to

~1-adrenoceptor activation involves an increased influx of calcium

through calcium channels. Prejunctional ~-adrenoceptors are involved in autoinhibitory feedback regulation of transmitter release from-noradrenergic neurones. In rat atria, the release of noradrenaline induced by sympathetic nerve stimulation is inhibited by both clonidine and methoxamine, and is enhanced (by disruption of noradrenaline-mediated autoinhibition) by both idazoxan and prazosin. prejunctional ~-adrenoceptors are of both e~- and ~-subtypes.

Thus, the

Drugs which produce

blockade of postjunctional ~-adrenoceptors could also produce an increase in neUrogenic release of noradrenaline due to blockade of prejunctional ~1-adrenoceptors, and this might result in more complex effects than would be anticipated.

Key words:

chronotropic responses, idazoxan, prazosin, Bay K 8644 calcium antagonists, transmitter release.

0022-2828[86/$50017+16 $03.00[0

(~)1986 Academic P~sslnc.(London) Limited

18

M.J.R~u~d eta/.

Introduction When Ahlquist (1) introduced the classification of adrenoceptors into the ~- and B-types, i t was clear that the cardiac adrenoceptors were predominantly of the B-type. Ahlquist's classification and terminology did not becomewidely accepted until the developmentof potent antagonists of B-adrenoceptors, the f i r s t being dichloroisoprenaline which was introduced by Powell and Slater (31) in 1958 at Eli Lilly.

Subsequently, pronethalol (7) and propranolol (8) were introduced by Black

and his colleagues at ICI and they were found to be of great therapeutic value in the treatment of many clinical disorders.

Since then, many other B-adrenoceptor drugs

have been developed, and the importance of these drugs in therapeutics dominated thinking about cardiac adrenoceptors to the extent that cardiac ~-adrenoceptors were largely overlooked. However,functional evidence for cardiac ~-adrenoceptors has been found in various species in the presence of B-adrenoceptor blockade. The production of positive inotropic effects by an ~-adrenoceptor agonist was f i r s t reported in rat ventricular strips by Wenzel and Su (49) in 1966. The effect was subsequently confirmed in guinea-pig atria (19), rabbit atria (5), chicken papillary muscle (6), frog ventricular strips (2) and humana t r i a l strips (36).

The

functional role of cardiac ~-adrenoceptors had been extensively reviewed (3, 4, 12, 30, 34, 35).

The postjunctional ~-adrenoceptors have been characterized as being of

the ~l-subtype.

The existence of cardiac ~1-adrenoceptors was confirmed by radio-

ligand studies in cardiac membranepreparations from rat (14, 20, 26, 32, 40, 50), guinea-pig (21), rabbit (26, 36) and dog (26). Although cardiac B-adrenoceptors subserve both inotropic and chronotropic responses, i t was originally thought that the ~-adrenoceptors subserved only inotropic responses. However, a positive chronotropic response to ~-adrenoceptor activation was reported, but only in isolated atria from rats made hypothyroid by pretreatment with propylthiouracil or thyroidectomy (23, 27, 38, 48).

Wagnerand

Brodde (48) reported only a t r i v i a l chronotropic response to phenylephrine in isolated right atrium from euthyroid rats after blockade of B-adrenoceptors. Since 1981 a number of observations have been made in euthyroid rats that led to the identification of cardiac al-adrenoceptors subserving positive chronotropic responses. This phenomenonhas been demonstrated in pithed rats (17, 18, 44, 45) and in rat isolated cardiac preparations (10, 24, 46, 47).

Cardiac~Adrenoceptors

19

The cardiac ~z-adrenoceptors subserving positive inotropic and chronotropic responses are postjunctional.

In addition, there are prejunctional ~-adrenoceptors

associated with the terminals of the autonomic nerves innervating the heart.

The

i n i t i a l subclassification of ~-adrenoceptors was based on their anatomical d i s t r i b u t i o n , those located postjunctionally being termed the ~z-subtype and those located prejunctionally were the ~2-subtype.

Subsequently, the use of agonists and

antagonists with relative selective a f f i n i t y f o r one or the other led

to the

identification of a population of postjunctional ~2-adrenoceptors in certain tissues and of prejunctional ~z-adrenoceptors in certain nerve teminals.

Evidencefor the

existence of prejunctional ~z-adrenoceptors associated with cardiac sympathetic nerves has been obtained in the rat in vivo (15, 22) and in isolated preparations of rat atria (43) and rabbit heart (41). The prejunctional ~-adrenoceptors subserve inhi,bition of transmitter release and are involved in an autoinhibitory feedback loop in noradrenergic transmission.

Disruption of the loop by blockade of ~-adrenoceptors results in a

greatly increased release of noradrenaline by a train of nerve impulses (33).

Thus

i t is necessary to take account of prejunctional as well as postjunctional effects of drugs proposed for blocking cardiac ~-adrenoceptors. Methods and Results Postjunctional cardiac ~-adrenoceptors in pithed rats Female Wistar rats weighing 200 to 300 g were pithed under halothane anaesthesia by passing a probe through the l e f t o r b i t into the spinal cord.

A carotid

artery was cannulated for measuring blood pressure and the ECG was used to trigger a cardiotachometer to provide a continuous measure of heart rate: these parameters were recorded on a Beckman polygraph. Phenylephrine (1-100 pg/kg, i . v . ) produced dose-dependent increases of blood pressure, and also increases in heart rate of up to 100 beats/min.

Propranolol

in doses of 0.01 to 0.3 mg/kg produced a dose-dependent reduction in the tachycardia response to phenylephrine, but the maximal blockade amounted to only a 50% attenuation of the response: increasing the dose of propranolol to 3 mg/kg produced no further reduction.

The residual response to phenylephrine in the presence of complete

B-adrenoceptor blockade was reduced and f i n a l l y abolished in a dose-dependentmanner by ~-adrenoceptor blocking drugs.

The IDso value for prazosin (2.6 • 0.6 pg/kg) was

M.J. Rand et aL

20

1.4% that of phentolamine (180 • 20 pg/kg) and 0.06% that Of yohimbine (4700 • 1100 pg/kg).

I t is clear, therefore, that part of the tachycardia response to phenyl-

ephrine involves activation of ~z-adrenoceptors. The combinede- and B-adrenoceptor antagonist labetalol, which, with respect to e-adrenoceptor blockade, is r e l a t i v e l y selective for the ~z-subtype, abolished entirely the tachycardia response to phenylephrine. Other agents that activate ~z-adrenoceptors also produced tachycardia in the presence of B-adrenoceptor blockade with propranolol and the responses were antagonized by prazosin.

Of those tested, the rank order of potency was adrenaline 9

noradrenaline 9 phenylephrine > methoxamine. The effect of methoxamineand i t s blockade by prazosin were also demonstrable in the absence of propranolol (Fig. 1): this was due to the fact that methoxaminehas both agonistic a c t i v i t y on ~z-adrenoceptors and antagonistic a c t i v i t y on B-adrenoceptors

n=4/C

50 increase in h e a r t rate (beats/rain)

10

100

METHO~(~a~g)

Fig. 1.

Increase in heart rate produced by methoxaminein pithed rats before (C) and

after administration of prazosin (Pz) in doses of 10 then 100 ~g/kg. The positive chronotropic responses to adrenaline, noradrenaline and phenylephrine when B-adrenoceptors were blocked by propranolol differed from those to the same agonists when ~z-adrenoceptors were blocked by prazosin in that the time course of the response to activation of ~-adrenoceptors was slower than that to activation of B-adrenoceptors. Thus, in the presence of propranolol, the peak responses to 3 and 10 ~g/kg of adrenaline were reached 34 • 2 s and 44 • 2 s,

Cardiac ~-Adrenoceptors respectively, after injection.

21

However, in the presence of prazosin, the correspond-

ing times were 12 • 2 s and 14 • 1 s, respectively.

Similar results were obtained

with noradrenaline. The time course of the response to phenylephrine was not s i g n i f icantly prolonged by propranolol, but was significantly shortened by prazosin.

The

difference in the time course of the response to activation of the two types of adrenoceptors may r e f l e c t different mechanisms of coupling of receptor activation to response, and the differences between agonists presumably reflect

differences in

their relative potencies for the two types of receptors. Postjunctional ~-adrenoceptors in rat isolated a t r i a Atria taken from female Wistar rats were mounted in an organ bath containing Krebs-Henseleit solution of the following composition (mM): NaCl, 118; KCI, 4.7; NaHC03 , 25; MgSo, , 0.45; KHzPO, , 1.03; CaCl2 , 2.5; D-(+)-glucose, 11.1.

The

solution was gassed with a mixture of 5% C02 and 95% O~ and maintained at 37~ Contractions were measuredwith an isometric strain gauge exerting a resting tension of 0.5 g.

The output of the strain gauge was recorded continuously on a Grass poly-

graph together with the rate of contractions which was obtained from a cardiotachometer coupler.

Concentration-response curves f o r phenylephrine were constructed

in a cumulative manner by increasing the concentration in steps of 0.5 log units. Subsequent curves were obtained 60 min after the f i r s t .

Drugs that were used to

investigate their effects on the responses to phenylephrine were added 20 min before constructing the curves.

In control experiments, in which no such drugs were used,

the second concentration-response curve was superimposable on the f i r s t . The concentration-response curve for the positive chronotropic effect of phenylephrine was not significantly affected by prazosin (10 riM), but was partly reduced by propranolol (0.3 ~M). However, in the presence of propranolol (0.3 ~M) the residual response was antagonized in a concentration-dependent manner by prazosin which already had a significant effect in a concentration of 1 nM.

In contrast,

yohimbine (10 nM) had no effect on the increase in rate of beating produced by phenylephrine in the presence of propranolol. These data confirm the observations in pithed rat indicating that :1-adrenoceptors subserving positive chronotropic responses are present in rat heart. The chronotropic response of isolated a t r i a to activation of ~1-adrenoceptors differed from that due to activation of B-adrenoceptors in the susceptibility

M.J. Rand et aL

22

of the former, but not the latter, to calcium channel blocking drugs. Thus, the responses to phenylephrinein the presence of propranolol were significantly reduced by verapamil (10 nM), nifedipine (10 nM), nicardipine (10 nM) and diltiazem (100 nM). In the presence of prazosin, when phenylephrineacts only on B-adrenoceptors, the calcium antagonists did not affect the responses. Furthermore,neither verapamil nor nifedipine affected responses to isoprenaline. The findings suggest that the cardiac ~1-adrenoceptors, but not the Badrenoceptors, are coupled to calcium channels that are blocked by the organic calcium channel blocking drugs (46). This postulate was tested by varying the calcium concentration in the Krebs-Henseleitsolution bathing the atria (normally 2.5 mM) and by using the calcium channel activator Bay K 8644. Increasing the calcium concentration to 3.5 mMenhanced, and decreasing i t to 1.25 mMdepressed the positive chronotropic responses to phenylephrinein the presence of propranolol.

Furthermore,the effect of verapamil in reducing the

response to phenylephrinewa~ counteracted by raising the calcium concentration, which demonstrates that verapamil was in fact acting as a calcium antagonist. The calcium channel activator Bay K 8644 (0.1 uM) had a positive chronotropic action in its own right and in addition i t significantly enhancedthe response to activation of the ~1-adrenoceptors by phenylephrinein the presence of B-adrenoceptor blockade (Fig. 2).

Beatklg rate of rat atria(beatslmln) Propranolol(O.3~uM)

present

9 Phenylephrlne(lO~uM)

TB

9

W

5 rnln L

-Fig. 2.

Rateof beating of rat isolated atria.

Increases in rate produced by

phenylephrine (10 pM, indicated by dots below record) and Bay K 8644 are shown by numbers abovethis tracing.

Washoutof the organ bath is indicated by W.

Cardiac =-Adrenoceptors

23

Taken together, the findings indicate strongly that cardiac postjunctional ~1-adrenoceptors are coupled to calcium channels that are sensitive to organic calcium antagonists and that the increased calcium entering the pacemakercells functions as the second messenger in mediating the chronotropic responses. Prejunctional cardiac ~-adrenoceptors Isolated atria were taken as mentioned above and mounted in a 2 ml bath in Krebs-Henseleit solution that contained sodium edetate (0.067 mM) and ascorbic acid (0.142 mM) to retard oxidation of noradrenaline, and atropine (1 ~M) to block cholinoceptors.

The intramural sympathetic nerves were stimulated through platinum

wire electrodes located on each side of the atrial preparation.

Stimulation was with

1 ms pulses at a supramaximal f i e l d strength (about 15 V/cm). The noradrenaline transmitter stores were radiolabelled by incubation for 30 min in [7,8-3H] noradrenaline (10 ~Ci/ml and 0.69 to 1.25 ~M depending on its specific a c t i v i t y ) .

The

tissue was then repeatedly washed with drug-free solution for 60 min during which a 'priming' stimulus of 30 pulses at I Hz was delivered to f a c i l i t a t e removal of nonspecifically bound [3H]-noradrenaline and its metabolites. Stimulation-induced release of transmitter noradrenaline was deduced by measuring the efflux of radioactivity from the atria into the bathing solution. Field stimulation with 1, 2, 4, 8 and 16 pulses at a frequency of 2 Hz resulted in increases in efflux of radioactivity. Whenexpressed as efflux per pulse, the value increased with the number of pulses, reaching a plateau level with g pulses. The selective ~1-adrenoceptor antagonist prazosin (0.1 and 3 ~M) and the selective ~2-adrenoceptor antagonist idazoxan (10 ~M) enhancedthe release of radioactivity elicited by stimulation, the effect being greatest on the plateau values with trains of 8 and 16 pulses.

The non-selective ~-adrenoceptor antagonist phentolamine (3 ~M)

produced much greater enhancementof the stimulation-induced efflux of radioactivity than either prazosin or idazoxan. Combinationof prazosin and idazoxan enhancedthe effluxes to the same extent as did phentolamine. The finding suggest that both ~zand ~2-adrenoceptors are involved in the autoinhibitory effect of noradrenaline on its subsequent release. Further evidence for the presence of prejunctional ~z-adrenoceptors was provided by the finding that methoxamine(10 uM) inhibited the stimulation-induced efflux of radioactivity, and its effect was blocked by prazosin (0.1 ~M).

24

M.J. Rand eta/.

Discussion The positive chronotropic responses elicited by activation of cardiac a-adrenoceptors was antagonizedby various a-adrenoceptoragonists in the pithed rat with an order of potency: prazosin > phentolamine > yohimbine, which is characteristic of that for az-adrenoceptors. Activation of the a-adrenoceptors by agonists gave an order of potency: adrenaline > noradrenaline > phenylephrine > methoxamine. The s ~ orders of potency have been reported for radioligand binding studies (30) and for studies on the positive inotropic response to activation of cardiac a-adrenoceptors (3, 49). The finding that az-adrenoceptors are involved in positive chronotropic responses in the euthyroid rat is in agreementwith that of Flavahanand McGrath (17), who showed that the positive chronotropic response to the selective az-adrenoceptor agonist amidephrinewas antagonizedby prazosin in the pithed rat.

Furthermore,the

chronotropic response to methoxamine, a relatively specific az-adrenoceptoragonist with ~-adrenoceptorblocking properties, was abolished by prazosin alone. This finding was further strengthened by the fact that the az-adrenoceptors in rat isolated atria were highly sensitive to a-adrenoceptorblockade by prazosin (1 nM), whereas yohimbine (10 nM) had no effect.

I t has also been shown in rat isolated papillary

muscle that prazosin inhibited the positive inotropic response to phenylephrinein the nanomolarrange (39). The time courses of the chronotropic responses to ax- and B-adrenoceptor activation by agonists differed in that the response following activation of azadrenoceptors developedmore slowly than did the response to B-adrenoceptoractivation. Similar findings with respect to chronotropic responses (18) in pithed rats and inotropic responses in rat l e f t atrium (48), ventricle (48) and papillary muscle (29) have also been reported. Calcium channel blocking drugs (verapamil, nifedipine, nicardipine and diltiazem) inhibited the positive chronotropic responses elicited by al-adrenoceptor activation.

Furthermore,the calcium antagonist D 600 antagonizedthe positive

inotropic effect produced by activation of a-adrenoceptorswith phenylephrinein rabbit papillary muscles more efficiently than that produced by activation of B-adrenoceptors with isoprenaline (16).

In contrast, calcium influx through calcium channels

does not appear to be involved in the response to B-adrenoceptoractivation since neither calcium antagonists nor Bay K 8644 affected the chronotropic response to

Cardiac =-Adrenoceptors

25

phenylephrine in the presence of prazosin, and the calcium antagonists had no effect on the chronotropic response to isoprenaline.

I t has been established that the

inotropic effect produced by ~z-adrenoceptoractivation is unrelated to changes in cyclic AMP levels (28, 34) which excludes the possibility that cyclic AMPmight act as the second messenger for cardiac responses produced by az-adrenoceptoractivation. I t appears that the positive chronotropic response to ~-adrenoceptoractivation results from an increased influx of calcium through channels that are sensitive to organic calcium antagonists. On the other hand, activation of cardiac B-adrenoceptors leads to stimulation of adenylate cyclase and an increase in cyclic AMP levels. The hypothesis that cardiac az-adrenoceptors in the pacemakercells are coupled to calcium channels was further reinforced by the following observations: that Bay K 8644 enhancedthe chronotropic responses to ~z-adrenoceptoractivation; that the effect of verapamil in reducing responses to phenylephrinewas reversed by raising the extracellular calcium concentration, and that the chronotropic response to ~z-adrenoceptoractivation was calcium dependent. Considerationof cardiac ~-adrenoceptors is not completewithout taking account of prejunctional as well as postjunctional receptors.

I t has been shownthat these consist of both ~z- and r

subtypes (15, 22, 41, 43). The importanceof cardiac postjunctional ~-adrenoceptors in cardiac physiology and pathophysiologyhas been pointed out by 0snes et al. (30).

I t is

likely that clinical investigators may turn their attention to blockadeof these receptors in attempts to ameliorate certain types of disorders (e.g., arrhythmias associated with ischaemia). Failure to take account of the effects of potential blocking drugs on other ~-adrenoceptorsmay lead to confusing results and may frustrate the clinical objectives.

In particular, the consequences for cardiac

noradrenergic transmission should be considered. The use of even highly selective ~-adrenoceptor antagonists to block the postjunctional receptors w i l l also result in partial blockadeof the autoinhibitory feedback control of release of transmitter noradrenaline by blocking prejunctional az-adrenoceptors. Hence,there w i l l be an increase in the amountof noradrenaline released by the samedegree of sympathetic drive.

The increased availability of noradrenalinemay to someextent competitively

overcome the postjunctional blockadeand, perhaps more seriously, could produce increased activation of cardiac B-adrenoceptors. I t follows, therefore, that any

26

M.J.I~d

et~.

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Acknowledgements Bay K 8644 was generously supplied by Bayer AG, West Germany.