Enhancement of Peripheral Alpha-Receptor Stimulation by Blockade of "Silent" Beta Receptors

Transcription

1 Enhancement of Peripheral Alpha-Receptor Stimulation by Blockade of "Silent" Beta Receptors By Thomas F. Burks, Ph.D., and Theodore Cooper, M.D., Ph.D. ABSTRACT Evidence is presented for the existence of "silent" peripheral beta adrenergic receptors with which alpha-receptor stimulants might combine without producing measurable vasodilation. Addition of propranolol (1 10~ 7 M) or MJ-1999 (5 10" G M) to Krebs solution perfusing canine isolated mesenteric arteries enhanced (1) the pressor responses to intra-arterially administered epinephrine, norepinephrine, and methoxamine and (2) the vasoconstrictor effects of perivascular sympathetic nerve stimulation. Enhancement of the pressor effects of epinephrine and norepinephrine was unaltered during cocaine or guanethidine potentiation. The doses of the beta-receptor blocking agents that enhanced pressor responses in isolated arteries were identical to those required to provide 80 to 100% blockade of the positive inotropic and chronotropic effects of epinephrine and norepinephrine in isolated rabbit hearts. Propranolol and MJ-1999 also enhanced the elevating effects of methoxamine on canine blood pressure. In higher concentrations, propranolol (1 10~* M) and MJ-1999 (1 10~!! M) produced alpha-receptor blockade in the isolated arteries, and propranolol, but not MJ-1999, produced myocardial depression in isolated rabbit hearts. These data are interpreted as indicating that alpha-receptor stimulants usually combine with both peripheral alpha and beta adrenergic receptors, and blockade of the peripheral beta receptors makes more stimulant available for alpha-receptor stimulation. ADDITIONAL KEY WORDS nerve stimulation angiotensin epinephrine norepinephrine propranolol 5-hydroxytryptamine MJ-1999 isolated rabbit hearts dogs It is generally accepted that certain stimulants of alpha adrenergic receptors, such as epinephrine, can combine with peripheral beta adrenergic receptors and initiate the events generally regarded as being due to beta-receptor stimulation. Other adrenergic stimulants, such as norepinephrine, are considered to have quite feeble peripheral betareceptor-stimulating activity (1). It would be From the Department of Pharmacology, The University of New Mexico School of Medicine, Albuquerque, New Mexico. This work was supported in part by U. S. Public Health Service Grants FR and HE from the National Heart Institute. Dr. Cooper's present address is National Heart Institute, Building 31, Bethesda, Maryland Accepted for publication September 20, isolated arteries methoxamine sympathetic nerves expected that prior administration of an agent that blocks beta receptors should enhance the pressor effects of an amine capable of stimulating both alpha and beta receptors; that this is true for epinephrine has been demonstrated by Ahlquist (2) and by Shanks (3). Levy and Ahlquist (4) have also shown that agents that block beta receptors can convert the depressor effects of ethylnorepinephrine to pressor effects. On the other hand, the pressor effects of agents possessing little or no ability to stimulate peripheral beta receptors, for example norepinephrine and methoxamine, would not be expected to be altered by the administration of chemicals that block beta receptors. There have been suggestions in the literature, however, that different types of 703

2 704 BURKS, COOPER effects produced by different agents result from the nature of actions or interactions that occur after drug-receptor combination rather than being only a consequence of an agent's combining with a single receptor type and thus initiating a response (5). If this were indeed the case, then it might be possible for certain agents to combine with both alpha and beta receptors but initiate actions measurably expressed as effects only on the alpha receptors. The beta receptors with which such agents combine but do not initiate a measurable pharmacological response could be termed "silent" beta receptors. This report presents the results of studies conducted primarily on isolated mesenteric arteries to test the above hypothesis by use of two agents that block beta receptors, propranolol and 4- (2-isopropylamino-l-hydroxyethyl) methanesulfonanilide hydrochloride (MJ-1999). These are believed to provide specific blockade of beta adrenergic receptors (6). Methods Isolated Mesenteric Arteries. Mongrel dogs of either sex, weighing 8 to 14 kg, were anesthetized with pentobarbital sodium (30 mg/kg iv), and isolated arteries were prepared in the manner described by Rogers et al. (7). After exposure of the small intestine through a midline abdominal incision, mesenteric arteries with branching arcades of smaller resistance arteries and with or without the periarterial sympathetic nerves were removed from the animals and mounted in an organ bath where they were perfused with Krebs bicarbonate solution (composition in grams per liter: NaCl, 6.93; KC1, 0.354; CaCL-2H.,O, 0.373; KHJPO.,, 0.163; MgSO 4, 0.143; NaHCO 3) 2.09; glucose, 1.80). The Krebs solution was aerated with 95% O,- 5% CO 2 and maintained at 37 C. The bath fluid [400 ml) was allowed to recirculate except in studies involving angiotensin or 5-hydroxytryptamine. The arteries were perfused by a Harvard peristaltic infusion pump (model ) or with a Holter microinfusion pump (model RD045), at constant flow rates of 20 to 80 ml/ min. Since flow was held constant throughout the experiment, changes in perfusion pressure were directly proportional to changes in arterial resistance. Perfusion pressure at the beginning of the experiments was 80 to 100 mm Hg; it was measured from a T tube between the pump and the artery by a Statham pressure transducer (P23 Db) and was recorded on an Offner Dynograph. In 10 preparations we stimulated the perivascular sympathetic nerves with a Grass model S4 stimulator and a frequency of 20 to 30 cps, a duration of 5 to 20 msec and a current of 6 to 15 v for 10 to 30 sec. In this paper, we use the term "agonist" to mean adrenergic drugs that can activate receptors in vascular smooth muscle to produce contraction or relaxation. The agonists were dissolved in purified water (which had no effect alone) and were injected into the arterial cannula in volumes of 0.01 to 0.1 ml. Unless otherwise noted, the other agents were added directly to the perfusion medium. Several test doses of the agonists were given in random order and several nerve stimulations performed until repeated stimulation caused reproducible responses; after establishment of control responses to agonists and to nerve stimulation, the other agents were added to the bath and allowed to recirculate, and the agonists were reapplied. The dose ranges employed were: epinetihrine 0.05 to 0.5 jxg; norepinephrine, 0.1 to 0.8 jxg; and methoxamine, 6 to 30 fig. To avoid artifacts produced by interactions among the agonists, the order of their administration was randomly determined before each experiment and then maintained throughout that experiment. Each agonist was also studied alone in at least one preparation to ascertain lack of interaction effects. Canine Blood Pressure. Mongrel dogs of either sex, weighing from 8 to 15 kg, were anesthetized with either alpha-chloralose (100 mg/ kg) or pentobarbital sodium (30 mg/kg iv). In each, the trachea was cannulated, but the dog breathed spontaneously. Both cervical vagi were sectioned, and blood pressure was measured from a femoral artery by a Statham pressure transducer (P23 Db) and recorded on an Offner Dynograph. Drugs were administered through a catheter in a femoral vein and were washed in with 2 to 5 volumes of saline. Heart rate was determined from the blood pressure recording. Each dog was given SU (1 mg/kg), a specific alpha-receptor-blocking agent (8). Pressor substances were given in random order in volumes of 1.0 to 2.0 ml before and after the animals were given either propranolol (0.5 mg/ kg) or MJ-1999 (2 mg/kg). The responses to occlusion of the common carotid arteries (30 or 60 sec) by an artery clamp were similarly tested. Isolated Rabbit Hearts. Dutch rabbits of either sex, weighing from 1.5 to 3 kg, were killed by cervical fracture, and the beating hearts were removed and flushed through the aorta with a saline solution. An aortic cannula was tied into place, and the hearts were perfused at a constant

3 BLOCKADE OF "SILENT" BETA RECEPTORS 705 flow established at 25 to 35 mm Hg with Locke- Ringer solution for isolated hearts (composition in grams per liter: NaCl, 9.00; KC1, 0.84; CaCL, 2H,,O, 0.48; NaHCO 3> 0.60; glucose, 4.00) warmed to 36 to 37 C and aerated by bubbling 95% CL-5% CO 2. Force of contraction was measured from a pin in the apex of the heart with a Grass force-displacement transducer (FTO3C) and recorded on an Offner Dynograph. Heart rates were determined from the mechanical contraction record. Bipolar surface ventricular electrograms were obtained from surface needle electrodes placed about 1 cm apart on the ventricle and were recorded on an Offner Dynograph. Stimulant drugs (epinephrine, norepinephrine, and calcium chloride) were injected in volumes of 1.0 ml into the aortic cannula; propranolol or MJ-1999 was dissolved in appropriate concentrations in the perfusion reservoir. The drugs and chemicals used were epinephrine (Suprarenin, Winthrop), norepinephrine (Levophed, Winthrop), methoxamine (Vasoxyl, Burroughs Wellcome), angiotensin (Hypertensin, Ciba), 5-hydroxytryptamine creatinine sulfate (Calbiochem), SU (Ciba), propranolol (Inderal, Ayerst), MJ-1999 (Sotalol, Mead Johnson), guanethidine (Ismelin, Ciba), cocaine hydrochloride, and phentolamine (Regitine, Ciba). Epinephrine and norepinephrine were calculated as the free bases; doses of all other agents were calculated as their salts or complexes. Statistical analyses were performed by use of Student's f-test, paired comparisons (9). Values of P equal to or less than 0.05 were considered significant. In each case, values of IV represent preparations from different animals. Results Isolated Mesenteric Arteries. In this preparation, vasoconstriction manifests itself as an increase in the perfusion pressure required to maintain constant flow; we refer to this as a "pressor response." Pressor responses to epinephrine, norepinephrine, methoxamine, and stimulation of perivascular sympathetic nerve were enhanced over their corresponding control values in the presence of propranolol (lxlo- 7 M) (Table 1, Fig. 1) or MJ-1999 (5 10-«M) (Table 1, Fig. 2). Dose levels of each beta-receptor-blocking agent from 10" 8 to 10~ 3 M were investigated, and the concentrations employed in the experiments presented in Table 1 produced optimal enhancement of the pressor responses to the alpha-receptor agonists. The beta-receptor- Circulalwn Research, Vol. I, November 1967 A x 10 C5 S I ec s~ "5 S i 'S 8* IS 25 I CO CO I s ; ^O5 00 Ol t '< CM d 0000 CD CM Tin t t cot- co CM ' ' in ' in in q q q q q q q q d d do do do VV V V VV VV IIII CD * II II CD 00 * CM II II II II 1O CO CM CO O ^ CO CQ CM CM <M CM CO i f CO ol l> 00 CO CO in in O CM CM IS I O hi, O ^ O O5 C O5 2 O5 ft "? o >, CM CO in co co co O5co O5co in co in in O c = c 'a'a II S J: J: u o 5 ^ J J a c o -7 "5 * a to z z Z Z 17 I "o 5 c o a.s OJ -U cj o c O QJ 3 CD ft o a 1 G5 I>, *T3 * i (

4 706 BURKS, COOPER (A) I min mmhg E NE FIGURE 1 E NE E NE A Pressor responses to intra-arterially administered angiotensin (A) (0,08 /ig), epinephrine (E) (0.1 ng), and norepinephrine (NE) (0.2 ng) in an isolated mesenteric artery. A = perfusion with control Krebs solution; B = perfusion with solution containing propranolol (1 10~ 7 M); and C = perfusion with propranolol (1 10-"i M). mmhg 1 min " JVJVJV. E NE M E NE M FIGURE Z E NE M Pressor responses to intra-arterially administered epinephrine (E) (0.2 ng), norepinephrine (NE) (0.4 fj.g), and meihoxamine (M) (18 ng) in an isolated mesenteric artery. A = control period; B = perfusion with MJ-1999 (5 10-e M); and C = perfusion with MJ-1999 (1 10~ s M). mmhg J 1 mi n E NE NE E FIGURE 3 E NE A Pressor responses to intra-arterially administered epinephrine (E) (0.2 ng), norepinephrine (NE) (0.5 ng), and angiotensin (A) (0.01 fig) in an isolated mesenteric artery. A = perfusion with control Krebs solution; B = perfusion with cocaine (1 x 10~ c ' M); C = perfusion with cocaine (1 10~ 6 M) plus propranolol (1 x 10~ 7 M); and D = perfusion with cocaine (1 10~ 6 M) plus propranolol (1 io-4 M).

5 nu aoiiicin. BLOCKADE OF "SILENT" BETA RECEPTORS 707 p q p p o o VV VV I2O EPINEPHRINE NOREPINEPHRINE 100 c,, s CM CO CM + CM CO mm Hg S s 40 2? "5 O5 20 I I i O5 O5 C5 1 i if 3 a.s *& 25 p p p p = O O O O o, VV VV i Ifi Ol IO CD i ( i I. f i co in n tf in * 00 CM 00 CM O! i CO CO CD t- (D 1- a nephrii 'a a nepliri: ihri S* 5" Noi a ihri: Noi S,S * o (M ol CM_ CO ' CD.5 a o c a DOSE FIGURE 4 Pressor effects of two dose levels each of epinephrine and norepinephrine on isolated mesenteric arteries during perfusion with control Krebs solution (A), solution containing cocaine (1 lo' 6 M) (B), and solution containing cocaine (1 10~ G M) plus propranolol (1 x 10~ 7 M) (C). Note that propranolol enhanced the pressor effects of epinephrine and norepinephrine in the presence of cocaine. Each point represents the mean data obtained from preparations from 4 dogs. blocking agents alone did not alter resistance. The order of effective enhancement by propranolol was epinephrine > norepinephrine > methoxamine > nerve stimulation for both absolute differences in pressor responses and percent increases in response. MJ-1999 enhanced methoxamine > epinephrine > norepinephrine > sympathetic nerve stimulation. In three preparations tested, guanethidine in a bath concentration of 3 xlo~ 8 M added to the enhancement of epinephrine pressor responses produced by MJ-1999 (5x10" M) (control mean response to epinephrine, 42 mm Hg; after MJ-1999, 67 mm Hg; after guanethidine, 153 mm Hg). In a single preparation tested, guanethidine (3 10~ 8 M) added to the propranolol (1 10~" M ) enhancement of the epinephrine response (control, 35 mm Hg; jiq

6 708 BURKS, COOPER after propranolol, 40 mm Hg; after guanethidine, 50 mm Hg). Guanethidine has been shown to enhance the pressor actions of catecholamines in this preparation by inhibiting catecholamine uptake mechanisms (7). The additive effect of guanethidine on the enhancement by propranolol and MJ-1999 of the pressor responses to epinephrine suggested that the beta-receptor-blocking agents were acting by some means other than diminution of adrenergic uptake mechanisms for their actions on the pressor responses studied (10). To provide more definitive evidence that propranolol and MJ-1999 were not inhibiting uptake, studies with cocaine were done. Control responses to epinephrine and norepinephrine were established, and cocaine in a concentration of 1 10" M (found in preliminary experiments to be the maximally effective concentration of cocaine) was added to the bath, and the doses of the agonists were again given until there was no further enhancement of their pressor actions. Then propranolol (1 10" 7 M ) or MJ-1999 (5 x 1() - M) was added to the bath and the agonists were readministered. The results are presented in Table 2 and Figure 3. In every case the beta-receptor-blocking agents enhanced the responses to epinephrine and norepinephrine in the presence of cocaine, and the absolute amounts of this enhancement agree closely to those produced in the absence of cocaine; the relevant data from the preceding tables have been compiled in Table 3. In one preparation each, propranolol and MJ-1999 were added to the bath before cocaine; the cocaine added to the enhancement of the responses produced by the beta-receptor-blocking agents. In four preparations, two dose levels each of epinephrine and norepinephrine were administered, after which propranolol was added to the bath and the agonists again given, then cocaine was added to the bath and the agonists readministered. The results of these experiments are illustrated in Figure 4. During the preliminary studies with various concentrations of the beta-receptorblocking agents, we noted that high concentrations of these substances in the bath fluid TABLE 3 Comparative Ability of Propianolol (1 10~ 7 MJ and MJ-1999 (5 10~ G >0 to Enhance Pressor Responses Alone or in the Presence of Cocaine (1 10~ r ' M) (from Tables 1 and 2) Pressor stimulus Epinephrine Norepinephrine Increase, propranolol alone (mm Hg) (% ) 25 ± ± Increase, propr: anolol in presence of cocaine (mm Hg) (%) 19 ± ± Increase, MJ-1999 alone (mm Hg1 (%) 28 ± ± Increase, MJ-1999 in presence of cocaine (%) (mm Hg) 27 ±5 32 ±8 TABLE 4 Ability of Propranolol (1-70-! M) and MJ-1999 (1 Z0-" M) to Antagonize Pressor Responses in Isolated Mesenteric Arteries Pressor stimulus Epinephrine Epinephrine Norepinephrine Norepinephrine Methoxamine Methoxamine No. of animals Mean control response (mm Hg) ^-receptor blocking agent propranolol MJ-1999 propranolol MJ-1999 propranolol MJ-1999 Mean response in presence of ^-receptor blocking agent (mm Hg) Difference ± SE 55 ± ± 4 55 ± ± 3 45 ± ± 4 p <0.01 < <0.01 < <0.05 < Mean percent decreases in responses* Percent decrease calculated as [(mean control response mean response after (3-receptor blocking agent)/ mean control response] 100.

7 BLOCKADE OF "SILENT" BETA RECEPTORS 709 O S 2 2 M CO oi" so 3 S 1 5? 111- Z 2 V =? en o Z si I + C-l O3 in oi I Ol Ol m en in V +1, ( CO CO ol V V V co in * t- in CO <M O Ol CO O) * CO a - a i v M s s 5 g 8 3 3* 3 I c c, CT is 1l 15 " -g t o o u. "o c a a c = I a + appeared to reduce the pressor responses of the alpha-receptor stimulants. This phenomenon was investigated in greater detail, and the results are presented in Table 4. Propranolol (1 10" 4 M) (Figs. 1, 3 and 5) and MJ-1999 (11(H M) (Fig. 2) significantly decreased the responses to epinephrine, norepinephrine, and methoxamine. In a single experiment, MJ-1999 (lxlo- 1 M) decreased the pressor response to nerve stimulation from a control value of 55 to 25 mm Hg, and in two preparations, propranolol (1 1CH M ) similarly decreased the responses to sympathetic nerve stimulation (means: before, 55 mm Hg; after, 10 mm Hg) (Fig. 5). As shown in Figure 5, guanethidine antagonized the effects of sympathetic nerve stimulation (7) but enhanced the effects of catecholamine administration. Phentolamine was given to offer a comparison of its alpha-receptor-blocking actions to those of high concentrations of propranolol and MJ Propranolol (1 1CH M) also decreased the pressor response to 5 fig 5-hydroxytryptamine (from 30 to 5 mm Hg). To ascertain the specificity of the decrease of the pressor responses to the above agents by propranolol and MJ-1999, angiotensin (0.01 to 0.05 ^ug) was tested before and after the addition of the beta-receptor-blocking agent to the bath. In four preparations, the pressor response to angiotensin was not decreased by MJ-1999 (lxl0~ a M) (mean of control responses to angiotensin, 58 mm Hg; after MJ-1999, 96 mm Hg). In two preparations the angiotensin pressor effects were not diminished by propranolol (1 10~ 4 M) (mean of control responses to angiotensin, 57 mm Hg; after propranolol, 112 mm Hg) (Fig. 1). The depressant effect of propranolol and MJ-1999 in high concentrations on responses to alpha-receptor stimulants was easily reversible. After 15 to 30 min of perfusion of the arteries with control Krebs solution, the responses to the alpha-receptor agonists were restored to their control values. Addition of guanethidine (3 10~ s M ) to the bath partly restored the responses to epinephrine (mean control, 57 mm Hg; after 1 10" 4 M propranolol, 22 mm Hg; after guanethidine, 55 mm Circulation Research. Vol. I. November 1967

8 710 BURKS, COOPER TABLE 6 Effects of an a-receptor-blocking Agent (SU-14542) and Two (3-Receptor-Blocking Agents (Propranolol and MJ-1999) on Canine Blood Pressure Responses to Epinephrine 3 Dogs in which ^-receptor-blocking agents enhanced responses to 1 fig/kg epinephrine Mean Mean response Mean response control after 1 mg/kg after ^-receptorblocking agent* response SU (mm Hg) (mm Hg) (mm Hg) Dogs in which ^-receptor-blocking agents antagonized responses to 1 /zg/kg epinepnrine Mean Mean response Mean response control after 1 mg/kg after )3-receptorblocking agentf response SU (mm Hg) (mm Hg) (mm Hg) *One dog received propranolol (0.5 mg/kg), 2 dogs received MJ-1999 (2 mg/kg). fone dog received propranolol (0.5 mg/kg), 2 dogs received MJ-1999 (2 mg/kg). mmhg 200-, 50-1 NE E S S NE E E S NE FIGURE 5 trf NE S E Pressor responses to intra-arterially administered norepinephrine (NE) (0.3 ng), epinephrine (E) (0.1 fig), and sympathetic nerve stimulation (S) (25 cps, 15 msec, 10 v 20 sec) in an isolated mesenteric artery. A = control period; B =perfusion with propranolol (1 10~ 7 M); C = perfusion with propranolol (1 10 'i M); D = perfusion with propranolol (1 10~'' M) plus guanethidine (3 10~ 8 M); and E = perfusion with propranolol (1 10-'< M), and guanethidine (3 x 10~ s M) plus phentolamine (5 x 10~ 8 M). Note the complete absence of a response to nerve stimulation in the presence of guanethidine (D). EteCTNOCRAH».ciMMVMMMM -, r l\jmvi\jvwvjvwvmm\,mm\n\mw E NE FIGURE 6 Effects on electrical and mechanical activity of perfusing isolated rabbit hearts with betareceptor-blocking agents. A = isolated rabbit heart perfused with control solution; B = same heart during perfusion with MJ-1999 (5 10~ 6 M); and C = same heart perfused with MJ-1999 (1 10- M). The heart shown in the lower panels received propranolol. D = perfusion with control heart solution; E = perfusion with propranolol (1 10~ 7 M); and F = perfusion with propranolol (1 10-i M).

9 BLOCKADE OF "SILENT" BETA RECEPTORS 711 Hg), but not the responses to sympathetic nerve stimulation (Fig. 5). Canine Blood Pressure. Since we believed that the effects of the beta-receptor-blocking agents on the actions of the pressor stimuli could more easily be assessed in the presence of attenuated responses to the stimuli, each dog was administered 1 mg/kg SU after the initial responses to the stimuli were recorded. Then the actions of epinephrine, norepinephrine, methoxamine, and bilateral carotid artery occlusion were again measured, the beta-receptor antagonists were given, and the pressor stimuli were reapplied. The results are presented in Table 5. Control experiments were performed to determine whether the effects of the alpha-receptorblocking agent SU diminished during the time required for completion of the above procedures. The effects of SU persisted undiminished for at least an hour longer than required for this experiment. Note that the pressor response to methoxamine was significantly increased by the beta-receptor-blocking agents. The qualitative effects of the betareceptor-blocking agents on responses to epinephrine deserve further attention and are detailed in Table 6. Of the six dogs tested, three showed an increased rise in blood pressure after the beta-receptor-blocking agents. The qualitative effects of propranolol and MJ-1999 were quite similar. The anesthetic agent employed (chloralose or pentobarbital) did not appear to alter the responses observed. The pressor responses to methoxamine were tested in two dogs that were not given SU After the control responses to methoxamine were established (mean, 102 mm Hg), one dog was given propranolol (0.5 mg/kg) and the other MJ-1999 (2 mg/ kg). Methoxamine was tested after administration of the beta-receptor-blocking agents, and the pressor responses were enhanced (mean, 125 mm Hg). Isolated Rabbit Hearts. These studies were included to compare the effects of propranolol and MJ-1999 on isolated hearts with their effects on isolated mesenteric arteries. The concentration of propranolol that blocked 80 to 100 of the positive inotropic and chronotropic responses to epinephrine and norepinephrine was 1 10" 7 M; that of MJ-1999 was 5 x 10" 6 M. These concentrations of the betareceptor antagonists did not alter the inotropic responses to calcium chloride. When a solution containing propranolol in a concentration of 1 lo^1 M was perfused through the isolated hearts, gross electrical disturbances were noted and contractile force was markedly decreased (from a mean contractile force of 11 g to a mean of 2 g). MJ-1999, tested in concentrations up to 1 10" 3 M showed no comparable effects. These phenomena are illustrated in Figure 6. There was no additive effect of MJ-1999 with propranolol in depressing the myocardium: MJ-1999 (lxlo~ 3 M ) plus propranolol (1 10~ 5 M ) did not produce any more myocardial depression than the slight effect (at 1 10~ 5 M ) of propranolol alone. The mean contractile force after 1 10" 3 M propranolol was 10.7 g (control, 11.0 g). Discussion These studies have demonstrated that two beta-adrenergic-receptor-blocking agents, propranolol and MJ-1999, can enhance the in vitro pressor responses to stimulants of alpha adrenergic receptors and indicate that the consequence of administering beta-receptorblocking drugs may be more complex than has previously been suspected. It appears likely that doses of the beta-receptor-blocking agents that block cardiac beta receptors also block peripheral beta receptors, including peripheral "silent" beta receptors, and thus enhance the vasoconstrictor effects of exogenously administered alpha-receptor stimulants or the effectiveness of neurogenic vasoconstrictor impulses. It has become well established from studies conducted in intact dogs and vascular beds of skeletal muscle that the pressor responses to epinephrine and ethylnorepinephrine, which are known to stimulate both alpha and beta receptors, are enhanced by the prior administration of beta-receptor-blocking agents (3, 4). Potentiation of epinephrine

10 712 BURKS, COOPER pressor responses by beta-receptor-blocking agents has been attributed to blockade of its peripheral vasodilator actions in skeletal muscle vascular beds (3). Blockade of pharmacologically silent beta receptors has not previously been considered as a factor contributing to enhancement of epinephrine pressor responses by beta-receptor-blocking agents. As with other vascular beta receptors, the silent receptors would presumably be any of three entities: (1) Those which would be activated by chemicals that relax vascular smooth muscle and would be susceptible to blockade by a specific group of substances known to block the effects of isoproterenol. (The influence of the activated beta receptors might be masked or diluted in our experimental systems, since in the absence of beta-receptor-blocking agents, the recorded event overwhelmingly reflects a constriction of vascular smooth muscle.) (2) These non-neuronal sites with which the alpha or beta receptor agonist combines, but at which the combination does not initiate any discernible response of vascular smooth muscle. (3) Those in which an agonist combines with a receptor, but the combination does not result in a sequence of events leading to smooth muscle relaxation, but perhaps to some other sequence of events. Combination of other agonists with this receptor could result in vascular muscle relaxation. Whichever of these three entities might pertain, generally or specifically, competitive blockade of these so-called silent sites would result in an increase in the quantity of agonist available to alpha-receptor sites, resulting in "enhancement" of pressor or constrictor responses. The latter would be true at doses of agonists below those that saturate alpha-receptor sites. It is imperative to keep in mind that the vascular response is not a necessary consequence of combination of the substance with the so-called beta-receptor site, since a number of chemical steps probably intervene between the initial drug-receptor combination and the muscular response. If this were not the case, the response to a non-beta-receptor agonist, such as a "pure" alpha-receptor agonist, would not be expected to show alteration in the presence of a pure beta-receptor blockade. Since methoxamine exhibits no "reversal" in the presence of alpha-receptor-blocking agents and produces neither positive inotropic nor chronotropic responses in mammalian hearts in situ or in vitro, it is generally considered to be a pure alpha-receptor stimulant. However, it was clearly more potent as a pressor substance in the presence of beta-receptorblocking agents. This suggests that methoxamine may interact with beta receptors in the canine peripheral vascular system and fulfills our criteria for interacting with silent beta receptors. Further, an N-tertiary butyl congener of methoxamine, butoxamine, has a known affinity for beta receptors, especially peripheral beta receptors (11). Hence, we have an explanation for the apparent incongruity in our data, namely that the methoxamine response is enhanced by betareceptor-blocking agents. It seems likely that methoxamine has an affinity for the betareceptor site, but the interaction does not result in a sequence of events leading to vascular smooth muscle relaxation (entities 2 or 3, above), and because of this failure to cause relaxation has been considered not to combine with beta-receptor sites. The above consideration also applies to norepinephrine, which in some test systems may possess a feeble ability to produce vasodilation after combination with peripheral beta receptors (3). Norepinephrine may have an affinity for beta receptors equal to that of epinephrine, but does not usually produce marked vasodilation, either because its powerful stimulant action on alpha receptors masks its slight vasodilatory properties (entity ], above) or because of its methoxamine-like ineffective combination with peripheral beta receptors (2 or 3, above). The action of norepinephrine is further complicated by the avidity with which this amine is taken up by the adrenergic nerve terminals. This could remove the norepinephrine from proximity to beta receptors before full development of its vasodilatory action. The experiments with Circulation Research, Vol. I, November 7967

11 BLOCKADE OF "SILENT" BETA RECEPTORS 713 cocaine demonstrated that the pressor activity of norepinephrine is still enhanced by the beta-receptor-blocking agents even when nerve terminal uptake is prevented. Epinephrine has long been known to produce vasodilation, especially in the presence of an alpha-receptor-blocking drug. Since it is known that epinephrine can stimulate beta receptors, the affinity of this substance for beta receptors (active or silent receptors) is not surprising. Epinephrine is also taken up by adrenergic nerve terminals but when such uptake is prevented by the use of cocaine, its pressor effects were still enhanced by betareceptor-blocking agents. Proper interpretation of the data obtained by use of the isolated artery preparation and canine blood pressure depends on an understanding of the pharmacological actions of the beta-receptor-blocking agents employed. Nakanao and Kusakari (12) found that in the perfused limb propranolol acts only as a beta-receptor-blocking agent. Stanton et al. (13) found in their test systems that MJ-1999 exhibits only specific beta-adrenergic-receptor blockade. Lish et al. (14) concluded that MJ is a potent agent that selectively blocks beta-adrenergic receptors and has no other significant peripheral action. The actions of MJ-1999 on catecholamine metabolic effects are consistent with those expected of a betareceptor-blocking agent (15). Aramendia and Kaumann (16), however, on the basis of studies with isolated atria of guinea pigs and with perfused hind limbs of dogs suggested that MJ-1999 blocks the release of norepinephrine from the sympathetic nerve endings. Since the effects of sympathetic nerve stimulation were enhanced by propranolol (1 lo" 7 M) and by MJ-1999 (5xl()- 8 M) and since the enhancement of the pressor responses to epinephrine and norepinephrine by the beta-receptor-blocking agents were unaltered in the presence of cocaine and guanethidine, we infer that at these concentrations of propranolol and MJ-1999, these agents do not effectively antagonize either release of norepinephrine from adrenergic nerve terminals or uptake of exogenous epi- Circulaiion Research, Vol. I. November 1967 nephrine or norepinephrine by the nerve terminals in isolated mesenteric arteries. We have been unable to find evidence that propranolol or MJ-1999 at doses which provide 80 to 100% blockade of the cardiac effects of catecholamines are anything other than specific beta-receptor-blocking agents in isolated mesenteric arteries. At these same dose ranges, propranolol provided greatest enhancement of the responses to epinephrine, which is known to have an affinity for beta receptors, and one might therefore infer that epinephrine exhibits greater affinity for silent beta receptors than does norepinephrine or methoxamine. But MJ-1999 enhanced the pressor responses to methoxamine more than those to epinephrine; thus it cannot be said on the basis of these studies that methoxamine is less able to combine with beta receptors than is epinephrine. These data suggest that epinephrine, norepinephrine, methoxamine and, by inference, isoproterenol may all possess similar affinities for peripheral beta receptors, but differ vastly in ability to initiate the biochemical events required to produce vascular muscle relaxation (isoproterenol > epinephrine > > norepinephrine > > methoxamine). These considerations add support to the hypothesis that the actions or interactions after drug-receptor combination can determine the ultimate nature of the drug effect. Methoxamine may combine with peripheral beta receptors with an affinity approaching that of epinephrine, but this combination is for some reason not noticeably expressed as the vasodilatory response as is that with epinephrine. These phenomena, as suggested by Long et al. (5), may apply to all autonomic drug receptors and should be taken into account in any consideration of autonomic drug effects. The combination of adrenergic stimulants with silent beta receptors does contribute to their total cardiovascular effects, and alterations of the combination alters their cardiovascular effects. The less pronounced enhancement of the effects of sympathetic nerve stimulation by the beta-receptor-blocking agents may reflect the well-known difficulty of altering such

12 714 BURKS, COOPER endogenous responses (with either alpha- or beta-receptor-blocking agents) or may indicate that the periarterial sympathetic nerve terminals are more intimately associated with alpha-adrenergic receptors than with beta receptors; there may be relatively fewer beta receptors (silent or otherwise) in the region of the nerve terminal than elsewhere in the vascular smooth muscle. The studies in isolated rabbit hearts were performed to help correlate the cardiac effects of propranolol and MJ-1999 with their peripheral actions, as demonstrated in this series of investigations. The doses of the beta-receptor-blocking agents required to provide 80 to 100% blockade of the positive inotropic and chronotropic effects of epinephrine and norepinephrine were exactly the same dose levels that maximally enhanced the peripheral pressor responses to the catecholamines (propranolol, 1 10~ 7 M; MJ-1999, M). The concentrations of propranolol and MJ-1999 required in the isolated rabbit hearts for beta-receptor-blockade closely agree with those found effective by other workers (6, 14, 17, 18). No evident myocardial depression was produced by the betareceptor-blocking agents with increasing concentrations until propranolol was perfused through the hearts at a concentration of 1 10" 4 M, the same concentration found to block peripheral alpha receptors. This effect on the heart may have resulted from the well-known local anesthetic or "quinidine-like" effect of propranolol (6, 17, 18), which was not exhibited by MJ-1999 (up to 1 lo" 3 M) in this study or in the studies of others (6, 17, 18). The fact that MJ-1999 did not produce myocardial depression at a concentration that antagonized the responses to catecholamines in isolated arteries adds support to the contention that the beta-receptor-blocking agents propranolol and MJ-1999 produce their inhibition of catecholamines in isolated arteries by some means other than a generalized smooth muscle depression, probably by blockade of alpha-adrenergic receptors. The ability of propranolol and MJ-1999 in high concentrations (1 10" 4 M and 1 1(H M, respectively) to antagonize the pressor effects of epinephrine, norepinephrine, methoxamine, 5-hydroxytryptamine, and sympathetic nerve stimulation probably reflects the feeble alpha-receptor-blocking ability of these compounds, requiring concentrations some 1,000 times higher than are required for beta-receptor blockade to also block alpha receptors. That the antagonism to the alphareceptor stimulants was the result of alphareceptor blockade and not the consequence of a general depression of the vascular smooth muscle was demonstrated by lack of reduction of the response to angiotensin. In the isolated mesenteric artery preparation, 5-hydroxytryptamine may act, at least in part, by directly or indirectly stimulating alpha-adrenergic receptors, and its pressor actions would be expected to be diminished by blockade of the alpha receptors. In various types of preparations the pressor actions of 5-hydroxytryptamine are antagonized by such alphareceptor-blocking agents as phentolamine, dibenamine, dihydroergotamine, and phenoxybenzamine (19). Shanks (20) found that propranolol in doses up to 1.0 mg/kg did not antagonize the pressor responses of phenylephrine and concluded that propranolol does not block alpha receptors; the dose of propranolol (lx 10~ 4 M) found to produce alpha-receptor blockade in isolated mesenteric arteries was many times that used by Shanks (20). Acknowledgments The authors are indebted to Dr. A. J. Plummer, Ciba Pharmaceutical Company, for generous supplies of phentolamine, guanethidine, and SU-14542; to Dr. Paul M. Lish, Mead Johnson Research Center, for supplies of MJ-1999; and to Dr. A. Sahagian-Edwards, Ayerst Laboratories, for a supply of propranolol. References 1. INES, I. R., AND NICKERSON', M.: Drugs acting on postganglionic adrenergic nerve endings and structures innervated by them. In The Pharmacological Basis of Therapeutics, edited by L. S. Goodman and A. Gilman. New York, Macmillan Co., 1965, pp AHLQUIST, R. P.: Beta adrenergic receptor blockade by naphthyl-isoproterenol (abstr.). Federation Proc. 22: 449, Circulation Research, Vol. I. Norernber 1967

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