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The plasma cyclic AMP response to catecholamines as potentiated by phentolamine in rats
European Journal of Pharmacology, 49 (1978) 169--176
169
© Elsevier/North-Holland Biomedical Press
THE PLASMA CYCLIC AMP RESPONSE TO CATECHOLAMINES AS POTENTIATED BY PHENTOLAMINE IN RATS SATOSHI KUNITADA and MICHIO UI *
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Received 26 August 1977, revised MS received 18 November 1977, accepted 1 February 1978
S. KUNITADA and M. UI, The plasma cyclic AMP response to catecholamines as potentiated by phentolamine in rats, European J. Pharmacol. 49 (1978) 169--176. Norepinephrine failed to increase plasma cyclic AMP when injected alone into fasted rats, in contrast with sharp increases elicited by isoproterenol, epinephrine or tyramine. In rats pretreated with 6-hydroxydopamine or cocaine, however, there was a significant increase in plasma cyclic AMP after norepinephrine injection, suggesting that the rapid neuronal catecholamine uptake was at least partly responsible for the lack of norepinephrine action. Phentolamine was very effective in enhancing the epinephrine-, norepinephrine- or tyramine-induced increase in plasma cyclic AMP but without effect on the isoproterenol-induced increase. Blockade of postsynaptic ~adrenoceptors, rather than of presynaptic receptors, is likely to be involved in the phentolamine potentiation, since it was even observed in rats treated with 6-hydroxydopamine or cocaine. A discussion is presented regarding the mechanism by which cyclic AMPfleneration is influenced by the ~- and f~-adrenoceptor interaction on effector cell membranes. Postsynaptic ~-adrenoceptor and ~-adrenoceptor interactions ~-Adrenergic stimulants
1. Introduction The s.c. injection of tyramine has been shown to lead to a rapid increase in plasma cyclic AMP due to the release of catecholamines from sympathetic nerve endings (Kunitada et al., 1978). This action of tyramine was markedly enhanced when ~-adrenergic receptors were blocked b y phentolamine. The purpose of the present paper is to show how phentolamine potentiates the plas-
* Correspondence to M.U., Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan.
Plasma cyclic AMP 6-Hydroxydopamine
ma cyclic AMP response stimulation.
Phentolamine Cocaine
to
/~-adrenergic
2. Materials and methods Male rats of a Wistar-derived strain were used after a 20-h fast. Blood specimens were withdrawn at intervals from the tail vein and analyzed for cyclic AMP by a sensitive radioimmunoassay (Honma et al., 1977) as described in the preceding paper. Theophylline, 10 mg/kg b o d y wt, was injected s.c. at time 0 into all of the rats for the purpose o f suppressing cyclic AMP phosphodiesterase. Sources of reagents were the same as those described in the preceding paper (Kunitada et al., 1978).
170
S. KUNITADA, M. UI
3. Results
3.1. Differential effects of phentolamine on increases in plasma cyclic AMP elicited by injection of catecholamines As in the previous study (Kunitada et al., 1978), the s.c. injection of phentolamine in a dose of 5 mg/kg b o d y weight caused a rapid increase in plasma cyclic AMP reaching a peak at 40 min and declining within 100 min after injection {fig. 1A and B). Fig. 1A further shows that the injection of epinephrine 100 min after the injection of phentolamine
800
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resulted in a tremendous increase in plasma cyclic AMP, whereas epinephrine injected into saline-treated rats elicited a much smaller increase. In contrast, the injection of isoproterenol in doses from 1 to 25 pg/kg gave rise to increases in plasma cyclic AMP which were similar whether the rat had been treated with phentolamine or not (fig. 1B). Peak levels of plasma cyclic AMP after the injection of these catecholamines and of norepinephrine and tyramine were plotted against the doses (fig. 2). It is seen that phentolamine was effective in potentiating epinephrine-induced and tyramine-induced increases in plasma cyclic AMP, but without effect on the isoproterenol-induced increase. In the case of norepinephrine, the increase in
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Fig. 1. Increases in plasma cyclic AMP by epinephrine and by isoproterenol in rats pretreated with phentolamine. In this and all o f the following figures, theophylline (10 mg]kg body weight) was injected s.c. into all rats at time O, and the results are expressed as mean +_ S.E.M. (symbols attached with vertical lines) with the number of observations following n =, or in parentheses. Saline (open symbols) or phentolamine (5 mg/kg, solid symbols) was injected s.c. at 20 rain. Epinephrine (panel A) or isoproterenol (panel B) was further injected s.c. at 120 rain in doses shown by smaller symbols in panels. Ordinate: plasma level of cyclic AMP (pmol/ml); abscissa: time (min).
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Fig. 2. Dose--response relationship for catecholamine-induced increases in plasma cyclic AMP. Epinephrine (panel A), isoproterenol (panel B), norepinephrine (panel C) or tyramine (panel D) was injected into rats treated with phentolamine (solid circles) or not treated (open circles). The time of injections and dose of phentolamine are the same as in fig. 1. Peak levels of plasma cyclic AMP after injection of catecholamines are plotted as a function of the doses used. Ordinate: plasma level of cyclic AMP (pmol/ml); abscissa: dose of catecholamines (pg/kg) or tyramine (mg/kg) (log scale).
P L A S M A CYCLIC A M P A F T E R P H E N T O L A M I N E
plasma cyclic AMP was only slight even when the rats had been treated with phentolamine. 3.2. Phentolamine potentiation of the action of norepinephrine to increase plasma cyclic AMP in cocaine-treated rats In rats not treated otherwise, norepinephrine injected in a dose of 100 pg/kg was without effect on the plasma level of cyclic AMP (fig. 3A). This is consistent with the results in fig. 2C. Norepinephrine however caused a sharp increase in plasma cyclic AMP when the rat had been injected i.v. with cocaine, an inhibitor of amine uptake mechanisms in the
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Fig. 3. Effect o f cocaine on c a t e c h o l a m i n e - i n d u c e d increases in plasma cyclic AMP. Cocaine, 10 mg/kg, was injected i.v. at time O. In panel A, n o r e p i n e p h r i n e ( 1 0 0 /~g/kg) was i n j e c t e d s.c. as s h o w n b y a n arrow. In p a n e l B, n o r e p i n e p h r i n e (as s h o w n b y a n a r r o w ) a n d c o c a i n e were injected into all rats. A t 20 min, saline or p h e n t o l a m i n e (0.5 m g / k g ) was i n j e c t e d s.c. In p a n e l C, i s o p r o t e r e n o l ( 5 / 2 g / k g ) was injected into all rats as s h o w n b y a n arrow. Ordinate: p l a s m a level o f cyclic A M P ( p m o l / r n l ) ; abscissa: t i m e (rain).
171
neuronal terminals (fig. 3A). The potency of cocaine alone to increase plasma cyclic AMP was rather small. In contrast to the norepinephrine-induced increase, the isoproterenol-induced increase in plasma cyclic AMP was unaffected by cocaine treatment (fig. 3C). Phentolamine was effective in further potentiating the increase in plasma cyclic AMP elicited by norepinephrine in cocaine-treated rats (fig. 3B), suggesting that phentolamine can enhance the plasma cyclic AMP response to norepinephrine by mechanisms somewhat distinct from those underlying the cocaine-induced potentiation. 3.3 Effects of 6-hydroxydopamine on plasma cyclic AMP responses to catecholamines and phentolamine The increase in plasma cyclic AMP induced by the simultaneous injection of norepinephrine and phentolamine was more marked in rats pretreated with 6-hydroxydopamine than in non-treated rats (fig. 4A). In contrast, there was no difference in the peak response of plasma cyclic AMP to isoproterenol (injected at 120 min in fig. 4A) between these two groups of rats. A higher dose (500 pg/kg) of norepinephrine was injected into 6-hydroxydopaminetreated rats in the experiments shown in fig. 4B. This dose of norepinephrine caused an about 4-fold increase in plasma cyclic AMP (open circles in fig. 4B); this increment was much larger than the increment induced by the same dose of the catecholamine in rats receiving no 6-hydroxydopamine (open circle above 500 pg in fig. 2C). Thus, the pretreatm e n t of rats with 6-hydroxydopamine enhanced the plasma cyclic AMP response to norepinephrine alone. In 6-hydroxydopaminetreated rats, the simultaneous injection of phentolamine and norepinephrine resulted in a more sharp, more rapid and more marked increase in plasma cyclic AMP than did the injection of norepinephrine alone (fig. 4B). Tyramine injected at 140 min (fig. 4B) caused no increase in plasma cyclic AMP in
172
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Fig. 4. Norepinephrine-induced increase in plasma cyclic AMP as p o t e n t i a t e d by phentolamine and 6hydroxydopamine. 6-Hydroxydopamine-treated rats were prepared by injecting 30 mg/kg of 6-hydroxydopamine intravenously 24 h before the experiment. In panel A, norepinephrine (100 pg/kg) and phentolamine (0.5 mg/kg) were injected s.c. at 20 rain, and isoproterenol (5 pg/kg) was injected s.c. at 120 min. In panel B, norepinephrine (500 pg/kg) and phentolamine were injected as in panel A at 20 rain and tyramine (2 mg/kg) was injected s.c. at 140 rain into 6 - h y d r o x y d o p a m i n e - t r e a t e d rats. Ordinate: plasma level of cyclic AMP (pmol/ml); abscissa: time (min).
6-hydroxydopamine-treated rats, showing that neuronal terminals are actually destroyed under these conditions (see Kunitada et al., 197S). Phentolamine caused a 2-fold increase in plasma cyclic AMP in 6-hydroxydopaminetreated rats (the first peak in fig. 5A); the increment was smaller than the increment (4-fold) observed in the rats not treated with 6-hydroxydopamine (the first peak in fig. 1A and B). The further injection of epinephrine into these 6-hydroxydopamine-treated rats gave rise to increases in plasma cyclic AMP which were more marked with phentolamine than without phentolamine (fig. 5A). For
Fig. 5. Failure of 6-hydroxydopamine to affect epinephrine-induced increases, and failure of phentolamine to affect glucagon-induced increases, in plasma cyclic AMP. Treatment of rats w i t h 6-hydroxydopamine was the same as in fig. 4. In panel A, s.c. injections of phentolamine (5 mg/kg) and epinephrine (30 pg/kg) were performed at 20 and 120 rain respectively. In panel B, normal rats were injected s.c. with phentolamine (0.5 mg/kg) and glucagon (20 pg/kg) at 15 and 20 rain respectively. Ordinate: plasma level of cyclic AMP (pmol/ml); abscissa: time (rain).
comparison, a similar experiment was repeated with rats receiving no 6-hydroxydopamine, the results are shown by triangles in fig. 5A. It is seen that the epinephrine-induced increase in plasma cyclic AMP as well as its enhancement by phentolamine also occurred regardless of whether the rats had been pretreated with 6-hydroxydopamine or not. Likewise, glucagon gave rise to an increase in plasma cyclic AMP in phentolamine-treated rats to the same extent as in non-treated rats. 3.4. Effect o f phenylephrine on isoproterenol-
induced increases in plasma cyclic AMP Phenylephrine (an a-adrenergic agent) was by itself essentially without effect on the plasma level of cyclic AMP (fig. 6). When phenylephrine was injected together with isopro-
PLASMA CYCLIC AMP AFTER PHENTOLAMINE
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terenol, the isoproterenol-induced increase in plasma cyclic AMP was only slightly inhibited.
173
plasma cyclic AMP following the injection of norepiriephrine. Cocaine is known to potentiate the action of catecholamines by two mechanisms: by blocking the neuronal uptake of catecholamines or by sensitizing postsynaptic responses to catecholamines (references in Greenberg and Innes, 1976). Under the conditions employed here, injected cocaine was likely to potentiate the plasma cyclic AMP response to catecholamines by the former mechanism because the action of isoproterenol, which is not taken up by neuronal terminals, was not affected by cocaine (fig. 3C). Evidence that the neuronal uptake of catecholamines is blocked in cocaine-treated rats has already been presented (Kunitada et al., 1978): tyramine, which should be taken up by nerve endings before releasing endogenous catecholamines, did not raise plasma cyclic AMP in the cocaine-treated rat. Likewise, the degeneration of sympathetic nerve endings caused by 6-hydroxydopamine was evidenced by the lack of the plasma cyclic AMP response to tyramine (fig. 4B, second arrow). Thus, it is concluded that rapid neuronal uptake is at least partly responsible for the inability of injected norepinephrine to increase plasma cyclic AMP by itself. 4.2. Mechanism by which a-adrenergic blockade potentiates the plasma cyclic AMP response to catecholamines
4. Discussion 4.1. Neuronal uptake o f injected norepinephrine as an explanation for the failure of norepinephrine to increase plasma cyclic AMP
Norepinephrine was incapable, by itself, of increasing plasma cyclic AMP significantly even at the highest dose (500 pg/kg) used; this was in sharp contrast with the marked increases induced by other catecholamines such as isoproterenol and epinephrine (fig. 2). When rats had been pretreated with cocaine (fig. 3A) or 6-hydroxydopamine (fig. 4B), however, there were significant increases in
An a-adrenoceptor-mediated negative feedback mechanism regulates catecholamine discharge from nerve endings (Langer, 1974; Starke et al., 1977). Blockade of presynaptic a-receptors prevents the feedback inhibition of catecholamine release (Enero et al., 1972; Cubeddu et al., 1974). Our findings that phentolamine enhanced the plasma cyclic AMP response to epinephrine (fig. 1A) but did not affect the response to isoproterenol (fig. 1B) might be accounted for in terms of the block of presynaptic receptors by phentolamine, because isoproterenol is not a trigger of the presynaptic negative feedback mecha-
174 nism. Alternatively, the blockade of aadrenoceptors on the effector cell membrane would favor the stimulation of ~-adrenoceptors by epinephrine, an a- and ~-adrenergic agonist, thereby leading to more production of cyclic AMP, but could not influence the activation of adenylate cyclase by a pure ~adrenergic agonist, isoproterenol. One of these two mechanisms, either the presynaptic or the postsynaptic one, appears to underlie the phentolamine-induced potentiation of plasma cyclic AMP responses depending on the experimental conditions. Phentolamine alone gave rise to an increase in plasma cyclic AMP which was smaller in 6-hydroxydopamine-treated rats (fig. 5A) than in non-treated rats (fig. 1A and B), indicating that the release of endogenous catecholamines from neuronal terminals was at least partly responsible for the phentolamineinduced increase in plasma cyclic AMP. The blockade of presynaptic a-adrenoceptors by phentolamine probably facilitated the normal tonic release of catecholamines, though alternative possibilities are not excluded; e.g., that hypotension induced by phentolamine led to increased adrenergic discharge. On the other hand, the injection of phentolamine caused a much greater increase in plasma cyclic AMP even when norepinephrine was injected into 6-hydroxydopamine-treated (fig. 4B) or cocaine-treated (fig. 3B) rats. Likewise, there was no difference in either the plasma cyclic AMP response to epinephrine or its enhancement by phentolamine between 6hydroxydopamine-treatedrats and non-treated rats (fig. 5A). It is therefore concluded that the action of injected norepinephrine and epinephrine to increase plasma cyclic AMP was enhanced by phentolamine as a result of the blockade of postsynaptic a-adrenoceptors rather than of presynaptic receptors. Failure of phentolamine to potentiate the action of a pure ~-agonist, isoproterenol, suggests that blockade of postsynaptic a-adrenoceptors is actually involved in the phentolamine-induced potentiation of the cyclic AMP responses. Possible mechanisms under-
S. KUNITADA,M. UI lying the interaction between postsynaptic a- and ~-adrenoceptors are discussed below. 4.3. Possible interactions between a- and {Jadrenoceptors on effector cells involved in adenylate eyclase activation
Increases in plasma cyclic AMP following injections of isoproterenol (Saitoh et al., 1976), epinephrine (Honma et al., 1977), tyramine (Kunitada et al., 1978) and norepinephrine (unpublished) into rats were overcome by simultaneous treatment of the animals with a ~-adrenolytic agent such as propranolol. /3-Adrenoceptor-mediated increases in plasma cyclic AMP would be enhanced by the blockade of postsynaptic a-adrenoceptors under one of the following three conditions. First: adenylate cyclase is activated via fl-receptors and inhibited via a-receptors. Under this condition, activation of adenylate cyclase resulting from the stimulation of ~-receptors by epinephrine or norepinephrine is always counteracted by its concurrent occupation of a-receptors. Blockade of a-receptors would then cause full activation of adenylate cyclase due to reversal of the counteraction. Second: a fraction of ~-receptors interacts with a-receptors in such a manner that the binding of agonists to the latter interferes with their binding to the former. Thus, this fraction of ~-receptors (the spare receptor) is unmasked by blockade of a-receptors. Third: a- and ~-receptors are located so closely to each other on effector cells that binding of agonists to a-receptors reduces their availability to ~-receptors. aBlockade would then make agonists more available to ~-receptors leading to more activation of adenylate cyclase. Any one of the three concepts is applicable to the present findings that a-blockade enhanced epinephrine-induced increases in plasma cyclic AMP but did not affect isoproterenol-induced increases. However, the influence of a-adrenoceptor stimulation on/~actions would be affected differently under the various conditions. For example, the
PLASMA CYCLIC AMP A F T E R PHENTOLAMINE
administration of a pure a-agonist (or the combined administration of epinephrine with a fl-adrenolytic agent) should reduce the basal (i.e., independent of H-receptors) activity of adenylate cycla.,;e by itself only if the first concept of "reversal of counteraction" were applicable. On the other hand, the isoproterenol-induced increase in cyclic AMP would not be attenuated by an a-agonist only if the potentiation by a-blockade of H-actions were explainable by the third concept of "increased agonist availability". Most of the data available concerning the potentiation of /3- (or a-) receptor-mediated actions induced by blockade of receptors of the other type can be accounted for in terms of the first or the second concept presented above, since ~- (or a-) actions were usually antagonized by a- (or ~-) actions. In the case of the ~-adrenoceptor-dependent increases in plasma cyclic AMP reported in the present paper, however, no evidence has been provided for antagonism by a-agonists; phenylephrine caused only a slight inhibition of fl-agonistinduced increases in plasma cyclic AMP (fig. 6). Thus, the action of phentolamine to favor the increases in plasma cyclic AMP elicited by epinephrine, norepinephrine or tyramine might be due to "increased agonist availability to H-receptors", though a mechanism unrelated to a-blockade (Wenkeovfi et al., 1975, 1976) is not fully excluded.
4.4. Lower potency o f norepinephrine than tyramine in raising the plasma cyclic AMP level The highest dose (500 t~g/kg) of norepinephrine gave rise to an about 12-fold increase in plasma cyclic AMP when presynaptic uptake was prevented by 6-hydroxydopamine and postsynaptic a-receptors were blocked by phentolamine (fig. 4B). This maximal response to norepinephrine was still much smaller than the response to tyramine which could be increased 30-fold in phentolamine-treated rats (fig. 2D). In the preceding paper tyramine was reported to elevate the plasma cyclic AMP
175
level due to the release of catecholamines from sympathetic nerve endings (Kunitada et al., 1978). Since norepinephrine is the predominant catecholamine released from sympathetic nerve endings, it seems strange that exogenously administered norepinephrine is less effective than tyramine in increasing plasma cyclic AMP. It might be conceivable, therefore, that dopamine or epinephrine released in a smaller amount by tyramine acts as a more potent activator of adenylate cyclase than does norepinephrine. Alternatively, differential responses of plasma cyclic AMP to endogenous and exogenous catecholamines could have resulted from an uneven distribution of adrenergic receptors on effector cells in relation to the adrenergic nerve endings (Rosell and Belfrage, 1975; Belfrage and Rosell, 1976); i.e., more cyclic AMP might be generated via stimulation of the receptors located in close contact with the nerve terminals than via stimulation of the receptors preferentially accessible to circulating catecholamines. This problem is currently under investigation in our laboratory.
References Belfrage, E. and S. Rosell, 1976, The role cf neuronal uptake at a- and ~-adrenoceptor sites in subcutaneous adipose tissue, Naunyn-Schmiedeb. Arch; Pharmacol. 294, 9. Cubeddu, L., S.Z. Langer and N. Weiner, 1974, The relationship between alpha receptor block, inhibition of norepinephrine uptake and the release and metabolism of 3H-norepinephrine, J. Pharmacol. Exptl. Therap. 188,368. Enero, M.A., S.Z. Langer, R.P. Rothlin and F.J.E. Stefano, 1972, Role of the a-adrenoceptor in regulating noradrenaline overflow by nerve stimulation, Brit. J. Pharmacol. 4 4 , 6 7 2 . Greenberg, R., and I.R. Innes, 1976, The role of bound calcium in supersensitivity induced by cocaine, Brit. J. Pharmacol. 57,329. Honma, M., T. Satoh, H. Takezawa and M. Ui, 1977, An ultrasensitive method for the simultaneous d e t e r m i n a t i o n of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue, Biochem. Med. 18, 257. Kunitada, S., M. Honma and M. Ui, 1978, Increases in plasma cyclic AMP dependent on endogenous
176 catecholamines, European J. Pharmacol. (in press). Langer, S.Z., 1974, Presynaptic regulation of catecholmine release, Biochem. Pharmacol. 23, 1793. Rosell, S. and E. Belfrage, 1975, Adrenergic receptors in adipose tissue and their relation to adrenergic innervation, Nature 253,738. Saitoh, Y., S. Morita, Y. Irie and H. Kohri, 1976. Evaluation of a new beta-adrenergic blocking agent, carteolol, based on metabolic responses in rats. II. Blockade by carteolol of the epinephrine- and isoproterenol-induced increases of tissue and blood cyclic AMP in vivo, Biochem. Pharmacol. 25,
S. KUNITADA, M. UI Starke, K., H.D. Taube and E. Borowski, 1977, Presynaptic receptor systems in catecholaminergic transmission, Biochem. Pharmacol. 26,259. Wenkeov~, J., E. Kuhn and M. Wenke, 1975, Adrenergic lipolysis in human adipose tissue in vitro, European J. Pharmacol. 30, 49. Wenkeov~, J., E. Kuhn and M. Wenke, 1976, Some adrenomimetic drugs affecting lipolysis in human adipose tissue in vitro, European J. Pharmacol. 35, 1.
169
© Elsevier/North-Holland Biomedical Press
THE PLASMA CYCLIC AMP RESPONSE TO CATECHOLAMINES AS POTENTIATED BY PHENTOLAMINE IN RATS SATOSHI KUNITADA and MICHIO UI *
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Received 26 August 1977, revised MS received 18 November 1977, accepted 1 February 1978
S. KUNITADA and M. UI, The plasma cyclic AMP response to catecholamines as potentiated by phentolamine in rats, European J. Pharmacol. 49 (1978) 169--176. Norepinephrine failed to increase plasma cyclic AMP when injected alone into fasted rats, in contrast with sharp increases elicited by isoproterenol, epinephrine or tyramine. In rats pretreated with 6-hydroxydopamine or cocaine, however, there was a significant increase in plasma cyclic AMP after norepinephrine injection, suggesting that the rapid neuronal catecholamine uptake was at least partly responsible for the lack of norepinephrine action. Phentolamine was very effective in enhancing the epinephrine-, norepinephrine- or tyramine-induced increase in plasma cyclic AMP but without effect on the isoproterenol-induced increase. Blockade of postsynaptic ~adrenoceptors, rather than of presynaptic receptors, is likely to be involved in the phentolamine potentiation, since it was even observed in rats treated with 6-hydroxydopamine or cocaine. A discussion is presented regarding the mechanism by which cyclic AMPfleneration is influenced by the ~- and f~-adrenoceptor interaction on effector cell membranes. Postsynaptic ~-adrenoceptor and ~-adrenoceptor interactions ~-Adrenergic stimulants
1. Introduction The s.c. injection of tyramine has been shown to lead to a rapid increase in plasma cyclic AMP due to the release of catecholamines from sympathetic nerve endings (Kunitada et al., 1978). This action of tyramine was markedly enhanced when ~-adrenergic receptors were blocked b y phentolamine. The purpose of the present paper is to show how phentolamine potentiates the plas-
* Correspondence to M.U., Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan.
Plasma cyclic AMP 6-Hydroxydopamine
ma cyclic AMP response stimulation.
Phentolamine Cocaine
to
/~-adrenergic
2. Materials and methods Male rats of a Wistar-derived strain were used after a 20-h fast. Blood specimens were withdrawn at intervals from the tail vein and analyzed for cyclic AMP by a sensitive radioimmunoassay (Honma et al., 1977) as described in the preceding paper. Theophylline, 10 mg/kg b o d y wt, was injected s.c. at time 0 into all of the rats for the purpose o f suppressing cyclic AMP phosphodiesterase. Sources of reagents were the same as those described in the preceding paper (Kunitada et al., 1978).
170
S. KUNITADA, M. UI
3. Results
3.1. Differential effects of phentolamine on increases in plasma cyclic AMP elicited by injection of catecholamines As in the previous study (Kunitada et al., 1978), the s.c. injection of phentolamine in a dose of 5 mg/kg b o d y weight caused a rapid increase in plasma cyclic AMP reaching a peak at 40 min and declining within 100 min after injection {fig. 1A and B). Fig. 1A further shows that the injection of epinephrine 100 min after the injection of phentolamine
800
Epinephrine
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resulted in a tremendous increase in plasma cyclic AMP, whereas epinephrine injected into saline-treated rats elicited a much smaller increase. In contrast, the injection of isoproterenol in doses from 1 to 25 pg/kg gave rise to increases in plasma cyclic AMP which were similar whether the rat had been treated with phentolamine or not (fig. 1B). Peak levels of plasma cyclic AMP after the injection of these catecholamines and of norepinephrine and tyramine were plotted against the doses (fig. 2). It is seen that phentolamine was effective in potentiating epinephrine-induced and tyramine-induced increases in plasma cyclic AMP, but without effect on the isoproterenol-induced increase. In the case of norepinephrine, the increase in
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Fig. 1. Increases in plasma cyclic AMP by epinephrine and by isoproterenol in rats pretreated with phentolamine. In this and all o f the following figures, theophylline (10 mg]kg body weight) was injected s.c. into all rats at time O, and the results are expressed as mean +_ S.E.M. (symbols attached with vertical lines) with the number of observations following n =, or in parentheses. Saline (open symbols) or phentolamine (5 mg/kg, solid symbols) was injected s.c. at 20 rain. Epinephrine (panel A) or isoproterenol (panel B) was further injected s.c. at 120 rain in doses shown by smaller symbols in panels. Ordinate: plasma level of cyclic AMP (pmol/ml); abscissa: time (min).
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Fig. 2. Dose--response relationship for catecholamine-induced increases in plasma cyclic AMP. Epinephrine (panel A), isoproterenol (panel B), norepinephrine (panel C) or tyramine (panel D) was injected into rats treated with phentolamine (solid circles) or not treated (open circles). The time of injections and dose of phentolamine are the same as in fig. 1. Peak levels of plasma cyclic AMP after injection of catecholamines are plotted as a function of the doses used. Ordinate: plasma level of cyclic AMP (pmol/ml); abscissa: dose of catecholamines (pg/kg) or tyramine (mg/kg) (log scale).
P L A S M A CYCLIC A M P A F T E R P H E N T O L A M I N E
plasma cyclic AMP was only slight even when the rats had been treated with phentolamine. 3.2. Phentolamine potentiation of the action of norepinephrine to increase plasma cyclic AMP in cocaine-treated rats In rats not treated otherwise, norepinephrine injected in a dose of 100 pg/kg was without effect on the plasma level of cyclic AMP (fig. 3A). This is consistent with the results in fig. 2C. Norepinephrine however caused a sharp increase in plasma cyclic AMP when the rat had been injected i.v. with cocaine, an inhibitor of amine uptake mechanisms in the
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Fig. 3. Effect o f cocaine on c a t e c h o l a m i n e - i n d u c e d increases in plasma cyclic AMP. Cocaine, 10 mg/kg, was injected i.v. at time O. In panel A, n o r e p i n e p h r i n e ( 1 0 0 /~g/kg) was i n j e c t e d s.c. as s h o w n b y a n arrow. In p a n e l B, n o r e p i n e p h r i n e (as s h o w n b y a n a r r o w ) a n d c o c a i n e were injected into all rats. A t 20 min, saline or p h e n t o l a m i n e (0.5 m g / k g ) was i n j e c t e d s.c. In p a n e l C, i s o p r o t e r e n o l ( 5 / 2 g / k g ) was injected into all rats as s h o w n b y a n arrow. Ordinate: p l a s m a level o f cyclic A M P ( p m o l / r n l ) ; abscissa: t i m e (rain).
171
neuronal terminals (fig. 3A). The potency of cocaine alone to increase plasma cyclic AMP was rather small. In contrast to the norepinephrine-induced increase, the isoproterenol-induced increase in plasma cyclic AMP was unaffected by cocaine treatment (fig. 3C). Phentolamine was effective in further potentiating the increase in plasma cyclic AMP elicited by norepinephrine in cocaine-treated rats (fig. 3B), suggesting that phentolamine can enhance the plasma cyclic AMP response to norepinephrine by mechanisms somewhat distinct from those underlying the cocaine-induced potentiation. 3.3 Effects of 6-hydroxydopamine on plasma cyclic AMP responses to catecholamines and phentolamine The increase in plasma cyclic AMP induced by the simultaneous injection of norepinephrine and phentolamine was more marked in rats pretreated with 6-hydroxydopamine than in non-treated rats (fig. 4A). In contrast, there was no difference in the peak response of plasma cyclic AMP to isoproterenol (injected at 120 min in fig. 4A) between these two groups of rats. A higher dose (500 pg/kg) of norepinephrine was injected into 6-hydroxydopaminetreated rats in the experiments shown in fig. 4B. This dose of norepinephrine caused an about 4-fold increase in plasma cyclic AMP (open circles in fig. 4B); this increment was much larger than the increment induced by the same dose of the catecholamine in rats receiving no 6-hydroxydopamine (open circle above 500 pg in fig. 2C). Thus, the pretreatm e n t of rats with 6-hydroxydopamine enhanced the plasma cyclic AMP response to norepinephrine alone. In 6-hydroxydopaminetreated rats, the simultaneous injection of phentolamine and norepinephrine resulted in a more sharp, more rapid and more marked increase in plasma cyclic AMP than did the injection of norepinephrine alone (fig. 4B). Tyramine injected at 140 min (fig. 4B) caused no increase in plasma cyclic AMP in
172
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Fig. 4. Norepinephrine-induced increase in plasma cyclic AMP as p o t e n t i a t e d by phentolamine and 6hydroxydopamine. 6-Hydroxydopamine-treated rats were prepared by injecting 30 mg/kg of 6-hydroxydopamine intravenously 24 h before the experiment. In panel A, norepinephrine (100 pg/kg) and phentolamine (0.5 mg/kg) were injected s.c. at 20 rain, and isoproterenol (5 pg/kg) was injected s.c. at 120 min. In panel B, norepinephrine (500 pg/kg) and phentolamine were injected as in panel A at 20 rain and tyramine (2 mg/kg) was injected s.c. at 140 rain into 6 - h y d r o x y d o p a m i n e - t r e a t e d rats. Ordinate: plasma level of cyclic AMP (pmol/ml); abscissa: time (min).
6-hydroxydopamine-treated rats, showing that neuronal terminals are actually destroyed under these conditions (see Kunitada et al., 197S). Phentolamine caused a 2-fold increase in plasma cyclic AMP in 6-hydroxydopaminetreated rats (the first peak in fig. 5A); the increment was smaller than the increment (4-fold) observed in the rats not treated with 6-hydroxydopamine (the first peak in fig. 1A and B). The further injection of epinephrine into these 6-hydroxydopamine-treated rats gave rise to increases in plasma cyclic AMP which were more marked with phentolamine than without phentolamine (fig. 5A). For
Fig. 5. Failure of 6-hydroxydopamine to affect epinephrine-induced increases, and failure of phentolamine to affect glucagon-induced increases, in plasma cyclic AMP. Treatment of rats w i t h 6-hydroxydopamine was the same as in fig. 4. In panel A, s.c. injections of phentolamine (5 mg/kg) and epinephrine (30 pg/kg) were performed at 20 and 120 rain respectively. In panel B, normal rats were injected s.c. with phentolamine (0.5 mg/kg) and glucagon (20 pg/kg) at 15 and 20 rain respectively. Ordinate: plasma level of cyclic AMP (pmol/ml); abscissa: time (rain).
comparison, a similar experiment was repeated with rats receiving no 6-hydroxydopamine, the results are shown by triangles in fig. 5A. It is seen that the epinephrine-induced increase in plasma cyclic AMP as well as its enhancement by phentolamine also occurred regardless of whether the rats had been pretreated with 6-hydroxydopamine or not. Likewise, glucagon gave rise to an increase in plasma cyclic AMP in phentolamine-treated rats to the same extent as in non-treated rats. 3.4. Effect o f phenylephrine on isoproterenol-
induced increases in plasma cyclic AMP Phenylephrine (an a-adrenergic agent) was by itself essentially without effect on the plasma level of cyclic AMP (fig. 6). When phenylephrine was injected together with isopro-
PLASMA CYCLIC AMP AFTER PHENTOLAMINE
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terenol, the isoproterenol-induced increase in plasma cyclic AMP was only slightly inhibited.
173
plasma cyclic AMP following the injection of norepiriephrine. Cocaine is known to potentiate the action of catecholamines by two mechanisms: by blocking the neuronal uptake of catecholamines or by sensitizing postsynaptic responses to catecholamines (references in Greenberg and Innes, 1976). Under the conditions employed here, injected cocaine was likely to potentiate the plasma cyclic AMP response to catecholamines by the former mechanism because the action of isoproterenol, which is not taken up by neuronal terminals, was not affected by cocaine (fig. 3C). Evidence that the neuronal uptake of catecholamines is blocked in cocaine-treated rats has already been presented (Kunitada et al., 1978): tyramine, which should be taken up by nerve endings before releasing endogenous catecholamines, did not raise plasma cyclic AMP in the cocaine-treated rat. Likewise, the degeneration of sympathetic nerve endings caused by 6-hydroxydopamine was evidenced by the lack of the plasma cyclic AMP response to tyramine (fig. 4B, second arrow). Thus, it is concluded that rapid neuronal uptake is at least partly responsible for the inability of injected norepinephrine to increase plasma cyclic AMP by itself. 4.2. Mechanism by which a-adrenergic blockade potentiates the plasma cyclic AMP response to catecholamines
4. Discussion 4.1. Neuronal uptake o f injected norepinephrine as an explanation for the failure of norepinephrine to increase plasma cyclic AMP
Norepinephrine was incapable, by itself, of increasing plasma cyclic AMP significantly even at the highest dose (500 pg/kg) used; this was in sharp contrast with the marked increases induced by other catecholamines such as isoproterenol and epinephrine (fig. 2). When rats had been pretreated with cocaine (fig. 3A) or 6-hydroxydopamine (fig. 4B), however, there were significant increases in
An a-adrenoceptor-mediated negative feedback mechanism regulates catecholamine discharge from nerve endings (Langer, 1974; Starke et al., 1977). Blockade of presynaptic a-receptors prevents the feedback inhibition of catecholamine release (Enero et al., 1972; Cubeddu et al., 1974). Our findings that phentolamine enhanced the plasma cyclic AMP response to epinephrine (fig. 1A) but did not affect the response to isoproterenol (fig. 1B) might be accounted for in terms of the block of presynaptic receptors by phentolamine, because isoproterenol is not a trigger of the presynaptic negative feedback mecha-
174 nism. Alternatively, the blockade of aadrenoceptors on the effector cell membrane would favor the stimulation of ~-adrenoceptors by epinephrine, an a- and ~-adrenergic agonist, thereby leading to more production of cyclic AMP, but could not influence the activation of adenylate cyclase by a pure ~adrenergic agonist, isoproterenol. One of these two mechanisms, either the presynaptic or the postsynaptic one, appears to underlie the phentolamine-induced potentiation of plasma cyclic AMP responses depending on the experimental conditions. Phentolamine alone gave rise to an increase in plasma cyclic AMP which was smaller in 6-hydroxydopamine-treated rats (fig. 5A) than in non-treated rats (fig. 1A and B), indicating that the release of endogenous catecholamines from neuronal terminals was at least partly responsible for the phentolamineinduced increase in plasma cyclic AMP. The blockade of presynaptic a-adrenoceptors by phentolamine probably facilitated the normal tonic release of catecholamines, though alternative possibilities are not excluded; e.g., that hypotension induced by phentolamine led to increased adrenergic discharge. On the other hand, the injection of phentolamine caused a much greater increase in plasma cyclic AMP even when norepinephrine was injected into 6-hydroxydopamine-treated (fig. 4B) or cocaine-treated (fig. 3B) rats. Likewise, there was no difference in either the plasma cyclic AMP response to epinephrine or its enhancement by phentolamine between 6hydroxydopamine-treatedrats and non-treated rats (fig. 5A). It is therefore concluded that the action of injected norepinephrine and epinephrine to increase plasma cyclic AMP was enhanced by phentolamine as a result of the blockade of postsynaptic a-adrenoceptors rather than of presynaptic receptors. Failure of phentolamine to potentiate the action of a pure ~-agonist, isoproterenol, suggests that blockade of postsynaptic a-adrenoceptors is actually involved in the phentolamine-induced potentiation of the cyclic AMP responses. Possible mechanisms under-
S. KUNITADA,M. UI lying the interaction between postsynaptic a- and ~-adrenoceptors are discussed below. 4.3. Possible interactions between a- and {Jadrenoceptors on effector cells involved in adenylate eyclase activation
Increases in plasma cyclic AMP following injections of isoproterenol (Saitoh et al., 1976), epinephrine (Honma et al., 1977), tyramine (Kunitada et al., 1978) and norepinephrine (unpublished) into rats were overcome by simultaneous treatment of the animals with a ~-adrenolytic agent such as propranolol. /3-Adrenoceptor-mediated increases in plasma cyclic AMP would be enhanced by the blockade of postsynaptic a-adrenoceptors under one of the following three conditions. First: adenylate cyclase is activated via fl-receptors and inhibited via a-receptors. Under this condition, activation of adenylate cyclase resulting from the stimulation of ~-receptors by epinephrine or norepinephrine is always counteracted by its concurrent occupation of a-receptors. Blockade of a-receptors would then cause full activation of adenylate cyclase due to reversal of the counteraction. Second: a fraction of ~-receptors interacts with a-receptors in such a manner that the binding of agonists to the latter interferes with their binding to the former. Thus, this fraction of ~-receptors (the spare receptor) is unmasked by blockade of a-receptors. Third: a- and ~-receptors are located so closely to each other on effector cells that binding of agonists to a-receptors reduces their availability to ~-receptors. aBlockade would then make agonists more available to ~-receptors leading to more activation of adenylate cyclase. Any one of the three concepts is applicable to the present findings that a-blockade enhanced epinephrine-induced increases in plasma cyclic AMP but did not affect isoproterenol-induced increases. However, the influence of a-adrenoceptor stimulation on/~actions would be affected differently under the various conditions. For example, the
PLASMA CYCLIC AMP A F T E R PHENTOLAMINE
administration of a pure a-agonist (or the combined administration of epinephrine with a fl-adrenolytic agent) should reduce the basal (i.e., independent of H-receptors) activity of adenylate cycla.,;e by itself only if the first concept of "reversal of counteraction" were applicable. On the other hand, the isoproterenol-induced increase in cyclic AMP would not be attenuated by an a-agonist only if the potentiation by a-blockade of H-actions were explainable by the third concept of "increased agonist availability". Most of the data available concerning the potentiation of /3- (or a-) receptor-mediated actions induced by blockade of receptors of the other type can be accounted for in terms of the first or the second concept presented above, since ~- (or a-) actions were usually antagonized by a- (or ~-) actions. In the case of the ~-adrenoceptor-dependent increases in plasma cyclic AMP reported in the present paper, however, no evidence has been provided for antagonism by a-agonists; phenylephrine caused only a slight inhibition of fl-agonistinduced increases in plasma cyclic AMP (fig. 6). Thus, the action of phentolamine to favor the increases in plasma cyclic AMP elicited by epinephrine, norepinephrine or tyramine might be due to "increased agonist availability to H-receptors", though a mechanism unrelated to a-blockade (Wenkeovfi et al., 1975, 1976) is not fully excluded.
4.4. Lower potency o f norepinephrine than tyramine in raising the plasma cyclic AMP level The highest dose (500 t~g/kg) of norepinephrine gave rise to an about 12-fold increase in plasma cyclic AMP when presynaptic uptake was prevented by 6-hydroxydopamine and postsynaptic a-receptors were blocked by phentolamine (fig. 4B). This maximal response to norepinephrine was still much smaller than the response to tyramine which could be increased 30-fold in phentolamine-treated rats (fig. 2D). In the preceding paper tyramine was reported to elevate the plasma cyclic AMP
175
level due to the release of catecholamines from sympathetic nerve endings (Kunitada et al., 1978). Since norepinephrine is the predominant catecholamine released from sympathetic nerve endings, it seems strange that exogenously administered norepinephrine is less effective than tyramine in increasing plasma cyclic AMP. It might be conceivable, therefore, that dopamine or epinephrine released in a smaller amount by tyramine acts as a more potent activator of adenylate cyclase than does norepinephrine. Alternatively, differential responses of plasma cyclic AMP to endogenous and exogenous catecholamines could have resulted from an uneven distribution of adrenergic receptors on effector cells in relation to the adrenergic nerve endings (Rosell and Belfrage, 1975; Belfrage and Rosell, 1976); i.e., more cyclic AMP might be generated via stimulation of the receptors located in close contact with the nerve terminals than via stimulation of the receptors preferentially accessible to circulating catecholamines. This problem is currently under investigation in our laboratory.
References Belfrage, E. and S. Rosell, 1976, The role cf neuronal uptake at a- and ~-adrenoceptor sites in subcutaneous adipose tissue, Naunyn-Schmiedeb. Arch; Pharmacol. 294, 9. Cubeddu, L., S.Z. Langer and N. Weiner, 1974, The relationship between alpha receptor block, inhibition of norepinephrine uptake and the release and metabolism of 3H-norepinephrine, J. Pharmacol. Exptl. Therap. 188,368. Enero, M.A., S.Z. Langer, R.P. Rothlin and F.J.E. Stefano, 1972, Role of the a-adrenoceptor in regulating noradrenaline overflow by nerve stimulation, Brit. J. Pharmacol. 4 4 , 6 7 2 . Greenberg, R., and I.R. Innes, 1976, The role of bound calcium in supersensitivity induced by cocaine, Brit. J. Pharmacol. 57,329. Honma, M., T. Satoh, H. Takezawa and M. Ui, 1977, An ultrasensitive method for the simultaneous d e t e r m i n a t i o n of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue, Biochem. Med. 18, 257. Kunitada, S., M. Honma and M. Ui, 1978, Increases in plasma cyclic AMP dependent on endogenous
176 catecholamines, European J. Pharmacol. (in press). Langer, S.Z., 1974, Presynaptic regulation of catecholmine release, Biochem. Pharmacol. 23, 1793. Rosell, S. and E. Belfrage, 1975, Adrenergic receptors in adipose tissue and their relation to adrenergic innervation, Nature 253,738. Saitoh, Y., S. Morita, Y. Irie and H. Kohri, 1976. Evaluation of a new beta-adrenergic blocking agent, carteolol, based on metabolic responses in rats. II. Blockade by carteolol of the epinephrine- and isoproterenol-induced increases of tissue and blood cyclic AMP in vivo, Biochem. Pharmacol. 25,
S. KUNITADA, M. UI Starke, K., H.D. Taube and E. Borowski, 1977, Presynaptic receptor systems in catecholaminergic transmission, Biochem. Pharmacol. 26,259. Wenkeov~, J., E. Kuhn and M. Wenke, 1975, Adrenergic lipolysis in human adipose tissue in vitro, European J. Pharmacol. 30, 49. Wenkeov~, J., E. Kuhn and M. Wenke, 1976, Some adrenomimetic drugs affecting lipolysis in human adipose tissue in vitro, European J. Pharmacol. 35, 1.