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Inhibition of the noradrenergic induction of pineal N-acetyltransferase by dibutyryl cyclic guanosine monophosphate and by ionophore X-537A
Neuroscience Letters, 2 (1976) 29--33 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
29
INHIBITION OF THE NORADRENERGIC INDUCTION OF PINEAL NACETYLTRANSFERASE BY DIBUTYI:tYL CYCLIC GUANOSINE MONOPHOSPHATE AND BY IONOPHORE X-537A
MICHAEL WILKINSON D~partement de Physiologie, Ecole de M~decine, Universit~ de Gen~ve, 1211 Genhve 4 (Switzerland) (Received January 13th, 1976) (Accepted January 20th, 1976)
SUMMARY
Utilizing rat pineals in vitro, it can be demonstrated that dibutyryl cyclic GMP and the calcium ionophore X-537A are both able to reversibly inhibit the elevation of pineal N-acetyltransferase induced by the ~-agonists norepinephrine or isoproterenol. These results are discussed in terms of the known hyposensitivy of the pineal gland to overstimulation by ~-agonists.
Sympathetic nerves innervating the rat pineal release norepinephrine which activates serotonin N-acetyltransferase (NAT), the rate-limiting enzyme in the biosynthesis of melatonin [ 1 ]. Much evidence suggests that norepinephrine elevates NAT by first binding to a ~-adrenergic receptor which in turn stimulates the formation of pineal adenosine-3',5'-cyclic monophosphate (cAMP) [3,10]. An intriguing property of this receptor is indicated by the observation that the responsiveness of NAT to induction by ~-agonists, in vivo and in vitro, varies considerably depending on the degree of previous stimulation [ 15 ]. Thus, repeated stimulation leads to subsensitivity whilst deprivation of agonist leads to supersensitivity of the receptors. These profound but reversible differences appear quickly (~24 h) and are conveniently demonstrated using an in vitro preparation in which very low concentrations of agonist may be employed [ 15]. When higher, non-physiological concentrations are used, the differences in response are abolished. Evidence is available indicating that central catecholamine receptors also exhibit the phenomenon of supersensitivity (see ref. 20 for example); other examples include smooth muscle [ 5], and the insulin receptor [6]. The mechanisms underlying the development of postsynaptic hyper- and hyposensitivity remain unknown. However, it has been postulated that hormone-induced intracellular elevations of cAMP in various cell types may be inhibited by guanosine-3',5'-cyclic monophosphate (cGMP) [7,19].Jt was of
30 interest, therefore, to investigate if the subsensitive response of pinealocytes to ~-agonists could be reproduced by exogenous cGMP added to pineals in vitro. Further, since calcium ions are a necessary requirement for endogenous elevations of cGMP [17,18] in certain tissues, we have also looked at the effects of the calcium ionophore X-537A, a substance known [8,11] to elevate intracellular calcium ion concentrations. Male rats (Wistar), of approximately 200 g body weight, were used except where indicated. They were sacrificed between two and three hours after the beginning of the light period. Pineals were dissected and placed directly into tissue culture dishes (60 × 15 mm) containing NCTC-135 culture medium (1.5 ml; 3 pineals per dish). The medium was modified by addition of pyruvate (1 mM), MEM non-essential amino acids, glucose (500 mg%), HEPES buffer (10 mM) and fungizone (1.25 pg/ml). All tissue culture products were obtained from GIBCO. Cultures were maintained at 37°C in a water saturated atmosphere of 95% air/CO2. NAT activity was measured by the method of Deguchi and Axelrod [4]. All reagents were commercially available. X-537A was a gift from Hoffmann-La Roche. Stock solutions of X-537A were in dimethyl sulfoxide (DMSO) (5 mg/ml), which, after dilution with culture medium gave a final concentration of 10 pg/ml; (DMSO 0.1%). Control cultures contained 0.1% DMSO. The dibutyryl derivative of cGMP (DBcGMP) was used in preference to the parent compound because of its greater resistance to degradation by phosphodiesterase. Preincubation of pineals with DBcGMP (1.0 mM) for periods of 30--40 min, followed by stimulation (4 h) with norepinephrine (NE; 10 -6 M) led to a highly significant (P < 0.001) inhibition of the rise of NAT (Fig. 1A). The glands fully recovered their sensitivity after washing out the DBcGMP. Butyric acid (10 .4 M and 10 -3 M), a possible degradation product of DBcGMP, had no inhibitory effect on NE-induced NAT, i.e., NAT levels in the presence or absence of butyric acid were approximately 1500 units. NAT induction by a more potent ~-agonist,/-isoproterenol (10 -7 M), was also inhibited by DBcGMP (1.0 mM; Fig. 1D). When the catecholamines were added concurrently with the DBcGMP, no significant inhibitory response was observed. Similarly, the use of higher concentrations of NE or isoproterenol abolished the effect of the DBcGMP. NAT induction by suboptimal doses of NE or isoproterenol was also significantly (P < 0.001) but reversibly inhibited by the calcium ionophore X-537A (10 ug/ml) (Fig. 1B and 1C respectively). The results indicate that the induction of NAT in vitro by small concentrations (~<10 -6 M) of ~-agonists may be reversibly inhibited by DBcGMP or by the calcium ionophore X-537A. The characteristics of this inhibition parallel some of the known features of pineal p-receptor hyposensitivity [15], i.e., the effect is reversible and is conveniently demonstrated in vitro using suboptimal concentrations of agonist. The similar results obtained using norepinephrine or isoproterenol indicate that the effect is at a postjunctional site, since it is known that the latter is not taken up by nerve endings [16]. At the
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Fig. 1. N-acetyltransferase activity in vitro. A: effects of stimulation with 10 -6 M norepinephrine (NE) (4 h, u ) and after preincubation with 1.0 mM DBcGMP (striped column}. B: 10 -6 M NE (3 h, u) and after preincubation with X-537A (10 ug/ml, checked column). C: 10 -~ M isoprotcrenol (2 h, cross-hatched column} and after preincubation with X-537A (10 ug/ml, vertically striped column). D: 10-7 ~/[ isoproterenol (2 h, cross-hatched column} and after preincubation with 1.0 mM DBcGMP (dotted column}. N-acetyltransferase units are picomoles of N-acetyltryptamine formed per gland per hour. Control values of NAT, without stimulation, were less than 100 units. Animals used in A were 350 g body weight and in B--D, approximately 200 g. N = number of pineals. Results expressed as mean -+ standard error; *P < 0.001; **P < 0.005 (Student's t-test). p r e s e n t t i m e it is n o t possible t o d e c i d e at precisely w h i c h p o i n t , b e t w e e n t h e /]-receptor a n d N A T elevation, t h e b l o c k a d e t a k e s place. T h e e x p e r i m e n t s suggest, h o w e v e r , t h a t b o t h c a l c i u m a n d cyclic G M P m a y be m e d i a t o r s in t h e r e s p o n s e o f pineal H-receptors t o o v e r s t i m u l a t i o n . T h e e v i d e n c e t h a t c a l c i u m and c G M P are closely i n t e r r e l a t e d in cell f u n c t i o n is e x t e n s i v e (see ref. 14). F o r e x a m p l e , in r a t p a r o t i d , t h e e f f e c t o f a c a l c i u m i o n o p h o r e is t o b l o c k t h e i s o p r o t e r e n o l - i n d u c e d rise in c A M P as well as t o elevate c G M P [2]. O u r results m a y also c o m p l e m e n t t h e w o r k o f P a r f i t t et al. [12] w h o r e p o r t e d an inhibit i o n o f t h e n o r a d r e n e r g i c s t i m u l a t i o n o f pineal N A T b y o u a b a i n or b y elevated p o t a s s i u m c o n c e n t r a t i o n s . High levels o f p o t a s s i u m chloride are k n o w n t o raise c G M P [ 17 ]. O u a b a i n , o n t h e o t h e r h a n d , b y elevating intracellular s o d i u m ion c o n c e n t r a t i o n s , brings a b o u t t h e a c c u m u l a t i o n o f c a l c i u m [9,13] w h i c h parallels o u r w o r k w i t h t h e i o n o p h o r e .
32 O u r e x p e r i m e n t s leave o p e n the q u e s t i o n o f w h e t h e r the p h e n o m e n o n o f h y p e r s e n s i t i v i t y o f r e c e p t o r s is in s o m e w a y related, in an antithetical way, to h y p o s e n s i t i v i t y a n d m a y also involve calcium ions. ACKNOWLEDGEMENTS T h e a u t h o r is grateful t o Madeleine Vuillet, K.B. R u f and D. de Ziegler f o r their help. T h e w o r k was s u p p o r t e d b y a g r a n t f r o m the Swiss N a t i o n a l Science F o u n d a t i o n (No. 3 . 0 1 6 . 7 3 ) t o Prof. K.B. Ruf. REFERENCES 1 Axelrod, J., The pineal gland: a neurochemical transducer, Science, 184 (1974) 1341-1348. 2 Butcher, F.R., The role of calcium and cyclic nucleotides in a-amylase release from slices of rat parotid: studies with ionophore A-23187, Metabolism, 24 (1975) 409--418. 3 Deguchi, T., Role of the beta adrenergic receptor in the elevation of cAMP and induction of serotonin N-acetyl transferase in rat pineal, Molec. Pharmacol., 9 (1973) 184-190. 4 Deguchi, T., and Axelrod, J., Sensitive assay for serotonin N-acetyltransferase activity in rat pineal, Analyt. Biochem., 50 (1972) 174--179. 5 Fleming, W.W., Supersensitivity in smooth muscle; introduction and historical perspective, Fed. Proc., 34 (1975) 1969--1970. 6 Gavin, J.R., Roth, J., Neville, D.M., de Meyts, P., and Buell, D.N., Insulin dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 84--88. 7 Goldberg, N.D., Haddox, M.K., Nicol, S.E., Glass, D.B., Sanford, C.H., Kuehl, F.A., and Estensen, R., Biologic regulation through opposing influences of cyclic GMP and cyclic AMP: the Yin Yang hypothesis, Advanc. Cyclic Nucleotide Res., 5 (1975) 307-330. 8 Hellman, B., Modifying actions of calcium ionophores on insulin release, Biochim. biophys. Acta (Amst.), 399 (1975) 157--169. 9 Hubbard, J.L., and Quastel, D.M.J., Micropharmacology of vertebrate neuromuscular transmission, Ann. Rev. Pharmacol., 13 (1973) 199--216. 10 Klein, D.C., and Weller, J.L., Adrenergic cAMP regulation of serotonin N-acetyltransferase and the temporal relationship of serotonin N-acetyltransferase activity to synthesis of 3H-N-acetyl serotonin and 3H-melatonin in cultured pineal, J. Pharmacol. exp. Ther., 186 (1973) 516--527. 11 Nordmann, J.J., and Currell, G.A., The mechanism of calcium ionophore-induced secretion from the rat neurohypophysis, Nature (Lond.), 253 (1975) 646--647. 12 Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K., and Marks, B.H., Blockade by ouabain or elevated potassium ion concentration of the adrenergic and cAMP-induced stimulation of pineal serotonin N-acetyl transferase activity, Molec. Pharmacol., 11 (1975) 1969--1970. 13 Rasmussen, H., Kurokawa, K., Mason, J., and Goodman, D.B.P., Cyclic AMP, calcium and cell activation. In R.V. Talmage and P.L. Munson (Eds.), Calcium, Parathyroid Hormone and the Calcitonins, Excerpta Medica, Amsterdam, 1972, pp. 492--501. 14 Rasmussen, H., Jensen, P., Lake, W., Friedmann, N., and Goodman, D.B.P., Cyclic nucleotides and cellular calcium metabolism, Advanc. Cyclic Nucleotide Res., 5 (1975) 375--394.
33
15 Romero, J.A., and Axelrod, J., Regulation of sensitivity to beta-adrenergic stimulation in induction of pineal N-acetyl transferase, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 1661--1665. 16 Romero, J.A., and Axelrod, J., Pineal beta-adrenergic receptor: regulation of sensitivity. In E. Usdin and W.E. Bunney, Jr (Eds.), Pre- and Post-Synaptic Receptors, Dekker, Inc., N e w York, 1975, pp. 265--282. 17 Schultz, G., Hardman, J.G., Schultz, K., Baird, C.E., and Sutherland, E.W., The importance of calcium ions for the regulation of c G M P levels, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 3889--3893. 18 Smith, R.J., and Ignarro, L.J., Bioregulation of lysosomal enzyme secretion from human neutrophils: roles of cGMP and calcium in stimulus-secretion coupling, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 108--112. 19 Stone, T.W., Taylor, D.E., and Bloom, F.E., cAMP and cGMP may mediate opposite neuronal responses in rat cerebral cortex, Science, 187 (1975) 845--847. 20 Zis, A.P., and Fibiger, H.C., Functional evidence fo rpostsynaptic supersensitivity of central noradrenergic receptors after denervation, Nature (Lond.), 256 (1975) 659--661.
29
INHIBITION OF THE NORADRENERGIC INDUCTION OF PINEAL NACETYLTRANSFERASE BY DIBUTYI:tYL CYCLIC GUANOSINE MONOPHOSPHATE AND BY IONOPHORE X-537A
MICHAEL WILKINSON D~partement de Physiologie, Ecole de M~decine, Universit~ de Gen~ve, 1211 Genhve 4 (Switzerland) (Received January 13th, 1976) (Accepted January 20th, 1976)
SUMMARY
Utilizing rat pineals in vitro, it can be demonstrated that dibutyryl cyclic GMP and the calcium ionophore X-537A are both able to reversibly inhibit the elevation of pineal N-acetyltransferase induced by the ~-agonists norepinephrine or isoproterenol. These results are discussed in terms of the known hyposensitivy of the pineal gland to overstimulation by ~-agonists.
Sympathetic nerves innervating the rat pineal release norepinephrine which activates serotonin N-acetyltransferase (NAT), the rate-limiting enzyme in the biosynthesis of melatonin [ 1 ]. Much evidence suggests that norepinephrine elevates NAT by first binding to a ~-adrenergic receptor which in turn stimulates the formation of pineal adenosine-3',5'-cyclic monophosphate (cAMP) [3,10]. An intriguing property of this receptor is indicated by the observation that the responsiveness of NAT to induction by ~-agonists, in vivo and in vitro, varies considerably depending on the degree of previous stimulation [ 15 ]. Thus, repeated stimulation leads to subsensitivity whilst deprivation of agonist leads to supersensitivity of the receptors. These profound but reversible differences appear quickly (~24 h) and are conveniently demonstrated using an in vitro preparation in which very low concentrations of agonist may be employed [ 15]. When higher, non-physiological concentrations are used, the differences in response are abolished. Evidence is available indicating that central catecholamine receptors also exhibit the phenomenon of supersensitivity (see ref. 20 for example); other examples include smooth muscle [ 5], and the insulin receptor [6]. The mechanisms underlying the development of postsynaptic hyper- and hyposensitivity remain unknown. However, it has been postulated that hormone-induced intracellular elevations of cAMP in various cell types may be inhibited by guanosine-3',5'-cyclic monophosphate (cGMP) [7,19].Jt was of
30 interest, therefore, to investigate if the subsensitive response of pinealocytes to ~-agonists could be reproduced by exogenous cGMP added to pineals in vitro. Further, since calcium ions are a necessary requirement for endogenous elevations of cGMP [17,18] in certain tissues, we have also looked at the effects of the calcium ionophore X-537A, a substance known [8,11] to elevate intracellular calcium ion concentrations. Male rats (Wistar), of approximately 200 g body weight, were used except where indicated. They were sacrificed between two and three hours after the beginning of the light period. Pineals were dissected and placed directly into tissue culture dishes (60 × 15 mm) containing NCTC-135 culture medium (1.5 ml; 3 pineals per dish). The medium was modified by addition of pyruvate (1 mM), MEM non-essential amino acids, glucose (500 mg%), HEPES buffer (10 mM) and fungizone (1.25 pg/ml). All tissue culture products were obtained from GIBCO. Cultures were maintained at 37°C in a water saturated atmosphere of 95% air/CO2. NAT activity was measured by the method of Deguchi and Axelrod [4]. All reagents were commercially available. X-537A was a gift from Hoffmann-La Roche. Stock solutions of X-537A were in dimethyl sulfoxide (DMSO) (5 mg/ml), which, after dilution with culture medium gave a final concentration of 10 pg/ml; (DMSO 0.1%). Control cultures contained 0.1% DMSO. The dibutyryl derivative of cGMP (DBcGMP) was used in preference to the parent compound because of its greater resistance to degradation by phosphodiesterase. Preincubation of pineals with DBcGMP (1.0 mM) for periods of 30--40 min, followed by stimulation (4 h) with norepinephrine (NE; 10 -6 M) led to a highly significant (P < 0.001) inhibition of the rise of NAT (Fig. 1A). The glands fully recovered their sensitivity after washing out the DBcGMP. Butyric acid (10 .4 M and 10 -3 M), a possible degradation product of DBcGMP, had no inhibitory effect on NE-induced NAT, i.e., NAT levels in the presence or absence of butyric acid were approximately 1500 units. NAT induction by a more potent ~-agonist,/-isoproterenol (10 -7 M), was also inhibited by DBcGMP (1.0 mM; Fig. 1D). When the catecholamines were added concurrently with the DBcGMP, no significant inhibitory response was observed. Similarly, the use of higher concentrations of NE or isoproterenol abolished the effect of the DBcGMP. NAT induction by suboptimal doses of NE or isoproterenol was also significantly (P < 0.001) but reversibly inhibited by the calcium ionophore X-537A (10 ug/ml) (Fig. 1B and 1C respectively). The results indicate that the induction of NAT in vitro by small concentrations (~<10 -6 M) of ~-agonists may be reversibly inhibited by DBcGMP or by the calcium ionophore X-537A. The characteristics of this inhibition parallel some of the known features of pineal p-receptor hyposensitivity [15], i.e., the effect is reversible and is conveniently demonstrated in vitro using suboptimal concentrations of agonist. The similar results obtained using norepinephrine or isoproterenol indicate that the effect is at a postjunctional site, since it is known that the latter is not taken up by nerve endings [16]. At the
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Fig. 1. N-acetyltransferase activity in vitro. A: effects of stimulation with 10 -6 M norepinephrine (NE) (4 h, u ) and after preincubation with 1.0 mM DBcGMP (striped column}. B: 10 -6 M NE (3 h, u) and after preincubation with X-537A (10 ug/ml, checked column). C: 10 -~ M isoprotcrenol (2 h, cross-hatched column} and after preincubation with X-537A (10 ug/ml, vertically striped column). D: 10-7 ~/[ isoproterenol (2 h, cross-hatched column} and after preincubation with 1.0 mM DBcGMP (dotted column}. N-acetyltransferase units are picomoles of N-acetyltryptamine formed per gland per hour. Control values of NAT, without stimulation, were less than 100 units. Animals used in A were 350 g body weight and in B--D, approximately 200 g. N = number of pineals. Results expressed as mean -+ standard error; *P < 0.001; **P < 0.005 (Student's t-test). p r e s e n t t i m e it is n o t possible t o d e c i d e at precisely w h i c h p o i n t , b e t w e e n t h e /]-receptor a n d N A T elevation, t h e b l o c k a d e t a k e s place. T h e e x p e r i m e n t s suggest, h o w e v e r , t h a t b o t h c a l c i u m a n d cyclic G M P m a y be m e d i a t o r s in t h e r e s p o n s e o f pineal H-receptors t o o v e r s t i m u l a t i o n . T h e e v i d e n c e t h a t c a l c i u m and c G M P are closely i n t e r r e l a t e d in cell f u n c t i o n is e x t e n s i v e (see ref. 14). F o r e x a m p l e , in r a t p a r o t i d , t h e e f f e c t o f a c a l c i u m i o n o p h o r e is t o b l o c k t h e i s o p r o t e r e n o l - i n d u c e d rise in c A M P as well as t o elevate c G M P [2]. O u r results m a y also c o m p l e m e n t t h e w o r k o f P a r f i t t et al. [12] w h o r e p o r t e d an inhibit i o n o f t h e n o r a d r e n e r g i c s t i m u l a t i o n o f pineal N A T b y o u a b a i n or b y elevated p o t a s s i u m c o n c e n t r a t i o n s . High levels o f p o t a s s i u m chloride are k n o w n t o raise c G M P [ 17 ]. O u a b a i n , o n t h e o t h e r h a n d , b y elevating intracellular s o d i u m ion c o n c e n t r a t i o n s , brings a b o u t t h e a c c u m u l a t i o n o f c a l c i u m [9,13] w h i c h parallels o u r w o r k w i t h t h e i o n o p h o r e .
32 O u r e x p e r i m e n t s leave o p e n the q u e s t i o n o f w h e t h e r the p h e n o m e n o n o f h y p e r s e n s i t i v i t y o f r e c e p t o r s is in s o m e w a y related, in an antithetical way, to h y p o s e n s i t i v i t y a n d m a y also involve calcium ions. ACKNOWLEDGEMENTS T h e a u t h o r is grateful t o Madeleine Vuillet, K.B. R u f and D. de Ziegler f o r their help. T h e w o r k was s u p p o r t e d b y a g r a n t f r o m the Swiss N a t i o n a l Science F o u n d a t i o n (No. 3 . 0 1 6 . 7 3 ) t o Prof. K.B. Ruf. REFERENCES 1 Axelrod, J., The pineal gland: a neurochemical transducer, Science, 184 (1974) 1341-1348. 2 Butcher, F.R., The role of calcium and cyclic nucleotides in a-amylase release from slices of rat parotid: studies with ionophore A-23187, Metabolism, 24 (1975) 409--418. 3 Deguchi, T., Role of the beta adrenergic receptor in the elevation of cAMP and induction of serotonin N-acetyl transferase in rat pineal, Molec. Pharmacol., 9 (1973) 184-190. 4 Deguchi, T., and Axelrod, J., Sensitive assay for serotonin N-acetyltransferase activity in rat pineal, Analyt. Biochem., 50 (1972) 174--179. 5 Fleming, W.W., Supersensitivity in smooth muscle; introduction and historical perspective, Fed. Proc., 34 (1975) 1969--1970. 6 Gavin, J.R., Roth, J., Neville, D.M., de Meyts, P., and Buell, D.N., Insulin dependent regulation of insulin receptor concentrations: a direct demonstration in cell culture, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 84--88. 7 Goldberg, N.D., Haddox, M.K., Nicol, S.E., Glass, D.B., Sanford, C.H., Kuehl, F.A., and Estensen, R., Biologic regulation through opposing influences of cyclic GMP and cyclic AMP: the Yin Yang hypothesis, Advanc. Cyclic Nucleotide Res., 5 (1975) 307-330. 8 Hellman, B., Modifying actions of calcium ionophores on insulin release, Biochim. biophys. Acta (Amst.), 399 (1975) 157--169. 9 Hubbard, J.L., and Quastel, D.M.J., Micropharmacology of vertebrate neuromuscular transmission, Ann. Rev. Pharmacol., 13 (1973) 199--216. 10 Klein, D.C., and Weller, J.L., Adrenergic cAMP regulation of serotonin N-acetyltransferase and the temporal relationship of serotonin N-acetyltransferase activity to synthesis of 3H-N-acetyl serotonin and 3H-melatonin in cultured pineal, J. Pharmacol. exp. Ther., 186 (1973) 516--527. 11 Nordmann, J.J., and Currell, G.A., The mechanism of calcium ionophore-induced secretion from the rat neurohypophysis, Nature (Lond.), 253 (1975) 646--647. 12 Parfitt, A., Weller, J.L., Klein, D.C., Sakai, K.K., and Marks, B.H., Blockade by ouabain or elevated potassium ion concentration of the adrenergic and cAMP-induced stimulation of pineal serotonin N-acetyl transferase activity, Molec. Pharmacol., 11 (1975) 1969--1970. 13 Rasmussen, H., Kurokawa, K., Mason, J., and Goodman, D.B.P., Cyclic AMP, calcium and cell activation. In R.V. Talmage and P.L. Munson (Eds.), Calcium, Parathyroid Hormone and the Calcitonins, Excerpta Medica, Amsterdam, 1972, pp. 492--501. 14 Rasmussen, H., Jensen, P., Lake, W., Friedmann, N., and Goodman, D.B.P., Cyclic nucleotides and cellular calcium metabolism, Advanc. Cyclic Nucleotide Res., 5 (1975) 375--394.
33
15 Romero, J.A., and Axelrod, J., Regulation of sensitivity to beta-adrenergic stimulation in induction of pineal N-acetyl transferase, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 1661--1665. 16 Romero, J.A., and Axelrod, J., Pineal beta-adrenergic receptor: regulation of sensitivity. In E. Usdin and W.E. Bunney, Jr (Eds.), Pre- and Post-Synaptic Receptors, Dekker, Inc., N e w York, 1975, pp. 265--282. 17 Schultz, G., Hardman, J.G., Schultz, K., Baird, C.E., and Sutherland, E.W., The importance of calcium ions for the regulation of c G M P levels, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 3889--3893. 18 Smith, R.J., and Ignarro, L.J., Bioregulation of lysosomal enzyme secretion from human neutrophils: roles of cGMP and calcium in stimulus-secretion coupling, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 108--112. 19 Stone, T.W., Taylor, D.E., and Bloom, F.E., cAMP and cGMP may mediate opposite neuronal responses in rat cerebral cortex, Science, 187 (1975) 845--847. 20 Zis, A.P., and Fibiger, H.C., Functional evidence fo rpostsynaptic supersensitivity of central noradrenergic receptors after denervation, Nature (Lond.), 256 (1975) 659--661.