Molecular and Cellular Endocrinology, 16 (1979) 147-156 0 Else~er~North-Ho~and Scientific Publishers, Ltd.
147
REVIEW
INHIBITiON OF ADE~ATE NEUROT~NSMr~ERS
CYCLASE BY HO~ONES
AND
Karl H. JAKOBS Pharmakologisches Institut der Universittit Heidelberg, Im Neuenheimer Feld 366.0-6900 Heidelberg [Federal Republic of Germany Received 16 August 1979
RECEPTOR-MEDIATED
ST~ULATION
OF ADE~ATE
CYCLASE
A large number of hormones and neurotransmitters stimulate the membranebound adenylate cyclase (EC 4.6.1 .l> in the course of eliciting their cellular responses. Hormonal factors that by interaction with their specitic receptors activate the enzyme include polypeptide hormones such as glucagon, ACTH, antidiuretic hormone and luteinizing hormone and locally acting hormones and neurotransmitters such as catecholamines (by fl-adrenergic receptors), histamine (by Hzreceptors), dopamine (e.g., in the caudate nucleus), prostaglandins (e.g., PGE, and PGIZ in platelets) and adenosine (e.g., in platelets and lymphocytes). It is now generally accepted that guanine nucleotides are involved in these receptor-mediated adenylate cyclase stimulations. Guanine nucleotides have been shown to affect the interaction of hormonal agonists with their receptors. GTP, GDP and stable GTP analogues have been found to reduce the apparent affinities of glucagon and prostaglandin E, to their receptors (Rodbell et al., 1971; Lin et al., 1977; Lefkowitz et al., 1977), and similarly, the apparent affinity of /3-adrenergic receptors towards fl-adrenergic agonists but not towards P-adrenergic antagonists has been shown to be reduced by guanine nucleotides (Maguire et al., 1976; Lefkowitz et al., 1976; Ross et al., 1977; W~li~s and Lefkowitz, 1978). This reduction of the apparent receptor affinity towards hormonal agonists has been attributed to an increased agonist dissociation rate induced by the guanine nucleotides. Aside from a role of these nucleotides in the receptor binding of hormones and neurotransmitters, guanine nucleotides function as intracellular effecters, which interact with a distinct regulatory moiety separate from the hormone receptor and the catalytic subunit of the adenylate cyclase system (Pfeuffer, 1977, 1979) and activate the enzyme synergistically with the hormones (for refs. see: Birnbaumer, 1977; Rodbell, 1978; Levitzki and Helmreich, 1979). Stimulatory hormones and neurotransmitters appear to increase adenylate cyclase activity by shifting the equilibrium of the enzyme from a low activity state, in which GDP is bound to the
Karl H. Jakobs
148
plasma
membrane
I
I
Fig. 1. Possible regulation of hormone-induced adenylate cyclase stimulation and jnhibition by guanine nucleotides through different functional sites and by sodium ions. H, hormone; R, receptor; LYand p, o(- and p-adrenergic agonist, each representing one example of stimulatory and inhibitory hormones, resp.; Nr , N2. NJ, functional guanine nucleotide sites; AC, catalytic subunit of the adenylate cyclase.
regulatory moiety, to a GTP-bound, high activity state (Cassel and Selinger, 1978). The question whether or not the receptor-linked nucleotide site (Nr in Fig. 1) is different from the adenylate cyclase-activating site (N2 in Fig. 1) is still undecided (Welton et al., 1977; Williams and Lefkowitz, 1978). Activation of the adenylate cyclase results in formation of an increased activity of a GTPase associated with the adenylate cyclase system (Cassel and Selinger, 1976), which appears to terminate the hormonal activation by hydrolysis of the bound GTP to GDP and Pi. That the guanine nucleotide binding protein is identical with the GTPase has been made likely by findings on the action of cholera toxin, which has been demonstrated in avian erythrocyte membranes to cause GTPase inhibition (Cassel and Selinger, 1977) and ADP-ribosylation of the GTP-binding protein (Cassel and Pfeuffer, 1978), but the final proof with complete purification of the protein has still not been presented.
RECEPTOR-MEDIATED
DECREASES IN CYCLIC AMP LEVELS
Aside from the hormonal agents, the receptors of which are coupled in a stimulatory manner to the adenylate cyclase, a large number of hormones and neurotransmitters has been found to lower the cyclic AMP level or to attenuate its increase due to stimulatory agents in intact cells and tissues. Such effects have been described, e.g., for ~-adrenergic agonists in human platelets (Salzman and Neri, 1969; Robison et al., 1971) and hamster fat cells (Hittelman and Butcher, 1973; Rosak and Hittelman, 1977), for adenosine and prostaglandin Er and E2 in rat fat cells (Fain, 1973), for muscarinic cholinergic agonists in rat myocardium (George et al., 1970), dog thyroid (Dumont et al., 1978) and rat parotis (Butcher et al., 1976), for opiates in neuronal cells (Collier and Roy, 1974; Sharma et al., 1975),
Inhibition
of adenylate cyclase
149
for thyroid hormones in mouse thyroid (Yu et al., 1976), for insulin in rat fat and liver cells (Butcher et al., 1968; Kuo and DeRenzo, 1969), for epidermal growth factor in tibroblasts (Anderson et al., 1979) and for somatostatin in rat anterior pituitary glands (Borgeat et al., 1974). The data obtained in intact cells suggested that the enzyme affected by these hormones and neurotransmitters is mainly the adenylate cyclase rather than cyclic nucleotide phosphodiesterase(s). Accordingly, it was proposed (Robison et al., 1967) that stimulatory (e.g., /.I-adrenergic) and inhibitory (e.g., a-adrenergic) receptors are coupled in an opposing manner to the adenylate cyclase. While occupancy of P-adrenergic receptors by the respective agonists causes adenylate cyclase stimulation, interaction of a-adrenergic agonists with their receptors should decrease the enzyme activity. Only insulin has been shown to increase the activity of a phosphodiesterase in rat fat cells (Loten and Sneyd, 1970) and liver (Loten et al., 1978). In dog thyroid, acetylcholine appears to increase the disappearance of cyclic AMP, possibly by increased degradation (Dumont et al., 1978). Except for an early communication, which showed inhibition of adenylate cyclase in cardiac homogenates by the choline@ agonist, carbachol (Murad et al., 1962), and for several occasional observations of insulin-induced inhibitions (Hepp, 197 1; Illiano and Cuatrecasas, 1972; Torres et al., 1978; Kiss, 1979), no clear evidence for hormone-induced adenylate cyclase inhibition in cell-free preparations had been presented until recently.
RECEPTOR-MEDIATED
INHIBITION
OF ADENYLATE
CYCLASE
Inhibition of platelet adenylate cyclase by cx-adrenergicagonists The first adenylate cyclase system to be studied extensively with regard to hormonal inhibition has been that in human platelets, where an o-adrenergic receptor-mediated decrease in cyclic AMP levels has been demonstrated (Salzman and Neri, 1969; Robison et al., 1971). In platelet lysates, epinephrine and norepinephrine decreased adenylate cyclase activity by about 50% (Jakobs et al., 1976). The catecholamine-induced enzyme inhibition occurred without an apparent lag phase and was immediately reversed by o-adrenergic blocking agents. There was a close correlation between a-adrenergic receptor-mediated adenylate cyclase inhibition and induction of platelet aggregation with regard to species specificity and potencies of a-adrenergic agonists and antagonists (Jakobs et al., 1978a; Lasch and Jakobs, 1979). This close correlation was further supported by binding studies on the platelet a-adrenergic receptor (Alexander et al., 1978; Newman et al., 1978; Jakobs and Rauschek, 1978). a-Adrenergic agonists inhibited the enzyme both in the absence and presence of adenylate cyclase stimulants such as prostaglandin El or adenosine (Jakobs et al., 1979b). Thereby, the Vmax of the enzyme was reduced without apparent changes in the affinities for its substrate, Mg . ATP, and the required free divalent cation (Mg’+) (Jakobs et al., 1978b).
150
Karl H. Jakobs
Factors required for adenylate cyclase inhibition In studies on the mechanism involved, it was found that the presence of GTP is absolutely necessary for inhibition of platelet adenylate cyclase by a-adrenergic agonists (Jakobs et al., 1978~). In the absence or presence of prostaglandin El, a-adrenergic agonists in a GTP-dependent manner reduced the enzyme activity. In the presence of cholera toxin, the GTP-induced activation was largely reversed by epinephrine. In contrast, stable GTP analogues such as guanylyl-5’-imidodiphosphate and guanosine-5’-O-(3-thiotriphosphate), which cause a persistently increased enzyme activity (Birnbaumer, 1977; Rodbell, 1978; Levitzki and Helmreich, 1979), reversed or prevented the hormone-induced adenylate cyclase inhibition (Jakobs, 1979). In studies on the inhibition of hamster fat cell adenylate cyclase by ol-adrenergic agonists and prostaglandins (E,, Es), an additional factor required besides GTP has been found. In addition to GTP, sodium ions were required for effective coupling of inhibitory receptors to the adenylate cyclase (Aktories et al., 1979a, b). Properties of hormone-induced adenylate cyclase inhibition In several systems besides human platelets and hamster fat cells, inhibition of adenylate cyclase as a consequence of interactions of hormones or neurotransmitters with their specific receptors has now been described. Such receptormediated decreases in cyclic AMP synthesis have been reported for ar-adrenergic agonists in rabbit platelets (Jakobs et al., 1976; Tsai and Lefkowitz, 1978) for adenosine in rat (Londos et al., 1978) and prostaglandin Es in human (Kather and Simon, 1979) fat cells, for a-adrenergic and choline@ agonists and opiates in neuroblastoma X glioma hybrid cells (Sabol and Nirenberg, 1979; Nathanson et al., 1978; Sharma et al., 1975; Blume and Boone, 1979), for cholinergic agonists in canine (Watanabe et al., 1978) and rabbit myocardium (Aktories and Jakobs, 1979; Jakobs et al., 1979a) and rat parotis (Oron et al., 1978) for thyroid hormones in bovine thyroid (Friedman et al., 1977) for a-adrenergic agonists (K.H.J. and R.A. Johnson, unpublished results) and somatostatin (Vinicor et al., 1977) in rat liver and for dopamine (DeCamilli et al., 1979) and somatostatin (Heindel et al., 1978) in pituitary adenoma cells. These hormone- or neurotransmitter-induced inhibitions of adenylate cyclase share several common properties. (a) The maximal inhibitions observed ranged between 40 and 60%. The inhibitory hormonal factors reduced the enzyme activities both in the absence and presence of stimulating hormones. (b) The inhibition by the agonists occurred without an apparent lag phase and was immediately reversed by antagonists (so far available). There was a close pharmacological correlation between data obtained studying receptor binding or physiological response and adenylate cyclase inhibition with regard to the potency order of hormonal agonists and antagonists, although usually somewhat higher concentrations were required in the cell-free system for half-maximal adenylate cyclase
Inhibition of adenylate cyclase
Table 1 Inhibitions systems
151
of adenylate cyclase by hormones and neurotransmitters:
Cell type
Hormone or neurotransmitter
requirements
Requirement a) for GTP
Na+
Human platelets Rabbit platelets N X G hybrid cells b)
o -adrenergic agonists a-adrenergic agonists cu-adrenergic agonists cholinergic agonists opiates
+ + + ? +
? ? ? ? +
Rat fat cells
adenosine insulin a-adrenergic agonists prostaglandin E 1, E2 prostaglandin E2
+ ? + + +
? ? + + ?
Rabbit myocardium Canine myocardium Rat liver
cholinergic agonists cholinergic agonists ol-adrenergic agonists insulin somatostatin
+ + + ? ?
+ ? + ? ?
Rat parotis Pituitary adenoma
cholinergic agonists dopamine somatostatin thyroid hormones (Ts, T4)
? ? ? (+)
? ? ? ?
Hamster fat cells Human fat cells
Bovine thyroid
in cell-free
a) +, requirement has been reported; ?, requirement for GTP or Na+ has not been studied or reported; (+), inhibition was increased by GTP. For refs. see text. b) N X G hybrid cells, neuroblastoma X glioma hybrid cells.
inhibition than for half-maximal physiological response or receptor occupancy. (c) Most of these forms of adenylate cyclase inhibition required the presence of GTP (Table 1). This fact resembles the GTP requirement for adenylate cyclase stimulation by hormones and neurotransmitters with the obvious difference that about 5- to lo-fold higher concentrations of GTP were required for half-maximal inhibition than for stimulation. So far studied, the enzyme inhibition was reversed or prevented by stable GTP analogues but not by cholera toxin plus GTP, both of which by different mechanisms can cause persistent adenylate cyclase activation. (d) Finally, as recently found in hamster fat cells for cr-adrenergic agonists and prostaglandins (Aktories et al., 1979a, b), in neuroblastoma X glioma hybrid cells for opiates (Blume and Boone, 1979) and in rat liver for cw-adrenergic agonists (K.H.J. and R.A. Johnson, unpublished results), sodium ions were required for effective coupling of inhibitory receptors to the adenylate cyclase, or these ions at least facilitated the hormone-induced inhibition, as found in rabbit myocardium for cholinergic agonists (Jakobs et al., 1979a).
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Karl H. Jakobs
Mechanisms possibly involved in hormone-induced adenylate cyclase inhibition
The similarities found in different systems suggest that a common mechanism is involved in adenylate cyclase inhibition by various hormones and neurotransmitters and that GTP and sodium ions play essential roles in the transduction process. The effects of GTP and sodium ions may be due to interactions at the receptor site. Sodium ions have been found to reduce the apparent affinity of agonists towards the receptors that appear to be involved in adenylate cyclase inhibition, as described for a-adrenergic (Tsai and Lefkowitz, 1978; U’Prichard and Snyder, 1978; Glossmann and Presek, 1979) and opiate receptors (Pert and Snyder, 1974), or to be required for the elicited physiological responses (Keryer et al., 1979). Similar effects of sodium ions have also been reported for receptors (P-adrenergic) involved in adenylate cyclase stimulation (U’Prichard et al., 1978). However, whereas sodium ions appear to be involved in hormone-induced adenylate cyclase inhibition, no such effects have been described for the hormone-dependent stimulation of the enzyme. Guanine nucleotides, which appear to be involved in adenylate cyclase stimulation and inhibition, cause a decrease in apparent receptor affinity both towards adenylate cyclase-stimulating hormones (Birnbaumer, 1977; Rodbell, 1978; Levitzki and Helmreich, 1979) and towards hormonal agonists that can induce adenylate cyclase inhibition, as described for a-adrenergic agonists in human platelets (Tsai and Lefkowitz, 1979) and rat liver (El-Refai et al., 1979) for cholinergic agonists in rat myocardium (Berrie et al., 1979) and for opiates in neuroblastoma X glioma hybrid cells (Blume, 1978). The decreases in apparent receptor affinities induced by sodium ions and guanine nucleotides appear to be due to an increased agonist dissociation rate. The fast dissociation of the agonist from the receptor may reform free receptor that can reenter the reaction sequence. The findings in myocardium that the apparent affinities of both /_?-adrenergic and cholinergic receptors towards their respective agonists were decreased by guanine nucleotides and that the cholinergic agonist, metacholine, antagonized the guanine nucleotideinduced decrease in Padrenergic receptor apparent affinity for isoproterenol (Watanabe et al., 1978) indicate that the effects of the guanine nucleotides on both types of receptors are mediated by a common regulatory component (Nr in Fig. 1). The findings further suggest that the adenylate cyclase inhibition might be due to such an antagonism at the nucleotide site controlling the receptor affinity. However, in adenylate cyclase studies, out of various guanine nucleotides only GTP caused an effective inhibitory coupling and stable GTP analogues prevented the inhibition, whereas in the binding studies the efficacies of GTP and its stable analogues were not different. Therefore, it seems conceivable that the GTP-dependent, hormone-induced adenylate cyclase inhibition is mediated at least partially if not completely by a nucleotide site separate from that controlling receptor affinity. There are several possible explanations for the findings that adenylate cyclase inhibition by hormones and neurotransmitters requires GTP and is prevented by
Inhibition of adenylate cyclase
153
stable GTP analogues but not by cholera toxin plus GTP. A hormonally-stimulated, increased hydrolysis of GTP by an increased GTPase activity, which is reduced by cholera toxin (Cassel and Selinger, 1977) may be involved. Stable GTP analogues would prevent this reaction, leading to a low activity state of the adenylate cyclase, and the increase in GTPase activity should overcome the cholera toxin-induced inhibition. Such a mechanism, however, would not explain why adenylate cyclase inhibition by hormones required GTP at higher concentrations than hormonal stimulation. It is rather feasible that the hormone-induced inhibition of adenylate cyclase is mediated by an up to now unidentified, separate inhibitory nucleotide site (Na in Fig. 1) (Yamamura et al., 1977; Cooper et al., 1979) which in equilibrium with the nucleotide site responsible for adenylate cyclase activation (N2 in Fig. 1) regulates enzyme activity. Such a regulatory site responsible for hormoneinduced adenylate cyclase inhibition may involve a phosphotransferase reaction, in which GTP but not stable GTP analogues can serve as a substrate. Although the present findings indicate functional differences in the proposed guanine nucleotide sites they do not exclude that one or the other of these functional sites are identical proteins with different properties when coupled to different components of the adenylate cyclase system.
CONCLUSIONS Aside from the questions concerning the mode of action of GTP and especially that of sodium ions in hormone-induced adenylate cyclase inhibition, there appear to be additional unresolved problems in this field, e.g., the mechanism by which insulin may decrease adenylate cyclase activity. Calcium ions and, thereby, the calcium-dependent regulator protein (CDR or calmondulin), adenosine and redoxregulated processes may also be involved in hormone-induced adenylate cyclase inhibitions, although there is so far no clear evidence for such interactions. Further studies on additional systems and with isolated components of the adenylate cyclase system are required to elucidate the regulatory processes leading to hormone-induced adenylate cyclase inhibition. Such studies may also finally give an insight into the question of the significance of decreased cyclic AMP synthesis besides other observed cellular events (e.g., increases in cytoplasmic calcium ions cyclic GMP level) for the overall concentration, triphosphoinositol turnover, physiological response of the cell to the hormonal stimulus.
ACKNOWLEDGEMENTS The author’s studies reported Deutsche Forschungsgemeinschaft.
herein
were
supported
by grants
from
the
154
Karl H. Jakobs
REFERENCES Aktories, K., and Jakobs, K.H. (1979) Hoppe-Seyler’s Z. Physiol. Chem. 360,223. Aktories, K., Jakobs, K.H., and Schultz, G. (1979a) Naunyn-Schiedeberg’s Arch. Pharmacol. 308, Suppl., R15. Aktories, K., Schultz, G., and Jakobs, K.H. (1979b) FEBS Lett. in press. Alexander, R.W., Cooper, B., and Handin, R.I. (1978) J. Clin. Invest. 61, 1136-1144. Anderson, W.B., Gallo, M., Wilson, J., Lovelace, E., and Pastan, I. (1979) FEBS Lett. 102, 329-332. Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.V., and Hulme, E.C. (1979) Biochem. Biophys. Res. Commun. 87,1000-1005. Birnbaumer, L. (1977) in: Receptors and Hormone Action, Vol. 1, Eds.: B.W. O’Malley and L. Birnbaumer (Academic Press, New York) pp. 485-547. Blume, A.J. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 75, 1713-1717. Blume, A.J., and Boone, G. (1979) Fed. Proc. 38, 628. Borgeat, P., Labrie, F., Drouin, J., Belanger, A., Immer, H., Sestanj, K., Nelson, V., Gotz, M., Schally, A.V., Coy, D.H., and Coy, E.J. (1974) Biochem. Biophys. Res. Commun. 56, 1052-1059. Butcher, R.W., Baird, C.E., and Sutherland, E.W. (1968) J. Biol. Chem. 243, 1705-1712. Butcher, F.R., Rudich, L., Emler, C., and Nemerovski, M. (1976) Mol. Pharmacol. 12, 862870. Cassel, D., and Pfeuffer, T. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 75, 2669-2673. Cassel, D., and Selinger, Z. (1976) Biochim. Biophys. Acta 452,538-551 Cassel, D., and Selinger, Z. (1977) Proc. Natl. Acad. Sci. (U.S.A.) 74, 3307-3311. Cassel, D., and Selinger, Z. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 75, 4155-4159. Collier, H.O.J., and Roy, A.C. (1974) Nature (London) 248, 24-27. Cooper, D.F., Schlegel, W., Lin, M.C., and Rodbell, M. (1979) Fed. Proc. 38,602. DeCamilli, P., Macconi, D., and Spada, A. (1979) Nature (London) 278, 252-254. Dumont, J.E., Boeynaems, J.M., Decoster, C., Erneux, C., Lamy, F., Lecocq, R., Mockel, J., Unger, J., and Van Sande, J. (1978) in: Advances in Cyclic Nucleotide Research, Vol. 9, Eds.: W.J. George and L.J. Ignarro (Raven, New York) pp. 723-734. El-Refai, M.F., Blackmore, P.F., and Extort, J.H. (1979) J. Biol. Chem. 254,4375-4386. Fain, J.N. (1973) Pharmacol. Rev. 25,67-118. Friedman, Y., Lang, M., and Burke, G. (1977) Endocrinology 101,858-868. George, W.J., Poison, J.B., O’Toole, A.G., and Goldberg, N.D. (1970) Proc. Natl. Acad. Sci. (U.S.A.) 66, 398-403. Glossmann, H., and Presek, P. (1979) NaunynSchmideberg’s Arch. Pharmacol. 306,67-73. Heindel, J.J., Williams, E., Robison, G.A., and Strada, S.S. (1978) J. Cycl. Nucl. Res. 4,453462. Hepp, K.D. (1971) FEBS Lett. 12,263-266. Hittelman, K.J., and Butcher, R.W. (1973) Biochim. Biophys. Acta 316,403-410. Illiano, G., and Cuatrecasas, P. (1972) Science 175,906-908. Jakobs, K.H. (1979) in: Proceedings of the 12th FEBS Meeting, Vol. 54: Cyclic Nucleotides and Protein Phosphorylation in Cell Regulation, Eds.: E.-G. Krause, L. Pinna and A. Wollenberger (Pergamon, Oxford) pp. 1 l-20. Jakobs, K.H., and Rauschek, R. (1978) Klin. Wschr. 56 (Suppl. I), 139-145. Jakobs, K.H., Saur, W., and Schultz, G. (1976) J. Cycl. Nucl. Res. 2, 381-392. Jakobs, K.H., Saur, W., and Schultz, G. (1978a) Naunyn-Schmiedeberg’s Arch. Pharmacol. 302, 285-291. Jakobs, K.H., Saur, W., and Schultz, G. (1978b) Mol. Pharmacol. 14,1073-1078. Jakobs, K.H., Saur, W., and Schultz, G. (1978c) FEBS Lett. 85,167-170.
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Jakobs, K.H., Aktories, K., and Schultz, G. (1979a) Naunyn-Schmiedeberg’s Arch. Pharmacol., in press. Jakobs, K.H., Saur, W., and Johnson, R.A. (1979b) Biochim. Biophys. Acta 583,409-421. Kather, H., and Simon, B. (1979) J. Clin. Invest. 64,609-612. Keryer, G., Herman, G., and Rossignol, B. (1979) FEBS Lett. 102,4-8. Kiss, Z. (1979) Eur. J. Biochem. 95,607-611. Kuo, F.J., and DeRenz0,C.E. (1969) J. Biol. Chem. 244,2252-2260. Lasch, P., and Jakobs, K.H. (1979) Naunyn-Schmiedeberg’s Arch. Pharmacol. 306, 119-125. Lefkowitz, R.J., Mullikin, D., and Caron, M.G. (1976) J. Biol. Chem. 251,4686-4692. Lefkowitz, R.J., Mullikin, D., Wood, C.L., Gore, T.B., and Mukherjee, C. (1977) J. Biol. Chem. 252,5295-5303. Levitzki, A., and Helmreich, E.J.M. (1979) FEBS Lett. 101, 213-219. Lin, M.C., Nicosia, S., Lad, P.M., and Rodbell, M. (1977) J. Biol. Chem. 252, 2790-2792. Londos, C., Cooper, D.M.F., Schlegel, W., and Rodbell, M. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 75,5362-5366. Loten, E.G., and Sneyd, J.G.T. (1970) Biochem. J. 120, 187-193. Loten, E.G., Assimacopoulos-Jeannet, F.D., Exton, J.H., and Park, C.R. (1978) J. Biol. Chem. 253,746-757. Maguire, M.E., Arsdale, P.M., and Gilman, A.G. (1976) Mol. Pharmacol. 12, 335-339. Murad, F., Chi, Y.-M., Rail, T.W., and Sutherland, E.W. (1962) J. Biol. Chem. 237, 1233-1238. Nathanson, N.M., Klein, W.L., and Nirenberg, M. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 75, 1788-1791. Newman, K.D., Williams, L.T., Bishopric, N.H., and Lefkowitz, R.J. (1978) J. Clin. Invest. 61, 395-402. Oron, Y., Kellogg, J., and Larner, J. (1978) FEBS Lett. 94, 331-334. Pert, C.B., and Snyder, S.H. (1974) Mol. Pharmacol. 10, 868-879. Pfeuffer, T. (1977) J. Biol. Chem. 252, 7224-7234. Pfeuffer, T. (1979) FEBS Lett. 101,85-89. Robison, G.A., Butcher, R.W., and Sutherland, E.W. (1967) Ann. N.Y. Acad. Sci. 139, 703723. Robison, G.A., Arnold, A., and Hartmann, R.C. (1971) Pharmacol. Res. Commun. 1, 325-332. Rodbell, M. (1978) in: Molecular Biology and Pharmacology of Cyclic Nucleotides, Eds.: G. Folco and R. Paoletti (Elsevier/North-Holland, Amsterdam) pp. l- 12. Rodbell, M., Krans, H.M.J., Pohl, S.L., and Birnbaumer, L. (1971) J. Biol. Chem. 246, 18721876. Rosak, C., and Hittelman, K.J. (1977) Biochim. Biophys. Acta 496,458-474. Ross, EM., Maguire, M.E., Sturgill, T.W., Biltonen, R.L., and Gilman, A.G. (1977) J. Biol. Chem. 252,5761-5775. Sabol, S.L., and Nirenberg, M. (1979) J. Biol. Chem. 254, 1913-1920. Salzman, E.W., and Neri, L.L. (1969) Nature (London) 224,609-610. Sharma, W.K., Nirenberg, M., and Klee, W.A. (1975) Proc. Natl. Acad. Sci. (U.S.A.) 72, 590594. Torres, H.N., Flawii, M.M., Hernaez, L., and Cuatrecasas, P. (1978) J. Membrane Biol. 43, l-18. Tsai, B.S., and Lefkowitz, R.J. (1978) Mol. Pharmacol. 14,540-548. Tsai, B.S., and Lefkowitz, R.J. (1979) Mol. Pharmacol. 16, 61-68. U’Prichard, D.C., and Snyder, S.H. (1978) 3. Biol. Chem. 253, 3444-3452. UPrichard, DC., Bylund, D.B., and Snyder, S.H. (1978) J. Biol. Chem. 253,5090-5102. Vinicor, F., Higdon, G., and Clark, Jr., C.M. (1977) Endocrinology 101, 1071-1077. Watanabe, A.M., McConnaughey, M.M., Strawbridge, R.A., Fleming J.W., Jones, L.R., and Besch, Jr., H.R. (1978) J. Biol. Chem. 253,4833-4836. Welton, A.F., Lad, P.M., Newby, A.C., Yamamura, H., Nicosia, S., and Rodbell, M. (1977) J.
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Biol. Chem. 252,5947-5950. Williams, L.T., and Lefkowitz, R.J. (1978) in: Receptor Binding Studies in Adrenergic Pharmacology (Raven, New York), pp. ill--126. Yamamura, H., Lad, P.M., and Rodbell, M. (1977) J. Biol. Chem. 252, 7964-7966. Yu, S., Friedman, Y., Richman, R., and Burke, G. (1976) J. Clin. Invest. 57, 745-755.
147
REVIEW
INHIBITiON OF ADE~ATE NEUROT~NSMr~ERS
CYCLASE BY HO~ONES
AND
Karl H. JAKOBS Pharmakologisches Institut der Universittit Heidelberg, Im Neuenheimer Feld 366.0-6900 Heidelberg [Federal Republic of Germany Received 16 August 1979
RECEPTOR-MEDIATED
ST~ULATION
OF ADE~ATE
CYCLASE
A large number of hormones and neurotransmitters stimulate the membranebound adenylate cyclase (EC 4.6.1 .l> in the course of eliciting their cellular responses. Hormonal factors that by interaction with their specitic receptors activate the enzyme include polypeptide hormones such as glucagon, ACTH, antidiuretic hormone and luteinizing hormone and locally acting hormones and neurotransmitters such as catecholamines (by fl-adrenergic receptors), histamine (by Hzreceptors), dopamine (e.g., in the caudate nucleus), prostaglandins (e.g., PGE, and PGIZ in platelets) and adenosine (e.g., in platelets and lymphocytes). It is now generally accepted that guanine nucleotides are involved in these receptor-mediated adenylate cyclase stimulations. Guanine nucleotides have been shown to affect the interaction of hormonal agonists with their receptors. GTP, GDP and stable GTP analogues have been found to reduce the apparent affinities of glucagon and prostaglandin E, to their receptors (Rodbell et al., 1971; Lin et al., 1977; Lefkowitz et al., 1977), and similarly, the apparent affinity of /3-adrenergic receptors towards fl-adrenergic agonists but not towards P-adrenergic antagonists has been shown to be reduced by guanine nucleotides (Maguire et al., 1976; Lefkowitz et al., 1976; Ross et al., 1977; W~li~s and Lefkowitz, 1978). This reduction of the apparent receptor affinity towards hormonal agonists has been attributed to an increased agonist dissociation rate induced by the guanine nucleotides. Aside from a role of these nucleotides in the receptor binding of hormones and neurotransmitters, guanine nucleotides function as intracellular effecters, which interact with a distinct regulatory moiety separate from the hormone receptor and the catalytic subunit of the adenylate cyclase system (Pfeuffer, 1977, 1979) and activate the enzyme synergistically with the hormones (for refs. see: Birnbaumer, 1977; Rodbell, 1978; Levitzki and Helmreich, 1979). Stimulatory hormones and neurotransmitters appear to increase adenylate cyclase activity by shifting the equilibrium of the enzyme from a low activity state, in which GDP is bound to the
Karl H. Jakobs
148
plasma
membrane
I
I
Fig. 1. Possible regulation of hormone-induced adenylate cyclase stimulation and jnhibition by guanine nucleotides through different functional sites and by sodium ions. H, hormone; R, receptor; LYand p, o(- and p-adrenergic agonist, each representing one example of stimulatory and inhibitory hormones, resp.; Nr , N2. NJ, functional guanine nucleotide sites; AC, catalytic subunit of the adenylate cyclase.
regulatory moiety, to a GTP-bound, high activity state (Cassel and Selinger, 1978). The question whether or not the receptor-linked nucleotide site (Nr in Fig. 1) is different from the adenylate cyclase-activating site (N2 in Fig. 1) is still undecided (Welton et al., 1977; Williams and Lefkowitz, 1978). Activation of the adenylate cyclase results in formation of an increased activity of a GTPase associated with the adenylate cyclase system (Cassel and Selinger, 1976), which appears to terminate the hormonal activation by hydrolysis of the bound GTP to GDP and Pi. That the guanine nucleotide binding protein is identical with the GTPase has been made likely by findings on the action of cholera toxin, which has been demonstrated in avian erythrocyte membranes to cause GTPase inhibition (Cassel and Selinger, 1977) and ADP-ribosylation of the GTP-binding protein (Cassel and Pfeuffer, 1978), but the final proof with complete purification of the protein has still not been presented.
RECEPTOR-MEDIATED
DECREASES IN CYCLIC AMP LEVELS
Aside from the hormonal agents, the receptors of which are coupled in a stimulatory manner to the adenylate cyclase, a large number of hormones and neurotransmitters has been found to lower the cyclic AMP level or to attenuate its increase due to stimulatory agents in intact cells and tissues. Such effects have been described, e.g., for ~-adrenergic agonists in human platelets (Salzman and Neri, 1969; Robison et al., 1971) and hamster fat cells (Hittelman and Butcher, 1973; Rosak and Hittelman, 1977), for adenosine and prostaglandin Er and E2 in rat fat cells (Fain, 1973), for muscarinic cholinergic agonists in rat myocardium (George et al., 1970), dog thyroid (Dumont et al., 1978) and rat parotis (Butcher et al., 1976), for opiates in neuronal cells (Collier and Roy, 1974; Sharma et al., 1975),
Inhibition
of adenylate cyclase
149
for thyroid hormones in mouse thyroid (Yu et al., 1976), for insulin in rat fat and liver cells (Butcher et al., 1968; Kuo and DeRenzo, 1969), for epidermal growth factor in tibroblasts (Anderson et al., 1979) and for somatostatin in rat anterior pituitary glands (Borgeat et al., 1974). The data obtained in intact cells suggested that the enzyme affected by these hormones and neurotransmitters is mainly the adenylate cyclase rather than cyclic nucleotide phosphodiesterase(s). Accordingly, it was proposed (Robison et al., 1967) that stimulatory (e.g., /.I-adrenergic) and inhibitory (e.g., a-adrenergic) receptors are coupled in an opposing manner to the adenylate cyclase. While occupancy of P-adrenergic receptors by the respective agonists causes adenylate cyclase stimulation, interaction of a-adrenergic agonists with their receptors should decrease the enzyme activity. Only insulin has been shown to increase the activity of a phosphodiesterase in rat fat cells (Loten and Sneyd, 1970) and liver (Loten et al., 1978). In dog thyroid, acetylcholine appears to increase the disappearance of cyclic AMP, possibly by increased degradation (Dumont et al., 1978). Except for an early communication, which showed inhibition of adenylate cyclase in cardiac homogenates by the choline@ agonist, carbachol (Murad et al., 1962), and for several occasional observations of insulin-induced inhibitions (Hepp, 197 1; Illiano and Cuatrecasas, 1972; Torres et al., 1978; Kiss, 1979), no clear evidence for hormone-induced adenylate cyclase inhibition in cell-free preparations had been presented until recently.
RECEPTOR-MEDIATED
INHIBITION
OF ADENYLATE
CYCLASE
Inhibition of platelet adenylate cyclase by cx-adrenergicagonists The first adenylate cyclase system to be studied extensively with regard to hormonal inhibition has been that in human platelets, where an o-adrenergic receptor-mediated decrease in cyclic AMP levels has been demonstrated (Salzman and Neri, 1969; Robison et al., 1971). In platelet lysates, epinephrine and norepinephrine decreased adenylate cyclase activity by about 50% (Jakobs et al., 1976). The catecholamine-induced enzyme inhibition occurred without an apparent lag phase and was immediately reversed by o-adrenergic blocking agents. There was a close correlation between a-adrenergic receptor-mediated adenylate cyclase inhibition and induction of platelet aggregation with regard to species specificity and potencies of a-adrenergic agonists and antagonists (Jakobs et al., 1978a; Lasch and Jakobs, 1979). This close correlation was further supported by binding studies on the platelet a-adrenergic receptor (Alexander et al., 1978; Newman et al., 1978; Jakobs and Rauschek, 1978). a-Adrenergic agonists inhibited the enzyme both in the absence and presence of adenylate cyclase stimulants such as prostaglandin El or adenosine (Jakobs et al., 1979b). Thereby, the Vmax of the enzyme was reduced without apparent changes in the affinities for its substrate, Mg . ATP, and the required free divalent cation (Mg’+) (Jakobs et al., 1978b).
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Karl H. Jakobs
Factors required for adenylate cyclase inhibition In studies on the mechanism involved, it was found that the presence of GTP is absolutely necessary for inhibition of platelet adenylate cyclase by a-adrenergic agonists (Jakobs et al., 1978~). In the absence or presence of prostaglandin El, a-adrenergic agonists in a GTP-dependent manner reduced the enzyme activity. In the presence of cholera toxin, the GTP-induced activation was largely reversed by epinephrine. In contrast, stable GTP analogues such as guanylyl-5’-imidodiphosphate and guanosine-5’-O-(3-thiotriphosphate), which cause a persistently increased enzyme activity (Birnbaumer, 1977; Rodbell, 1978; Levitzki and Helmreich, 1979), reversed or prevented the hormone-induced adenylate cyclase inhibition (Jakobs, 1979). In studies on the inhibition of hamster fat cell adenylate cyclase by ol-adrenergic agonists and prostaglandins (E,, Es), an additional factor required besides GTP has been found. In addition to GTP, sodium ions were required for effective coupling of inhibitory receptors to the adenylate cyclase (Aktories et al., 1979a, b). Properties of hormone-induced adenylate cyclase inhibition In several systems besides human platelets and hamster fat cells, inhibition of adenylate cyclase as a consequence of interactions of hormones or neurotransmitters with their specific receptors has now been described. Such receptormediated decreases in cyclic AMP synthesis have been reported for ar-adrenergic agonists in rabbit platelets (Jakobs et al., 1976; Tsai and Lefkowitz, 1978) for adenosine in rat (Londos et al., 1978) and prostaglandin Es in human (Kather and Simon, 1979) fat cells, for a-adrenergic and choline@ agonists and opiates in neuroblastoma X glioma hybrid cells (Sabol and Nirenberg, 1979; Nathanson et al., 1978; Sharma et al., 1975; Blume and Boone, 1979), for cholinergic agonists in canine (Watanabe et al., 1978) and rabbit myocardium (Aktories and Jakobs, 1979; Jakobs et al., 1979a) and rat parotis (Oron et al., 1978) for thyroid hormones in bovine thyroid (Friedman et al., 1977) for a-adrenergic agonists (K.H.J. and R.A. Johnson, unpublished results) and somatostatin (Vinicor et al., 1977) in rat liver and for dopamine (DeCamilli et al., 1979) and somatostatin (Heindel et al., 1978) in pituitary adenoma cells. These hormone- or neurotransmitter-induced inhibitions of adenylate cyclase share several common properties. (a) The maximal inhibitions observed ranged between 40 and 60%. The inhibitory hormonal factors reduced the enzyme activities both in the absence and presence of stimulating hormones. (b) The inhibition by the agonists occurred without an apparent lag phase and was immediately reversed by antagonists (so far available). There was a close pharmacological correlation between data obtained studying receptor binding or physiological response and adenylate cyclase inhibition with regard to the potency order of hormonal agonists and antagonists, although usually somewhat higher concentrations were required in the cell-free system for half-maximal adenylate cyclase
Inhibition of adenylate cyclase
Table 1 Inhibitions systems
151
of adenylate cyclase by hormones and neurotransmitters:
Cell type
Hormone or neurotransmitter
requirements
Requirement a) for GTP
Na+
Human platelets Rabbit platelets N X G hybrid cells b)
o -adrenergic agonists a-adrenergic agonists cu-adrenergic agonists cholinergic agonists opiates
+ + + ? +
? ? ? ? +
Rat fat cells
adenosine insulin a-adrenergic agonists prostaglandin E 1, E2 prostaglandin E2
+ ? + + +
? ? + + ?
Rabbit myocardium Canine myocardium Rat liver
cholinergic agonists cholinergic agonists ol-adrenergic agonists insulin somatostatin
+ + + ? ?
+ ? + ? ?
Rat parotis Pituitary adenoma
cholinergic agonists dopamine somatostatin thyroid hormones (Ts, T4)
? ? ? (+)
? ? ? ?
Hamster fat cells Human fat cells
Bovine thyroid
in cell-free
a) +, requirement has been reported; ?, requirement for GTP or Na+ has not been studied or reported; (+), inhibition was increased by GTP. For refs. see text. b) N X G hybrid cells, neuroblastoma X glioma hybrid cells.
inhibition than for half-maximal physiological response or receptor occupancy. (c) Most of these forms of adenylate cyclase inhibition required the presence of GTP (Table 1). This fact resembles the GTP requirement for adenylate cyclase stimulation by hormones and neurotransmitters with the obvious difference that about 5- to lo-fold higher concentrations of GTP were required for half-maximal inhibition than for stimulation. So far studied, the enzyme inhibition was reversed or prevented by stable GTP analogues but not by cholera toxin plus GTP, both of which by different mechanisms can cause persistent adenylate cyclase activation. (d) Finally, as recently found in hamster fat cells for cr-adrenergic agonists and prostaglandins (Aktories et al., 1979a, b), in neuroblastoma X glioma hybrid cells for opiates (Blume and Boone, 1979) and in rat liver for cw-adrenergic agonists (K.H.J. and R.A. Johnson, unpublished results), sodium ions were required for effective coupling of inhibitory receptors to the adenylate cyclase, or these ions at least facilitated the hormone-induced inhibition, as found in rabbit myocardium for cholinergic agonists (Jakobs et al., 1979a).
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Mechanisms possibly involved in hormone-induced adenylate cyclase inhibition
The similarities found in different systems suggest that a common mechanism is involved in adenylate cyclase inhibition by various hormones and neurotransmitters and that GTP and sodium ions play essential roles in the transduction process. The effects of GTP and sodium ions may be due to interactions at the receptor site. Sodium ions have been found to reduce the apparent affinity of agonists towards the receptors that appear to be involved in adenylate cyclase inhibition, as described for a-adrenergic (Tsai and Lefkowitz, 1978; U’Prichard and Snyder, 1978; Glossmann and Presek, 1979) and opiate receptors (Pert and Snyder, 1974), or to be required for the elicited physiological responses (Keryer et al., 1979). Similar effects of sodium ions have also been reported for receptors (P-adrenergic) involved in adenylate cyclase stimulation (U’Prichard et al., 1978). However, whereas sodium ions appear to be involved in hormone-induced adenylate cyclase inhibition, no such effects have been described for the hormone-dependent stimulation of the enzyme. Guanine nucleotides, which appear to be involved in adenylate cyclase stimulation and inhibition, cause a decrease in apparent receptor affinity both towards adenylate cyclase-stimulating hormones (Birnbaumer, 1977; Rodbell, 1978; Levitzki and Helmreich, 1979) and towards hormonal agonists that can induce adenylate cyclase inhibition, as described for a-adrenergic agonists in human platelets (Tsai and Lefkowitz, 1979) and rat liver (El-Refai et al., 1979) for cholinergic agonists in rat myocardium (Berrie et al., 1979) and for opiates in neuroblastoma X glioma hybrid cells (Blume, 1978). The decreases in apparent receptor affinities induced by sodium ions and guanine nucleotides appear to be due to an increased agonist dissociation rate. The fast dissociation of the agonist from the receptor may reform free receptor that can reenter the reaction sequence. The findings in myocardium that the apparent affinities of both /_?-adrenergic and cholinergic receptors towards their respective agonists were decreased by guanine nucleotides and that the cholinergic agonist, metacholine, antagonized the guanine nucleotideinduced decrease in Padrenergic receptor apparent affinity for isoproterenol (Watanabe et al., 1978) indicate that the effects of the guanine nucleotides on both types of receptors are mediated by a common regulatory component (Nr in Fig. 1). The findings further suggest that the adenylate cyclase inhibition might be due to such an antagonism at the nucleotide site controlling the receptor affinity. However, in adenylate cyclase studies, out of various guanine nucleotides only GTP caused an effective inhibitory coupling and stable GTP analogues prevented the inhibition, whereas in the binding studies the efficacies of GTP and its stable analogues were not different. Therefore, it seems conceivable that the GTP-dependent, hormone-induced adenylate cyclase inhibition is mediated at least partially if not completely by a nucleotide site separate from that controlling receptor affinity. There are several possible explanations for the findings that adenylate cyclase inhibition by hormones and neurotransmitters requires GTP and is prevented by
Inhibition of adenylate cyclase
153
stable GTP analogues but not by cholera toxin plus GTP. A hormonally-stimulated, increased hydrolysis of GTP by an increased GTPase activity, which is reduced by cholera toxin (Cassel and Selinger, 1977) may be involved. Stable GTP analogues would prevent this reaction, leading to a low activity state of the adenylate cyclase, and the increase in GTPase activity should overcome the cholera toxin-induced inhibition. Such a mechanism, however, would not explain why adenylate cyclase inhibition by hormones required GTP at higher concentrations than hormonal stimulation. It is rather feasible that the hormone-induced inhibition of adenylate cyclase is mediated by an up to now unidentified, separate inhibitory nucleotide site (Na in Fig. 1) (Yamamura et al., 1977; Cooper et al., 1979) which in equilibrium with the nucleotide site responsible for adenylate cyclase activation (N2 in Fig. 1) regulates enzyme activity. Such a regulatory site responsible for hormoneinduced adenylate cyclase inhibition may involve a phosphotransferase reaction, in which GTP but not stable GTP analogues can serve as a substrate. Although the present findings indicate functional differences in the proposed guanine nucleotide sites they do not exclude that one or the other of these functional sites are identical proteins with different properties when coupled to different components of the adenylate cyclase system.
CONCLUSIONS Aside from the questions concerning the mode of action of GTP and especially that of sodium ions in hormone-induced adenylate cyclase inhibition, there appear to be additional unresolved problems in this field, e.g., the mechanism by which insulin may decrease adenylate cyclase activity. Calcium ions and, thereby, the calcium-dependent regulator protein (CDR or calmondulin), adenosine and redoxregulated processes may also be involved in hormone-induced adenylate cyclase inhibitions, although there is so far no clear evidence for such interactions. Further studies on additional systems and with isolated components of the adenylate cyclase system are required to elucidate the regulatory processes leading to hormone-induced adenylate cyclase inhibition. Such studies may also finally give an insight into the question of the significance of decreased cyclic AMP synthesis besides other observed cellular events (e.g., increases in cytoplasmic calcium ions cyclic GMP level) for the overall concentration, triphosphoinositol turnover, physiological response of the cell to the hormonal stimulus.
ACKNOWLEDGEMENTS The author’s studies reported Deutsche Forschungsgemeinschaft.
herein
were
supported
by grants
from
the
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