Is cyclic AMP an obligatory second messenger for luteinizing hormone?

Molecular and Cellular Endocrinology, 69 (1990) Cll-Cl5 Elsevier Scientific Publishers Ireland, Ltd.

MOLCEL

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02263

At the Cutting

Is cyclic AMP an obligatory

Edge

second messenger

for luteinizing

hormone?

Brian A. Cooke Department

of Biochemistry,

Royal Free Hospital School of Medicine,

Key words: Luteinizing hormone; Cyclic AMP; pase A,; Leydig cell; Steroidogenesis

Calcium;

G protein;

The text book dogma regarding trophic hormone action is that luteinizing hormone (LH), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH) and human chorionic gonadotrophin (hCG) stimulate their target cell responses via the second messenger cyclic AMP. Indeed the criteria for Sutherland’s second messenger theory are satisfied in many respects, i.e. all these hormones increase cyclic AMP in their target cells, and their responses are potentiated at submaximal levels of hormone by phosphodiesterase inhibitors. In addition, cyclic AMP analogues can mimic all the stimulatory effects of the hormones. Doubts about this dogma arise especially for one of these hormones, LH, from the well-documented finding that the levels of hormone which result in at least 50% of maximum steroidogenesis in rat Leydig and ovarian cells cause no detectable changes in cyclic AMP levels [l-4]. This discrepancy has always been assumed to be due to a lack of sensitivity of the assay for cyclic AMP; indeed evidence has been presented to show that activation of cyclic AMP-dependent protein kinase can be detected with no measurable change in the levels of cyclic AMP [4]. However, although the sensitivity of the cyclic AMP assay has increased by several orders of magnitude since the original observations, and highly sensitive cell

Address for correspondence: Brian A. Cooke, Department of Biochemistry, Royal Free Hospital School of Medicine, University of London, Rowland Hill Street, London NW3 2PF. U.K. 0303-7207/90/$03.50

0 1990 Elsevier Scientific

Publishers

Ireland,

Universrty of London, London N W3 2PF, U.K.

Ion channel;

Chloride

channel;

Arachidonic

acid;

Phospholi-

preparations are now available, low levels of LH are still steroidogenic, without detectable changes in cyclic AMP. So what is the evidence for other LH-induced second messengers? There is no doubt that cyclic AMP can stimulate steroidogenesis, but how important is it physiologically? This short review will attempt to answer these questions in the light of recent studies which indicate that calcium and calcium-mediated events are required for the control of steroidogenesis, and suggest that cyclic AMP plays a sensitizing role in steroidogenesis. The Role of carbohydrate residues in LH and hCG The high degree of glycosylation of LH and hCG has been found to be linked to the ability of these hormones to increase cyclic AMP levels in Leydig and ovarian cells; the ‘deglycosylated’ (70% after anhydrous hydrogen fluoride treatment) forms of these hormones have either no effect or at best a very small effect on cyclic AMP production [5-71. The ability of deglycosylated hCG/LH to stimulate steroidogenesis does, however, depend on the species and cell type; in the mouse Leydig tumour MA10 cells a very weak stimulation of steroidogenesis occurs [7] whereas in rat Leydig cells they cause near-maximum steroidogenesis [8,9]. Addition of lectins (wheat germ agglutinin) partially restores the ability of the deglycosylated hormones to stimulate cyclic AMP [9,10]. Deglycosylation does not impair the ability of these hormones to bind to the LH receptor, in fact quite Ltd.

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the reverse; an increase in both the binding [7,9] and also the rate of internalization of the LH receptor occurs [9]. These studies demonstrate that glycosylation is necessary to achieve full cyclic AMP production but is not required for binding to the LH receptor. Differences in the ability to stimulate steroidogenesis in different Leydig cells may reflect the presence of other signalling pathways.

and opsin receptors) do have 20-30s sequence homology with each other in their third transmembrane loops [14]. The isolated LH receptor is, therefore, very different from the other receptors that activate adenylate cyclase via G,. It does, however, have functional activity because the LH receptor cDNA from rat ovaries has been expressed in embryonic kidney cells and binding to hCG and its ability to stimulate cyclic AMP production has been demonstrated [ll].

The LH receptor Calcium The LH receptor has recently been sequenced and cloned from rat ovarian tissue [ll] and from porcine Leydig cells [12]. It consists of a 26 residue signal peptide, a 341 residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein family, and a 333 membrane spanning region which displays a low sequence similarity (approximately 20%) with members of the GTP binding protein coupled receptor family. Hydropathy analysis suggests the existence of seven transmembrane domains. It is well established that binding of agonists to the fi-adrenergic receptor (BAR) promotes interaction with the GTP binding protein G, leading to activation of adenylate cyclase. Deletion mutagenesis and molecular replacements have been used to identify the sites of interaction between the receptor and G, [13]. These indicate that most of the putative extracellular hydrophobic domain of the PAR can be deleted without affecting the G-protein coupling. Deletions primarily at the N- and C-terminals within the region predicted to form the third intracellular loop of the receptor, however, result in a complete loss of G-protein coupling and adenylate cyclase stimulation by the mutant receptor. These and other studies identify this loop to be the site of coupling of the PAR to the G-protein. Similar conclusions have been reached for the muscarinic receptor. In the predicted third intracellular loop of the LH receptor only three of the amino acids are the same as in the PAR. The cloned LH receptor therefore does not have a G-protein amino acid binding sequence homologous with the PAR or any other receptor which couples to G proteins. The latter (e.g. PAR, muscarinic acetyl choline

Extracellular calcium is required for maximum LH-stimulated steroidogenesis in testicular Leydig cells; testosterone production is decreased by 50% in calcium-free medium [15]. Both LH- and cyclic AMP-mediated increases in intracellular calcium and calcium influx and efflux have been demonstrated in Leydig and ovarian cells [15-201. Additional evidence for the role of calcium in steroidogenesis is the steroidogenic effect of LH releasing hormone (LHRH) analogues which can stimulate up to 60% of the maximal LH-stimulated testosterone production in rat Leydig cells; LHRH has no effect on cyclic AMP and its action is clearly via calcium-dependent processes [21]. Earlier [22] and more recent studies [23] in our laboratory with calmodulin inhibitors have clearly shown that dose-related inhibition of cyclic AMP formation occurs in the presence of LH, forskolin or cholera toxin, indicating a calcium/calmodulin-regulated control of adenylate cyclase in rat Leydig cells. Of special relevance to the role of cyclic AMP in steroidogenesis is the biphasic effect of the calmodulin inhibitor, calmidazolium, on steroidogenesis [23]. At high levels (> 5 PM) inhibition occurs; however, over a dose range of 1-4 PM, a marked stimulation occurs in the absence of added LH, which reaches the same maximum as LH-stimulated levels. These doses of calmidazolium which stimulate testosterone cause a decrease of basal cyclic AMP. Both inhibition and stimulation are unaffected by the presence of phosphodiesterase inhibitors, thus confirming that they are independent of cyclic AMP. The stimulatory action of calrnidazolium on testosterone production could be explained by an increase in intracellular calcium resulting from the inhibition

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of calmodulin-dependent calcium/ magnesium ATPase which is required to pump calcium from the cell. It would thus appear that calcium/calmodulin can stimulate steroidogenesis in the absence of elevated cyclic AMP levels, and that stimulation of Leydig cell cyclic AMP production is also calcium/calmodulin dependent. Synergism between LH and cyclic AMP It is well established that the phosphodiesterase inhibitors, e.g. methyl isobutyl xanthine (MIX) will stimulate both basal and submaximal LHstimulated testosterone production in Leydig and ovarian cells. The effect is very marked; with submaximal levels of LH in Leydig cells a 3-6-fold increase in testosterone production is obtained when MIX is added. Recent studies have shown that with very low levels of dibutyryl cyclic AMP and LH, which by themselves do not stimulate steroidogenesis, there is a marked synergistic effect on testosterone synthesis when they are added together [24]. This low level of dibutyryl cyclic AMP gives a left shift of the dose-response curve for LH very similar to that obtained with MIX. These observations again suggest that LH can act via non-cyclic AMP-dependent pathways and that cyclic AMP can act synergistically with the second messenger(s) formed by these pathways. Ion channels If LH does regulate testosterone via a calciumdependent mechanism, how is calcium controlled and what is its mode of action? No conclusive reports have been made of measurements of the calcium mobilizer, inositol 1,4,5trisphosphate. As seen above, calcium efflux and influx at the cell plasma membrane occur in Leydig cells and this may be one of the main ways in which calcium is regulated, i.e. via calcium ion channels. Other ion channels that have been demonstrated are those involving K+ and Cl-. It has been shown by patch clamp studies that calcium-dependent K+ and Clcurrents exist in adult Leydig cells [25]; the K+ outward membrane current was demonstrated in low (1O-9-1O-8 M) calcium media whereas with higher calcium concentrations (10-7-10-6 M) a

Cl- current was recorded. The authors sugggested that an increase in intracellular calcium activates a Cl- current. However, they were not able to demonstrate any effects of hCG on either current. Our recent experiments indicate that Cl- channels may be very important in the action of LH [24]. We have found that omission of Cl- from the incubation medium of isolated Leydig cells markedly stimulates submaximal LH-controlled steroidogenesis. Furthermore, the addition of SITS (4-acetamido-4’-isothiocyanatostilbene-2,2’-disulphonic acid), the Cl- channel blocker, inhibited LH-stimulated testosterone production with an ED,, of 90 pM. Of especial interest is that this inhibition is only obtained with levels of LH which stimulate no or low production of cyclic AMP. In the presence of high levels of LH or with other ligands that increase cyclic AMP levels (dibutyryl cyclic AMP, forskolin). no inhibition with SITS occurred. These results suggest that the mechanism of LH action at very low levels of cyclic AMP depend on a Cl- channel, whereas in the presence of high levels of cyclic AMP this effect is overridden. Role of protein kinase C In addition to the stimulatory action of LH and hCG on cyclic AMP and steroidogenesis in Leydig and ovarian cells, these gonadotrophins also cause refractoriness or desensitization of these same responses. Evidence from in vitro and in vivo work suggests a failure of communication between the LH receptor and the adenylate cyclase (see review, [26]). A feature of the LH-induced desensitization of Leydig cells is that although the cells become refractory to further LH stimulation they have a high continuous production of ‘basal’ cyclic AMP which is approximately 50% of the levels obtained with maximum stimulating levels of LH, and is not due to residual receptor-bound LH. The desensitizing effect of LH cannot be mimicked with cyclic AMP analogues, again indicating a noncyclic AMP-dependent pathway for LH. The desensitizing effects of LH (but not the effect on basal production) can be mimicked by protein kinase C activators such as phorbol esters indicating that this enzyme may be involved. In further studies carried out on the effects of inhibi-

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tors of this enzyme (sphingosine or psychosine), it was found that preincubation of Leydig cells with either inhibitor resulted in inhibition of LHstimulated cyclic AMP production [27]. Maximum inhibition occurred with 1 PM inhibitor suggesting that Leydig cells are very sensitive to these protein kinase C inhibitors. It has also been shown that sphingosine and psychosine inhibit luteal cell function [28]. Thus stimulation (with phorbol esters) and inhibition (with sphingosine and psychosine) of protein kinase C lead to inhibition of adenylate cyclase activity. This apparent paradox may be explained by the fact that the effect of the inhibitors was found to dependent upon the time of addition with respect to LH [27], i.e. they only inhibited during the first 15 min of stimulation with LH. This implies that the initial activation of the adenylate cyclase may be dependent upon the activation of protein kinase C. Continued stimulation under conditions which lead to desensitization may cause additional protein kinase C-mediated phosphorylation leading to uncoupling of the adenylate cyclase. Release and action of arachidonic metabolites

acid and its

Arachidonic acid can be released from phospholipids by calcium-mediated activation of phospholipase A, (PLA,) and/or phospholipase C (PLC) followed by hydrolysis of diaclglycerol by diacylglycerol lipase. It is then further metabolized via the cyclooxygenase and lipoxygenase pathways to prostaglandins or leukotrienes respectively. Our studies indicate that the products of the lipoxygenase pathway (but not the cyclooxygenase pathway) are involved in the control of steroidogenesis [22,29], and that LH stimulates the release of arachidonic acid from Leydig cells [30]. Furthermore, inhibition of PLA, (by dexamethasone or mepacrine) inhibits LH-stimulated steroidogenesis without affecting LH-induced cyclic AMP production [31]. Dibutyryl cyclic AMP- and forskolin-stimulated testosterone production were also inhibited. 22 R-hydroxycholesterol-stimulated testosterone production was not inhibited by quinacrine or dexamethasone, showing that they were not exerting their inhibit-

ing effects steroidogenic

by decreasing enzymes.

the

activity

of

the

Conclusions The evidence suggests that physiological levels of LH activate a transducing system(s) that does not involve the formation of cyclic AMP. This primarily results in the mobilization of calcium from intracellular stores and/or by opening of plasma membrane calcium channels. Other channels, in particular the Cl- channel, are also involved. In view of the evidence for the release of arachidonic acid, the effects of inhibition of PLA, an lipoxygenases and the known regulatory role of calcium in this pathway, this ion may be involved in the formation of arachidonic acid metabolites which may act as second messengers for the control of steroidogenesis. At high levels of LH, cyclic AMP formation is stimulated and it may then play a cooperative role in steroidogenesis by enhancing the essential calcium-regulated pathways especially via PLA,. Finally, the LH receptor has been shown to be different from the other receptors that couple to G, and stimulate adenylate cyclase, and in addition to its effects on steroidogenesis, LH may regulate adenylate cyclase via calcium/calmodulin and protein kinase C. References [1] Moyle, W.R. and Ramachandran, J. (1973) Endocrinology 93, 127-134. [2] Catt, K.J. and Dufau, M.L. (1973) Nature New Biol. 244, 219-221. [31 Rommerts, F.F.G., Cooke, B.A., van der Kemp, J.W.C.M. and van der Molen, H.J. (1973) FEBS Lett. 33, 114-118. F.H.A. (1976) [41 Cooke, B.A., Lindh, M.L. and Janszen, Biochem J. 160, 439-446. 151 Moyle, W.R., Bahl O.P. and Mars, L. (1975) J. Biol. Chem. 250, 9163-9169. 161 Sairam, M.R. and Schiller, P.W. (1979) Arch. Biochem. Biophys. 197, 294-301. [71 Sairam, M.R. (1989) FASEB J. 3, 1915-1926. PI Platts, E.A., Sairam, M.R., Schulster, D. and Cooke, B.A. (1988) in Molecular and Cellular Endocrinology of the Testis (Cooke, B.A. and Sharpe, R.M., eds.), Serono Symposia Vol. 50, pp. 59-63, Raven Press, New York. M.R. and Cooke, B.A. (1989) J. [91 Rose, M.P., Sairam, Endocrinol. Suppl. 123, Abstract 64. M.A., Shimogashi, Y. and Wakabayashi, K.L. UOI Hattori, (1989) Mol. Cell. Endocrinol. 66, 207-214.

Cl5 [ll] McFarland, K.C. et al. (1989) Science 245, 494-499. [12] Loosfelt, H. et al. (1989) Science 245, 525-528. [13] Strader, C.D., Signal, I.S. and Dixon, R.A.F. (1989) Trends Pharmacol. Sci. Suppl. 4 (Sub-types of Muscarinic Receptors), 26-30. [14] Tubo, T. et al. (1986) Nature 323, 411-416. [15] Janszen, F.H.A., Cooke, B.A., van Driel, M.J.A. and van der Molen, H.J. (1976) B&hem. J. 160, 433-437. [16] Sullivan, M.H.F. and Cooke, B.A. (1986) B&hem. J. 236, 45-51. [17] Platts, E.A., Schulster, D. and Cooke, B.A. (1988) 5th European Workshop on the Molecular and Cellular Endocrinology of the Testis, Abstract B7. [18] Veldhuis, J.D. and Klase, P.A. (1982) Endocrinology 111, l-6. [19] Veldhuis, J.D. (1987) Endocrinology 120, 445-449. [20] Asem, E.K., Molnar, M. and Hertelendy, F. (1987) Endocrinology 120, 853-859. 1211 Cooke, B.A. and Sullivan, M.H.F. (1985) Mol. Cell. Endocrinol. 41, 115-122.

[22] Sullivan, M.H.F. and Cooke, B.A. (1985) Biochem. J. 232, 55-59. [23] Choi, M.S.K. and Cooke, B.A. (1989) J. Endocrinol. Suppl. 123, Abstract 66. [24] Choi, M.S.K. and Cooke, B.A. (submitted for publication). [25] Duchatelle, P. and Jaffre (1987) FEBS Lett. 217, 11-15. [26] Rommerts, F.F.G. and Cooke, B.A. (1988) in New Comprehensive Biochemistry: Hormones and their Actions (Cooke, B.A., King R.J. and van der Molen, H.J., eds.), Vol. 2, pp. 163-180.. Elsevier, Amsterdam. [27] Rose, M.P. and Band, A. (1988) B&hem. Sot. Trans. 17. 510-511. [28] Sender Baum, M.G. and Ahren, K.E.B. (1988) Mol. Cell. Endocrinol. 60, 127-135. [29] Dix, C.J., Habberfield, A.D., Sullivan, M.H.F. and Cooke, B.A. (1984) Biochem. J. 219, 529-537. [30] Chaudry, L., Schulster, D. and Cooke, B.A. (1989) Biothem. Sot. Trans. 17, 752-753. [31] Abayasekara, D.R.E., Band, A.M. and Cooke, B.A. (1990) Mol. Cell. Endocrinol. 70 (in press).