Luteinizing hormone-releasing hormone rapidly elicits an opening of ...

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Phurmucdogy. I George Square, Edinburgh EH8 9JZ,. U. K. Several groups ... ization-induced release (Hopkins & Walker, 1978) and the verapinoid inhibitors of ...
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Luteinizing hormone-releasing hormone rapidly elicits an opening of membrane Ca2+ channels which is suppressed by protein kinase C RORY MITCHELL, NICOLA MINAUR. MELANIE JOHNSON, SALLY-ANN OGlER and GEORGE FINK M . R.C. Bruin Metuholism Unit. University Depurtment of Phurmucdogy. I George Square, Edinburgh EH8 9JZ, U .K . Several groups have demonstrated that the stimulation by luteinizing hormone-releasing hormone (LHRH) of luteinizing hormone (LH) secretion from the anterior pituitary is dependent on extracellular Ca'+ (Marian & Conn, 1979; Pickering & Fink, 1979; Naor et ul., 1980). Hopkins & Walker (1978); Marian & Conn (1979) and Naor et a/. (1980) showed that La'+ inhibited LHRH-induced (but not basal) LH secretion and organic Ca'+ -channel blockers of the verapinoid series are also effective (Marian & Conn, 1979; Naor et d.,1980). Therefore Ca2+has been extensively proposed as the second messenger mediating LHRH action (Marian & Conn. 1979). Nevertheless, LH RH-induced LH release appears rather less sensitive to removal of extracellular Ca2+than depolarization-induced release (Hopkins & Walker, 1978) and the verapinoid inhibitors of release described were generally only partial (Marian & Conn, 1979; Naor et ul.. 1980). These findings suggest that LH RH can mobilize intracellular as well as extracellular Ca'+. This is consistent with evidence that LHRH can evoke phosphoinsitide hydrolysis (Schrey, 1985). thus generating both inositol phosphates, that may mobilize intracellular Ca'+, and diacylglycerol, that may activate protein kinase C (PKC). I t has been suggested that the mobilization by phosphoinositide linked receptors of intra- and extra-cellular Ca'+ is intimately linked (Putney, 1986). We therefore aimed to assess whether elements of the phosphoinositide response, particularly activation of PKC, participate in causing the LHRH-induced influx of 45Ca-+. described briefly by Hopkins & Walker (1978). COB Wistar rats were maintained under controlled lighting (light 05 :00-19 : OOh) and temperature (22 C) and given tree access to Diet 41 B and tap water. Oestrous cycles were assessed by daily vaginal smears. Anterior pituitaries were chopped into 500 pm prisms and suspended in a pre-warmed and pre-oxygenated Ca2+-uptake buffer with 0.1o/' bovine serum albumin and 35% Percoll. The buffer contained (concentrations in mM): NaCl 154, KCI 5.4, CaCI, 1.5, glucose 11.0 and Hepes 6.0 [buffered to pH 7.4 with solid Tris (hydroxymethy1)-aminomethane base]. Aliquots of tissue suspension were added to equal volumes of buffer, then preincubated at 37'C and gassed with 100% O2for approx. 20 min before initiation of the experiment. Pre-warmed buffer containing 4'Ca'+ to give a final concentration of 2pM was added and then after precise time intervals, '"Ca'+ uptake was halted by quenching with 3 ml of an ice-cold wash. This was uptake buffer containing 2 mM-EGTA instead of Ca", Abbreviations used: LHRH. luteinizing hormone-releasing hormone: LH. luteinizing hormone: PKC, protein kinase C.

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similar to those used by other workers to halt 4'Ca2+uptake in smooth muscle. After quenching, the contents of each tube were filtered and the tissue received another 3 ml of the relevant wash immediately, as an acute wash. The filtering was carried out on a Millipore filter block under vacuum, using Millipore cellulose acetate filters supported on GF/B. Three further 2 min washes followed. The "Ca'+ uptake was measured by liquid scintillation counting. The amount of % i 2 +accumulated by unstimulated tissue increased linearly in a time-dependent fashion. but the excess uptake due to l00nM-LHRH or 6 0 m ~ - K +was maximal within 30s (the incubation time used for further studies. LHRH increased 4'Ca'+ influx in a concentrationdependent fashion from 0.1 to 100 n M . Maximal responses to 100 nM-LHRH (as percentage increase over controls) varied with tissue from different stages of the oestrous cycle: pro-oestrus, 99 f 14; oestrus, 124 f 15; met-oestrus. 74 10; di-oestrus, 35 f 7; male, 40 k 9 ( n = 6 8 , means k s.E.M.). Control unstimulated uptake was no different in tissue from these various sources. The activators of PKC, phorbol 12-myristate 13-acetate ( 100 nM) and 1 -oleoyl 2-acetyl rac-glycerol (63 PM) (incubated with tissue for IOmin before uptake) drastically inhibited the LHRH response with no effect on basal "Ca" uptake. In oestrus tissue the response to IOOnM-LHRH in the presence of these activators was reduced to 30 f 10% and --4 f 7% respectively (n = 5 , means f s.E.M.). Similar effects were observed in male tissue where concentrationdependent inhibition of the response to IOOnM-LHRH by phorbol 12-myristate 13-acetate was observed. This was maximal by l O O n M and was not mimicked by 10pM-4/jphorbol. LHRH receptor properties in tissue subjected to these PKC activators were investigated using equilibrium binding of [''51]buserelin (Mitchell et al., 1985). Neither affinity nor number of sites were altered (male tissue). suggesting that the locus of PKC action must be at some post-receptor site. PKC activation clearly therefore does not act to mediate the opening of membrane Ca'+ channels by LHRH but rather acts as a regulatory inhibitor of this effect. Whether other consequences of the phosphoinositide response, such as the production of inositol phosphates, are responsible for Ca'+ channel opening, is not clear. Hopkins. C. R. & Walker. A. M. (1978) Mol. Cdl. Endoc,rinol. 12. 1x9 208 Marian. J. & Conn, P. M. (1979) Mol. Phurmuc,ol. 16. 196 201 Mitchell, R.. Ogier. S.-A.. Johnson, M.. Cleland. A,, Bennie, J. & Fink, G. (1985) in Ncwroendocrine Molecular Biology, (Fink, G . . Harmar. A. J . & McKerns. K. W. eds.). p. 91 100. Plenum Publishing Corporation, New York Naor. 2.. Leifer. A. M. & Catt, K. J. (1980) Endocrinology 107. 1438 1445 Pickering. A. J.-M. C. & Fink, G . (1979). J . Endwrinol. 81. 223 234 Putney. J. W. (1986) Cell Cukiuni 7. 1 12 Schrey. M. P. (19x5) Biocheni. J . 226. 563 569 Received 27 August 1986