On the regulation of rat testicular steroidogenesis. Short term effect of luteinizing hormone and cycloheximide in vivo and Ca2+ in vitro on steroid production in cell-free systems.

An attempt has been made to correlate the rapid effect of luteinizing hormone on testicular steroid production in vivo with testicular steroid concentrations and in vitro steroid production rates in testis tissue preparations. Within 20 min after intravenous administration of 25 mug luteinizing hormone, increases were observed in testosterone concentrations in testicular venous plasma and in whole testis tissue and in pregnenlone concentrations isolated testis mitochondrial fractions. Testosterone production by whole testis homogenates and pregnenolone production by isolated mitochondrial fractions were significantly increased within 5 min after in vivo administration of luteinizing hormone. Injection of cycloheximide 10 min prior to luteinizing hormone prevented the stimulating effect of luteinizing hormone to steroid levels in testicular venous plasma and testis tissue and on steroid production rates by preparations of rat testis tissue. Cycloheximide treatment of control animals did not significantly alter testosterone concentrations and testosterone production rates vitro, although mitochondrial pregnenolone concentrations and production rates were decreased. Testosterone production by whole testis homogenates as well as the pregnenolone production by isolated mitochondrial fractions obtained from luteinizing hormone treated testes and control glands showed a biphasic time curve A period (5-10 min) of high steroid production was followed by a period lower steroid production. Addition of 25 mug luteinizing hormone or 10(-8)--10(-5) M adenosine 3':5'-monophosphate (cyclic AMP) to the incubation medium had no effect pregnenolone production by isolated mitochondrial fractions. Administration of leuteinizing hormone in vivo markedly enhance the stimulating effect of Ca2+ on testosterone production by whole testis homogenates and on pregnenolone production by isolated mitochondrial fractions.     

Differential effects of calcium on progesterone production in small and large bovine luteal cells

We studied the effects of calcium (Ca2+) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 x 10(5) small and 5 x 10(3) large cells for 2 and 4 h respectively. Removal of Ca2+ from the medium did not influence basal P production in the small cells (P greater than 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E2 (PGE2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F2 alpha (PGF2 alpha) was impaired (P less than 0.05) by low Ca2+ concentrations. LH and PGE2-stimulated cAMP production was not altered by low extracellular Ca2+ concentrations, and PGF2 alpha had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P less than 0.05) in Ca2(+)-deficient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca2(+)-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca2+, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release and/or action, suppressed (P less than 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P less than 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca2+. The inhibitory effect of Ca2+ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca2(+)-calmodulin/protein kinase C-dependent mechanisms.     

5,6-Epoxyeicosatrienoic acid mobilizes Ca2+ in anterior pituitary cells

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.     

Mechanisms of secretory responses to gonadotropin-releasing hormone and phorbol esters in cultured pituitary cells. Participation of protein kinase C and extracellular calcium mobilization

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of gonadotropin releasing hormone (GnRH) and phorbol esters in cultured pituitary cells. During incubation in normal medium, GnRH stimulated LH release with an ED50 of 0.35 nM. Incubation in Ca2+-deficient medium (Ca2+-free, 10 microM) substantially decreased but did not abolish the LH responses to GnRH. The extracellular Ca2+-dependent component of GnRH action could be mimicked by high K+ concentrations, consistent with the presence of voltage-sensitive calcium channels (VSCC) in pituitary gonadotrophs. Ca2+ channel agonist (Bay K 8644) and antagonist (nifedipine) analogs, respectively, enhanced or partially inhibited LH responses to GnRH and also to K+, the latter confirming the participation of two types of VSCC (dihydropyridine-sensitive and -insensitive) in K+-induced secretion. Phorbol esters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), 4 beta-phorbol-12,13-dibenzoate, and 4 beta-phorbol-12,13-diacetate, stimulated LH release with ED50s of 5, 10, and 1000 nM, respectively, and with about 70% of the efficacy of GnRH. Phorbol ester-stimulated LH secretion was decreased but not abolished by progressive reduction of [Ca2+]e in the incubation medium, and the residual LH response was identical with that elicited by GnRH in Ca2+-deficient medium. TPA increased [Ca2+]i to a peak after 20 s in normal medium but not in the absence of extracellular Ca2+, indicating that protein kinase C (Ca2+/phospholipid-dependent enzyme) promotes calcium entry but can also mediate secretory responses without changes in calcium influx and [Ca2+]i. The extracellular Ca2+-dependent action of TPA on LH release was blocked by Co2+. However, nifedipine did not alter TPA action on [Ca2+]i and LH release. These observations indicate that protein kinase C can participate in GnRH-induced LH release that is independent of Ca2+ entry, but also promotes the influx of extracellular Ca2+ through dihydropyridine-insensitive Ca2+-channels.     

Regulation of testicular function by insulin and transforming growth factor-beta

Hyperinsulinism is associated with disorders of androgen production in humans. We have studied the effects of insulin and insulin-like growth factor-1 on androgen production in vitro using a crude preparation of mouse Leydig cells incubated with luteinizing hormone in a serum-free medium. We found a positive correlation between testosterone production and the luteinizing hormone dose over 3 hours. Exposure of the cells for 1 hour to insulin (1 micrograms/ml) prior to the addition of luteinizing hormone significantly augmented the amount of testosterone produced in response to the gonadotropin when added after this preincubation. In contrast, prior exposure of the cells to proinsulin (30 micrograms/ml), insulin-like growth factor-1 (30 ng/ml), or epidermal growth factor-1 (1 micrograms/ml) did not influence the testosterone response to luteinizing hormone. Transforming growth factor-beta reduced the testosterone response to luteinizing hormone. Transforming growth factor-beta (1,000 pg/ml) blocked the insulin augmentation of luteinizing hormone-stimulated testosterone production. We conclude that insulin has an endocrine effect on testosterone production by mouse Leydig cells in vitro. Furthermore, the Leydig cell response to insulin is itself sensitive to interaction with transforming growth factor-beta which may operate as part of the paracrine control of Leydig cell function.     

The role of calcium in luteinizing hormone-releasing hormone agonist (ICI 118630)-stimulated steroidogenesis in rat Leydig cells.

The luteinizing hormone-releasing hormone (LHRH) agonist ICI 118630 was found to increase testosterone production in purified rat testis Leydig cells in a concentration- and time-dependent manner, but no consistent changes in cyclic AMP levels were detectable. The stimulation of steroidogenesis by LHRH agonist was found to be dependent on the concentration of Ca2+ in the incubation medium; at least 1 mM was required. The calcium ionophore A23187 mimicked the effects of the LHRH agonist on steroidogenesis, and addition of both compounds together did not further increase testosterone production. The calcium ionophore caused a small increase in cyclic AMP which was independent of the concentration of the ionophore and of the calcium concentrations. The evidence obtained in this study indicates that LHRH agonist-stimulated steroidogenesis in rat testis Leydig cells is primarily mediated by calcium and not cyclic AMP.     

Effect of protein-synthesis inhibitors on testosterone production in rat testis interstitial tissue and Leydig-cell preparations.

Luteinizing-hormone-stimulated testosterone biosynthesis was inhibited by cycloheximide during incubation of rat testis intersitial tissue in vitro and also by puromycin and cycloheximide during incubation of Leydig-cell preparations, but not by chloramphenicol. These results suggest that a protein regualtor(s) formed by cytoplasmic protein synthesis is involved in steroidogenesis in the rat testis. The specific effect of cycloheximide and puromycin on protein synthesis rather than on other non-specific processes is suggested by the inhibition of protein synthesis and steroidogenesis with different doses of the inhibitors and the lack of effect of cycloheximide on luteinizing-hormone-induced adenosine 3':5'-cyclic monophosphate production. Stimulation of testosterone production by luteinizing hormone during superfusion of interstitial tissue was detectable within 10-20 min and reached a maximum of 120 min, and thereafter slowly decreased. Cycloheximide added at maximum steroid production caused a rapid decrease in testosterone synthesis which followed first-order kinetics (half-life 13 min), thus indicating that the protein regulator(s) has a short half-life. No effect of cycloheximide, puromycin or chloramphenicol on testosterone production in the absence of added luteinizing hormone was found, suggesting that the basal production of testosterone is independent of protein synthesis.     

Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes

The effect of Ca²⁺ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca²⁺ in the range of 0.1–0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca²⁺ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fractions, incubated in the presence of initial Ca²⁺ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.Pregnenolone production from endogenous substrates in mitochondrial fractions isolated in 0.25 M sucrose from control and luteinizing hormone treated testes incubated in the absence of added succinate and Ca²⁺, was maintained during 10–20 min.After longer incubation times no further steroid synthesis took place. Addition of 0.5 mM Ca²⁺ to the incubation medium at time zero slightly stimulated initial pregnenolone production rates in control mitochondrial fractions, but had no effect during prolonged incubations. Addition of 0.5 mM Ca²⁺ to mitochondrial fractions isolated from luteinizing hormone treated glands showed no effect either on initial production rate or during prolonged incubations.Pregnenolone production rates were maintained during 90 min in the presence of 20 mM succinate in the incubation medium. Under such conditions production rates during the first 20 min in mitochondrial fractions obtained from luteinizing hormone treated glands were approx. 3 times higher than in relevant control samples. Addition of 0.5 mM Ca²⁺ to the incubation medium containing 20 mM succinate markedly stimulated initial pregnenolone production rates in control mitochondrial fractions, but gave only a small stimulation of succinate-supported production rates in luteinizing hormone treated testicular mitochondrial fractions. These results indicate that Ca²⁺ in vitro can mimic the trophic effect of luteinizing hormone in vivo on mitochondrial pregnenolone production.Ageing of mitochondrial protein for 60 min at 33°C resulted in a marked increase in pregnenolone production rates in mitochondrial fractions obtained from control testes. The same treatement hardly influenced production rates in mitochondrial fractions isolated from luteinizing hormone treated testes. Ageing may have an effect on the ultrastructure of freshly prepared mitochondria, causing a change in the amount of cholesterol readily available for the enzyme complex.The gluco- and mucoprotein specific agent Ruthenium red (50–2000 ng/ml) did not inhibit pregnenolone production in either control or hormone treated testicular mitochondrial fractions, incubated in the absence of added Ca²⁺. the presence of 200–2000 ng Ruthenium red per ml incubation mixture.The present results have been discussed in relation to the possible involvement of Ca²⁺ in the molecular mechanism of short-term action of luteinizing hormone on testicular androgen production.     

Effect of Ca2+, ruthenium red and ageing on pregnenolone production by mitochondrial fractions from normal and luteinizing hormone treated rat testes.

The effect of Ca2+ in vitro on pregnenolone production rates under various incubation conditions by mitochondrial fractions isolated from testes of normal rats and of rats after in vivo treatment with luteinizing hormone has been investigated. Concentrations of Ca2+ in the range of 0.1-0.5 mM stimulated succinate supported pregnenolone production in mitochondrial fractions from both control and luteinizing hormone treated testes. When mitochondrial fractions were isolated in 0.25 M sucrose without additions, Ca2+ in vitro increased succinate supported pregnenolone production rates in mitochondrial fractions isolated from control testes to a greater extent than in mitochondrial fractions, from luteinizing hormone treated testes. Production rates in control mitochondrial fraction, incubated in the presence of initial Ca2+ concentrations of 0.7 mM and higher were almost similar to production rates in relevant luteinizing hormone treated mitochondria.     

Discordant regulation by luteinizing hormone of ornithine decarboxylase activity and testosterone production in isolated rat testicular cells invitro

Possible functional relationship between luteinizing hormone-stimulated ornithine decarboxylase and testosterone production was examined in rat testicular interstitial cells $\text{in}\text{vitro}$. Although luteinizing hormone enhanced both ornithine decarboxylase activity and testosterone production at a similar physiological dose range, we found dissociation in the two responses in terms of their temporal aspect and the way they were affected by an irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, and protein synthesis inhibitor cycloheximide. The results suggest that there appears to be no causal coupling between luteinizing hormone-stimulated enzyme activity and testicular steroidogenesis.     

Correlation of protein kinase activation and testosterone production after stimulation of Leydig cells with luteinizing hormone.

The effect of different doses of luteinizing hormone on activation of protein kinases, cyclic AMP and testosterone production was studied in purified rat testis Leydig-cell preparations in the presence of 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor). In addition, the nature of the protein kinases present in these cells and other tissues was investigated. The following results were obtained. 1. With all the amounts of luteinizing hormone used (0.1-1000 ng/ml), both activation of protein kinase and stimulation of testosterone production were demonstrated. With the lowest amount of luteinizing hormone (0.1 ng/ml), an 8.4+/-0.9% (S.E.M.,n=6) stimulation of protein kinase activation occurred, increasing to 100% with 1000 ng/ml, compared with 3.2+/-1.0%(S.E.M.,n=7) and 100% stimulation of testosterone production with 0.1 and 100 ng/ml respectively. 2. With amounts of luteinizing hormone up to 1 ng/ml (which gave half-maximal stimulation of testosterone production) no detectable increases in net cyclic AMP production were obtained. With higher amounts of luteinizing hormone, cyclic AMP production increased, but maximal production was not reached with 1000 ng/ml. 3. Two isoenzymic forms of protein kinase were present in Leydig cells and seminiferous tubules; type I was eluted with 0.075 M-and type II with 0.22-0.25 m-NaCl from DEAE-cellulose columns. 4. The protein kinase activity was not affected by the presence of erythrocytes in the Leydig-cell preparation, but varied depending on the type of histone used as substrate (histone F2b greater than mixed greater than histone F1).     

The effects of calcium ionophore A23187 on interstitial cell steroidogenesis

The present study was performed to evaluate the effects of calcium ionophore A23187 on adenosine 3',5'-monophosphate (cyclic AMP) and testosterone production in rat interstitial cells. Interstitial cells were incubated in Krebs-Ringer solution with varying amounts of luteinizing hormone, pregnenolone, or A23187. Cyclic AMP and testosterone were measured in the incubation medium after 4 h incubation. A23187 (0.01--10 microgram/ml) caused progressive increases of cyclic AMP formation (from 0.18 +/- 0.02 (S.E.) pmol/10(6) cells for the control of 0.42 +/- 0.02 pmol/10(6) cells, P less than 0.025), while testosterone production remained unaltered. When varying amounts of A23187 were added concomitantly with luteinizing hormone (5 IU/l), A23187 inhibited luteinizing hormone-induced steroidogenesis in a dose-dependent manner, but it had no effect on luteinizing hormone-induced cyclic AMP formation. When pregnenolone (10(-6) M) was added to the cells, testosterone formation increased from 1.50 +/- 0.22 to 8.46 +/- 1.65 ng/10(6) cells. A23187 (1 microgram/ml) had no discernable effect on the conversion of pregnenolone to testosterone. The main effect of increased cytosol calcium on steroidogenesis seems to be at the steps beyond adenylate cyclase-cyclic AMP. These results suggest that calcium is important for the conversion of cholesterol to pregnenolone, while the steps beyond pregnenolone are relatively independent of Ca2+.     

Improved sensitivity of the mouse interstitial cell testosterone assay with the addition of forskolin.

Modification of the mouse interstitial cell testosterone assay by the addition of 1.5 microM forskolin to the incubation medium has improved the sensitivity of this luteinizing hormone bioassay from approximately 100 to 3 pg/tube of NIH rLH RP-2. Luteinizing hormone can be clearly detected in 1 microL of serum from rats castrated 1 week previously and 5 microL of serum from intact rats. Parallelism was noted between dilution curves of serum from intact and castrated rats, and the luteinizing hormone standard curve. Luteinizing hormone detected in serum samples from 30 intact rats by the improved bioassay and by radioimmunoassay was significantly correlated (r = 0.85). Secretion patterns of circulating luteinizing hormone in individual rats were similar when detected by either bioassay or radioimmunoassay. Thus, with the addition of forskolin, the mouse interstitial cell testosterone assay has been improved so that luteinizing hormone can be detected in small volumes of serum or plasma from male rats.     

Effects of veratridine on Ca fluxes and the release of oxytocin and vasopressin from the isolated rat neurohypophysis

Uptake of radioactive calcium, 45Ca efflux, and hormone release from the isolated rat neurohypophysis were monitored in vitro after the addition of veratridine to the incubation medium. Veratridine dramatically increased hormone release, but the release was not sustained and had declined by about 90% after 2 h. Removal of external Na+ prevented hormone release as did addition to the incubation medium of tetrodotoxin or the calcium antagonists D600 and Mn2+ ions. Veratridine increased 45Ca uptake into the isolated neurohypophysis and the increase could be prevented by addition of tetrodotoxin or D600 to the medium. Efflux of 45Ca was not changed by addition of veratridine. The results underline the importance of both Na+ and Ca+2 channels in the regulation of secretion of neurosecretory products.     

Cloned ovarian cells from sheep: Cholesterol dependency in progesterone synthesis

The TV4 cell line is derived from sheep ovarian tissues trypsinized for 60 min and developed from a clone after serial dilutions. The TV4 cells had a doubling time of 24 h in B2 medium with 10% fetal calf serum and 10% BSA. TV4 cells synthesized progesterone (P4) in the presence of cholesterol. As the concentration of cholesterol increased (0, 92.5 and 125 mg/l), synthesis of P4 increased (P<0.01) from 1.05 +/- 0.20 to 30.6 +/- 3.03 ng/ml. Kinetics of P4 production were determined; a linear production response (y = 5.816 + 1.05 x, y = ng/ml, x = hour of incubation; R(2) = 0.97) was observed with up to 35 ng/ml of P4 obtained by 30 h of incubation. Follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, or FSH and testosterone did not have any effect on estradiol-17beta (E2) or P4 production. Aromatase activity measured by RIA and HPLC following incubation with either nonradiolabeled or labeled testosterone was undetectable. In conclusion, this study established a cell line from the sheep ovary which has a high ability of divide and produce progesterone.     

Atrial natriuretic factor regulates steroidogenic responsiveness and cyclic nucleotide levels in mouse Leydig cells in vitro

The effects of synthetic atrial natriuretic factor (ANF) on the regulation of mouse Leydig cell steroidogenesis have been studied in vitro. ANF in nanomolar concentration increased testosterone production by more than 30-fold over basal levels. Concomitantly, cyclic guanosine monophosphate levels were increased 35-fold; cyclic adenosine monophosphate levels fell minimally (15-20%). ANF at low concentration (1 X 10(-11) M) inhibited testosterone production by luteinizing hormone-stimulated cells, while at higher concentration (greater than 2 X 10(-9) M) ANF stimulated steroidogenesis beyond the level attained by luteinizing hormone alone. These results indicate that ANF can exert stimulatory effects on testosterone steroidogenesis in vitro, and that the mechanism may involve an intracellular messenger other than cyclic adenosine monophosphate.     

Regulation by calcium of prolactin and growth hormone mRNA sequences in primary cultures of rat pituitary cells

Addition of Ca2+ to primary cultures of female pituitary cells incubated in serum-free medium lacking added Ca2+ yielded no effects on levels of prolactin or growth hormone mRNA, assayed by cytoplasmic dot hybridization. However, incubation of the cells in serum-free medium containing sufficient ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to reduce medium Ca2+ levels below the 10-40 microM present as a trace contaminant yielded a decrease in the levels of both mRNAs. The decrease was dose-dependent at extracellular Ca2+ concentrations below 1.0 microM, had an apparent half-maximum at about 0.3 microM, and did not appear to plateau with increasing incubation times. Following 2-3-day incubations of cells in low Ca2+, a reduction of prolactin mRNA (23-70-fold) consistently greater than the reduction of growth hormone mRNA (9-15-fold) was observed. Similar effects of reduced extracellular Ca2+ were obtained with primary cultures of male pituitary cells. The specificity of these effects of lowered extracellular Ca2+ was demonstrated by the following observations. The decreases in these mRNAs were substantially reversible by readdition of Ca2+ to the incubation medium. Reduction of extracellular Ca2+ led to no detectable changes in cellular ribosomal RNA levels or over-all RNA synthesis. In male pituitary cells, the level of another metal-regulated mRNA, that for metallothionein, was not decreased by a reduction of extracellular Ca2+ that caused a 40-fold decrease in levels of prolactin and growth hormone mRNA. Hence, Ca2+ exhibits specificity in its regulation of pituitary prolactin and growth hormone gene expression.     

Role of calcium in the modulation of ornithine decarboxylase activity in isolated pig granulosa cells in vitro

We examined the role of Ca(2+) in the control of basal and hormone-stimulated ornithine decarboxylase activity in isolated pig granulosa cells maintained under chemically defined conditions in vitro. Omission of Ca(2+) from the incubation medium (measured Ca(2+) concentration 5mum) decreased basal enzymic activity, and significantly (P<0.01) impaired the response to maximally stimulating doses of either lutropin or follitropin. No significant alteration occurred in the concentration of either gonadotropin required to elicit half-maximal effects. The addition of EGTA (1.27-2.0mm) to chelate residual extracellular Ca(2+) further decreased hormone-induced rises in ornithine decarboxylase activity. Despite the presence of 1.27mm concentrations of extracellular Ca(2+), the administration of presumptive Ca(2+) antagonists, believed to impair trans-membrane Ca(2+) influx [verapamil (10-100mum), nifedipine (1-100mum) or CoCl(2) (1mm)] suppressed hormone-stimulated ornithine decarboxylase activity. The inhibitory effects of verapamil or of Ca(2+) omission from the medium were not overcome by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.25mm), or by cholera toxin, or by an exogenously supplied cyclic AMP analogue, 8-bromo cyclic AMP. Conversely, micromolar concentrations of a putative bivalent-cation ionophore, A23187, increased significantly the stimulation of ornithine decarboxylase activity by saturating concentrations of lutropin or 8-bromo cyclic AMP. Thus the present observations implicate Ca(2+) ions in the modulation of hormone action and cellular function in normal ovarian cells.     

Dependence of secretory responses to gonadotropin-releasing hormone on diacylglycerol metabolism. Studies with a diacylglycerol lipase inhibitor, RHC 80267

The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.     

Evidence for a role of protein kinase C in luteinizing hormone synthesis and secretion. Impaired responses to gonadotropin-releasing hormone in protein kinase C-depleted pituitary cells

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of phorbol esters and gonadotropin-releasing hormone (GnRH) in normal and protein kinase C (Ca2+/phospholipid-dependent enzyme)-depleted pituitary cell cultures. LH secretory responses of normal pituitary cells to GnRH were reduced but not abolished in Ca2+-deficient medium, consistent with the existence of extracellular Ca2+-dependent and -independent components of GnRH action. Both of these components could be elicited by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). The LH secretory responses to TPA and GnRH were additive only at low doses and converged to a common maximum at high concentrations of the agonists in the presence or absence of extracellular Ca2+. The release of stored LH by GnRH and TPA was accompanied by secretion of newly synthesized LH from 2 to 5 h during stimulation by either of the agonists. LH synthesis was increased in a progressive and dose-dependent manner by GnRH and TPA, and the ratio between newly synthesized and released hormone was near 1:2. TPA caused rapid and complete translocation of cytosolic protein kinase C to the particulate fraction of pituitary cells, followed by a progressive decrease in total enzyme content to approximately 10% after 6 h. Partial recovery of the cytosolic enzyme (to 20%) occurred after washing and reincubation for 15 h. Such kinase C-depleted cells showed prominent, dose-dependent reductions in the actions of GnRH and TPA on LH release and synthesis in both normal and Ca2+-deficient media. These observations support the hypothesis that protein kinase C participates in LH biosynthesis and secretion in pituitary gonadotrophs and is involved in the actions of GnRH upon these processes.