Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.     

Activation of protein kinase C potentiates cyclic AMP production and stimulates steroidogenesis in differentiated ovarian granulosa cells

Gonadotropin-stimulated steroidogenesis in the differentiating ovarian granulosa cell is mediated through the activation of cAMP-dependent protein kinase, and is also modulated by calcium-dependent mechanisms. Granulosa cells contain calcium-activated, phospholipid-dependent protein kinase (C kinase), and show an increase in phosphatidylinositol turnover in response to GnRH agonist analogs. To evaluate the role of C kinase in ovarian steroidogenesis, the potent phorbol ester, TPA, and the permeant diacylglycerol, OAG, were used to activate C kinase in granulosa cells from PMSG-treated immature rats. Both TPA and OAG caused dose-dependent stimulation of progesterone production without affecting intra- or extracellular cAMP levels. However, the maximum steroid responses to these compounds were less than those stimulated by cAMP. The ED50 for TPA-stimulated progesterone production was 3 nM, which is close to the known Km for activation of C kinase. Stimulation of steroidogenesis was only observed with biologically-active phorbol esters and permeant diacylglycerols such as OAG and DOG. Exposure of granulosa cells to phospholipase C also increased progesterone production in a dose-dependent manner without changing the cAMP content. Although TPA and OAG did not increase basal cAMP production, both agents enhanced the cAMP responses stimulated by hCG and forskolin; likewise, phospholipase C alone did not change cAMP production but caused a dose-dependent increase in the cAMP responses to hCG and forskolin. These results demonstrate that activation of C kinase promotes steroidogenesis in ovarian granulosa cells, and potentiates the activation of adenylate cyclase by hCG and forskolin. Such findings support the possibility that the calcium, phospholipid-dependent enzyme could be involved in the regulation of progesterone production by hormonal ligands such as gonadotropins and GnRH.     

Cyclic AMP inhibits secretion from bovine adrenal chromaffin cells evoked by carbamylcholine but not by high K+

The role of cAMP in the control of secretion from bovine adrenal chromaffin cells was examined using the adenylate cyclase activator, forskolin. Treatment of chromaffin cells with forskolin resulted in a rise in cAMP levels. Forskolin inhibited catecholamine release elicited by carbamylcholine or nicotine but had no effect on secretion evoked by 55 mM K+. Inhibition of carbamylcholine-stimulated release by forskolin was half-maximal at 10 microM forskolin. The inhibition by forskolin of secretion evoked by carbamylcholine was at a step distal to the rise in intracellular free calcium concentration ([Ca2+]i), since this rise was not inhibited by forskolin, which itself produced a small rise in [Ca2+]i. The results suggest that secretion evoked by carbamylcholine is due to the activation of an additional second messenger pathway acting with the rise in [Ca2+]i. This additional pathway may be the target for cAMP action.     

Phospholipase A2 activation by melittin causes amylase release from exocrine pancreas

Phospholipase A2-induced deacylation of membrane phospholipids is associated with changes in membrane fluidity. The importance of this reaction in the pancreatic amylase secretory process was tested using melittin, a phospholipase A2 stimulating peptide. Phospholipase A2 activity (using [3H]arachidonic acid release as an index) and amylase secretion were both increased in a time- and concentration-dependent manner by melittin. Phospholipids prelabelled with [3H]oleic acid or [14C]linoleic acid also released radioactive free fatty acids in response to melittin. Prostaglandin synthesis was not involved in the melittin response, since inhibitors of arachidonic acid oxidation (indomethacin, 5,8,11,14-eicosatetraynoic acid) did not alter the ability of melittin to release [3H]arachidonic acid or amylase. When melittin was co-applied with carbachol, cholecystokinin octapeptide, or vasoactive intestinal peptide, amylase secretion was additive. The effect of melittin on both fatty acid and amylase release was dependent on extracellular calcium, though melittin's effects were not dependent on the intracellular accumulation of second messengers such as calcium or cAMP. The data suggest that activation of phospholipase A2 by melittin results in the triggering of the secretory process in exocrine pancreas by a different mechanism than that for other pancreatic secretagogues.     

Role of calcium and cAMP in the action of adrenocorticotropin on aldosterone secretion

When the dose-response curve of adrenocorticotropin (ACTH)-induced aldosterone secretion is compared to that of ACTH-induced intracellular cAMP, the ED50 for intracellular cAMP is more than 10 times as high as that for aldosterone production. In contrast, the dose-response curve of forskolin-induced aldosterone secretion correlates well with that for forskolin-induced intracellular cAMP. ACTH, but not forskolin, increases calcium influx into glomerulosa cells without inducing the mobilization of calcium from an intracellular pool. The effect of ACTH on calcium influx is dose-dependent and ED50 is 3.5 X 10(-11) M. In a perifusion system, the effect of 1 nM ACTH on aldosterone secretion is much greater than that of 1 microM forskolin, even though these two stimulators induce identical increases in the intracellular cAMP. Perifusion with combined A23187 (50 nM) and forskolin (1 microM) stimulates aldosterone secretion to a value comparable to that induced by 1 nM ACTH. Likewise, BAY K 8644 (1 nM), which induces a comparable increase in calcium influx, potentiates the effect of 1 microM forskolin. When the intracellular [Ca2+] is fixed at either 100 or 300 nM, forskolin-stimulated intracellular cAMP content is identical, but ACTH-stimulated intracellular cAMP content at 100 nM [Ca2+]i is 60% of that at 300 nM [Ca2+]i. Both the ACTH- and forskolin-induced aldosterone secretion rate is higher at 300 nM than at 100 nM [Ca2+]i. These results indicate that ACTH stimulates calcium influx, that calcium potentiates ACTH-induced but not forskolin-induced cAMP generation, and that Ca2+ and cAMP act as synarchic messengers in ACTH-mediated aldosterone secretion.     

Effects of luteinizing hormone-releasing hormone agonist and calcium upon adenylyl cyclase activity of human corpus luteum membranes

We have investigated the ability of the agonist analog of luteinizing hormone-releasing hormone (LH-RH), D-Trp6-LH-RH (LH-RHa), and of CaCl2 to inhibit directly gonadotropin stimulation of adenylyl cyclase in a cell-free system prepared from human corpus luteum. In the presence of a submaximally effective concentration of hCG, addition of 10(-5)M final concentration of LH-RHa did not alter the gonadotropin-stimulated enzyme activity, nor did LH-RHa alone show any effect upon basal levels of the enzyme. The failure to inhibit adenylyl cyclase would indicate that the LH-RHa does not affect gonadotropin receptor binding or cAMP synthesis and/or degradation in this membrane system, suggesting that the luteolytic effects of LH-RH are unlikely to involve a direct antigonadotropic activity at the level of the human corpus luteum. In great contrast to LH-RHa, addition of CaCl2 resulted in a dose-dependent inhibition of hCG-stimulable adenylyl cyclase. Thus, in the presence of either a maximally or submaximally effective concentration of hCG, inhibition was significant at 0.5 mM CaCl2 added in excess of ATP (2 mM) and EDTA (1 mM), being about 90% upon addition of 2.5 mM CaCl2. We also found that calcium reduced enzyme stimulation by forskolin and the GTP analog, guanyl 5'-yl imidodiphosphate [GMP-P(NH)P] in a dose-related manner and that activation by NaF was less sensitive to inhibition by calcium. Accordingly, at 2.5 mM CaCl2, guanyl nucleotide and forskolin stimulations were inhibited 96% and 86%, respectively, while NaF stimulation was reduced by 40%. Because previous studies have shown that calcium does not impair gonadotropin binding activity, the calcium-dependent inhibition of gonadotropin responsiveness reported here would imply an alteration in the functional coupling of the components of the luteal adenylyl cyclase system. These data suggest that calcium may play a role in the regulation of gonadotropin action in the human corpus luteum.     

Involvement of cAMP and calcium in the induction of ornithine decarboxylase activity in an osteoblast cell line

The role of cAMP and calcium in the induction of ornithine decarboxylase (ODC, E.C.4.1.1.17) activity in the osteogenic sarcoma cell line, UMR 106-01, was studied, with particular interest for parathyroid hormone (PTH). PTH and forskolin dose-dependently induced the ODC activity and the cAMP production. Protein synthesis is involved in the effect of PTH and forskolin on ODC activity but not on cAMP production. Using quin2 we showed that 20 nM PTH and 10 microM forskolin increased the intracellular ionized calcium concentration ([Ca2+]i), thereby offering the possibility for calcium to play a role as cellular mediator in the action of PTH and forskolin in bone. Data obtained with A23187 showed that solely an increase of the [Ca2+]i is not sufficient to stimulate basal or potentiate PTH- and forskolin-induced ODC activity. However, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced ODC activity point to a specific role for calcium. Moreover, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced cAMP production indicate that the involvement of calcium in the induction of ODC activity is primarily located at another site than the adenylate cyclase. These data indicate that calcium is involved in the control of basal ODC activity. Furthermore, these data suggest that both cAMP and calcium are involved in the induction of ODC activity by PTH and forskolin. More precisely, ODC activity in UMR 106-01 cells can be induced by PTH and forskolin via a calcium-dependent cAMP messenger system.     

Potentiating role of cyclic AMP in pancreatic enzyme secretion, demonstrated by means of forskolin

The role of cyclic AMP in the regulation of enzyme secretion by the rabbit pancreas has been investigated by means of forskolin, an activator of the catalytic subunit of adenylate cyclase. Forskolin increases the cyclic AMP level in isolated pancreatic acini in a dose-dependent way. Basal amylase release, however, remains unchanged. Forskolin potentiates the increase in amylase release induced by the C-terminal octapeptide of cholecystokinin (CCK-8). Potentiation is already apparent at hormone concentrations which are only marginally effective in stimulating amylase secretion. CCK-8 alone does not raise the cellular cAMP level, but it potentiates the forskolin-induced increase. In relative terms, potentiation is higher with decreasing concentration of forskolin. These results indicate that cAMP alone does not play a direct role in CCK-stimulated pancreatic enzyme secretion in the rabbit, but it potentiates enzyme secretion already stimulated through a cAMP-independent process.     

Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.     

Chloroquine inhibition of cholera toxin

Cholera toxin (CT) stimulated adenylate cyclase and a phospholipase which elevated cellular levels of 3',5'-cyclic adenosine monophosphate (cAMP) and arachidonic acid (AA). The AA was quickly converted to prostaglandins (PGs) via the cyclo-oxygenase pathway. Chloroquine exerted minimal inhibition of cAMP levels in CT-treated cells, although CT-induced release of [3H]AA and PGs was blocked completely when the drug was added in concentrations as low as 0.1 mM (50 micrograms/ml). Inhibition of [3H]AA release was complete when chloroquine was added before or within 30 min after CT. The capacity of chloroquine to inhibit either phospholipase C (PLC) or phospholipase A2 (PLA2) could explain the antisecretory activity of this drug.     

Mode of action of 2-(diethylamino)-7-ethoxychromone on human platelets.

The in vitro effect of 2-(diethylamino)-7-ethoxychromone (RC39XVIII) on human platelet aggregation induced by several agonists and on thromboxane B2 formation, granule release and intracellular cAMP elevation has been studied. The chromosome-derivative exerts a dose-dependent inhibitory effect on aggregation produced by U46619, arachidonic acid, thrombin, collagen and ADP. RC39XVIII inhibits aggregation, TxB2 formation and granule release in parallel. Moreover the drug potentiates cAMP accumulation induced by iloprost and forskolin. The drug also inhibits soluble cAMP phosphodiesterase in a dose-dependent manner. No effect on adenylate cyclase activity measured in platelet membranes was evident.     

Effects of chronic hypogonadism on corticosterone secretion and cyclic AMP production in male rat adrenocortical cells

AIM: To determine the secretion of corticosterone (CCS) both in vivo and in vitro during different intervals after orchidectomy in male rats. METHODS: Three- and 12-month-old rats had been orchidectomized 0, 3, 6, or 9 months before decapitation. RESULTS: Orchidectomy increased the concentrations of plasma CCS, the basal release of CCS, and the adenosine 3', 5'-cyclic monophosphate (cAMP) production in rat zona fasciculata reticularis (ZFR) cells. The forskolin/3-isobutyl-l-methylxanthine-stimulated releases of CCS and cAMP production by ZFR cells were higher in rats with chronic hypogonadism. The CCS release from ZFR cells of orchidectomized rat was not altered by 8-bromo-cAMP treatment. Orchidectomy enhanced the stimulatory effect of deoxycorticosterone on CCS release in ZFR cells. CONCLUSION: These results suggest that orchidectomy-related increases of CCS secretion in rats are associated with an increase of adenylate cyclase activity, cAMP generation, and 11-beta-hydroxylase activity in ZFR cells.     

Interleukin-1 beta- and forskolin-induced synthesis and secretion of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by transforming growth factor-beta 2.

Interleukin-1 beta and forskolin induce prostaglandin E2 release as well as 14-kDa group II phospholipase A2 gene expression and secretion of the enzyme from rat glomerular mesangial cells. We now report that pretreatment of mesangial cells with transforming growth factor-beta 2 prior to stimulation with interleukin-1 beta or forskolin inhibits the induced release of prostaglandin E2. At the same time the secretion of group II phospholipase A2, measured both as enzyme activity with sn-2-labeled phosphatidylethanolamine as substrate and as enzyme protein in immunoblot experiments, is dose-dependently inhibited by pretreatment of the cells with transforming growth factor-beta 2. Analyses of enzyme activity and enzyme protein levels in the cells indicated that this is not due to inhibition of enzyme secretion with a concomitant increase in cellular levels of the enzyme. Rather, pretreatment of the cells with transforming growth factor-beta 2 largely prevented both the interleukin-1 beta- and the forskolin-induced synthesis of phospholipase A2. This is the first report indicating an inhibition of group II phospholipase A2 gene expression by transforming growth factor-beta 2. In line with those results, transforming growth factor-beta 2 did not induce the synthesis and secretion of group II phospholipase A2. However, under conditions where the interleukin-1 beta-induced expression of group II phospholipase A2 is fully suppressed by transforming growth factor-beta 2, the growth factor itself stimulated prostaglandin E2 synthesis by a mechanism apparently not involving group II phospholipase A2. The immunochemical identification of the inducible and secretable phospholipase A2 from rat mesangial cells as a group II enzyme was confirmed by purification and N-terminal amino acid sequence determination.     

Cellular signals regulating the release of ANF.

Atrial natriuretic factor (ANF), a peptide hormone that regulates salt and water balance and blood pressure, is synthesized, stored, and secreted from mammalian myocytes. Stretching of atrial myocytes stimulates ANF secretion, but the cellular processes involved in linking mechanical distension to ANF release are unknown. We reported that phorbol esters, which mimic the action of diacylglycerol by acting directly on protein kinase C and the Ca2+ ionophore A23187, which introduces free Ca2+ into the cell, both increase basal ANF secretion in the isolated perfused rat heart. Phorbol ester also increased responsiveness to Ca2+ channel agonists, such as Bay k8644, and to agents that increase cAMP, such as forskolin and membrane-permeable cAMP analogs. In neonatal cultured rat atrial myocytes, protein kinase C activation by 12-O-tetradecanoylphorbol 13-acetate stimulated ANF secretion, whereas the release was unresponsive to changes in intracellular Ca2+. Endothelin, which stimulates phospholipase C mediated hydrolysis of phosphoinositides and activates protein kinase C, increased both basal and atrial stretch-induced ANF secretion from isolated perfused rat hearts. Similarly, phorbol ester enhanced atrial stretch-stimulated ANF secretion, while the increase in intracellular Ca2+ appeared to be negatively coupled to the stretch-induced ANF release. Finally, phorbol ester stimulated ANF release from the severely hypertrophied ventricles of hypertensive animals but not from normal rat myocardium. These results suggest that the protein kinase C activity may play an important role in the regulation of basal ANF secretion both from atria and ventricular cells, and that stretch of atrial myocytes appears to be positively modulated by phorbol esters.     

Isoproterenol-induced desensitization of mucin release in isolated rat submandibular acini

Release of [14C]glucosamine-labelled mucins was studied in vitro using well-characterised preparations of rat submandibular acini. Mucin release was stimulated by forskolin, an activator of the catalytic subunit of adenylate cyclase, and 3-isobutyl-1-methylxanthine (IBMX), a cyclic nucleotide phosphodiesterase inhibitor. Both stimulated in a dose-dependent manner to the same maximum as that seen with isoproterenol. Neither forskolin nor IBMX added in the presence of isoproterenol increased secretion above the maximum in response to isoproterenol alone, suggesting a similar mechanism of action, mediated by cyclic AMP. Prior exposure of acini to isoproterenol (10 microM) for 45 min, followed by washout resulted in (a) persistent increase in basal secretion which was abolished by propranolol and (b) reduced stimulation of mucin secretion in response to either a second isoproterenol challenge, noradrenaline or forskolin. Thus, exposure of rat submandibular acini in vitro desensitizes the cells to subsequent stimulation. Although this mimics the decreased beta-adrenergic secretory responses seen in submandibular cells from cystic fibrosis patients, results suggest that the isoproterenol-induced desensitization is at the level of beta-receptor and adenylate cyclase, rather than distal to cyclic AMP.     

Cytoplasmic Ca2+ in platelets is controlled by cyclic AMP: antagonism between stimulators and inhibitors of adenylate cyclase

Activation of platelets by thrombin rapidly increases cytoplasmic free calcium, [Ca2+]i, measured by Quin -2, and induces secretion. Stimulators of adenylate cyclase (i.e. PGI2, PGD2, forskolin) suppressed or reversed the increase of [Ca2+]i. Inhibitors of adenylate cyclase (i.e. epinephrine, ADP), added before or after thrombin, counteracted PGI2, PGD2 and forskolin and thereby increased [Ca2+]i and restored secretion. Responses to epinephrine (via alpha-2 adrenoreceptors) and ADP were independent of extracellular Ca2+, but required maintained occupancy of thrombin receptors and intact cAMP-phosphodiesterase activity. These results indicate that cAMP serves as an inhibitory second-messenger that antagonizes the mobilization of Ca2+, an activator second-messenger.     

Somatostatin inhibits pepsinogen secretion via a cyclic AMP-independent pathway.

Frog esophageal mucosa contains peptide glands which release pepsinogen in response to a variety of secretagogues and serves as a model to examine the inhibitory action of somatostatin. The pepsinogen secretion in response to bethanechol was inhibited by somatostatin in a noncompetitive fashion. The maximal response induced by bethanechol was reduced and the EC50 for bethanechol was increased in the presence of somatostatin. On the other hand, somatostatin showed essentially no effect on pepsinogen release evoked by ionophore A23187, dibutyryl cAMP or by forskolin in the presence of atropine. Atropine was included in the incubation mixture to eliminate the effect of acetylcholine released by forskolin from the intrinsic cholinergic neurons also present in the mucosa. Somatostatin did not exert any significant effect on the basal or the forskolin-stimulated cAMP accumulation in the mucosa, nor the basal or the forskolin-stimulated adenylate cyclase activity in the membranes of the peptic cells isolated from the mucosa. Thus, these results seem to suggest that somatostatin inhibits pepsinogen secretion from frog esophageal mucosa by a cAMP-independent pathway.     

Inhibition of basal and hormone-stimulated adenylate cyclase in adipocyte ghosts by the prostaglandin endoperoxide prostaglandin H2.

The prostaglandin endoperoxide PGH2 (15-hydroxy-9alpha, 11alpha-peroxidoprosta-5,13-dienoic acid), at a concentration of 2.8 x 10(-5) M inhibited basal adenylate cyclase activity 11% and epinephrine-stimulated activity 30 to 35%. PGH2 inhibited epinephrine-stimulated enzyme activity in the presence of 10 mM theophylline, 2.5 mM adenosine 3':5'-monophosphate (cAMP), or in the absence of inhibitors or substrates of the cAMP phosphodiesterase. When the cAMP phosphodiesterase was assayed directly using 62 nM and 1.1 muM cAMP, PGH2 did not affect the 100,000 x g particulate cAMP phosphodiesterase from fat cells. The inhibition of adenylate cyclase by PGH2 was readily reversible. A 6-min preincubation of ghost membranes with PGH2, followed by washing, did not alter subsequent epinephrine-stimulated adenylate cyclase activity. During epinephrine stimulation, the PGH2 inhibition was apparent on initial rates of cAMP synthesis, and the addition of PGH2 to the enzyme system at any point during an assay markedly reduced the rate of cAMP synthesis. Between 2.8 x 10(-7) M and 2.8 x 10(-5) M, PGH2 inhibited epinephrine-stimulated enzyme activity in a concentration-dependent manner. The stimulation of adenylate cyclase by thyroid-stimulating hormone, glucagon, and adrenocorticotropic hormone as well as by epinephrine was antagonized by PGH2, suggesting that PGH2 may be an endogenous feedback regulator of hormone-stimulated lipolysis in adipose tissue.     

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3',5'-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17beta-HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats.