Control of thyroid secretion: effects of stimulators of protein kinase C, thyrotropin, and calcium mobilization on secretion of iodinated compounds from sheep thyroid cells.

We have compared and contrasted the abilities of TSH and agents capable of discretely activating the cAMP-dependent protein kinase, protein kinase C, or calcium mobilization to influence the secretion of iodinated compounds from cells prelabeled with iodide and blocked from further organification with methimazole. We found that calcium mobilization induced by A23187, protein kinase C activation induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) and TSH all stimulated the secretion of iodinated compounds. The effects of TSH were mimicked by forskolin and those of TPA by a synthetic diacylglycerol, sn-1,2-dioctanoylglycerol. The effects of TPA were partially inhibited by staurosporine whereas those of TSH were not. Epidermal growth factor and norepinephrine were without effect on thyroid secretion. The effects of A23187 and TPA were synergistic. The effects of TSH and TPA were not and the increased secretion induced by either agent was partially prevented by the combination. Preincubation of cells with TSH desensitized the cells to further stimulation by TSH but the stimulatory effects of TPA were unaffected. Exposure of cells to medium without calcium also induced loss of iodinated compounds which was partially prevented by TSH or forskolin but not TPA. TSH did not stimulate the rapid production of inositol trisphosphate production. We conclude that the mechanisms by which TSH (through stimulation of cAMP) and stimulators of other intracellular pathways exert their effects on secretion of iodocompounds, differ. Activation of protein kinase C and acute production of inositol trisphosphate do not appear to be involved in the mechanism of action of TSH in stimulating thyroid secretion but calcium mobilization is implicated.     

Peroxide formation and glucose oxidation in calf thyroid slices: regulation by protein kinase-C and cytosolic free calcium

We have determined the effects of tetradecanoyl phorbol acetate (TPA) and of the calcium ionophore A23187 on two thyroid responses to TSH previously reported to be cAMP-independent. We observed that TPA and A23187, at doses of 1.0 microM, stimulated both hydrogen peroxide generation and glucose oxidation in calf thyroid slices. A subthreshold dose of A23187 (0.1 microM) added to a submaximal dose of TPA (0.5 microM) acted synergistically, stimulating H2O2 production to the same degree as a maximally effective dose of TSH (50 mU/ml). Forskolin (25 microM), a direct stimulator of adenylate cyclase, actually inhibited both glucose oxidation and hydrogen peroxide generation. Lithium chloride (25 mM) had no effect on either response, either in the basal state or with TSH stimulation. The calcium channel antagonist verapamil (50 microM) decreased the basal activity of glucose oxidation and peroxide generation but did not substantially inhibit the effect of TSH on H2O2 generation under the conditions studied. These data support the concept that TSH induces changes in the thyroid phosphatidylinositol metabolism which activates protein kinase-C (c-kinase) and raises cytosolic free calcium. These events appear to act in concert to mediate certain metabolic responses in differentiated thyroid tissue.     

The control of testicular androgen production in the goldfish: effects of activators of different intracellular signalling pathways

The putative roles of different signal transduction pathways in the regulation of testicular androgen production in goldfish were investigated. In addition to the role of the gonadotropin-adenylate cyclase pathway, which was studied using human chorionic gonadotropin and forskolin, we determined the effects of changes in intracellular calcium content and protein kinase C activation on androgen production using calcium ionophore A23187 and phorbol 12-myristate 13-acetate (PMA), respectively. Testis fragments incubated in vitro respond to hCG in a time- and dose-dependent manner with a resultant increase in the secretion of testosterone (T) and 11-ketotestosterone (11-KT). Although ineffective alone, PMA (400 nM) and A23187 (4000 nM) stimulate a small but significant increase (3-fold above basal) in T production. This response is minor compared to the up to 200-fold increase in T secretion observed in response to either hCG or forskolin. PMA (25-400 nM) alone and A23187 (250-4000 nM) alone inhibit the stimulatory actions of hCG on T production. Unlike PMA, the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect on hCG-stimulated T production. PMA and A23187 did not influence the effects of forskolin on T production, suggesting that the compounds exert their effects prior to adenylate cyclase activation. In summary, the present studies suggest that in addition to the stimulatory actions of the adenylate cyclase second messenger system, changes in intracellular calcium content and protein kinase C activation may modulate testicular androgen production in the goldfish.     

Stimulation of the adenosine 3',5'-monophosphate and the Ca2+ messenger systems together reverse dopaminergic inhibition of prolactin release

Dopaminergic inhibition of PRL release stimulated by agents that affect cytosolic Ca2+ concentrations, C-kinase activity, and cAMP levels was studied in perifused rat anterior pituitary cells cultured on cytodex beads. We used A23187 (20 microM) to increase intracellular Ca2+, the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 50 nM) to stimulate C-kinase, forskolin (10 microM) to increase intracellular cAMP, and 8-bromo-cAMP to mimic cAMP. Dopamine (10 microM) inhibited PRL release to 20-60% of the basal release within 10 min. After 30 min of preincubation with dopamine, the absolute amount of release stimulated by 100 nM TRH was strongly inhibited, although the pattern of release, a quick burst followed by sustained release at a lower rate, was the same in the presence or absence of dopamine. A23187 (20 microM) caused a rapid burst of PRL release that subsided within 10 min, and TPA (50 nM) caused a sustained release that began within 4 min and continued for at least 30 min. TPA and A23187 combined caused a rapid burst of release followed by a sustained phase of release similar to that caused by TRH. Preincubation with dopamine inhibited the absolute amount of PRL release caused by A23187 alone, TPA alone, or the two combined, although, as with TRH, the pattern of release remained the same. Forskolin (1 or 10 microM) or 8-bromo-cAMP (3 mM) induced a 1.5- to 2-fold increase in PRL release, and this was completely prevented by dopamine. Preincubation with both dopamine and 8-bromo-cAMP or forskolin restored the amount of release stimulated by TPA alone or TPA and A23187 in the presence of dopamine to the level of release stimulated by these agents in the absence of dopamine. Therefore, activating either the cAMP messenger system or the Ca2+ system alone will not abolish dopaminergic inhibition, but activating the two together will. These results suggest that dopamine blocks release by inhibiting both adenylate cyclase and a step in the Ca2+ messenger system.     

Generation of hydrogen peroxide in cultured porcine thyroid cells: synergistic regulation by cytoplasmic free calcium and protein kinase C

This study set out to elucidate the mechanism by which H2O2 generation is regulated in cultured porcine thyroid cells. We monitored continuously the effects of the calcium ionophore A23187 and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on H2O2 generation, using homovanillic acid and horseradish peroxidase. A23187 and TPA stimulated H2O2 generation. A23187 increased cytoplasmic free calcium and TPA activated protein kinase C. Generation of H2O2 is therefore regulated by cytoplasmic free calcium and protein kinase C. Exposure to A23187 or TPA augmented further the stimulation of H2O2 generation by TPA or A23187 respectively. Thus A23187 and TPA, by increasing cytoplasmic free calcium and activating protein kinase C respectively, synergistically activate H2O2 generation.     

A modulatory role for cyclic AMP in the control of thyrotrophin release: studies with forskolin and dibutyryl cyclic AMP

We have studied the effects of cyclic AMP (cAMP) on TSH secretion by cultured rat pituitary cells, using forskolin and dibutyryl cAMP (dbcAMP) to raise the cellular cAMP content by different mechanisms. Forskolin (10 mumol/l), a stimulator of adenylate cyclase, raised the cAMP content within 10 min, but had a more delayed effect on TSH release, with no significant stimulation for at least 6 h, but a clear dose-dependent effect at 24 h. Incubation with dbcAMP likewise increased TSH release after 6-24 h. By contrast, high cellular cAMP levels induced by either forskolin or dbcAMP augmented the TSH response to TRH at an early stage, before any detectable change in unstimulated TSH release. Pretreatment of cells with forskolin led to a parallel upward shift in the subsequent TRH dose-response curve, without a significant change in median effective dose or any change in cellular TSH content. These findings suggest that cAMP acts to increase the availability of TSH for acute release by TRH by modulation of an intracellular releasable hormone pool, and indicate synergistic interactions between the adenylate cyclase system and the phospholipid-calcium stimulus-release coupling mechanism of TRH.     

Protein kinase C activators and calcium-mobilizing agents synergistically increase GH, LH, and TSH secretion from anterior pituitary cells

A series of studies was designed to determine the effects of protein kinase C activators on TSH, LH, and GH release from anterior pituitary cells. A 15-min incubation of cultured pituitary cells with synthetic diacylglycerol or phorbol myristate acetate, stimulators of protein kinase C, increased GH, LH, and TSH release. Similarly phospholipase C, which liberates endogenous diacylglycerol, stimulated GH, LH, and TSH secretion. The potentiation of the effects of protein kinase C activators is achieved by calcium mobilization in various cell types. The results of the present studies show that calcium ionophore A23187 or calcium channel activator maitotoxin potentiate diacylglycerol-, phorbol ester-, or phospholipase C-induced GH, LH, or TSH release. These findings suggest that activation of protein kinase C by diacylglycerol and mobilization of calcium may be synergistically involved in the regulation of GH, LH, and TSH release.     

Role of the calcium-calmodulin system in the adenylate cyclase activity of vascular smooth muscle

In order to evaluate the relationship between the calcium-calmodulin system and the adenylate cyclase activity of vascular smooth muscle, we examined the effects of several calcium effectors on the basal and stimulated adenylate cyclase activity. Thoracic aortae were removed from Wistar rats and the tissues were homogenated with cold homogenizing buffer containing 1 nM EDTA. Membrane protein fraction of the smooth muscle was prepared by centrifugation at 37,000 g. In this procedure, endogenous guanine nucleotides and contractile proteins remained. The protein fraction was incubated with 2 mM EGTA, 50 microM trifluoperazine, 0.1 microM A23187 or 25 microM calmodulin under basal and stimulated (50 microM isoproterenol, 100 microM GTP and 50 microM forskolin) conditions. The adenylate cyclase activity was determined by a method modified in our laboratory using double isotope counting. Trifluoperazine reduced the basal adenylate cyclase activity significantly (p less than 0.01) as well as the stimulated enzyme activities. A23187 did not affect the basal enzyme activity, but elevated the isoproterenol stimulated enzyme activity significantly (p less than 0.05). EGTA did not affect the basal and stimulated adenylate cyclase activities. Calmodulin elevated the basal enzyme activity significantly (p less than 0.02), but did not affect the stimulated enzyme activities. These results suggest that the calcium-calmodulin system is necessary for maintenance of the adenylate cyclase activity of vascular smooth muscle cells. The calmodulin acting site is considered to be the catalytic subunit, and stimulation of the enzyme is accelerated by calcium ion.     

Growth hormone-releasing factor secretion from fetal hypothalamic cell cultures is modulated by forskolin, phorbol esters, and muscimol

The regulation of GRF secretion was studied using a fetal rat hypothalamic cell culture system. The cells were subjected to short term release experiments on days 10-18 after plating, and GRF secretion was assessed by RIA. The identity of GRF immunoreactivity in the incubation medium was confirmed by reverse phase liquid chromatographic analysis. Depolarization of the cells with 56 mM K+ evoked a 4-fold increase in basal GRF release. When cultures were pretreated for 6 days with the adenylate cyclase activator forskolin, basal GRF release was augmented in subsequent release experiments to levels 2-fold greater than those in the control cultures. In nonpretreated cultures, forskolin (1-100 microM) and the protein kinase C activator phorbol 12-myristate 13-acetate (10 nM-1 microM), stimulated basal GRF release in a dose-dependent fashion. The Ca2+ channel blocker verapamil (100 microM) significantly inhibited the GRF response to both forskolin and phorbol 12-myristate 13-acetate. The gamma-aminobutyric acid (GABA) agonist muscimol (0.1-10 microM) inhibited forskolin-stimulated, but not K+ stimulated, GRF release in a dose-dependent manner. This inhibition was reversed by the GABA antagonists bicuculline and picrotoxinin. Muscimol (10 microM) slightly suppressed basal GRF release. The present findings suggest that GRF secretion can be evoked by agents known to increase intracellular cAMP levels or activate protein kinase-C. They also support a role for GABA in the inhibitory control of GRF secretion.     

Adenylate cyclase system responsive to thyroid stimulating hormone (TSH) of porcine thyroid cells in primary monolayer cultures. Potential effect of forskolin on TSH-mediated adenylate cyclase stimulation

The TSH-responsive adenylate cyclase system was studied using porcine thyroid cells in a primary monolayer culture. Isolated porcine thyroid cells treated with collagenase were inoculated into 96 wells at the density of 5 X 10(4) viable cells/0.25 ml Ham F-12 containing 10% fetal bovine serum and cultured for 4 days in a humidified atmosphere with 5% CO2. Adenylate cyclase activities in the cells treated or non-treated with protein synthesis inhibitor were assayed in Hanks/20 mM Hepes buffer (pH 7.4) containing 1% BSA, 1 mM IBMX and various stimulants at 37 degrees C for 30 or 60 min. The reaction was stopped by adding ice-cold TCA, and cAMP content in the extract was measured by radioimmunoassay after treatment with water-saturated ether. The cultured thyroid cells had an adenylate cyclase system responsive to TSH, cholera toxin and forskolin. TSH (50 mU/ml) stimulated the activity about eight fold over the basal activity. Cholera toxin (1 microgram/ml) and forskolin (100 microM), however, were much stronger activators of the adenylate cyclase system. In the cells pretreated with cyclo-heximide (5 micrograms/ml) up to 24 hours, cAMP formation by TSH was potentiated 200 approximately 170% compared to that in non-treated cells, suggesting a suppression of an inhibitory mechanism dependent upon new protein synthesis. In contrast, forskolin (100 microM)-stimulation was greatly reduced to 30% of the control after 24-hour treatment. Cholera toxin (1 microgram/ml)-stimulation was significantly lessened or slightly reduced by the treatment. Although the ability of forskolin to act synergistically with TSH or cholera toxin was observed in non-treated cells, it was clearly unaffected and demonstrated in the cells treated with protein synthesis inhibitor. The mechanism(s) and site(s) of forskolin action still remain unclear. However, these observations are compatible with a two-site model of forskolin action. The direct activating site of forskolin appears to reside in a protein which is closely associated with the catalytic unit of adenylate cyclase system and has a relatively shorter half-life than other components of the system. The potential action of forskolin may reside in a more stable complex of an activated stimulatory guanine nucleotide binding component and catalytic unit of the adenylate cyclase system. Based on these results, it is likely that the primary monolayer culture of porcine thyroid cells is a good model to investigate the adenylate cyclase system in the thyroid, and that forskolin may potentiate the TSH-mediated stimulation of adenylate cyclase.     

Somatostatin inhibits corticotropin-releasing factor-stimulated adrenocorticotropin release, adenylate cyclase, and activation of adenosine 3',5'-monophosphate-dependent protein kinase isoenzymes in AtT20 cells

The mechanisms by which somatostatin (SRIF) inhibits CRF-induced ACTH secretion from AtT20 cells were characterized by comparing the effects of SRIF on cAMP production, adenylate cyclase activity, and activation of cAMP-dependent protein kinase isoenzymes with its effects on ACTH release. In isolated membranes, CRF (100 nM) stimulated adenylate cyclase activity 4- to 5-fold. SRIF inhibited CRF-stimulated adenylate cyclase in a concentration-dependent manner. However, maximal inhibition was 50%. SRIF did not inhibit basal adenylate cyclase or forskolin-stimulated cyclase in the absence of guanine nucleotides and had only small effects on forskolin-stimulated cyclase when assayed in the presence of guanine nucleotides. CRF (100 nM) induced small rises (2-fold) in intracellular cAMP levels which produced maximal ACTH release. SRIF inhibited basal and CRF-stimulated ACTH release in a concentration-dependent manner, and there was a good correlation between inhibition of ACTH release and inhibition of the activation of cAMP-dependent protein kinases in these cells. Thus, the effect of SRIF on CRF-induced ACTH release appeared to result from its effect on inhibition of adenylate cyclase. In the presence of 3-methylisobutylxanthine (MIX), CRF increased cAMP levels 20-fold and activated a greater proportion of cAMP-dependent protein kinase, but did not stimulate ACTH release more than CRF alone. Under these conditions, SRIF (100 nM) inhibited cAMP accumulation by 90%. ACTH release was also inhibited, but higher concentrations of SRIF were required to block ACTH release compared to cells incubated in the absence of MIX. Sufficient cAMP levels were achieved so that activation of cAMP-dependent protein kinases was only partially blocked. There was still sufficient cAMP to activate cAMP-dependent protein kinase to an extent equal to that seen with CRF without MIX. Similar effects of SRIF on cAMP accumulation and protein kinase activation were seen when cells were stimulated with forskolin. Our results demonstrate that SRIF inhibits ACTH release from AtT20 cells by inhibiting hormone-sensitive adenylate cyclase and thereby prevents the activation of cAMP-dependent protein kinases. However, under conditions where cAMP-dependent protein kinases are still sufficiently active to induce ACTH secretion, high concentrations of SRIF can inhibit ACTH release by a mechanism independent of cAMP-dependent protein kinase.     

Dopamine inhibits prolactin release when cyclic adenosine 3',5'-monophosphate levels are elevated

We purified lactotrophs from pituitary tumors induced by estrogen in ovariectomized female Fischer 344 rats from 80% of the population before to more than 90% after purification through a continuous Percoll density gradient. The percentage of lactotrophs was evaluated by immunofluorescence. The patterns of PRL release stimulated by 100 nM TRH, 20 microM A23187 (a Ca++ ionophore), 50 nM 12-O-tetradecanoyl-phorbol-13-acetate (a C-kinase activator), or combinations of these agents, or inhibited by 10 microM dopamine were similar in perifused primary cultures of tumor lactotrophs to patterns in cultures of anterior pituitary cells from female retired breeders used previously. In particular, dopamine completely inhibited the release stimulated by forskolin. Intracellular cAMP concentrations and PRL accumulation in the medium were measured in monolayer cultures of purified tumor lactotrophs. In 9 separate experiments, forskolin (10 microM) increased intracellular cAMP concentrations more than 60-fold above control after 30 min of incubation. Preincubation (30 min) with dopamine (10 microM) reduced the cAMP accumulation caused by forskolin, but levels were still at least 20-fold above basal levels in most experiments. PRL release was stimulated 2-fold with forskolin alone, but there was no stimulation of PRL release by forskolin in the presence of dopamine even though cAMP levels were elevated above basal. Therefore, a decrease in cAMP levels is not necessary to inhibit PRL release, and dopamine must have a mechanism for inhibiting PRL release in addition to inhibiting adenylate cyclase.     

Sensitization of adrenocortical cell adenylate cyclase activity to ACTH by angiotensin II and activators of protein kinase C

Exposure of bovine adrenocortical cells to optimal concentrations of angiotensin II (A II) resulted in an almost 2-fold enhancement of cellular cAMP accumulation in response to steroidogenic concentrations of ACTH. This effect was dose-dependent and transient, with a maximum after 4-6 min of treatment with A II. Activators of protein kinase C such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and 1,2-dioctanoyl-sn-glycerol mimicked that effect in a sustained fashion. The ACTH-sensitized state of the adrenocortical adenylate cyclase system induced by TPA exhibited also an enhanced response to forskolin. On the other hand, previous treatment of the cells by pertussis toxin suppressed any further effect of TPA. It is suggested that, following A II exposure, the Gi inhibitory components of the adrenocortical cell adenylate cyclase system may be inactivated, leading to increased response to ACTH. This process may involve protein kinase C activation, subsequent to intracellular generation of lipidic messengers resulting from accelerated phosphoinositide breakdown induced by angiotensin.     

Possible involvement of adenylyl cyclase-cAMP-protein kinase a pathway in somatostatin inhibition of growth hormone release from chicken pituitary cells.

Somatostatin (SRIF) reduces growth hormone releasing hormone (GRF)-stimulated growth hormone (GH) release from avian and mammalian adenohypophyseal cells. The present studies examined the intracellular mechanisms mediating SRIF inhibition of GRF-stimulated GH release from chicken pituitary cells. Increases (P less than 0.05) in GH release were observed in the presence of (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) a cAMP analog, 8-bromo-cAMP; (4) the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX) combined with GRF; (5) a tumor-promoting phorbol ester and protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA); (6) a diacylglycerol analog, 1,2-dioctanoyl-glycerol (DiC8); and (7) a calcium ionophore, A23187, alone and in combination with PMA. Somatostatin (10 ng/ml) reduced the release of GH stimulated by GRF, forskolin, and 8-bromo cAMP and the GRF-provoked release of GH in the presence of IBMX (P less than 0.05). Somatostatin, however, did not influence GH release in the presence of the protein kinase C activators, PMA or DiC8, or the calcium ionophore A23187. These data suggest that SRIF inhibits GRF-provoked GH release by reducing the ability of the cAMP-protein kinase A but not of the calcium or protein kinase C intracellular message pathways to stimulate GH release.     

Triiodothyronine inhibits phorbol ester-induced thyrotropin release from elutriated bovine thyrotrophs

The inhibitory effect of T3 on TSH release was studied on a population of thyrotroph-enriched cells prepared from bovine pituitary glands by centrifugal elutriation. The cells (2.0 X 10(5)/ml) were cultured for 2 days and then exposed to TRH, phorbol-12 myristate-13 acetate (PMA), or calcium ionophore (A23187) with or without 100 nM T3 for two different preincubation periods, 3 h and 24 h. Cytosolic TSH and release of TSH into the medium were measured by a specific RIA for bovine TSH. TRH (10 nM, 100 nM), PMA (100 nM, 1 microM, 10 microM), and A23187 (100 nM, 1 microM, 10 microM) increased TSH release in a dose-dependent manner. One-hundred nanomolar TRH, 10 microM PMA, and 10 microM A23187 increased TSH release maximally from 176 +/- 6 microU/ml (mean +/- SD, n = 4) to 240 +/- 40, 308 +/- 39, and 228 +/- 16, respectively. PMA and A23187 interacted synergistically in the release of TSH. Cytosolic TSH was not affected by TRH or A23187. PMA (100 nM) together with A23187 resulted in a decrease in cytosolic TSH. PMA alone (1 and 10 microM) decreased cytosolic TSH content to 84% and 77%, respectively, of the control level, suggesting that PMA enhances release of TSH. One-hundred nanomolar T3 had no effect on the basal release of TSH when given for 3 h, but resulted in a 47% decrease when administered for 24 h. The inhibitory effect of T3 on TRH-induced TSH release was found when the cells were preincubated with T3 for 24 h, but not for 3 h. In contrast, PMA-induced TSH release was significantly inhibited to 74% of induced levels by preincubation with T3 even for 3 h, and further inhibition occurred with an increase in preincubation time. These data suggest that the effectiveness of T3 depends on the mode of stimulation, and that the more immediate reaction observed with PMA induction may result from the interaction of T3 with the protein kinase C pathway.     

Forskolin, an activator of adenylate cyclase, stimulates pancreatic insulin, glucagon, and somatostatin release in the dog: studies in vitro

Forskolin, a direct activator of adenylate cyclase, stimulates cAMP production in islet cells. The effects of forskolin on the release of somatostatin, glucagon, and insulin were studied using the isolated, perfused dog pancreas. It was found that concentrations ranging from 0.075 microM-1 microM stimulated the secretion of somatostatin, glucagon, and insulin in a dose-related manner. The effects of 0.15 microM and of 0.6 microM forskolin were modulated by the prevailing glucose level with higher D and B and lower A cell responses at high (11 mM) than at low (2.8 mM) or zero glucose. In the absence of extracellular Ca++, forskolin (1 microM) possessed no stimulatory effect on pancreatic hormone secretion. Perfusion of 1 microM atropine, 1 microM propranolol, and 1 microM phentolamine had no effect on forskolin-mediated (0.3 microM) hormone output from pancreas. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (25 microM) elicited qualitatively similar hormone response to forskolin. In conclusion, the experiments demonstrate that forskolin is a potent, reversible, stimulus of pancreatic hormone secretion. Its effects are apparently not mediated via the sympathetic or parasympathetic nerve endings in pancreas. Forskolin may prove to be a valuable pharmacological tool in probing the role of the adenylate cyclase-cAMP system in pancreatic hormone secretion.     

Effects of calmodulin inhibitors on amylase secretion from rat pancreatic acini.

To investigate the role of calmodulin in stimulus-secretion coupling in pancreatic acinar cells, we studied the effects of W-7, a calmodulin inhibitor, and KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II), on amylase secretion from rat pancreatic acini. Calmodulin inhibitor (W-7, 100 microM) and Ca2+/CaM kinase II inhibitor (KN-62, 10 microM) reduced amylase secretion stimulated by cholecystokinin (CCK) or carbachol. W-7 and KN-62 also inhibited amylase secretion stimulated by both calcium ionophore (A23187) and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA). To clarify the role of calmodulin in the interaction of intracellular mediators, pancreatic acini were permeabilized with streptolysin O. Following permeabilization, amylase secretion was stimulated by submicromolar free Ca2+, and this Ca(2+)-dependent amylase secretion was enhanced by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), TPA or cyclic adenosine 3',5'-monophosphate (cAMP). W-7 and KN-62 had no effects on amylase secretion stimulated by Ca2+ alone, but inhibited the enhancement in Ca(2+)-dependent amylase secretion by GTP gamma S, TPA or cAMP. These data suggest that calmodulin plays an important role in Ca(2+)-dependent amylase secretion from pancreatic acinar cells and in the interaction between Ca2+ and other intracellular messengers.     

Comparison of patterns of prolactin release in GH4C1 cells and primary pituitary cultures

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA, an activator of C-kinase), the cation ionophore A23187, forskolin (an activator of adenylate cyclase) and thyrotropin-releasing hormone (TRH) on prolactin release from anterior pituitary cells in primary culture were investigated and compared to the effects of these same agents on prolactin release from GH4C1 cells. In both GH4C1 cells and primary pituitary cultures, 100 nM TRH increased prolactin release 3- to 5-fold within 4 min after the stimulation started. This peak response was followed by a fall to a sustained increased rate of release approximately 1.5-fold above the basal rate. The decline after the early peak was slower in primary cultures than in GH4C1 cells. Addition of 20 microM A23187 to primary cultures caused a rapid 2- to 4-fold increase in release that fell to basal values within 12 min after the stimulation started. In GH4C1 cells, A23187 caused a rise in prolactin release of less than 2-fold that was sustained longer than the rise seen in primary cultures. Perifusion of either type of cells with 50 nM TPA caused a rapid 2- to 2.5-fold increase in release that also was sustained for 30 min or more in both types of cells. Perifusion with combined TPA and A23187 caused a 3- to 5-fold increase in rate of release from each cell type that declined rapidly to a 2-fold sustained release in primary cultures, and that declined more slowly in GH4C1 cells. Forskolin, 1 microM, had only a small effect by itself, but potentiated the effect of TPA or combined TPA and A23187 in both types of cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormone secretagogues increase cytosolic calcium by increasing cAMP in corticotropin-secreting cells.

Corticotropin (ACTH)-releasing factor, vasoactive intestinal peptide, and catecholamines--hormones that stimulate ACTH secretion and cAMP generation--increased cytosolic calcium in AtT-20 cells. The increase in intracellular calcium is presumably a consequence of the stimulated cAMP synthesis, since forskolin, an activator of the catalytic unit of adenylate cyclase, and the cAMP analog 8-bromoadenosine 3',5'-cyclic monophosphate (8Br-cAMP) also increased the cytosolic levels of this ion. Pretreatment with somatostatin, a neuropeptide that inhibits stimulation of the adenylate cyclase system and the secretion of ACTH blocked the increase of cytosolic calcium. The effect of 8Br-cAMP, which bypasses the cyclase, was not inhibited by somatostatin pretreatment. The source of the increased calcium appears to be mainly extracellular. This is indicated by the inability of the secretagogues to increase cytosolic calcium in a medium deprived of this ion or in the presence of blockers of voltage-gated calcium channels. The involvement of calcium channels in the calcium rise evoked by the secretagogues was supported by experiments using the whole-cell patch-clamp technique. In these experiments 8Br-cAMP increased voltage-dependent calcium currents. These results suggest the following chain of events in the receptor-mediated elevation of cytosolic calcium and the concomitant release of ACTH from AtT-20 cells: hormone-receptor binding----cAMP synthesis----protein kinase activation----calcium channel activation----increase in cytosolic calcium----many steps----ACTH release. Phorbol myristate acetate, a compound which does not stimulate cAMP generation but enhances the release of ACTH in AtT-20 cells, decreased the cytosolic calcium level.     

Selectivity of quercetin inhibition on stimulated amylase release in rat pancreatic acini

Quercetin (Q) has been shown to inhibit Ca2+-dependent processes. The present study evaluates the effect of Q on amylase release stimulated by various agonists in dispersed rat pancreatic acini. Q inhibited amylase release stimulated by an optimal concentration of carbachol. The inhibition was dependent on Q concentration. Preincubation with Q was not necessary. Maximal inhibition (up to 60% of control) was reached at 50 microM of Q and was completely reversible. Full responsiveness of the acini to agonist stimulation was reestablished as early as 5 min upon the removal of Q. At 50 microM, Q inhibited stimulated amylase release by optimal concentrations of tetradecanoylphorbol-13-acetate (TPA) (10(-6) M), A23187 (3 x 10(-6) M), cholecystokinin C-terminal octapeptide (CCK-OP) (10(-9) M) and carbachol (3 x 10(-6) M), but not by vasoactive intestinal polypeptide (VIP) (3 x 10(-7) M). Instead, Q promoted amylase release stimulated by VIP. The inhibition of amylase secretion by Q occurred only at near optimal, optimal, and supraoptimal concentrations of TPA, A23187, CCK-OP, and carbachol. The potentiation effect of Q on VIP-stimulated amylase secretion was, however, seen at all concentrations of VIP used (10(-8) to 10(-6) M). Quercetin also inhibited protein kinase C activity from rat pancreas in a dose-dependent manner. Maximal inhibition (approximately 85%) was seen at 100 microM of Q. These results provide further support that the intermediary steps for stimulated enzyme secretion in pancreatic acini by TPA, A23187, CCK-OP, and carbachol involve calmodulin and/or protein kinase C, whereas VIP does not.(ABSTRACT TRUNCATED AT 250 WORDS)