The effect of nifedipine on CRF-41 and AVP-induced ACTH release in vitro

The effect of nifedipine on CRF-41- and AVP-induced ACTH release was examined using monolayer cultured rat anterior pituitary cells and pituitary halves. Nifedipine inhibited ACTH release induced by synthetic rat CRF-41 in two systems. In pituitary halves, CRF-41 significantly stimulated both ACTH release and cyclic AMP accumulation. Nifedipine inhibited CRF-41-induced ACTH release and the inhibitory effect of nifedipine on CRF-41-induced ACTH release was accompanied by parallel decrease of cyclic AMP levels in pituitary halves. Nifedipine did not inhibit AVP-induced ACTH release in pituitary halves, and AVP did not significantly affect cyclic AMP accumulation in pituitary halves. These results suggest that CRF-41 stimulates ACTH release through the intracellular cyclic AMP system and calcium-calmodulin system which are accelerated by the influx of extracellular calcium ions. Moreover, it is suggested that the calcium required for AVP-induced ACTH release is derived primarily from intracellular rather than extracellular sources.     

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.     

Factors influencing the release of cyclic AMP from mouse thyroid tissue stimulated by TSH in vitro

The accumulation of cyclic AMP (cAMP) in mouse thyroid tissue in response to TSH in the presence of 1 mM theophylline was accompanied by a release of the nucleotide from the tissue into the incubation medium. This cAMP release was almost rectilinearly related to the time of exposure to TSH, and rectilinearly related to the log concentration of TSH in the range 0.1-5 mU/ml. The cAMP release proved to be independent of the pre-incubation time up to 4 h, and took place also in the absence of methylxanthines when the cAMP level was low. The total cAMP accumulation in response to TSH was augmented by different inhibitors of protein synthesis but the fraction of the nucleotide that was retained intracellularly was increased only by puromycin. Dipyridamole had an effect similar to that of puromycin. Depolarization or treatment with ouabain did not change the distribution of cAMP between tissue and medium. It is concluded that the release of cAMP from thyroid tissue stimulated by TSH may take place under physiological conditions, that it seems to be regulated by the actual concentration of TSH, and that it may be of significance for the regulation of the intracellular cAMP level.     

Inhibitory effects of cyclic AMP on vasopressin release from the rat neural lobe in vitro.

The basal release of vasopressin from the isolated neural lobe of the rat decreased in the presence of exogenous cAMP, 8Br-cAMP, diB-cAMP, theophylline, SQ 20,009 and RO20-1724. The concentration-related decrease in vasopressin release, in the presence of phosphodiesterase inhibitors, was accompanied by a progressive increase in cAMP concentration in the neural lobe. The findings suggest a local modulation of vasopressin release from the neural lobes.     

Modification of basal and GRF-stimulated cyclic AMP levels and growth hormone release by phospholipid metabolic enzyme inhibitors

The relative importance of several phospholipid pathways in cyclic AMP (cAMP) metabolism and growth hormone (GH) release was determined by an indirect, pharmacological approach in cultured anterior pituitary cells. The diglyceride lipase inhibitor RHC-80267 (30-100 microM) had no significant effect on cAMP levels but markedly inhibited basal and growth hormone-releasing factor-(GRF) stimulated GH secretion. A phospholipase A2 inhibitor quinacrine (30 microM) increased cellular cAMP content while decreasing GH release. Indomethacin, which reduces cyclooxygenase activity, affected neither cAMP levels nor GRF-enhanced GH release; this drug (30-100 microM) did reduce basal GH release. The lipoxygenase inhibitors nordihydroguaiaretic acid and BW-755c both reduced basal and GRF-stimulated GH release in a concentration-dependent manner. Both agents had various effects on cAMP levels. These results suggest that phospholipid metabolism, through both the cyclooxygenase and lipoxygenase pathways, contributes to basal GH release, while the lipoxygenase route predominates in GRF-stimulated GH release in vitro. Interestingly, cAMP metabolism can be dissociated from GH release with some of these probes, indicating an action of phospholipid metabolites distal or lateral to the cAMP-generating system.     

Intervention of the calcium-calmodulin system in the release of growth hormone induced by somatocrinin

This study was carried out to evaluate the role of the calcium-calmodulin system in cellular mediation of somatocrinin action by cells producing growth hormone. We compared GH release to intra- and extracellular cAMP levels, after incubation of pituitary cells in monolayer culture with somatocrinin alone or somatocrinin associated with exogenous calmodulin, or specific calmodulin inhibitor (W13). Calmodulin (10(-6) M) decreases somatocrinin - induced cAMP levels, maintaining elevated GH levels. W13 (10(-5) M) increases somatocrinin - induced cAMP and GH levels. Thus GH release induced by somatocrinin is modulated by two interrelated systems, adenylcyclase - cAMP system and Ca++-calmodulin system.     

Parallel stimulation of cyclic AMP accumulation and LH and FSH release by analogs of LH-RH in vitro

Dose-response studies with des-(pyro) Glu-LH-RH, [Phe³] LH-RH, [Phe⁵] LH-RH [0-methyl-Tyr⁵] LH-RH, [Ile⁷] LH-RH and des-Gly¹⁰-LH-RH-ethylamide show concomitant stimulation of adenohypophyseal adenosine 3',5'-monophosphate (cyclic AMP) concentration, and LH and FSH release in rat anterior pituitary gland in vitro.Des-His²-des-Gly¹⁰-LH-RH-ethylamide, and inhibitor of the response to LH-RH in vivo, had no effect on cyclic AMP accumulation or on LH release in vitro. The present finding of parallel changes of intracellular cyclic AMP levels and hormonal release with LH-RH analogs having a wide spectrum of biological activity adds strong support for a role of the cyclic nucleotide as mediator of the action of LH-RH in the anterior pituitary gland.     

Adrenaline inhibition of insulin release: role of cyclic AMP.

Catecholamines inhibit adenylate cyclase in pancreatic B-cells, but the importance of the resulting fall in cAMP concentration for the decrease in insulin release remains controversial. Adrenaline caused a dose-dependent inhibition (EC50 = 5.7 nM) of insulin release by mouse islets incubated in a medium containing 15 mM glucose. Supplementation of the medium with 500 microM dibutyryl-cAMP or 1 microM forskolin potentiated the effect of glucose on release and attenuated the inhibition by 1 and 10 nM adrenaline; the EC50 value was increased 2-fold. The inhibitory action of 100 nM or 1 microM adrenaline was, however, not affected. This apparent change in adrenaline potency was not simply due to the larger rate of release since it was not observed when the effect of glucose was potentiated by cytochalasin-B. However, when the same rate of insulin release as that produced by 15 mM glucose alone was achieved by combining 10 mM glucose and 250 microM dibutyryl-cAMP, the inhibitory potency of adrenaline was unaffected. Intracellular microelectrodes were used to determine whether the changes in B-cell membrane potential brought about by adrenaline are mediated by a fall in cAMP levels. Addition of dibutyryl-cAMP or forskolin to a medium containing 10 or 15 mM glucose increased the Ca(2+)-dependent electrical activity triggered by the sugar. However, this did not prevent adrenaline from hyperpolarizing the membrane transiently and causing a steady-state decrease in the intensity of the electrical activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro release and biosynthesis of tumor ACTH in ectopic ACTH producing tumors.

Tumor tissues obtained from 4 patients with the ectopic ACTH syndrome were studied for release and synthesis of tumor ACTH, using an in vitro incubation system. The effect of various agents on release of tumor ACTH was evaluated in three cases; beta-MSH released and adenosine 3',5'-monophosphate (cyclic AMP) formed in the tissue were determined in one. Biosynthetic experiments using labeled amino acid incorporation were performed in two cases. Secretion of tumor ACTH was significantly stimulated in all cases by crude rat median eminence extract which was also effective in stimulating beta-MSH secretion associated with elevated tissue cyclic AMP levels in one. Addition of cyclic AMP and dibutyryl cyclic AMP caused a significant increase in release of both tumor ACTH and beta-MSH in one. Biogenic amines (norepinephrine and serotonin) markedly elevated tussie cyclic AMP levels without a corresponding increase of hormone release in one. Incorporation experiments revealed that 3H- or 14C-phenylalanine was incorporated into immunoreactive ACTH of a larger molecular size (big ACTH) in both cases by chromatographic procedures. However, biological activity of big ACTH was found to be undetectable by an in vivo steroidogenic assay. A mild tryptic digestion of the big forms resulted in the appearance of little ACTH to which the major radioactive peak shifted. These data suggest that the mechanism of release of tumor ACTH and beta-MSH is very similar to that of the pituitary, and that intracellular cyclic AMP may in part play some role in release of both hormones. It is also suggested that some ectopic ACTH producing tumors predominantly synthesize big ACTH, a possible precursor of ACTH, with less bioactivity.     

Calmodulin involvement on the Ca++-dependent release of LHRH and SRIF in vitro

Mediobasal hypothalamic (MBH) slices of male adult rats were superfused at 37 degrees C with oxygenated Hepes-buffer Locke medium. Bacitracin (2 X 10(-5) M) was added to prevent enzymatic degradation of LHRH and SRIF. 6 min pulse of K+ (56 mM), veratridine (15 microM) or the ionophore A 23187 (10(-5) M), markedly stimulated the release of both neuropeptides. Trifluoperazine, a calmodulin inhibitor, decreased the K+-evoked LHRH and SRIF release in a dose-dependent manner; it was also effective in inhibiting the veratridine-induced neuropeptides release. Phenytoin, a calmodulin-dependent kinase inhibitor, also decreased in a dose-dependent manner the K+-induced LHRH and SRIF release; the basal release of both neuropeptides remained unaffected by either treatment. The ionophore-stimulated release of both neuropeptides was significantly inhibited as well. These data demonstrate that a Ca++-calmodulin kinase system may be involved in the mechanism of depolarization-induced LHRH and SRIF release from hypothalamic nerve terminals.