Biomedical subjects

L Vallar

Publications and source records attributed to L Vallar.

45 records · Page 3Linked to original sources

PubMed1987

Guanine nucleotides induce Ca2+-independent insulin secretion from permeabilized RINm5F cells.

The role of guanine nucleotides in insulin secretion was investigated in electrically permeabilized RINm5F cells. Ca2+ stimulated insulin release (EC50 approximately 2 microM Ca2+). The GTP stable analog, GTP gamma S, elicited insulin secretion at vanishingly low Ca2+ concentrations (less than 10(-11) M), slightly potentiated the response to intermediate Ca2+ levels, but exerted less than additive effects at maximal Ca2+ concentrations. The GDP analog, GDP beta S, inhibited both GTP gamma S- and Ca2+-stimulated secretion. The action of GTP gamma S was not mediated by cAMP, as the latter only enhanced Ca2+-induced secretion. In contrast, 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted insulin release at nonstimulatory Ca2+ levels as well as potentiating the Ca2+ response. GTP analogs stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), as assessed by inositol phosphate generation. However, this could not fully explain guanine nucleotide-induced secretion because: GTP gamma S-stimulated PtdInsP2 breakdown was totally dependent on Ca2+ and abolished at Ca2+ below 10(-11) M; at these Ca2+ levels, activators of protein kinase C were weak or ineffective secretagogues; the GTP analog Gpp(NH)p was much less effective than GTP gamma S in activating PtdInsP2 hydrolysis, while fully mimicking the effect on Ca2+-independent secretion. Both GTP gamma S-induced PtdInsP2 hydrolysis and insulin release were insensitive to pertussis toxin and cholera toxin. The findings point to a guanine nucleotide-regulated site in the activation of insulin secretion different from the known transmembrane signalling systems.

Animals↗

PubMed1987

Regulation of exocytosis in electrically permeabilized insulin-secreting cells. Evidence for Ca2+ dependent and independent secretion.

The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca2+ (EC50 = 2.4 microM). The stable GTP analogue GTP gamma S enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (less than 10 pM) Ca2+ concentrations. At optimal Ca2+ (10 microM) the effect of GTP gamma S was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A2. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca2+ dependent effects, (ii) phospholipase C was not activated in the absence of Ca2+ and insulin secretion due to the phorbol ester TPA displayed a different Ca2+ dependency, (iii) arachidonic acid did not elicit Ca2+ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca2+ or TPA is attenuated by the inhibitory guanine nucleotide GDP beta S, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.

Animals↗

PubMed1987

Effect of calcium on adenylate cyclase of rat anterior pituitary gland.

The effect of free calcium (Ca2+) on adenylate cyclase (AC) activity of rat anterior pituitary gland have been investigated in order to shed some light on the interrelationships between the two second messengers (cAMP and calcium) which operate in pituitary cells. Anterior pituitary homogenates or crude membranes preparations (obtained using buffers free of divalent cation chelators) were assayed and the concentrations of Ca2+ in the assay mixture containing EGTA were calculated by a computer program for each addition of CaCl2. A wide range of Ca2+ concentrations (from 2 X 10(-9) to 6 X 10(-4)M) was spanned. Ca2+ was found to markedly inhibit pituitary AC and the mathematical analysis of data indicated the presence of two inhibition The two KiS were: 1.78 +/- 0.48 X 10(-7) M and 2.47 +/- 0.52 X 10(-4) M for the homogenates and 1.71 +/- 0.45 X 10(-7) M and 3.15 +/- 0.85 X 10(-4) M for the membrane preparations. No stimulation of the enzyme could be detected at any Ca2+ concentration tested. Furthermore, because of our experimental conditions it is unlikely that there was substantial loss of endogenous calmodulin, or other calcium binding protein(s) required to mediate AC stimulation by calcium. The lack of a calcium-calmodulin stimulation of pituitary AC was confirmed by experiments with anticalmodulin drugs (trifluoperazine and calmidazolium, R24571) and experiments with EGTA-washed membranes in the presence of exogenous calmodulin. At any Ca2+ concentration, the same AC activity was observed in the presence and in the absence of anticalmodulin drugs or added calmodulin. The mechanism of pituitary AC inhibition by Ca2+ was investigated focusing on a range of Ca2+ concentrations near the Ki for the high affinity calcium site and thus similar to the intracellular Ca2+ concentrations. Ca2+ was found to act as a competitive inhibitor of the Mg2+ activation of AC and as a noncompetitive inhibitor with respect to the MgATP2-, the substrate of the enzyme. The effects of Ca2+ on AC were also studied in cell populations and tissues extremely rich in PRL-secreting cells (cell fractions purified from rat anterior pituitaries and human prolactinomas). The pattern of Ca2+ action was found to be nearly superimposable on that observed in total pituitary.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

PubMed1987

Lack of desensitization of adenomatous somatotrophs to growth-hormone releasing hormone in acromegaly.

The study was undertaken to investigate whether GHRH causes desensitization in GH-secreting adenomas in analogy to the normal situation. For this purpose, GH secretion after repeated GHRH administration to acromegalic patients in vivo and cultured adenomatous somatotrophs in vitro was studied. Six acromegalic patients and 6 normal subjects received 3 consecutive 50 micrograms GHRH rapid iv injections at 2-h intervals. Blood samples were drawn at 0, 15, 30, 60, and 120 min after each dose. The acromegalic patients had variable responses to the first injection and in each patient the second and third injections elicited serum GH responses that were quite similar to those after the first one. The GH increment, evaluated as net incremental area under the curve (mean +/- SE; nanograms per ml/120 min) was 2660 +/- 1501 after the first injection, 2176 +/- 1378 after the second injection, and 1978 +/- 1191 after the third injection; P = NS. On the contrary, in normal subjects a marked elevation of GH was observed only after the first injection (net incremental area under the curve, mean +/- SE after the first injection: 710 +/- 154; after the second injection: 6 +/- 39 and after the third injection: 108 +/- 28, P less than 0.01 vs. the first dose). The effect of in vitro GHRH pretreatment on the subsequent response to GHRH was evaluated in monlayer cultures from 12 GH-secreting adenomas. At 10(-8) M, GHRH significantly stimulated GH release from 8 adenomas. The GHRH pretreatment that was effective in inducing desensitization in cultured rat anterior pituitary cells, i.e. preexposure of the cultured cells to 10(-8) M GHRH for 4 h at 37 C, did not abolish the GH response to the subsequent challenge with GHRH (mean percent stimulation: 150 +/- 14% in untreated cells vs. 153 +/- 30% in pretreated cells; P = NS). Only in one adenoma, did GHRH-promoted desensitization occur. No modification was found in GHRH unresponsive adenomas. In the adenomas, not only the efficacy but also the potency of GHRH on GH release after GHRH pretreatment was of the same order of magnitude as in untreated cells. No desensitization to GHRH action occurred when the pretreatment time was prolonged up to 8 h and the GHRH concentration in the preincubation medium was increased to 10(-7) M.(ABSTRACT TRUNCATED AT 400 WORDS)

Acromegaly↗

PubMed1986

Morphological studies on mixed growth hormone (GH)- and prolactin (PRL)-secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule.

A morphological study was carried out on five mixed GH- and PRL-secreting pituitary adenomas, surgically removed from acromegalic patients with hyperprolactinemia, in order to verify whether the two hormones were contained in the same cell or in different cells. Double labeling with the protein A-gold immunotechnique was used to visualize the ultrastructural localization of the two hormones on ultrathin sections of the tumors. By means of this high resolution technique we found in all adenomas the presence of numerous (from 50-80% of the whole cell population) mammosomatotrophs, i.e. cells containing simultaneously PRL and GH. The occurrence of cells producing only GH (in four tumors) or only PRL (in one tumor) was also observed. In mixed cells GH and PRL were segregated in the same mixed granule. In one tumor granules positive only for GH together with mixed granules were found in the same cell. Immunofluorescence studies, at the light microscopic level, allowed us to clearly identify mammosomatotrophs only in two tumors. Double labeling using the gold immunotechnique appears therefore to be the most suitable experimental approach to detect the existence of mixed cells in plurihormonal adenomas. Our results support the idea that the frequency of mixed adenomas with mixed cells may be higher than that believed previously. The simultaneous presence of two hormones in the same secretory granule could explain why, in patients having mixed tumors, factors able to stimulate or inhibit the release of one hormone can also stimulate or inhibit the secretion of the other.

Adenoma↗

PubMed1985

In vitro studies on TSH secretion and adenylate cyclase activity in a human TSH-secreting pituitary adenoma. Effects of somatostatin and dopamine.

We have studied the in vitro TSH secretion and the adenylate cyclase (AC) activity of a human pituitary adenoma surgically removed from a hyperthyroid patient showing high serum TSH levels. The tumor appeared almost homogeneously constituted by cells positive for an anti-TSH-beta antiserum and showing the ultrastructural characteristics of the adenomatous thyrotrophs. Adenoma fragments released in vitro a large amount of TSH (148.4 microU/mg prot/30 min), alpha-subunit (35.5 ng/mg prot/30 min) and TSH-beta (10.1 ng/mg prot/30 min). The effects of somatostatin (GHRIH) and dopamine (DA) on the hormone release have been tested in vitro. Both agents markedly inhibited the release of intact TSH and TSH-beta whereas the release of alpha-subunit was less affected. The two agents were effective at concentrations higher than 10(-8)M. The ability of GHRIH and DA in modulating the AC activity was investigated in membrane fraction preparations. GHRIH inhibited AC at concentrations higher than 10(-7)M. The maximal inhibition was 32% at 10(-5)M. Conversely, DA slightly stimulated AC activity. This effects was not mimicked by the dopaminergic ergot CH 29-717, which was completely ineffective on the enzyme. These results suggest that: 1) in this TSH-secreting pituitary adenoma a normal secretory response to the inhibiting agents (GHRIH and DA) is present; 2) different mechanisms of transduction of the GHRIH and DA signals (cAMP dependent and cAMP independent) could be operating in this tumor.

Adenoma↗

PubMed1984

Presence of an adenylate cyclase dually regulated by somatostatin and human pancreatic growth hormone (GH)-releasing factor in GH-secreting cells.

We have investigated the effects of SRIF and human pancreatic GH-releasing factor-44 (hpGRF-44) on adenylate cyclase (AC) activity of male rat anterior pituitaries (in which somatotrophs are present in large proportion) and of human GH-secreting pituitary adenomas (which are almost homogeneously constituted by somatotrophs). The adenoma's responsiveness to both agents in terms of secretion was previously demonstrated in in vitro experiments. SRIF inhibited in a dose-dependent fashion the GH release from monolayer cultures of the tumors. The inhibition ranged from 32-66% at the maximal effective concentration (10(-6) M). hpGRF-44 stimulated GH release in a dose-dependent fashion. The stimulation was 78-172% at 10(-7) M. SRIF and hpGRF-44 markedly affected AC activity in both systems. SRIF elicited a pronounced inhibition of the enzyme activity in a dose-dependent manner. The inhibition was about 40% in the rat and ranged from 16-49% in adenomas at the maximal effective concentration (10(-5) M SRIF). The inhibitory effect was GTP-dependent. hpGRF-44 markedly stimulated AC activity. The stimulation was dose dependent and GTP dependent. The stimulation was about 650% in the rat and 26-350% in adenomas at the maximal effective concentration (10(-6) M). These results suggest the presence of a dually regulated (by SRIF and hpGRF-44) AC in GH-secreting cells; an involvement of cAMP in the intracellular mechanisms transducing the signals of SRIF and hpGRF-44 in somatotrophs.

Adenylyl Cyclases↗

PubMedDate not supplied

Combined effects of vasoactive intestinal peptide and dopamine on adenylate cyclase in prolactin-secreting cells.

VIP stimulates adenylate cyclase activity of male and female rat anterior pituitaries and human prolactinomas, while dopamine inhibits the enzyme activity of female rat pituitaries and prolactinomas. A dopamine inhibited cyclase can be detected also in male rats provided the enzyme activity is increased by VIP. The analysis of the dose-response curves for one agent (VIP or dopamine) in the absence or in the presence of the other indicates that the two agents exhibit a different pattern of interaction in the different systems. In fact, in female rat pituitaries and in human prolactinomas, the curves for dopamine +/- VIP and for VIP +/- dopamine were parallel, indicating that the two agents exherted their effects independently from one another. On the contrary, in male rat pituitaries, the curves were definitively non parallel, that is, the inhibitory effect of dopamine was greatly amplified by VIP. In no case was the apparent affinity (EC50) of one agent modified by the presence of the other. It is concluded that two different modes of interaction between stimulatory and inhibitory neurohormones might exist at the level of adenylate cyclase from anterior pituitary cells.

Adenoma↗

PubMedDate not supplied

Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas.

Gs and Gi are guanine nucleotide-binding, heterotrimer proteins that regulate the activity of adenylate cyclase, and are responsible for transferring stimulatory and inhibitory hormonal signals, respectively, from cell surface receptors to the enzyme catalytic unit. These proteins can be directly activated by agents such as GTP and analogues, fluoride and magnesium. Decreased amounts of Gs and Gi, and even the absence of Gs, have been described, whereas an altered Gs has been reported in a cultured cell line (UNC variant of S49 lymphoma cells), but has never been observed in human disease states. We have found a profoundly altered Gs protein in a group of human growth hormone-secreting pituitary adenomas, characterized by high secretory activity and intracellular cyclic AMP levels. In the membranes from these tumours no stimulation of adenylate cyclase activity by growth hormone-releasing hormone, by GTP or by fluoride was observed. Indeed, the last two agents caused an inhibition, probably mediated by Gi. In contrast, adenylate cyclase stimulation by Mg2+ was enormously increased. This altered pattern of adenylate cyclase regulation was reproduced when a cholate extract of the tumour membranes (which contains G proteins) was reconstituted with Gs-free, cyc- S49 cell membranes. Inasmuch as secretion from somatotrophic cells is known to be a cAMP-dependent function, the alteration of Gs could be the direct cause of the high secretory activity of the tumours in which it occurs.

Adenoma↗