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R Goberna

Publications and source records attributed to R Goberna.

At least 37 records · Page 2Linked to original sources
PubMed1994

Pancreastatin inhibits insulin-stimulated glycogen synthesis but not glycolysis in rat hepatocytes.

The effect of rat pancreastatin on glycogen synthesis and glycolysis rate was studied in insulin-stimulated rat hepatocytes. We have determined the incorporation of [U-14C]glucose into glycogen as a measurement of the rate of glycogen synthesis; and the production of lactate as a measurement of the rate of glycolysis. Rat pancreastatin by itself did not affect either the rate of glycogen synthesis or glycolysis in rat hepatocytes from 6 h fasted rats. However, pancreastatin inhibited about 45% the insulin-stimulated glycogen synthesis whereas it enhanced the rate of glycolysis of insulin-stimulated hepatocytes about 25%. These effects were found to be dependent on pancreastatin concentration from 10(-11) M to 10(-7) M. Maximal effect was achieved at 10(-8) M and the half-maximal effect was observed at 0.3 nM. Pancreastatin decreased the rate of glycogen synthesis in a wide range of insulin concentrations (10(-12) - 10(-8) M). However, the effect on insulin-stimulated glycolysis was only observed at high concentrations of pancreastatin and insulin. These results suggest a role of pancreastatin in the possible mechanisms involved in insulin resistance.

Animals↗
PubMed1994

Pancreastatin activates pertussis toxin-sensitive guanylate cyclase and pertussis toxin-insensitive phospholipase C in rat liver membranes.

We have recently found the calcium dependent glycogenolytic effect of a pancreastatin on rat hepatocytes and the mobilization of intracellular calcium. To further investigate the mechanism of action of pancreastatin on liver we have studied its effect on guanylate cyclase, adenylate cyclase, and phospholipase C, and we have explored the possible involvement of GTP binding proteins by measuring GTPase activity as well as the effect of pertussis toxin treatment of plasma liver membranes on the pancreastatin stimulated GTPase activity and the production of cyclic GMP and myo-inositol 1,4,5-triphosphate. Pancreastatin stimulated GTPase activity of rat liver membranes about 25% over basal. The concentration dependency curve showed that maximal stimulation was achieved at 10(-7)M pancreastatin (EC50 = 3 nM). This stimulation was partially inhibited by treatment of the membranes with pertussis toxin. The effect of pancreastatin on guanylate cyclase and phospholipase C were examined by measuring the production of cyclic GMP and myo-inositol 1,4,5-triphosphate respectively. Pancreastatin increased the basal activity of guanylate cyclase to a maximum of 2.5-fold the unstimulated activity at 30 degrees C, in a time- and dose-dependent manner, reaching the maximal stimulation above control with 10(-7) M pancreastatin at 10 min (EC50 = 0.6 nM). This effect was completely abolished when rat liver membranes had been ADP-ribosylated with pertussis toxin. On the other hand, adenylate cyclase activity was not affected by pancreastatin. Phospholipase C activity of rat liver membranes was rapidly stimulated (within 2-5 min) at 30 degrees C by 10(-7) M pancreastatin, reaching a maximum at 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1994

Diminished insulin receptors on erythrocyte ghosts in nonobese patients with essential hypertension independent of hyperinsulinemia.

Hypertension is associated with insulin resistance and dyslipidemia in a syndrome named X. Epidemiologic evidence also supports a link between hyperinsulinemia and blood pressure (BP), independent of obesity and non-insulin-dependent diabetes mellitus. To assess the possible role of insulin receptors in this syndrome, we studied insulin binding by erythrocyte ghosts in patients with moderate essential hypertension with or without fasting or postglucose hyperinsulinemia. We measured plasma glucose and insulin before and at 30, 60, and 120 min after administration of 75 g glucose in 62 hypertensive patients and 20 matched normotensive controls. Both groups had comparable age (mean 45 years) and waist/hip ratios (mean 0.88). Patients undergoing antihypertensive treatment did not receive antihypertensive medication for 3 weeks. Patients with fasting or postglucose hyperglycemia were excluded from the study. Insulin binding to erythrocyte ghosts was significantly decreased (p < 0.001) to almost half the values of controls (6.5% specific binding) in both patients with hyperinsulinemic (3.2% specific binding) and those with normoinsulinemic (3.9% specific binding) hypertension. Scatchard analysis demonstrated that this was due to a lesser number of insulin receptors. These data indicate that patients with essential hypertension can show decreased erythrocyte insulin receptors without detectable hyperinsulinemia.

Adult↗
PubMed1994

Receptors for pancreastatin in rat liver membranes: molecular identification and characterization by covalent cross-linking.

High affinity binding sites for pancreastatin were identified for the first time, and their molecular characterization was performed with rat liver membranes. Using rat 125I-pancreastatin, we have studied the interaction of pancreastatin with liver membranes. Cross-linking of the tracer to the membranes was performed using the bifunctional reagent dithiobis(succinimidyl propionate). Analysis of binding under equilibrium conditions indicated the existence of one class of binding sites, with a Bmax of 15 fmol/mg of protein and an apparent Kd of 0.2 nM. The cross-linking of 125I-pancreastatin to liver membranes revealed a single band of M(r) 40,000, corresponding to the 125I-pancreastatin-receptor complex. The labeling of this complex was inhibited in the presence of rat pancreastatin (10(-10) to 10(-7) M) and in the presence of guanyl-5'-ylimidodiphosphate (10(-7) to 10(-4) M). Pretreatment of rat liver membranes with pertussis toxin did not affect pancreastatin binding or the inhibition by guanyl-5'-ylimidodiphosphate of pancreastatin binding. The specificity of pancreastatin binding was further assessed by displacement experiments with pancreastatin from other species and vasopressin. The binding of the pancreastatin-receptor complexes to Sepharose coupled to different lectins showed the glycoprotein nature of the pancreastatin receptor. These results strongly suggest that rat liver possesses a specific pancreastatin receptor, a glycoprotein of M(r) 35,000 that is coupled to a pertussis toxin-insensitive G protein in the plasma membrane.

Animals↗
PubMed1993

Pancreastatin increases free cytosolic Ca2+ in rat hepatocytes, involving both pertussis-toxin-sensitive and -insensitive mechanisms.

Freshly isolated rat hepatocytes, loaded with the Ca2+ probe Fluo-3, responded to homologous pancreastatin with a sudden increase in free cytosolic Ca2+ ([Ca2+]i) as well as glucose release. Addition of rat pancreastatin (0.1 microM) to hepatocytes resulted in an increase in [Ca2+]i from 150 nM to 700 nM, which declined back to nearly basal values within 2-3 min. Half-maximal and maximal effects were observed at 0.3 and 100 nM pancreastatin respectively. The increase in [Ca2+]i induced by vasopressin and noradrenaline was very similar in extent (from 150 to 800 nM) to that produced by pancreastatin. Neither the alpha 1-adrenergic blocker prazosin nor the vasopressin antagonist V1 modified the increase in [Ca2+]i induced by pancreastatin. Pig pancreastatin and its 33-49 C-terminal fragment produced about 65 and 75% of the effect of homologous pancreastatin respectively. Glucose production correlated with changes in [Ca2+]i in the same order of potency: vasopressin > rat pancreastatin > pig 33-49 pancreastatin > pig 1-49 pancreastatin. The effect of pancreastatin on [Ca2+]i was decreased by 50% when Ca2+ was omitted from the medium, and totally abolished when hepatocytes were depleted of internal Ca2+ stores by preincubation without Ca2+ and with 2 mM EGTA. When hepatocytes were preincubated for 5 min with PMA, the effects of ATP and noradrenaline were prevented, and those of vasopressin and pancreastatin remained unchanged. The pretreatment of hepatocytes with pertussis toxin diminished the response to pancreastatin and vasopressin. These results suggest that pancreastatin is a new Ca(2+)-mobilizing glycogenolytic hormone acting through a specific receptor which may involve both pertussis-toxin-sensitive and -insensitive GTP-binding regulatory proteins.

Adenosine Triphosphate↗
PubMed1993

Pancreastatin (33-49) enhances the priming effect of glucose in the rat pancreas.

Short-term exposure to glucose increases insulin secretion during subsequent stimulation. We investigated the effect of the new regulatory peptide pancreastatin on this priming effect of glucose in the perfused rat pancreas. Pancreastatin (33-49) at a concentration of 10(-8) M inhibited insulin release when stimulated by glucose at a concentration of 16.7 mM. However, after a second pulse of 16.7 mM glucose, pancreastatin potentiated the priming effect of glucose on insulin secretion. The modulation of insulin secretion by pancreastatin results in a potentiation of the priming effect of glucose in the rat pancreas, suggesting a role for pancreastatin in the adaptation of the B cell to glucose-stimulated insulin secretion.

Animals↗
PubMed1993

Sensitivity of insulin-secreting RIN m5F cells to undergoing apoptosis by the protein kinase C inhibitor staurosporine.

This study shows the sensitivity of insulin-secreting RIN m5F cells to undergoing apoptosis without modifying intracellular free calcium concentrations. The culture of cells in the absence of serum during 6 h failed to produce spontaneous DNA fragmentation. However, when cultures were carried out in the presence of the putative protein kinase C inhibitor staurosporine (1 microM), cells underwent apoptosis. Tumor-promoting phorbol ester failed to inhibit this effect. The presence of the chemotherapeutic drug bleomycin (0.6 mg/ml) in the culture medium reproduced the same pattern of cleaved DNA in nucleosomal pieces. Lower doses of staurosporine (0.1-1 nM) inhibited DNA synthesis but were unable to trigger apoptosis in 6 h culture. Higher doses of staurosporine (0.1-1 microM), which abolished DNA synthesis almost completely, were needed to trigger apoptosis. Short incubation of RIN m5F cells in the presence of staurosporine did not produce any change in the concentration of intracellular calcium or in the integrity of the plasma membrane. These results suggest the involvement of protein kinase C in RIN m5F cell survival.

Alkaloids↗
PubMed1992

Glycogenolytic effect of pancreastatin in isolated rat hepatocytes is mediated by a cyclic-AMP-independent Ca(2+)-dependent mechanism.

We have studied the effect of pig pancreastatin on glucose and lactate production in freshly isolated rat hepatocytes. Pancreastatin stimulated the rate of glucose output, whereas, in contrast with glucagon, it failed to modify the rate of lactate production. The effective concentration of pancreastatin was in the range 0.1-100 nM, with half-maximal rate close to 1 nM. The ability of pancreastatin to increase glucose output was abolished by chelation of the calcium in the medium. By itself, pancreastatin did not increase cyclic AMP (cAMP) levels and had no influence on cAMP levels in glucagon-stimulated hepatocytes. Our results point out a possible role of pancreastatin in glycogenolysis. This appears to be mediated by a cAMP-independent Ca(2+)-dependent mechanism.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Stimulatory effect of vasoactive intestinal peptide (VIP) on cyclic AMP production in rat peritoneal macrophages.

Vasoactive intestinal peptide (VIP) stimulated cyclic AMP production in rat peritoneal macrophages. The stimulatory effect of VIP was dependent on time, temperature and cell concentration, and was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). At 15 degrees C, the response occurred in the 0.1-1000 nM range of VIP concentrations. Half maximal stimulation of cellular cyclic AMP (ED50) was obtained at 1.2 +/- 0.5 nM VIP, and maximal stimulation (about 3-fold basal level) was obtained between 100-1000 nM. The cyclic AMP system of rat peritoneal macrophages showed a high specificity for VIP. The order of potency observed in inducing cyclic AMP production was VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, pancreastatin and octapeptide of cholecystokinin did not modify cyclic AMP levels at concentrations as high as 1 microM. The beta-adrenergic agonist isoproterenol increased the cyclic AMP production and show additive effect with VIP. Somatostatin inhibits the accumulation of cyclic AMP in the presence of both vasoactive intestinal peptide and isoproterenol. The finding of a VIP-stimulated cyclic AMP system in rat peritoneal macrophages, together with the previous characterization of high-affinity receptors for VIP in the same cell preparation, strongly suggest that VIP may be involved in the regulation of macrophage function.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes.

Apoptosis of freshly isolated rat hepatocytes was induced by either the omission of fetal bovine serum in the culture medium or addition of the protein kinase C inhibitors polymyxin B or staurosporine. The time-course of DNA breakdown into oligonucleosome-sized fragments and the activity of protein kinase C was determined. Hepatocytes were found to be sensitive to bleomycin which induced a high degree of DNA breakdown even within 30 min incubation. Both staurosporine and polymyxin B induced DNA degradation in hepatocytes after three hours incubation, an effect that was partially prevented by phorbol myristate acetate (PMA). After eight hours incubation, PMA failed to counteract this action and itself produced the apoptosis of rat hepatocytes. The results suggest the involvement of protein kinase C in hepatocyte survival.

Alkaloids↗
PubMed1992

Somatostatin inhibition of VIP- and isoproterenol-stimulated cyclic AMP production in rat peritoneal macrophages.

The dual regulation of cyclic AMP levels in rat peritoneal macrophages incubated with somatostatin, vasoactive intestinal peptide (VIP), and isoproterenol was studied. Somatostatin exerted a non-competitive inhibition of the stimulatory effect of VIP and isoproterenol on cyclic AMP production. In addition, somatostatin inhibited basal cyclic AMP levels. Our results suggest that somatostatin and VIP may modulate the immune response acting, through cyclic AMP, on macrophage functions.

Animals↗
PubMed1992

Pancreastatin increases cytosolic Ca2+ in insulin secreting RINm5F cells.

We have investigated the effect of pancreastatin on cytosolic Ca2+ concentration in the insulin secreting cell line RINm5F. Changes in [Ca2+]i induced by pancreastatin were detected by Fluo-3 fluorescence using both flow cytometry and batch analysis measurements, and turned out to be from 90 to 315 nM equivalent to 80% of that caused by ATP, which increased [Ca2+]i from 90 nM to 400 nM. This effect of pancreastatin did not depend on extracellular calcium and was not mediated by alpha-adrenergic receptors since it was not prevented by the alpha-blocker yohimbine. It is concluded that pancreastatin has a role in the homeostasis of free cytosolic calcium in the insulin secreting cell line Rinm5F.

Adenosine Triphosphate↗
PubMed1992

Chronic ethanol intake inhibits both the vasoactive intestinal peptide binding and the associated cyclic AMP production in rat enterocytes.

1. Chronic ethanol intake during 6, 8, 10 or 12 weeks resulted in a decrease of 125I-vasoactive intestinal peptide (VIP) binding to rat enterocytes. 2. Native peptide displaced 125I-VIP binding to enterocytes, exhibiting a IC50 at about 4 nM native VIP in control and ethanol-treated animals. 3. The number of binding sites in ethanol-treated animals were significantly diminished when compared to control animals. This reduction is observed in both the high-affinity and the low-affinity binding sites. 4. Increasing concentrations of native VIP produced a similar cyclic AMP rise in enterocytes from control or ethanol-treated rats during 6 weeks. However, after 8 weeks of ethanol treatment, a significant decrease in cyclic AMP production stimulated by VIP was observed.

Animals↗
PubMed1992

Pancreastatin and its 33-49 C-terminal fragment inhibit glucagon-stimulated insulin in vivo.

1. Pancreastatin, a 49 amino acid peptide derived from chromogranin A, has been shown to have an inhibitory effect on insulin secretion in the perfused pancreas and isolated islets. 2. We have studied the effect of pancreastatin on glucagon-stimulated insulin release and the hyperglycemic of glucagon effect in vivo. 3. When administered in the mesenteric vein, pancreastatin inhibited the increase in insulin levels induced by glucagon stimulation, thereby potentiating the hyperglycemic effect of glucagon. 4. This study describes a regulatory role of pancreastatin on glucagon-induced insulin release in vivo.

Animals↗
PubMed1992

Glucogenolytic and hyperglycemic effect of 33-49 C-terminal fragment of pancreastatin in the rat in vivo.

The effects of the 33-49 C-terminal fragment of pancreastatin on glycogen content, glycemia, insulinemia and glucagonemia were studied in the rat in vivo. It was found that after intramesenteric vein injection of the peptide, the glycogen content of liver decreased compared with control group injected with saline-1 < % BSA. Blood glucose levels were increased by the C-terminal fragment of pancreastatin. This study shows that the 33-49 C-terminal fragment of pancreatasin could play a role in glucose metabolism not mediated by insulin or glucagon.

Animals↗
PubMed1991

Characterization of functional receptors for vasoactive intestinal peptide (VIP) in rat peritoneal macrophages.

Functional vasoactive intestinal peptide (VIP) receptors have been characterized in rat peritoneal macrophages. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Scatchard analysis of binding data suggested the presence of two classes of binding sites: a class with high affinity (kd = 1.1 +/- 0.1 nM) and low capacity (11.1 +/- 1.5 fmol/10(6) cells), and a class with low affinity (kd = 71.6 +/- 10.2 nM) and high capacity (419.0 +/- 80.0 fmol/10(6) cells). Structural requirements of these receptors were studied with peptides structurally or not structurally related to VIP. Several peptides inhibited 125I-VIP binding to rat peritoneal macrophages with the following order of potency: VIP greater than rGRF greater than hGRF greater than PHI greater than secretin. Glucagon, insulin, somatostatin, pancreastatin and octapeptide of cholecystokinin (CCK 26-33) were ineffective. VIP induced an increase of cyclic AMP production. Half-maximal stimulation (ED50) was observed at 1.2 +/- 0.5 nM VIP, and maximal stimulation (3-fold above basal levels) was obtained between 0.1-1 microM. Properties of these binding sites strongly support the concept that VIP could behave as regulatory peptide on the macrophage function.

Animals↗
PubMed1991

Characterization of melatonin binding sites in the harderian gland and median eminence of the rat.

The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using [125I]melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37 degrees C after 30-60 min incubation. Binding was also dependent on protein concentration (up to 1.5 mg/ml). The specific binding of [125I]melatonin was saturable, exhibiting only one class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8 fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of [125I]melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the [125I]melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland.

Animals↗