UNIPROT:P01275 - FACTA Search

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Query: UNIPROT:P01275 (glucagon)
26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Isolation and culture techniques for hepatocytes from whole livers of the cynomolgus monkey, Macaca fascicularis, are described. Hepatocytes were isolated by two-step perfusion of livers, using collagenase with hyaluronidase; fructose and trypsin inhibitor were included to reduce cell loss. Yields from a single liver average 4 X 10(9) cells with viabilities of 90.8 +/- 5.7%. Cells, plated on collagen substrates, were assessed for changes in morphology and various marker enzyme activities over a period of 7 d in culture. Cells exhibited a morphology similar to that observed for this species in vivo; little change in attached and spread cells was observed over the length of time monitored. Enzyme activities for catalase, succinate dehydrogenase, and tyrosine aminotransferase were observed to decrease significantly (though considerable activity remained), whereas acid phosphatase and 5'-nucleotide phosphodiesterase remained unchanged. Activity of cytochrome P-450 reductase was observed to increase slightly for the first 2 d, then decrease to about 60% of initial levels. Activity of alpha-mannosidase was stable for 4 d but was observed to be increased at Day 7. Cells were observed to retain metabolic responsiveness, demonstrated by glucose production by both gluconeogenesis and glycogenolysis in response to glucagon stimulation. The monkey hepatocytes obtained by methods described here thus retain hepatocellular morphology and activity through at least 1 wk in culture without medium or culture modification.
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PMID:Isolation and culture of hepatocytes from the cynomolgus monkey (Macaca fascicularis). 197 77

In heart failure, an increase in the activity of the sympathetic nervous system takes place to maintain perfusion pressure to vital organs, resulting in increased levels of noradrenaline in the blood of these patients. This permanent stimulation produces a down-regulation of cardiac beta-adrenoceptors. Since noradrenaline acts primarily on the cardiac beta 1-adrenoceptor subtype, beta 1-adrenoceptors decrease in number, whereas the beta 2-adrenoceptor subpopulation remains unchanged in most instances. Consequently, the positive inotropic response to beta-adrenoceptor agonists is diminished. However, there is also a decrease in the positive inotropic effect of beta 2-adrenoceptor agonists, histamine and cAMP-phosphodiesterase inhibitors such as milrinone, whereas the positive inotropic effect of cAMP-independent Na(+)-channel activators such as DPI 206-106 and the effects of cardiac glycosides are not diminished. These observations suggest a more generalised alteration of the cAMP-adenylate cyclase system in the failing heart. Stimulatory guanine nucleotide-binding protein (Gs) couples receptors to adenylate cyclase that stimulate cAMP formation, such as beta-adrenoceptors, histamine receptors and glucagon receptors. In the failing human heart, Gs content has been reported to remain unchanged as compared with that in non-failing myocardium. However, there is a 35%-40% increase in inhibitory guanine nucleotide-binding proteins (Gi), which are involved in the receptor-mediated inhibition of adenylate cyclase. Taken together, two defects of the cAMP-adenylate cyclase system have been identified: an increase in Gi content and a decrease in the number of beta-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Alterations of the cAMP-adenylate cyclase system in the failing human heart. Consequences for the therapy with inotropic drugs]. 197 43

Glucagon exerts positive inotropic and chronotropic effects in the heart. Like its glycogenolytic effect in liver cells, the cardiac effects of glucagon are often correlated with adenylyl cyclase stimulation. Therefore, cyclic AMP-dependent phosphorylation of L-type Ca2+ channels might be involved in the inotropic effect of glucagon. There have been no reports, however, of the effects of glucagon on the cardiac Ca2+ current (ICa). Also, the physiological effects of glucagon could involve mechanisms other than stimulation of adenylyl cyclase. Here we show that glucagon enhances ICa in frog and rat ventricular myocytes. The effect of glucagon in rats resulted from a stimulation of adenylyl cyclase. In frogs, however, the effect of glucagon on ICa was smaller and occurred at a concentration tenfold lower than in rats, and adenylyl cyclase was not modified. In addition, cAMP potentiated the effect of glucagon on ICa in frog ventricle, which correlated with the observed inhibition by glucagon of low-Km cAMP phosphodiesterase activity. Therefore, this is an example of a hormone that affects cardiac function in a similar way to a variety of synthetic cardiotonic compounds, such as milrinone and Ro-20-1724. Inhibition of phosphodiesterase activity by glucagon may be essential in animals in which glucagon increases cardiac contractility but does not effectively stimulate adenylyl cyclase.
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PMID:Glucagon stimulates the cardiac Ca2+ current by activation of adenylyl cyclase and inhibition of phosphodiesterase. 215 95

Using experimentally derived data for the activities and kinetic constants of hepatocyte cyclic AMP phosphodiesterase isoenzymes together with the derived changes in adenylate cyclase activity, due to stimulation and subsequent desensitization by glucagon, a computer model was established to simulate hepatocyte cyclic AMP metabolism. The established ability of glucagon to activate the 'dense-vesicle' cyclic AMP phosphodiesterase by eliciting its cyclic AMP-dependent phosphorylation was shown on the model to be capable of eliciting a profound reduction in the glucagon-stimulated increase in intracellular cyclic AMP. This was consistent with experimentally derived observations using the compound ICI 118233 which was used to inactivate the 'dense-vesicle' enzyme selectively. The non-hydrolysable adenosine agonist N6 (phenylisopropyl)-adenosine (PIA), which prevents glucagon pre-treatment of hepatocytes blocking the ability of insulin to stimulate the peripheral plasma membrane cyclic AMP phosphodiesterase, is shown here to accentuate the ability of insulin to decrease glucagon-elevated intracellular cyclic AMP concentrations. This effect was obliterated using the compound ICI 63197, a selective inhibitor of the peripheral plasma membrane phosphodiesterase. Computer modelling studies, taking into account experimentally derived actions in insulin in activating the peripheral plasma membrane phosphodiesterase, confirmed the potential of this enzyme to decrease intracellular cyclic AMP concentrations. Modelling of the putative effect of an insulin 'mediator' in activating the two cyclic GMP-stimulated cyclic AMP phosphodiesterase isoenzymes was shown to elicit a decrease in intracellular cyclic AMP concentrations which was comparable to that caused by insulin's action on intact hepatocytes. The relative contribution of each phosphodiesterase form to the metabolism of hepatocyte intracellular cyclic AMP, together with an assessment of the potential effect of inhibition and activation of specific species, was evaluated using the computer model. These experimental and stimulation studies indicate that alterations in the phosphodiesterase activity of the 'dense-vesicle' enzyme, the peripheral plasma membrane enzyme, the cyclic GMP-stimulated cyclic AMP isoforms and the IBMX-insensitive PDE-MQ-II can elicit profound effects upon hepatocyte intracellular cyclic AMP concentrations.
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PMID:The use of selective inhibitors and computer modelling to evaluate the role of specific high affinity cyclic AMP phosphodiesterases in the hormonal regulation of hepatocyte intracellular cyclic AMP concentrations. 217 3

We have utilized the vibrating probe technique to examine transport by individual chloride cells in the short-circuited fish opercular epithelium. Variability in the steady state and in response to rapid perturbations, including fast-acting hormones and ion replacement, was analyzed. Negative short-circuit currents, corresponding to chloride secretion, were associated with the apical crypts of all but five of 386 chloride cells sampled. Average chloride cell short-circuit current and conductance were 2.7 +/- 0.1 nA and 87.7 +/- 3.8 nS, respectively, or 19 mA cm-2 and 620 mS cm-2 (resistance = 1.6 omega cm2) when normalized to apical crypt surface area. Exposure to 1 microM epinephrine rapidly inhibited the tissue short-circuit current by inhibiting the current pumped by all chloride cells, i.e. all chloride cells have adrenergic receptors. The time course of inhibition for each cell mirrored that of the whole tissue. Reversal of epinephrine inhibition of the tissue short-circuit current by glucagon and phosphodiesterase inhibition was by reversal of epinephrine's inhibition of individual chloride cells, and not by turning on cells which were previously inactive or uninhibited, or by stimulating nonchloride cells. A great amount of variability existed among chloride cells in the ability of these agents to reverse epinephrine-inhibited current. Likewise, considerable variability in the response of chloride cell conductance to these perturbations was observed, and in many instances a clear dissociation between current and conductance was noted. In the steady state, variability among cells in a single tissue always defined a linear relationship between chloride cell current and conductance with zero-current conductance intercept at zero. Equivalent circuit modeling indicates that the leak conductance of chloride cells within a single tissue always contributes the same proportion to the total individual chloride cell conductance, such that the ratio between the conductances of the active and leak pathways of chloride cells is constant. The leak pathway is almost certainly dominated by a sodium-selective paracellular pathway. The results suggest that these cells control the permeability of their paracellular pathway. A possible mechanism for this control is discussed.
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PMID:Vibrating probe analysis of teleost opercular epithelium: correlation between active transport and leak pathways of individual chloride cells. 241 Jun 19

Glucagon increases the rate of glycogenolysis in in vitro cultures of hepatic tissue from the axolotl Ambystoma mexicanum. The hormone causes an increase in the concentration of cyclic AMP in the tissue which is followed by activation of glycogen phosphorylase and subsequent breakdown of glycogen and release of glucose from the tissue. Insulin counteracts the glycogenolytic effect of glucagon by inhibiting the increase in tissue cyclic AMP concentration brought about by glucagon. This inhibitory effect of insulin is not seen in the presence of the phosphodiesterase inhibitor IBMX and so it appears that the initial action of insulin is a stimulation of cyclic AMP phosphodiesterase activity which lowers the tissue concentration of cyclic AMP and so counters the actions of hormones that act by raising the tissue concentration of cyclic AMP. This model for the mode of action of insulin is supported by the finding that insulin also interferes with the glycogenolytic actions of adrenaline, a second hormone which acts by raising tissue cyclic AMP concentrations.
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PMID:Glucagon and insulin regulate in vitro hepatic glycogenolysis in the axolotl Ambystoma mexicanum via changes in tissue cyclic AMP concentration. 241 34

Inoculation of golden Syrian hamsters with Venezuelan encephalitis (VE) virus results in a sustained diminution in glucose-stimulated insulin release that is correctable by cyclic (c) AMP analogs and phosphodiesterase inhibitors. This suggested the importance of directly measuring cAMP content in VE-infected and control islets in response to insulin secretagogues. The basal cAMP content of VE-infected islets (0.14 +/- 0.02 pmol/micrograms islet DNA) was approximately half that of control islets (0.27 +/- 0.02 pmol/micrograms islet DNA) (P less than 0.05). In the presence of 10 microM glucagon (and 3 mM glucose), the rate of cAMP generation in VE-infected islets was only half that of control islets. With 10 mM alpha-ketoisocaproic acid, the rates of cAMP generation were indistinguishable between control and experimental groups. In response to 20 mM glucose and 3-isobutyl-1-methylxanthine (IBMX) (a phosphodiesterase inhibitor), cAMP generation in VE-infected islets was 81% (NS) of the control rate. When a more specific phosphodiesterase inhibitor, RO 20-1724, was used with 20 mM glucose, cAMP generation in the infected islets was only 44% (P less than 0.001) of the control value. Insulin secretion over the perifusion period paralleled the cAMP levels. In the presence of 10 mM alpha-ketoisocaproic acid, there was no difference in insulin secretion between VE-infected and control islets, while there was a statistically significant (P less than 0.05) difference with 10 microM glucagon or 20 mM glucose (in 1 mM RO 20-1724). These data point to a defect in the cAMP generation system of VE-infected islets, although additional factors involved in insulin secretion may also be impaired by the virus.
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PMID:Virus-induced alterations in cyclic adenosine monophosphate generation in hamster islets of Langerhans. 241 63

The effects of submaximal doses of AlF4- to mobilize hepatocyte Ca2+ were potentiated by glucagon (0.1-1 nM) and 8-p-chlorophenylthio-cAMP. A similar potentiation by glucagon of submaximal doses of vasopressin, angiotensin II, and alpha 1-adrenergic agonists has been previously shown (Morgan, N. G., Charest, R., Blackmore, P. F., and Exton, J. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4208-4212). When hepatocytes were pretreated with the protein kinase C activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), the effects of AlF4- to mobilize Ca2+, increase myo-inositol 1,4,5-trisphosphate (IP3), and activate phosphorylase were attenuated. Treatment of hepatocytes with PMA likewise inhibits the ability of vasopressin, angiotensin II, and alpha 1-adrenergic agonists to increase IP3 and mobilize Ca2+ (Lynch, C. J., Charest, R., Bocckino, S. B., Exton, J. H., and Blackmore, P. F. (1985) J. Biol. Chem. 260, 2844-2851). In contrast, the ability of AlF4- or angiotensin II to lower cAMP or inhibit glucagon-mediated increases in cAMP was unaffected by PMA. The ability of AlF4- to lower cAMP was attenuated in hepatocytes from animals treated with islet-activating protein, whereas Ca2+ mobilization was not modified. These results suggest that the lowering of cAMP induced by AlF4- and angiotensin II was mediated by the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase, whereas Ca2+ mobilization was not. Addition of glucagon, forskolin, or 8CPT-cAMP to hepatocytes raised IP3 and mobilized Ca2+. Both effects were blocked by PMA pretreatment, whereas cAMP and phosphorylase a levels were only minimally affected by PMA. The mobilization of Ca2+ induced by cAMP in hepatocytes incubated in low Ca2+ media was not additive with that induced by maximally effective doses of vasopressin, angiotensin II, or alpha 1-adrenergic agonists, indicating that the Ca2+ pool(s) affected by agents which increase cAMP is the same as that affected by Ca2+-mobilizing hormones which do not increase cAMP. These findings support the proposal that AlF4- mimics the effects of the Ca2+-mobilizing hormones in hepatocytes by activating a guanine nucleotide-binding regulatory protein (Np) which couples the hormone receptors to a phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phosphodiesterase. They also suggest that Np, PIP2 phosphodiesterase, or a factor involved in their interaction is activated following phosphorylation by cAMP-dependent protein kinase and inhibited after phosphorylation by protein kinase C.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate. 242 66

Treatment of intact hepatocytes with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) potentiated the ability of glucagon to increase intracellular cyclic AMP concentrations. This effect was dose-dependent upon TPA, exhibiting an EC50 of 0.39 ng/ml and such activation was observed at both saturating and sub-saturating concentrations of glucagon. However, this stimulatory effect of TPA was completely abolished by the presence of the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine, when TPA now inhibited the glucagon-stimulated increase in intracellular cyclic AMP concentrations. It is suggested that, as well as inhibiting glucagon-stimulated adenylate cyclase activity, TPA also inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. Treatment of either hepatocyte homogenates or purified cyclic AMP phosphodiesterase with TPA failed to show any direct inhibitory effect of TPA on activity showing that TPA did not exert any direct inhibitory action on phosphodiesterase activity. However, homogenates made from hepatocytes that had been pre-treated with TPA did show a reduced cyclic AMP phosphodiesterase activity. It is suggested that TPA might inhibit cyclic AMP phosphodiesterase activity through phosphorylation by C-kinase.
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PMID:The phorbol ester TPA inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. 243 Aug 36

Vasoactive intestinal peptide (VIP) was originally isolated from porcine duodenum and considered to be a gut hormone. Recent evidence indicates that it may also be involved in reproductive functions. In this study, a possible action of VIP on steroidogenesis by cultured testicular cells was investigated. Neonatal testicular cells were treated in vitro with hormones for 3 days and medium steroid or cAMP content was measured by radioimmunoassay. Treatment of cultured cells with VIP (10(-9) to 10(-6) M) increased the production of testosterone, progesterone, and pregnenolone in a dose-dependent fashion. Testosterone production in response to 10(-6) M VIP was about 5-10% of that maximally induced by LH. Addition of methyl-isobutyl-xanthine, a phosphodiesterase inhibitor, to the VIP-containing cultures significantly enhanced production of testosterone by 13-fold, of progesterone by 9-fold, and of pregnenolone by 2.5-fold as compared to treatment with VIP alone. Additional experiments also showed a dose-dependent stimulation of cAMP production by VIP. The VIP-related hormones PHM-27, secretin, and glucagon also stimulated progesterone and testosterone production with a potency order (PHM-27 greater than secretin greater than glucagon) consistent with that observed for other VIP receptor-mediated actions. A direct stimulatory effect of VIP on Leydig cells was indicated in studies on steroidogenesis by testicular cells separated on a metrizamide density gradient. In these studies, VIP stimulated androgen production in an LH-responsive subpopulation of testis cells but failed to affect steroid production in non-LH-responsive cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Vasoactive intestinal peptide stimulates androgen biosynthesis by cultured neonatal testicular cells. 243 Aug 45

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