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Biomedical subjects

E L Watson

Publications and source records attributed to E L Watson.

At least 37 records · Page 2Linked to original sources
PubMed1989

Monovalent ion enhancement of beta-adrenergic-stimulated adenylate cyclase activity in mouse parotid gland.

Sodium activated basal adenylate cyclase at all concentrations of sodium examined (5-100 mM) and independently of GTP. Stimulation of adenhylate cyclase by the beta-adrenergic agonist, isoproterenol, was enhanced at all concentrations (5-100 mM) of sodium ions tested in the presence of GTP. Maximal enzyme activation under all conditions occurred between 25 and 50 mM NaCl. Enhancement of forskolin-activated adenylate cyclase by sodium did not require GTP nor was it affected by guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), a competitive inhibitor of GTP. The selectivity of adenylate cyclase for monovalent cations was Na+ congruent to K+. Lithium chloride produced an inhibition of hormone-activated adenylate cyclase. Sodium ions also enhanced isoproterenol- and forskolin-activated adenylate cyclase of submandibular gland membranes. In contrast to mouse parotid and submandibular glands, activation of mouse liver and brain adenylate cyclase activities by forskolin and isoproterenol was not enhanced by sodium ions. The tissue differences were not related to differences in potency of the agonists. These results suggest (1) that sodium ions may have a selective and positive regulatory role in hormonal activation of adenylate cyclase in mouse exocrine tissue, and (2) that sodium ions enhance hormonal activation of enzyme by interacting at a site on the adenylate cyclase complex which is independent of the hormone receptor (Rs) and the stimulatory guanine nucleotide binding protein (Ns).

Adenylyl Cyclases↗
PubMed1987

Calcium-dependent protein kinase reactions associated with parotid gland secretory granule membranes.

Rat parotid secretory granule membranes were examined for the presence of calcium-dependent protein kinase activities and kinase substrates. Protein kinase C (C-kinase), which is stimulated by certain phospholipids, was present in the membranes, as indicated by its ability to catalyze the phosphorylation of histone. Two substrates for protein kinase C were seen in the granule membranes. The cytosolic fraction from the cell contained kinase activity, which was stimulated by phosphatidylserine and which caused the phosphorylation of two granule membrane polypeptides. In addition, when both granule membranes and cytosol were incubated together, phosphorylation of the cytosolic substrates was inhibited, indicating that the granule membrane substrates were phosphorylated preferentially. The results indicate that the granule membranes may react with cytosolic protein kinase C activity in a way which would direct an intracellular calcium and diacylglycerol signal toward the granule membrane. Since these signals occur during stimulation by various agonists, the mechanism may contribute to secretion.

Animals↗
PubMed1986

Forskolin activation of adenylate cyclase in mouse parotid membranes.

In mouse parotid membranes forskolin activated adenylate cyclase four-fold; maximal activation of the enzyme occurred with 10 microM forskolin. Activation was not dependent on the guanyl nucleotide GTP nor on the inhibitory guanine nucleotide 5'-0-(2-Thiodiphosphate), GDP beta S. In contrast, stimulation of adenylate cyclase by isoproterenol required GTP and was antagonized by GDP beta S in a dose-dependent manner. These results indicate that the guanyl-binding protein of mouse parotid adenylate cyclase is not a requisite for forskolin activation and lends support for direct interaction of forskolin at the catalytic subunit.

Adenylyl Cyclases↗
PubMed1986

Calmodulin activation and calcium regulation of parotid gland adenylate cyclase.

The effect of Ca2+ on the adenylate cyclase activity associated with membranes prepared from mouse parotid gland has been examined. Ca2+ stimulated then inhibited adenylate cyclase activity, with values for half-maximal stimulation and inhibition of 0.6 and 10 microM, respectively. Maximal activation (1.4-fold) was observed at 2 microM free Ca2+. These membranes contained 1.2 microgram calmodulin/mg protein. Exogenous calmodulin (0.2-1.2 microgram) activated, in a concentration-dependent manner, adenylate cyclase activity, with maximal activation being 2.5-fold at 12 micrograms calmodulin. Preparation of membranes in 2 mM ethyleneglycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacet ic acid (EGTA) resulted not only in a significant decrease in calmodulin levels (0.5 microgram calmodulin/mg protein) but also in a loss of the ability of Ca2+ to stimulate the enzyme. Exogenous calmodulin restored the ability of Ca2+ to stimulate the adenylate cyclase activity associated with EGTA-treated membranes. Trifluoperazine (50 microM) blocked the ability of Ca2+ to activate adenylate cyclase activity in control membranes. The effect of trifluoperazine could be reversed by exogenous calmodulin (0.5 or 5.0 micrograms). These data indicate that calmodulin mediates the activation of parotid gland adenylate cyclase by Ca2+ and that Ca2+, at concentrations which stimulate and inhibit amylase secretion, can activate and inhibit adenylate cyclase activity.

Adenylyl Cyclases↗
PubMed1985

Augmentation of cholinergic-mediated amylase release by forskolin in mouse parotid gland.

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated 45Ca2+ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10(-8)M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 X 10(-7) M) or hydroxylamine (50 microM) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism.

Amylases↗
PubMed1983

Diphenylhydantoin: effects on amylase release, cyclic nucleotides and ion fluxes in mouse parotid acini.

The effects of diphenylhydantoin were investigated on isolated mouse parotid acini. Diphenylhydantoin inhibited carbachol-stimulated amylase release at concentrations of 5 X 10(-4) M and greater; isoproterenol-stimulated amylase release was not affected. The inhibition of carbachol-stimulated amylase release was attributable to inhibition of 45Ca2+ uptake. The ability of diphenylhydantoin to inhibit carbachol-stimulated cyclic GMP (c-GMP) accumulation was also related to inhibition of 45Ca2+ uptake. Diphenylhydantoin (5 X 10(-4)M) alone, was found to stimulate c-GMP accumulation, an effect related to enhanced 45Ca2+ efflux. Diphenylhydantoin alone, however, failed to enhance amylase release. These findings suggest that the effects of diphenylhydantoin on carbachol-stimulated amylase release and on c-GMP accumulation are related to its effects on transmembrane calcium movements.

Amylases↗
PubMed1982

Does cyclic GMP mediate amylase release from mouse parotid acini?

In mouse parotid acini both cholinergic and beta-adrenergic agonists increased intracellular levels of cyclic-GMP (c-GMP) as well as amylase release. The derivative of c-GMP, 8-bromo-c-GMP, mimicked the effects of cholinergic and beta-adrenergic stimulation on amylase release. Nitroprusside (NP), hydroxylamine (HA) and sodium azide (NaA) increased c-GMP levels and also enhanced amylase release in a dose-dependent manner; cyclic-AMP (c-AMP) levels were not affected. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (MIX) enhanced the effects of carbachol on both c-GMP accumulation and amylase release. These results suggest that c-GMP may mediate the actions of cholinergic agonists and at least partially mediate the actions of beta-adrenergic agonists on mouse parotid enzyme release.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Effect of sodium ions on cyclic AMP and cyclic GMP levels in mouse parotid acini.

The ability of the beta-adrenergic agonist, isoproterenol, to elevate intracellular levels of cyclic-AMP (c-AMP) and cyclic GMP (c-GMP) in mouse parotid acini was dependent upon the extracellular sodium concentration. In the absence of extracellular sodium isoproterenol-stimulated c-GMP and c-AMP levels were significantly reduced; carbachol-stimulated c-GMP levels were not affected. Monensin, a sodium ionophore, mimicked the effects of isoproterenol in elevating c-GMP levels; this effect was abolished in the absence of extracellular sodium. Monensin did not mimic the effects of isoproterenol in elevating c-AMP levels. The data presented suggests that sodium ions may play a role in beta-adrenergic regulation of cyclic nucleotide levels in mouse parotid gland and that the mechanisms involved in regulation of c-AMP and c-GMP levels appear to be different.

Adrenergic beta-Agonists↗
PubMed1982

Cytochemical adenylate cyclase: localization in dispersed parotid acinar cells.

Rat and mouse parotid acinar cells have been dispersed into small acinar groups and individual cells. Amylase release in response to secretagogues has been determined. Cytochemical localization of adenylate cyclase in these cells has demonstrated isoproterenol-stimulated adenylate cyclase in the same location as the reported in tissue slices, at the extracellular aspect of the lumenal plasma membrane: adenylate cyclase activity was also visualized in completely separate cells, restricted to the lumenal pole of the cell. These results support the validity of other localizations of this enzyme in intact tissue blocks, and illustrate cytochemical similarity of cells in dispersed preparations to cells in tissue slices. The specialized location of plasma membrane adenylate cyclase at the locus of secretory granule exocytosis is retained even without intercellular junctions.

Adenylyl Cyclases↗
PubMed1981

Effects of monensin on amylase release from mouse parotid acini.

Isolated mouse parotid acinar cells (acini) were prepared by enzyme digestion, divalent cation depletion, and mechanical shearing. Acini were found to be morphologically intact, i.e., 95% viable as judged by trypan blue exclusion. Amylase release by the cholinergic agonist carbachol, by the beta-adrenergic agonist isoproterenol, and by monensin was similar to responses obtained in mouse parotid fragments. Monensin-stimulated amylase release was associated with enhanced 22Na+ uptake and 45Ca2+ efflux; monensin did not affect 45Ca2+ uptake. In the absence of extracellular Na+, the response to monensin (50 microM) was reduced from 162 +/- 33.5 to 12.4 +/- 0.5%; monensin also failed to stimulate 45Ca2+ efflux. Similar results were obtained with isoproterenol (10(-6) M). The results suggest that Na+ ions may play a role in amylase release possibly by releasing Ca2+ from internal stores.

Amylases↗
PubMed1979

Calcium mediation of cholinergic-stimulated amylase release from mouse parotid gland.

Amylase release from mouse parotid fragments was stimulated independently by cholinergic and beta-adrenergic agents. The cholinergic agonist, carbachol, significantly increased release of amylase only in Ca2+ containing medium whereas isoproterenol-stimulated amylase release was unaffected by Ca2+ removal. The ionophore, A23187, mimicked the effect of cholinergic stimulation when Ca2+ was present in the medium. Uptake of 45Ca2+ into tissue fragments was enhanced by carbachol and A23187 but not by isoproterenol; atropine blocked the effect of carbachol. Diphenylhydantoin (DPH) and verapamil partially inhibited carbachol-stimulated amylase release and 45Ca2+ uptake, whereas diazoxide potentiated these effects; in all cases there was good parallelism between 45Ca2+ uptake and amylase release. It was concluded that the primary step in the release of amylase from mouse parotid gland in response to cholinergic agents is an increased influx of Ca2+.

Amylases↗
PubMed1978

Effects of ionophores A23187 and X537A on vascular smooth muscle activity.

The ionophore A23187 initiated contractions in both dog coronary artery and rabbit aortic strips in a dose-dependent manner whereas X537A contracted rabbit aortic strips and relaxed KCl-induced contractions in dog coronary artery. Diphenylhydantoin (DPH) and verapamil ccompletely abolished KCl-induced responses in both vascular tissues. DPH (10(-4) M) partially prevented A23187-induced responses in both tissues and verapamil (33 micrometer) partially prevented A23187-induced responses in dog coronary artery but not in rabbit aorta. Phenoxybenzamine (10(-5) M), but not practolol, significantly reduced X537A-induced contractions in rabbit aortic strips but did not affect A23187-induced contractions in either tissue. The inability of DPH and verapamil to completely block the A23187 contractions leads one to conclude that these agents do not completely block calcium influx, or that A23187 does not work solely by increasing the permeability of the cell membrane to calcium. The effect of X537A on rabbit aorta, however, may be mediated at least partially, via release of catecholamines.

Animals↗