Biomedical subjects

F Dowd

Publications and source records attributed to F Dowd.

18 recordsLinked to original sources

PubMed2006

Regulator of G-protein signaling 2 (RGS2) inhibits androgen-independent activation of androgen receptor in prostate cancer cells.

Hormones acting through G protein-coupled receptors (GPCRs) can cause androgen-independent activation of androgen receptor (AR) in prostate cancer cells. Regulators of G-protein signaling (RGS) proteins, through their GTPase activating protein (GAP) activities, inhibit GPCR-mediated signaling by inactivating G proteins. Here, we identified RGS2 as a gene specifically downregulated in androgen-independent prostate cancer cells. Expression of RGS2, but not other RGS proteins, abolished androgen-independent AR activity in androgen-independent LNCaP cells and CWR22Rv1 cells. In LNCaP cells, RGS2 inhibited G(q)-coupled GPCR signaling. Expression of exogenous wild-type RGS2, but not its GAP-deficient mutant, significantly reduced AR activation by constitutively activated G(q)Q209L mutant whereas silencing endogenous RGS2 by siRNA enhanced G(q)Q209L-stimulated AR activity. RGS2 had no effect on RGS-insensitive G(q)Q209L/G188S-induced AR activation. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) was found to be involved in RGS2-mediated regulation of androgen-independent AR activity. In addition, RGS2 functioned as a growth suppressor for androgen-independent LNCaP cells whereas androgen-sensitive LNCaP cells with RGS2 silencing had a growth advantage under steroid-reduced conditions. Finally, RGS2 expression level was significantly decreased in human prostate tumor specimens. Taken together, our results suggest RGS2 as a novel regulator of AR signaling and its repression may be an important step during prostate tumorigenesis and progression.

Adenocarcinoma↗

PubMed1995

Enhanced hypo-osmoregulation induced by warm-acclimation in antarctic fish is mediated by increased gill and kidney Na+/K(+)-ATPase activities.

Serum osmolality and serum inorganic ion concentrations were studied in two antarctic fish species, Trematomus bernacchii and T. newnesi, during 5 weeks of acclimation to 4 degrees C and compared with control values for groups acclimated to -1.5 degrees C. Acclimation to 4 degrees C significantly decreased the serum osmolality of both species, thereby increasing their seawater-to-extracellular fluid (ECF) osmotic gradient. The decline in osmolality with acclimation to 4 degrees C was accompanied by significant and rapid losses of Na+ and Cl- during the first 14 days of acclimation and was maintained throughout the study period. At day 35 of acclimation, the lipid composition and microsomal Na+/K(+)-ATPase specific activities at 4 degrees C and 37 degrees C were determined in membranes from gill, kidney, liver and muscle tissues. No warm-induced decrease in fatty acid unsaturation was found in the tissues of either species. In the gills and kidneys of both species, the Na+/K(+)-ATPase activities assayed at 4 degrees C were increased after acclimation to 4 degrees C. The Na+/K(+)-ATPase activities at 37 degrees C increased at the higher acclimation temperature in T. newnesi kidneys and T. bernacchii gills, but in both species there was no compensation to temperature in the liver, regardless of assay temperature. Muscle Na+/K(+)-ATPase activity decreased in response to warm-acclimation in T. bernacchii and T. newnesi assayed at 4 degrees C and 37 degrees C, respectively. During acclimation to 4 degrees C, the discontinuity in the Arrhenius plot of the Na+/K(+)-ATPase activities of T. newnesi gill moved to a lower temperature, whereas that of kidney remained unchanged. The results indicate that acclimation to 4 degrees C induced a decrease in serum osmolality which resulted from the positive compensation of Na+/K(+)-ATPase in osmoregulatory tissues. The enhancement in Na+/K(+)-ATPase activity at 4 degrees C suggests that energy expenditure in antarctic fish may be lessened, in part, by maintaining a reduced seawater-to-ECF osmotic gradient.

Acclimatization↗

PubMed1991

The effect of HCO3- on anion-stimulated ATPase from rat parotid granules.

ATPase from isolated secretory granules was stimulated in a concentration-dependent manner by HCO3- above 0.9 mM. Maximal stimulation was found at about 16 mM HCO3- and was about half of that with sulphite (SO3(2-)). The activation site(s) appeared to be similar to at least one class of SO3(2-) sites, HCO3(-)-stimulate ATPase was inhibited by SITS. Furthermore, maximal stimulation with SO3(2-) was not enhanced with HCO3-. At low Mg2+ concentrations, Ca2+ stimulated granule ATPase. At higher concentrations of Mg2+ (0.5 mM and above), Ca2+ at 0.1 mM or less had little effect on HCO3(-)-ATPase, and Ca2+ at 4 mM inhibited HCO3(-)-ATPase. At concentrations of Ca2+ above 0.44 mM, the enzyme was partially stimulated in the absence of Mg2+ and presence of HCO3-. Mitochondrial contamination did not account for the presence of ATPase in the isolated granule fraction. The granule ATPase may be regulated by HCO3- and calcium and this could be related to changes in the granule environment during exocytosis.

Adenosine Triphosphatases↗

PubMed1987

The effect of calcium and cyclic AMP on amylase release in digitonin-permeabilized parotid gland cells.

Rat parotid cells were permeabilized with digitonin to examine their secretory dynamics. Cells were isolated by a modification of the method previously described by Hootman [1985). J. Biol. Chem. 260, 4186-4194) in which alpha-chymotrypsin was included. The final preparation consisted of approx. 40-60% single cells. The cells were 85-90% viable by trypan blue exclusion and secreted amylase when stimulated with isoproterenol. Digitonin (2 or 5 microM) was sufficient for permeabilization while 2 microM digitonin was somewhat more effective in maintaining cell integrity as indicated by lactate dehydrogenase release. Digitonin had minimal effects on intracellular granules in the whole cell and was, thus, relatively selective. The response of digitonin-permeabilized cells to calcium (without secretagogues) in the incubation medium was monitored by amylase release. For a wide range of applied free calcium concentrations (1 X 10(-7) M to 10(-4) M) a statistically significant increase in amylase secretion was observed. Control cells did not release amylase to a similar extent without secretagogue. Cyclic AMP (50 microM) significantly enhanced amylase secretion from digitonin-treated cells at all concentrations of free calcium tested. Neither calcium nor cyclic AMP alone was sufficient to stimulate maximal amylase release. Our results provide direct evidence for a model in which calcium and cyclic AMP work on separate pathways as interacting regulators of exocytosis.

Adenosine Triphosphate↗

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↗

PubMed1985

Comparison of cyclic AMP-dependent protein kinases from salivary glands of four species.

Cyclic AMP-dependent protein kinase activity, subcellular distribution, and isozyme profile were compared in rabbit, rat, guinea pig, and mouse in both parotid and submandibular glands. Glands were homogenized under hypotonic conditions and the following fractions isolated: 600 g pellet, 27,000 g pellet, and 27,000 g supernatant. The specific activity of the enzyme was similar in the eight glands and was highest in the 27,000 g supernatant. The average activity in the 27,000 g supernatant was approximately 75% of the total gland activity, although there was considerable variability between tissues and species. After being washed with isotonic buffer, this percentage was increased to an average of 84%. When isozyme patterns of the kinase were examined, the rabbit parotid was unique in that it contained a high percentage of isozyme I as isolated on DEAE cellulose columns.

Animals↗

PubMed1984

Reserpinization: effects on parotid gland function.

Chronic reserpinization of mice and rabbits affected parotid gland function; cholinergic and beta-adrenergic-stimulated amylase release were significantly reduced, and cyclic-AMP and cyclic-GMP levels were significantly elevated above control-stimulated levels. Amylase released by dibutyryl-c-AMP was also reduced, suggesting a defect in stimulus-secretion coupling subsequent to c-AMP formation.

Amylases↗

PubMed1984

Isoproterenol-induced amylase release in rabbit parotid acini: relation of protein phosphorylation, cyclic AMP and related kinase activity to changes in secretory rate.

Isoproterenol-induced amylase release from rabbit parotid acini was examined in relation to cyclic AMP (cAMP) concentrations, cAMP-dependent protein kinase (cAMP-PK) activity ratios and protein phosphorylation. Initial stimulation of amylase release by isoproterenol was preceded by increases in cAMP, cAMP-PK activity ratios and phosphorylation of a 34,000 MW (major) and a 30,000 MW (minor) protein in the microsomal fraction. When propranolol was added, decreases in cAMP concentrations and cAMP-PK activity ratios preceded the reduction in amylase release. Detailed analysis was performed on the 34,000 MW protein. The relation of dephosphorylation of protein 34 and reduction in amylase release was complex. Slight dephosphorylation occurred before or concurrently with the decrease in amylase release; however, maximal dephosphorylation was preceded by maximal inhibition of amylase release. When secretion of amylase was reinstituted by isoproterenol or forskolin, increases in cAMP and cAMP-PK activity ratios occurred before or in concert with amylase release but rephosphorylation of protein 34 occurred after the start of amylase release. Photoaffinity labeling studies using [32P]-8-azidoadenosine-3',5'-cyclic monophosphate indicated that proteins 34 and 30 were not regulatory subunits of cAMP-PK or their breakdown products. Although these data are consistent with phosphorylation of proteins 34 or 30 being required for triggering initial secretion, maximum dephosphorylation was not essential for inhibition of secretion. Furthermore, initiation of amylase release by the gland after a short period of quiescence did not depend on prior phosphorylation of protein 34. These data may indicate the absence of a requirement of amylase release for phosphorylation of protein 34.

Adenosine Triphosphate↗

PubMed1984

Role of calmodulin-dependent phosphorylation in chronic sulpiride-induced striatal dopamine receptor supersensitivity.

In the present study we investigated several pharmacological and biochemical parameters in striatal preparations of rats which were treated chronically with a selective D2 receptor antagonist, sulpiride. Chronic sulpiride treatment in rats (50 mg/kg s.c. for 20 days) potentiated stereotyped responses by apomorphine (0.1-2.5 mg/kg). In association with the dopaminergic behavior supersensitivity, we observed a significant increase in the number of specific [3H]spiperone binding sites (D2 receptors) in the striatum without affecting the ligand binding affinity constant. We further observed a marked increase in the sensitivity of the protein kinase to calcium (0.1-0.5 mM) and calmodulin (1 micrograms) in these rats. The D1 receptor functions which are represented by the basal and dopamine-stimulated adenylate cyclase and the cyclic AMP-dependent protein kinase activity were not changed after chronic sulpiride treatment. In vitro, pretreatment of striatal particulates with the Ca++-chelating agent ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (1.2 mM) to remove endogenous Ca++ and calmodulin or the addition of Ca++ and calmodulin to the striatal particulates did not affect the binding affinities of dopamine agonists and antagonists to the receptors. Therefore, the increased sensitivity of the calmodulin-dependent system seen in chronic sulpiride-treated rats correlates with the increased number of D2 receptors in striatal dopamine receptor supersensitivity.

Adenylyl Cyclases↗

PubMed1983

Isolation and characteristics of bovine pituitary secretory granules.

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measureable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both growth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10(-5) M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.

Animals↗

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↗

PubMed1981