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M Bushfield

Publications and source records attributed to M Bushfield.

29 records · Page 2Linked to original sources
PubMed1990

Tissue levels, source, and regulation of 3'-AMP: an intracellular inhibitor of adenylyl cyclases.

Tissue levels of 3'-AMP were measured in several rat tissues and the sensitivities of the respective adenylyl cyclases were compared with respect to "P" site-mediated inhibition by 3'-AMP2'-deoxy-3'AMP (2'd3'-AMP), and 2',5'-dideoxyadenosine. IC50 values for these P site inhibitors of adenylyl cyclases varied widely among tissues, e.g., with skeletal muscle being least sensitive to 3'-AMP (IC50 greater than 170 microM) and brain being most sensitive (IC50 approximately 10 microM). These differences were noted when activation was with Mn2+ but diminished with Mn2+ plus forskolin and conceivably may reflect the distribution of different isozymes of adenylyl cyclase. 3'-AMP levels also varied significantly among rat tissues, with spleen having the highest levels (approximately 280 nmol/g), kidney, liver, heart, and brain having decreasing 3'-AMP content, and skeletal muscle levels being immeasureably low (less than 0.1 nmol/g). When rats were made diabetic with streptozotocin, the 3'-AMP content of livers increased from approximately 47 nmol/g in control animals to approximately 84 nmol/g, a change largely reversed by maintenance of diabetic animals with insulin. The data suggest that tissue 3'-AMP levels may be regulated and in certain tissues may be sufficient to inhibit adenylyl cyclase in vivo. Three potential sources of 3'-AMP and 2'd3'-AMP, the most potent naturally occurring P site inhibitors of adenylyl cyclase, were examined. No evidence was found for the formation of either nucleotide from the respective cyclic nucleotide by a unique cyclic nucleotide phosphodiesterase or from the respective nucleoside by a hypothetical adenosine 3'-kinase and ATP. Substantial 3'-AMP and 2'd3-AMP were formed by spleen and liver homogenates from the respective oligonucleotides (RNA, mRNA, and DNA) in a time- and protein-dependent manner. The data imply the existence of enzymes in these tissues to catalyze the formation of 3'-AMP and 2'd3'-AMP from nucleic acids and suggest that these activities may account for the formation of P site agonists under in vivo conditions. The data suggest that these P site inhibitors are a potential link between fluctuations in nucleic acid metabolism and altered sensitivity of membrane-bound adenylyl cyclase to stimulatory signals.

Adenosine Monophosphate↗
PubMed1989

Cation and structural requirements for P site-mediated inhibition of adenylate cyclase.

The cation and structural requirements of the intracellular inhibitory "P" site of adenylate cyclase were investigated in human platelet membranes, bovine sperm particles, and detergent-solubilized and purified preparations from rat and bovine brain. Sensitivity of adenylate cyclase to P site-mediated inhibition was enhanced by reversible and irreversible activators of the enzyme. The most effective sensitization of the platelet and brain adenylate cyclases was observed with Mn2+ and upon proteolysis with inhibin in the presence of guanosine 5'-O-(3-thiotriphosphate). These resulted in IC50 values for (2',5'dideoxy-adenosine (2',5'-dd-Ado) and 2'-deoxy-3'-AMP of approximately 1-2 microM. The data were consistent with the ideas that P site-mediated inhibition of adenylate cyclase is dependent on divalent cation and is a function of enzyme activity. A number of nucleosides and nucleotides were synthesized and used to define structural requirements for P site-mediated inhibition of a detergent-solubilized adenylate cyclase from rat brain. The data suggest a strict requirement for an intact adenine moiety and a beta-glycosidic linkage for the ribosyl moiety. 2'-Deoxy-and especially 2',5'-dideoxy-ribosyl moieties enhanced sensitivity and a strong preference for phosphate at the 3'-position was exhibited. Substitutions at the 5'-ribose position impaired sensitivity. The order of potency and IC50 values of the more potent adenosine analogs were 2',5'-dideoxy-3'-AMP (congruent to 0.1 microM) greater than 2'-deoxy-3'-AMP (congruent to 1 microM) greater than 2',5'-dd-Ado (congruent to 3 microM) greater than 3'-AMP (congruent to 9 microM) greater than 2'-deoxy-adenosine (congruent to 15 microM) greater than adenosine (congruent to 80 microM). Large substitutions at the 3'-ribose position were tolerated, e.g., dApdN di- and dAp(dN)4 penta-nucleotides and succinyl- and p-fluoro-sulfonyl-benzoyl- moieties. The purified adenylate cyclase from bovine brain was inhibited by P site agonists with IC50 values of 34 and 45 microM for 2'-deoxy-3'-AMP and 2',5'-dd-Ado, respectively. The data imply, first, that the locus of the P site is the catalytic subunit of adenylate cyclase and, second, that the increased sensitivity observed with Mn2+ is due to an effect of the cation on the catalytic subunit. In contrast with adenylate cyclases from other mammalian tissues, the enzyme from bovine sperm exhibited only weak sensitivity to P site agonists; 2'-deoxy-3'-AMP congruent to 2',5'-dd-Ado greater than adenosine, each with IC50 values greater than 1000 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗
PubMed1987

Effects of protein kinase C activation on human platelet cyclic AMP metabolism.

Treatment of intact human platelets with the tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), specifically inhibited PGD2-induced cyclic AMP formation without affecting the regulation of cyclic AMP metabolism by PGI2, PGE1, 6-keto-PGE1, adenosine or adrenaline. This action of PMA was: (i) concentration-dependent; (ii) not mediated by evoked formation or release of endogenous regulators of adenylate cyclase activity (thromboxane A2 or ADP); (iii) mimicked by 1,2-dioctanoylglycerol (DiC8) but not by 4 alpha-phorbol 12,13-didecanoate (which does not activate protein kinase C); (iv) attenuated by Staurosporine. These results indicate that activation of protein kinase C in platelets may provide a regulatory mechanism to abrogate the effects of the endogenous adenylate cyclase stimulant PGD2 without compromising the effects of exogenous stimulants of adenylate cyclase (PGI2, 6-keto-PGE1, adenosine).

Adenosine Diphosphate↗
PubMed1987

Possible mechanisms of the potentiation of blood-platelet activation by adrenaline.

Vasopressin and adrenaline in combination exert synergistic effects on platelet activity. This study investigated the effects of sub-threshold concentrations of adrenaline (0.1-1 microM) on vasopressin (10 nM-1 microM)-induced platelet aggregation, ATP secretion, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Potentiation of vasopressin-induced aggregation and ATP secretion by adrenaline was accompanied by enhanced elevation of [Ca2+]i and [32P]phosphatidic acid formation. The stimulatory effects of adrenaline on vasopressin-induced platelet activation were mimicked by the combination of the Ca2+ ionophore, ionomycin, and the protein kinase C activator, phorbol 12-myristate 13-acetate, but not by either of these agents alone. These results suggest that the potentiation of vasopressin-induced platelet activation by adrenaline is mediated via enhancement of inositol phospholipid hydrolysis and elevation of [Ca2+]i.

Adenosine Triphosphate↗
PubMed1985

Inhibition of platelet-activating-factor-induced human platelet activation by prostaglandin D2. Differential sensitivity of platelet transduction processes and functional responses to inhibition by cyclic AMP.

It has been proposed that cyclic AMP inhibits platelet reactivity: by preventing agonist-induced phosphoinositide hydrolysis and the resultant formation of 1,2-diacylglycerol and elevation of cytosolic free Ca2+ concentration [( Ca2+]i); by promoting Ca2+ sequestration and/or extrusion; and by suppressing reactions stimulated by (1,2-diacylglycerol-dependent) protein kinase C and/or Ca2+-calmodulin-dependent protein kinase. We used the adenylate cyclase stimulant prostaglandin D2 to compare the sensitivity to cyclic AMP of the transduction processes (phosphoinositide hydrolysis and elevation of [Ca2+]i) and functional responses (shape change, aggregation and ATP secretion) that are initiated after agonist-receptor combination on human platelets. Prostaglandin D2 elicited a concentration-dependent elevation of platelet cyclic AMP content and inhibited platelet-activating-factor(PAF)-induced ATP secretion [I50 (concn. causing 50% inhibition) approximately 2 nM], aggregation (I50 approximately 3 nM), shape change (I50 approximately 30 nM), elevation of [Ca2+]i (I50 approximately 30 nM) and phosphoinositide hydrolysis (I50 approximately 10 nM). A 2-fold increase in cyclic AMP content resulted in abolition of PAF-induced aggregation and ATP secretion, whereas maximal inhibition of shape change, phosphoinositide hydrolysis and elevation of [Ca2+]i required a greater than 10-fold elevation of the cyclic AMP content. This differential sensitivity of the various responses to inhibition by cyclic AMP suggests that the mechanisms underlying PAF-induced aggregation and ATP secretion differ from those underlying shape change. Thus a major component of the cyclic AMP-dependent inhibition of PAF-induced platelet aggregation and ATP secretion is mediated by suppression of certain components of the activation process that occur distal to the formation of DAG or elevation of [Ca2+]i.

Adenosine Triphosphate↗
PubMed1985

Regulation of platelet cytosolic free calcium by cyclic nucleotides and protein kinase C.

PAF elicits a rapid, concentration-dependent elevation of platelet cytosolic free calcium ([Caf]), measured by quin2. Elevation of [Caf] is transient, and the rate of reversal increases with agonist concentration. Adenylate cyclase stimulants (PGI2, PGD2) and 8-bromo cAMP; a guanylate cyclase stimulant (sodium nitroprusside) and 8-bromo cGMP; and a protein kinase C stimulant (phorbol myristate acetate) block the elevation of [Caf] induced by PAF, and accelerate its reversal. These results suggest that cAMP, cGMP and 1,2-diacylglycerol (DAG) could act as second messengers to regulate [Caf] in platelets. As PAF is known to stimulate platelet phosphoinositide hydrolysis (ergo DAG formation) but fails to elevate platelet cAMP or cGMP, it is proposed that DAG, via activation of protein kinase C, may act as an endogenous modulator of platelet [Caf]: an action that contributes to the role of DAG as a bi-directional regulator of platelet reactivity.

Aminoquinolines↗
PubMed1985

Endogenous and pharmacological mechanisms for the regulation of human platelet cytosolic free Ca2+.

Because they inhibit the processes that promote elevation of [Ca2+]i and augment the processes that promote removal of Ca2+ from the cytosol, receptor antagonists, agents that mimic or elevate cAMP, cGMP or 1,2-Diacylglycerol (DG), and both inorganic and organic Ca2+ channel blockers can be considered to act as 'Ca2+ antagonists' on human platelets. Agonist-induced elevation of [Ca2+]i is associated with phosphoinositide hydrolysis. Unlike agents that mimic or elevate cAMP, cGMP or DG, receptor antagonists and organic Ca2+ influx, mobilisation of internal Ca2+ and inositol lipid hydrolysis. Lanthanides apparently inhibit only Ca2+ influx. Thus La3+ but not Verapamil or Diltiazem block receptor-operated Ca2+ channels on human platelets. The endogenous processes that promote extrusion or sequestration of cytosolic Ca2+ may be augmented by cAMP, cGMP, DG and by Ca2+. DG, via activation of protein kinase C, may serve as a bi-directional regulator of platelet reactivity.

Aminoquinolines↗
PubMed1984

Thyrotropin-releasing hormone-stimulated [3H]inositol metabolism in GH3 pituitary tumor cells. Studies with lithium.

GH3 pituitary tumor cells were labeled to isotopic equilibrium with [3H]inositol. Thyrotropin-releasing hormone (TRH), which has been shown to stimulate inositol phospholipid metabolism in these cells, enhanced the accumulation of [3H]inositol-derived radioactivity in the cell's acid-soluble fraction. Separation of the [3H]inositol metabolites by ion-exchange chromatography revealed that TRH induced a rapid rise in the cellular content of [3H]inositol mono-, bis-, and trisphosphate. The latter two metabolites accumulated in a multiphasic manner with an initial peak 5-10 sec after TRH addition. This was followed by a short-lived decline and a secondary rise which left the metabolite levels elevated for at least 50 min. The GH3 cell [3H]inositol monophosphate and [3H] inositol content also rose in response to TRH, but the latter accumulated with a considerably slower time course than the phosphorylated derivatives. None of these responses could be mimicked by the calcium ionophore A23187. Incubation of GH3 cells with TRH in the presence of lithium led to an enhanced accumulation of [3H]inositol monophosphate and, to a lesser extent, of [3H]inositol bis- and trisphosphate. This accumulation rose in a linear fashion with time for at least 20 min, by which point 50% of the [3H]inositol-containing phospholipids had been depleted. When lithium was added 30 min after TRH, [3H]inositol monophosphate accumulated at the same rate as was found when TRH and lithium were added together, indicating that the TRH-induced phospholipid response in GH3 cells does not desensitize. Under normal conditions, approximately equal amounts of the three [3H]inositol phosphates were formed within 5 sec of TRH addition. However, when TRH was added to cells grown chronically in lithium-containing medium, or to cells incubating at a subphysiological temperature (25 degrees), greater than 90% of the metabolites formed were the bis- or trisphosphates. This indicates that the primary event stimulated by TRH is the breakdown by phospholipase C of phosphatidylinositol 4,5-bisphosphate and, perhaps also, of phosphatidylinositol 4-phosphate.

Animals↗