guanosine-triphosphate and 2--deoxy-3--adenosine-monophosphate

ATP-sensitive K+ (KATP) channels are present at high density in membranes of cardiac cells, where they regulate cardiac function during metabolic impairment. The present study analyzes the effects of extracellular ATP (ATPc), a P2-purinergic agonist that can be released under various conditions in the myocardial cell bed, on KATP current (IK-ATP) in rat ventricular myocytes. Under the whole-cell patch-clamp configuration at a physiological level of intracellular ATP, applying ATPc in the micromolar range did not activate IK-ATP. However, dialyzing the cell with a low-ATP (100 mumol/L) pipette solution elicited a slowly, quasilinearly increasing IK-ATP that was markedly enhanced by applying ATPe in the presence of a Purinergic antagonist. The effect was reversible on washing out the agonist. The IK-ATP enhancement was inhibited by cholera toxin treatment of the myocytes, suggesting that a Gs protein was involved to mediate the effect. Experiments on excised patches allowed us to exclude a membrane-delimited G protein-dependent pathway. Rather, the results suggested that ATPe activates the adenylyl cyclase, since its inhibition by 2'-deoxyadenosine 3'-monophosphate and SQ-22536, which respectively interact with the purine and catalytic site of the cyclase, strongly reduced the ATPe-induced IK-ATP enhancement, whereas neither compound affected IK-ATP in inside-out patches. Inhibition of cAMP-dependent protein kinase by protein kinase inhibitor peptide 5-24 did not alter the purinergic effect. The findings suggests that ATPe triggers the activation of adenylyl cyclase, which causes a subsarcolemmal ATP depletion sufficient to enhance IK-ATP as it develops during low-ATP dialysis of rat ventricular myocytes.

Topics: Adenine; Adenosine Triphosphate; Adenylyl Cyclase Inhibitors; Animals; Benzopyrans; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Colforsin; Cromakalim; Deoxyadenine Nucleotides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Extracellular Space; GTP-Binding Proteins; Guanidines; Guanosine Triphosphate; Heart Ventricles; Male; Patch-Clamp Techniques; Peptide Fragments; Pinacidil; Potassium Channels; Pyrroles; Rats; Rats, Wistar; Uncoupling Agents

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'

Topics: Adenosine; Adenylyl Cyclase Inhibitors; Brain; Colforsin; Deoxyadenine Nucleotides; Dideoxyadenosine; Dideoxynucleosides; Egtazic Acid; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Magnesium; Manganese; Receptors, Purinergic; Structure-Activity Relationship; Thionucleotides