Inhibition of glucose transport by cyclic GMP in cardiomyocytes.

Recent evidence points to a potential role of cyclic GMP (cGMP) in the control of cardiac glucose utilization. The present work examines whether the glucose transport system of cardiac myocyte is a site of this cGMP-dependent regulation. Treatment of isolated rat cardiomyocytes (for 10 min) with the membrane-permeant cGMP analogue 8-(4-chlorophenylthio)-cGMP (8-p-CPT-cGMP, 200 microM) caused a decrease in glucose transport in non-stimulated (basal) myocytes, as well as in cells stimulated with insulin or with the mitochondrial inhibitor oligomycin B by up to 40%. An inhibitory effect was also observed with another cGMP analogue (8-bromo-cGMP), and in cells stimulated by hydrogen peroxide or anoxia. In contrast, 8-p-CPT-cAMP (200 microM), or the beta-adrenergic agonist isoprenaline (which increases cAMP levels) did not depress glucose transport, and even potentiated the effect of insulin. Blockade of endogenous cGMP formation with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) significantly increased basal and insulin-dependent glucose transport (by 25%), whereas addition of the guanylate cyclase activator 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazol (YC-1, 30 microM) produced a depression of glucose transport (by 20%). Confocal laser scanning microscopic studies revealed that cGMP partially prevents the insulin-induced redistribution of the glucose transporter GLUT4 from intracellular stores to the cell surface. These observations suggest that the glucose transport system of cardiomyocytes represents a metabolic target of inhibition by cGMP, and that this regulation occurs at the level of the trafficking of glucose transporters.

Purinergic inhibition of glucose transport in cardiomyocytes.

ATP is known to act as an extracellular signal in many organs. In the heart, extracellular ATP modulates ionic processes and contractile function. This study describes a novel, metabolic effect of exogenous ATP in isolated rat cardiomyocytes. In these quiescent (i.e. noncontracting) cells, micromolar concentrations of ATP depressed the rate of basal, catecholamine-stimulated, or insulin-stimulated glucose transport by up to 60% (IC50 for inhibition of insulin-dependent glucose transport, 4 microM). ATP decreased the amount of glucose transporters (GLUT1 and GLUT4) in the plasma membrane, with a concomitant increase in intracellular microsomal membranes. A similar glucose transport inhibition was produced by P2 purinergic agonists with the following rank of potencies: ATP approximately ATPgammaS approximately 2-methylthio-ATP (P2Y-selective) > ADP > alpha,betameATP (P2X-selective), whereas the P1 purinoceptor agonist adenosine was ineffective. The effect of ATP was suppressed by the poorly subtype-selective P2 antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid but, surprisingly, not by the nonselective antagonist suramin nor by the P2Y-specific Reactive Blue 2. Glucose transport inhibition by ATP was not affected by a drastic reduction of the extracellular concentrations of calcium (down to 10(-9) M) or sodium (down to 0 mM), and it was not mimicked by a potassium-induced depolarization, indicating that purinoceptors of the P2X family (which are nonselective cation channels whose activation leads to a depolarizing sodium and calcium influx) are not involved. Inhibition was specific for the transmembrane transport of glucose because ATP did not inhibit (i) the rate of glycolysis under conditions where the transport step is no longer rate-limiting nor (ii) the rate of [1-14C]pyruvate decarboxylation. In conclusion, extracellular ATP markedly inhibits glucose transport in rat cardiomyocytes by promoting a redistribution of glucose transporters from the cell surface to an intracellular compartment. This effect of ATP is mediated by P2 purinoceptors, possibly by a yet unknown subtype of the P2Y purinoceptor family.

Luminometric measurement of subnanomole amounts of key metabolites in extracts from isolated heart muscle cells.

In principle, luminometry allows very sensitive metabolite measurements as shown with standards in aqueous solutions (e.g., buffers). However, components of complex biological samples may largely interfere with luminometric reactions. We now describe a procedure by which subnanomole amounts of intermediary metabolites (malate, glucose 6-phosphate) can be measured by luminometry in extracts from isolated mammalian cells, namely rat heart muscle cells. Basically, measurements occur in two steps: (i) Enzymatically catalyzed reactions involving the metabolite to be measured lead to the stoichiometric production of NAD(P)H; (ii) the oxidation of this NAD(P)H in a luciferase/reductase system results in light production which is proportional to the original concentration of the metabolite. The reaction scheme is thus as follows: (1) Metabolite (malate, glucose 6-phosphate) + NAD(P)+ --> X + NAD(P)H + H+; (2) NAD(P)H + O2 + RCOH --> NAD(P)+ + RCOOH + H2O + hnu. The cardiomyocytes used are previously subjected to an ethanolic extraction in which the cellular NAD(P)H is destroyed by acidification. Subsequent evaporation of the extracts allows to neutralize and to concentrate the samples. This contributes, along with other experimental maneuvers, to increasing the sensitivity of the method. With this procedure, we were able to detect amounts of approximately 70 pmol of malate and approximately 90 pmol of glucose 6-phosphate in cardiomyocyte samples. In addition, the calculated cellular concentrations of malate and glucose 6-phosphate (101.1 +/- 4.5, and 202.8 +/- 26.1 microM, respectively, in the absence of exogenous substrate) correspond to values previously reported for heart tissue. In principle, the procedure described could be applied to the measurement of any ethanol-extractable metabolite that can be converted in reactions involving NAD(P)+.

The effect of anoxia on cardiomyocyte glucose transport does not involve an adenosine release or a change in energy state.

The action of anoxia on glucose transport was investigated in isolated resting rat cardiomyocytes. Incubation of these cells in the absence of oxygen for 30 min resulted in a 4- to 5-fold increase in glucose transport (with a lag period of 5-10 min). Up to 40 min of anoxia failed to alter the cellular concentrations of ATP, phosphocreatine, and creatine. Adenosine deaminase (1.5 U/ml), the A1-adenosine receptor antagonist 1,3-diethyl-8-phenylxanthine (1 microM), or the A2-selective antagonist 3,7-dimethyl-1-propargylxanthine (20 microM) had no effect on anoxia-dependent glucose transport. Moreover, adenosine (10-300 microM, added under normoxia) did not stimulate glucose transport. Wortmannin (1 microM) did not influence the effect of anoxia, but completely suppressed that of insulin. On the other hand, the effects of anoxia and insulin were not additive. These results indicate (i) that the effect of anoxia on cardiomyocyte glucose transport is not mediated by a change in energy metabolism, nor by an adenosine release; (ii) that it probably does not involve a phosphatidylinositol 3-kinase, in contrast to the effect of insulin, and (iii) that the signal chains triggered by anoxia or insulin may converge downstream of this enzyme, or, alternatively, that anoxic conditions may impair the action of the hormone.