A photoaffinity probe for the cardiac adenosine transporter.

We have developed and utilized a photoaffinity probe to identify the adenosine transporter in cardiac sarcolemmal (SL) vesicles. The probe is an azidosalicylate derivative of adenosine made by reacting adenosine with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA). Following synthesis and radiolabeling of the probe (ASA-adenosine), 125I-ASA-adenosine was purified by high pressure liquid chromatography. Iodine-125-ASA-adenosine, upon irradiation with uv light, covalently labeled a 65 kDa protein in bovine cardiac SL vesicles. Labeling of this protein was greatly diminished in the presence of nonradiolabeled adenosine, 5'-amino adenosine, or guanosine (inhibitors of purine nucleoside transport) but not by glucose (which does not inhibit transport). We conclude that the cardiac adenosine transporter is a protein with an apparent Mr of 65 kDa.

Guanosine metabolism in adult rat cardiac myocytes: inhibition by acyclovir and analysis of a metabolic pathway.

The metabolic fate of transported guanosine was examined in adult rat cardiac myocytes. Freshly isolated cells were incubated with 50 microM 8-[3H]-guanosine and the purine nucleoside phosphorylase (PNP) inhibitor acyclovir, and the nucleotide products extracted and examined for radiolabel distribution. Acyclovir inhibited guanosine incorporation into the 5'-nucleotide pool up to 66%. The drug did not inhibit guanosine transport. Other experiments using 5'-[3H]-guanosine and 8-[14C]-guanosine in concert as metabolic tracers showed both tritium and radiocarbon in the guanine nucleotide products. We concluded from this study that both a kinase (probably adenosine kinase) and the enzyme pair purine nucleoside phosphorylase/hypoxanthine-guanine phosphoribosyltransferase are responsible for guanosine salvage in heart cells.

Differing significance of Na(+)-Ca2+ exchange in the regulation of cytosolic Ca2+ in rat exocrine gland acini and cardiac myocytes.

In order to compare the importance of Na(+)-Ca2+ exchange in the regulation of cytosolic Ca2+ concentration (Ca2+i), acini obtained from rat pancreas and submandibular glands as well as cardiac myocytes were loaded with Na+ by inhibition of Na(+)-K+ ATPase activity then loaded with fura-2. In the exocrine tissues, incubation in K(+)-free buffer or with ouabain had no substantial effect on resting Ca2+i or on the changes in Ca2+i following exposure to carbachol as compared with acini incubated under control conditions. In contrast, rat cardiac myocytes, treated identically, showed marked changes in Ca2+i under resting and stimulated conditions as compared with controls. We conclude that the Na(+)-Ca2+ exchange systems of rat pancreatic and submandibular gland acini contribute little to the overall regulation of Ca2+i at rest during cholinergic stimulation.

Myocardial glucose utilization. Failure of adenosine to alter it and inhibition by the adenosine analogue N6-(L-2-phenylisopropyl)adenosine.

The effects of adenosine and the nonmetabolizable adenosine analogue N6-(L-2-phenylisopropyl)adenosine (PIA) on glucose transport or metabolism were determined in purified myocardial sarcolemmal vesicles, isolated cardiocytes, and perfused hearts. Adenosine (100 microM) did not affect hexose transport in myocytes. Also, adenosine deaminase, added to metabolize adenosine to inosine, did not alter transport of hexose into myocytes regardless of whether or not insulin was present. In contrast, PIA effectively inhibited 3-O-methyl-D-glucose uptake in myocytes even during insulin stimulation. PIA inhibited D-glucose-specific transport in both rat and bovine cardiac sarcolemmal vesicles (Ki = 26 microM at [D-glucose] = 5 mM). However, insulin did not affect glucose transport in sarcolemmal vesicles, which implies that receptor-coupled processes probably are not intact in this preparation. Thus, inhibition of PIA may not be receptor mediated. Also, PIA inhibited binding of cytochalasin B to bovine cardiac sarcolemmal vesicles, which supports the idea that PIA inhibits glucose flux by binding to the glucose transporter. To determine if adenosine altered glucose metabolism rather than transport, we measured the rate of 3H2O production from metabolism of D-[2-3H]glucose in paced rat hearts ([D-glucose] = 5.5 mM, [pyruvate] = 0.2 mM) perfused with a range of PIA or adenosine concentrations with or without 0.01 microM insulin. Adenosine (0.01-100 microM) in the presence or absence of insulin increased coronary flow but did not change glycolytic rates. Similar results were obtained with PIA (no insulin) rather than adenosine in the perfusate. However, with glucose as the only exogenous substrate, 100 microM PIA inhibited glycolysis by approximately 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic characterization and radiation-target sizing of the glucose transporter in cardiac sarcolemmal vesicles.

Stereospecific glucose transport was assayed and characterized in bovine cardiac sarcolemmal vesicles. Sarcolemmal vesicles were incubated with D-[3H]glucose or L-[3H]glucose at 25 degrees C. The reaction was terminated by rapid addition of 4 mM HgCl2 and vesicles were immediately collected on glass fiber filters for quantification of accumulated [3H]glucose. Non-specific diffusion of L-[3H]glucose was never more than 11% of total D-[3H]glucose transport into the vesicles. Stereospecific uptake of D-[3H]glucose reached a maximum level by 20 s. Cytochalasin B (50 microM) inhibited specific transport of D-[3H]glucose to the level of that for non-specific diffusion. The vesicles exhibited saturable transport (Km = 9.3 mM; Vmax = 2.6 nmol/mg per s) and the transporter turnover number was 197 glucose molecules per transporter per s. The molecular sizes of the cytochalasin B binding protein and the D-glucose transport protein in sarcolemmal vesicles were estimated by radiation inactivation. These values were 77 and 101 kDa, respectively, and by the Wilcoxen Rank Sum Test were not significantly different from each other.

Cardiac myocyte guanosine transport and metabolism.

Guanosine transport and metabolism were examined in adult rat cardiac myocytes. Myocytes transported guanosine via saturable [Km = 18 microM, maximum velocity (Vmax) = 3.61 pmol.mg-1.s-1] and nonsaturable (rate constant = 1.47 X 10(-2] processes. The saturable process was inhibited by nitrobenzyl-thioinosine, inosine [inhibition constant (Ki) = 180 microM], and adenosine (Ki = 112 microM). Extracellular guanosine taken up by myocytes was slowly phosphorylated to guanine nucleotides. The majority of guanosine (98%) existed as free intracellular guanosine after 60 s. Countertransport of nucleosides could not be demonstrated in these cells at physiological concentrations in the presence of up to a 10-fold gradient of nucleoside. These studies indicate that adult rat cardiac myocytes can be used to assess myocardial guanosine transport separate from its metabolism. Comparable inhibition of guanosine and adenosine transport by each other and by inosine support the hypothesis that guanosine and adenosine are transported by a common carrier.

Forskolin inhibition of hexose transport in cardiomyocytes.

The effects of insulin, forskolin, isoproterenol, and epinephrine on 3-O-methylglucose (hexose) transport and cell cyclic AMP levels were determined in adult rat cardiomyocytes. Insulin stimulated hexose transport in these cells an average of 2.5-fold. Initial hexose transport rates at 1 mM hexose were 3.75 X 10(-2) nmol/mg cell protein/second in the absence of insulin, and 8.25 X 10(-2) nmol/mg cell protein/second in the presence of 12.3 microM insulin. Forskolin at 5 microM nearly abolished hexose transport within 3 s of exposure, but did not increase cell cyclic AMP concentrations within 9 s. The apparent Ki for hexose transport inhibition was about 0.3 microM forskolin. Epinephrine and isoproterenol at 50 microM increased cell cyclic AMP 4-fold during 9 s exposure, but did not affect hexose transport. Treatment of cells with these catecholamines of forskolin for up to 99 s increased cell cyclic AMP, but only forskolin inhibited hexose transport. We conclude from these results that forskolin acts on hexose transport independent of its action on adenyl cyclase, and that cyclic AMP does not inhibit or stimulate hexose transport.

Myocyte and endothelial injury with ischemia reperfusion in isolated rat hearts.

We determined the time course of ischemic injury, the effects of reperfusion, and the protective effects of prostacyclin, oxygen radical scavengers, and diltiazem on myocardial myocyte and endothelial cell functions in isolated rat hearts. Left ventricular power and coronary microvascular permeability were used as indexes of myocyte and endothelial cell function, respectively. Neither 5- nor 10-min ischemia reperfusion significantly changed power or permeability. However, with reperfusion following 20 and 30 min of ischemia, power was reduced 50 and 60% and permeability increased 70 and 90%. In 30-min ischemic hearts the ischemia-induced increase in permeability was apparent after 4 min reperfusion and further exacerbated at 20 min. Hypoxic reperfusion did not prevent increased permeability. Prostacyclin or a combination of superoxide dismutase, catalase, and mannitol also did not prevent increased permeability, and the radical scavengers did not ameliorate depressed power. In contrast, perfusion with diltiazem during ischemia reperfusion blunted the reduction in power and prevented the increase in permeability. We conclude that ischemia reperfusion causes similar time course of injury to myocytes and endothelial cells; reperfusion contributes to endothelial injury, and diltiazem affords protection to both cell types.

Rat cardiac myocyte adenosine transport and metabolism.

Based on the importance of myocardial adenosine and adenine nucleotide metabolism, the adenosine salvage pathway in ventricular myocytes was studied. Accurate estimates of transport rates, separate from metabolic flux, were determined. Adenosine influx was constant between 3 and 60 s. Adenosine metabolism maintained intracellular adenosine concentrations less than 10% of the extracellular adenosine concentrations and thus unidirectional influx could be measured. Myocytes transported adenosine via saturable [Michaelis constant = 6.2 +/- 2.1 microM and maximal velocity (Vmax) = 9.58 +/- 0.98 X 10(-1) pmol X mg protein-1 X s-1] and nonsaturable (rate constant = 1.8 X 10(-3)/s) processes. A minimum estimate of the Vmax of myocytic adenosine kinase (2 pmol X mg protein-1 X s-1) indicated the saturable component of adenosine influx was independent of adenosine kinase activity. Saturable transport was inhibited by nitrobenzylthioinosine and verapamil (inhibitor constant = 17 +/- 5 microM). Extracellular adenosine taken up by myocytes was rapidly phosphorylated to adenine nucleotides. Not all extracellular adenosine, though, was phosphorylated on entering myocytes, since free, as opposed to protein-bound, intracellular adenosine was detected after digitonin extraction of cells in the presence of 1 mM ethylene-diaminetetraacetic acid.

Erythrocyte adenosine transport: effects of Ca2+ channel antagonists and ions.

On the basis of observations of adenosine-Ca2+ competition, we assessed the effects on erythrocyte adenosine transport of Ca2+ channel antagonists, mono- and divalent cations, and Cl- and Cl- transport inhibitors. The Ca2+ channel antagonists, diltiazem and verapamil, competitively inhibited adenosine influx (Ki = 158 +/- 17.4 and 13.5 +/- 1.3 microM at 10 microM adenosine, respectively), despite no apparent effect on transport by Ca2+, Mg2+, Na+, or K+. Verapamil also inhibited uridine efflux (Ki = 1.7 +/- 0.3 microM at 84-100 microM intracellular uridine). The absence of Cl- decreased adenosine influx rates from 0.615 +/- 0.013 to 0.386 +/- 0.008 nmol X s-1 X ml intracellular H2O-1. The Cl- transport inhibitors, diisothiocyanostilbene disulfonate (10 microM), furosemide (1 mM), and NO-3 (145 mM), decreased adenosine influx rates to 0.301 +/- 0.008, 0.325 +/- 0.013, and 0.430 +/- 0.009 nmol X s-1 X ml intracellular H2O-1, respectively. These studies indicate that the Ca2+ channel antagonists inhibit adenosine release and uptake and therefore may modulate adenosine-mediated events. Additionally, they suggest that adenosine and anion transport systems are linked or share common features.

Myocardial nucleotide transport.

A thorough consideration of the evidence for striated muscle cell transmembrane nucleotide movement provides only equivocal support for adenine nucleotide specific translocation across cell membranes. It is obvious that nucleotide-derived adenosine is taken up into cells in preference to free adenosine, and it is important to understand why this is so. The potential importance of released nucleotides to cell regulation justify studies to determine the source and release mechanism.

Hyaluronidase reversal of increased coronary vascular resistance in ischemic rat hearts.

Testicular hyaluronidase prevents increased coronary vascular resistance (CVR) during prolonged myocardial ischemia. The mechanism is unknown, but edema and contracture both have been suggested to increase CVR. Additionally, the extent of contracture has been inversely related to ATP levels. Therefore, isolated perfused ischemic rat hearts were treated with hyaluronidase, following a 25% increase in CVR, to determine whether 1) increased CVR was reversed, 2) edema or contracture was reduced, and 3) tissue ATP levels were increased. Three hours of low-flow ischemia decreased coronary flow (CF) from 17.4 +/- 0.13 to 12.6 +/- 0.2 ml X min-1 X g dry tissue-1. During the subsequent 2 h of ischemia, CF of vehicle-treated hearts continued to decline to 8.0 +/- 0.76 ml X min-1 X g dry tissue-1, whereas CF of hyaluronidase-treated hearts increased to 15.6 +/- 1.17 ml X min-1 X g dry tissue-1. These changes in CF persisted during postischemic perfusion. Furthermore, restoration of coronary vascular resistance by hyaluronidase was associated with a 19% reduction in tissue water compared with control ischemic hearts but not with a reduction in cardiac contracture or an increase in tissue ATP. These results suggest that treatment of ischemic hearts with hyaluronidase reverses increased CVR through a reduction in tissue edema.

Myocardial adenosine salvage rates and restoration of ATP content following ischemia.

The isolated perfused rat heart was utilized to determine the maximum rate of adenosine incorporation into adenine nucleotides and the effect of ischemia on this rate. In aerobic hearts, the rates of [8-14C]adenosine incorporation into nucleotides in nanomoles/minute per gram dry tissue were ATP 34 +/- 2, ADP 6 +/- 0.4, AMP 3 +/- 0.3, and IMP, 1 +/- 0.2. Following ischemia these values were not significantly different except for the rate of incorporation into IMP, which doubled. The extent of adenosine deamination with one pass through the coronary vasculature was the same in aerobic and postischemic hearts: 2% and 7% of the perfusate adenosine was converted to hypoxanthine and inosine, respectively. These percentages were similar at 50, 100, and 200 micron adenosine. Perfusion of aerobic hearts for 5 h with adenosine did not change ATP concentrations. Therefore, [8-14C]adenosine incorporation into ATP in these hearts appeared to represent ATP turnover. In contrast, 5 h perfusion of postischemic hearts with adenosine restored ATP concentrations to control values. The synthesis rate calculated from the increase in ATP concentration was comparable to the synthesis rate calculated from [8-14C]adenosine incorporation. Thus, incorporation of [8-14C]adenosine into ATP in postischemic hearts represented net ATP synthesis.

Energy metabolism in the ischemic heart.

A reduction in myocardial oxygen supply during ischemia, not only leads to reduced aerobic ATP production but does not stimulate glycolytic ATP synthesis. The residual aerobically synthesized ATP comes primarily from continued inefficient (i.e., compared to glucose in terms of moles of ATP produced per mole of O2 consumed) oxidation of fatty acids. This leads to elevated tissue levels of long chain fatty acyl-CoA and fatty acyl-carnitine. Both are potentially cell damaging metabolic intermediates. Restriction of glycolysis is due to inhibition of glyceraldehyde-3-phosphate dehydrogenase by accumulated metabolites, such as H+, lactate and NADH. The reduced production of ATP leads to decreased levels of high energy phosphate stores which in turn may impair myocardial mechanical function.

Myocardial ATP synthesis and mechanical function following oxygen deficiency.

The relationship between oxygen deficiency-reduced high energy phosphate levels and their resynthesis upon return to aerobic conditions was investigated in the isolated perfused rat heart. Any net adenosine triphosphate (ATP) hydrolysis during anoxia tended to impair ATP resynthesis with subsequent aerobic perfusion. Thirty minutes of ischemia reduced myocardial ATP 50%, and with restoration of aerobic conditions ATP increased to only 60% of control levels. The major source of postischemic and postanoxic ATP was adenosine 5'-monophosphate and adenosine 5'-disphosphate. Loss of purine base from oxygen-deficient cells limited restoration of ATP. The inclusion of adenosine, ATP, or creatine phosphate (CP) in the perfusate did not enhance postischemic tissue adenine-nucleotide concentrations. Postischemic and postanoxic CP concentrations returned to control values and were independent of ischemic and anoxic ATP and CP concentrations. Complete resynthesis of CP suggests that cellular energy-producing pathways were functional. Ventricular performance was directly related to tissue ATP concentration in aerobic control, postischemic, and postanoxic hearts. Thus, loss of adenine nucleotides during oxygen deficiency may impair subsequent aerobic synthesis of ATP and mechanical function.

Effect of hyaluronidase and methylprednisolone on myocardial function, glucose metabolism, and coronary flow in the isolated ischemic rat heart.

Ischemia in the isolated perfused rat heart resulted in an increase in coronary vascular resistance. Studies were undertaken to determine the effect of hyaluronidase and methylprednisolone on this increase in resistance as well as on glycolytic rate and mechanical function of ischemic hearts. Neither hyaluronidase nor methylprednisolone affected the rate of glucose utilization in working perfused control or ischemic rat hearts. However, both agents prevented a reduction in coronary flow during a 2-hour ischemic period. Associated with the higher coronary flows were higher tissue concentrations of creatine phosphate and lower concentrations of lactate. These agents also prevented accumulation of tissue water in the ischemic hearts. Such changes would appear to be beneficial to the ischemic heart, although mechanical function of post-ischemic hearts was not enhanced by the presence of either hyaluronidase or methylprednisolone. The results, however, suggest that the reduction in myocardial infarct size noted with hyaluronidase and methylprednisolone may be due to their prevention of further reduction of coronary flow in marginally eschemic tissue.

Rate-limiting steps of carbohydrate and fatty acid metabolism in ischemic hearts.

Control of glycolysis and fatty acid oxidation in ischemic myocardium was studied in isolated working rat hearts. Coronary flow was reduced to the whole heart. In ischemic tissue, oxygen consumption, glycolysis and fatty acid oxidation all decreased in proportion to the restriction in coronary flow. Inhibition of glycolysis developed at the level of glyceraldehyde-3-phosphate dehydrogenase. Restricted flux through this step appeared to result from accumulation of lactate, H+ and NADH. The rate of glycolysis was inversely related to accumulation of lactate. Additions of high levels of lactate to the perfusate inhibited glycolysis in aerobic, anoxic and ischemic hearts. The mechanism of this effect of lactate in anaerobic hearts is unknown, but does not appear to be related to pH changes. Oxidation of fatty acids was restricted at the level of beta-oxidation and high levels of both long-chain acyl CoA and carnitine derivatives accumulated.