Myocardial metabolism of exogenous FDP is consistent with transport by a dicarboxylate transporter.

The extent to and the mechanism by which fructose-1,6-bisphosphate (FDP) crosses cell membranes are unknown. We hypothesized that its transport is either via band 3 or a dicarboxylate transporter. The question was addressed in isolated Langendorff rat hearts perfused under normoxic conditions. Groups of hearts received the following metabolic substrates (in mM): 5 FDP; 5 FDP + either 5, 10, or 20 fumarate; 10 FDP and either 5, 10, or 20 fumarate; or 5 FDP + 2 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS), a band 3 inhibitor. FDP uptake and metabolism were measured as production of [(13)C]lactate from [(13)C]FDP or (14)CO(2) and [(14)C]lactate from uniformly labeled [(14)C]FDP in sample perfusates. During 30 min of perfusion, FDP metabolism was 12.4 +/- 2.6 and 31.2 +/- 3.0 micromol for 5 and 10 mM FDP, respectively. Addition of 20 mM fumarate reduced FDP metabolism over a 30-min perfusion period to 3.1 +/- 0.6 and 6.3 +/- 0.5 micromol for 5 and 10 mM FDP groups, respectively. DNDS did not affect FDP utilization. These data are consistent with transport of FDP by a dicarboxylate transport system.

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.

Guanosine metabolism in adult rat cardiac myocytes: ribose-enhanced GTP synthesis from extracellular guanosine.

The metabolic fate of transported guanosine was examined in adult rat cardiac myocytes. Freshly isolated cells were incubated with 10 microM or 100 microM [3H]guanosine and the nucleotide products extracted and examined for radiolabel distribution. The data presented show significant incorporation of guanosine into the 5'-nucleotide pool, and a marked stimulation of that incorporation by ribose. An average of 233 pmol/mg cell protein extracellular guanosine was incorporated into the cellular 5'-nucleotides over 90 min at both 10 microM and 100 microM external nucleoside. This appeared primarily as GTP (approx. 204 pmol/mg cell protein in 90 min). Only guanine nucleotides contained radiolabel; adenine nucleotides and IMP remained unlabelled even after 90 min incubation of the cells with [3H]guanosine. Addition of 5 mM ribose to the medium stimulated guanosine incorporation into 5'-nucleotides 1.6-fold (380 pmol/mg protein vs 234 pmol/mg over 90 min at 10 microM guanosine), but did not enhance the amount of guanosine transported into the cells. Intracellular guanosine concentrations exceeded those of the incubation medium at both external guanosine concentrations studied. More [3H]guanosine was salvaged at 100 microM than at 10 microM external guanosine (562 vs 380 pmol/mg protein in 90 min), but only if ribose was present in the medium. We conclude from these studies that guanosine is salvaged by heart muscle, and that at high guanosine levels the rate of guanosine salvage appears dependent on the availability of phosphoribosylpyrophosphate within the cells. At lower guanosine levels in the presence of ribose, cell guanine concentrations limit the rate of guanosine incorporation into 5'-nucleotides.

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)

Lactate does not enhance anoxia/reoxygenation damage in adult rat cardiac myocytes.

Accumulation of lactate in myocardial cells has been proposed as a primary trigger of ischemic damage in heart. This hypothesis was tested using isolated cardiac myocytes from adult rats. Cells were subjected to anoxia/reoxygenation protocols in the presence or absence of lactate at two extracellular pH values. Reductions in total rods and increased numbers of shortened rods ("contracted" cells) were evident in cell populations exposed to anoxia and in reoxygenated populations in the absence of glucose at pH values of 6.9 and 7.3. Although lower pH reduced cell adenine nucleotide contents over those seen at higher pH, neither 10 mM nor 50 mM lactate enhanced nucleotide loss or caused extra morphological damage under any condition in this study. Therefore, under conditions simulating those in ischemic heart, cell damage could not be attributed to high lactate concentrations.

Evidence for high molecular weight Na-Ca exchange in cardiac sarcolemmal vesicles.

Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na-Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na-Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (Mr) of approximately 226,000 +/- 20,000 SD (n = 6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 1:1 with mobile phase buffer containing 50 mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na-Ca exchange activity. Na-Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na-Ca exchange activity were 91 +/- 7 and 68 +/- 11%, respectively. In the peak fraction, Na-Ca exchange specific activity increased two- to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimum Mr of 224,000 Da. Specific 45Ca2+-binding SL proteins with Mr of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose. These data suggest that native cardiac Na-Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na-Ca exchange protein(s) remains incomplete.

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.

The effects of hyaluronidase on interstitial hydration, plasma protein exclusion, and microvascular permeability in the isolated perfused rat heart.

Hyaluronic acid, a principal glycosaminoglycan of the cardiac interstitium, may have a role in interstitial hydration, interstitial plasma protein exclusion and microvascular transport process (Wiederhielm, 1976b). We have investigated whether hyaluronidase reduces myocardial hyaluronate concentrations and thereby alters these several physical aspects in the isolated rat heart. Studies were conducted in ischemic, as well as aerobic hearts because of the reported therapeutic efficacy of the enzyme in myocardial ischemia. Two hours of perfusion with hyaluronidase significantly reduced myocardial hyaluronate content. Additionally, hyaluronidase decreased interstitial volume of both aerobic and otherwise edematous ischemic hearts, and prevented ischemic induced increased coronary vascular resistance in ischemic hearts. However, hyaluronidase did not effect the albumin interstitial exclusion volume or microvascular albumin and sorbitol exchange in aerobic hearts. In ischemic hearts, the enzyme did not prevent nor enhance the increase in microvascular permeability which occurred. We conclude that hyaluronate is neither a determinant of interstitial protein exclusion nor microvascular permeability, but plays an important role in interstitial hydration.

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.