Biomedical subjects
Publications and source records attributed to R Ramasamy.
There is considerable interest in using merozoite proteins in a vaccine against falciparum malaria. Observations that antibodies to merozoite surface proteins block invasion are a basis for optimism. This article draws attention to important and varied aspects of how antibodies to Plasmodium falciparum merozoites affect red blood cell invasion.
Metabolic interventions that promote glucose use during ischemia have been shown to protect ischemic myocardium and improve functional recovery on reperfusion. We evaluated whether the cardioprotection afforded by high glucose during low-flow ischemia is associated with changes in the sarcolemmal content of glucose transporters, specifically GLUT-4. Isolated rat hearts were paced at 300 beats/min and perfused under normal glucose (5 mM) or high glucose (10 mM) conditions in buffer containing 0.4 mM albumin, 0.4 mM palmitate, and 70 mU/l insulin and subjected to 50 min of low-flow ischemia and 60 min of reperfusion. To determine the importance of insulin-sensitive glucose transporters in mediating cardioprotection, a separate group of hearts were perfused in the presence of cytochalasin B (10 microM), a preferential inhibitor of insulin-sensitive glucose transporters. Ischemic contracture during low-flow ischemia and creatine kinase release on reperfusion was decreased, and the percent recovery of left ventricular function with reperfusion was enhanced in hearts perfused with high glucose (P < 0.03). Hearts perfused with high glucose exhibited increased GLUT-4 protein expression in the sarcolemmal membrane compared with control hearts under baseline conditions, and these changes were additive with low-flow ischemia. In addition, high glucose did not affect the baseline distribution of sarcolemmal GLUT-1 and blunted any changes with low-flow ischemia. These salutary effects were abolished when glucose transporters are blocked with cytochalasin B. These data demonstrate that protection of ischemic myocardium by high glucose is associated with increased sarcolemmal content of the insulin-sensitive GLUT-4 and suggest a target for the protection of jeopardized myocardium.
Amyloid beta-peptide-binding alcohol dehydrogenase (ABAD) is a member of the family of short chain dehydrogenase/reductases whose distinctive properties include the capacity to bind amyloid beta-peptide and enzymatic activity toward a broad array of substrates including n-isopropanol and beta-estradiol. In view of the wide substrate specificity of ABAD and its high activity on l-beta-hydroxyacyl-CoA derivatives, we asked whether it might also catalyze the oxidation of the ketone body d-3-hydroxybutyrate. This was indeed the case, and oxidation proceeded with K(m) of approximately 4.5 mm and V(max) of approximately 4 nmol/min/mg protein. When placed in medium with d-beta-hydroxybutyrate as the principal energy substrate, COS cells stably transfected to overexpress wild-type ABAD (COS/wtABAD) better maintained 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, cellular energy charge, and morphologic phenotype compared with COS/vector cells. Using a severe model of metabolic perturbation, transgenic mice with targeted neuronal expression of ABAD subjected to transient middle cerebral artery occlusion showed strokes of smaller volume and lower neurologic deficit scores in parallel with increased brain ATP and decreased lactate, compared with nontransgenic controls. These data suggest that ABAD contributes to the protective response to metabolic stress, especially in the setting of ischemia.
The malaria vector Anopheles tessellatus is able to take several blood meals in a gonotrophic cycle. The fecundity is largely dependent on the first blood meal and is not generally increased by subsequent blood meals during a gonotrophic cycle. Larval rearing densities influenced adult body size. There is an inverse relationship between wing length and larval rearing densities. Smaller mosquitoes produced from larvae reared at higher densities had reduced body reserves of protein, lipid and carbohydrates. At emergence, ovarian development in An. tessellatus is in the previtellogenic stage and it remained at this stage until the intake of a blood meal. The number of ovarian follicles is related to wing length and, irrespective of adult body size, An. tessellatus developed oocytes to maturity with a single blood meal. This is attributed to the availability of metabolic reserves above the threshold level required for further development of oocytes. Mosquitoes that took more than one blood meal had largely digested their previous blood meal and had ongoing vitellogenesis. Blood meals subsequent to the first one apparently contribute mainly to increasing metabolic reserves. The stimulus for a second and third blood meal in An. tessellatus appears to be completion of the digestion of the previous blood meal. There was no evidence that multiple blood meals taken in the first gonotrophic cycle influenced fecundity significantly in the second cycle.
In Sri Lanka, malaria is transmitted mainly by Anopheles culicifacies Giles sensu lato (Diptera: Culicidae). In India, this nominal taxon comprises sibling species A, B, C, D and E, distinguished by their chromosome morphology. Species B (identified by polytene chromosome sequence Xab, 2g1 + h1) is not such an efficient vector of malaria as other members of the An. culicifacies complex in India. All specimens of An. culicifacies s.l. examined from Sri Lanka possess Xab, 2g1 + h1 polytenes, previously interpreted as species B, despite their important vector status. Recently, species E was described from Rameshwaram Island (Tamil Nadu, India) between Sri Lanka and the Indian mainland, where both species B and E are sympatric. Species B and E share polytene sequence Xab, 2g1 + h1 but differ by the mitotic Y-chromosome being acrocentric in species B, submetacentric in species E, the latter implicated as vector of vivax malaria. From May 1999 to January 2000, we surveyed Y-chromosomes of male progeny from An. culicifacies Xab, 2g1 + h1 females collected from cattle bait in diverse malarious districts of Sri Lanka: Badulla, Monaragala, Puttalam and Trincomalee. Karyotypes of readable quality were obtained from 42/83 families examined, with overall proportions 24% acrocentric and 76% submetacentric Y-chromosome carriers, both types being sympatric in at least 3/4 localities sampled. By analogy with the situation on Rameshwaram Island, we interpret these observations to demonstrate widespread presence of two members of the An. culicifacies complex in Sri Lanka, their karyotypes being compatible with species B and E, the latter predominant and having greater vector potential.
BACKGROUND: We have previously demonstrated that perfusion of isolated hearts with high concentrations of glucose results in increased glycolysis during ischemia, diminished ischemic injury, and improved functional recovery with reperfusion. OBJECTIVE: To evaluate a possible mechanism by which glucose conferred this protection. We examined the hypothesis that increased exogenous glucose concentrations results in increased concentrations of fructose-2,6-bisphosphate, a potent activator of phosphofructokinase-1, and thus increases glycolysis. METHODS: Perfused rabbit hearts were subjected to 60 min of low-flow ischemia. Control hearts were perfused with buffer containing 0.4 mmol/l palmitate, 5 mmol/l glucose, and 70 mU/l insulin, and treated hearts were perfused with buffer containing 0.4 mmol/l palmitate, 15 mmol/l glucose and 210 mU/l insulin. RESULTS: Ischemic contracture was attenuated by perfusion of high concentrations of glucose (high glucose) (P < 0.05 compared with control). Glucose uptake and lactate production were greater in hearts perfused with high glucose, as was the ATP concentration at the end of ischemia (P < 0.05 compared with controls). Exogenous glucose uptake and lactate production correlated well with fructose-2,6-bisphosphate content (P = 0.007). CONCLUSIONS: Enhancement of glycolysis in hearts perfused with high glucose may be the result of stimulation of phosphofructokinase-1 by fructose-2,6-bisphosphate. Accordingly, this may serve as an important mechanism by which cardioprotection may be achieved.
Nicotinic acid (niacin) has been shown to decrease myocyte injury. Because interventions that lower the cytosolic NADH/NAD(+) ratio improve glycolysis and limit infarct size, we hypothesized that 1) niacin, as a precursor of NAD(+), would lower the NADH/NAD(+) ratio, increase glycolysis, and limit ischemic injury and 2) these cardioprotective benefits of niacin would be limited in conditions that block lactate removal. Isolated rat hearts were perfused without (Ctl) or with 1 microM niacin (Nia) and subjected to 30 min of low-flow ischemia (10% of baseline flow, LF) and reperfusion. To examine the effects of limiting lactate efflux, experiments were performed with 1) Ctl and Nia groups subjected to zero-flow ischemia and 2) the Nia group treated with the lactate-H(+) cotransport inhibitor alpha-cyano-4-hydroxycinnamate under LF conditions. Measured variables included ATP, pH, cardiac function, tissue lactate-to-pyruvate ratio (reflecting NADH/NAD(+)), lactate efflux rate, and creatine kinase release. The lactate-to-pyruvate ratio was reduced by more than twofold in Nia-LF hearts during baseline and ischemic conditions (P < 0.001 and P < 0.01, respectively), with concurrent lower creatine kinase release than Ctl hearts (P < 0.05). Nia-LF hearts had significantly greater lactate release during ischemia (P < 0.05 vs. Ctl hearts) as well as higher functional recovery and a relative preservation of high-energy phosphates. Inhibiting lactate efflux with alpha-cyano-4-hydroxycinnamate and blocking lactate washout with zero flow negated some of the beneficial effects of niacin. During LF, niacin lowered the cytosolic redox state and increased lactate efflux, consistent with redox regulation of glycolysis. Niacin significantly improved functional and metabolic parameters under these conditions, providing additional rationale for use of niacin as a therapeutic agent in patients with ischemic heart disease.
Asymmetric dimethylarginine (ADMA), a compound detectable in human plasma, is an endogenous inhibitor of NO synthase. Endothelial dysfunction is an early event in atherogenesis, and large-vessel atherosclerosis is a major cause of morbidity and mortality in patients with type 2 diabetes mellitus. Fifty patients with type 2 diabetes mellitus were studied at baseline and 5 hours after ingestion of a high-fat meal. Plasma ADMA measured by using high-performance liquid chromatography increased from 1.04+/-0.99 to 2.51+/-2.27 micromol/L (P:<0.0005). Brachial arterial vasodilation after reactive hyperemia, a NO-dependent function, measured by high-resolution ultrasound, decreased from 6.9+/-3.9% at baseline to 1.3+/-4.5% (P:<0.0001). These changes occurred in association with increased plasma levels of triglycerides and very low density lipoprotein triglycerides, with reduced low density lipoprotein cholesterol and high density lipoprotein cholesterol, and with no changes in total cholesterol. The increase in plasma ADMA in response to a high-fat meal was significantly and inversely related to the decrease in percent vasodilation. In 10 of the subjects studied with a similar protocol on another day, no significant changes in the brachial artery flow responses or in plasma ADMA were observed 5 hours after ingestion of a nonfat isocaloric meal. The data suggest that ADMA may contribute to abnormal blood flow responses and to atherogenesis in type 2 diabetics.
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Nutrient stimulation of pancreatic beta-cells increases the cellular reduced pyridine nucleotide content, but the specific role of cytosolic redox state in glucose-induced insulin release (GIIR) remains undetermined. The role of cytosolic redox state has been assessed (as reflected by the lactate/pyruvate ratio) in nutrient- and non-nutrient-induced insulin release using a recently established glucose-sensitive clonal beta-cell line (BRIN-BD11). Long-term exposure to the NAD+ precursor vitamin nicotinic acid (NA, 100 microM) was used to promote a more oxidized state in the cytosol. Glucose (2-16 mM) evoked a dose-dependent rise in the cytosolic NADH/NAD+ ratio which was linearly related to the extent of GIIR. NA suppressed the glucose-induced rise in the NADH/NAD+ ratio and concomitantly reduced GIIR by 44%. It also inhibited, by 47%, the average glucose-induced rise in cytosolic free Ca2+ concentration ([Ca2+]i, assessed by fura-2 microfluorometry from single cells). The latter effect was not accounted for by a reduction in the activity of voltage-sensitive Ca2+ channels, inasmuch as both high K+- and tolbutamide-induced [Ca2+]i rises remained insensitive to NA exposure. NA did not affect insulin release evoked by any of the depolarizing agents, indicating that steps in the stimulus-secretion coupling cascade distal to Ca2+ influx are insensitive to changes in the cytosolic redox state. It is concluded that GIIR is partially controlled by the cytosolic redox state. Moreover, the impairment in GIIR, caused by a shift toward a more oxidized state in the cytosol, originates from an attenuated [Ca2+]i response. The latter is likely mediated by the influence of cytosolic redox state on specific metabolic pathways (NADH shuttle systems and/or the malonyl-CoA pathway), leading ultimately to enhancement of the activity of ATP-sensitive K+ channels.
The gene for a 45 kDa merozoite surface protein (MSA-2) of the human malaria parasite Plasmodium falciparum was PCR amplified and cloned into eukaryotic expression vectors VR1012 and pcDNA3 to yield plasmids P1 and P2, respectively. The coding sequences for two N-terminal fragments of the 185 kDa merozoite surface protein (MSA-1) gene were similarly PCR amplified and cloned into vectors VR1020 and VR1012 to yield plasmids P3 and P4, respectively. The MSA-1 signal peptide sequence, present in P4, was replaced with the human tissue plasminogen activator signal sequence in P3. The four plasmids expressed the cloned genes under the control of the cytomegalovirus promoter and carried 3' bovine growth hormone termination/poly A signals. P1, P3 and P4 also contained the cytomegalovirus intron A enhancer sequence. MSA-1 expression was more readily detected than MSA-2 in Cos cells transfected with P3/P4 and P1/P2 respectively. The MSA-2 gene was also cloned into the phagemid pBluescript IISK+ with and without a 3' poly A tail composed of 35 A residues. MSA-2 was synthesised in HeLa cells infected with a recombinant vaccinia virus carrying T7 RNA polymerase when MSA-2 recombinant pBluescript was transfected into the cells. Inoculation with P1 intramuscularly or intradermally and with P2 intradermally into rabbits led to the production of antibodies to MSA-2 detectable by immunofluorescence and Western blotting. Antibodies were also produced against MSA-1 after intramuscular/intradermal inoculation with P3 and P4. Inoculation of rabbits with MSA-2 mRNA yielded better antibody titres when a poly A tail was present. Antibody levels were maintained for > 9 weeks after the final immunisation. However the immune sera failed to inhibit in vitro parasite growth.
Three model peptides containing B-epitopes from conserved, non-repetitive regions of the merozoite surface antigens, MSA2 and MSA1, and the erythrocyte binding protein EBP of Plasmodium falciparum were synthesised. The peptides incorporated GPG spacers and C residues at the N and C termini, and were polymerised by oxidation to form cystine bridges. Multiple copies of essentially the same peptide sequences were also synthesised on a branching lysyl matrix to form a tetrameric multiple antigen peptide. Rabbits were immunised with the polymerised and multiple antigen peptides, in alum followed by Freund's adjuvant, and the antibody responses examined by IFA and ELISA. Reproducible antibody responses were obtained against the MSA1 and EBP but not MSA2 peptides. IgG antibody levels detected by ELISA after three injections of antigen in alum, increased significantly after further immunisation in Freund's adjuvant. IgG levels were largely maintained for at least 23 weeks after the final immunisation. IgM antibodies, generally detectable only after immunisation in Freund's adjuvant, were absent 23 weeks later. Antibody titres against the native protein on fixed parasites, assayed by IFA, were three to five orders of magnitude lower than the corresponding ELISA titres against the peptides. Antibody-dependent inhibition of P. falciparum growth in vitro could not be demonstrated with the immune rabbit sera. The MSA1 and EBP peptides elicited cross-reactive antibodies. The results suggest that the selected non-repetitive sequences are conformationally constrained in the native proteins and only a small proportion of the anti-peptide antibodies bind to the native proteins. The significance of the findings for the development of peptide vaccines and the use of peptides in immunoassays is discussed.
It has been previously suggested that alterations in sodium homeostasis, leading to calcium overload may play a part in the mediation of cardiac ischemic injury. It has been demonstrated that the Na+-H+ exchanger plays an important role with regard to the regulation of intracellular sodium during ischemia and reperfusion and that inhibition of the Na+-H+ exchanger during ischemia protects hearts from ischemic injury. Studies using chemically-induced diabetic animals have suggested that the cardiac Na+-H+ exchanger in the diabetic heart is impaired and is responsible for limiting the increase in sodium during ischemia. The extent to which the Na+-H+ exchanger contributes to increases in intracellular sodium during ischemia in diabetic hearts is unclear as direct measurements of exchanger activity have not been made in genetically diabetic hearts. Therefore, this paper aims to address the following issues: (a) is the Na+-H+ exchanger impaired in a genetically diabetic rat heart: (b) does this impairment result in lower [Na]i or [Ca]i during ischemia; and (c) does Na+-H+ exchanger inhibition limit injury and functional impairment in diabetic hearts during ischemia and reperfusion? These issues were examined by inhibiting the Na+-H+ exchanger with ethylisopropylamiloride (EIPA) in isolated perfused hearts from both genetically diabetic (BB/W) and non-diabetic rats. Levels of intracellular sodium, intracellular calcium, intracellular pH and high energy phosphates (using 23Na,19F, 31P NMR spectroscopies, respectively) during global ischemia and reperfusion were also measured. The impact of diabetes on Na+-H+ exchanger activity was assessed by measuring pH recovery of these hearts after an acid load. Creatine kinase release during reperfusion was used as a measure of ischemic injury. This study demonstrated that the Na+-H+ exchanger is impaired in diabetic hearts. Despite this impaired activity, inhibition of Na+-H+ exchanger protected diabetic hearts from ischemic injury and was associated with attenuation of the rise in sodium and calcium, and limitation of acidosis and preservation of ATP during ischemia. The data presented here favor the use of Na+-H+ exchanger inhibitors to protect ischemic myocardium in diabetics. Also, the data provides possible mechanisms for the altered susceptibility of diabetic hearts to ischemic injury.
Repetitive brief ischemic episodes (ischemic preconditioning, PC) result in transient intracellular acidosis and protect the heart from subsequent ischemic injury, potentially through a protein kinase C (PKC)-dependent mechanism. We hypothesized that repetitive brief acidification of the heart without concomitant ischemia would also protect the heart from ischemic injury via a PKC-dependent mechanism. Isolated rat hearts underwent 30 min of global ischemia following control perfusion (CTL), or after PC or repetitive acidosis (RA), in the presence of absence of chelerythrine, a specific PKC inhibitor. Intracellular pH, PCr and ATP were measured using 31P NMR spectroscopy, while intracellular sodium [Na]i was measured using 23Na spectroscopy. Na,K-ATPase activity was measured prior to ischemia and on reperfusion. Both PC and RA resulted in transient acidification prior to ischemia. Ischemic injury, as assessed by creatinine kinase (CK) release on reperfusion, was reduced in both the PC and RA hearts [63+/-14 and 16+/-4 IU/g dry weight (dw) respectively, v 705+/-72 IU/gdw for control P<0.001], and was associated with improved functional recovery on reperfusion. PC and RA each significantly reduced Na,K-ATPase activity prior to ischemia (8.18+/-0.47 and 7.76+/-0.54 micromol ADP/h/mg protein) when compared to control (11.05+/-0.54 micromol ADP/h/mg protein P<0.05), limited the rate of ATP depletion during ischemia, and resulted in more rapid normalization of [Na]i on reperfusion. Chelerythrine resulted in intermediate CK release in PC and RA hearts (443+/-48 and 375+/-72 IU/gdw, P<0.001 v PC, P<0.01 v control), but did not alter the rate of ATP depletion or [Na]i kinetics in either PC or RA hearts. PC and RA each protect the ischemic heart, having in common ATP preservation during ischemia and more rapid normalization of [Na]i on reperfusion. These effects, not modulated by protein kinase C, are consistent with the hypothesis that ATP preservation during ischemia provides enhanced substrate for sodium efflux via the Na,K-ATPase on reperfusion.
OBJECTIVE: We have previously demonstrated that zopolrestat, an inhibitor of the enzyme aldose reductase, reduces ischemic injury in hearts from diabetic and non-diabetic rats. To further explore potential cardioprotective mechanisms of zopolrestat, we measured changes in intracellular sodium, calcium, and Na+,K(+)-ATPase activity in zopolrestat treated hearts during ischemia and reperfusion. METHODS: Hearts from acute diabetic (Type I) and age-matched control rats were isolated and retrogradely perfused. Hearts had either control perfusion or exposure to 1 microM zopolrestat for 10 min, followed by 20 min of global ischemia and 60 min of reperfusion. Changes in intracellular sodium and calcium were measured using 23Na and 19F magnetic resonance spectroscopy, respectively, while the activity of Na+,K(+)-ATPase was measured using biochemical assays. RESULTS: Zopolrestat blunted the rise in [Na]i during ischemia in both diabetic hearts and non-diabetic hearts. The end-ischemic [Na]i was 21.3 +/- 2.6 mM in the zopolrestat treated diabetics and 25.9 +/- 2.3 in zopolrestat treated non-diabetics, versus 31.6 +/- 2.6 mM and 32.9 +/- 2.8 mM in the untreated diabetics and untreated non-diabetics, respectively, (P = 0.002). Similarly, the rise in [Ca]i at the end of ischemia was significantly reduced in zopolrestat treated diabetic and non-diabetic hearts (P = 0.005). Zopolrestat increased the activity of Na-,K(+)-ATPase in diabetic hearts under baseline conditions (11.70 +/- 0.95 versus 7.28 +/- 0.98 mumol/h/mg protein, P = 0.005) as well as during ischemia and reperfusion. Similar changes in Na+,K(+)-ATPase activity were also observed in non-diabetic hearts. CONCLUSIONS: The data provide additional support to the protective effects of zopolrestat and suggest that a possible mechanism of action may be associated with the attenuation of the rise in [Na]i and [Ca]i during ischemia and reperfusion.
INTRODUCTION: Fasting protects the ischemic heart from injury and infarction. Previous studies have shown that hearts from fasted animals have greater glycogen utilization and a lower cytosolic redox state (NADH/NAD+) during global ischemia. While the mechanisms of increased glycogen utilization in fasted animals have not been elucidated, animals that hibernate or are tolerant of anoxia are known to increase the tissue content of the active form of glycogen phosphorylase, phosphorylase a. Therefore, this study was designed to (a) determine whether hearts from fasted animals have increased activity of glycogen phosphorylase during ischemia and (b) define those mechanisms responsible for this increase. METHODS: Hearts isolated from either fed or fasted (24 h) rats were perfused and freeze-clamped at baseline, and after 1 and 10 min of ischemia, for measurement of phosphorylase activity, phosphorylase kinase activity, and glucose-6-phosphate concentrations. RESULTS: Fasting increased the phosphorylase a/b ratio under both baseline and ischemic conditions. This increase was not accompanied by an increase in the activity of phosphorylase kinase, either with maximal [Ca2+] or under physiologic [Ca2+]. Glucose 6-phosphate concentrations were lower in hearts from fasted animals under baseline, but not ischemic, conditions. CONCLUSIONS: Fasting enhances glycogen utilization during ischemia by increasing the active form of glycogen phosphorylase. This increase is not due to a change in phosphorylation by phosphorylase kinase nor end-product inhibition by G-6P. While the precise mechanism of increased glycogen phosphorylase activity in fasted animals is not clear, one likely explanation may be the lower cytosolic redox state demonstrated in the myocardium of fasted animals.
The 40-50 kDa merozoite surface antigen (MSA2) is a candidate molecule for use in a malaria vaccine. The gene for MSA2 from the 3D7 isolate of Plasmodium falciparum was amplified by polymerase chain reaction and cloned into the bacterial expression vector pGEX-3X to obtain a fusion protein of MSA2 with Schistosoma japonicum glutathione S-transferase. The recombinant fusion protein was used to immunize rabbits. After four injections, the sera had Western blotting and immunofluorescence titres of 10(-6). Immune sera, and immunoglobulin (Ig)G, F(ab)'2, F(ab) prepared from the immune sera, were assessed for their effects on the growth of 3D7 parasites in vitro by microscopy and a [3H]-hypoxanthine incorporation assay. The antibodies did not significantly inhibit red blood cell invasion and parasite growth when added to cultures as 10% v/v serum or as immunoglobulin preparations at concentrations up to 200 microg ml(-1). However, in the presence of IgG or F(ab)'2, but not F(ab), antibodies to MSA2, the proportions of red blood cells invaded by more than one merozoite increased significantly. Multiple invasion is attributed to merozoites cross-linked by bivalent antibodies, attaching to and subsequently invading the same red cell. These observations have a bearing on the evasion of host immune responses by the parasite and the use of full-length recombinant MSA2 protein in a malaria vaccine.
Myocardial ischemia results in an increase in intracellular sodium concentration ([Na]i), which may lead to cellular injury via cellular swelling and calcium overload. Because protein kinase C (PKC) has been shown to reduce Na-K-ATPase activity, we postulated that pharmacological inhibition of PKC would directly increase Na-K-ATPase activity, reduce [Na]i during ischemia, and provide protection from ischemic injury. Isolated rat hearts were subjected to 30 min of global ischemia with and without the specific PKC inhibitor chelerythrine. Intracellular pH, ATP, and [Na]i were assessed using 31P and 23Na NMR spectroscopy, whereas Na-K-ATPase and PKC activity were determined using biochemical assays. Na/H exchanger activity was determined using the ammonium prepulse technique under nonischemic conditions. Chelerythrine increased Na-K-ATPase activity (13.76 +/- 0.89 vs. 10.89 +/- 0.80 mg ADP. h(-1). mg protein(-1); P = 0.01), reduced PKC activity in both the membrane and cytosolic fractions (39% and 28% of control, respectively), and reduced creatine kinase release on reperfusion (48 +/- 5 IU/g dry wt vs. 689 +/- 63 IU/g dry wt; P = 0.008). The rise in [Na](i) during ischemia was significantly reduced in hearts treated with chelerythrine (peak [Na](i) chelerythrine: 21.5 +/- 1.2 mM; control: 31.9 +/- 1.2 mM; P < 0.0001), without an effect on either acidosis (nadir pH 6.16 +/- 0.05 for chelerythrine vs. 6.08 +/- 0.04 for control), the rate of ATP depletion or Na/H exchanger activity. These data support the hypothesis that pharmacological inhibition of PKC before ischemia induces cardioprotection by reducing intracellular sodium overload via an increase in Na-K-ATPase activity.