Showing papers by "Dipak K. Das published in 1996"

Phospholipase D Plays a Role in Ischemic Preconditioning in Rabbit Heart

Abstract: Background Activation of protein kinase C (PKC) is thought to be a critical step in ischemic preconditioning. Many receptor agonists activate PKC via stimulation of phospholipase C (PLC), which degrades membrane phospholipids to diacylglycerol (DAG), an important PKC cofactor. However, adenosine receptors, critical components of the prototypical preconditioning pathway, are not thought to couple to PLC in the cardiomyocyte. We therefore tested whether ischemic preconditioning or adenosine might instead activate phospholipase D (PLD) to produce DAG. Methods and Results PLD activity was measured in isolated rabbit hearts. Ischemic injury was evaluated in either isolated rabbit hearts or dispersed myocytes. PLD activity doubled from a control level of 74.8±10.0 to 140.0±11.5 μmol·min −1 ·g −1 ( P N 6 -(2-phenylisopropyl)-adenosine [(R)-PIA] for 20 minutes. When sodium oleate, which activates PLD, was administered to isolated hearts before a 30-minute coronary occlusion, infarct size (15.6±2.0% of the risk zone) was significantly smaller than in untreated hearts (30.4±2.2%; P Conclusions We conclude that PLD stimulation is involved in the protection of ischemic preconditioning in the rabbit heart.

The role of protein kinase C in ischemic/reperfused preconditioned isolated rat hearts

Abstract: Protein kinase C (PKC) has been implicated in the preconditioning-induced cardiac protection in ischemic/reperfused myocardium. We studied the effect of PKC inhibition with calphostin C (25, 50, 100, 200, 400, and 800 nM), a potent and specific inhibitor of PKC, in isolated working nonpreconditioned and preconditioned ischemic/reperfused hearts. In the nonpreconditioned groups, all hearts underwent 30 min of normothermic global ischemia followed by 30 min of reperfusion. In the preconditioned groups, hearts were subjected to four cycles of ischemic preconditioning by using 5 min of ischemia followed by 10 min reperfusion, before the induction of 30 min ischemia and reperfusion. At low concentrations of calphostin C (25, 50, and 100 nM), the PKC inhibitor had no effect on the incidence or arrhythmias or postischemic cardiac function in the nonpreconditioned ischemic/reperfused groups. With 200 and 400 nM of calphostin C, a significant increase in postischemic function and a reduction in the incidence of arrhythmias were observed in the nonpreconditioned ischemic/reperfused groups. Increasing the concentration of calphostin C to 800 NM, the recovery of postischemic cardiac function was similar to that of the drug-free control group. In preconditioned hearts, lower concentrations (< 100 nM) of calphostin C did not change the response of the myocardium to ischemia and reperfusion in comparison to the preconditioned drug-free myocardium. Two hundred and 400 nM of the PKC inhibitor further reduced the incidence of ventricular fibrillation (VF) from the preconditioned drug-free value of 50% to 0 (p < 0.05) and 0 (p < 0.05), respectively, indicating that the combination of the two, preconditioning and calphostin C, affords significant additional protection. Increasing the concentration of calphostin C to 800 nM blocked the cardioprotective effect of preconditioning (100% incidence of VF). The recovery of cardiac function was similarly improved at calphostin C doses of 200 and 400 nM and was reduced at 800 nM (p < 0.05). With 200 and 400 nM of calphostin C, both cytosolic and particulate PKC activity were reduced by approximately 40 and 60%, respectively, in both preconditioned and preconditioned/ischemic/reperfused hearts. The highest concentration of calphostin C (800 nM) resulted in almost a complete inhibition of cytosolic (100%) and particulate (85%) PKC activity correlated with the abolition of preconditioning-induced cardiac protection. In conclusion, calphostin C protects the ischemic myocardium obtained from intact animals, provides significant additional protection to preconditioning at moderate doses, and blocks the protective effect of preconditioning at high concentrations. The dual effects of calphostin C appear to be strictly dose and "enzyme inhibition" related.

Molecular cloning, sequencing and expression analysis of a fatty acid transport gene in rat heart induced by ischemic preconditioning and oxidative stress.

Abstract: In this study, ischemia and oxidative stress-inducible gene expression in heart was examined by subtractive hybridization technique. Total RNA was isolated from ventricular muscle fragments of normal and oxidative stress-induced hearts. Poly (A)+ RNA was purified followed by the construction of a plasmid cDNA library. This was followed by the subtractive screening of oxidative stress-induced cDNA library. The positive colonies were amplified and the plasmid isolated. An aliquot was subjected to restriction cutting with Bam HI and EcoRI; the fragments corresponding to cDNA insert were separated by electrophoresis, radiolabeled by random-primed DNA synthesis, and used as probes in standard Northern blotting experiments. An aliquot containing the plasmid from the confirmed positives was then subjected to bidirectional partial DNA sequencing (using M13 and T7/T3α primers) by the chain-extension/chain termination method. These sequences were subjected to a computerized search for homologies against all sequences in the updated worldwide Gen Bank and EMBL sequence databases followed by restriction mapping and reading frame identification. Out of 24 putative positive colonies screened, one clone was matched with > 97% homology with FAT gene that has been implicated in binding or transport of long chain fatty acids. cDNA probe synthesized from this clone identified two major transcripts of 4.8 and 2.9 kb. Additional experiments were then performed where isolated perfused rat hearts were subjected to the following treatments: (1)5 min ischemia; (2) 10 min ischemia; (3) 20 min ischemia; (4) 5 min ischemia followed by 10 min reperfusion (ischemic preconditioning); and (5) 5 min ischemia followed by 10 min reperfusion, repeated four times (4 × preconditioning). RNAs were extracted from these hearts and hybridized with the FAT cDNA probe. The results indicated that FAT gene was induced by oxidative stress, ischemic preconditioning, but not by ischemia. (Mol Cell Biochem 160/161:241–247, 1996)

Lanthanum Provides Cardioprotection by Modulating Na+-Ca2+ Exchangea

Abstract: Intracellular Ca2+ overloading is a well-recognized feature in myocardial ischemic and reperfusion injury.' The exact mechanism for Caz+ influx into the cell remains controversial, but it is generally accepted that both Ca2+ slow channels and Nat-Ca2+ exchangers play a role in the pathophysiology of myocardial ischemic and reperfusion injury.2 It is known that acidosis induced by anaerobic metabolism leads to enhanced exchange of intracellular H+ for Nat which in turn becomes instrumental for the intracellular Ca2+ accumulation. This hypothesis is well supported by the observations that inhibitors of Na+-H+exchangers such as amiloride can inhibit the accumulation of Ca2+.' Approximately 41% of the total exchangeable Ca2 is localized in a kinetic compartment defined by rapid compartment, and this Ca2' pool is rapidly displaced by lanthanum (La3+), a known inhibitor of Ca2+-binding protein^.^ La3+ is also a potent inhibitor of CaZf entry into mitochondria via the uniport ~ y s t e m . ~ La3+ has also been found to nonspecifically block the Ca2+ channels. In this study, we examined the effects of La3+ on myocardial rhythm disturbances associated with ischemia and reperfusion. In an attempt to examine its mechanism of actions, we also examined the myocardial contents of Na+, K+, Ca2* and Mg2+.