kb-r7943 and Ischemia

Several studies indicate that an increase in the extracellular potassium (K+) concentration is a factor exerting a damaging effect on cochlear hair cells (HCs). The present study was designed to examine the effects of high extracellular K+ concentrations on the HCs under normoxic and ischemic conditions. Organotypic cultures of the organ of Corti of newborn rats were exposed to normoxia and ischemia at K+ concentrations of 5-70 mM in artificial perilymph for 3-4h. The number of IHCs and OHCs in the apical, medial and basal parts of the cochlea were counted 24h later. The work resulted in two main findings: (1) extracellular K+ concentrations of 30-70 mM had no effect on the HCs under normoxic conditions; (2) under ischemic conditions, a clear HC loss, mainly in the medial and basal cochlear parts, was observed at 5 mM K+ as previously reported. In contrast, a high extracellular K+ concentration strongly attenuated the HC loss. This effect nearly completely disappeared by the addition of both eosin, an inhibitor of the plasma membrane calcium ATPase (PMCA), and linopirdine, an inhibitor of the KCNQ4 channel, indicating that a normal activity of the PMCA and the KCNQ4 channels are key factors for HC survival under ischemia and depolarizing conditions.

Topics: Animals; Animals, Newborn; Calcium; Calcium-Transporting ATPases; Hair Cells, Auditory; Indoles; Ischemia; KCNQ Potassium Channels; Organ Culture Techniques; Potassium; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Voltage-Gated; Pyridines; Rats; Rats, Wistar; Sodium-Calcium Exchanger; Thiourea

Reverse-mode activation of the Na+-Ca2+ exchanger (NCX) at the time of reperfusion following ischemia contributes to Ca2+ overload and cardiomyocyte injury. The aim of the present study was to determine whether increased NCX in the myocardium that survived after infarction enhances its vulnerability to ischemia/reperfusion injury. Rabbits were divided into post-MI and sham groups and underwent ligation of the left circumflex coronary artery and sham operation, respectively. Two weeks later, hearts were isolated and perfused with crystalloid in the Langendorff mode with monitoring of left ventricular (LV) pressure. NCX level in the myocardium was determined by Western blotting. Myocardial stunning was induced by 5 episodes of 5-min global ischemia/5-min reperfusion. Using separate groups of hearts, myocardial infarction was induced by 30-min global ischemia/2-h reperfusion with or without treatment with 0.3 microM KB-R7943, a reverse-mode selective blocker of NCX. Heart weight-to-body weight ratio was 20% larger and NCX protein level was 60% higher in the post-MI group than in the sham group. However, there were no significant differences between severities of myocardial stunning after the repetitive ischemia/ reperfusion (18 +/- 7 vs. 25 +/- 2% reduction in LV developed pressure) and between infarct sizes after 30-min ischemia (59.1 +/- 4.1 vs. 63.0 +/- 4.5% of risk area) in the post-MI and sham groups. KB-R7943 limited infarct size in the post-MI group by 53%, and the extent of this protection was not different from that we have reported for hearts without previous infarcts (i.e. 45% reduction of infarct size). These results suggest that enhanced NCX expression does not necessarily increase myocardial vulnerability to myocardial stunning and infarction.

Topics: Animals; Anti-Arrhythmia Agents; Blotting, Western; Heart Ventricles; Ischemia; Myocardial Infarction; Myocardium; Necrosis; Organ Size; Oxygen; Rabbits; Reperfusion Injury; Sodium-Calcium Exchanger; Thiourea; Time Factors

Sarcoplasmic reticulum (SR) dysfunction is one of the multiple alterations that occurs in ischemia-reperfused hearts. Because SR function is regulated by phosphorylation of phospholamban (PLB), a SR protein phosphorylated by cAMP-dependent protein kinase (PKA) at Ser(16)and Ca(2+)-calmodulin-dependent protein kinase (CaMKII) at Thr(17), the phosphorylation of these residues during ischemia and reperfusion was examined in Langendorff-perfused rat hearts. Ser(16)phosphorylation increased significantly after 20 min of ischemia from 2.5+/-0.6% to 99.8+/-25.5% of maximal isoproterenol-induced site-specific phosphorylation and decreased to control values immediately after reperfusion. Thr(17)phosphorylation transiently increased at 2-5 min of ischemia and at 1 min of reperfusion (R1, 166.2+/-28.2%). The ischemia-induced increase in Ser(16)phosphorylation was significantly diminished in hearts from catecholamine-depleted animals and/or after beta-blockade and abolished in the presence of the PKA-inhibitor, H-89. Thr(17)phosphorylation at the beginning of ischemia was blunted by nifedipine, whereas at R1 it was significantly diminished by perfusion with 0 m m Ca(2+)in the presence of EGTA and by the Na(+)/Ca(2+)exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit CaMKII, decreased Thr(17)phosphorylation at R1 and significantly prolonged half relaxation time. The results demonstrated a dissociation between the phosphorylation of PLB sites, being phosphorylation of Ser(16)dependent on the beta-adrenergic cascade during ischemia and phosphorylation of Thr(17)on Ca(2+)influx both, at the beginning of ischemia and reperfusion. Phosphorylation of Thr(17)at the onset of reflow may provide the cell a mechanism to cope with Ca(2+)overload, transiently favoring the recovery of relaxation during early reperfusion.

Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Catecholamines; Cyclic AMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Heart; Ischemia; Male; Myocardium; Phosphorylation; Rats; Rats, Wistar; Receptors, Adrenergic, beta; Reperfusion Injury; Sarcoplasmic Reticulum; Serine; Sodium-Calcium Exchanger; Thiourea; Threonine; Time Factors

We investigated whether the preischemic or postischemic treatment with KB-R7943, a novel and selective Na+/Ca2+ exchange inhibitor, has renal protective effects in mice with ischemic acute renal failure (ARF). Ischemic ARF was induced by clamping the left renal pedicle for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function was markedly diminished 24 h after reperfusion. Preischemic treatment with KB-R7943 attenuated the ARF-induced renal dysfunction. The ischemia/reperfusion-induced renal dysfunction was also overcome by postischemic treatment with KB-R7943. Histopathologic examination of the kidneys of ARF mice revealed severe renal damage such as tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. Histologically evident damage and Ca2+ deposition in necrotic tubular epithelium were improved by preischemic treatment with KB-R7943. In addition, preischemic treatment with KB-R7943 significantly suppressed the increment of endothelin-1 (ET-1) content in the kidney at 2, 6, and 24 h after reperfusion. These findings suggest that Ca2+ overload via the reverse mode of Na+/Ca2+ exchange, followed by renal ET-1 overproduction, plays an important role in the pathogenesis of the ischemia/reperfusion-induced ARF. KB-R7943 may prove to be an effective therapeutic agent for cases of ischemic ARF in humans.

Topics: Acute Kidney Injury; Animals; Calcium; Endothelin-1; Ischemia; Kidney; Male; Mice; Reperfusion Injury; Sodium-Calcium Exchanger; Thiourea