ABSTRACT
Effect of strophanthidin (Str) on intracellular calcium concentration ([Ca2+]i) was investigated on isolated ventricular myocytes of guinea pig. Single ventricular myocytes were obtained by enzymatic dissociation technique. Fluorescent signal of [Ca2+]i was detected with confocal microscopy after incubation of cardiomycytes in Tyrode' s solution with Fluo3-AM. The result showed that Str increased [Ca2+]i in a concentration-dependent manner. The ventricular myocytes began to round-up into a contracture state once the peak level of [Ca2+]i was achieved in the presence of Str (10 micromol L(- 1)), but remained no change in the presence of Str (1 and 100 nmol L(-1)). Tetrodotoxin (TTX), nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str (1 and 100 nmol L(-1)) , but had no obvious effects on the action of Str (10 micromol L(-1)). The elevation of [Ca2+]i caused by Str at all of the detected concentrations was partially antagonized by rynodine (10 micromol L(-1)) or the removal of Ca2+ from Tyrode's solution. In Na+, K+ -free Tyrode' s solution, the response of cardiomycytes in [Ca2+]i elevation to Str (10 micromol L(-1)) was attenuated, while remained no change to Str (1 and 100 nmol L(-1)). TTX, nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str at all of the detected concentrations in Na+, K+ -free Tyrode's solution. The study suggests that the elevation of [Ca2+]i by Str at the low (nomomolar) concentrations is partially mediated by the extracellular calcium influx through Ca2+ channel or a "slip mode conductance" of TTX sensitive Na+ channel. While the effect of Str at high (micromolar) concentrations was mainly due to the inhibition of Na+, K+ -ATPase. Directly triggering the release of intracellular Ca2+ from sarcoplasmic reticulum (SR) by Str may be also involved in the mechanism of [Ca2+]i elevation.
Subject(s)
Animals , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester , Pharmacology , Aequorin , Pharmacology , Calcium , Metabolism , Calcium Channel Blockers , Pharmacology , Calcium Channels , Metabolism , Fura-2 , Pharmacology , Guinea Pigs , Myocardium , Pathology , Nifedipine , Pharmacology , Ryanodine , Pharmacology , Sarcolemma , Metabolism , Pathology , Sarcoplasmic Reticulum , Metabolism , Sodium-Calcium Exchanger , Sodium-Potassium-Exchanging ATPase , Strophanthidin , Pharmacology , Tetrodotoxin , Pharmacology , Thapsigargin , PharmacologyABSTRACT
<p><b>AIM</b>To study the involvements of nuclear factor of activated T-cells (NFATc) and NF-kappaB in calcineurin-mediated ischemic brain damage in vivo.</p><p><b>METHODS</b>The rat transient forebrain ischemia conducted through 15 min ischemia followed by 8, 24, and 72 h reperfusion was induced using the four-vessel method. The rats were divided randomly into five groups; sham control group, ischemia/reperfusion (I/R) group, CsA treated groups (for 8, 24, and 72 h reperfusion). Western blotting was performed to detect changes of FasL, NFATc, I-kappaB-alpha, and phospho-I-kappaB-alpha protein expression, and gel shift assays for NFAT FasL-DNA binding activities.</p><p><b>RESULTS</b>Western blotting showed that the expressions of both FasL and NFATc protein were significantly increased in the hippocampus of rat subjected to transient forebrain ischemia in comparison with those of the sham control group, which were markedly reduced by CsA. The I-kappaB-alpha protein showed no changes in all groups, and phospho-I-kappaB-alpha protein was not observed in this study. Proximal and distal FasL promoter NFAT sites bind NFAT proteins from the hippocampal neurons subjected to transient forebrain ischemia, and DNA-binding activities increased significantly compared with those of the sham control group. CsA markedly inhibited these changes.</p><p><b>CONCLUSION</b>NFATc may be involved in calcineurin-mediated ischemic brain damage and transcription factor NF-kappaB may not be involved.</p>