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Aim: To investigate the influence of resveratrol (Res), a nutritional antioxidant, on the inhibition of apoptosis in rat primary neuron cultures., Methods: The cultured cortical neurons of neonatal Sprague-Dawley rats were pretreated with Res (0.1, 1.0, and 10.0 micromol/L) and oxygen-glucose deprivation/reperfusion (OGD/RP) with oxygen and glucose were initiated at d 10 in vitro. Neuronal apoptosis was determined by flow cytometry, and morphological changes of neurons were observed by an electron microscope. For the mechanism studies, the intracellular free calcium concentration ([Ca2+]i) and the transcription of caspases-3 and -12 in neurons were detected by Fura 2/AM loading and real-time RT-PCR, respectively., Results: OGD/RP insult could induce an increase in the apoptotic rate of neurons (from 11.1% to 49.0%), and elicit an obvious morphological change in neurons; pretreatments with Res (0.1, 1.0, and 10.0 micromol/L, respectively) significantly reduced the elevated rate of apoptosis to 41.7%, 40.8%, and 37.4%, respectively, and ameliorated the neuronal morphological injury. Similarly, the OGD/RP insult obviously elicited the elevated levels of the [Ca2+]i and the expressions of caspases-3 and -12 mRNA in neurons. Res pretreatments markedly depressed the neuronal abnormal elevation of [Ca2+]i and the overexpression of caspases-3 and -12 mRNA in a concentration-dependent manner., Conclusion: Res can attenuate the rat cortical neuronal apoptosis induced by OGD/RP. The mechanisms are, at least partly, due to the inhibition of the calcium overload and the overexpression of caspases-3 and -12 mRNA.

Cells of the human promyelocytic cell line HL-60 can be controllably induced to terminally differentiate into either granulocytes or monocyte/macrophages. HL-60 promyelocytes and terminally differentiated macrophages express a K(+)-selective ion channel which is activated by intracellular free Ca2+ concentrations above 10(-7) M. Because of its voltage independence, this channel can be distinguished from the voltage- and Ca(2+)-activated family of outward-rectifying channels. The channel is selective for K+ against Na+ and is blocked by Ba2+, thus it may be similar to the Ca(2+)-activated K+ channel previously described in human macrophages. In its sensitivity to block by charybdotoxin, this channel also resembles a Ca(2+)-activated K+ channel of lymphocytes, which plays a role in activation-dependent hyperpolarization. In contrast to promyelocytes and macrophages, functional expression of the Ca(2+)-activated K+ channel is suppressed to nearly undetectable levels in granulocytes derived from HL-60 cells by retinoic acid-induced differentiation. These data suggest that signals which produce elevation of intracellular Ca2+ will hyperpolarize promyelocytes and differentiated macrophages by activating this conductance; however, signals which elevate free Ca2+ in granulocytes must act on other effectors, which may produce a different final influence on membrane potential.

Macrophages derived from phorbol ester-induced human leukemic (HL-60) cells exhibit a voltage-activated inward rectifying potassium conductance which was modulated by macrophage colony-stimulating factor (Wieland, S. J., Chou, R. H., and Gong, Q. H. (1990) J. Cell. Physiol. 142, 643-651). Roles of intracellular messengers in this regulatory mechanism were investigated. Intracellular dialysis with inositol 1,3,4,5-tetrakisphosphate (IP4) or inositol 1,4,5-trisphosphate during tight-seal whole cell recording produced a rapid increase in the inward rectifying conductance. Changes in intracellular Ca2+ levels alone did not reproduce the stimulatory effect of these modulators. Intracellular dialysis with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) resulted in profound inhibition of this conductance. These data suggest a novel cellular function for inositol polyphosphates, particularly IP4, and show antagonistic modulation with GTP gamma S on a human macrophage inward rectifier.

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A2 (PLA2) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca(2+)-induced Ca2+ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na+ currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA2 contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA2 by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca2+ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na+ currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.

The action of a 16,300 mol. wt phospholipase A2 with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage- and Ca2(+)-activated K+ current; the sensitivity for voltage activation of the K+ current was enhanced by increasing free Ca2+ in the recording pipette from 10(-8) M to 2 x 10(-6) M. In contrast, peripheral blood lymphocytes possessed voltage-activated K+ currents that were inhibited by increasing intracellular Ca2+. Exposure of either preparation to B. fasciatus toxin (0.2-5 x 10(-6) M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of -140 mV to -40 mV. However, the sensitivity for voltage activation of the K+ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K- currents in human lymphocytes, suggesting a specific increase in intracellular Ca2- levels in both cell types. The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca2+ levels. Exposure of either lymphocytes or epithelial cells to toxin (10(-6) M) resulted in a transient increase in Ca2+. However, while the Ca2+ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca2(+)-regulated K+ channels may reflect an irreversible action of the B. fasciatus phospholipase A2 on a Ca2+-dependent regulatory process.