Gossypol, a polyphenolic dialdehyde toxin isolated from cotton seed, has anti-cancer properties and has recently shown some success in the treatment of glioma. Its effects on brain neurons and blood vessels are poorly understood. In this work we examined the effects of gossypol on cytosolic Ca concentration ([Ca ] ) of mouse brain bEND.3 endothelial cells. Cell viability tests revealed that after 3 hour and 18 hour exposures, 10 µmol/L gossypol caused 23% and 65% cell death, respectively; 3 µmol/L gossypol caused no and 21% cell death, respectively. [Ca ] was raised concentration-dependently by 1-10 µmol/L gossypol. We then explored the Ca signalling triggered by 3 µmol/L gossypol, which inflicted minimal toxicity: the Ca signal was composed largely of Ca influx and to a small extent, intracellular Ca release. Such Ca influx was much larger than store-operated Ca influx triggered by maximal Ca pool depletion. The Ca influx triggered by 3 and 10 µmol/L gossypol caused NO release and cell death, respectively. Gossypol also triggered influx of Mn and Na , but not Ni and Co . Gossypol-triggered Ca signal was inhibited only by 14% and 37% by 100 µmol/L La and 10 µmol/L nimodipine, respectively; and not suppressed at all by 5 mmol/L Ni . Gossypol-triggered Ca signal was suppressed by 78% by 30 µmol/L ruthenium red, suggesting gossypol may act on TRPV channels. Our results suggest gossypol triggered opening of a non-selective cation pore, possibly a member of the TRPV family.

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Gamma-linolenic acid (GLA), a natural fatty acid obtained from oils of various vegetables and seeds, has been demonstrated as an anticancer agent. In this work, we investigated the anticancer effects of GLA on breast cancer BT-474 cells. GLA at 30 μM, a concentration reportedly within the range of circulating concentrations in clinical studies, caused apoptotic cell death. GLA caused an elevation in mitochondrial Ca2+ level and a decrease in mitochondrial membrane potential. GLA treatment depleted cyclopiazonic acid (CPA)-sensitive Ca2+ store and triggered substantial Ca2+ influx. Intracellular Ca2+ release triggered by GLA was suppressed by 3 μM xestospongin C (XeC, IP3 receptor-channel blocker) and 100 μM ryanodine (ryanodine receptor-channel blocker), suggesting that the Ca2+ release was via IP3 receptor-channel and ryanodine receptor-channel. Increased expressions of p-eIF2α and CHOP were observed in GLA-treated cells, suggesting GLA-treated cells had increased expressions of p-eIF2α and CHOP, which suggest endoplasmic reticulum (ER) stress. In addition, GLA elicited increased production of reactive oxygen species. Taken together, our results suggest a basal level of GLA induced apoptotic cell death by causing Ca2+ overload, mitochondrial dysfunction, Ca2+ store depletion, ER stress, and oxidative stress. This is the first report to show that GLA caused Ca2+ store depletion and ER stress. GLA-induced Ca2+ store depletion resulted from opening of IP3 receptor-channel and ryanodine receptor-channel.

Propofol, a general anesthetic administered intravenously, may cause pain at the injection site. The pain is in part due to irritation of vascular endothelial cells. We here investigated the effects of propofol on Ca 2+ transport and pain mediator release in human umbilical vein endothelial cells (EA.hy926). Propofol mobilized Ca 2+ from cyclopiazonic acid (CPA)-dischargeable pool but did not cause Ca 2+ release from the lysosomal Ca 2+ stores. Propofol-elicited Ca 2+ release was suppressed by 100 mM ryanodine, suggesting the participation of ryanodine receptor channels. Propofol did not affect ATP-triggered Ca 2+ release but abolished the Ca 2+ influx triggered by ATP; in addition, propofol also suppressed store-operated Ca 2+ entry elicited by CPA. Ca 2+ clearance during CPA-induced Ca 2+ discharge was unaffected by a low Na + (50 mM) extracellular solution, but strongly suppressed by 5 mM La 3+ (an inhibitor of plasmalemmal Ca 2+ pump), suggesting Ca 2+ extrusion was predominantly through the plasmalemmal Ca 2+ pump. Propofol mimicked the effect of La 3+ in suppressing Ca 2+ clearance. Propofol also stimulated release of pain mediators, namely, reactive oxygen species and bradykinin. Our data suggest propofol elicited Ca 2+ release and repressed Ca 2+ clearance, causing a sustained cytosolic [Ca 2+ ]i elevation. The latter may cause reactive oxygen species and bradykinin release, resulting in pain.

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