Effects of mercuric chloride exposure on the glutamate uptake by cultured retinal pigment epithelial cells.
The cytotoxicity of mercuric chloride and the effects of mercuric chloride on glutamate and calcium uptake and the factors regulating glutamate uptake were studied in retinal pigment epithelium (RPE) cell cultures. RPE cells isolated from pig eyes and human RPE cell line (D407) cells were cultured to confluency and further subcultured according to the test protocol in question. The cytotoxicity caused by 15 min of exposure to mercuric chloride (0.01--1000 microM) was evaluated by WST-1 assay based on the activity of mitochondrial dehydrogenases. [(3)H]Glutamate uptake was measured after the cells were exposed to 0.1--100 microM mercuric chloride and the selected regulators of protein kinase C (PKC) pathway: PKC activator SC10, PKC inhibitor chelerythrine chloride, phospholipase A(2)/C inhibitor manoalide, tyrosine kinase inhibitor lavendustin A, competitive NMDA receptor antagonist AP7 and IP(3) receptor antagonist heparin. Intracellular calcium was monitored with Fluo-3 probe starting immediately after the exposure to 1--1000 microM mercuric chloride. Mercuric chloride showed concentration-dependent effects on cell viability, on glutamate uptake and on intracellular calcium concentration. The results give some support to the concept that glutamate uptake is affected by PKC. The PKC inhibitor chelerythrine chloride decreased glutamate uptake by 25%, but the PKC activator SC10 could partly prevent the inhibitory effect of mercuric chloride. Lavendustin A, manoalide and heparin had smaller, but statistically significant, effects. All these substances act on mediators which can regulate the activity of PKC. However, PKC is not likely to be the only regulator of glutamate uptake. The rise observed in [Ca(2+)](i) may initiate various cellular events during mercury intoxication.
Toimela
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TA
Tampere University Medical School, FIN-33014 University of Tampere, Finland.
Tähti
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D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics; D018929:Cell Culture Techniques; D004202:Disinfectants / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000493:pharmacokinetics*; D006801:Humans; D008627:Mercuric Chloride / Q000633:toxicity*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects*; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D013552:Swine
D004202:Disinfectants; D018698:Glutamic Acid; D008627:Mercuric Chloride; D011493:Protein Kinase C; D002118:Calcium