EFFECT OF ETHANOL ON ENERGY STATUS AND INTRACELLULAR CALCIUM OF SERTOLI CELLS - A STUDY USING IMMOBILIZED PERFUSED CELLS

The effects of ethanol on ATP, O2 consumption, and cytosolic ionized C a2+ (Ca-i(2+)) were studied in Sertoli cells isolated from the testes of 18- to 21-day-old rats. The cells were immobilized in agarose gel t hreads and perfused with Dulbecco's Modified Eagle's Medium. Intracell ular ATP was determined by P-31 nuclear magnetic resonance spectroscop y and enzymatic assay, Ca-i(2+) was measured with the photoprotein aeq uorin, cell viability was assessed by trypan blue exclusion, and O2 co nsumption was monitored with a Clark electrode. Ethanol was used with or without pretreatment with the alcohol dehydrogenase inhibitor 4-met hylpyrazole (MP). Perfusing the cells for 90 min with 500 mm ethanol p roduced a 50% reduction in the P-31 nuclear magnetic resonance betaATP signal, and pretreatment with 15 mm MP enhanced this decline of the b etaATP peak to 75%. Enzymatic measurements of ATP revealed that exposu re to 500 mm ethanol reduced the ATP levels from 52 +/- 5 to 38 +/- 3 nmol/10(6) cells with MP pretreatment and to 28 +/- 4 nmol/10(6) cells without MP pretreatment (n = 5). Basal O2 consumption was 5.2 +/- 0.5 nmol/min.10(6) cells (n = 5), and it was reduced by ethanol or ethano l plus MP to 4 +/- 0.4 and 3.1 +/- 0.2, respectively (n = 5). The basa l concentration of Ca-i(2+) in Sertoli cells was 98 +/- 0.7 nm (n = 32 ). During perfusion with 500 mm ethanol, Ca-i(2+) increased to 208 +/- 98 nm (n = 5) and was not modified further by the presence of MP. Per fusing the cells for 90 min with 500 mm ethanol with or without MP cau sed a decrease in cell viability from 93 +/- 2 to 76 +/- 3 and 67 +/-3 , respectively (n = 8). Exposure to 5 mm acetaldehyde produced only a minimal reduction in ATP, with no observable effect at lower concentra tions, suggesting that the significant reductions in ATP, O2 consumpti on, and cell viablity evoked by ethanol were not caused by acetaldehyd e. These data suggest that ethanol is toxic to Sertoli cells, and its toxicity is not a result of ethanol metabolism.