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 Ca2+ (Ca2+i) were studied in Sertoli cells isolated from the testes of 18- to 21-day-old rats. The cells were immobilized in agarose gel threads and perfused with Dulbecco's Modified Eagle's Medium. Intracellular ATP was determined by 31P nuclear magnetic resonance spectroscopy and enzymatic assay, Cai2+ was measured with the photoprotein aequorin, cell viability was assessed by trypan blue exclusion, and O2 consumption was monitored with a Clark electrode. Ethanol was used with or without pretreatment with the alcohol dehydrogenase inhibitor 4-methylpyrazole (MP). Perfusing the cells for 90 min with 500 mM ethanol produced a 50% reduction in the 31P nuclear magnetic resonance beta ATP signal, and pretreatment with 15 mM MP enhanced this decline of the beta ATP peak to 75%. Enzymatic measurements of ATP revealed that exposure 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 ethanol plus MP to 4 +/- 0.4 and 3.1 +/- 0.2, respectively (n = 5). The basal concentration of Cai2+ in Sertoli cells was 98 +/- 0.7 nM (n = 32). During perfusion with 500 mM ethanol, Cai2+ increased to 208 +/- 98 nM (n = 5) and was not modified further by the presence of MP. Perfusing the cells for 90 min with 500 mM ethanol with or without MP caused 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 concentrations, suggesting that the significant reductions in ATP, O2 consumption, and cell viability evoked by ethanol were not caused by acetaldehyde. These data suggest that ethanol is toxic to Sertoli cells, and its toxicity is not a result of ethanol metabolism.