title: Protection against methoxyacetic-acid-induced spermatocyte apoptosis with calcium channel blockers in cultured rat seminiferous tubules: possible mechanisms.
abstract: A calcium-mediated mechanism underlying spermatocyte apoptosis induced by 2-methoxyethanol (2-ME) has been previously proposed. This hypothesis was tested in vitro in the present study using cultured juvenile (25 days old) and adult rat seminiferous tubules (JRST and ARST, respectively) with methoxyacetic acid (MAA, the active metabolite of 2-ME). In JRST, spermatocyte degeneration was morphologically obvious 19 hr after a 5-hr exposure to 5 mM MAA. The lesion was unaffected by the presence or absence of extratubular Ca2+. However, MAA-induced cell death was significantly prevented by cotreatment with the dihydropyridines (DHP) nifedipine (50 microM) and nicardipine (20 microM), as well as verapamil (50 microM) and TMB-8 (50 microM), all of which are able to inhibit calcium movement through plasma membranes. However, neither ryanodine, dantrolene, nor cyclosporin A and ruthenium red, which inhibit Ca2+ mobilization from intracellular stores (endoplasmic reticulum and mitochondria), affected the MAA-induced cell death. Inhibition of calcium mobilization through IP3-sensitive pathways by blocking the product of IP3 with manoalide, neomycin, and U73122 did not block the MAA-induced lesion. The protective effects of 50 microM nifedipine and 50 microM TMB-8 were also observed in ARSTs treated with 10 mM MAA for 5 hr. However, when rat testicular sections were immunohistochemically stained with monoclonal antibodies specific for the alpha 1 (the DHP receptor) or the alpha 2 subunits of DHP-sensitive calcium channels, no positive staining was found. Finally, in an attempt to see whether the intracellular free calcium concentrations ([Ca2+]i) in germ cells were increased after the MAA treatment, intact seminiferous tubules were loaded with indo-1 and were measured using laser-scanning confocal microscopy. No detectable increase in the signal in MA A-sensitive spermatocytes was observed, while a 34-54% increase in the signal could be detected in the same cell types when tubules were exposed to 10 microM of the calcium ionophore 4-bromo-A23187 for 5 min. Collectively, these data suggest that the protective effect of calcium channel blockers against the MAA-induced spermatocyte apoptosis is probably not through their blocking effect on DHP-sensitive calcium channels. We postulate alternate mechanisms based on stabilization of cells membranes, or interactions with calmodulin or protein kinase C.
authors.affiliation: National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
authors.forename: L H
authors.initials: LH
authors.lastname: Li
authors.forename: R N
authors.initials: RN
authors.lastname: Wine
authors.forename: D S
authors.initials: DS
authors.lastname: Miller
authors.forename: J M
authors.initials: JM
authors.lastname: Reece
authors.forename: M
authors.initials: M
authors.lastname: Smith
authors.forename: R E
authors.initials: RE
authors.lastname: Chapin
mesh_terms: D015761:4-Aminopyridine / Q000494:pharmacology; D000085:Acetates / Q000633:toxicity*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D008297:Male; D026902:Potassium Channel Blockers; D051381:Rats; D017207:Rats, Sprague-Dawley; D012671:Seminiferous Tubules / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013090:Spermatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
chemical_list: D000085:Acetates; D002121:Calcium Channel Blockers; D026902:Potassium Channel Blockers; D013757:Tetraethylammonium Compounds; D019789:Tetraethylammonium; D015761:4-Aminopyridine; C013598:methoxyacetic acid; D002118:Calcium