Stimulation of Ca2+ uptake into epididymal bull spermatozoa by analogues of amiloride

Certain amiloride analogues 3',4'-dichlorobenzamil 2',4'-dimethylbenzamil and alpha',2'-benzobenzamil hydrochloride (ATBB) stimulate calcium accumulation and motility by epididymal bovine spermatozoa. This stimulation can be seen at a range of 0.1-0.4 mM, while at higher concentration there is inhibition of calcium uptake by these amiloride analogues. The amiloride derivative 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CBDMB), which bears a 4-chlorobenzyl substituent on the 5-amino nitrogen atom, did not stimulate calcium uptake. The amiloride analogue 3',4'-dichlorobenzamil inhibits the Na+/Ca2(+)-exchange activity in isolated plasma membrane vesicles, and the stimulatory effect of 3',4'-dichlorobenzamil on calcium uptake into epididymal sperm could be seen in Na(+)-free medium. Thus, the stimulation of Ca2+ accumulation in the cells caused by 3',4'-dichlorobenzamil is not a result of inhibiting the Na(+)-dependent Ca2+ clearance. There is no stimulation of Ca2+ uptake into ejaculated cells by adding 3',4'-dichlorobenzamil, which is not due to the presence of the calcium-transport inhibitor (caltrin) in these cells [Rufo, G.A., Schoff, P.K. & Lardy, H.A. (1984) J. Biol. Chem. 259, 2547-2552]. The stimulatory effect of 3',4'-dichlorobenzamil on Ca2+ uptake is inhibited by the voltage-dependent Ca2(+)-channel blockers nifedipin and diltiazem. This indicates that the stimulation of Ca2+ uptake by the amiloride analogues is due to the activation of a voltage-dependent Ca2+ channel of the plasma membrane.