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

Certain amiloride analogues 3′,4′‐dichlorobenzamil 2′,4′‐dimethylbenzamil and α′,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 Ca^2‐ 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.