Tetanus toxin-mediated cleavage of cellubrevin inhibits proton secretion in the male reproductive tract

Our laboratory has previously shown that the vacuolar H+-ATPase, located in a subpopulation of specialized cells establishes a luminal acidic environm ent in the epididymis and proximal part of the vas deferens (Breton S, Smit h PJS, Lui B, and Brown D. Nat Med 2: 470-472, 1996). Low luminal pH is cri tical for sperm maturation and maintenance of sperm in a quiescent state du ring storage in these organs. In the present study we examined the regulati on of proton secretion in the epididymis and vas deferens. In vivo microtub ule disruption by colchicine induced an almost complete loss of H+-ATPase a pical polarity. Endocytotic vesicles, visualized by Texas red-dextran inter nalization, contain H+-ATPase, indicating active endocytosis of the pump. C ellubrevin, an analog of the vesicle soluble N-ethyl malemide-sensitive fac tor attachment protein (SNAP) receptor (V-SNARE) synaptobrevin, is highly e nriched in H+-ATPase-rich cells of the epididymis and vas deferens, and tet anus toxin treatment markedly inhibited bafilomycin-sensitive proton secret ion by 64.3 +/- 9.0% in the proximal vas deferens. Western blotting showed effective cleavage of cellubrevin by tetanus toxin in intact vas deferens, demonstrating that the toxin gained access to cellubrevin. These results su ggest that H+-ATPase is actively endocytosed and exocytosed in proton-secre ting cells of the epididymis and vas deferens and that net proton secretion requires the participation of the V-SNARE cellubrevin.