Phorbol esters and synthetic diacylglycerols inhibit gamma-aminobutyric acid uptake in human spermatozoa: an effect mediated by protein kinase C?

Protein kinase C (PKC) and protein kinase A (PKA) are present in human spermatozoa and probably involved in the cascade systems leading to acrosomal exocytosis. We have investigated the possibility that these two protein kinases are involved in the regulation of gamma-aminobutyric acid (GABA) uptake in human spermatozoa. Swim-up preparations of human spermatozoa were exposed to phorbol 12-myristate 13 acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), which are activators of PKC, and to dibutyryl cyclic AMP (dbcAMP), a PKA activator, and subsequently incubated with radiolabelled GABA. Accumulated intracellular GABA was measured in a scintillation counter. Both PMA and OAG caused inhibition of GABA uptake while dbcAMP was without effect. The inhibitory effect on GABA uptake observed following exposure to the PKC activators could, however, not be counteracted by preincubation of the sperm samples with the PKC inhibitors staurosporine or H-7. The inhibitory effect on GABA uptake following addition of PMA was enhanced by prolonged exposure to the reagent and by increased capacitation prior to addition of the reagent. A small numerical increase in the percentage of acrosome-reacted cells was observed following exposure of the sperm cells to the highest concentrations of PMA, OAG and dbcAMP. No sperm motility parameters were affected by the treatment protocols as measured using computer-assisted semen analysis (CASA).