On the presence of 3H-GABA uptake mechanism in bovine spermatozoa

In the present study, existence of 3H-GABA uptake mechanism in bovine spermatozoa and the modulation of 3H-GABA transport by GABA itself were evaluated. The hypothesis was tyrosine phosphorylation affects transporter (GAT) function. 3H-GABA uptake assays were performed on bovine spermatozoa and it resulted to be temperature- and time-dependent and Km was 1.48 μM. Uptake was inhibited by the metabolic inhibitor ouabain and different blockers of GAT-1 (β-alanine, l-DABA, nipecotic acid, tiagabine). Extracellular GABA up-regulated GABA transport, while the addition of SKF89976A, a high affinity inhibitor of the rat brain GABA transporter, reduced GABA uptake. Tyrosine phosphorylation affects transporter function since genistein, a broad-spectrum tyrosine kinase inhibitor, decreased 3H-GABA uptake. Reduction in uptake did not occur in the presence of daidzein, an inactive genistein analogue. Furthermore, the genistein-mediated reduction in transport could be prevented by the tyrosine phosphatase inhibitor pervanadate. The action of these drugs on GABA transport is likely mediated through the GABA transporter GAT-1 since SKF89976A blocked a majority of GABA uptake. Wash-out experiments indicated that the genistein effect was reversible. When the experiments were conducted using “in vitro” capacitated spermatozoa there was no detectable uptake. Present results demonstrate that the carrier-mediated GABA uptake system in bovine spermatozoa modulates its function in response to extracellular GABA, that changes in lipid distribution and membrane composition which occur during capacitation eliminates GABA uptake and suggest the involvement of tyrosine phosphorylation in GABA transport.