IDENTIFICATION AND CHARACTERIZATION OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS IN RAT TESTIS

PCR analysis and immunoblotting with isoform specific antibodies was u sed to identify the presence of type I, II and III inositol 1,4,5-tris phosphate receptors (InsP(3)Rs) in rat testis. PCR analysis also revea led that rat testis express both forms of the S1 splice variant (S1(+) and S1(-)), but only the S2(-) form of the S2 splice variant of the t ype I InsP(3) receptor. PCR analysis was also used to identify InsP(3) R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [H-3]-Ins P(3) binding was found to be 9 times lower for testicular microsomes t han for cerebellar microsomes, with a B-max of 1.4 pmoles/mg protein c ompared to 12.5 pmoles/mg protein for cerebellar microsomes. The K-d f or InsP(3) binding to its receptor in testicular microsomes was 60 +/- 10 nM which was similar to that found for cerebellar microsomes (80 20 nM). InsP(3)-induced Ca2+ release (IlCR) in testicular microsomes was found to have an EC50 (concentration which causes a half-maximal r esponse) of 0.5 +/- 0.03 mu M, also similar to that seen for cerebella r microsomes (0.3 mu M). Maximal IICR occurred at about 20 mu M InsP(3 ), with up to 4% of total intracellular Ca2+ stores being mobilized as compared to between 10-30% for cerebellar microsomes. Time resolved I ICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate cons tant of 0.15 s(-1) at 30 mu M InsP(3). The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (similar to 1 s(-1)) and taken together with the binding data support the proposal that the receptor density/Ca2+ store is similar to 8 time s lower than seen in cerebellar microsomal vesicles. The pharmacologic al properties as assessed using heparin and InsP(3) analogues also con firmed similar behaviour for testicular InsP(3)Rs and cerebellar InsP( 3)Rs.