Identification and characterization of inositol 1,4,5-trisphosphate receptors in rat testis

Abstract

PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP 3Rs) in rat testis. PCR analysis also revealed 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 type I InsP 3 receptor. PCR analysis was also used to identify InsP 3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [ 3H]-Ins P 3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a B max of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The K d for 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 Ca 2+ release (IICR) in testicular microsomes was found to have an EC 50 (concentration which causes a half-maximal response) of 0.5 ± 0.03 μM, also similar to that seen for cerebellar microsomes (0.3 μM). Maximal IICR occurred at about 20 μM InsP 3, with up to 4% of total intracellular Ca 2+ stores being mobilized as compared to between 10–30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s −1 at 30 μM InsP 3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (∼1 s −1) and taken together with the binding data support the proposal that the receptor density/Ca 2+ store is ∼8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP 3 analogues also confirmed similar behaviour for testicular InsP 3Rs and cerebellar InsP 3Rs.