Effect of 1α,25-dihydroxyvitamin D3 in plasma membrane targets in immature rat testis: Ionic channels and gamma-glutamyl transpeptidase activity

Abstract

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is critical for the maintenance of normal reproduction since reduced fertility is observed in vitamin D-deficient male rats. The aim of this study was to investigate the effect of 1,25D(3) in 30-day-old rat testicular plasma membrane targets (calcium uptake and gamma-glutamyl transpeptidase (GGTP) activity), as well as to highlight the role of protein kinases in the mechanism of action of 1,25D(3). The results demonstrated that 1,25D(3) induced a fast increase in calcium uptake in rat testis through a nongenomic mechanism of action. This effect was dependent on PKA, PKC and MEK. Moreover, ionic channels, such as ATP- and Ca(2+)-dependent K(+) channels and Ca(2+)-dependent Cl(-) channels, are involved in the mechanism of action. The use of BAPTA-AM showed that [Ca(2+)](i) was also implicated, and the incubation with digoxin produced an increase in (45)Ca(2+) uptake indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, 1,25D(3) was able to increase the GGTP activity. Considered together, our results indicate a PKA/PKC/MEK-dependent 1,25D(3) pathway as well as ionic involvement leading to (45)Ca(2+) uptake in immature rat testis. These findings demonstrate that 1,25D(3) stimulates calcium uptake and increases GGTP activity which may be involved in male reproductive functions.

1,25(OH)2 vitamin D3 signalling on immature rat Sertoli cells: gamma-glutamyl transpeptidase and glucose metabolism

“…Sertoli cells in either control or treated group were incubated in serum free medium with or without 1,25-D 3 (1 nM) or curcumin (1 nM) or PMA (100 nM) for 1, 3, 6 and 24 h. In some experiments inhibitors (CM, CH, H-89, KT, dibAMPc, ST, Ro) were added 30 min before the incubation time and maintained for the whole incubation period. In all cases, the inhibitors' concentrations used were those that have previously resulted in inhibition in similar assays (Menegaz et al 2009, Menegaz et al 2010, Menegaz et al 2011Zanatta et al 2011a, Zanatta et al 2013Rosso et al 2012).…”

“…Since our previous studies showed that 1 nM 1,25-D 3 has a positive effect on GGT activity in rat testis (Zanatta et al 2011a), the same concentration was chosen to carry out these experiments in isolated Sertoli cells. Sertoli cells in either control or treated group were incubated in serum free medium with or without 1,25-D 3 (1 nM) or curcumin (1 nM) or PMA (100 nM) for 1, 3, 6 and 24 h. In some experiments inhibitors (CM, CH, H-89, KT, dibAMPc, ST, Ro) were added 30 min before the incubation time and maintained for the whole incubation period.…”

“…The absorbance of the samples was determined in a plate reader (Tecan Infinite 200 PRO, LAMEB I/CCB/UFSC) at 530 nm. The results were calculated as U/L./ μg protein expressed as % of control (Zanatta et al 2011a). …”

“…The lactate levels were estimated by the lactate oxidase method (Hutchesson et al 1997) and the results were calculated as mg of lactate/μg of protein expressed as % of control (Zanatta et al 2011a). …”

“…(1,25-D 3 ), the active form of vitamin D 3 , has biological effects in 38 tissues, including rodent and human testis (Norman et al 2001b;Blomberg Jensen et al 2010;Zanatta et al 2011a;. The classical mechanism of action of 1,25-D 3 is through a nuclear receptor (VDRnuc), where the hormone interacts with its receptor and induces VDRnuc heterodimerization with retinoid receptor X, resulting in the regulation of gene expression (Bouillon et al 1995).…”