Cyclic adenosine 3',5'-monophosphate-independent regulation of cytosolic calcium in Sertoli cells

We have examined the cAMP-independent regulation of cytosolic calcium concentration in rat Sertoli cells using the effect of vasoactive hormones, known as testicular paracrine regulators operating via the non-cAMP pathway, on cytosolic calcium. Calcium concentrations were estimated with dual excitation fluorimetry, using freshly isolated, fura-2/AM-loaded cells. No increase in the cellular cAMP concentration was detected after stimulation with angiotensin II (AII), vasopressin, PGF(2 alpha) or atrial natriuretic peptide. Whereas both AII and vasopressin evoked a rise in cytosolic calcium from a basal level of 81.4 +/- 4 to 142.5 +/- 18 and 154.4 +/- 11 nM, respectively, PGF(2 alpha) had only a minimal effect (98 +/- 5 nM), and atrial natriuretic peptide no effect (86.6 +/- 9 nM). The effect of AII on calcium was blocked by the the selective AT(2), but not by the AT(1), receptor antagonist, indicating the selective presence on Sertoli cells of AT(2) AII receptor. Similarly, the vasopressin-induced calcium response was blocked by vasopressin V-1, but not by V-2 receptor antagonist, consistent with the presence of V-1 receptor subtype in these cells. Removal of extracellular calcium or blockade of calcium channels did not inhibit the calcium increase due to AII and vasopressin, suggesting the involvement of intracellular calcium. Thapsigargin increased the basal cytosolic calcium concentration to 137 +/- 10 nM. Depletion of intracellular calcium stores with thapsigargin before stimulation with AII or vasopressin abolished both the AII-mediated and the vasopressin-mediated calcium rise in the presence as well as the absence of extracellular calcium, indicating that the increase in calcium is predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. This study indicates that calcium homeostasis of Sertoli cells might also be regulated by cAMP-independent metabolism apart from the well known cAMP dependent pathway. Furthermore, our findings support the idea that angiotensin and vasopressin might be important paracrine regulators of Sertoli cells functions.