INOSITOL LIPID-MEDIATED SIGNALING IN RESPONSE TO ENDOTHELIN AND ATP IN THE MAMMALIAN TESTIS

The testis is a complex organ in which local control is achieved by signalling between its constituent cells. Herein we describe the responses of cultured rat testicular cells and a mouse Sertoli cell-line to stimulation by endothelin and ATP, and elsewhere we have shown that rat peritubular myoid cells possess phosphoinositidase C-coupled V-1a-vasopressin receptors identical to those of liver (Howl, J. et al, 1995, Endocrinology 136: 2206-2213). 1. Peritubular myoid cells from pre-pubertal rats responded through ET(A) receptors with PtdIns(4,5)P-2 hydrolysis [EC(50) for endothelin-1 (ET-1) similar to 0.4 nM], elevation of intracellular [Ca2+], and tyrosine phosphorylation of a variety of cellular proteins. They also showed enhanced adenylate cyclase activity, with an EC(50) for ET-1 of similar to 3 nM, also through ET(A) receptors. Pharmacological elevation of [cAMP] did not immediately change the ET-1-stimulated formation of inositol phosphates, but attenuated the response after several hours. 2. Pre-pubertal rat Sertoli cells showed no detectable responses to ET-1, but responded to FSH with elevated [cAMP] and to ATP with PtdIns(4,5)P-2 hydrolysis. PtdIns(4,5)P-2 hydrolysis was equally responsive to ATP and UTP, and so appears to be activated by P-2U-purinergic receptors. This response was enhanced by protein kinase C inhibition and attenuated by PKC activation. 3. Despite its lack of effect on rat Sertoli cells in primary culture, ET-1 provoked PtdIns(4,5)P-2 hydrolysis in the TM4 murine Sertoli cell line (EC(50) similar to 0.6 nM), and this response was negatively regulated by protein kinase C activation. 5. No receptor-stimulated activation of phosphoinositase C was detected in 'germ cell' populations, but the non-specific G protein activator AlF4- provoked inositol phosphate accumulation in these cells, so demonstrating their potential to respond through yet to be identified G protein-coupled receptors with phosphoinositidase C activation. 6. Immunoblotting studies showed the presence in rat testis of phosphoinositidase C-beta 1 and the alpha-subunits(s) of the G-protein(s) G(q) and/or G(11). These studies show that testicular myoid and Sertoli cells use at least three G protein-coupled receptors (V-1a-vasopressins, ET(A)-endothelin and P-2U, purinergic) to signal through phosphoinositidase C activation, that ET-1 can activate multiple signalling pathways in myoid cells, and that the ET-1-stimulated phosphoinositidase C responses of myoid and Sertoli cells have different regulatory characteristics.