EFFECTS OF EXTRACELLULAR NUCLEOTIDES IN THE PITUITARY - ADENOSINE-TRIPHOSPHATE RECEPTOR-MEDIATED INTRACELLULAR RESPONSES IN GONADOTROPE-DERIVED ALPHA-T3-1 CELLS

We have recently identified gonadotropes as target cells for ATP actio n via ATP receptors of the P2U subtype. The present studies have used gonadotrope-derived alpha T3-1 cells to examine the possible signaling mechanisms subserving ATP action in gonadotropes. Addition of ATP pro duced a biphasic intracellular Ca2+ (Ca-i(2+)) response: a transient s pike followed by a small plateau. Removal of extracellular Ca2+ or dep olarization with KCl abolished the plateau but had no effect on the sp ike. The plateau was also blocked by cadmium Dr nifedipine but not nic kel. Pretreatment with GnRH or thapsigargin but not ryanodine inhibite d the subsequent Ca-i(2+) response to ATP. Pertussis toxin had no effe ct on ATP-induced Ca-i(2+) response, whereas the phospholipase C inhib itor U73122 reduced the response. These observations suggest that the Ca-i(2+) response is mediated by a pertussis toxin-insensitive and pho spholipase C-coupled G-protein and reflects Ca2+ release from the GnRH - and thapsigargin-sensitive Ca2+ pool followed by Ca2+ influx through high voltage-gated Ca2+ channels. Activation of these ATP receptors h ad no apparent effects on the cAMP and cGMP signaling systems. Treatme nt with ATP gamma S caused the translocation of protein kinase C (PKC) epsilon but not PKC zeta and PKC alpha to the particulate fraction. Th ese data not only characterize the ATP receptor-mediated intracellular signaling in alpha T3-1 cells and render further evidence for a media tor role for nucleotides in gonadotrope function but also provide the first direct demonstration of PKC translocation by ATP receptors.