DIFFERENTIAL INVOLVEMENT OF CALCIUM CHANNELS AND PROTEIN-KINASE-C ACTIVITY IN GNRH-INDUCED PHOSPHOLIPASE-C, PHOSPHOLIPASE-A(2) AND PHOSPHOLIPASE-D ACTIVATION IN A GONADOTROPE CELL-LINE (ALPHA-T3-1)

The mode of action of GnRH on pituitary gonadotropes involves metaboli sm of phospholipids, protein kinase-C (PKC) and voltage sensitive Ca2. channels (VSCC) activation. We have studied the differential role of PKC and VSCC on the coupling of the GnRH receptor with phospholipases -C (PLC), -A(2) (PLA(2)) and -D (PLD) activities in a gonadotrope cell line (alpha T3-1), by measuring the production of inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respective ly. We demonstrated that in these cells GnRH stimulated through a spec ific receptor, IPs formation, a rapid and sustained diacylglycerol gen eration, consequently AA release and a delayed PEt production in a dos e-dependent manner. In contrast to GnRH-induced PLC activity, the PLA( 2) and PLD stimulation by the neuropeptide involved Ca2+ mobilization via VSCC activation. BAY-K8644 a VSCC agonist significantly potentiate d, while the VSCC antagonist nitrendipine markedly inhibited GnRH-indu ced AA release and PEt production. TPA, a phorbol eater which induced a rapid and important redistribution of PKC, although unable to elicit PLC or PLA(2) stimulation, specifically provoked PLD activation in a PKC-dependent but Ca2+-independent manner. The PKC stimulation by TPA significantly inhibited the GnRH-stimulated IPs and AA formation, whil e it potentiated the GnRH-evoked PEt production. This negative feed-ba ck of PKC on GnRH-induced PLC and PLA, activities was reversed when PK C was either down regulated after long TPA treatments or inhibited by the PKC inhibitors, staurosporine or GF109203X. The GnRH-induced PEt f ormation was markedly diminished in PKC depleted cells or after PKC in hibition. Under such conditions, both agonist and antagonist bf VSCC b ecame less effective in modulating the remaining GnRH-evoked PEt forma tion. These results suggest that PKC, in coordination with Ca2+, plays a key role in regulating the cross-talk between the multiple phosphol ipases implicated in the GnRH signal transduction.