A Ca2+ calmodulin dependent kinase rather than protein kinase C is involved in up-regulation of the LHRH receptor.
Stimulation of protein kinase C (PKC) by phorbol ester (PMA) was reported previously to increase total binding of the peptide in whole rat pituitary cells. The effect could be obtained in cells from intact, not from spayed animals, suggesting a different level of spontaneous phosphorylation in both conditions. In the present work, endogenous PKC was desensitized in pituitary cells sampled from intact or 3 weeks castrated male rats and maintained in primary culture. Desensitization was induced by overnight incubation with 1 microM PMA. The maximum number of plasma membrane LHRH receptors (Bmax) present on cells from in intact animals was higher (+ 98 +/- 9%) when binding was performed at 0.5 degrees C instead of 21 degrees C as already observed in non PKC-desensitized cells. PMA (100 nM) was ineffective to increase Bmax, suggesting effectiveness of enzyme desensitization. In contrast, ionomycin 1 microM increased Bmax (53 +/- 10%). This increment was inhibited by W7, a calmodulin inhibitor, with an IC50 = 1 +/- 0.35 10(-6) M. No temperature dependency of the Bmax was observed in cells from castrated rats as already shown in the absence of PKC desensitization. Under these conditions, a Bmax decrease of 34 +/- 6% and 36.5 +/- 7.5% respectively was observed in the presence of H7, a PKC inhibitor, or of W7 (IC50 = 1 +/- 0.5 10(-5) M and IC50 = 0.8 +/- 0.2 10(-6) M). We conclude that a Ca2+ calmodulin dependent protein kinase rather than PKC itself is responsible for unmasking LHRH receptors.
Leblanc
P
P
Unité de Dynamique des Systèmes Neuroendocriniens, INSERM U 159, Paris, France.
L'Héritier
A
A
Kordon
C
C
D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002369:Castration; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006358:Hot Temperature; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D010903:Pituitary Gland, Anterior / Q000201:enzymology*; D011493:Protein Kinase C / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011966:Receptors, LHRH / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015854:Up-Regulation
D002147:Calmodulin; D047428:Protein Kinase Inhibitors; D011966:Receptors, LHRH; D015759:Ionomycin; D011494:Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate