Growth hormone increases calcium uptake in rat fat cells by a mechanism dependent on protein kinase C

Growth hormone (GH ; 500 ng/ml) rapidly doubled cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) in rat adipocytes as determined with the Ca 2+ indicator fura 2. No response was seen in Ca 2+ -free medium, suggesting that the increase in [Ca 2+ ] i was due to Ca 2+ influx. GH also doubled the influx of Mn 2+ as inferred from the rate of fluorescence quenching. Depolarization with 30 mM K + also increased [Ca 2+ ] i , and the increase in [Ca 2+ ] i due to either GH or 30 mM K + was blocked by 100 nM nimodipine, suggesting that GH increases [Ca 2+ ] i by activating voltage-sensitive L-type Ca 2+ channels. GH increased [Ca 2+ ] i even when K + channels were blocked, suggesting that activation of Ca 2+ uptake was not secondary to closure of K + channels and consequent depolarization. A diacylglycerol (DAG) analogue, 1,2-dioctanoyl-sn-glycerol (50 μM), duplicated, and the protein kinase C (PKC) inhibitors calphostin C (100 nM), chelerythrine (1 μM), and bis-indolylmaleimide (250 nM) inhibited the effects of GH on [Ca 2+ ] i . Xanthogenate tricyclodecan-9-yl (D609), a specific inhibitor of phospholipase C (PLC), abolished the increase in [Ca 2+ ] i due to GH but not to DAG. The results suggest that GH increases [Ca 2+ ] i by activation of PLC, release of DAG, and activation of a Ca 2+ -independent isoform of PKC. PKC-catalyzed phosphorylation of either the Ca 2+ channels or a protein that regulates them may account for the influx of Ca 2+ produced by GH.