Cytosolic phospholipase C activity: I. Evidence for coupling with cytosolic guanine nucleotide-binding protein, Gi alpha.
In a previous report we showed that glucocorticoid inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Gi alpha levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Gi alpha. In order to establish the nature of the coupling between cytosolic Gi alpha and cytosolic PLC we examined the effects of G-protein activators, and inhibitors on cytosolic PLC activity from rat splenocytes and the rat lymphoma cell line Nb 2, with [3H] PI and [3H]PIP2 as substrates. 1) Neither GTP nor its nonhydrolyzable analogue, GTP gamma S, at 100 microM had any effect on the calcium stimulated as well as the basal PLC activity. 2) However, affinity purified antibodies to Gi alpha 1 and Gi alpha 2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP2 as substrate, soluble PLC activity was inhibited 50-70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. 3) Administration of Gi alpha 1 antisense oligonucleotides to splenocytes for 48 h produced 25-40% decrease in cytosolic Gi alpha 1 levels compared to control. The soluble PLC activity with both PI and PIP2 as substrates was also reduced by 25-50% compared to control conditions. This suggest that cytosolic Gi alpha is associated with the activation of splenocyte soluble PLC. 4) Pertussis toxin administered in vivo significantly reduced cytosolic Gi alpha immunoreactivity and soluble PLC activity when PI was used as substrate, providing additional evidence that cytosolic Gi alpha is associated with the activation of soluble PLC. 5) Another agent that has been used extensively to define G-protein coupled processes is NaF/AlCl3. NaF (5 mM; with or without AlCl3) inhibited soluble PLC activity with PIP2 as substrate, in contrast to the stimulatory effect that has been reported in the activation of membrane PLC. 6) Because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperizine (50 microM, TFP), an inhibitor of protein phosphatase 2B; TFP (50 microM) significantly inhibited soluble PLC activity when PI was used as substrate. These results suggest a direct involvement of cytosolic Gi alpha in the activation of soluble PLC from splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Gi alpha coupled PLC is addressed in the second paper.
Akompong
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Laboratory of Neuroendocrinology, Rockefeller University, New York, New York 10021.
Spencer
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RL
McEwen
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BS
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics; D037342:Pertussis Toxin; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012969:Sodium Fluoride / Q000494:pharmacology; D012995:Solubility; D013154:Spleen / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000276:immunology / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000906:Antibodies; D006150:Guanine Nucleotides; D016376:Oligonucleotides, Antisense; D010566:Virulence Factors, Bordetella; D012969:Sodium Fluoride; D037342:Pertussis Toxin; D010749:Phosphoprotein Phosphatases; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D043265:Phosphatidylinositol Diacylglycerol-Lyase