title: How far does phospholipase C activity depend on the cell calcium concentration? A study in intact cells.
abstract: The dependence of phospholipase C activity on the cytosolic Ca2+ concentration ([Ca2+]i) was studied in intact liver cells treated with the Ca2+-mobilizing hormone vasopressin, or not so treated. Phospholipase C (PLC) activity was estimated from the formation of [3H]inositol trisphosphate (InsP3) and the degradation of [3H]phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The [Ca2+]i of the cells was clamped from 29 to 1130 nM by quin2 loading. This wide concentration range was obtained by loading the hepatocytes with a high concentration of the Ca2+ indicator in low-Ca2+ medium or by using the Ca2+ ionophore ionomycin in medium containing Ca2+. In resting cells, in which [Ca2+]i was 193 nM, treatment with 0.1 microM-vasopressin which stimulates liver PLC maximally, tripled InsP3 content and raised [Ca2+]i to 2 microM within 15 s. Lowering [Ca2+]i partially decreased cell InsP3 content as well as the ability of vasopressin to stimulate InsP3 formation maximally. At 29 nM, the lowest Ca2+ concentration obtained in isolated liver cells, basal InsP3 content was 64% of that measured in control cells. Addition of vasopressin no longer affected [Ca2+]i, but significantly increased InsP3 by 200%, although less than in the controls (300%). The maintenance of the greater part of the PLC response at constant [Ca2+]i indicated that, in the liver, InsP3 formation does not result from an increase in [Ca2+]i. The effects of lowering [Ca2+]i were reversible. When low cell [Ca2+]i was restored to a normal value, resting InsP3 content and the ability of vasopressin to stimulate InsP3 formation maximally by 300% were also restored. Raising [Ca2+]i from 193 to 1130 nM had little effect on the InsP3 content or the vasopressin-mediated increase in InsP3. In agreement with the stimulation of PLC activity by vasopressin, cell [3H]PtdInsP2 and total PtdInsP2 were degraded by application of this hormone for 15 s. In contrast, when [Ca2+]i was lowered to 29 nM, basal [3H]PtdInsP2 and total PtdInsP2 were increased by about 30%, [3H]PtdInsP2 was further increased by vasopressin, but total PtdInsP2 was not changed. These results show that, in intact hepatocytes, PLC is little affected by [Ca2+]i concentrations above 193 nM, but is partially dependent on Ca2+ below that value. They suggest that, in addition to activating PLC activity, vasopressin might stimulate PtdInsP2 synthesis, presumably via phosphatidylinositol-phosphate kinase, and that this pathway might predominate in cells with low [Ca2+]i.
authors.forename: D
authors.initials: D
authors.lastname: Renard
authors.forename: J
authors.initials: J
authors.lastname: Poggioli
authors.forename: B
authors.initials: B
authors.lastname: Berthon
authors.forename: M
authors.initials: M
authors.lastname: Claret
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D002469:Cell Separation; D005260:Female; D006868:Hydrolysis; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D000634:Aminoquinolines; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D001127:Arginine Vasopressin; D010738:Type C Phospholipases; D002122:Calcium Chloride; C035739:Quin2; D002118:Calcium