Low CO 2 Stimulates Inositol Phosphate Turnover and Increased Inositol 1,4,5-Trisphosphate Levels in Piglet Cerebral Microvascular Smooth Muscle Cells

Abstract

Abstract

In contrast to hypercapnic dilation, hypocapnia-induced cerebral vasoconstriction does not involve prostanoids in newborn pigs. The hypothesis that increased pH or decreased CO₂ tension increases inositol phosphate turnover in piglet cerebral microvascular smooth muscle (SM) cells was addressed to begin to assess the possibility that this second-messenger system is involved in hypocapnia-induced cerebral vasoconstriction. Cerebral microvascular SM cells in primary culture prelabeled with [³H]-myoinositol were stimulated for 30 sec with artificial cerebrospinal fluid of increased or normal pH, (7.80 vs 7.40), constant PCO₂ 36 mm Hg. Following extraction from cells, radiolabeled inositol phosphates were separated by HPLC. These metabolic alkalosis studies were repeated using an inositol 1,4,5-trisphosphate (lns[1,4,5]P₃ protein-binding assay (PBA). Respiratory alkalosis using aCSF with pH 7.60, PCO₂ 20 mm Hg versus control pH 7.40, PCO₂ 36 mm Hg was similarly tested with PBS measurement of Ins(1,4,5)P₃. aCSFs of control pH 7.40, and PCO₂s of 70, 36, or 25 mm Hg were studied both by [³H]-myoinositol (HPLC) and PBA to further determine the importance of CO₂ tension, in the presence of fixed pH, on Ins(1,4,5)P₃ production. When PCO₂ was constant, inositol phosphate turnover (as measured by [³H]-lns[1,4,5]P₃ accumulation) increased when pH was increased from 7.40 to 7.80 at 30 sec of stimulation. Mean [³H]-Ins(1,4,5)P₃ accumulation at pHs of 7.40 and 7.80, constant PCO₂ of 36 mm Hg, were 2.9 ± 0.7 and 4.1 ± 0.8 cpm/μg protein, respectively. Ins(1,4,5)P₃ levels for pH of 7.40 or 7.80 and constant PCO₂ of 36 mm Hg, were 25.4 ± 1.8 and 38 ± 8 pmol/well, respectively, by PBA. Respiratory alkalosis also increased Ins(1,4,5)P₃ levels. For pH of 7.40, PCO₂ 36 mm Hg and pH 7.60, PCO₂ 20 mm Hg, Ins(1,4,5)P₃ levels were 37.6 ± 16 and 64.1 ± 25 pmol/well, respectively. Decreasing CO₂ tension (from 70 mm Hg to 25 mm Hg) in the presence of fixed pH 7.40 failed to increase Ins(1,4,5)P₃ levels. The present data demonstrate that decreased CO₂ tension stimulates an increase in Ins(1,4,5)P₃ production in piglet cerebral microvascular smooth muscle cells. Increasing pH via lower PCO₂ increases the level of Ins(1,4,5)P₃ even more than increasing pH with fixed base, but extracellular pH appears to be important since decreased PCO₂ without changing extracellular pH had no effect. We conclude that the inositol phosphate second messenger system in cerebral microvascular smooth muscle responds appropriately to acute alkalosis to be involved in hypocapnia-induced cerebral vasoconstriction.