pH o,pH i,and P CO 2 in Stimulation of IP 3 and [Ca 2+ ] c in Piglet Cerebrovascular Smooth Muscle

Abstract

Hypocapnia produces cerebral vasoconstriction. The mechanisms involved in hypocapnia-induced elevation of vascular smooth muscle tone remain unclear. We addressed the hypothesis that, in cerebrovascular smooth muscle, increases in extracellular pH (pH_o) cause increases in Ins(1,4,5)P₃ and cytosolic calcium ([Ca²⁺]_c). Superfused primary cultures of piglet cerebral microvascular smooth muscle cells were exposed to artificial CSF (aCSF) of control (pH_o 7.4, PCO₂ 36 mm Hg), metabolic alkalosis (pH_o 7.7, PCO₂ 36 mm Hg), or respiratory alkalosis (pH_o 7.7, PCO₂ 19 mm Hg). Intracellular pH (pH_i) and [Ca²⁺]_c were measured, using BCECF and fura-2, respectively, with dual wavelength spectroscopy. Ins(1,4,5)P₃ was determined by a protein binding assay. Both metabolic and respiratory acidosis treatments increased pH, from the control value of about 7.2 to 7.35. Metabolic and respiratory alkalosis increased Ins(1,4,5)P₃, as we showed previously. Metabolic and respiratory alkalosis increased [Ca²⁺]_c about 80% and 110%, respectively. Neither Ins(1,4,5)P₃ nor [Ca²⁺]_c increased in cells treated with aCSF that produced control pH_o with increased pH_i (7.3). In contrast, when pH_o increased (7.7), but pH_i was maintained at control (7.2), Ins(1,4,5)P₃ increased from 123 pmol/well to 307 pmol/well and [Ca²⁺]_c increased 46%. However, the increase of [Ca²⁺]_c was less than with either respiratory or metabolic alkalosis. Thus, hypocapnia-induced cerebral vasoconstriction could involve production of Ins(1,4,5)P₃ with resultant elevation in [Ca²⁺]_c. While the Ins(1,4,5)P₃ signal appears to be dependent on an increase in extracellular pH, a role for intracellular pH cannot be completely excluded.