Extracellular pH, Ca 2+ Influx, and Response of Vascular Smooth Muscle Cells to 5-Hydroxytryptamine

Abstract

Background and Purpose —Cerebral vascular smooth muscle cells (VSMCs) contract on extracellular pH (pH o ) increases and relax on pH o decreases. These changes in tone are believed to result from changes in [Ca 2+ ] i , although the responsible mechanisms are not fully understood. VSMCs also contract in response to 5-hydroxytryptamine (5-HT), which increases [Ca 2+ ] i via both Ca 2+ release and influx. We hypothesized that examining effects of pH o decreases on 5-HT–induced [Ca 2+ ] i changes would allow us to identify mechanisms whereby pH o influences tone. Accordingly, we compared [Ca 2+ ] i increases in cerebral VSMCs, evoked by 5-HT, with increases evoked by increased pH o and examined 5-HT–dependent [Ca 2+ ] i increases at normal and decreased pH o . Methods —We monitored [Ca 2+ ] i, , using the Ca 2+ -sensitive dye fura 2, in cultured rat cerebral VSMCs obtained by enzymatic digestion of middle cerebral arteries and their branches (passages 1 to 3) grown on glass coverslips and superfused with physiological saline. Results —Increasing pH o from 7.3 to 7.8 increased [Ca 2+ ] i , and these increases were prevented in Ca 2+ -free solutions. Decreasing pH o from 7.3 to 6.9 did not alter [Ca 2+ ] i unless [Ca 2+ ] i was first raised by treatment with 5-HT (10 μmol/L). 5-HT resulted in biphasic [Ca 2+ ] i increases characterized by transient peaks blocked by the Ca 2+ -ATPase inhibitor thapsigargin (10 nmol/L) and prolonged plateaus blocked by the Ca 2+ channel blocker Ni 2+ (1 mmol/L). Acidification did not alter the transient peaks but significantly reduced 5-HT–induced Ca 2+ influx. Conclusions —We conclude that increasing pH o induces Ca 2+ influx in rat cerebral VSMCs and decreasing pH o inhibits 5-HT–stimulated Ca 2+ entry but not intracellular Ca 2+ release.