Effects of hypoxia on [Ca2+]i, pHi and myosin light chain phosphorylation in guinea-pig taenia caeci.

Abstract

1. Hypoxia (achieved by bubbling with N2 instead of O2) reduces the force of a KCl (40 mM)-induced contracture to approximately 10% of the control value in guinea-pig taenia caeci. The underlying mechanism of this relaxation in response to hypoxia was investigated by measuring the major cell signalling parameters, intracellular Ca2+ concentration ([Ca2+]i) and myosin regulatory light chain (LC20) phosphorylation (MLC-P1), as well as intracellular pH (pHi), a factor often suggested to mediate hypoxic relaxation of muscle. 2. [Ca2+]i, measured using the ratiometric fluorescent dye fura-2, increased when 40 mM KCl was added to physiological saline solution (PSS) (peak value assigned 100%), and the steady state after 15 min was 92.8%. There were no detectable decreases in [Ca2+]i during hypoxia. 3. MLC-Pi, measured using isoelectric focusing-polyacrylamide gel electrophoresis and identified using Western blotting, increased from 9% of the total LC20 in Ca(2+)-free PSS to a peak value of 51% in 40 mM KCl-PSS. The steady-state value in hypoxia of 43% was not significantly different from that in control oxygenated conditions at the same point in time. 4. pHi, measured using the ratiometric fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF), under quiescent conditions (Ca(2+)-free PSS) was 7.23 and increased to 7.36 with 40 mM KCl. After imposition of hypoxia pHi remained unchanged despite the known increase in both lactate content and production. 5. As [Ca2+]i and MLC-Pi, key factors in activation, were not decreased by hypoxia and changes in pHi were minor, hypoxic relaxation in guinea-pig taenia caeci appears to be directly related to energy limitation rather than any oxygen-sensing mechanism.