MECHANISM OF CARBACHOL-EVOKED CONTRACTIONS OF GUINEA-PIG ILEAL SMOOTH-MUSCLE CLOSE TO FREEZING-POINT

1 The effect of lowering the temperature to near freezing-point upon the contractions and [H-3]-inositol phosphate responses to carbachol were investigated in longitudinal smooth muscle from the guinea-pig ileum. 2 The peak amplitude of the contraction to a single application of 100 muM carbachol was the same at 37-degrees-C and temperatures near freezing-point. However, the sensitivity to carbachol was reduced upon lowering the temperature and the time to peak contraction was increased from 5-10 s to 2-10 min. Even when the temperature was maintained near freezing-point, washing off carbachol produced a relaxation and eventual return of tension to basal levels. 3 Incubating the tissue in 140 mm K+, calcium-free solution or in calcium channel antagonists significantly reduced the carbachol-induced contraction to 10-30% of the control at 37-degrees-C and also at 3-degrees-C. Thus the majority of the activator calcium required for contraction entered the tissue via voltage-dependent calcium channels (VDCs) at both 37-degrees-C and 3-degrees-C. 4 The contractions produced by high potassium solutions were less at temperatures close to freezing-point than those at 37-degrees-C suggesting that voltage-dependent calcium entry was inhibited as the temperature was lowered. 5 A small part of the contractile response to 100 muM carbachol was resistant to the removal of extracellular calcium at both 37-degrees-C and 3-degrees-C and this component was increased under depolarizing conditions. This suggests that the release of stored calcium contributes to a minor degree to contraction at both 37-degrees-C and 3-degrees-C. 6 Although 100 muM carbachol produced a statistically significant rise in several [H-3]-inositol phosphate isomers at both 37-degrees-C and 3-degrees-C, the production of [H-3]-inositol phosphates was less at 3-degrees-C than at 37-degrees-C and the increase in their production caused by carbachol was much slower. 7 These results suggest that the carbachol-induced contraction at 3-degrees-C utilizes both calcium entry through VDCs and calcium release from intracellular stores, as at 37-degrees-C. The components of the responses dependent upon intracellular calcium release at 37-degrees-C and at temperatures near freezing-point were similar. However, the production of [H-3]-inositol phosphates, including the calcium-mobilizing second messenger inositol (1,4,5) trisphosphate (Ins(1,4,5)P3), is reduced at such low temperatures.