Mechanism of carbachol‐evoked contractions of guinea‐pig ileal smooth muscle close to freezing point

Abstract

1 The effect of lowering the temperature to near freezing-point upon the contractions and [3H]-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 tLM carbachol was the same at 37°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 5-10s to 2-10min. 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°C and also at 3°C. Thus the majority of the activator calcium required for contraction entered the tissue via voltage-dependent calcium channels (VDCs) at both 37C and 3°C. 4 The contractions produced by high potassium solutions were less at temperatures close to freezingpoint than those at 37°C suggesting that voltage-dependent calcium entry was inhibited as the temperature was lowered. 5 A small part of the contractile response to 100 AM carbachol was resistant to the removal of extracellular calcium at both 37°C and 3°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°C and 3°C.6 Although 100 JLM carbachol produced a statistically significant rise in several [3H]-inositol phosphate isomers at both 37°C and 3°C, the production of [3H]-inositol phosphates was less at 3°C than at 37°C and the increase in their production caused by carbachol was much slower. 7 These results suggest that the carbachol-induced contraction at 3°C utilizes both calcium entry through VDCs and calcium release from intracellular stores, as at 37°C. The components of the responses dependent upon intracellular calcium release at 37°C and at temperatures near freezing-point were similar. However, the production of [3H]-inositol phosphates, including the calcium-mobilizing second messenger inositol (1,4,5) trisphosphate (Ins(1,4,5)P3), is reduced at such low temperatures.

Receptor signaling mechanisms underlying muscarinic agonist‐evoked contraction in guinea‐pig ileal longitudinal smooth muscle

“…One might expect a more straightforward function of M 3 activation, for example, the Ca 2+ released from the store directly activates the contractile proteins. The direct function would be rather important for an initial brief phase of contractions to high concentrations of a full agonist such as carbachol, or tension generation by agonists in the absence of extracellular Ca 2+ (Brading & Sneddon, 1980; Blackwood & Bolton, 1993).…”

“…Thus, the M 3 ‐mediated Ca 2+ store release might be supposed as the major cause of the muscarinic agonist‐evoked contractions. However, the contractile response to muscarinic agonists, regardless of the full or partial type, is severely inhibited by voltage‐dependent Ca 2+ channel blockers (Brading & Sneddon, 1980; Takayanagi et al ., 1990; Blackwood & Bolton, 1993; Hishinuma et al ., 1997). This means that the muscarinic contraction depends largely on Ca 2+ entry via Ca 2+ channels opened by membrane depolarization.…”

Control of intestinal motility by the Cav1.2 L‐type calcium channel in mice

“…Ca 2ϩ released from intracellular stores is believed to be the main trigger for smooth muscle contraction. However, contractions evoked by cholinergic agonists were reduced by L-type Ca 2ϩ channel blockers in intestinal muscle (12,13). Thus, it is unclear to what extent muscarinic-mediated contractions depend on a L-type Ca 2ϩ channel in intestinal smooth muscle.…”

Muscarinic cationic current in gastrointestinal smooth muscles: signal transduction and role in contraction

“…4b), 140 m m K + medium, and Ca 2+ ‐free solution showed that the M 3 contractions involve multiple mechanisms, all of which lead to rises in [Ca 2+ ]c, including voltage‐dependent Ca 2+ entry due to both spike discharge and sustained activation of VDCCs by maintained depolarization, voltage‐independent Ca 2+ entry, and intracellular Ca 2+ release. Analogous properties have been described for the muscarinic contractions in various intact tissues co‐expressing M 2 and M 3 receptors including guinea‐pig stomach, ileum and taenia caeci and rat stomach and ileum (Brading & Sneddon, 1980; Parekh & Brading, 1991; Blackwood & Bolton, 1993; Elorriaga, Anselmi, Hernandez, D'ocon & Ivorra, 1996; Hishinuma et al. , 1997; Unno et al.…”

“…Indeed, the muscarinic contractile responses are reduced or almost abolished by VDCC antagonists in all tissues tested (e.g. Parekh & Brading, 1991; Blackwood & Bolton, 1993; Hishinuma, Hongo, Matsumoto, Narita & Kurokawa, 1997; Unno et al. , 2003a).…”