Extracellular Cl- regulates electrical slow waves and setting of smooth muscle membrane potential by interstitial cells of Cajal in mouse jejunum

Abstract

What is the central question of this study? The aim was to investigate the roles of extracellular chloride in electrical slow waves and resting membrane potential of mouse jejunal smooth muscle by replacing chloride with the impermeant anions gluconate and isethionate. What is the main finding and its importance? The main finding was that in smooth muscle cells, the resting Cl^- conductance is low, whereas transmembrane Cl^- movement in interstitial cells of Cajal (ICCs) is a major contributor to the shape of electrical slow waves. Furthermore, the data confirm that ICCs set the smooth muscle membrane potential and that altering Cl^- homeostasis in ICCs can alter the smooth muscle membrane potential. Intracellular Cl^- homeostasis is regulated by anion-permeable channels and transporters and contributes to excitability of many cell types, including smooth muscle and interstitial cells of Cajal (ICCs). Our aims were to investigate the effects on electrical activity in mouse jejunal muscle strips of replacing extracellular Cl^- (Cl^-_o ) with the impermeant anions gluconate and isethionate. On reducing Cl^-_o , effects were observed on electrical slow waves, with small effects on smooth muscle membrane voltage (E_m ). Restoration of Cl^- hyperpolarized smooth muscle E_m proportional to the change in Cl^-_o concentration. Replacement of 90% of Cl^-_o with gluconate reversibly abolished slow waves in five of nine preparations. Slow waves were maintained in isethionate. Gluconate and isethionate substitution had similar concentration-dependent effects on peak amplitude, frequency, width at half peak amplitude, rise time and decay time of residual slow waves. Gluconate reduced free ionized Ca²⁺ in Krebs solutions to 0.13 mm. In Krebs solutions containing normal Cl^- and 0.13 mm free Ca²⁺ , slow wave frequency was lower, width at half peak amplitude was smaller, and decay time was faster. The transient hyperpolarization following restoration of Cl^-_o was not observed in W/W^v mice, which lack pacemaker ICCs in the small intestine. We conclude that in smooth muscle cells, the resting Cl^- conductance is low, whereas transmembrane Cl^- movement in ICCs plays a major role in generation or propagation of slow waves. Furthermore, these data support a role for ICCs in setting smooth muscle E_m and that altering Cl^- homeostasis in ICCs can alter smooth muscle E_m .

Keywords: chloride transport; gastrointestinal motility; pacemaker potentials.

Fig. 1

The effect of replacing extracellular chloride, [Cl⁻]_o with gluconate on slow wave activity recorded from mouse jejunal smooth muscle layer. Replacement of [Cl⁻]_o by gluconate significantly altered slow wave properties in a concentration dependent manner (see Fig. 2). A1, A2-C, Representative traces of intracellular recording made in [Cl⁻]_o/[gluconate⁻]_o: 13.3/120 mM (A1, A2); 39.9/93.4 mM (B); and 120/13.3 mM (C), respectively. Control [Cl⁻]_o was 134 mM. In 5 out of 9 experiments, slow waves were abolished (A1). In the remaining 4 experiments, small amplitude residual slow wave activity was observed (A2). The horizontal bar over each trace indicates the perfusion period of low [Cl⁻]_o solution. Expanded time scales are shown at the bottom of A1, A2, B, and C with different time points before and after the low [Cl⁻]_o perfusion.

Fig. 2

Measurement of electrical slow wave properties upon replacing extracellular chloride, [Cl⁻]_o with gluconate. A–H, Summarized data from N=4–9 experiments illustrating the effects of reduced [Cl⁻]_o on electrical properties: A transient (< 1min) concentration-dependent hyperpolarization in membrane potential (E_m) was observed in the solution change (A, ΔE_m). At steady state (after 4–5 min of perfusion), no change was observed in E_m (B, ΔE_m). On returning to normal Krebs solution, a transient, concentration-dependent hyperpolarization in E_m (C, ΔE_m) was observed. (D) shows the concentration dependence on a logarithmic scale. Slow waves that remained at steady state (after 4–5 min of perfusion) were analyzed, concentration-dependent reduction in slow wave amplitude (E); decreases in the instantaneous frequency (F); shortening of slow wave width (G, half-width); slower rise times of 10%–90% of peak amplitude (H); and reduction in the decay times of 90%–10% of peak amplitude (I) were seen (see text for details). These effects were reversible on washout. Values are mean ± STDEV (N=4–9, *P < 0.05, ANOVA with Bonferroni’s correction for multiple comparisons).

Fig. 3

The effect of replacing extracellular chloride, [Cl⁻]_o with isethionate on slow wave activity recorded from mouse jejunal smooth muscle layer. Replacement of [Cl⁻]_o by isethionate had different effects from gluconate on slow wave activity (see Fig. 1). A–C, Representative traces of intracellular recording made in [Cl⁻]_o/[isethionate⁻]_o: 13.3/120 mM (A); 39.9/93.4 mM (B); and 119.8/13.3 mM (C), respectively. Control [Cl⁻]_o was 134 mM. The horizontal bar over each trace indicates the perfusion period of low [Cl⁻]_o solution. Expanded time scales are shown at the bottom of A, B and C with different time points before and after the low [Cl⁻]_o perfusion. A significant concentration dependent alteration in slow wave properties was seen (see Fig. 4).

Fig. 4

Measurement of electrical slow wave properties upon replacing extracellular chloride, [Cl⁻]_o with isethionate. A–H, Summarized data from N=5–7 experiments illustrating the effects of reduced [Cl⁻]_o on electrical properties: Replacement of [Cl⁻]_o with isethionate had no transient effects in membrane potential, E_m (A, ΔE_m). At steady state (after 4–5 min of perfusion), a concentration-dependent depolarization in E_m was observed (B, ΔE_m). Upon returning to normal Krebs solution, a transient, concentration-dependent hyperpolarization in E_m (C, ΔE_m) was observed. (D) shows the concentration dependence on a logarithmic scale. Slow waves that remained at steady state (after 4–5 min of perfusion) were analyzed, concentration-dependent reduction in slow wave amplitude (E); decreases in the instantaneous frequency (F); shortening of slow wave width (G, half-width); slower rise times at 10%–90% of peak amplitude (H); and reduction in the decay times at 90%–10% of peak amplitude (I) were seen (see text for details). These effects were reversible on washout. Values are mean ± STDEV (N=5–7, *P < 0.05, ANOVA with Bonferroni’s correction for multiple comparisons).

Fig. 5

The calcium binding capacity of the sodium salts of gluconate (Na-gluc) and isethionate (Na-ise) in low chloride Krebs solutions as measured using Ca²⁺ ion-selective electrode. Gluconate and isethionate reduced free Ca²⁺ in Krebs solution. Histogram data showing a significant decrease in free Ca²⁺ (see text for details). Values are mean ± SEM (N=5, *P < 0.05, ANOVA with Bonferroni’s correction for multiple comparisons).

Fig. 6

The effect on slow wave activity of reducing [Ca²⁺]_o in Krebs to the level of [Ca²⁺]_o obtained by adding gluconate (A) and isethionate (B) (see Fig. 5). Lowering [Ca²⁺]_o did not replicate the effect of replacing [Cl⁻]_o with gluconate or isethionate on electrical activity in mouse jejunal smooth muscle layer. A–B, Representative traces of intracellular recordings upon perfusion with 0.13 mM and 0.54 mM [Ca²⁺]_o. The horizontal bar over each trace indicates the perfusion period of low [Ca²⁺]_o solution. Expanded time scales are shown below the traces. C–I, Summarized data illustrating the effects of reduced [Ca²⁺]_o on slow wave properties: At 0.13 mM [Ca²⁺]_o, (equivalent to the level found in 13.3 mM Cl⁻, 120.3 mM gluconate Krebs) decreases in the instantaneous frequency (F), shortening of slow wave width (G) and a decrease in the decay times from 90 to 10% of peak amplitude (I) were observed. These effects were reversible on washout. No change was observed in membrane potential (C) and the rest of the slow wave properties (E, H). No effects were observed at 0.54 mM [Ca²⁺]_o (equivalent to 13.3 mM Cl⁻, 120.3 mM isethionate Krebs; panel D–I). Values are mean ± SEM (N=4–5, *P < 0.05, ANOVA with Bonferroni’s correction for multiple comparisons).

Fig. 7

The effect of a 90% reduction in [Cl⁻]_o on the spontaneous electrical activity recorded from the mutant W/W^v, an ICC deficient mouse. Gluconate but not isethionate caused a rapid, tetrodotoxin insensitive, hyperpolarizing effect on membrane potential (E_m) in W/W^v mice. A–C, Representative traces with sodium salts of gluconate (A), gluconate in the presence of TTx (B), and isethionate (C). The horizontal bar over each trace indicates the perfusion period of low [Cl⁻]_o solution with and without TTx. Expanded time scales are shown below the traces. D, E, Summarized data illustrating the effect of reduced [Cl⁻]_o on E_m. Replacement of Cl⁻ with gluconate (4–5 min) caused a significant hyperpolarization in E_m that was reversed on washout and not different after TTx (0.5 μM, D). Isethionate did not cause the same effect (E). Data for individual experiments are shown, whiskers represent the means ± STDEV (N=3–6, *P < 0.05, ANOVA with Bonferroni’s correction for multiple comparisons).

Fig. 8

The effect of low [Cl⁻]_o gluconate Krebs solution corrected for the effects of Ca²⁺ chelation and the effect of replacement of HCO₃⁻ with HEPES. Replacement of [Cl⁻]_o by gluconate after correcting the free Ca²⁺ concentration to 1mM significantly altered slow wave properties (see Table 4). Representative traces of intracellular recording made in [Cl⁻]_o: 33.3 mM, 119.8 gluconate (A1, A2). In 1 out of 9 experiments, slow waves were abolished (A1). In the remaining 8 experiments, residual slow wave activity was observed (A2). The horizontal bar over each trace indicates the perfusion period of low [Cl⁻]_o solution. Expanded time scales are shown at the bottom of A1, A2. Replacement of HCO₃⁻ with HEPES in Krebs solution gassed with 100% O₂ resulted in small but significant changes in the slow wave properties and a small depolarization in the membrane potential (see Table 4). The horizontal bar over each trace indicates the perfusion period with HCO₃⁻ -free solution. Expanded time scales are shown below.