Hydrogen sulfide regulates the colonic motility by inhibiting both L-type calcium channels and BKCa channels in smooth muscle cells of rat colon

Abstract

Objective: To examine the hypothesis that hydrogen sulfide (H2S) regulates the colonic motility by modulating both L-type voltage-dependent calcium channels and large conductance Ca2+-activated K+ (BKCa) channels.

Methods: Immunohistochemistry was performed on rat colonic samples to investigate the localization of the H2S-producing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). The contractions of proximal colonic smooth muscle were studied in an organ bath system. The whole-cell patch-clamp technique was used to record both L-type calcium currents (ICa,L) and BKCa currents in colonic smooth muscle cells (SMCs) isolated from male Wistar rats.

Results: Immunohistochemistry revealed the presence of CBS and CSE in mucosa, smooth muscle cells and myenteric neurons. The H2S donor NaHS inhibited spontaneous contractions of the longitudinal muscle and circular muscle strips in a dose-dependent manner, and the inhibitory effects were not blocked by tetrodotoxin. NaHS inhibited the peak ICa,L in colonic SMCs at a membrane potential of 0 mV. The current-voltage (I-V) relationship of L-type calcium channels was modified by NaHS, and the peak of the I-V curve was shifted to the right. NaHS (200 μΜ) evoked a significant rightward shift of the steady-state activation curve and inhibited the inactivation of L-type calcium channels. Furthermore, NaHS reversibly decreased the peak ICa,L in a dose-dependent manner. Likewise, BKCa channels were significantly inhibited by NaHS, and the addition of NaHS caused a time- and dose-dependent reduction in the BKCa current.

Conclusion: The relaxant effect of H2S on colonic muscle strips may be associated with the direct inhibition of H2S on L-type calcium channels. H2S may be involved in the regulation of calcium homeostasis in colonic SMCs of rat colon.

Fig 1. Immunohistochemical localization of CBS (A) and CSE (B) in the rat proximal colon.

Magnification 20×.

Fig 2. Effect of NaHS on spontaneous contraction of colonic muscle strips.

(A and C) NaHS inhibited the spontaneous contractions of longitudinal muscle (LM) and circular muscle (CM) in a concentration-dependent manner, which was still recorded in the presence of TTX (1 μM). (B and D) Summarized results of LM and CM before and after application of NaHS in the presence and absence of TTX. (n = 7 for each group, ^* P<0.05 vs. control)

Fig 3. Effect of Nifedipine on I _Ca,L in colonic SMCs.

(A) Original traces of whole cell recordings in response to a series of depolarizing voltage pulses from a holding potential of −50 mV to +20 mV in 10 mV steps before (control) and after application of Nifedipine(1 μM). (B) The representative effects of Nifedipine on the I-V relationship of I _Ca,L.

Fig 4. Effect of NaHS on peak I _Ca,L.

(A)Representative traces of I _Ca,L elicited by a single depolarized step pulse from −50 mV to 0 mV before (control) and after application of NaHS(200 and 400 μM). (B) Summarized data showing the density of the currents at 0 mV (n = 7 for each group, *P<0.05 vs. control).

Fig 5. Effect of NaHS on the I-V relationship and dynamic characteristics of I _Ca,L.

(A) Original traces of whole cell recordings in response to a series of depolarizing voltage pulses from a holding potential of −50 mV to +20 mV in 10 mV steps before (control) and after application of the H₂S donor NaHS (200 and 400 μM). (B) The representative effects of NaHS (200 and 400 μM) on the I-V relationship of I _Ca,L. (C and D) Effect of NaHS(200 μM) on the steady-state activation of I _Ca,L and the steady-state inactivation of I _Ca,L(n = 6 for each group).

Fig 6. Concentration-dependent property and time course of NaHS on I _Ca,L.

(A) A dose response relationship of NaHS-induced inhibition on peak I _Ca,L at 0 mV. (B) Effect of NaHS(200 μM) on peak I _Ca,L at 0 mV with a washout period after application of the test compound (n = 6 for each group, P<0.05 vs. control).

Fig 7. Effect of Iberiotoxin (IbTx) on I _BK,Ca in colonic SMCs.

(A) Original traces of whole cell recordings in response to a series of depolarizing voltage pulses from a holding potential of −80 mV to +60 mV in 20 mV steps before (control) and after the application of IbTx (100 nM). (B) The representative effects of IbTx on the I-V relationship of I _BK,Ca.

Fig 8. Inhibitory effect of NaHS on I _BK,Ca.

(A) Original traces of whole cell recordings in response to a series of depolarizing voltage pulses from a holding potential of −80 mV to +60 mV in 20 mV steps before (control) and after application of NaHS (200 and 400 μM) (B) The representative effects of NaHS at different concentrations on the I-V relationship of I _BK,Ca. (C) Summarized data showing the density of the currents at +60 mV. (D and E) Representative traces elicited by a single depolarized step pulse from −80 mV to +60 mV and the time course of NaHS induced inhibition on I _BK,Ca (n = 8 for each group, *P<0.05 vs. control).