KV7 channels regulate muscle tone and nonadrenergic noncholinergic relaxation of the rat gastric fundus

Abstract

Voltage-dependent type 7 K+ (KV7) channels play important physiological roles in neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators in the rat gastric fundus and the expression of KV7 channels in this tissue. Muscle tone and electrical field stimulation (EFS)-evoked relaxations of precontracted longitudinal muscle strips of the rat gastric fundus were investigated under nonadrenergic noncholinergic conditions by organ bath studies. Gene expression was studied by real-time PCR and tissue localization of channels was investigated by immunohistochemistry. The KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean pD2 and Emax of 5.4 and 48% of the maximal U46619-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed U46619-precontracted strips, with pD2s of 4.7 and 4.4 and Emax of 93% and 91% of the maximal relaxation induced by papaverine, respectively. XE-991 concentration-dependently inhibited retigabine-induced relaxation with a pIC50 of 6.2. XE-991 and DMP-543, another KV7 channel blocker, increased by 13-25% or reduced by 11-21% the relaxations evoked by low- or high-frequency EFS, respectively. XE-991 also reduced the relaxation induced by vasoactive intestinal polypeptide (VIP) by 33% of controls. Transcripts encoded by all KV7 genes were detected in the fundus, with 7.4 and 7.5 showing the highest expression levels. KV7.4 and 7.5 channels were visualized by confocal immunofluorescence in both circular and longitudinal muscle layers. In conclusion, in the rat proximal stomach, KV7 channels appear to contribute to the resting muscle tone and to VIP- and high-frequency EFS-induced relaxation. KV7 channel activators could be useful relaxant agents of the gastric smooth muscle.

Graphical abstract

Fig. 1

Motor effects induced by XE-991 on longitudinal muscle strips of the rat gastric fundus under NANC conditions. (A) Representative tracings showing the concentration-dependent contracting effects of XE-991 (0.5–100 μM). (B) Mean concentration–response curve for contractions induced by XE-991 (0.5–100 μM). Contractions were measured as peak amplitudes. Peak amplitudes are expressed as percentages of a maximal amplitude parameter measured from the maximal U46619-induced contraction point reached before the addition of any test substance to the maximal papaverine (300 μM)-induced relaxation point reached at the end of each experiment. Each point represents the mean ± s.e.m. of responses observed in nine strips.

Fig. 2

Motor effects induced by retigabine and flupirtine on U46619 (0.1 μM)-precontracted longitudinal muscle strips of the rat gastric fundus under NANC conditions. (A) and (B) Representative tracings showing the concentration-dependent relaxant effects of retigabine (1–100 μM) and flupirtine (1–100 μM), respectively. The effect of ω-conotoxin GVIA (30 nM) on retigabine (100 μM)-induced relaxation is also shown. (C) Mean concentration–response curves for relaxations induced by retigabine (1–100 μM) and flupirtine (4–100 μM). Relaxations were measured as peak amplitudes. Peak amplitudes are expressed as percentages of a maximal amplitude parameter measured from the maximal U46619-induced contraction point reached before the addition of any test substance to the maximal papaverine (300 μM)-induced relaxation point reached at the end of each experiment. Each point represents the mean ± s.e.m. of responses observed in six strips.

Fig. 3

Effects of XE-991 (0.5–20 μM) on retigabine (30 μM)-induced submaximal relaxation of U46619 (0.1 μM)-precontracted longitudinal muscle strips of the rat gastric fundus. Mean retigabine (30 μM)-induced relaxations observed in the absence (0 XE-991, controls) or presence of XE-991 (0.5–20 μM). Relaxations were measured as peak amplitudes. Peak amplitudes are expressed as percentages of a maximal amplitude parameter measured from the maximal U46619-induced contraction point reached before the addition of any test substance to the maximal papaverine (300 μM)-induced relaxation point reached at the end of each experiment. Each point represents the mean ± s.e.m. of responses observed in three strips. Significant differences between test and control responses: ***P < 0.001.

Fig. 4

Effects of XE-991 on the NANC relaxant responses induced by EFS (2 or 13 Hz, 120 mA, 1 ms, pulse trains of 2 min) of U46619 (0.1 μM)-precontracted longitudinal muscle strips of the rat gastric fundus. (A) Representative tracings showing the effects of XE-991 (20 μM). The submaximal relaxation amplitudes induced by EFS (2 Hz) were expressed as percentages of a maximal amplitude parameter measured from the maximal U46619-induced contraction point reached before the addition of any test substance to the maximal papaverine (300 μM)-induced relaxation point reached at the end of each experiment (parameter indicated with a in the panel). Similarly, the submaximal AUCs (mm min) of the relaxant responses induced by EFS (13 Hz) were divided by this same maximal amplitude parameter (mm) and expressed as min. On the contrary, each maximal relaxation amplitude induced by EFS (13 Hz) was expressed as a percentage of its own maximal amplitude parameter, measured from the U46619-induced contraction point reached at the start of each EFS-induced relaxation to the maximal papaverine (300 μM)-induced relaxation point reached at the end of each experiment (parameters indicated with b and c in the panel, respectively). (B) Mean NANC relaxations evoked by 2 Hz (left graph) or 13 Hz EFS (middle and right graphs) observed in the absence (0 XE-991, time controls) or presence of XE-991 (10–50 μM). Relaxations were measured as peak amplitudes (left and middle graphs) or AUCs (right graph) and are expressed as percentages of control responses. Each point represents the mean ± s.e.m. of responses observed in eight (0, 10 and 20 μM XE-991) or nine (50 μM XE-991) strips. Significant differences between test and control responses: *P < 0.05, **P < 0.01,***P < 0.001.

Fig. 5

Effects of XE-991 on the relaxant responses induced by NaNO₂ (300 μM) in 0.1 N HCl or VIP (10 nM) of U46619 (0.1 μM)-precontracted longitudinal muscle strips of the rat gastric fundus. (A) Representative tracings showing the effects of XE-991 (20 μM). (B) Mean relaxations evoked by NaNO₂ (left graph) or VIP (middle and right graphs) observed in the absence (0 XE-991, time controls) or presence of XE-991 (20 μM). Relaxations were measured as peak amplitudes (left and middle graphs) or AUCs (right graph), normalized for the analysis in a similar way to that described in the legend of Fig. 3 and are expressed as percentages of control responses. Each point represents the mean ± s.e.m. of responses observed in seven (20 μM XE-991) or nine (0 XE-991, time controls) strips. Significant differences between test and control responses: *P < 0.05, **P < 0.01, ***P < 0.001. ###P < 0.001 vs. time controls.

Fig. 6

Expression of K_V7 channel genes in the rat gastric fundus. (A) Agarose gel electrophoresis of RT-PCR products obtained from cDNA amplification of rat gastric fundus mRNA using K_V7-selective primers. Amplicon sizes and primer sequences are shown in Table 1. For a comparison, the expression of K_V7.1 and K_V7.2–7.5 channel genes in rat heart and brain, respectively, was also studied. (B) Quantification of transcripts for K_V7 channel subtypes by use of real-time quantitative PCR. Data are expressed as 2^−Δct relative to GAPDH gene expression, as described in Section 2. Each bar is the mean ± s.e.m. of four separate determinations.

Fig. 7

Expression of K_V7.4 and K_V7.5 channel subunits in the rat gastric fundus. Low (A) and high (B) magnification confocal images of rat gastric fundus cryosections stained for the nuclear marker chromomycin A3 (in green), for K_V7.4 or K_V7.5 (in red), as indicated; merged images are shown in the rightmost panels. The scale bar is 100 μm in (A), and 10 μm in (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)