Pharmacological characterization of 5-HT4 receptors mediating relaxation of canine isolated rectum circular smooth muscle

Abstract

This study aimed to characterize for the first time in vitro 5-HT4 receptors in the canine gastrointestinal tract. For this purpose, we used circular muscle strips of the canine isolated rectum. In the presence of methysergide (60 microM), 5-HT induced relaxation of methacholine (1 microM)-precontracted muscle strips, yielding a monophasic sigmoidal concentration-relaxation curve (pEC50 7.2+/-0.07). Tetrodotoxin (0.3 microM) did not affect the curve to 5-HT, suggesting the inhibitory 5-HT receptor is located on the smooth muscle. Granisetron (0.3 microM) did also not affect the curve to 5-HT, which excludes the 5-HT3 receptor mediating the relaxation to 5-HT. The presence of methysergide rules out the involvement of 5-HT1, 5-HT2 or 5-HT7 receptors. 5-HT, the selective 5-HT4 receptor agonists R076186, prucalopride (R093877) and SDZ HTF-919 and the 5-HT4 receptor agonists cisapride and 5-MeOT relaxed the muscle strips with a rank order of potency R076186 = 5-HT > cisapride > prucalopride > or = SDZ HTF-919 > 5-MeOT. The selective 5-HT4 receptor antagonists GR 125487, RS 39604 and GR 113808 competitively antagonized the relaxations to 5-HT, yielding pK(B) estimates of 9.7, 7.9 and 9.1, respectively. The selective 5-HT4 receptor antagonist SB 204070 shifted the curve to 5-HT rightward and depressed the maximal response (apparent pA2 10.6). GR 113808 (10 nM) produced a parallel rightward shift of the curve to the selective 5-HT4 receptor agonists R076186 (pA2 8.8). It is concluded that 5-HT induces relaxation of the canine rectum circular muscle through stimulation of a single population of smooth muscle 5-HT4 receptors. For the first time, a nonhuman species was shown to exhibit relaxant 5-HT4 receptors in the large intestine.

Figure 1

Concentration-relaxation curves to 5-HT, 5-MeOT and cisapride (left panel) and to 5-HT, R076186, prucalopride and SDZ HTF-919 (right panel) in the canine isolated rectal circular smooth muscle. The curves shown superimposed on the mean experimental data points represent simulations using the Hill-equation and the parameters for midpoint location (with horizontal standard error bars), upper asymptote location (with vertical standard error bars) and the Hill slope, that were obtained from the iterative fitting procedure.

Figure 2

Left panels: Concentration-relaxation curves to 5-HT in the absence and presence of GR 113808 (a), RS 39604 (b), and GR 125487 (c) in the canine isolated rectal circular smooth muscle. The curves shown superimposed on the mean experimental data points are simulations using the Hill-equation and the parameters for midpoint location (with horizontal standard error bars), upper asymptote location (with vertical standard error bars) and the Hill slope that were obtained from the iterative fitting procedure. Right panels: The Schild plots for GR 113808 (a), RS 39604 (b) and GR 125487 (c). The line shown superimposed on the experimental data points was obtained by stimulating Schild regression analysis with the Schild slope constrained to unity, providing the X-axis intercept representing the pK_B.

Figure 3

The concentration-relaxation curves to 5-HT in the absence and presence of SB 204070 in the canine isolated rectal circular smooth muscle. The curves shown superimposed on the mean experimental data points are simulations using the Hill-equation and the parameters for midpoint location (with horizontal standard error bars), upper asymptote location (with vertical standard error bars) and the Hill slope that were obtained from the iterative fitting procedure.

Figure 4

The concentration-relaxation curves to R076186 in the absence and presence of GR 113808 using the canine isolated rectal circular smooth muscle preparation. The curves shown superimposed on the mean experimental data points are simulations using the Hill-equation and the parameters for midpoint location (with horizontal standard error bars), upper asymptote location (with vertical standard error bars) and the Hill slope that were obtained from the iterative fitting procedure.