Noradrenaline inhibits pacemaker currents through stimulation of beta 1-adrenoceptors in cultured interstitial cells of Cajal from murine small intestine

Abstract

1. Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous inward currents (pacemaker currents) responsible for the production of slow waves in gastrointestinal smooth muscle. The effects of noradrenaline on the pacemaker currents in cultured ICCs from murine small intestine were investigated by using whole-cell patch-clamp techniques at 30 degrees C. 2. Under current clamping, ICCs had a mean resting membrane potential of -58+/-5 mV and produced electrical slow waves. Under voltage clamping, ICCs produced pacemaker currents with a mean amplitude of -410+/-57 pA and a mean frequency of 16+/-2 cycles min(-1). 3. Under voltage clamping, noradrenaline inhibited the amplitude and frequency of pacemaker currents and increased resting currents in the outward direction in a dose-dependent manner. These effects were reduced by intracellular GDP beta S. 4. Noradrenaline-induced effects were blocked by propranolol (beta-adrenoceptor antagonist). However, neither prazosin (alpha(1)-adrenoceptor antagonist) nor yohimbine (alpha(2)-adrenoceptor antagonist) blocked the noradrenaline-induced effects. Phenylephrine (alpha(1)-adrenoceptor agonist) had no effect on the pacemaker currents, whereas isoprenaline (beta-adrenoceptor agonist) mimicked the effect of noradrenaline. Atenolol (beta(1)-adrenoceptor antagonist) blocked the noradrenaline-induced effects, but butoxamine (beta(2)-adrenoceptor antagonist) did not. In addition, BRL37344 (beta(3)-adrenoceptor agonist) had no effect on pacemaker currents. 5. 9-(Tetrahydro-2-furanyl)-9H-purine-6-amine (SQ-22536; adenylate cyclase inhibitor) and a myristoylated protein kinase A inhibitor did not inhibit the noradrenaline-induced effects and 8-bromo-cAMP had no effects on pacemaker currents. 8-Bromo-cGMP and SNAP inhibited pacemaker currents and these effects of SNAP were blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a guanylate cyclase inhibitor). However, ODQ did not block the noradrenaline-induced effects. 6. Neither tetraethylammonium (a voltage-dependent K(+) channel blocker), apamin (a Ca(2+)-dependent K(+) channel blocker) nor glibenclamide (an ATP-sensitive K(+) channel blocker) blocked the noradrenaline-induced effects. 7. The results suggest that noradrenaline-induced stimulation of beta(1)-adrenoceptors in the ICCs inhibits pacemaker currents, and that this is mediated by the activation of G-protein. Neither adenylate cyclase, guanylate cyclase nor a K(+) channel-dependent pathway are involved in this effect of noradrenaline.

Figure 1

Concentration-dependent effect of noradrenaline on pacemaker currents in cultured ICCs of the murine small intestine. (a–c) The slow waves of ICCs exposed to noradrenaline (0.1, 1 and 5 μM) at a holding potential of −70 mV. Noradrenaline inhibited spontaneous pacemaker currents in a concentration-dependent manner in ICCs and induced increased resting currents in the outward direction. (d–f) A summary of the inhibitory effects of noradrenaline on pacemaker currents in ICCs. Each column represents the mean±s.e. (n=9–12/group). ^*P<0.05, significantly different from the controls.

Figure 2

Effects of α- and β-adrenoceptor antagonists on the response to noradrenaline and of α- and β-adrenoceptor agonists on pacemaker currents. (a) Prazosin (5 μM) and (c) yohimbine (5 μM), selective α₁- and α₂-adrenoceptor antagonists, did not block the noradrenaline-mediated inhibition of pacemaker currents. (b) Phenylephrine (2 μM), an α₁-adrenoceptor agonist, had no effects on pacemaker currents. (d) Propranolol (5 μM), a nonselective β-adrenoceptor antagonist, significantly blocked the noradrenaline-induced inhibition of pacemaker currents and (e) isoprenaline (2 μM), a β-adrenoceptor agonist, mimicked the inhibitory effects of noradrenaline on pacemaker currents. Pacemaker currents were recorded from separate cells.

Figure 3

Effects of β₁- and β₂-adrenoceptor antagonists on the response to noradrenaline and the effect of a β₃-adrenoceptor agonist on pacemaker currents. (a) Atenolol (5 μM), a selective β₁-adrenoceptor antagonist, significantly blocked the noradrenaline-mediated inhibition of pacemaker currents. (b) However, butoxamine (5 μM), a β₂-adrenoceptor antagonist, did not block the noradrenaline-mediated inhibition of pacemaker currents. (c) BRL37344 (5 μM), a β₃-adrenoceptor agonist, also had no effects on pacemaker currents. Pacemaker currents were recorded from separate cells.

Figure 4

Effects of GDPβS on the response to noradrenaline. (a) Pacemaker currents of ICCs exposed to noradrenaline (2 μM) in the presence of GDPβS (1 mM) in the pipette. GDPβS partially blocked the noradrenaline-mediated inhibition of pacemaker currents. The effect of noradrenaline in the presence of GDPβS but not in the pipette is summarized in (b) and (c). Each column represents the mean±s.e. (n=7).

Figure 5

Effects of adenylate cyclase and a protein kinase A inhibitor on the noradrenaline-mediated inhibition of pacemaker currents. (a) Effect of noradrenaline on pacemaker currents in the presence of SQ-22536 (10 μM). SQ-22536 did not block the noradrenaline-mediated inhibition of pacemaker currents. (b) Effect of noradrenaline on pacemaker currents in the presence of mPKAI (1 μM). mPKAI did not block the noradrenaline-mediated inhibition of pacemaker currents. (c) Effect of 8-bromo-cAMP (100 μM) on pacemaker currents. 8-bromo-cAMP had no effect on pacemaker currents. Pacemaker currents were recorded from separate cells.

Figure 6

Effects of a guanylate cyclase inhibitor on the noradrenaline-mediated inhibition of pacemaker currents. (a) Effect of 8-bromo-cGMP (100 μM) on pacemaker currents. 8-Bromo-cGMP inhibited pacemaker currents. (b) Effect of SNAP (100 μM) on pacemaker currents. (c) Effect of SNAP in the presence of ODQ (10 μM). SNAP also inhibited the pacemaker current and this effect was antagonized by the pretreatment with ODQ. (d) Effect of noradrenaline on pacemaker currents in the presence of ODQ (10 μM). ODQ did not block the noradrenaline-mediated inhibition of pacemaker currents. Pacemaker currents were recorded from separate cells.

Figure 7

Effects of K⁺ channel blockers on noradrenaline-mediated inhibition of pacemaker currents. (a) TEA (10 mM) (a voltage-dependent K⁺ channel blocker), (b) apamin (0.1 μM) (a Ca²⁺-dependnet K⁺ channel blocker) and (c) glibenclamide (10 μM) (an ATP-sensitive K⁺ channel blocker) did not block the noradrenaline-mediated inhibition of pacemaker currents. (d) Slow waves of ICCs exposed to noradrenaline (2 μM) in the current-clamping mode (I=0). Noradrenaline abolished the generation of slow waves, but did not induce the hyperpolarization of membrane potential. Pacemaker currents were recorded from separate cells.