TREK-1 channels do not mediate nitrergic neurotransmission in circular smooth muscle from the lower oesophageal sphincter

Abstract

Background and purpose: The ionic mechanisms underlying nitrergic inhibitory junction potentials (IJPs) in gut smooth muscle remain a matter of debate. Recently, it has been reported that opening of TWIK-related K(+) channel 1 (TREK-1) K(+) channels contributes to the nitrergic IJP in colonic smooth muscle. We investigated the effects of TREK-1 channel blockers on nitrergic neurotransmission in mouse and opossum lower oesophageal sphincter (LOS) circular smooth muscle (CSM).

Experimental approach: The effects of TREK-1 channel blockers were characterized pharmacologically in murine and opossum gut smooth muscle using conventional intracellular and tension recordings.

Key results: In LOS, L-methionine depolarized the resting membrane potential (RMP) but did not inhibit the nitrergic IJP. Cumulative application of theophylline hyperpolarized the RMP and inhibited the nitrergic IJP concentration dependently. The induced membrane hyperpolarization was prevented by pre-application of caffeine, but not by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. 8-Br-cAMP significantly hyperpolarized membrane potential and increased the amplitude of the nitrergic IJP. In opossum LOS muscle strips, L-methionine increased resting tone but had no effect on nerve-mediated LOS relaxation. On the other hand, theophylline markedly inhibited tone. In CSM from mouse proximal colon, L-methionine caused modest inhibition of nitrergic IJPs.

Conclusions and implications: TREK-1 channels were not involved in the nitrergic IJP in LOS CSM. Not only does L-methionine have no effect on the nitrergic IJP or LOS relaxation, but the effect of theophylline appears to be due to interruption of Ca(2+)-releasing pathways (i.e. caffeine-like effect) rather than via blockade of TREK-1 channels.

Figure 1

Effects of TREK-1 K⁺ channel blockers L-methionine and theophylline on the resting membrane potential (RMP) and on the purinergic and nitrergic inhibitory junction potentials (IJPs) in the presence of atropine (3 µM), guanethidine (3 µM) and substance P (1 µM) to ensure non-adrenergic, non-cholinergic, non-tachykinergic conditions. (A) Experimental recording demonstrating that L-methionine (1 mM) depolarized RMP, but theophylline (2.5 mM) hyperpolarized the RMP. (B,C) IJPs depicted in panel A, on an expanded time scale, before and after application of the channel blockers,. L-methionine did not have any significant effects on IJPs. However, theophylline significantly inhibited the fast inhibitory junction potential and abolished the sIJP.

Figure 2

Cumulative concentration–response of theophylline. (A) Time course of effects of cumulative application of theophylline (0.03–3.0 mM) at intervals of 8 min, on electrical properties. Theophylline (0.03–1.0 mM) hyperpolarized resting membrane potential (RMP) in a concentration-dependent fashion, but subsequent bath application of theophylline (3 mM) surprisingly depolarized the RMP. The effects of theophylline were reversible and completely recovered 30 min after washing out. (B) Inhibitory junction potentials (IJPs) depicted in panel A displayed at an expanded time scale. (B, panels h,i) Overlapped IJPs before, during and after the application of theophylline.

Figure 3

Statistical analysis of concentration–response curves for the theophylline effect on (A) resting membrane potential (RMP), and (B) fast inhibitory junction potential and slow inhibitory junction potential (sIJP). The amplitude of sIJP was significantly increased by theophylline only at the 0.1 mM concentration.

Figure 4

Failure of tetraethylammonium (TEA), a Ca²⁺-activated large-conductance K⁺ channels (BK) channel blocker, to prevent the inhibitory effects of theophylline on electrical properties. (A) Continuous recording of the effects of pre-application of TEA (2 mM, 5 min) on the inhibition induced by theophylline (3 mM, 5 min). (B, panels a–c) Inhibitory junction potentials (IJPs) from panel A at an expanded time scale in control, TEA and TEA plus theophylline. TEA failed to prevent the hyperpolarization and abolition of nitrergic IJPs, suggesting that the inhibitory effects of theophylline were not due to the opening of BK channels.

Figure 5

Effects of 8-Br-cAMP on resting membrane potential (RMP), unitary potentials and nitrergic biphasic inhibitory junction potentials (IJPs) in the presence of apamin. Panel A demonstrates that 8-Br-cAMP (1 mM, 10 min) hyperpolarized RMP by about 7 mV over control. (B, panels a–c) Nitrergic IJPs from panel A, on an expanded time scale, in control, 5 min and 10 min after application of 8-Br-cAMP. (B, panel d) Superimposed nitrergic biphasic IJPs in comparison. 8-Br-cAMP increased the amplitude of the biphasic IJPs.

Figure 6

The inhibitory effects of theophylline on the resting membrane potential were not prevented by pre-application of the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ). (A) Full experimental recording of electrical properties before and after bath application of ODQ (20 µM, 15 min) and theophylline (3 mM, 5 min). (B, panels a–e) Inhibitory junction potentials (IJPs) depicted in panel A. displayed at an expanded time scale in control (a), after application of agents (b–d) and recovery (e). Potency of ODQ was validated by further application of N-nitro-L-arginine methyl ester (L-NAME) (100 µM, 10 min), which had no further effect on the IJP. Failure of ODQ to prevent the hyperpolarization excludes the possibility that the hyperpolarization is due to the intracellular accumulation of cGMP resulting from the inhibition of phosphodiesterase by theophylline. The asterisk (*) represents perfusion interference.

Figure 7

Pre-application of caffeine prevented the resting membrane potential (RMP) hyperpolarization induced by theophylline. In panel A, an experimental recording is shown of the effects of theophylline (3 mM, 5 min) in the presence of caffeine (5 mM, 5 min). Caffeine hyperpolarized the RMP and abolished nitrergic inhibitory junction potentials (IJPs). Further application of theophylline produced RMP depolarization rather than hyperpolarization. (B, panels a–c) IJPs (on an expanded time scale) before and after administration of caffeine and theophylline. (B, panel d) Overlapped IJPs in comparison.

Figure 8

Effects of N-nitro-L-arginine methyl ester (L-NAME) (A) and L-methionine (B) on long-lasting slow inhibitory junction potential (sIJP) in murine proximal colon circular smooth muscle. (A, panel a) and (B, panel a) depict original tracings, demonstrating that both L-NAME and L-methionine depolarize resting membrane potential. (A, panel b) and (B, panel b) show inhibitory junction potentials (IJPs) (on an expanded time scale) induced by four-pulse nerve stimulation before and after drug administration. L-NAME virtually abolishes the long-lasting sIJP, whereas L-methionine causes only partial inhibition.

Figure 9

Effects of L-methionine on lower oesophageal sphincter (LOS) basal tone (A) and of nerve-mediated relaxation (B) in opossum LOS. L-methionine significantly increased basal tone but did not inhibit LOS relaxation induced by electrical field stimulation. Subsequent application of N-nitro-L-arginine methyl ester (L-NAME) abolished LOS relaxation and unmasked a contraction. L-NAME also inhibited the spontaneous fluctuations in the basal LOS tone. The arrow in (A) represents the time of application of L-methionine. The arrows in (B) represent the onset of electrical field stimulation. These results are consistent with the electrophysiological data obtained in murine LOS.