The KCNQ channel inhibitor XE991 suppresses nicotinic acetylcholine receptor-mediated responses in rat intracardiac ganglion neurons.

BACKGROUND: XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone) is reportedly a potent and selective Kv7 (KCNQ) channel inhibitor. This study aimed to evaluate how XE991 affects nicotinic responses in intracardiac ganglion neurons. METHODS: We studied how the KCNQ channel inhibitor XE991 could affect nicotinic responses in acutely isolated rat intracardiac ganglion neurons using a perforated patch-clamp recording configuration and Ca2+ imaging. RESULTS: XE991 reversibly and concentration-dependently inhibited the nicotine (10 µM)-induced current with an IC50 of 14.4 µM. The EC50 values for nicotine-induced currents in the absence and presence of 10 µM XE991 were 8.7 and 12.0 µM, respectively. Because XE991 suppressed the maximum response of the nicotine concentration-response curve, the inhibitory effect of this drug appears to be noncompetitive. In addition, linopirdine reduced the amplitude of 10 µM nicotine-induced currents with an IC50 value of 16.9 µM. The inorganic KCNQ channel inhibitor Ba2+ affected neither the nicotine-induced current nor the inhibitory effect of XE991 on the nicotinic response. The KCNQ activator flupirtine at a concentration of 10 µM slightly but markedly inhibited the nicotine-induced current. Finally, XE991 inhibited the nicotine-induced elevation of intracellular calcium concentration and the nicotine-induced firing of action potentials. CONCLUSION: We propose that XE991 inhibits nicotinic acetylcholine receptors in intracardiac ganglion neurons, which in turn attenuate nicotine-induced neuronal excitation.

Histamine depolarizes rat intracardiac ganglion neurons through the activation of TRPC non-selective cation channels.

The cardiac plexus, which contains parasympathetic ganglia, plays an important role in regulating cardiac function. Histamine is known to excite intracardiac ganglion neurons, but the underlying mechanism is obscure. In the present study, therefore, the effect of histamine on rat intracardiac ganglion neurons was investigated using perforated patch-clamp recordings. Histamine depolarized acutely isolated neurons with a half-maximal effective concentration of 4.5 µM. This depolarization was markedly inhibited by the H1 receptor antagonist triprolidine and mimicked by the H1 receptor agonist 2-pyridylethylamine, thus implicating histamine H1 receptors. Consistently, reverse transcription-PCR (RT-PCR) and Western blot analyses confirmed H1 receptor expression in the intracardiac ganglia. Under voltage-clamp conditions, histamine evoked an inward current that was potentiated by extracellular Ca2+ removal and attenuated by extracellular Na+ replacement with N-methyl-D-glucamine. This implicated the involvement of non-selective cation channels, which given the link between H1 receptors and Gq/11-protein-phospholipase C signalling, were suspected to be transient receptor potential canonical (TRPC) channels. This was confirmed by the marked inhibition of the inward current through the pharmacological disruption of either Gq/11 signalling or intracellular Ca2+ release and by the application of the TRPC blockers Pyr3, Gd3+ and ML204. Consistently, RT-PCR analysis revealed the expression of several TRPC subtypes in the intracardiac ganglia. Whilst histamine was also separately found to inhibit the M-current, the histamine-induced depolarization was only significantly inhibited by the TRPC blockers Gd3+ and ML204, and not by the M-current blocker XE991. These results suggest that TRPC channels serve as the predominant mediator of neuronal excitation by histamine.

Excitatory effect of bradykinin on intrinsic neurons of the rat heart.

The heart receives sympathetic and parasympathetic innervation through the intrinsic cardiac nervous system. Although bradykinin (BK) has negative inotropic and chronotropic properties of cardiac contraction, the direct effect of BK on the intrinsic neural network of the heart is still unclear. In the present study, the effect of BK on the intracardiac ganglion neurons isolated from rats was investigated using the perforated patch-clamp technique. Under current-clamp conditions, application of 0.1 µM BK depolarized the membrane, accompanied by repetitive firing of action potentials. When BK was applied repeatedly, the second responses were considerably less intense than the first application. The BK action was fully inhibited by the B2 receptor antagonist Hoe-140, but not by the B1 receptor antagonist des-Arg9-[Leu8]-BK. The BK response was mimicked by the B2 agonist [Hyp3]-BK. The BK-induced depolarization was inhibited by the phospholipase C inhibitor U-73122. BK evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca2+ markedly increased the BK-induced currents, suggesting an involvement of Ca2+-permeable non-selective cation channels. The muscarinic agonist oxotremorine-M (OxoM) also elicited the extracellular Ca2+-sensitive cationic currents. The OxoM response did not exhibit rundown with repeated agonist application. The amplitude of current evoked by 1 µM OxoM was comparable to that induced by 0.1 µM BK. Co-application of 0.1 µM BK and 1 µM OxoM elicited the current whose peak amplitude was almost the same as that elicited by OxoM alone, suggesting that BK and OxoM activate same cation channels. BK also reduced the amplitude of M-current, while the M-current inhibitor XE-991 affected neither resting membrane potential nor the BK-induced depolarization. From these results, we suggest that BK regulates excitability of intrinsic cardiac neurons by both an activation of non-selective cation channels and an inhibition of M-type K+ channels through B2 receptors.