David V. Miller

Intraluminal acid induces oesophageal shortening via capsaicin-sensitive neurokinin neurons

Objective: Intraluminal acid evokes reflex contraction of oesophageal longitudinal smooth muscle (LSM) and consequent oesophageal shortening. This reflex may play a role in the pathophysiology of oesophageal pain syndromes and hiatus hernia formation. The aim of the current study was to elucidate further the mechanisms of acid-induced oesophageal shortening. Design: Intraluminal acid perfusion of the intact opossum smooth muscle oesophagus was performed in vitro in the presence and absence of neural blockade and pharmacological antagonism of the neurokinin 2 receptor, while continuously recording changes in oesophageal axial length. In addition, the effect of these antagonists on the contractile response of LSM strips to the mast cell degranulating agent 48/80 was determined. Finally, immunohistochemistry was performed to look for evidence of LSM innervation by substance P/calcitonin gene-related peptide (CGRP)-containing axons. Results: Intraluminal acid perfusion induced longitudinal axis shortening that was completely abolished by capsaicin desensitization, substance P desensitization, or the application of the neurokinin 2 receptor antagonist MEN10376. Compound 48/80 induced sustained contraction of LSM strips in a concentration-dependent fashion and this was associated with evidence of mast cell degranulation. The 48/80-induced LSM contraction was antagonized by capsaicin desensitization, substance P desensitization and MEN10376, but not tetrodotoxin. Immunohistochemistry revealed numerous substance P/CGRP-containing neurons innervating the LSM and within the mucosa. Conclusions: This study suggests that luminal acid activates a reflex pathway involving mast cell degranulation, activation of capsaicin-sensitive afferent neurons and the release of substance P or a related neurokinin, which evokes sustained contraction of the oesophageal LSM. This pathway may be a target for treatment of oesophageal pain syndromes.

Proteinase-activated receptor-2 activation evokes oesophageal longitudinal smooth muscle contraction via a capsaicin-sensitive and neurokinin-2 receptor-dependent pathway

Background  Intraluminal acid evokes sustained oesophageal longitudinal smooth muscle (LSM) contraction and oesophageal shortening, which may play a role in oesophageal pain and the aetiology of hiatus hernia. In the opossum model, this reflex has been shown to involve mast cell activation and release of neurokinins from capsaicin‐sensitive neurons. The aim of this study was to determine whether proteinase‐activated receptor‐2 (PAR‐2) activation evokes reflex LSM contraction via similar mechanisms.

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

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).

Impaired platelet-derived growth factor receptor expression and function in cultured lower esophageal sphincter circular smooth muscle cells from W/Wv mutant mice

We have previously demonstrated that lower esophageal sphincter (LES) circular smooth muscle (CSM) is functionally impaired in W/W(v) mutant mice that lack interstitial cells of Cajal, and speculated that this could be due to altered smooth muscle differentiation. Platelet-derived growth factor (PDGF) is involved in the maturation and differentiation of smooth muscle. To determine whether PDGF expression and (or) function is altered in W/W(v) mutant mice, PDGF-Rβ expression was measured using RT-PCR, qPCR, and immunocytochemistry, and Ca(2+) imaging and perforated patch clamp recordings performed in isolated LES CSM cells. RT-PCR and immunocytochemistry showed significantly reduced PDGF-Rβ expression in the LES from mutant as opposed to wild-type mice. Quantitative comparison of CSM cell numbers in histological specimens revealed a significantly increased average cell size in the mutant tissue. The specific PDGF-Rβ ligand, PDGF-BB, caused a significant increase in intracellular Ca(2+) in cells from the wild-type mice compared with the mutants. Using a ramp protocol, PDGF-BB caused a 2-fold increase in outward K(+) currents in cells from the wild-type mice, whereas no significant increase was measured in the cells from the mutants. We conclude that the expression and function of PDGF-Rβ in LES CSM from W/W(v) mice is impaired, providing further evidence that LES CSM is abnormal in W/W(v) mutants.

Diversity of K+ channels in circular smooth muscle of opossum lower esophageal sphincter

We previously demonstrated that a balance of K+ and Ca2+-activated Cl- channel activity maintained the basal tone of circular smooth muscle of opossum lower esophageal sphincter (LES). In the current studies, the contribution of major K+ channels to the LES basal tone was investigated in circular smooth muscle of opossum LES in vitro. K+ channel activity was recorded in dispersed single cells at room temperature using patch-clamp recordings. Whole-cell patch-clamp recordings displayed an outward current beginning to activate at -60 mV by step test pulses lasting 400 ms (-120 mV to +100 mV) with increments of 20 mV from holding potential of -80 mV ([K+]I = 150 mM, [K+]o = 2.5 mM). However, no inward rectification was observed. The outward current peaked within 50 ms and showed little or no inactivation. It was significantly decreased by bath application of nifedipine, tetraethylammonium (TEA), 4-aminopyridine (4-AP), and iberiotoxin (IBTN). Further combination of TEA with 4-AP, nifedipine with 4-AP, and IBTN with TEA, or vice versa, blocked more than 90% of the outward current. Ca2+-sensitive single channels were recorded at asymetrical K+ gradients in cell-attached patch-clamp configurations (100.8+/-3.2 pS, n = 8). Open probability of the single channels recorded in inside-out patch-clamp configurations were greatly decreased by bath application of IBTN (100 nM) (Vh = -14.4+/-4.8 mV in control vs. 27.3+/-0.1 mV, n = 3, P < 0.05). These data suggest that large conductance Ca2+-activated K+ and delayed rectifier K+ channels contribute to the membrane potential, and thereby regulate the basal tone of opossum LES circular smooth muscle.