Can Eosinophilic Esophagitis Cause Achalasia and Other Esophageal Motility Disorders?

Eosinophilic esophagitis (EoE), a disorder identified by its esophageal mucosal features, often is associated with esophageal motility abnormalities, which are manifestations of esophageal muscle dysfunction. Those motility abnormalities sometimes normalize with treatments that reduce esophageal eosinophilia, suggesting that eosinophils can cause reversible esophageal motility disturbances, perhaps by releasing myoactive and neuroactive eosinophil products. Although achalasia uncommonly is associated with EoE as currently defined, most achalasia patients have evidence of an abnormal accumulation of eosinophils and/or their degranulation products in the esophageal muscularis propria, a location inaccessible to routine endoscopic evaluation. Achalasia is an idiopathic condition resulting from destruction of neurons in the myenteric plexus of the esophagus, and degranulating eosinophils release toxic proteins capable of destroying those neurons, thereby causing the irreversible motility abnormalities of achalasia. This report reviews data on the association of esophageal eosinophilia with achalasia and other esophageal motility abnormalities. Based on this review, we propose that EoE, like eosinophilic gastroenteritis, might have mucosal-predominant and muscle-predominant forms with different clinical manifestations. A muscle-predominant form of EoE could underlie a variety of reversible and irreversible esophageal motility disorders, including achalasia. The concept that esophageal motility abnormalities might develop from a muscle-predominant form of EoE warrants serious consideration and further investigation.

Trypsin-induced biphasic regulation of tone in the porcine lower esophageal sphincter.

The lower esophageal sphincter (LES) plays an important role in coordinated esophageal motility. The present study aimed to elucidate how trypsin affects LES contractility. Porcine LES circular smooth muscle strips were prepared. Contractile responses to trypsin were assessed. Trypsin (300nM) induced a transient contraction. At concentrations of 1µM or higher, trypsin induced biphasic responses, consisting of a transient contraction followed by a transient relaxation. Pretreatment with either 1µM tetrodotoxin or carbenoxolone had no effect on these responses. In contrast, trypsin-induced responses were completely blocked by pretreatment with the serine protease inhibitor. Pretreatment with 10µM FSLLRY-NH2, a PAR2 antagonist, significantly inhibited trypsin-induced biphasic responses. Trypsin (1µM)-induced contractions were partially inhibited by pretreatment with 10µM Y-27632. In addition, trypsin (10µM)-induced relaxation was partially inhibited by pretreatment with 10µM Y-27632, 10µM PD98059 or 10µM SB203580. Trypsin-induced relaxation was abolished by increasing the extracellular K(+) concentration to 40mM, but not by pretreatment with l-arginine methyl ester. Furthermore, trypsin-induced relaxation was partially inhibited by pretreatment with 10µM glibenclamide or 1µM 4-aminopyridine. Trypsin causes biphasic regulation of LES tone by directly acting on smooth muscle. Rho-associated protein kinase (ROK) is involved in trypsin-induced contraction, whereas ROK, ERK1/2, p38MAPK, and membrane hyperpolarization are involved in relaxation. The regulation of LES tone by trypsin may play a role in esophageal motility.

Dominant role of interstitial cells of Cajal in nitrergic relaxation of murine lower oesophageal sphincter.

Oesophageal achalasia is a disease known to result from reduced relaxation of the lower oesophageal sphincter (LES). Nitric oxide (NO) is one of the main inhibitory transmitters. NO-sensitive guanylyl cyclase (NO-GC) acts as the key target of NO and, by the generation of cGMP, mediates nitrergic relaxation in the LES. To date, the exact mechanism of nitrergic LES relaxation is still insufficiently elucidated. To clarify the role of NO-GC in LES relaxation, we used cell-specific knockout (KO) mouse lines for NO-GC. These include mice lacking NO-GC in smooth muscle cells (SMC-GCKO), in interstitial cells of Cajal (ICC-GCKO) and in both SMC/ICC (SMC/ICC-GCKO). We applied oesophageal manometry to study the functionality of LES in vivo. Isometric force studies were performed to monitor LES responsiveness to exogenous NO and electric field stimulation of intrinsic nerves in vitro. Cell-specific expression/deletion of NO-GC was monitored by immunohistochemistry. Swallowing-induced LES relaxation is strongly reduced by deletion of NO-GC in ICC. Basal LES tone is affected by NO-GC deletion in either SMC or ICC. Lack of NO-GC in both cells leads to a complete interruption of NO-induced relaxation and, therefore, to an achalasia-like phenotype similar to that seen in global GCKO mice. Our data indicate that regulation of basal LES tone is based on a dual mechanism mediated by NO-GC in SMC and ICC whereas swallow-induced LES relaxation is mainly regulated by nitrergic mechanisms in ICC.

Impaired platelet-derived growth factor receptor expression and function in cultured lower esophageal sphincter circular smooth muscle cells from W/W(v) 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-Rß 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-Rß 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-Rß 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-Rß in LES CSM from W/W(v) mice is impaired, providing further evidence that LES CSM is abnormal in W/W(v) mutants.

Evidence for altered circular smooth muscle cell function in lower esophageal sphincter of W/Wv mutant mice.

Nitrergic neurotransmission to gut smooth muscle is impaired in W/W(v) mutant mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). In addition, these mice have been reported to have smaller amplitude unitary potentials (UPs) and a more negative resting membrane potential (RMP) than control mice. These abnormalities have been attributed to absence of ICC-IM, but it remains possible that they are due to alterations at the level of the smooth muscle itself. Amphotericin-B-perforated patch-clamp recordings and Ca(2+) imaging (fura 2) were compared between freshly isolated single circular smooth muscle cells (CSM) from W/W(v) mutant and control mice lower esophageal sphincter (LES). There was no significant difference in seal resistance, capacitance, or input resistance in response to applied electrotonic current pulses between CSM cells from W/W(v) mutants and controls. Compared with control mice, RMP was more negative and UPs significantly smaller in CSM cells from mutant mice LES. Administration of caffeine induced an inward current in cells from both mutant and control mice, but the current density was significantly larger in cells from W/W(v) mutants. Membrane potential hyperpolarization induced by sodium nitroprusside was larger in cells from control mice vs. W/W(v) mutants. In addition, intracellular Ca(2+) transients induced by caffeine were significantly increased in cells from mutants. These findings indicate that LES CSM is abnormal in W/W(v) mutant mice. Thus some physiological functions attributed to ICC-IM based on experiments in smooth muscle of ICC deficient mice may need to be reconsidered.

Responses of human sling and clasp fibers to cholecystokinin (CCK) and gastrin through CCK receptors.

BACKGROUND AND AIM: Cholecystokinin (CCK) and gastrin exert their influences via CCK receptors. This research was conducted to look at the responses of the sling and clasp fibers forming the human lower esophageal sphincter (LES) to CCK and gastrin, and the role of CCK receptors in the responses. METHODS: Muscle strips of sling and clasp fibers from the LES were obtained from patients undergoing subtotal esophagectomy. Isometric tension responses of the strips to CCK-8 and gastrin-17 were studied, and the maximum effect (E(max)) for each agonist was derived. CCK-A receptor antagonist, CR1409 and CCK-B antagonist, CR2945 were applied to sling and clasp fibers and their pK(B) values were calculated. RESULTS: Sling fibers produced significant contractions following exposure to CCK-8 and gastrin-17, while clasp fibers had less responses to the two agents. CR1409 and CR2945 inhibited responses of sling to CCK-8 in a concentration-dependent fashion. The inhibition effects of the two antagonists on clasp fibers were not measurable because there was a mild contraction of the fiber in response to CCK-8. CONCLUSION: The contractions generated by sling fibers following exposure to CCK and gastrin are greater than that produced by clasp fibers. CCK-A receptors are more important for the generation of contractions by the sling fibers, whereas both CCK-A and CCK-B receptors are involved in the functional regulation of the clasp fibers. [Corrections added after online publication 28 April 2008: in the Background and Aims section of the preceding abstract, all instances of 'CKK' were corrected to 'CCK'. In the final sentence of the abstract 'CCKA'was corrected to 'CCK-A'. In the article title '(CKK)' was corrected to '(CCK)'.].

Proteins of interstitial cells of Cajal and intestinal smooth muscle, colocalized with caveolin-1.

The murine jejunum and lower esophageal sphincter (LES) were examined to determine the locations of various signaling molecules and their colocalization with caveolin-1 and one another. Caveolin-1 was present in punctate sites of the plasma membranes (PM) of all smooth muscles and diffusely in all classes of interstitial cells of Cajal (ICC; identified by c-kit immunoreactivity), ICC-myenteric plexus (MP), ICC-deep muscular plexus (DMP), ICC-serosa (ICC-S), and ICC-intramuscularis (IM). In general, all ICC also contained the L-type Ca(2+) (L-Ca(2+)) channel, the PM Ca(2+) pump, and the Na(+)/Ca(2+) exchanger-1 localized with caveolin-1. ICC in various sites also contained Ca(2+)-sequestering molecules such as calreticulin and calsequestrin. Calreticulin was present also in smooth muscle, frequently in the cytosol, whereas calsequestrin was present in skeletal muscle of the esophagus. Gap junction proteins connexin-43 and -40 were present in circular muscle of jejunum but not in longitudinal muscle or in LES. In some cases, these proteins were associated with ICC-DMP. The large-conductance Ca(2+)-activated K(+) channel was present in smooth muscle and skeletal muscle of esophagus and some ICC but was not colocalized with caveolin-1. These findings suggest that all ICC have several Ca(2+)-handling and -sequestering molecules, although the functions of only the L-Ca(2+) channel are currently known. They also suggest that gap junction proteins are located at sites where ultrastructural gap junctions are know to exist in circular muscle of intestine but not in other smooth muscles. These findings also point to the need to evaluate the function of Ca(2+) sequestration in ICC.

Potassium channel diversity within the muscular components of the feline lower esophageal sphincter.

We hypothesized that regional differences in electrophysiological properties exist within the musculature of the feline lower esophageal sphincter (LES) and that they may potentially contribute to functional asymmetry within the LES. Freshly isolated esophageal smooth muscle cells (SMCs) from the circular muscle and sling regions within the LES were studied under a patch clamp. The resting membrane potential (RMP) of the circular SMCs was significantly more depolarized than was the RMP of the sling SMCs, resulting from a higher Na+ and Cl- permeability in circular muscle than in sling muscle. Large conductance Ca2+-activated K+ (BKCa) set the RMP at both levels, since specific BKCa inhibitors caused depolarization; however, BKCa density was greatest in the circular region. A significant portion of the outward current was due to non-BKCa, especially in sling muscle, and likely delayed rectifier K+ channels (KDR). There was a large reduction in outward current with 4-aminopyridine (4-AP) in sling muscle, while BKCa blockers had a limited effect on the voltage-activated outward current in sling muscle. Differences in BKCa:KDR channel ratios were also manifest by a leftward shift in the voltage-dependent activation curve in circular cells compared to sling cells. The electrophysiological differences seen between the circular and sling muscles provide a basis for their different contributions to LES activities such as resting tone and neurotransmitter responsiveness, and in turn could impart asymmetric drug responses and provide specific therapeutic targets.