Achalasia-Specific Quality of Life After Pneumatic Dilation or Laparoscopic Heller Myotomy With Partial Fundoplication: A Multicenter, Randomized Clinical Trial.

OBJECTIVES: Achalasia is a chronic, progressive, and incurable esophageal motility disease. There is clinical uncertainty about which treatment should be recommended as first-line therapy. Our objective was to evaluate the effectiveness of pneumatic dilation compared with laparoscopic Heller myotomy with partial fundoplication in improving achalasia-specific quality of life. METHODS: This was a prospective, multicenter, randomized trial at five academic hospitals in Canada. Fifty previously untreated adults with a clinical diagnosis of primary achalasia, confirmed by manometric testing, were enrolled between November 2005 and March 2010, and followed for 5 years after treatment. Randomization was stratified by site, in random blocks of size four and with balanced allocation. Patients were treated with pneumatic dilation or laparoscopic Heller myotomy with partial fundoplication. The primary outcome was the difference between the treatments in the mean improvement of the achalasia severity questionnaire (ASQ) score at 1 year from baseline. Prespecified secondary outcomes included general and gastrointestinal quality of life, symptoms, esophageal physiology measures (lower esophageal sphincter relaxation and pressure, esophageal emptying, abnormal esophageal acid exposure), complications, and incidence of retreatment. Functional and imaging studies were performed blinded and all outcome assessors were blinded. RESULTS: There were no significant differences between treatments in the improvement of ASQ score at 1 year from baseline (27.5 points in the Heller myotomy arm vs. 20.2 points in the pneumatic dilation arm; difference 7.3 points, 95% confidence interval -4.7 to 19.3; P=0.23). There were no differences between treatments in improvement of symptoms, general and gastrointestinal quality of life, or measures of esophageal physiology. Improvements in ASQ score diminished over time for both interventions. At 5 years, there were no differences between treatments in improvement of ASQ score, symptoms, and general or gastrointestinal quality of life. There were no serious adverse events. No patient who received Heller myotomy required retreatment, whereas five patients treated initially with pneumatic dilation required retreatment. CONCLUSIONS: Treatment with pneumatic dilation or laparoscopic Heller myotomy similarly improves achalasia-specific disease severity at 1 year. Either of the therapeutic approaches can be used as first-line therapy for previously untreated adults with achalasia.

Interstitial cells of Cajal: pacemaker cells of the pancreatic duct?

OBJECTIVES: Ramon y Cajal discovered interstitial cells in the pancreas associated with intrinsic nerves. It was our aim to provide evidence for or against the hypothesis that the pancreatic duct harbors interstitial cells of Cajal (ICCs) that may function as pacemakers for duct motility. METHODS: We used immunohistochemistry using c-Kit as the ICC marker and protein gene product 9.5 for nerves. Electron microscopy further characterized the cells and their interrelationships. RESULTS: c-Kit-positive cells were associated with smooth muscle cells and nerve fibers of the duct wall and were rich in mitochondria, rough endoplasmic reticulum, and intermediate filaments; they possessed occasional caveolae and had a discontinuous basal lamina. They were connected by small gap junctions to each other and to smooth muscle cells. c-Kit-positive cells around large blood vessels were similar. c-Kit-positive cells within acini were similar in structure but were not associated with smooth muscle cells. CONCLUSIONS: The c-Kit-positive cells around the main duct were identified as ICCs and have the morphological criteria to likely function as pacemaker cells for the previously observed spontaneous rhythmic pancreatic duct contractions. Interstitial cells of Cajal around the large blood vessels likely affect vessel wall rhythmicity.

Nitric oxide activation of a potassium channel (BK(Ca)) in feline lower esophageal sphincter.

AIM: To assess the effect of nitric oxide (NO) on the large conductance potassium channel (BK(Ca)) in isolated circular (CM) and sling (SM) muscle cells and muscle strips from the cat lower esophageal sphincter (LES) to determine its regulation of resting tone and relaxation. METHODS: Freshly enzymatically-digested and isolated circular smooth muscle cells were prepared from each LES region. To study outward K+ currents, the perforated patch clamp technique was employed. To assess LES resting tone and relaxation, muscle strips were mounted in perfused organ baths. RESULTS: (1) Electrophysiological recordings from isolated cells: (a) CM was more depolarized than SM (-39.7 ± 0.8 mV vs -48.1 ± 1.6 mV, P < 0.001), and maximal outward current was similar (27.1 ± 1.5 pA/pF vs 25.7 ± 2.0 pA/pF, P > 0.05); (b) The NO donor sodium nitroprusside (SNP) increased outward currents only in CM (25.9 ± 1.9 to 46.7 ± 4.2 pA/pF, P < 0.001) but not SM (23.2 ± 3.1 to 27.0 ± 3.4 pA/pF, P > 0.05); (c) SNP added in the presence of the BK(Ca) antagonist iberiotoxin (IbTX) produced no increase in the outward current in CM (17.0 ± 2.8 vs 13.7 ± 2.2, P > 0.05); and (d) L-NNA caused a small insignificant inhibition of outward K+ currents in both muscles; and (2) Muscle strip studies: (a) Blockade of the nerves with tetrodotoxin (TTX), or BK(Ca) with IbTX had no significant effect on resting tone of either muscle; and (b) SNP reduced tone in both muscles, and was unaffected by the presence of TTX or IbTX. CONCLUSION: Exogenous NO activates BK(Ca) only in CM of the cat. However, as opposed to other species, exogenous NO-induced relaxation is predominantly by a non-BK(Ca) mechanism, and endogenous NO has minimal effect on resting tone.

Distinct modulation of Kv1.2 channel gating by wild type, but not open form, of syntaxin-1A.

SNARE proteins, syntaxin-1A (Syn-1A) and SNAP-25, inhibit delayed rectifier K(+) channels, K(v)1.1 and K(v)2.1, in secretory cells. We showed previously that the mutant open conformation of Syn-1A (Syn-1A L165A/E166A) inhibits K(v)2.1 channels more optimally than wild-type Syn-1A. In this report we examined whether Syn-1A in its wild-type and open conformations would exhibit similar differential actions on the gating of K(v)1.2, a major delayed rectifier K(+) channel in nonsecretory smooth muscle cells and some neuronal tissues. In coexpression and acute dialysis studies, wild-type Syn-1A inhibited K(v)1.2 current magnitude. Of interest, wild-type Syn-1A caused a right shift in the activation curves of K(v)1.2 without affecting its steady-state availability, an inhibition profile opposite to its effects on K(v)2.1 (steady-state availability reduction without changes in voltage dependence of activation). Also, although both wild-type and open-form Syn-1A bound equally well to K(v)1.2 in an expression system, open-form Syn-1A failed to reduce K(v)1.2 current magnitude or affect its gating. This is in contrast to the reported more potent effect of open-form Syn-1A on K(v)2.1 channels in secretory cells. This finding together with the absence of Munc18 and/or 13-1 in smooth muscles suggested that a change to an open conformation Syn-1A, normally facilitated by Munc18/13-1, is not required in nonsecretory smooth muscle cells. Taken together with previous reports, our results demonstrate the multiplicity of gating inhibition of different K(v) channels by Syn-1A and is compatible with versatility of Syn-1A modulation of repolarization in various secretory and nonsecretory (smooth muscle) cell types.

Insulin regulates islet alpha-cell function by reducing KATP channel sensitivity to adenosine 5'-triphosphate inhibition.

Glucose regulates pancreatic islet alpha-cell glucagon secretion directly by its metabolism to generate ATP in alpha-cells, and indirectly via stimulation of paracrine release of beta-cell secretory products, particularly insulin. How the cellular substrates of these pathways converge in the alpha-cell is not well known. We recently reported the use of the MIP-GFP (mouse insulin promoter-green fluorescent protein) mouse to reliably identify islet alpha- (non-green cells) and beta-cells (green cells), and characterized their ATP-sensitive K(+) (K(ATP)) channel properties, showing that alpha-cell K(ATP) channels exhibited a 5-fold higher sensitivity to ATP inhibition than beta-cell K(ATP) channels. Here, we show that insulin exerted paracrine regulation of alpha-cells by markedly reducing the sensitivity of alpha-cell K(ATP) channels to ATP (IC(50) = 0.18 and 0.50 mM in absence and presence of insulin, respectively). Insulin also desensitized beta-cell K(ATP) channels to ATP inhibition (IC(50) = 0.84 and 1.23 mM in absence and presence of insulin, respectively). Insulin effects on both islet cell K(ATP) channels were blocked by wortmannin, indicating that insulin acted on the insulin receptor-phosphatidylinositol 3-kinase signaling pathway. Insulin did not affect alpha-cell A-type K(+) currents. Glutamate, known to also inhibit alpha-cell glucagon secretion, did not activate alpha-cell K(ATP) channel opening. We conclude that a major mechanism by which insulin exerts paracrine control on alpha-cells is by modulating its K(ATP) channel sensitivity to ATP block. This may be an underlying basis for the proposed sequential glucose-insulin regulation of alpha-cell glucagon secretion, which becomes distorted in diabetes, leading to dysregulated glucagon secretion.

Electrophysiological characterization of pancreatic islet cells in the mouse insulin promoter-green fluorescent protein mouse.

We recently reported a transgenic [mouse insulin promoter (MIP)-green fluorescent protein (GFP)] mouse in which GFP expression is targeted to the pancreatic islet beta-cells to enable convenient identification of beta-cells as green cells. The GFP-expressing beta-cells of the MIP-GFP mouse were functionally indistinguishable from beta-cells of normal mice. Here we characterized the ionic channel properties and exocytosis of MIP-GFP mouse islet beta- and alpha-cells. Beta-cells displayed delayed rectifying K+ and high-voltage-activated Ca2+ channels and exhibited Na+ currents only at hyperpolarized holding potential. Alpha-cells were nongreen and had both A-type and delayed rectifier K+ channels, both low-voltage-activated and high-voltage-activated Ca2+ channels, and displayed Na+ currents readily at -70 mV holding potential. Alpha-cells had ATP-sensitive K+ channel (KATP) channel density as high as that in beta-cells, and, surprisingly, alpha-cell KATP channels were more sensitive to ATP inhibition (IC50=0.16+/-0.03 mM) than beta-cell KATP channels (IC50=0.86+/-0.10 mM). Whereas alpha-cells were rather uniform in size [2-4.5 picofarad (pF)], beta-cells varied vastly in size (2-12 pF). Of note, small beta-cells (<4.5 pF) showed little exocytosis, whereas medium beta-cells (5-8 pF) exhibited vigorous exocytosis, but large beta-cells (>8 pF) had weaker exocytosis. We found no correlation between beta-cell size and their Ca2+ channel density, suggesting that Ca2+ influx may not be the cause of the heterogeneity in exocytotic responses. The MIP-GFP mouse therefore offers potential to further explore the functional heterogeneity in beta-cells of different sizes. The MIP-GFP mouse islet is therefore a reliable model to efficiently examine alpha-cell and beta-cell physiology and should greatly facilitate examination of their pathophysiology when the MIP-GFP mice are crossed with diabetic models.

Ion channel diversity in the feline smooth muscle esophagus.

We have characterized ion-channel identity and density differences along the feline smooth muscle esophagus using patch-clamp recording. Current clamp recording revealed that the resting membrane potential (RMP) of esophageal smooth muscle cells (SMC) from the circular layer at 4 cm above the lower esophageal sphincter (EBC4; LES) were more depolarized than at 2 cm above LES. Higher distal Na(+) permeability (but not Cl(-) permeability) contributes to this RMP difference. K(+) channels but not large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels contribute to RMP at both levels, because nonspecific K(+)-channel blockers depolarize all SMC. Depolarization of SMC under voltage clamp revealed that the density of voltage-dependent K(+) channels (K(V)) was greatest at EBC4 due to increased BK(Ca.) Delayed rectifier K(+) channels (K(DR)), compatible with subtype K(V)1.2, were present at both levels. Differences in K(Ca)-to-K(DR) channel ratios were also manifest by predictable shifts in voltage-dependent inactivation at EBC4 when BK(Ca) channels were blocked. We provide the first evidence for regional electrophysiological differences along the esophageal body resulting from SMC ion channel diversity, which could allow for differential muscular responses to innervation and varied muscular contribution to peristaltic contractions along the esophagus.