Axial stretch: A novel mechanism of the lower esophageal sphincter relaxation

Swallow and esophageal distension-induced relaxations of the lower esophageal sphincter (LES) are associated with an orad movement of the LES because of a concurrent esophageal longitudinal muscle contraction. We hypothesized that the esophageal longitudinal muscle contraction induces a cranially directed mechanical stretch on the LES and therefore studied the effects of a mechanical stretch on the LES pressure. In adult opossums, a silicon tube was placed via mouth into the esophagus and laparotomy was performed. Two needles with silk sutures were passed, 90 degrees apart, through the esophageal walls and silicon tube, 2 cm above the LES. The tube was withdrawn, and one end of each of the four sutures was anchored to the esophageal wall and the other end exited through the mouth to exert graded cranially directed stretch on the LES by using pulley and weights. A cranially directed stretch caused LES relaxation, and with the cessation of stretch there was recovery of the LES pressure. The degree an d duration of LES relaxation increased with the weight and the duration of stretch, respectively. The mean LES relaxation in all animals was 77.7 +/- 4.7%. The required weight to induce maximal LES relaxation differed in animals (714 +/- 348 g). N(G)-nitro-L-arginine, a nitric oxide inhibitor, blocked the axial stretch-induced LES relaxation almost completely (from 78 to 19%). Our data support the presence of an axial stretch-activated inhibitory mechanism in the LES. The role of axial stretch in the LES relaxation induced by swallow and esophageal distension requires further investigation.     

MAPK mediates PKC-dependent contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Esophageal (ESO) circular muscle contraction and lower esophageal sphincter (LES) tone are PKC dependent. Because MAPKs may be involved in PKC-dependent contraction, we examined ERK1/ERK2 and p38 MAPKs in ESO and LES. In permeabilized LES muscle cells, ERK1/2 antibodies reduced 1,2-dioctanoylglycerol (DG)- and threshold ACh-induced contraction, which are PKC dependent, but not maximal ACh, which is calmodulin dependent. LES tone was reduced by the ERK1/2 kinase inhibitor PD-98059 and by the p38 MAPK inhibitor SB-203580. In permeable ESO cells, ACh contraction was reduced by ERK1/ERK2 and p38 MAPK antibodies and by PD-98059 and SB-203580. ACh increased MAPK activity and phosphorylation of MAPK and of p38 MAPK. The 27-kDa heat shock protein (HSP27) antibodies reduced ACh contraction. HSP27 and p38 MAPK antibodies together caused no greater inhibition than either one alone. p38 MAPK and HSP27 coprecipitated after ACh stimulation, suggesting that HSP27 is linked to p38 MAPK. These data suggest that PKC-dependent contraction in ESO and LES is mediated by the following two distinct MAPK pathways: ERK1/2 and HSP27-linked p38 MAPK.     

PGF(2alpha)-induced contraction of cat esophageal and lower esophageal sphincter circular smooth muscle

Lower esophageal sphincter (LES) tone depends on PGF(2alpha) and thromboxane A(2) acting on receptors linked to G(i3) and G(q) to activate phospholipases and produce second messengers resulting in muscle contraction. We therefore examined PGF(2alpha) signal transduction in circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (Eso) and LES. In Eso, PGF(2alpha)-induced contraction was inhibited by antibodies against the alpha-subunit of G(13) and the monomeric G proteins RhoA and ADP-ribosylation factor (ARF)1 and by the C3 exoenzyme of Clostridium botulinum. A [(35)S]GTPgammaS-binding assay confirmed that G(13), RhoA, and ARF1 were activated by PGF(2alpha). Contraction of Eso was reduced by propranolol, a phospholipase D (PLD) pathway inhibitor and by chelerythrine, a PKC inhibitor. In LES, PGF(2alpha)-induced contraction was inhibited by antibodies against the alpha-subunit of G(q) and G(i3), and a [(35)S]GTPgammaS-binding assay confirmed that G(q) and G(i3) were activated by PGF(2alpha). PGF(2alpha)-induced contraction of LES was reduced by U-73122 and D609 and unaffected by propranolol. At low PGF(2alpha) concentration, contraction was blocked by chelerythrine, whereas at high concentration, contraction was blocked by chelerythrine and CGS9343B. Thus, in Eso, PGF(2alpha) activates a PLD- and protein kinase C (PKC)-dependent pathway through G(13), RhoA, and ARF1. In LES, PGF(2alpha) receptors are coupled to G(q) and G(i3), activating phosphatidylinositol- and phosphatidylcholine-specific phospholipase C. At low concentrations, PGF(2alpha) activates PKC. At high concentration, it activates both a PKC- and a calmodulin-dependent pathway.     

Variability in the muscle composition of rat esophagus and neural pathway of lower esophageal sphincter relaxation

Several studies from our laboratory show that axial stretch of the lower esophageal sphincter (LES) in an oral direction causes neurally mediated LES relaxation. Under physiological conditions, axial stretch of the LES is caused by longitudinal muscle contraction (LMC) of the esophagus. Because longitudinal muscle is composed of skeletal muscle in mice, vagal-induced LMC and LES relaxation are both blocked by pancuronium. We conducted studies in rats (thought to have skeletal muscle esophagus) to determine if vagus nerve-mediated LES relaxation is also blocked by pancuronium. LMC-mediated axial stretch on the LES was monitored using piezoelectric crystals. LES and esophageal pressures were monitored with a 2.5-Fr solid-state pressure transducer catheter. Following bilateral cervical vagotomy, the vagus nerve was stimulated electrically. LES, along with the esophagus, was harvested after in vivo experiments and immunostained for smooth muscle (smooth muscle α-actin) and skeletal muscle (fast myosin heavy chain). Vagus nerve-stimulated LES relaxation and esophageal LMC were reduced in a dose-dependent fashion and completely abolished by pancuronium (96 μg/kg) in six rats (group 1). On the other hand, in seven rats, LES relaxation and LMC were only blocked completely by a combination of pancuronium (group 2) and hexamethonium. Immunostaining revealed that the longitudinal muscle layer was composed of predominantly skeletal muscle in the group 1 rats. On the other hand, the longitudinal muscle layer of group 2 rats contained a significant amount of smooth muscle (P < 0.05). Our study shows tight coupling between axial stretch on the LES and relaxation of the LES, which suggests a cause and effect relationship between the two. We propose that the vagus nerve fibers that cause LMC induce LES relaxation through the stretch-sensitive activation of inhibitory motor neurons.     

Distinct kinases are involved in contraction of cat esophageal and lower esophageal sphincter smooth muscles

Contraction of smooth muscle depends on the balance of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) activities. Because MLCK activation depends on the activation of calmodulin, which requires a high Ca²⁺ concentration, phosphatase inhibition has been invoked to explain contraction at low cytosolic Ca²⁺ levels. The link between activation of the Ca²⁺-independent protein kinase C (PKC) and MLC phosphorylation observed in the esophagus (ESO) (Sohn UD, Cao W, Tang DC, Stull JT, Haeberle JR, Wang CLA, Harnett KM, Behar J, and Biancani P. Am J Physiol Gastrointest Liver Physiol 281: G467–G478, 2001), however, has not been elucidated. We used phosphatase and kinase inhibitors and antibodies to signaling enzymes in combination with intact and saponin-permeabilized isolated smooth muscle cells from ESO and lower esophageal sphincter (LES) to examine PKC-dependent, Ca²⁺-independent signaling in ESO. The phosphatase inhibitors okadaic acid and microcystin-LR, as well as an antibody to the catalytic subunit of type 1 protein serine/threonine phosphatase, elicited similar contractions in ESO and LES. MLCK inhibitors (ML-7, ML-9, and SM-1) and antibodies to MLCK inhibited contraction induced by phosphatase inhibition in LES but not in ESO. The PKC inhibitor chelerythrine and antibodies to PKC, but not antibodies to PKCII, inhibited contraction of ESO but not of LES. In ESO, okadaic acid triggered translocation of PKC from cytosolic to particulate fraction and increased activity of integrin-linked kinase (ILK). Antibodies to the mitogen-activated protein (MAP) kinases ERK1/ERK2 and to ILK, and the MAP kinase kinase (MEK) inhibitor PD-98059, inhibited okadaic acid-induced ILK activity and contraction of ESO. We conclude that phosphatase inhibition potentiates the effects of MLCK in LES but not in ESO. Contraction of ESO is mediated by activation of PKC, MEK, ERK1/2, and ILK. protein kinase C; myosin light chain kinase; phosphatase; integrin-linked kinase     

Crural diaphragm inhibition during esophageal distension correlates with contraction of the esophageal longitudinal muscle in cats

Esophageal distension causes simultaneous relaxation of the lower esophageal sphincter (LES) and crural diaphragm. The mechanism of crural diaphragm relaxation during esophageal distension is not well understood. We studied the motion of crural and costal diaphragm along with the motion of the distal esophagus during esophageal distension-induced relaxation of the LES and crural diaphragm. Wire electrodes were surgically implanted into the crural and costal diaphragm in five cats. In two additional cats, radiopaque markers were also sutured into the outer wall of the distal esophagus to monitor esophageal shortening. Under light anesthesia, animals were placed on an X-ray fluoroscope to monitor the motion of the diaphragm and the distal esophagus by tracking the radiopaque markers. Crural and costal diaphragm electromyograms (EMGs) were recorded along with the esophageal, LES, and gastric pressures. A 2-cm balloon placed 5 cm above the LES was used for esophageal distension. Effects of baclofen, a GABA(B) agonist, were also studied. Esophageal distension induced LES relaxation and selective inhibition of the crural diaphragm EMG. The crural diaphragm moved in a craniocaudal direction with expiration and inspiration, respectively. Esophageal distension-induced inhibition of the crural EMG was associated with sustained cranial motion of the crural diaphragm and esophagus. Baclofen blocked distension-induced LES relaxation and crural diaphragm EMG inhibition along with the cranial motion of the crural diaphragm and the distal esophagus. There is a close temporal correlation between esophageal distension-mediated LES relaxation and crural diaphragm inhibition with the sustained cranial motion of the crural diaphragm. Stretch caused by the longitudinal muscle contraction of the esophagus during distension of the esophagus may be important in causing LES relaxation and crural diaphragm inhibition.     

The mechanism of action of bombesin on cat lower esophageal sphincter

The effect of bombesin on the tone and the responses of strips from the lower esophageal sphincter (LES) to field electrical stimulation (FES) (2 Hz, 0.2 ms, supramaximal current intensity, 20 s duration) was studied. Bombesin dose-dependently increased the LES tone. The threshold for this effect was 10(-14) M and was particularly pronounced with a concentration of 10(-8) M. The response reached maximum between the 3rd and the 5th min after application, persisted for 15-20 min, and was followed by a slight time-dependent decrease. Bombesin increased FES-produced relaxation of LES by 39% as compared to the control. The potentiating effect of bombesin on the LES relaxation was also observed after cholinergic and adrenergic receptor blockade. It is concluded that bombesin may modulate the release of cholinergic, adrenergic and noncholinergic, nonadrenergic inhibitory neurotransmitters.     

Distension of the esophagogastric junction augments triggering of transient lower esophageal sphincter relaxation

Patients with gastroesophageal reflux disease show an increase in esophagogastric junction (EGJ) distensibility and in frequency of transient lower esophageal sphincter relaxations (TLESR) induced by gastric distension. The objective was to study the effect of localized EGJ distension on triggering of TLESR in healthy volunteers. An esophageal manometric catheter incorporating an 8-cm internal balloon adjacent to a sleeve sensor was developed to enable continuous recording of EGJ pressure during distension of the EGJ. Inflation of the balloon doubled the cross-section of the trans-sphincteric portion of the catheter from 5 mm OD (round) to 5 × 11 mm (oval). Ten healthy subjects were included. After catheter placement and a 30-min adaptation period, the EGJ was randomly distended or not, followed by a 45-min baseline recording. Subjects consumed a refluxogenic meal, and recordings were made for 3 h postprandially. A repeat study was performed on another day with EGJ distension status reversed. Additionally, in one subject MRI was performed to establish the exact position of the balloon in the inflated state. The number of TLESR increased during periods of EGJ distension with the effect being greater after a meal [baseline: 2.0(0.0-4.0) vs. 4.0(1.0-11.0), P=0.04; postprandial: 15.5(10.0-33.0) vs. 22.0(17.0-58.0), P=0.007 for undistended and distended, respectively]. EGJ distension augments meal-induced triggering of TLESR in healthy volunteers. Our data suggest the existence of a population of vagal afferents located at sites in/around the EGJ that may influence triggering of TLESR.     

Demonstration of mucous mechanoreceptors in the lower esophageal sphincter. Comparison with muscle mechanoreceptors

In anaesthetized cats, vagal unitary discharges originating from the Lower Oesophageal Sphincter (L.O.S.) were recorded in nodose ganglia by means of glass microelectrodes. Numerous mechanoreceptors located both in mucosa and muscular layers were found in L.O.S. The mucus mechanoreceptors (high threshold receptors) were activated by strong compressions and distensions, by rapid passage of liquid through the oesophagus and by striking the mucosa. The muscular mechanoreceptors (low threshold receptors) responded to contraction and distension of L.O.S. Both receptors were connected to nonmyelinated fibres (conduction velocity: 0.9-1.4 m/sec).     

Role of cADPR in sodium nitroprusside-induced opossum esophageal longitudinal smooth muscle contraction

Nitric oxide (NO) relaxes most smooth muscle, including the circular smooth muscle (CSM) of the esophagus, whereas in the adjacent longitudinal smooth muscle (LSM), it causes contraction. The second messenger pathways responsible for this NO-induced LSM contraction are unclear, given that these opposing effects of NO are both cGMP dependent. In intestinal LSM, but not CSM, cADP ribose (cADPR)-dependent pathways participate in Ca(2+) mobilization and muscle contraction; whether similar differences exist in the esophagus is unknown. The purpose of this study was to determine whether cADPR plays a role in the NO-mediated contraction of opossum esophageal LSM. Standard isometric tension recordings were performed using both LSM and CSM strips from opossum distal esophagus that were hung in 10-ml tissue baths perfused with oxygenated Krebs solution. cADPR produced concentration-dependent contraction of LSM strips with an EC(50) of 1 nM and peak contraction of 57 +/- 18% of the 60 mM KCl-induced contraction. cADPR had no effect on CSM strips at concentrations up to 10(-6) M. The EC(50) of cADPR caused contraction (18 +/- 2% from initial resting length) of isolated LSM cells. Sodium nitroprusside (SNP; 300 muM) induced contraction of LSM strips that averaged 67 +/- 5% of the KCl response. cADPR antagonists 8-bromo-cADPR and 8-amino-cADPR, as well as ryanodine receptor antagonists ryanodine and tetracaine, significantly inhibited the SNP-induced contraction. In conclusion, in the opossum esophagus, 1) cADPR induces contraction of LSM, but not CSM, and 2) NO-induced contraction of LSM appears to involve a cADPR-dependent pathway.     

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.     

Dendroaspis natriuretic peptide is the most potent natriuretic peptide to cause relaxation of lower esophageal sphincter

Atrial natriuretic peptide (ANP) causes relaxation in the opossum lower esophageal sphincter. The effects of dendroaspis natriuretic peptide (DNP) and other natriuretic peptides in the lower esophageal sphincter were not known. We measured the relaxation of transverse strips from the guinea pig lower esophageal sphincter caused by DNP, ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and a natriuretic peptide receptor-C agonist des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23) amide (cANF(4-23)) in vitro. In resting strips of the guinea pig lower esophageal sphincter DNP and BNP caused marked relaxations. Furthermore, in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips, DNP caused marked and BNP caused moderate, concentration-dependent relaxations. ANP as well as CNP caused mild relaxations. In contrast, cANF(4-23) did not cause relaxation. The relative potencies for natriuretic peptides to cause relaxation were DNP>BNP>ANP>=CNP in both sarafotoxin S6c and carbachol-contracted lower esophageal sphincter strips. The DNP and BNP-induced relaxations were not affected by tetrodotoxin or atropine, suggesting that the natriuretic peptide-induced response was not neutrally mediated. In conclusion, these results demonstrate that natriuretic peptides cause the relaxation of the guinea pig lower esophageal sphincter. DNP is the most potent natriuretic peptide to cause lower esophageal sphincter relaxation, which might be mediated by natriuretic peptide receptor-A or a novel DNP-selective natriuretic peptide receptor.     

Effect of lower esophageal sphincter tone and crural diaphragm contraction on distensibility of the gastroesophageal junction in humans

Previous studies of distensibility of the gastroesophageal junction (GEJ) in humans have not tried to distinguish between the effects of muscle action and passive elastic tissue properties of the GEJ. We studied 15 healthy subjects (ages 23-67 yr, 11 men/4 women) by using a catheter with a highly complaint bag positioned manometrically at the GEJ. The bag was distended with air at a rate of 20 ml/min while intrabag pressure was recorded. Distensions were performed during normal breathing, with breath held at maximum inspiration (MI) to activate the diaphragmatic crura, and with midesophageal balloon distension (BD) to relax the lower esophageal sphincter. In 10 subjects, distensions were performed after atropine injection (12 microg/kg iv). Pressure-volume curves and incremental distensibility values were calculated and compared among the different conditions. Both MI and BD significantly altered the slopes of the pressure-volume curves, whereas no effect was seen with atropine. Maximum distensibility was seen at the volume increment of 5-10 ml and was reduced with larger volumes. Distensibility measurements for the various test conditions tended to converge at the largest volume increment, suggesting that distensibility at this degree of distension was more related to the passive elastic properties of the GEJ. On the basis of these findings, we conclude that there can be significant active muscular contributions to recordings of distensibility at the GEJ, variations that must be controlled for during different study conditions.     

Deglutitive upper esophageal sphincter relaxation: a study of 75 volunteer subjects using solid-state high-resolution manometry

This study aimed to use a novel high-resolution manometry (HRM) system to establish normative values for deglutitive upper esophageal sphincter (UES) relaxation. Seventy-five asymptomatic controls were studied. A solid-state HRM assembly with 36 circumferential sensors spaced 1 cm apart was positioned to record from the hypopharynx to the stomach. Subjects performed ten 5-ml water swallows and one each of 1-, 10-, and 20-ml volume swallows. Pressure profiles across the UES were analyzed using customized computational algorithms that measured 1) the relaxation interval (RI), 2) the median intrabolus pressure (mIBP) during the RI, and 3) the deglutitive sphincter resistance (DSR) defined as mIBP/RI. The automated analysis succeeded in confirming bolus volume modulation of both the RI and the mIBP with the mean RI ranging from 0.32 to 0.50 s and mIBP ranging from 5.93 to 13.80 mmHg for 1- and 20-ml swallows, respectively. DSR was relatively independent of bolus volume. Peak pharyngeal contraction during the return to the resting state postswallow was almost 300 mmHg, again independent of bolus volume. We performed a detailed analysis of deglutitive UES relaxation with a novel HRM system and customized software. The enhanced spatial resolution of HRM allows for the accurate, automated assessment of UES relaxation and intrabolus pressure characteristics, in both cases confirming the volume-dependent effects and absolute values of these parameters previously demonstrated by detailed analysis of concurrent manometry/fluoroscopy data. Normative values were established to aid in future clinical and investigative studies.     

Ultrastructural analysis of spinal primary afferent fibers within the circular muscle of the cat lower esophageal sphincter

We have analyzed the ultrastructural characteristics and environment of spinal primary afferent fibers that run within the circular muscle of the cat lower esophageal sphincter. These were selectively labeled by anterogradely transported cholera toxin B subunit conjugated with horseradish peroxidase. Most of the labeled fibers were perpendicular to the muscle cells but some ran sinuously or parallel to the muscle cells. All the labeled fibers were unmyelinated and exhibited relatively rare varicosities. Most of the fibers were in large nerve fiber bundles surrounded by perineurium and probably project to the mucosa. Only some fibers that were in small nerve fiber bundles with no perineurium ran parallel to the musculature and established close relationships with smooth muscle cells. They might be a small subpopulation of the spinal tension receptors, most of the other spinal tension receptors being located in the myenteric plexus area, between the circular and longitudinal muscle. Accepted: 2 December 1999     

Calcitonin gene-related peptide: a sensory and motor neurotransmitter in the feline lower esophageal sphincter

The effect of calcitonin gene-related peptide (CGRP) on the feline lower esophageal sphincter (LES) was determined and correlated with its anatomic distribution as determined by immunohistochemistry. Intraluminal pressures of the esophagus and LES were recorded in anesthetized cats. In separate cats, gastroesophageal junctions were removed after locating the LES manometrically and stained for CGRP-like immunoreactivity (LI) and substance P-LI (SP-LI) by indirect immunohistochemistry. CGRP-LI in the LES was most prominent in large nerve fascicles between the circular and longitudinal muscle layers and only rarely seen in nerve fibers within the circular muscle. The myenteric plexus contained numerous CGRP-LI nerve fibers but cell bodies were not seen. Many CGRP-LI nerve fibers in the myenteric plexus and occasional varicose nerves in the circular muscle demonstrated colocalization with SP-LI. Colocalization of CGRP-LI with SP-LI was also seen in the perivascular nerves of the submucosal and intramural blood vessels and in varicose fibers in the lamina propria of the gastric fundic mucosa. In the esophagus, CGRP-LI nerves extended through the muscularis mucosa and penetrated the squamous epithelium to the lumen. CGRP, given intra-arterially caused a dose-dependent fall in basal LES pressure, with a threshold dose of 10(-8) g/kg (2.63 pmol/kg). At the maximal effective dose, 5 x 10(-6) g/kg (1.31 x 10(3) pmol/kg), CGRP produced 61.0 +/- 6.0% decrease in basal LES pressure. At this dose, mean systemic blood pressure fell by 40.9 +/- 7.8%. The LES relaxation induced by a submaximal dose of CGRP (10(-6) g/kg, 262.7 pmol/kg), 50.3 +/- 3.2% relaxation was partially inhibited by tetrodotoxin (26.9 +/- 10.8% relaxation, P less than 0.025). The inhibitory effect of CGRP was not affected by cervical vagotomy, hexamethonium, atropine, propranolol, or naloxone. The LES contractile response to the D90 of SP (5 x 10(-8) g/kg, 37.1 pmol/kg) was not altered by CGRP 10(-8) or 10(-6) g/kg and the CGRP relaxation effect was not altered by the threshold dose of substance P (5 X 10(-9) g/kg, 3.71 pmol/kg). CONCLUSIONS: (1) CGRP-LI is present at the feline LES and is primarily seen in large nerve fascicles which pass from the intermuscular plane and through the circular muscle layer to the submucosa and in mucosal nerves. (2) CGRP colocalizes with SP-LI in some varicose nerve fibers of the circular muscle of the esophagus, LES and fundus, in perivascular nerves of the submucosal and intramucosal blood vessels, and in nerves of the lamina propria of the gastric fundus. (3) The luminal penetration of CGRP-LI nerves in the squamous mucosa of the esophagus suggests a sensory func     

Functional and molecular analysis of L-type calcium channels in human esophagus and lower esophageal sphincter smooth muscle

Excitation of human esophageal smooth muscle involves the release of Ca(2+) from intracellular stores and influx. The lower esophageal sphincter (LES) shows the distinctive property of tonic contraction; however, the mechanisms by which this is maintained are incompletely understood. We examined Ca(2+) channels in human esophageal muscle and investigated their contribution to LES tone. Functional effects were examined with tension recordings, currents were recorded with patch-clamp electrophysiology, channel expression was explored by RT-PCR, and intracellular Ca(2+) concentration was monitored by fura-2 fluorescence. LES muscle strips developed tone that was abolished by the removal of extracellular Ca(2+) and reduced by the application of the L-type Ca(2+) channel blocker nifedipine (to 13 +/- 6% of control) but was unaffected by the inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA). Carbachol increased tension above basal tone, and this effect was attenuated by treatment with CPA and nifedipine. Voltage-dependent inward currents were studied using patch-clamp techniques and dissociated cells. Similar inward currents were observed in esophageal body (EB) and LES smooth muscle cells. The inward currents in both tissues were blocked by nifedipine, enhanced by Bay K8644, and transiently suppressed by acetylcholine. The molecular form of the Ca(2+) channel was explored using RT-PCR, and similar splice variant combinations of the pore-forming alpha(1C)-subunit were identified in EB and LES. This is the first characterization of Ca(2+) channels in human esophageal smooth muscle, and we establish that L-type Ca(2+) channels play a critical role in maintaining LES tone.     

Changes in mechanism of PACAP-induced relaxation in longitudinal muscle of the distal colon of Wistar rats with age

Mechanisms of relaxation of longitudinal muscle of the distal colon induced by exogenously added pituitary adenylate cyclase activating peptide (PACAP) were studied in 2- to 30-week-old Wistar rats. Exogenous PACAP induced very significant relaxation of the longitudinal muscle in 2-week-old rats, but this effect decreased significantly with age. The cyclic AMP-cyclic AMP-dependent protein kinase (PKA) pathway and the tyrosine kinase-small conductance Ca2+-activated K+ channel (SK channel) pathway were found to be involved in the mechanism of PACAP-induced relaxation. In 2-week-old rats, PACAP-induced relaxation was significantly inhibited by tetrodotoxin (TTX). Since relaxation was also significantly inhibited by NG-nitro-L-arginine (N5-nitro-amidino-L-2,5-diamino-pentanoic acid: L-NOARG), the neurogenic effect of PACAP seems to be mediated mainly through nitric oxide neurons. In 8-week-old rats, L-NOARG and TTX had little effect on PACAP-induced relaxation, suggesting that the relaxant effect in 8-week-old rats is a direct action on longitudinal smooth muscle cells. Changes in the mechanisms of PACAP-induced relaxation with age were examined in the distal colon in relation to changes in the neurogenic and the direct effects of PACAP. The neurogenic effect in the exogenous PACAP-induced relaxation of the longitudinal muscle of the Wistar rat distal colon is dominant in tissue isolated from 2-week-old and lost in tissue isolated from 8-week-old rats.     

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.