Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh

1994 ◽  
Vol 266 (5) ◽  
pp. G767-G774 ◽  
Author(s):  
P. Biancani ◽  
K. M. Harnett ◽  
U. D. Sohn ◽  
B. Y. Rhim ◽  
J. Behar ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Lower Esophageal Sphincter ◽  
Calcium Release ◽  
Circular Muscle ◽  
Dependent Manner ◽  
Differential Signal ◽  
Esophageal Sphincter ◽  
Resting Tone ◽  
Dependent Pathway

Lower esophageal sphincter (LES) basal tone and contraction in response to maximally effective doses (Emax) of acetylcholine (ACh) may be mediated by different intracellular transduction pathways. In the basal state resting tone, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation and levels of diacylglycerol (DAG) (C. Hillemeier, K. N. Bitar, and P. Biancani, unpublished data) are higher in LES circular muscle than in esophageal muscle, which does not maintain tone. In vitro resting tone and spontaneously elevated formation of Ins(1,4,5)P3 in LES circular muscle strips decrease in a dose-dependent manner in response to the phospholipase C antagonist 1-[6-([(17-beta)-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione (U-73122). Basal Ins(1,4,5)P3 formation, however, is submaximal, since it can be increased by cholinergic stimulation. These data suggest that LES tone is associated with partial activation of phospholipase C. We therefore tested submaximal doses of Ins(1,4,5)P3 and DAG in permeabilized LES muscle cells and found that they act synergistically; their interaction depends on calcium release and is mediated through a protein kinase C (PKC)-dependent pathway. In contrast, we have previously shown that contraction induced by Emax of ACh is mediated through calmodulin-dependent mechanisms (14). To investigate these differences, we tested high and low doses of ACh. Contraction induced by high doses of ACh was inhibited by calmodulin but not by PKC antagonists, as previously reported, but low ACh doses were preferentially inhibited by PKC antagonists. Similarly, low Ins(1,4,5)P3 concentrations activated a PKC-dependent pathway, whereas contraction induced by Emax of Ins(1,4,5)P3 was calmodulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanylate cyclase inhibitors: effect on tone, relaxation, and cGMP content of lower esophageal sphincter

1992 ◽  
Vol 263 (1) ◽  
pp. G97-G101 ◽  
Author(s):  
J. A. Murray ◽  
C. Du ◽  
A. Ledlow ◽  
P. L. Manternach ◽  
J. L. Conklin
Keyword(s):  
Membrane Potential ◽  
Lower Esophageal Sphincter ◽  
Guanylate Cyclase ◽  
Circular Muscle ◽  
Resting Membrane Potential ◽  
Base Line ◽  
Sphincter Muscle ◽  
Dependent Manner ◽  
Esophageal Sphincter ◽  
Resting Tone

Relaxation of the lower esophageal sphincter (LES) results from activation of its intrinsic innervation. This relaxation is associated temporally with an increase in the guanosine 3',5'-cyclic monophosphate (cGMP) content of the muscle. This study tests the hypothesis that variations in the production of cGMP mediate resting LES tone and nerve-induced relaxation. We examined the effects of guanylate cyclase inhibitors, such as cystamine and methylene blue (MB), on the resting tone, resting membrane potential, electrical field stimulation (EFS)-induced relaxation, and cGMP content of circular smooth muscle from the LES of the opossum. Strips of sphincter muscle were placed in a tissue bath and stretched to 125% resting length. Both cystamine and MB increased the resting tone of LES muscle in a concentration-dependent manner (EC50 = 1.1 +/- 0.2, n = 12, and 1.6 +/- 0.4 mM, n = 10, respectively). The increase in tone by cystamine was not blocked by tetrodotoxin, atropine, or propranolol. Cystamine (1 mM) did not alter the resting membrane potential of circular muscle cells of the LES. The removal of extracellular Ca2+ by the addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 4 mM) and nifedipine (1 microM) shortened the duration but not the amplitude of the response to cystamine. Pretreatment with caffeine (5 mM) in the presence of EGTA and nifedipine to deplete intracellular Ca2+ stores blocked the increase in tone by cystamine. Cystamine (1 mM) failed to inhibit LES relaxation induced by EFS. Carbachol, at a concentration that induced a similar increase in base-line tone, attenuated the nerve-mediated relaxation. Cystamine did not alter basal cGMP levels, but inhibited the rise in cGMP induced by EFS. The data indicate that cystamine increases LES tone but does not inhibit EFS-induced relaxation, even though it inhibits EFS-induced increases in cGMP content. The increase in tone is dependent on the presence of intracellular Ca2+ stores.

Feline lower esophageal sphincter sling and circular muscles have different functional inhibitory neuronal responses

2006 ◽  
Vol 290 (1) ◽  
pp. G23-G29 ◽  
Author(s):  
Marie-Claude L'Heureux ◽  
Ahmad Muinuddin ◽  
Herbert Y. Gaisano ◽  
Nicholas E. Diamant
Keyword(s):  
Lower Esophageal Sphincter ◽  
Circular Muscle ◽  
Electrical Field Stimulation ◽  
Cholinergic Receptor ◽  
Cholinergic Innervation ◽  
Inhibitory Influence ◽  
Cholinergic Stimulation ◽  
Esophageal Sphincter ◽  
Low Tone ◽  
Resting Tone

The lower esophageal sphincter (LES) has a circular muscle component exhibiting spontaneous tone that is relaxed by nitric oxide (NO) and a low-tone sling muscle that contracts vigorously to cholinergic stimulation but with little or no evidence of NO responsiveness. This study dissected the responses of the sling muscle to nitrergic innervation in relationship to its cholinergic innervation and circular muscle responses. Motor responses were induced by electrical field stimulation (EFS; 1–30 Hz) of muscle strips from sling and circular regions of the feline LES in the presence of cholinergic receptor inhibition (atropine) or NO synthase inhibition [ NG-nitro-l-arginine (l-NNA) ± atropine]. This study showed the following. First, sling muscle developed less intrinsic resting tone compared with circular muscle. Second, with EFS, sling muscle contracted (most at ≤10 Hz), whereas circular muscle relaxed >50% by 5 Hz. Third, on neural blockade with atropine or l-NNA ± atropine, 1) sling muscle, although predominantly influenced by excitatory cholinergic stimulation, had a small neural NO-mediated inhibition, with no significant non-NO-mediated inhibition and 2) circular muscle, although little affected by cholinergic influence, underwent relaxation predominantly by neural release of NO and some non-NO inhibitory influence (at higher EFS frequency). Fourth, the sling, precontracted with bethanecol, could relax with NO and some non-NO inhibition. Finally, the tension range of both muscles is similar. In conclusion, sling muscle has limited NO-mediated inhibition to potentially augment or replace sling relaxation effected by switching off its cholinergic excitation. Differences within the LES sling and circular muscles could provide new directions for therapy of LES disorders.

IL-1β signaling in cat lower esophageal sphincter circular muscle

2006 ◽  
Vol 291 (4) ◽  
pp. G672-G680 ◽  
Author(s):  
Weibiao Cao ◽  
Ling Cheng ◽  
Jose Behar ◽  
Piero Biancani ◽  
Karen M. Harnett
Keyword(s):  
Lower Esophageal Sphincter ◽  
P38 Mapk ◽  
Circular Muscle ◽  
Circular Smooth Muscle ◽  
Sequential Activation ◽  
Cox 2 ◽  
Esophageal Sphincter ◽  
Sb 203580

In a cat model of acute experimental esophagitis, resting in vivo lower esophageal sphincter (LES) pressure and in vitro tone are lower than in normal LES, and the LES circular smooth muscle layer contains elevated levels of IL-1β that decrease the LES tone of normal cats. We now examined the mechanisms of IL-1β-induced reduction in LES tone. IL-1β significantly reduced acetylcholine-induced Ca2+ release in Ca2+-free medium, and this effect was partially reversed by catalase, demonstrating a role of H2O2 in these changes. IL-1β significantly increased the production of H2O2, and the increase was blocked by the p38 MAPK inhibitor SB-203580, by the cytosolic phospholipase A2 (cPLA2) inhibitor AACOCF3, and by the NADPH oxidase inhibitor apocynin, but not by the MEK1 inhibitor PD-98059. IL-1β significantly increased the phosphorylation of p38 MAPK and cPLA2. IL-1β-induced cPLA2 phosphorylation was blocked by SB-203580 but not by AACOCF3, suggesting sequential activation of p38 MAPK-phosphorylating cPLA2. The IL-1β-induced reduction in LES tone was partially reversed by AACOCF3 and by the Ca2+-insensitive PLA2 inhibitor bromoenol lactone (BEL). IL-1β significantly increased cyclooxygenase (COX)-2 and PGE2 levels. The increase in PGE2 was blocked by SB-203580, AACOCF3, BEL, and the COX-2 inhibitor NS-398 but not by PD-98059 or the COX-1 inhibitor valeryl salicylate. The data suggested that IL-1β reduces LES tone by producing H2O2, which may affect Ca2+-release mechanisms and increase the synthesis of COX-2 and PGE2. Both H2O2 and PGE2 production depend on sequential activation of p38 MAPK and cPLA2. cPLA2 activates NADPH oxidases, producing H2O2, and may produce arachidonic acid, converted to PGE2 via COX-2.

Opposing roles of K+and Cl−channels in maintenance of opossum lower esophageal sphincter tone

2000 ◽  
Vol 279 (6) ◽  
pp. G1226-G1234 ◽  
Author(s):  
Yong Zhang ◽  
David V. Miller ◽  
William G. Paterson
Keyword(s):  
Lower Esophageal Sphincter ◽  
Circular Muscle ◽  
Isometric Tension ◽  
Niflumic Acid ◽  
Resting Membrane Potential ◽  
Channel Blockers ◽  
Microelectrode Recordings ◽  
Basal Tone ◽  
Esophageal Sphincter

The ionic basis underlying the maintenance of myogenic tone of lower esophageal sphincter circular muscle (LES) was investigated in opossum with the use of standard isometric tension and conventional intracellular microelectrode recordings in vitro. In tension recording studies, nifedipine (1 μM) reduced basal tone to 27.7 ± 3.8% of control. The K+channel blockers tetraethylammonium (TEA, 2 mM), charybdotoxin (100 nM), and 4-aminopyridine (4-AP, 2 mM) enhanced resting tone, whereas apamin and glibenclamide were without affect. Cl−channel blockers DIDS (500 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (500 μM), as well as niflumic acid (0.1–300 μM), decreased basal tone, but tamoxifen was without effect. Intracellular microelectrode recordings revealed ongoing, spontaneous, spike-like action potentials (APs). Nifedipine abolished APs and depolarized resting membrane potential (RMP). Both TEA and 4-AP significantly depolarized RMP and augmented APs, whereas niflumic acid dose-dependently hyperpolarized RMP and abolished APs. These data suggest that, in the opossum, basal tone is associated with continuous APs and that K+and Ca2+-activated Cl−channels have important opposing roles in the genesis of LES tone.

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

2004 ◽  
Vol 82 (11) ◽  
pp. 1006-1017 ◽  
Author(s):  
Anne Marie F Salapatek ◽  
Junzhi Ji ◽  
Ahmad Muinuddin ◽  
Nicholas E Diamant
Keyword(s):  
Lower Esophageal Sphincter ◽  
Circular Muscle ◽  
Outward Current ◽  
Resting Membrane Potential ◽  
Delayed Rectifier ◽  
Electrophysiological Properties ◽  
Voltage Dependent ◽  
Esophageal Smooth Muscle Cells ◽  
Esophageal Sphincter ◽  
Resting Tone

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 BKCainhibitors caused depolarization; however, BKCadensity 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 BKCablockers had a limited effect on the voltage-activated outward current in sling muscle. Differences in BKCa:KDRchannel 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.Key words: esophagus, esophageal motility, gastroesophageal reflux, KCa, KDR, LES tone.

Inhibition of lower esophageal sphincter circular muscle by female sex hormones.

1978 ◽  
Vol 234 (3) ◽  
pp. E243 ◽  
Author(s):  
R S Fisher ◽  
G S Roberts ◽  
C J Grabowski ◽  
S Cohen
Keyword(s):  
Lower Esophageal Sphincter ◽  
Sex Hormones ◽  
Circular Muscle ◽  
Response Curves ◽  
Circular Smooth Muscle ◽  
Female Sex ◽  
Female Sex Hormones ◽  
Esophageal Sphincter ◽  
Muscle Responses

To determine the effect of estrogenic and progesteronic activity on lower esophageal sphincter (LES) circular muscle, studies were performed on 20 adult opossums. Does-response curves on circular smooth muscle strips from the LES were constructed for gastrin and acetylcholine alone, and with 17beta-estradiol and/or progesterone added. Each female hormone significantly decreased the maximal LES muscle responses to gastrin and acetylcholine. A combination of 17beta-estradiol and progesterone abolished the response to gastrin. In contrast, the male sex hormone, dihydrotestosterone, had no effect. In conclusion, administration of estrogen and progesteron, but not dihydrotestosterone, in vitro reduced LES muscle responses to gastrin and acetylcholine. These studies suggest that the female sex hormones can alter LES function and potentially may be of importance in the pathogenesis of heartburn of pregnancy.

Role of chloride ions in lower esophageal sphincter tone and relaxation

1992 ◽  
Vol 263 (1) ◽  
pp. G115-G126 ◽  
Author(s):  
J. K. Saha ◽  
J. N. Sengupta ◽  
R. K. Goyal
Keyword(s):  
Lower Esophageal Sphincter ◽  
Channel Blocker ◽  
Chloride Ions ◽  
Disulfonic Acid ◽  
Gamma Aminobutyric Acid ◽  
Inhibitory Neurotransmitter ◽  
Cl Channel ◽  
Esophageal Sphincter ◽  
Resting Tone

Studies were performed in strips of opossum lower esophageal sphincter (LES) muscle in vitro. External Cl(-)-free Krebs solution (0[Cl-]o) inhibited resting tone. Treatment with the Cl- channel blocker diphenylamine-2-carboxylate (DPC, 0.3-100 microM) caused a concentration-dependent relaxation of LES muscle, as did treatment with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1 microM-3 mM), a Cl(-)-HCO3- exchange blocker, and bumetanide (0.3-100 microM), a blocker of the Na(+)-K(+)-2Cl- cotransport. DIDS and bumetanide are also known to cause Cl- channel block. The calculated pD2 and Emax values for DPC, DIDS, and bumetanide were 5.24 +/- 0.28 (n = 5), 3.38 +/- 0.16 (n = 5), and 4.49 +/- 0.23 M (n = 5), and 78.80 +/- 5.38, 74.80 +/- 6.54, and 83.70 +/- 10.20%, respectively. The neuronal Cl- channel activators gamma-aminobutyric acid and glycine had no effect on the resting tone. DPC, DIDS, and bumetanide appear to have acted directly on smooth muscle rather than indirectly through the release of inhibitory neurotransmitters because LES relaxation by these agents was not influenced by tetrodotoxin (10 microM), which blocks action potentials in nerves, or by omega-conotoxin (1 microM), which inhibits the release of neurotransmitters from nerve terminals. LES muscle relaxed by exposure to 0[Cl-]o, DPC, DIDS, and bumetanide contracted with the addition of carbachol (30 microM); muscle so treated was resistant to the inhibitory neurotransmitter-mediated relaxation ordinarily induced by electrical field stimulation (EFS, 0.12-32 Hz). This effect was not nonselective, as the EFS-resistant muscle relaxed fully with isoproterenol (0.1-100 microM). HCO(3-)-free Krebs in the nominal absence of CO2 did not affect the resting tone and its relaxation. The Ca2+ channel blocker nifedipine decreased resting tone but did not antagonize the relaxation of carbachol-contracted muscle induced by either EFS or isoproterenol. These studies suggest that Cl- may play an important role in LES tone and relaxation due to inhibitory neurotransmitter released from intramural nerves.

VIP-induced alterations in cAMP and inositol phosphates in the lower esophageal sphincter

1990 ◽  
Vol 259 (2) ◽  
pp. G239-G244 ◽  
Author(s):  
S. M. Szewczak ◽  
J. Behar ◽  
G. Billett ◽  
C. Hillemeier ◽  
B. Y. Rhim ◽  
...  
Keyword(s):  
Lower Esophageal Sphincter ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Circular Muscle ◽  
Tissue Samples ◽  
Cyclic Monophosphate ◽  
Circular Smooth Muscle ◽  
Esophageal Sphincter ◽  
Camp Levels

Adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and inositol phosphate (IP) levels were measured in thin tissue samples from the circular smooth muscle of the cat lower esophageal sphincter (LES) at 37 degrees C during vasoactive intestinal peptide (VIP)-induced relaxation. On exposure of in vitro LES circular muscle strips to 10(-6) M VIP at the same temperature, relaxation of spontaneous resting tone begins within 3-6 s, is half maximal at 30 s, and maximal at 1 min. VIP-induced changes in cAMP, cGMP, and IP metabolite levels were measured at 5 and 30 s after the addition of 10(-6) M VIP. At 5 s cAMP levels increased significantly with respect to time-matched unstimulated controls, whereas inositol 1,4,5-trisphosphate (1,4,5-IP3) decreased and these changes remained constant at 30 s. cGMP levels were unchanged at either 5 or 30 s after exposure to 10(-6) M VIP. These data suggest that VIP-induced relaxation is temporally linked to decreased 1,4,5-IP3 as well as increased cAMP levels.

H2O2: a mediator of esophagitis-induced damage to calcium-release mechanisms in cat lower esophageal sphincter

2005 ◽  
Vol 288 (6) ◽  
pp. G1170-G1178 ◽  
Author(s):  
Weibiao Cao ◽  
Karen M. Harnett ◽  
Ling Cheng ◽  
Michael T. Kirber ◽  
Jose Behar ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Lower Esophageal Sphincter ◽  
Calcium Release ◽  
Circular Muscle ◽  
Activity Measurement ◽  
Free Medium ◽  
High Concentration ◽  
Normal Cells ◽  
Intracellular Ca ◽  
Esophageal Sphincter

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular Ca2+ stores. We therefore measured cytosolic Ca2+ in response to a maximally effective dose of ACh in fura 2-AM-loaded lower esophageal sphincter (LES) circular muscle cells and examined the contribution of H2O2 to the reduction in Ca2+ signal. In normal cells, the ACh-induced Ca2+ increase was the same in normal-Ca2+ and Ca2+-free medium and was abolished by the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C inhibitor U-73122, confirming that the initial ACh-induced contraction depends on Ca2+ release from intracellular stores through production of inositol trisphosphate. In LES cells, the ACh-induced Ca2+ increase in normal-Ca2+ medium was significantly lower in esophagitis than in normal cells and was further reduced (∼70%) when the cells were incubated in Ca2+-free medium. This reduction was partially reversed by the H2O2 scavenger catalase. H2O2 measurements in LES circular muscle showed significantly higher levels in esophagitis than in normal cells. When normal LES cells were incubated with H2O2, the ACh-induced Ca2+ increase was significantly reduced in normal-Ca2+ and Ca2+-free medium and was similar to that observed in animals with esophagitis. The initial ACh-induced contraction was also reduced in normal cells incubated with H2O2. H2O2, when applied to cells at sufficiently high concentration, produced a visible and prolonged Ca2+ signal in normal cells. H2O2-induced cell contraction was also sensitive to depletion of stores by thapsigargin (TG); conversely, H2O2 reduced TG-induced contraction, suggesting that TG and H2O2 may operate through similar mechanisms. Ca2+-ATPase activity measurement indicates that H2O2 and TG reduced Ca2+-ATPase activity, confirming similarity of mechanism of action. We conclude that H2O2 may be at least partly responsible for impairment of Ca2+ release in acute experimental esophagitis by inhibiting Ca2+ uptake and refilling Ca2+ stores.

Calcium source diversity in feline lower esophageal sphincter circular and sling muscle

2004 ◽  
Vol 286 (2) ◽  
pp. G271-G277 ◽  
Author(s):  
Ahmad Muinuddin ◽  
Leila Neshatian ◽  
Herbert Y. Gaisano ◽  
Nicholas E. Diamant
Keyword(s):  
Lower Esophageal Sphincter ◽  
Calcium Release ◽  
Circular Muscle ◽  
Cyclopiazonic Acid ◽  
Smooth Muscle Contraction ◽  
Atpase Inhibitor ◽  
Continuous Release ◽  
Spontaneous Tone ◽  
Calcium Source ◽  
Esophageal Sphincter

Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca2+used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca2+-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca2+channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca2+from Ins(1,4,5)P3-sensitive stores is important in tone generation in both muscles. In Ca2+-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca2+stores with CPA or inhibition of Ins(1,4,5)P3-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca2+sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca2+entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.

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