Biomedical subjects

J Behar

Publications and source records attributed to J Behar.

At least 37 records · Page 2Linked to original sources

PubMed1997

Leukotrienes in acetylcholine-induced contraction of esophageal circular smooth muscle in experimental esophagitis.

BACKGROUND & AIMS: Phospholipase A2 (PLA2) participates in acetylcholine (ACh)-induced contraction of esophageal circular smooth muscle. Because PLA2, arachidonic acid, and its metabolites are involved in inflammatory responses, their role after induction of experimental esophagitis was examined. METHODS: Experiments were performed in esophageal smooth muscle cells (ESO) isolated by enzymatic digestion from the circular layer of normal and esophagitis animals. Content of peptidoleukotrienes (leukotriene [LT] C4, LTD4, and LTE4) was measured in esophageal circular muscle tissue. RESULTS: The cytosolic PLA2 antagonist trifluoromethyl ketone analogue of arachidonic acid inhibited ACh-induced contraction of normal and esophagitis ESO. Inhibition by secreted PLA2 antagonists AM5 and MJ33 was significantly greater in esophagitis ESO. The lipoxygenase inhibitor nordihydro-guaiaretic acid and the LTD4 antagonist ICI 198,615 inhibited ACh-induced contraction of esophagitis but not of normal ESO. Secreted PLA2 and LTD4 contracted normal ESO more than esophagitis ESO. However, in esophagitis, ESO contraction was increased by threshold diacylglycerol concentration. Resting levels of LTs were greater in esophagitis than in normal circular esophageal muscle and increased in response to ACh in esophagitis but not in normal esophageal muscle. CONCLUSIONS: Esophagitis shifts the signal transduction pathway activated by ACh. Esophagitis increased the contribution of secreted PLA2 and of LTs to ACh-induced contraction.

Acetylcholine↗

PubMed1997

Gallbladder relaxation in patients with pigment and cholesterol stones.

BACKGROUND & AIMS: Gallbladders with cholesterol stones show a defective contraction in response to agonists. The aim of this study was to investigate the muscle relaxation of human gallbladders with cholesterol or black pigment gallstones. METHODS: Gallbladder relaxation was measured in vitro using muscle strips and single muscle cells. Relaxation was expressed as percent inhibition of either basal active tension in strips or maximal cell contraction induced by diacylglycerol. The production of cyclic nucleotides was determined using a 125I-labeled radioimmunoassay kit. RESULTS: Frequency-dependent relaxation evoked by electrical field stimulation was significantly lower in gallbladders with cholesterol stones than in gallbladders with pigment stones. Relaxation and adenosine 3',5'-cyclic monophosphate (cAMP) production induced by isoproterenol, vasoactive intestinal peptide, and forskolin were also significantly decreased in gallbladders with cholesterol stones. However, the relaxation in response to 8-bromo-cAMP, nitric oxide (NO), and the NO donor S-nitroso-N-acetylpenicillamine (SNAP), which circumvent plasma membrane receptors and directly activate intracellular mechanisms, was similar in gallbladders with cholesterol and pigment stones. Guanosine 3',5'-cyclic monophosphate production induced by NO and SNAP was also similar. CONCLUSIONS: Human gallbladder muscle from specimens with cholesterol stones show an impaired relaxation and lower cAMP production compared with specimens with pigment stones. The muscle defect(s) responsible for this impairment seem to be in the plasma membranes.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Daily omeprazole surpasses intermittent dosing in preventing relapse of oesophagitis: a US multi-centre double-blind study.

INTRODUCTION: Relapse of erosive oesophagitis occurs in almost all patients if treatment is stopped after initial healing. AIM: To assess the potential of different therapeutic regimens of omeprazole to prevent relapse of erosive reflux oesophagitis after initial healing with omeprazole. PATIENTS AND METHODS: Patients whose active erosive reflux oesophagitis (grade > or = 2) had healed (grade 0 or 1) after 4-8 weeks of open-label omeprazole 40 mg daily (phase I) were eligible to join a multi-centre, 6-month double-blind, placebo-controlled maintenance study (phase II), which included endoscopy, symptom assessments, serum gastrin measurements, and gastric fundic biopsies. During phase I, endoscopy was performed at weeks 0, 4, and 8. At the end of phase I, 429 of 472 patients (91%) were healed, and there were significant reductions in heartburn, dysphagia and acid regurgitation. Of the 429 patients who healed, 406 joined phase II and were randomized to one of three groups: 20 mg omeprazole daily (n = 138), 20 mg omeprazole for 3 consecutive days each week (n = 137), or placebo (n = 131). During phase II, endoscopy was performed at months 1, 3, and 6 or at symptomatic relapse. RESULTS: The percentages of patients still in endoscopic remission at 6 months were 11% for placebo, 34% for omeprazole 3-days-a-week, and 70% for omeprazole daily. Both omeprazole regimens were superior to placebo in preventing recurrence of symptoms (P < 0.001); however, omeprazole 20 mg daily was superior to omeprazole 20 mg 3-days-a-week (P < 0.001). Compared to baseline, omeprazole therapy resulted in no significant differences among treatment groups in the distribution of gastric endocrine cells. CONCLUSIONS: These results show that after healing of erosive oesophagitis with 4-8 weeks of omeprazole, relapse of oesophagitis and recurrence of reflux symptoms can be prevented in 70% of patients with a maintenance regimen of 20 mg daily, but that intermittent dosing comprising 3 consecutive days each week significantly compromises efficacy.

Anti-Ulcer Agents↗

PubMed1997

Different pathways mediate cholecystokinin actions in cholelithiasis.

Smooth muscle from gallbladders with cholesterol stones exhibits impaired response to cholecystokinin (CCK). This study investigated whether the impaired response is mediated by different signal-transduction pathways responsible for CCK-induced contraction in prairie dog and human gallbladders with cholesterol stones. Gallbladder muscle cells were isolated enzymatically to study contraction. Protein kinase C (PKC) activity was measured by examining the phosphorylation of a specific substrate peptide from myelin basic protein Ac-MBP-(4-14). Gallbladder muscle cells from high-cholesterol-fed prairie dogs contracted less in response to CCK octapeptide (CCK-8) than those from the control group. However, inositol-1,4,5-trisphosphate (IP3), diacylglycerol, and guanosine 5'-O-(3-thiotriphosphate) induced the same magnitudes of contraction in these two groups. In control prairie dog and human gallbladders, the maximal contraction caused by 10(-8) M CCK-8 was blocked by the calmodulin antagonist CGS9343B but not by the PKC inhibitor H-7. Conversely, in gallbladders with cholesterol stones from prairie dogs or human patients, the maximal contraction induced by 10(-8) M CCK-8 was blocked by H-7 and chelerythrine but not by CGS9343B. In these gallbladders CCK-8 caused a significant PKC translocation from the cytosol to the membrane. High CCK concentrations may activate the calmodulin-dependent pathway in functionally normal gallbladder muscle and the PKC-dependent pathway in muscle from gallbladders with cholesterol stones. The defect of gallbladder muscle after cholesterol feeding and stones might reside in the steps before G protein activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Experimental esophagitis affects intracellular calcium stores in the cat lower esophageal sphincter.

We previously showed that lower esophageal spincter (LES) tone depends on spontaneous production of inositol 1,4,5-trisphosphate (IP3) and release of intracellular Ca2+ and that acute experimental esophagitis reduces LES tone and IP3 production, suggesting damage to mechanisms responsible for release of Ca2+ from intracellular stores. In the present investigation, we examined the possibility that mechanisms responsible for Ca2+ storage or uptake may also be damaged. LES circular muscle cells were isolated by enzymatic digestion. Contraction was measured in response to IP3 and thapsigargin, which enhances release of Ca2+ from intracellular stores, and in response to calmodulin and to diacylglycerol. In addition, normal cells were incubated in thapsigargin to assess the effect of depletion of intracellular Ca2+ stores on contractile response. Contraction in response to IP3 and thapsigargin was reduced in experimental esophagitis, but contraction in response to calmodulin or diacylglycerol was not. Acetylcholine (ACh)-induced contraction of normal cells was inhibited by the calmodulin antagonist CGS-9343B but not by 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7). In contrast, in cells from animals with esophagitis or in thapsigargin-treated cells from normal animals, ACh-induced contraction was inhibited by H-7 and not by CGS-9343B. We conclude that experimental esophagitis may damage intracellular Ca2+ stores in the LES and change the intracellular contractile pathways activated by ACh from calmodulin dependent in normal cells to protein kinase C dependent in esophagitis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Different protein kinase C isozymes mediate lower esophageal sphincter tone and phasic contraction of esophageal circular smooth muscle.

Circular muscle of the esophagus (ESO) is normally relaxed and contracts phasically in response to neural stimuli. In contrast, lower esophageal sphincter (LES) circular muscle maintains spontaneous tone and relaxes in response to neural stimuli. We have previously shown that in vitro, spontaneous LES tone and contraction of ESO in response to acetylcholine (ACh) are antagonized by protein kinase C (PKC) inhibitors, suggesting that PKC activation is responsible for these functions. In the current study, Western blot analysis of LES and ESO revealed PKC-alpha, -betaII, and -gamma isozymes in LES circular muscle, but only PKC-betaII translocated from the cytosolic to the membrane fraction in response to ACh. In contrast, ESO contained PKC-betaII, -gamma, and -epsilon, and only PKC-epsilon translocated to the membrane fraction in response to ACh. In LES single cells isolated by enzymatic digestion and permeabilized by saponin, 1-2-dioctanoylglycerol-mediated contraction was inhibited by preincubation with PKC-betaII antiserum but not by other PKC antisera. In esophageal cells, contraction was inhibited by the PKC-epsilon antiserum but not by antisera against other PKC isozymes. N-Myristoylated peptides derived from the pseudosubstrate sequences of PKC isozymes were used to inhibit saponin, 1-2-dioctanoylglycerol-induced contraction of LES and ESO smooth muscle cells. Contraction of LES cells was reduced by the alpha beta gamma pseudosubstrate but not by the alpha, delta, or epsilon pseudosubstrate. Contraction of ESO cells was reduced by the epsilon pseudosubstrate but not by the alpha, delta, or alpha beta gamma pseudosubstrate. We conclude that different types of contractile activity in the ESO and LES are mediated by different PKC isozymes. LES contraction is mediated by the calcium-dependent PKC-betaII, whereas contraction of ESO is mediated by the calcium-independent PKC-epsilon.

Animals↗

PubMed1997

Acute experimental esophagitis activates a second signal transduction pathway in cat smooth muscle from the lower esophageal sphincter.

In single cells, isolated by enzymatic digestion from the circular muscle layer of the lower esophageal sphincter (LES), acute experimental esophagitis (AE) alters signal transduction in response to a maximally effective dose of acetylcholine. In normal LES contraction was inhibited by M3 >> M1 or M2 antagonists. In AE inhibition by M2 antagonists increased significantly so that contraction was inhibited by M3 > M2 > M1 antagonists. In normal cells permeabilized by saponin, contraction was antagonized by antibodies against Gq/11, by the phosphatidylinositol-specific phospholipase C (PI-PLC) antagonist U 73122, but not by the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609, or by the phospholipase D pathway inhibitor propranolol. In AE contraction was reduced by Gq/11 and Gi3 antibodies and by U73122, propranolol and D609. After thapsigargin treatment of normal cells to reduce intracellular Ca++ stores, contraction was inhibited by M2 and M3 antagonists, by antibodies against Gq/11 and Gi3, by U73122, D609 and propranolol, suggesting that depletion of Ca++ stores reproduces the changes induced by AE. We conclude that in normal LES smooth muscle cells acetylcholine-induced contraction is mediated by M3 receptors linked to Gq/11 and PI-PLC, whereas in AE, contraction through this pathway is reduced, perhaps because of reduction in Ca++ stores, and a second pathway is activated by M2 receptors linked to Gi3, PC-PLC and phospholipase D.

Acetylcholine↗

PubMed1997

Cellular dysplasia in acquired cystic renal disease: comparison of histomorphometrically gauged nuclear parameters in normal kidneys, renal cell carcinomas and acquired cystic kidneys.

Nuclear parameters were assessed by computer-assisted image analysis in the cells of abnormal epithelial formations in the acquired cystic kidneys of two dialysis patients, the proximal and distal tubules of a normal kidney and two well differentiated renal cell carcinomas. One acquired cystic kidney contained many small clear celled foci and am 0.9 cm-size clear celled lesion and the second one a papillary microadenoma. The clear celled lesion was cytologically indistinguishable from the carcinomas. The histomorphometrically gauged nuclear parameters were maximal and minimal ferret diameters, averaged ferret diameter, aspect ratio, shape factor, area, volume and specific length and width. Statistical evaluation evidenced that the nuclear area, volume, aspect ration and shape factor allowed for the distinction between benign and malignant epithelial structures. The medians of the nuclear parameters of atrophic tubules, cysts, clear celled foci, papillary adenoma and clear celled lesion in the two acquired cystic kidneys deviation from those of normal renal tubules and in, increasing order of disparity, approached those of the carcinomas.

Carcinoma, Renal Cell↗

PubMed1996

Membrane cholesterol alters gallbladder muscle contractility in prairie dogs.

The cause and effect relationship between membrane cholesterol and gallbladder muscle contractility was examined by altering membrane cholesterol to phospholipid mole ratio using cholesterol-rich or cholesterol-free liposomes. Gallbladder single muscle cells, from prairie dogs that were fed either a regular or high-cholesterol (1.2%) diet, were isolated enzymatically with collagenase. Plasma membranes of gallbladder muscle were purified in sucrose gradient. Cholesterol was measured using the cholesterol oxidase method. Phospholipids were measured with the method of G.R. Bartlett (J. Biol. Chem. 234: 466-468, 1959). The results of this experiment are 1) after high-cholesterol feeding, cholesterol contents and cholesterol/ phospholipid mole ratio in plasma membranes of gallbladder muscle increased 90%, and muscle cell contraction in response to cholecystokinin octapeptide decreased 58%; 2) similar changes were observed when normal gallbladder muscle cells were incubated with cholesterol-rich liposomes for 2 h; and 3) the changes induced either in vivo or in vitro were reversed when muscle cells were subsequently incubated with cholesterol-free liposomes for 2-6 h. We conclude that gallbladder muscle may incorporate excess cholesterol into its plasma membrane when exposed to a cholesterol-rich environment, that excess membrane cholesterol impairs muscle contractility, and that these changes appear to be reversible.

Animals↗

PubMed1996

Histologic analysis of the periprosthetic tissues of long-term surviving cemented total hip arthroplasties.

Qualitative and semiquantitative features of the interfacial membranes of five long-term (> 16 years) surviving cemented total hip arthroplasties (four revision cases and one autopsy case) were compared with those of thirty short-term surviving (< 15 years) cemented hip prostheses. Cement granulomas, micron-sized polyethylene particles-induced giant-celled granulomas, sheets of submicron-sized polyethylene particles-laden macrophages, and aggregated, metallic particles-laden macrophages were scattered in the fibrous tissue of all interfacial membranes. Quantitatively, characteristics of the interfacial membranes of the two groups differed from one another. The dominant species of prosthetic debris in the interfacial membranes of the short-term surviving joint replacements was derived from the polyethylene acetabular socket, and, correspondingly, giant-celled granulomas and macrophagic sheets predominated. Metallic particles and the macrophagic reaction thereto dominated in the interfacial membranes of the long-term surviving arthroplasties, and large cement and polyethylene chunks typically were incorporated in the fibrous tissue of the membranes without an accompanying macrophagic response. In long-term surviving hip arthroplasties, metallic particles may be at least as important as polymeric detritus in stimulating the formation of the bone-resorbing, granulomatous interfacial membrane, which is the hallmark of aseptically loosened arthroplasties. Differences in mechanical settings may account for unlike modes and rates of generation of prosthetic breakdown products, explaining the disparate survivorship of different patients' artificial joints.

Bone Cements↗

PubMed1995

Direct G protein activation reverses impaired CCK signaling in human gallbladders with cholesterol stones.

Human gallbladders were used to investigate the mechanisms of the impaired contraction induced by cholecystokinin (CCK) associated with cholesterol stones. Single muscle cells were isolated enzymatically with collagenase. Inositol 1,4,5-trisphosphate was measured by high-performance liquid chromatography. Diacylglycerol was assayed by thin-layer chromatography. CCK stimulation showed decreased muscle contraction and production of inositol 1,4,5-trisphosphate and diacylglycerol in gallbladders with cholesterol stones compared with those with pigment stones. Exogenous calmodulin induced maximal contraction of 22.4 +/- 0.5 and 21.0 +/- 0.6% in gallbladders with cholesterol and pigment stones, respectively. Similar findings were observed with a synthetic diacylglycerol analogue. Two G protein activators, aluminum fluoride and guanosine 5'-O-(3-thiotriphosphate), evoked similar responses in these two types of gallbladders, with maximal contractions of 21.3 +/- 0.4 and 23.3 +/- 0.5%, respectively, in those with cholesterol stones and 20.9 +/- 0.8 and 22.6 +/- 0.4%, respectively, in those with pigment stones. These results suggest that receptor-dependent ligands like CCK cannot fully activate the intracellular pathways, which, however, can be fully stimulated by circumventing receptors with G protein activators or second messengers. After G protein activation, the pathways appear to be functionally intact. The defect might then reside in the receptor or in the interaction between receptors and G proteins.

Aluminum Compounds↗

PubMed1995

The histologic features of the interfacial membrane of intramedullary nails.

Thirty-six interfacial membranes collected at the time of removal of intramedullary L316 stainless steel nails were studied histologically. The membranes consisted of bland fibrous tissue in a minority of cases. Most often, the nails were enclosed within a synovial-like membrane. Palisading macrophages and fibroblasts abutted on the metallic surface of the nails. Foreign body giant-celled granulomas were scattered in the midzone of the membranes, mono- and polykaryonic macrophages having phagocytozed small metallic particles, necrotic bony debris, and, sometimes, lipidic compounds. Aggregates of hemosiderin-containing macrophages occasionally marked the sites of previous hemorrhages. When present in the retrieved specimen, the bone underlying the membrane was undergoing remodeling. Interfacial motion, consequent on dissimilar stiffness of the bone and nail, as well as deposition of metallic and bony debris, are likely responsible for the formation of the synovial-like interfacial membrane.

Bone Nails↗

PubMed1995

Agonist-independent, muscle-type-specific signal transduction pathways in cat esophageal and lower esophageal sphincter circular smooth muscle.

Smooth muscle cells isolated from the circular muscle layer of cat esophagus and lower esophageal sphincter (LES) exhibit distinct contractile intracellular signal transduction pathways in response to acetylcholine. To determine whether these contractile pathways are muscle type dependent, the authors examined the signal transduction pathways utilized by substance P and bombesin, which in other tissues, use different signal transduction pathways, and by the GTP analog, guanosine 5'-O-3-thiotriphosphate (GTP gamma S), which activates all available G proteins. Western blot analysis of esophageal and LES circular muscle revealed the presence of Gq-G11 (42 kD), Gi1-Gi2 (40 kD) and Go-Gi3 (40 kD) types of G proteins. The responses of esophageal cells to bombesin and substance P were blocked by 1) a Gi3 protein antibody, 2) the inhibitor of specific phosphatidylcholine-phospholipase C (PLC) D609 potassium tricyclo-[5.2.1.0(2.6)]-decyl-(9[8])-xanthogenate, 3) inhibition of phosphatidic acid phosphohydrolase by propranolol, 4) the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and 5) incubation in Ca(++)-free medium. Conversely, the responses of LES muscle cells to bombesin and substance P were blocked by 1) a Gq-G11 antibody, 2) a phosphatidylinositol-specific PLC antagonist U-73122 (1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione), 3) the calmodulin inhibitor CGS9343B (1,3-Dihydro-1-[1-((4-methyl-4H,6H-pyrrolo[1,2-a]-[4,1]benzoxazepin++ +-4 - yl)methyl-4-piperindinyl]-2H-benzimidazol-2-one maleate) and 4) incubation in Sr++. After permeabilization by saponin, inositol 1,4,5-trisphosphate contracted LES but not esophageal cells. The inositol 1,4,5-trisphosphate receptor antagonist heparin and depletion of intracellular Ca++ stores by thapsigargin or A23187 4-Benzoxazolecarboxylic acid, 5-(methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol- 2-yl)ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-, [6s-[6.alpha. (2S*,3S*),8.beta. (R*), 9.beta., 11. alpha.]]-(9Cl), blocked bombesin- and substance P-induced contraction of LES but not of esophageal muscle. In addition, contraction in response to GTP gamma S, which activates all G proteins, was blocked in esophageal cells by a Gi3-protein antibody, propranolol, D609 and H7. In LES muscle cells, the response to GTP gamma S was blocked by a Gq protein antibody, U-73122 and CGS934B. These data demonstrate that, in esophageal muscle, different agonists activate the same Gi3 protein, phosphatidylcholine-specific phospholipases and protein kinase C-dependent pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Distinct muscarinic receptors and signal transduction pathways in gallbladder muscle.

Acetylcholine (ACh) caused a dose-dependent contraction of gallbladder muscle cells in either a normal (1.9 mM) Ca2+, zero-Ca2+ or 4 mM Sr2+ medium, with a maximal contraction about 21 +/- 1% at 10(-6) M. Pirenzepine, methoctramine and p-fluoro-hexahydro-sila-difenidol (the M1, M2 and M3 antagonist, respectively) alone had no inhibitory effect on ACh-induced contraction in normal Ca2+ medium, which was blocked by the combination of methoctramine and p-F-HHSiD. In the 4 mM Sr2+ medium, methoctramine dose dependently inhibited ACh-induced contraction and shifted the ACh dose-response curve to the right. The contraction induced by ACh was further blocked by 10(-4) M propranolol (phosphatidic acid phosphohydrolase inhibitor that prevents the production of diacylglycerol from phospholipase D activation), 10(-5) M H-7 and chelerythrine (the protein kinase C inhibitors) by 64%, 75% and 77%, respectively. In contrast, in the zero-Ca2+ medium, p-fluoro-hexahydro-sila-difenidol dose-dependently inhibited ACh-induced contraction and shifted the ACh dose-response curve to the right. The action of ACh was further blocked by 10(-6) M U-73122 (phospholipase C inhibitor) and 10(-5) M CGS 9343B (calmodulin antagonist) by 95% and 77%, respectively. In conclusion, ACh contracts the gallbladder muscle by stimulating the M2 and M3 muscarinic receptors. The M2 receptors are linked to Ca2+ influx, activation of phospholipase D and protein kinase C-dependent pathway, whereas the M3 receptors are preferentially associated with the activation of phospholipase C, intracellular Ca2+ release and calmodulin-dependent pathway.

Acetylcholine↗

PubMed1994

Cholecystokinin-coupled intracellular signaling in human gallbladder muscle.

BACKGROUND/AIMS: It has been shown that cholecystokinin (CCK) contracts the gallbladder muscle by utilizing intracellular calcium, but the intracellular pathways have not been elucidated. The present study was designed to characterize the signal transduction pathways that mediate CCK-induced contraction of human gallbladder muscle. METHODS: Single muscle cells were isolated from human gallbladders by enzymatic digestion with collagenase. Permeable cells were obtained by incubation with saponin. Protein kinase C (PKC) activity was determined by measuring the phosphorylation of a specific substrate peptide from myelin basic protein, Ac-MBP(4-14). RESULTS: The inositol-1,4,5-trisphosphate (IP3) antagonist heparin blocked the contractions induced by CCK. The PKC inhibitor H-7 blocked the contractions caused by low, but not high, concentrations of CCK and IP3. In contrast, the calmodulin inhibitor CGS9343B blocked the contractions induced by high, but not low, doses of CCK and IP3. Furthermore, exogenously activated calmodulin blocked the PKC-mediated contraction induced by diacylglycerol. Direct measurements of PKC activity showed that low, but not high, CCK concentrations caused PKC translocation. CONCLUSIONS: CCK contracts the gallbladder muscle via IP3-mediated calcium release. CCK activates the PKC pathway at low concentrations, whereas it activates the calmodulin pathway at high concentrations, which in turn inhibits the activation of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Calcium requirements for acetylcholine-induced contraction of cat esophageal circular muscle cells.

Esophageal circular muscle cells isolated by enzymatic digestion contracted in response to acetylcholine (ACh) and in response to the protein kinase C (PKC) agonist 1,2-dioctanoylglycerol (1,2 DAG). Both responses were blocked by PKC antagonists but not by calmodulin antagonists. Furthermore, specific PKC activity, measured in the particulate fraction of the muscle, increased in response to cholinergic stimulation, suggesting that ACh-induced contraction is mediated by a PKC-dependent pathway. ACh-induced contraction decreased with decreasing extracellular Ca2+ and was blocked in Ca(2+)-free physiological salt solution (PSS). Similarly, contraction by the nonhydrolyzable GTP analogue guanosine 5'-O-(3-thiotriphosphate) was blocked by removal of Ca2+ from the PSS. Diacylglycerol production in response to ACh was reduced when extracellular Ca2+ was reduced from 2 to 0.5 mM and was abolished in Ca(2+)-free PSS. The response to 1,2-DAG, however, did not significantly change as extracellular Ca2+ or cytosolic Ca2+ was reduced to zero. Heparin (10 micrograms/ml), thapsigargin (3 microM), or the Ca2+ ionophore A-23187 (3 microM) had no effect on 1,2-DAG or ACh-induced contraction in permeable cells. The data suggest that contraction in response to ACh is mediated by influx of extracellular Ca2+ and a PKC-dependent pathway. Ca2+ may be required mainly to activate the phospholipases responsible for production of diacylglycerol, since contraction of esophageal muscle cells in response to 1,2-DAG is Ca2+ independent.

Acetylcholine↗

PubMed1994

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

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)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1994

Role of 100-kDa cytosolic PLA2 in ACh-induced contraction of cat esophageal circular muscle.

We have shown that acetylcholine (ACh)-induced contraction of esophageal circular muscle cells is mediated by activation of protein kinase C (PKC). We now examine the role of phospholipase A2 (PLA2). ACh increases [3H]arachidonic acid release in esophageal but not in lower esophageal sphincter (LES) muscle. In addition, ACh-induced contraction of esophageal but not of LES cells was reduced by the PLA2 antagonist dimethyleicosadienoic acid and by antiserum to a 100-kDa cytosolic PLA2 (cPLA2). These data suggest that the 100-kDa cPLA2 plays a role in ACh-induced contraction of esophageal but not of LES muscle. In esophageal cells, arachidonic acid produced by PLA2 caused little contraction by itself but potentiated contraction induced by the PKC agonist diacylglycerol (DAG). The free fatty acids linoleic acid and linolenic acid also potentiated DAG-induced contraction. Indomethacin and nordihydroguaiaretic acid had no effect on arachidonic acid-induced potentiation of DAG. The potentiation of DAG-induced contraction by arachidonic acid was inhibited by the PKC inhibitor H-7, but it was not affected by the calmodulin inhibitor CGS-9343B. We conclude that a 100-kDa cPLA2 participates in ACh-induced esophageal contraction by producing arachidonic acid and potentiating DAG-induced activation of a PKC-dependent pathway.

Acetylcholine↗