Effect of unsaturated fatty acid on muscarinic current in guinea pig gastric antral circular myocytes. (73/4387)

AIM: To investigate the effect of ectogenesis unsaturated fatty acid on carbachol-induced muscarinic current (ICCh) and its mechanism. METHODS: Using the whole-cell patch-clamp technique, ICCh was recorded in single smooth muscle cell isolated from the antral circular smooth muscles of guinea-pig stomach. RESULTS: Arachidonic acid (AA) was added in external perfusing solution and AA inhibited ICCh to 46 %+/-8 %, 23 %+/-5 %, and 3.8 %+/-0.9 % at 1, 3, and 5 micromol/L. Another unsaturated fatty acid, linoleic acid (LA) also inhibited ICCh in a dose-dependant manner. LA inhibited ICCh to 69 %+/-10 %, 35 %+/-5 %, and 7.4 %+/-1.2 % at 1, 5, and 10 micromol/L, respectively. The same concentration (5 micromol/L) of AA, LA, and oleic acid (OA) suppressed ICCh to 3.8 %+/-0.9 %, 35 %+/-5 %, and 67 %+/-9 %, respectively. The inhibitory potency sequence of these unsaturated fatty acids was AA>LA>OA. After 10-15 min of pretreatment with H-7 (a protein phosphorylation C inhibitor) 100 micromol/L or indomethacin (a cyclooxygenase inhibitor) 10 micromol/L, ICCh was inhibited by 5 micromol/L of AA to 5.5 %+/-0.7 % and 3.0 %+/-1.0 %, respectively. CONCLUSION: The unsaturated fatty acids directly inhibited ICCh, and the inhibitory potency was related to the number of double bonds in fatty acid chain.  (+info)

Modulation of smooth muscle phenotype in vitro by homologous cell substrate. (74/4387)

We have developed a novel cell culture system that supports the shortening of smooth muscle cells. Primary rat airway smooth muscle cells were plated on an ethanol-fixed, confluent monolayer of homologous smooth muscle cells (homologous cell substrate, HCS). Cells grown on HCS exhibited morphological and functional characteristics consistent with a differentiated phenotype. Cells on HCS were spindle shaped with a well-defined long axis, whereas cells grown on glass were larger and irregularly shaped. Smooth muscle-specific alpha-actin immunostained diffusely in cells on HCS, whereas it appeared as stress fibers in cells on glass. Agonists recruited a greater fraction of HCS cells to contract, resulting in greater changes in cell area or length on average, but the maximal capacity of shortening of individual cells was similar between the groups. Unlike cells on glass, cells on HCS shortened to methacholine. HCS was reversible and persisted over several passages. Agonists stimulated intracellular Ca(2+) oscillations in cells on HCS, whereas they elicited biphasic peak and plateau transients in cells on glass. HCS modulates smooth muscle cell phenotype in vitro.  (+info)

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes. (75/4387)

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.  (+info)

Neurohumoral control of gastrointestinal motility. (76/4387)

Neurohumoral substances and their receptors play a major part in the complex regulation of gastrointestinal motility and have therefore been the predominant targets for drug development. The numerous receptors involved in motility are located mainly on smooth muscle cells and neuronal structures in the extrinsic and intrinsic parts of the enteric nervous system. Within this system, receptor agonists and antagonists interacts directly to modify excitatory or inhibitory signals. In view of this complexity it is not surprising that our knowledge about the mechanisms of actions of the various neurohormones and drugs affecting gut motility has been rather fragmented and incomplete. However, recently substantial progress has been achieved, and drug therapy for gut dysmotility is emerging, based primarily on neurohumoral receptors. This paper presents a selective review of the neurohumoral regulatory mechanisms of gastrointestinal motility. In this context, the physiology and pharmacology of the smooth muscle cells, gastrointestinal motility and dysmotility, the enteric nervous system, gastrointestinal reflexes, and serotonin is presented. Further investigation and understanding of the transmitters and receptors involved in especially the reflex activation of peristalsis is crucial for the development of novel therapies for motility disorders.  (+info)

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis. (77/4387)

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.  (+info)

Low-molecular-weight fucoidan promotes therapeutic revascularization in a rat model of critical hindlimb ischemia. (78/4387)

The therapeutic potential of low-molecular-weight (LMW) fucoidan, a sulfated polysaccharide extracted from brown seaweed devoid of direct antithrombin effect, was investigated in vitro and in a model of critical hindlimb ischemia in rat. In vitro results showed that LMW fucoidan enhanced fibroblast growth factor (FGF)-2-induced [(3)H]thymidine incorporation in cultured rat smooth muscle cells. Intravenous injection in rats of LMW fucoidan significantly increased the stromal-derived factor (SDF)-1 level from 1.2 +/- 0.1 to 6.5 +/- 0.35 ng/ml in plasma. The therapeutic effect of LMW fucoidan (5 mg/kg/day), FGF-2 (1 micro g/kg/day), and LMW fucoidan combined with FGF-2 was assessed 14 days after induction of ischemia by 1) clinical evaluation of claudication, 2) tissue blood flow analysis, 3) histoenzymology of muscle metabolic activity, and 4) quantification of capillary density. Both LMW fucoidan and FGF-2 similarly improved residual muscle blood flow (62.5 +/- 6.5 and 64.5 +/- 4.5%, respectively) compared with the control group (42 +/- 3.5%, p < 0.0001). The combination of FGF-2 and LMW fucoidan showed further significant improvement in tissue blood flow (90.5 +/- 3%, p < 0.0001). These results were confirmed by phosphorylase activity, showing muscle regeneration in rats treated with the combination of FGF-2 and LMW fucoidan. Capillary density count increased from 9.6 +/- 0.7 capillaries/muscle section in untreated ischemic controls to 14.3 +/- 0.9 with LMW fucoidan, 14.5 +/- 0.9 with FGF-2, and 19.1 +/- 0.9 in combination (p < 0.001). Thus, LMW fucoidan potentiates FGF-2 activity, mobilizes SDF-1, and facilitates angiogenesis in a rat model. This natural compound could be of interest as an alternative for conventional treatment in critical ischemia.  (+info)

Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells. (79/4387)

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate proteolysis of the extracellular matrix and other extracellular proteins, including growth factors and their receptors. The aberrant expression of these genes is common in most cancers. We profiled the RNA levels of every human MMP and TIMP in a variety of cell types (fibroblast, endothelial, hematopoietic, carcinoma, melanoma, and glioma) using quantitative PCR, with the aim of identifying novel expression patterns. Almost all members of the membrane-type (MT-) MMP and TIMP families were elevated in glioma lines compared to carcinomas. In clinical glioma specimens, there were positive correlations between glioma grade and RNA levels of MT-1, MT-2, and MT-6 MMP, TIMP-1 and TIMP-2, and for several growth factors and receptors. These findings suggest that advanced malignant gliomas have elevated levels of membrane-associated MMPs and TIMPs, which may potentially regulate vascularization and invasion. Concurrent elevation of signaling molecules suggests potential bidirectional relationships that enhance tumor aggressiveness.  (+info)

Activation of Ca2+-activated Cl- current by depolarizing steps in rabbit urethral interstitial cells. (80/4387)

Interstitial cells were isolated from strips of rabbit urethra for study using the amphotericin B perforated-patch technique. Depolarizing steps to -30 mV or greater activated a Ca2+ current (ICa), followed by a Ca2+-activated Cl- current, and, on stepping back to -80 mV, large Cl- tail currents were observed. Both currents were abolished when the cells were superfused with Ca2+-free bath solution, suggesting that Ca2+ influx was necessary for activation of the Cl- current. The Cl- current was also abolished when Ba2+ was substituted for Ca2+ in the bath or the cell was dialyzed with EGTA (2 mM). The Cl- current was also reduced by cyclopiazonic acid, ryanodine, 2-aminoethoxydiphenyl borate (2-APB), and xestospongin C, suggesting that Ca2+-induced Ca2+ release (CICR) involving both ryanodine and inositol 1,4,5-trisphosphate receptors contributes to its activation.  (+info)