Effects of intracellular Na+, K+ and Cl- on Ca(2+)-regulated exocytosis activated by 10 microM acetylcholine (ACh) were studied in guinea-pig antral mucous cells which are permeabilized by nystatin treatment. Ca(2+)-regulated exocytotic events were modulated by [Na+]i, [K+]i and [Cl-]i via mediation of PTX-sensitive G proteins. Increases in [Na+]i and PTX inhibit G protein (G(Na)), which suppressed the exocytosis. Increases in [K+]i caused the exchange of G proteins (from G(Na) to G(K)) to increase, and GK evoked activation of the exocytosis and was inhibited by PTX. Increases in [Cl-]i and PTX inhibit G protein (G(Cl)), which stimulates exocytotic events. Based on these observations, the exocytosis in antral mucous cells were modulated by intracellular ions, concentration of which were increased or decreased by cell volume changes caused by Ach.
Acetylcholine/pharmacology ; Animal ; Cell Membrane Permeability/drug effects ; Exocytosis/physiology* ; Exocytosis/drug effects ; Gastric Mucosa/metabolism ; Gastric Mucosa/cytology ; Guinea Pigs ; Hypertonic Solutions/pharmacology ; Ionophores/pharmacology ; Nystatin/pharmacology ; Pertussis Toxins/pharmacology ; Potassium/pharmacokinetics* ; Pyloric Antrum/metabolism* ; Pyloric Antrum/cytology ; Sodium Chloride/pharmacokinetics* ; Vasodilator Agents/pharmacology

To investigate the function of exogenous unsaturated fatty acids in hyposmotic membrane stretch enhancement of muscarinic current (ICCh) in antral circular smooth muscle cells of guinea pig, we recorded the membrane current with the conventional whole cell patch-clamp technique. I(CCh) elicited by 50 micromol/L carbachol (CCh) at the holding potential of 20 mV under isosmotic condition was taken as control. Hyposmotic membrane stretch increased I(CCh) to 226.0+/-21.0%. When the cells were pretreated with 5 micromol/L arachidonic acid (AA), linoleic acid (LA) or oleic acid (OA), I(CCh)was inhibited to 3.8+/-0.6%, 35.2+/-0.8% and 66.6+/-0.6% respectively. Hyposmotic membrane stretch increased I(CCh) to 106.0+/-2.5%, 173.2+/-6.8% and 222.1+/-11.0% of the control respectively. Five micromol/L AA inhibited hyposmotic membrane stretch-enhanced I(CCh) by 51.2+/-3.8%, while the control I(CCh) under isosmotic condition was inhibited by 96.2+/-1.6%. The results suggest that unsaturated fatty acids inhibited I(CCh) and the inhibitory effect is more significant when the unsaturation degree is increased. However, the unsaturated fatty acids are not involved in the increase of I(CCh) induced by hyposmotic membrane stretch.
Animals ; Fatty Acids, Unsaturated ; pharmacology ; Guinea Pigs ; Membrane Potentials ; drug effects ; Myocytes, Smooth Muscle ; cytology ; physiology ; Osmotic Pressure ; Patch-Clamp Techniques ; Pyloric Antrum ; cytology ; physiology ; Receptors, Muscarinic ; physiology ; Sodium Chloride

This study was designed to isolate Ca2+-activated K+ current (IKCa) and elucidate its physiological significance in freshly isolated interstitial cells of Cajal (ICCs) of guinea-pig stomach. Single ICC was freshly isolated by enzymatically dissociating from myenteric border of gastric antrum free of circular muscles, and conventional whole-cell voltage clamp technique including immunohistochemical techniques were employed to characterize the cells: In myenteric border of gastric antrum, ICC-MY (ICCs from myenteric border) were detected by immunohistochemical reactivity, and single ICC-MY which has many branches was immunohistochemically c-Kit positive. Under K+-rich and 0.1 mM ethylene glycol-bis (2-aminoethyl ether)-N,N,N',N'-tetraacetic acid pipette solution, ICC produced spontaneous inward current (-256+/-92.2 pA). When step-depolarizing pulse from -80 to +80 mV was applied at holding potential (Vh) of -80 mV, voltage-dependent outward currents were recorded with superimposed spontaneous transient outward currents (STOCs). Both STOCs and outward currents were reversibly affected by tetraethylammonium chloride (TEA) and iberiotoxin (IbTX); 2 mM TEA and 200 nM IbTX completely abolished STOCs and significantly inhibited outward K+ current over the whole potential range tested for current/voltage (I/V) relationship. In addition, TEA delayed repolarization phase of spontaneous inward current. The present results indicate the presence of IKCa in a single ICC, and it might be involved in regulation of repolarizing phase of spontaneous inward current in guinea-pig stomach.
Animals ; Calcium/*metabolism ; Guinea Pigs ; Membrane Potentials/drug effects ; Patch-Clamp Techniques ; Peptides/pharmacology ; Potassium Channels/drug effects/*physiology ; Proto-Oncogene Proteins c-kit/*metabolism ; Pyloric Antrum/cytology/*physiology ; Tetraethylammonium/pharmacology

To investigate the relationship between cytoskeleton and hyposmotic membrane stretch-induced increase in muscarinic current, the role of actin microfilament in hyposmotic membrane stretch-induced increase in muscarinic current was studied with the whole-cell patch clamp technique in guinea-pig gastric myocytes. In this study, the muscarinic current was induced by carbachol (50 micromol/L) or GTPgammaS (0.5 mmol/L). The results showed that hyposmotic superfusate (202 mOsmol/L) increased carbachol-induced current (I(CCh)) by 145+/-27% and increased GTPgammaS-induced current by 183+/-30%; but in the presence of cytochalasin-B (Cyt-B, 20 micromol/L), an actin cytoskeleton disruptor, hyposmotic membrane stretch increased I(CCh) by 70+/-6%. However, hyposmotic membrane stretch induced increase in I(CCh) was potentiated to 545+/-81% by phalloidin (20 micromol/L), an actin microfilament stabilizer. The results demonstrated that hyposmotic membrane stretch increased the muscarinic currents induced by carbachol or GTPgammaS and that the actin microfilament is involved in the process in guinea-pig gastric myocytes.
Actin Cytoskeleton ; physiology ; Animals ; Carbachol ; pharmacology ; Female ; Guinea Pigs ; Male ; Membrane Potentials ; drug effects ; Myocytes, Smooth Muscle ; physiology ; Osmotic Pressure ; Patch-Clamp Techniques ; Pyloric Antrum ; cytology ; Receptors, Muscarinic ; physiology

OBJECTIVETo observe the effect of Banxia Xiexin Decoction (BXD) on expression of the interstitial cells of Cajal (ICCs) and stem cell factor (SCF) in the antrum of rats with diabetes mellitus (DM).

METHODSTotally sixty healthy male SD rats were randomly divided into the control group, the model group, the BXD group, and the Western medicine group (WM, treated by domperidone), 15 in each group. Diabetic rat models were established by a single intraperitoneal injection of streptozotocin (STZ, 55 mg/kg). Those in the BXD group were perfused with BXD at the daily dose of 5.4 g/kg. An equal volume of distilled water was given by gastrogavage to those in the WM group and the control group for 8 successive weeks. The body weight and blood glucose of all rats were detected, and the gastric residual rates were detected with semisolid nutrient paste by gastrogavage. The expression of positive ICCs and SCF were observed by immunohistochemical method and quantified image analyzer.

RESULTSCompared with the control group,the body weight reduced, blood glucose and gastric residual rates increased, and the mean optical density of positive ICCs and SCF significantly decreased in the model group (P < 0.05). Compared with the model group,symptoms such as polydipsia, polyphagia, polyuria were relieved, spirits improved, the body weight and mean optical densities of positive ICCs and SCF significantly increased (P < 0.05), and gastric residual rates significantly decreased in the BXD group and the WM group (P < 0.05). The blood glucose significantly decreased (P < 0.05) in the BXD group. The mean optical density of positive ICCs was higher in the BXD group than in the WM group (P < 0.05).

CONCLUSIONSBXD could promote the expression of positive ICCs and SCF. It could improve the gastric motility in DM rats by partially inverting abnormal changes of gastric antral ICCs and SCF.

Animals ; Diabetes Mellitus, Experimental ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Interstitial Cells of Cajal ; metabolism ; Male ; Pyloric Antrum ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cell Factor ; metabolism