thapsigargin and methoctramine

The interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal (GI) tract. In the present study, the effects of Dangkwisoo‑san (DS) on pacemaker potentials in cultured ICCs from the small intestine of the mouse were investigated. The whole‑cell patch‑clamp configuration was used to record pacemaker potentials from cultured ICCs and the increase in intracellular Ca2+ concentration ([Ca2+i) was analyzed in cultured ICCs using fura‑2‑acetoxymethyl ester. The generation of pacemaker potentials in the ICCs was observed. DS produced pacemaker depolarizations in a concentration dependent manner in current clamp mode. The 4‑diphenylacetoxy‑N‑methyl‑piperidine methiodide muscarinic M3 receptor antagonist inhibited DS‑induced pacemaker depolarizations, whereas methoctramine, a muscarinic M2 receptor antagonist, did not. When guanosine 5'‑[β‑thio] diphosphate (GDP‑β‑S; 1 mM) was in the pipette solution, DS marginally induced pacemaker depolarizations, whereas low Na+ solution externally eliminated the generation of pacemaker potentials and inhibited the DS‑induced pacemaker depolarizations. Additionally, the nonselective cation channel blocker, flufenamic acid, inhibited the DS‑induced pacemaker depolarizations. Pretreatment with Ca2+‑free solution and thapsigargin, a Ca2+‑ATPase inhibitor in the endoplasmic reticulum, also eliminated the generation of pacemaker currents and suppressed the DS‑induced pacemaker depolarizations. In addition, [Ca2+]i analysis revealed that DS increased [Ca2+]i. These results suggested that DS modulates pacemaker potentials through muscarinic M3 receptor activation in ICCs by G protein‑dependent external and internal Ca2+ regulation and external Na+. Therefore, DS were observed to affect intestinal motility through ICCs.

Topics: Animals; Calcium-Transporting ATPases; Cells, Cultured; Diamines; Female; Gastrointestinal Motility; Guanosine Diphosphate; Interstitial Cells of Cajal; Intestine, Small; Male; Mice; Mice, Inbred BALB C; Pain; Phytotherapy; Piperidines; Plants, Medicinal; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Thapsigargin; Thionucleotides

We studied the effect of carbachol on pacemaker currents in cultured interstitial cells of Cajal (ICC) from the mouse small intestine by muscarinic stimulation using a whole cell patch clamp technique and Ca2+-imaging. ICC generated periodic pacemaker potentials in the current-clamp mode and generated spontaneous inward pacemaker currents at a holding potential of-70 mV. Exposure to carbachol depolarized the membrane and produced tonic inward pacemaker currents with a decrease in the frequency and amplitude of the pacemaker currents. The effects of carbachol were blocked by 1-dimethyl-4-diphenylacetoxypiperidinium, a muscarinic M(3) receptor antagonist, but not by methotramine, a muscarinic M(2) receptor antagonist. Intracellular GDP-beta-S suppressed the carbachol-induced effects. Carbachol-induced effects were blocked by external Na+-free solution and by flufenamic acid, a non-selective cation channel blocker, and in the presence of thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum. However, carbachol still produced tonic inward pacemaker currents with the removal of external Ca2+. In recording of intracellular Ca2+ concentrations using fluo 3-AM dye, carbachol increased intracellular Ca2+ concentrations with increasing of Ca2+ oscillations. These results suggest that carbachol modulates the pacemaker activity of ICC through the activation of non-selective cation channels via muscarinic M(3) receptors by a G-protein dependent intracellular Ca2+ release mechanism.

Topics: Animals; Anti-Inflammatory Agents; Biological Clocks; Calcium; Calcium-Transporting ATPases; Carbachol; Cation Transport Proteins; Cells, Cultured; Diamines; Flufenamic Acid; Guanosine Diphosphate; Intestine, Small; Membrane Potentials; Mice; Mice, Inbred BALB C; Neuromuscular Depolarizing Agents; Patch-Clamp Techniques; Piperidines; Plant Roots; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Thapsia; Thapsigargin; Thionucleotides

Activation of muscarinic receptors with carbachol has no effect on intracellular Ca2+ concentration in cerebellar granule cell cultures. Only after elevating intracellular Ca2+ concentrations using either 40 mM KCl or activating glutamatergic receptors was carbachol able to increase intracellular Ca2+. The response lasted about 10 s, and the median increase in intracellular Ca2+ with either 100 microM or 300 microM carbachol was about 85 nM. Carbachol at 30 microM elicited an increase in intracellular calcium that was half maximal. After a 16 min or 32 min delay following cell depolarization, responses to carbachol were only found in about 20% of the cells studied and the median increase in intracellular Ca2+ was about 14 nM. (-)-Quinuclidinylxanthene-9-carboxylate hemioxalate (QNX) at 10 nM (a muscarinic m3 antagonist), but not methoctramine at 5 microM (a muscarinic m2 antagonist), attenuated the action of 100 microM carbachol. This carbachol-mediated response was elicited in the absence of extracellular Ca2+ and in the presence of 10 microM dantrolene, but was blocked by 1 microM thapsigargin. Lastly, treatments of cerebellar granule cell cultures with carbachol (100 microM) for up to 12 h results in a desensitization of the carbachol-mediated release of stored Ca2+. Thus, carbachol acts on muscarinic m3 receptors to induce the release of Ca2+ from IP3-sensitive Ca2+ stores that must be filled by a prior period of high intracellular Ca2+.

Topics: Animals; Calcium; Carbachol; Cells, Cultured; Cerebellum; Dantrolene; Diamines; Fura-2; Glutamates; Glutamic Acid; Microscopy, Fluorescence; Neurons; Parasympatholytics; Quinuclidines; Rats; Receptors, Glutamate; Receptors, Muscarinic; Terpenes; Thapsigargin; Xanthenes