calcimycin and barium-chloride

Bone marrow mesenchymal stem cells (MSCs) are used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not fully understood. The present study was to investigate the functional ionic channels in undifferentiated mouse bone marrow MSCs using whole cell patch-voltage clamp technique, RT-PCR, and Western immunoblotting analysis. We found that three types of ionic currents were present in mouse MSCs, including a Ca(2+)-activated K(+) current (I(KCa)), an inwardly rectifying K(+) current (I(Kir)), and a chloride current (I(Cl)). I(Kir) was inhibited by Ba(2+), and I(KCa) was activated by the Ca(2+) ionophore A-23187 and inhibited by the intermediate-conductance I(KCa) channel blocker clotrimazole. I(Cl) was activated by hyposmotic (0.8 T) conditions and inhibited by the chloride channel blockers DIDS and NPPB. The corresponding ion channel genes and proteins, KCa3.1 for I(KCa), Kir2.1 for I(Kir), and Clcn3 for I(Cl), were confirmed by RT-PCR and Western immunoblotting analysis in mouse MSCs. These results demonstrate that three types of functional ion channel currents (i.e., I(Kir), I(KCa), and I(Cl)) are present in mouse bone marrow MSCs.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Barium Compounds; Blotting, Western; Bone Marrow Cells; Calcimycin; Cell Size; Cells, Cultured; Chloride Channels; Chlorides; Clotrimazole; Intermediate-Conductance Calcium-Activated Potassium Channels; Ionophores; Membrane Potentials; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Nitrobenzoates; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

In order to investigate how ethanol inhibits the contractility of gallbladder smooth muscle, the effects of different ethanol concentrations were examined on gallbladder contractile responses to stimulants which act on the smooth muscle by different mechanisms. A low concentration (25 mM) of ethanol significantly inhibited the contractile response to phorbol-12,13-dibutyrate (PDBu), but not that to KCl. It also did not significantly affect contractile response to Ca2+ ionophore A23187 in the presence of verapamil or that to Ba2+ in a medium without Ca2+. On the other hand, a higher concentration (200 mM) of ethanol significantly inhibited the contractile responses to PDBu, KCl, Ca2+ ionophore A23187 and BaCl2. These results together with our recent finding that PDBu-induced contraction of guinea pig gallbladder is completely dependent on activation of the voltage-dependent Ca2+ channel suggest that low concentrations of ethanol selectively inhibit the signal transduction of gallbladder smooth muscle cells in the pathway from activation of protein kinase C to that of the voltage-dependent Ca2+ channel, while high concentrations of ethanol inhibit the intracellular common pathway (probably in the cytoskeletal apparatus).

Topics: Animals; Barium Compounds; Calcimycin; Calcium; Central Nervous System Depressants; Chlorides; Drug Interactions; Enzyme Activation; Ethanol; Gallbladder; Guinea Pigs; In Vitro Techniques; Ionophores; Male; Muscle Contraction; Muscle, Smooth; Phorbol 12,13-Dibutyrate; Potassium Chloride; Protein Kinase C; Solutions; Stimulation, Chemical

Catecholamine secretory vesicle core proteins (chromogranins) contain an activity that inhibits catecholamine release, but the identity of the responsible peptide has been elusive. Size-fractionated chromogranins antagonized nicotinic cholinergic-stimulated catecholamine secretion; the inhibitor was enriched in processed chromogranin fragments, and was liberated from purified chromogranin A. Of 15 synthetic peptides spanning approximately 80% of chromogranin A, one (bovine chromogranin A344-364 [RSMRLSFRARGYGFRGPGLQL], or catestatin) was a potent, dose-dependent (IC50 approximately 200 nM), reversible secretory inhibitor on pheochromocytoma and adrenal chromaffin cells, as well as noradrenergic neurites. An antibody directed against this peptide blocked the inhibitory effect of chromogranin A proteolytic fragments on nicotinic-stimulated catecholamine secretion. This region of chromogranin A is extensively processed within chromaffin vesicles in vivo. The inhibitory effect was specific for nicotinic cholinergic stimulation of catecholamine release, and was shared by this chromogranin A region from several species. Nicotinic cationic (Na+, Ca2+) signal transduction was specifically disrupted by catestatin. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. This small domain within chromogranin A may contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells and neurons.

Topics: Amino Acid Sequence; Animals; Antibodies, Blocking; Autocrine Communication; Barium Compounds; Calcimycin; Calcium; Cattle; Chlorides; Chloroquine; Chromaffin Cells; Chromogranin A; Chromogranins; Dose-Response Relationship, Drug; Homeostasis; Humans; Immunoblotting; Molecular Sequence Data; Nicotine; Nicotinic Antagonists; Norepinephrine; Oligopeptides; PC12 Cells; Peptide Fragments; Potassium Chloride; Rats; Receptors, Purinergic P2; Signal Transduction; Sodium; Species Specificity; Substance P

To study the effect of regeneration on the release of creatine kinase (CK) and prostaglandin E2 from muscle, extensor digitorum longus muscles of mice were injected with 50 microliters of BaCl2 solution in saline (1.2% wt/vol). Injected muscles showed almost complete degeneration at 3 days postinjection but had regenerated to approximately the same fiber cross-sectional area as contralateral control muscles by 12 days postinjection. These muscles released reduced amounts of intracellular CK compared with contralateral control muscles in response to excessive isometric contractile activity or treatment with the calcium ionophore A-23187 (20 microM) in vitro. However, regenerating muscles contained a lower total CK activity than contralateral control muscles, which accounted for the reduced CK efflux after experimental damage. Regenerating muscles released an increased amount of prostaglandin E2 compared with control muscles after both damaging stimuli. We conclude that regenerated muscles show no reduced susceptibility to contraction-induced damage (as assessed by CK release) but show an elevated release of prostaglandin E2 in response to contractile activity or an increase in intracellular calcium.

Topics: Animals; Barium Compounds; Calcimycin; Chlorides; Creatine Kinase; Dinoprostone; Electric Stimulation; Histocytochemistry; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscles; Regeneration

The factors which influence the exocytosis of mucins are not well characterized. Since the physical properties of mucins may be affected significantly by the co-secretion of electrolytes and water, we studied the relationship between ion movement and mucin secretion in T84 cells, a human colonic adenocarcinoma cell line which has been well characterized with respect to apical chloride secretion. Secretion of mucin was assessed by immunoassay of mucin appearing in the medium within 30 min of stimulation. Cells were grown on plastic in DMEM/Ham's F12 medium and experiments were carried out at 70% confluence. Mucin secretion was stimulated by the calcium ionophore A23187, or A23187 plus vasoactive intestinal polypeptide. Stimulated mucin secretion was not affected by loop diuretics (furosemide (1 x 10(-3) M) or bumetanide (1 x 10(-4) M)), with or without the addition of ouabain (5 x 10(-5) M) and amiloride (1 x 10(-5) M), making it unlikely that transcellular chloride movements in necessary for mucin secretion. However, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; (1 x 10(-5) and 5 x 10(-5) M) and three potassium channel blockers BaCl2 (1 x 10(-3) and 5 x 10(-3) M), tetraethylammonium chloride (1 x 10(-2) M) and quinine (5 x 10(-4) M) inhibited mucin secretion. A DIDS-sensitive chloride channel or chloride/bicarbonate exchanger and a Ca2(+)-dependent potassium channel may play important roles in mucin secretion. Since plasma membranes are sparingly permeable to DIDS, the DIDS-sensitive site is likely to be on the apical plasma membrane, perhaps at an initiation locus for exocytosis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Adenocarcinoma; Amiloride; Barium; Barium Compounds; Calcimycin; Cell Line; Chlorides; Colonic Neoplasms; Furosemide; Humans; Kinetics; Mucins; Ouabain; Potassium Channels; Quinine; Stilbenes; Tetraethylammonium; Tetraethylammonium Compounds; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

1 The effects of quinine sulphate, tetramethylammonium chloride (TMA) and tetraethylammonium chloride (TEA) (all blockers of the Ca2+-activated K+ channels) on the relaxations induced by acetylcholine (ACh), calcium ionophore A23187 and sodium nitrite were studied in helical strips of rabbit aorta. 2 The strips were contracted to a moderate stable tone with phenylephrine (10(-7) M). ACh (4 X 10(-9) to 10(-6) M) as well as A23187 (10(-8) to 3 X 10(-7) M) reduced this tone in a concentration- and endothelium-dependent manner. 3 Pretreatment of the tissues with quinine (2.5 X 10(-5) to 10(-4) M) for 60 min produced a concentration-dependent inhibition of the relaxation induced by ACh. Also 90 min incubation of the strips with TMA (3 X 10(-3) to 6.5 X 10(-2) M) or TEA (10(-3) to 3 X 10(-2) M) inhibited the ACh-evoked relaxation in a manner similar to quinine. 4 Quinine (10(-4) M, 60 min), TMA (6.5 X 10(-2) M, 90 min) or TEA (3 X 10(-2) M, 90 min) produced 5 to 10 fold reductions in the relaxant EC50 values of A23187 and ACh and depressed (by 40 to 95%) the maximal relaxations to the ionophore and ACh. 5. On a molar basis, quinine was more effective than the two tetraalkylammonium ions in reducing the endothelium-dependent relaxations of the aortic strips induced by ACh or A23187. The inhibitory actions were reversible after 60 to 90 min washout. 6. Exposure of the strips to either quinine (10-4M, 60 min), TMA (6.5 x 10-2 M, 90 min) or TEA (3 X 10-2 M, 90 min), however, did not influence significantly the relaxations evoked by sodium nitrite, a direct smooth muscle relaxant. 7. These results suggest that stimulation of the Ca2+-activated K' channels could be, at least partially, responsible for the endothelium-dependent relaxations induced by ACh or A23 187. Their activation might not be required for the endothelium-independent relaxant effects of sodium nitrite.

Topics: 4-Aminopyridine; Aminopyridines; Animals; Aorta, Thoracic; Barium; Barium Compounds; Calcimycin; Chlorides; Endothelium, Vascular; Female; In Vitro Techniques; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Quaternary Ammonium Compounds; Quinine; Rabbits; Sodium Nitrite; Tetraethylammonium Compounds

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Adenosine Triphosphate; Adrenal Glands; Animals; Barium; Barium Compounds; Calcimycin; Cattle; Cells, Cultured; Chlorides; Chromaffin System; Epinephrine; Kinetics; Nicotine; Norepinephrine; Potassium Chloride