ISSN:
1432-2013
Schlagwort(e):
Ca2+ entry
;
Carbachol
;
Atropine
;
Lanthanum
;
Barium
;
Lidocaine
;
NPPB
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Medizin
Notizen:
Abstract We investigated the inhibitory action of K+ channel blockers on carbachol-stimulated Ca2+ entry into human Cl−-secretory colonic epithelial cells (HT-29/B6). Digital imaging of the fluorescent calcium indicator dye fura-2 was performed to monitor effects of K+ channel blockers on cytosolic calcium in resting and carbachol-stimulated HT-29/B6 cells. Stimulation with the muscarinic agonist carbachol (100 μM) caused a clearly biphasic intracellular calcium (Cai response: Cai was stimulated from resting levels (85±3 nM, n=100) to a sudden transient peak (821±44 nM) followed by a sustained plateau (317±12 nM). The maintained elevation was dependent on external Ca2+ and represented a new steady state between Ca2+ entry and exit across the plasma membrane. A monophasic Ca2+ response was induced in the absence of external Ca2+ and after the initial peak Cai returned to baseline. The Cai plateau was reduced to resting levels by either the muscarinic antagonist atropine (1 μM) or the inorganic Ca2+ channel blocker lanthanum (effective concentration for 50% inhibition of Ca1 plateau EC50=68±18 nM), but it was unaffected by the organic Ca2+ channel blockers verapamil and nifedipine. Barium, lidocaine and 4-nitro-2-(3-phenylpropylamino)benzoate (NPPB), well-known blockers of basolateral K+ channels of HT-29/B6 cells, rapidly and reversibly reduced carbachol-stimulated Ca2+ entry. The Cai plateau was calculated to be 50% inhibited by barium (96±2 μM), lidocaine (74±3 μM) and NPPB (27±10 μM). The Cai plateau was transiently increased by 1 μM and 10 μM NPPB to 50% and 34%, respectively, probably via hyperpolarization of the membrane potential by blockade of Cl− channels (so that the membrane potential approached V K). The resting Cai was transiently increased by 50 μM and 300 μM NPPB to 308±13 nM and 447±153 nM, respectively, suggesting that NPPB induced a Ca2+ release from internal Ca stores. We conclude that carbachol-stimulated Ca2+ entry into HT-29/B6 cells (a) requires muscarinic receptor occupation, (b) is highly sensitive to lanthanum and (c) is dependent on membrane potential and therefore inhibited by channel blockers that depolarize the cell potential. Also, the sensitivity of Cai levels to K+ channel blockers indicates that there are feedback relationships among rates of Ca2+ entry, activity of Ca2+-activated K+ and Cl− channels and membrane potential.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF00381512
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