Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP |
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Authors: | K -G Fischer J Leipziger P Rubini-Illes R Nitschke R Greger |
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Institution: | 1. Abteilung IV Nephrologie, Medizinische Klinik, Albert-Ludwigs-Universit?t, Hugstetter Strasse 55, D-79106, Freiburg, Germany 2. Physiologisches Institut der Albert-Ludwigs-Universit?t, Hermann-Herder-Strasse 7, D-79104, Freiburg, Germany
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Abstract: | In HT29 colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca2+ activity (Ca2+]i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P
3-) mediated Ca2+ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca2+ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin
10 μmol/l = FOR) on the Ca2+ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and
reduced cytosolic Ca2+ (Ca2+]i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of
FOR was abolished when the cells were depolarized by a high-K+ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, Ca2+]i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced
Ca2+]i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V
m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V
m was depolarized by 15–54 mV by various increments in the bath K+ concentration. This led to corresponding reductions in Ca2+]i. Irrespective of the cause of depolarization (high K+ or FOR) there was a significant correlation between the change in V
m and change in Ca2+]i. These data indicate that the cAMP-mediated attenuation of Ca2+ influx is caused by the depolarization produced by this second messenger.
Received: 12 March 1996/Accepted: 2 April 1996 |
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Keywords: | Ca2+ influx Fura-2 cAMP Forskolin Carbachol HT29 Second messenger Patch-clamp technique |
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