Attenuation of stimulated Ca2+ influx in colonic epithelial (HT29) cells by cAMP.
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 P3-) 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 monophosphate (cAMP) (forskolin 10 micromol/l = FOR) on the Ca2+ transient in the presence of CCH (100 micromol/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 (Vm) of the patch-clamped cells significantly and reversibly from -54 +/- 7.4 to -27 +/- 5.9 mV (n = 6). In additional experiments Vm 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 Vm 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.
Fischer
K G
KG
Physiologisches Institut der Albert-Ludwigs-Universität, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Leipziger
J
J
Rubini-Illes
P
P
Nitschke
R
R
Greger
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D019073:HT29 Cells; D006801:Humans
D005456:Fluorescent Dyes; D005576:Colforsin; D000255:Adenosine Triphosphate; D002217:Carbachol; D000242:Cyclic AMP; D002118:Calcium; D016257:Fura-2