Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells.
We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.
Bischof
G
G
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Illek
B
B
Reenstra
W W
WW
Machen
T E
TE
D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002217:Carbachol / Q000494:pharmacology*; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004573:Electrolytes / Q000378:metabolism; D019833:Genistein; D007211:Indoles / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D007529:Isoflavones / Q000494:pharmacology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D004573:Electrolytes; D007211:Indoles; D007529:Isoflavones; D013729:Terpenes; D019284:Thapsigargin; D002217:Carbachol; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D000252:Calcium-Transporting ATPases; D002118:Calcium; C000543:cyclopiazonic acid