Uncoupling of phospholipase C from receptor regulation of [Ca2+]i in T84 colonic cells by prolonged exposure to phorbol dibutyrate.
The T84 colonic cell line, a cultured Cl- secretory cell, elevates intracellular free Ca2+ [( Ca2+]i) in a concentration-dependent manner when exposed to carbachol or histamine. As determined with a fluorescence microscope imaging system, exposure of T84 cells to 100 microM carbachol or histamine resulted in an immediate [Ca2+]i rise of approximately 50-80 nM in all cells. Preincubation of monolayers for 1 h or longer with 0.4 microM phorbol 12,13-dibutyrate (PDB) reduced the number of cells which responded to histamine or carbachol and reduced the magnitude of the increase in the responding cells. This effect reached its maximum after 2 h and persisted for at least 24 h of PDB incubation. Binding of quinuclidinyl benzilate, a cholinergic receptor antagonist, indicated that down-regulation of external receptors was not an explanation for this effect. Examination of phospholipase C activity in T84 cell membranes showed increased basal activity in PDB-treated compared with control cells. Measurement of inositol phosphates generated by intact cells using myo-[3H]inositol incorporation or receptor binding assays showed that 2 h of incubation with PDB elevated basal levels of inositol 1,4,5-trisphosphate and prevented any further carbachol-induced generation of inositol trisphosphate. Probably as a consequence, both total cell calcium and Ca2+ ionophore-releasable calcium were decreased after 2 h of PDB incubation. Membrane-associated protein kinase C activity was elevated after a 2 h exposure to PDB but was below the level of detection after 24 h with PDB. Protein kinase C antagonists neither duplicated nor blocked the uncoupling of carbachol receptors induced by long term treatment with PDB. The results suggest that prolonged PDB incubation caused uncoupling and elevation of phospholipase C activity from cholinergic and histaminergic receptor regulation resulting in increased basal levels of inositol 1,4,5-trisphosphate. Protein kinase C apparently is not involved directly in the mechanism that leads to these effects.
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Department of Medicine (Gastroenterology Division), Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Homaidan
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F
Watson
A
A
Bredt
D S
DS
Sharp
G W
GW
Zahniser
D
D
Donowitz
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M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D003106:Colon / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D006632:Histamine / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000633:toxicity*; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats; D010738:Type C Phospholipases / Q000378:metabolism*
D005456:Fluorescent Dyes; D015240:Phorbol 12,13-Dibutyrate; D011813:Quinuclidinyl Benzilate; D006632:Histamine; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2