Protein kinase C activity does not mediate the inhibitory effect of carbachol on chloride secretion by T84 cells

Abstract

Carbachol induces calcium-dependent chloride secretion and activates protein kinase C in T84 cells. However, prolonged stimulation with carbachol or direct activation of protein kinase C inhibits subsequent calcium-dependent chloride secretion. Furthermore, the ability of carbachol to elevate inositol tetrakisphosphate levels may be linked to inhibition of chloride secretion. Here we demonstrate that protein kinase C activation increases levels of inositol tetrakisphosphates (1,3,4,6- and 3,4,5,6-isomers) in T84 colonic epithelia. Furthermore, this corresponds to an inhibition of chloride secretion. However, protein kinase C is unlikely to mediate the analogous effects of carbachol. Neither the ability of carbachol to inhibit calcium-dependent chloride secretion nor its effects on inositol 3,4,5,6-tetrakisphosphate levels were reversed by staurosporine. Carbachol also has quantitatively and qualitatively different effects on inositol tetrakisphosphate isomers than protein kinase C activators. Thus protein kinase C activity can increase levels of various inositol tetrakisphosphate isomers within T84 cells but does not mediate carbachol-induced increases in these putative messengers. These data further support the hypothesis that inositol 3,4,5,6-tetrakisphosphate is a negative second messenger, uncoupling epithelial chloride secretion from changes in intracellular calcium.

Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl? cotransporter

“…In respiratory epithelia, phorbol esters acutely activate CFTR, and recently, PKC-dependent phosphorylation of CFTR was shown to modulate regulation of channel activity by protein kinase A (PKA) (Jia et al, 1997). However, in several epithelial cell lines, sustained activation of PKC by PMA has been shown to cause progressive inhibition of cAMP-elicited Cl Ϫ secretion (Kachintorn et al 1992;Matthews et al, 1993Matthews et al, , 1995Reenstra, 1993;Trapnell et al, 1991;Traynor-Kaplan et al, 1994;Vaandrager et al, 1992). This effect was initially postulated to result from PKCdependent downregulation of steady state CFTR mRNA levels and protein expression (Trapnell et al, 1991).…”

Insulin and IGF-I inhibit calcium-dependent chloride secretion by T84 human colonic epithelial cells

“…However, we were unable to distinguish whether activation of a PI 3-kinase signaling pathway alone could lead to an inhibition of chloride secretion. This ambiguity arose because EGF has also been shown to elevate the levels of Ins (3,4,5,6)P 4 in T84 cells, albeit to a much lesser extent than observed with carbachol (35,37). The fact that insulin is unable to generate Ins (3,4,5,6)P 4 in T84 cells (Fig.…”

“…Incubations were terminated by the addition of 1 ml of ice-cold methanol. Labeled inositol phosphates were extracted and quantitated by HPLC as previously described (37).…”