okadaic-acid and inositol-3-4-5-6-tetrakisphosphate

We have studied the regulation of Ca(2+)-dependent chloride (Cl(Ca)) channels in a human pancreatoma epithelial cell line (CFPAC-1), which does not express functional cAMP-dependent cystic fibrosis transmembrane conductance regulator chloride channels. In cell-free patches from these cells, physiological Ca(2+) concentrations activated a single class of 1-picosiemens Cl(-)-selective channels. The same channels were also stimulated by a purified type II calmodulin-dependent protein kinase (CaMKII), and in cell-attached patches by purinergic agonists. In whole-cell recordings, both Ca(2+)- and CaMKII-dependent mechanisms contributed to chloride channel stimulation by Ca(2+), but the CaMKII-dependent pathway was selectively inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)). This inhibitory effect of Ins(3,4,5,6)P(4) on Cl(Ca) channel stimulation by CaMKII was reduced by raising [Ca(2+)] and prevented by inhibition of protein phosphatase activity with 100 nm okadaic acid. These data provide a new context for understanding the physiological relevance of Ins(3,4,5,6)P(4) in the longer term regulation of Ca(2+)-dependent Cl(-) fluxes in epithelial cells.

Topics: Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Membrane; Cells, Cultured; Chloride Channels; Cyclic AMP; Dose-Response Relationship, Drug; Electrophysiology; Epithelial Cells; Gene Expression Regulation; Humans; Inositol Phosphates; Ionophores; Kinetics; Models, Biological; Okadaic Acid; Patch-Clamp Techniques; Protein Isoforms; Tumor Cells, Cultured; Up-Regulation; Uridine Triphosphate

1. We have studied the regulation of whole-cell chloride current in T84 colonic epithelial cells by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1, 2-bisdeoxy-1,2-bisfluoro-Ins(3,4,5,6)P4 (i.e. F2-Ins(3,4,5,6)P4). 2. Calmodulin-dependent protein kinase II (CaMKII) increased chloride current 20-fold. This current (ICl,CaMK) continued for 7 +/- 1.2 min before its deactivation, or running down, by approximately 60 %. This run-down was prevented by okadaic acid, whereupon ICl,CaMK remained near its maximum value for >= 14.3 +/- 0.6 min. 3. F2-Ins(3, 4,5,6)P4 inhibited ICl,CaMK (IC50 = 100 microM) stereo-specifically, since its enantiomer, F2-Ins(1,4,5,6)P4 had no effect at >= 500 microM. Dose-response data (Hill coefficient = 1.3) showed that F2-Ins(3,4,5,6)P4 imitated only the non-co-operative phase of inhibition by Ins(3,4,5,6)P4, and not the co-operative phase. 4. Ins(3,4,5,6)P4 was prevented from blocking ICl,CaMK by okadaic acid (IC50 = 1.5 nM) and microcystin (IC50 = 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P4 to function. The IC50 values indicate that more than one species of phosphatase was required. One of these may be PP1, since F2-Ins(3,4,5,6)P4-dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.

Topics: Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cell Communication; Cell Line; Chloride Channels; Colon; Electric Stimulation; Electrophysiology; Enzyme Inhibitors; Epithelial Cells; Humans; Inositol Phosphates; Membrane Potentials; Okadaic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Signal Transduction; Stereoisomerism