The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as short-circuit current (Isc). Luminal ATP stimulated a transient increase in the release of mucins and of Isc corresponding to a secretory Cl- current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED50 of approximately 40 microM. Maximal peak Isc after stimulation with ATP was approximately 35 microA/cm2 with an apparent ED50 of about 0.4 mM. ATP-dependent Isc was at least in part due to Cl- secretion since removal of Cl- from the medium reduced the peak Isc by 40% and the Isc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP > ADP > AMP > or = adenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P2-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl- channels. P2-receptor-stimulated mucin and Isc release was strongly inhibited by a 30 min preincubation with the classical K+ channel blockers quinine (1 mM), quinidine (1 mM), and Ba2+ (3 mM). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K+ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P2 receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.

Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.

No abstract

The ability of all cells to maintain their volume during an osmotic challenge is dependent on the regulated movement of salt and water across the plasma membrane. We demonstrate the phosphorylation-dependent gating of a nonselective conductance in Caco-2 cells during cellular shrinkage. Intracellular application of exogenous purified rat brain protein kinase C (PKC) resulted in the activation of a current similar to that activated during shrinkage with a Na(+)-to-Cl- permeability ratio of approximately 1.7:1. To prevent possible PKC- and/or shrinkage-dependent activation of cystic fibrosis transmembrane regulator (CFTR), which is expressed at high levels in Caco-2 cells, a functional anti-peptide antibody, anti-CFTR505-511, was introduced into the cells via the patch pipette. Anti-CFTR505-511, which is directed against the Walker motif in the first nucleotide binding fold of CFTR, prevented the PKC/shrink-age current activation. The peptide CFTR505-511 also induced current inhibition, suggesting the possible involvement of a regulatory element in close proximity to the channel that shares sequence homology with the first nucleotide binding fold of CFTR and whose binding to the channel is required for channel gating.

No abstract