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.
Our laboratory has recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates membrane polyphosphoinositide turnover and increases intracellular calcium concentration ([Ca2+]i) in Caco-2 cells. The role of binding to the vitamin D receptor (VDR) in the regulation of these rapid biochemical events, however, remains unclear. The present studies were, therefore, conducted using analogues of 1,25(OH)2D3, which differ markedly in their affinities for the VDR, to assess and compare their effects on [Ca2+]i and on inositol 1,4,5-trisphosphate (IP3) formation. Competitive binding studies performed with both intact cells and high-salt cytosolic extracts from Caco-2 cells demonstrated that 1,25(OH)2D3 and 1,24-(OH)2-22-ene-24-cyclopropyl-D3 (BT) have high affinities for the VDR; 25(OH)-16-ene,23-yne-D3 (AT), however, has a much lower affinity (approximately 1,000-fold less) for the VDR. Despite these large differences in binding affinities for the VDR, AT and BT produced similar concentration-dependent increases in [Ca2+]i and in IP3 formation while 1,25(OH)2D3 was approximately 10-fold less active. These results indicate that the structural requirements for the rapid action of these secosteroids on signal transduction in Caco-2 cells are different from those for receptor binding and transcriptional regulation.
Intestinal chloride (Cl-) secretion can be induced by the heat-stable enterotoxin (STa) from Escherichia coli via generation of cGMP. We investigated the regulatory pathway responsible for cGMP-mediated Cl- secretion in the human colonic carcinoma cell line Caco-2 using whole-cell voltage clamp techniques. Cyclic GMP or cAMP induced a 5-fold increase in Cl- conductance (gCl) in the presence of intracellular ATP and 3-isobutyl-1-methylxanthine. Current activation by cGMP persisted in the presence of the type I cGMP-dependent protein kinase (PKG) inhibitor, KT5823, but was inhibited by the specific peptide inhibitor of the cAMP-dependent protein kinase A (PKA), PKI5-24. The stimulatory effects of cGMP and cAMP on gCl were not additive. The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by intracellular ATP and by cAMP-dependent phosphorylation. In order to determine whether CFTR was involved in the cGMP-dependent increase in gCl, we tested the effect of intracellularly injected anti-CFTR505-511 antibodies previously shown to inhibit CFTR function. Antibodies introduced into individual cells via the patch pipette completely inhibited cGMP-dependent current activation. Cyclic GMP also failed to activate gCl in cystic fibrosis cells. Taken together, these studies demonstrate that activation of the CFTR via PKA-dependent phosphorylation accounts for the cGMP-mediated increase in Cl- secretion in Caco-2 cells.
1. Neurokinin A (NKA) is a mammalian tachykinin distributed principally in the nervous system, including the myenteric innervation of the gut. 2. NKA may be involved in neurogenic inflammation and as a modulatory factor in the diarrhoea associated with mucosal inflammation of inflammatory bowel disease (ulcerative colitis). 3. We evaluated the effect of NKA on the short-circuit current ISC, assumed to reflect electrogenic chloride secretion, across muscle-stripped rat colonic mucosa mounted in Ussing chambers. 4. Serosal addition of NKA produced a concentration-dependent (0.1-100 nM) increase in ISC with an EC50 (half-maximal effective concentration) value of 7.5 nM. The maximum (mean +/- S.E.M.) increase in ISC (microA/cm2) for NKA was 111 +/- 10. 5. Tetrodotoxin (0.5 microM) and bumetanide (10 microM), but not atropine (1.0 microM), hexamethonium (100 microM) or pyrilamine (10 microM), significantly inhibited NKA-induced increases in ISC. 6. The response to NKA was attenuated by 45 min pre-treatment with antisera raised against vasoactive intestinal polypeptide (VIP). Moreover, prior desensitization to VIP attenuated the effect of NKA. 7. These studies suggest that NKA increases ISC in rat colon, in part, through a non-cholinergic neural mechanism involving VIP.
This study examined the role of cholinergic receptors in mediating the chloride secretory response evoked by stimulation of enteric neurons in muscle-stripped segments of mucosa from guinea pig distal colon set up in flux chambers. Basal short-circuit current was not altered by tetrodotoxin, hexamethonium or muscarinic antagonists. The neurally evoked response was not altered by hexamethonium, but was reduced by muscarinic antagonists with a rank order of potency: atropine = 4-diphenyl acetoxy-N-methylpiperidine greater than pirenzepine. 1,1-Dimethyl-4-phenyl-piperazinium iodide increased resting short-circuit current above basal levels. Carbachol and bethanechol evoked a concentration-dependent increase in short-circuit current that was reduced by tetrodotoxin. The effects of carbachol were antagonized by hexamethonium, 4-diphenyl acetoxy-N-methylpiperidine and pirenzepine. Competition for [3H]quinuclidinyl benzilate binding indicated a rank order of potency of 4-diphenyl acetoxy-N-methylpiperidine greater than pirenzepine greater than carbachol greater than bethanechol. These studies indicate that cholinergic and noncholinergic transmission play a significant role in regulation of chloride transport in the guinea pig distal colon. Both nicotinic and muscarinic receptors on neurons that project to the mucosa as well as epithelial muscarinic receptors are mediators of the chloride secretory response.
