1,25(OH)2 vitamin D3 stimulates membrane phosphoinositide turnover, activates protein kinase C, and increases cytosolic calcium in rat colonic epithelium.

The hormonal form of vitamin D, 1,25(OH)2 vitamin D3 [1,25(OH)2D3], regulates colonic calcium absorption and colonocyte proliferation and differentiation. In this study, we have examined the effect of 1,25(OH)2D3 on membrane phosphoinositide turnover, protein kinase C activation, and regulation of intracellular calcium concentration [( Ca+2]i) in isolated rat colonic epithelium. In a concentration-dependent manner, 1,25(OH)2D3 stimulated breakdown of membrane phosphoinositides within 15 s, generating diacylglycerol and inositol 1,4,5-triphosphate (IP3). 1,25(OH)2D3 rapidly activated colonic protein kinase C, with maximal translocation of activity from the cytosol to the membrane occurring within 1 min of exposure to the secosteroid. Studies performed in isolated colonocytes with the fluorescent dye fura-2 demonstrated that 10(-8) M 1,25(OH)2D3 caused a rapid rise in [Ca+2]i which then transiently decreased before rising to a new plateau value. When these experiments were performed in a calcium-free buffer, an increase in [Ca+2]i was observed, but both the transient and secondary rise were diminished in magnitude, suggesting that 1,25(OH)2D3 may stimulate both release of intracellular calcium stores and calcium influx. 1,25(OH)2D3 stimulated [3H]thymidine uptake in rat colonocytes, 4 h after an in vivo injection. These studies indicate that 1,25(OH)2D3 exerts a rapid influence on membrane phosphoinositide metabolism which may mediate certain of the secosteroid's effects on colonocyte calcium transport and proliferation.

Correction of enhanced Na(+)-H+ exchange of rat small intestinal brush-border membranes in streptozotocin-induced diabetes by insulin or 1,25-dihydroxycholecalciferol.

Diabetes was induced in rats by administration of a single i.p. injection of streptozotocin (50 mg/kg body wt). After 7 d, diabetic rats were further treated with insulin or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] for an additional 5-7 d. Control, diabetic, diabetic + insulin, and diabetic + 1,25(OH)2D3 rats were then killed, their proximal small intestines were removed, and villus-tip epithelial cells were isolated and used to prepare brush-border membrane vesicles. Preparations from each of these groups were then analyzed and compared with respect to their amiloride-sensitive, electroneutral Na(+)-H+ exchange activity, using 22Na uptake as well as acridine orange techniques. The results of these experiments demonstrated that (a) H+ gradient-dependent 22Na uptake as well as Na+ gradient-dependent transmembrane H+ fluxes were significantly increased in diabetic vesicles compared to their control counterparts, (b) kinetic studies demonstrated that this enhanced 22Na uptake in diabetes was a result of increased maximal velocity (Vmax) of this exchanger with no change in apparent affinity (Km) for Na+, (c) serum levels of 1,25(OH)2D3 were significantly lower in diabetic animals compared with their control counterparts; and (d) insulin or 1,25(OH)2D3 treatment restored the Vmax alterations to control values, without any significant changes in Km, concomitant with significantly increasing the serum levels of 1,25(OH)2D3 in diabetic animals. These results indicate that Na(+)-H+ activity is significantly increased in proximal small intestinal luminal membranes of streptozotocin-induced diabetic rats. Moreover, alterations in the serum levels of 1,25(OH)2D3 may, at least in part, explain this enhanced antiporter activity and its correction by insulin.

1,25-Dihydroxyvitamin D3 and 12-O-tetradecanoyl phorbol 13-acetate cause differential activation of Ca(2+)-dependent and Ca(2+)-independent isoforms of protein kinase C in rat colonocytes.

Considerable evidence that alterations in protein kinase C (PKC) are intimately involved in important physiologic and pathologic processes in many cells, including colonic epithelial cells, has accumulated. In this regard, phorbol esters, a class of potent PKC activators, have been found to induce a number of cellular events in normal or transformed colonocytes. In addition, our laboratory has demonstrated that the major active metabolite of vitamin D3, 1,25(OH)2D3, also rapidly (seconds-minutes) activated PKC and increased intracellular calcium in isolated rat colonocytes. These acute responses, however, were lost in vitamin D deficiency and partially restored with the in vivo repletion of 1,25(OH)2D3. The Ca(2+)-independent or novel isoforms of PKC expressed in the rat colon and the isoform-specific responses of PKC to acute treatment with phorbol esters or 1,25(OH)2D3 have not been previously characterized. Moreover, the effects of vitamin D status on PKC isoform expression, distribution, and response to agonists are also unknown. In the present experiments, in addition to PKC-alpha, rat colonocytes were found to express the novel isoforms delta, epsilon, and zeta by Western blotting using isoform-specific PKC antibodies. The tumor-promoting phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate, caused time- and concentration-dependent translocations of all these isoforms except PKC-zeta. In vitamin D deficiency, there were no alterations in colonic PKC isoform expression but significant changes in the subcellular distribution of PKC-alpha, -delta, and -zeta. Acute treatment of colonocytes from D-sufficient, but not D-deficient, rats with 1,25(OH)2D3 caused a rapid transient redistribution of only PKC-alpha from the soluble to the particulate fraction. The alterations in PKC isoform distribution and PKC-alpha responsiveness to 1,25(OH)2D3 in vitamin D deficiency were partially, but significantly, restored with 5-7 d in vivo repletion of this secosteroid. Both 12-O-tetradecanoyl phorbol 13-acetate and 1,25(OH)2D3 activated endogenous PKC, as assessed by inhibition of myristoylated alanine-rich C kinase substrate back-phosphorylation by exogenous PKC. These studies indicate that PKC-alpha, -delta, and/or -epsilon likely mediate important phorbol ester-stimulated events described in the rat colon. In contrast, PKC-alpha is implicated in the rapid (s-min) PKC-dependent events initiated by 1,25(OH)2D3 in rat colonocytes.

1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation.

Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-gamma as well as c-Src in rat colonocytes, and indicate that PLC-gamma is a direct substrate of secosteroid-activated c-Src in these cells.

Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat.

To determine whether supplemental dietary calcium and/or vitamin D deficiency are involved in modulating colon cancer induced by 1,2-dimethylhydrazine (DMH), Sprague-Dawley rats were fed diets containing either: (a) a normal content of calcium (0.87%) and phosphorus (0.60%) with 2.2 IU of vitamin D3 per g of feed (group A); (b) the same diet as group A, but with calcium and phosphorus increased to 1.80 and 0.80%, respectively (group B); or (c) a vitamin D-deficient diet with supplemental calcium (1.80%) and phosphorus (0.80%) (group C). After 6 weeks on their respective diets, one-half the animals in each group were given s.c. injections of either vehicle or DMH (20 mg/kg body weight/week) for 26 weeks. Animals were then sacrificed and the incidence of tumors as well as the number of tumors per tumor-bearing rat were determined. Colonic mucosal polyamine levels were measured after 15 weeks of exposure to vehicle or DMH, before development of histologically recognizable neoplasms. The results of these experiments demonstrated that neither calcium supplementation alone nor supplemental calcium in conjunction with vitamin D deficiency altered the incidence of colonic cancer induced by this carcinogen. Supplemental calcium, however, significantly decreased the number of rats with multiple tumors and reduced tumor size. Moreover, vitamin D deficiency abolished these protective effects of calcium on colon cancer in this experimental model. DMH treatment increased polyamine levels in the premalignant colonic mucosa in group A rats. This carcinogen-induced effect was blunted by high dietary calcium. Vitamin D-deficient, calcium-supplemented rats (group C) showed an increase in N1-acetylspermidine, but not the other polyamines, with DMH treatment.

1,2-Dimethylhydrazine-induced alterations in protein kinase C activity in the rat preneoplastic colon.

Recently, a number of studies in experimental animals and humans have suggested that alterations in the activity of protein kinase C (PKC) may be involved in the malignant transformation process. To determine whether such alterations in this kinase were present before the development of 1,2-dimethylhydrazine (DMH)-induced colon cancers, rats were given s.c. injections of this procarcinogen (20 mg/kg body weight/week) or diluent for 10 or 15 weeks. Animals were sacrificed after these time periods and colonic epithelium was harvested from each group. The activity and distribution of PKC in the cytosolic and membrane fractions of these preparations as well as 1,2-diacylglycerol mass and phosphoinositide turnover were then examined and compared in the presence and absence of 10 nM 1,25-dihydroxycholecalciferol, an agent which has previously been found to influence these biochemical parameters in the normal rat colonic epithelium. The results of these studies demonstrate that: (a) the percentage of PKC activity in the membrane fraction was significantly greater in DMH-treated animals compared to their control counterparts at 10 and 15 weeks; (b) the total PKC activity was similar at 10 weeks, but markedly reduced in the colonic mucosa of the DMH-treated group at 15 weeks; (c) 1,2-diacylglycerol mass and phosphoinositide turnover were increased in the colonic mucosa of rats administered this carcinogen at both time points; and (d) in control, but not in DMH-treated animals, in vitro addition of 1,25-dihydroxycholecalciferol increased PKC activity, 1,2-diacylglycerol mass and phosphoinositide turnover at each of the times studied. Based on these findings, it would appear that alterations in PKC activity may play a role in the malignant transformation process of the colon in animals administered DMH.

K-ras mutations in 1,2-dimethylhydrazine-induced colonic tumors: effects of supplemental dietary calcium and vitamin D deficiency.

Recent studies from our laboratory have demonstrated that dietary supplemental calcium had no significant effect on the incidence of 1,2-dimethylhydrazine-induced colonic tumors, but did decrease the number of rats with multiple tumors and reduced tumor size. Moreover, concomitant vitamin D deficiency appeared to abolish these protective effects of calcium on colonic tumors in this experimental model. To date, however, the mechanism(s) involved in these phenomena remain unclear. In order to address these important issues, 1,2-dimethylhydrazine-induced colonic tumors from animals on control, Ca(2+)-supplemented, vitamin D-sufficient, and Ca(2+)-supplemented, vitamin D-deficient diets were examined for the presence of ras oncogene mutations. DNA was extracted from each of these tumors. Targeted areas of K-ras and H-ras genes were amplified by the polymerase chain reaction and analyzed for point mutations using allele-specific oligonucleotide hybridization and subsequent DNA sequencing. The results of these studies demonstrated that: (a) approximately one-third of 1,2-dimethylhydrazine-induced colonic carcinomas in the control group had K-ras G to A mutations; (b) no mutations, however, were detected in the cancers of the calcium-supplemented group; (c) concomitant vitamin D deficiency abolished the antimutagenic effect of dietary calcium supplementation (e.g., approximately one-third of cancers in this group again had detectable K-ras mutations); and (d) no H-ras point mutations were detected in colonic tumors from any group. These findings suggest that alterations in K-ras mutations may be one possible mechanism by which calcium and vitamin D status influence colonic carcinogenesis in this experimental model.

1 alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol, a noncalcemic analogue of 1 alpha,25-dihydroxyvitamin D3, inhibits azoxymethane-induced colonic tumorigenesis.

Vitamin D3 and its metabolites, particularly 1 alpha,25-dihydroxyvitamin D3 (1 alpha, 25(OH)2D3), have received increasing attention as potential anticarcinogens in the prevention of cancers in a number of organs, including the colon. These agents, however, have the potential to induce hypercalcemia, thus limiting their practical use for these purposes. In the present studies it was, therefore, of interest to determine whether dietary supplementation with 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (RO24-5531), a recently synthesized apparently noncalcemic analogue of 1 alpha,25(OH)2D3, inhibited colon cancer induced by azoxymethane (AOM). Rats were placed on a standard diet or fed this diet with supplemental RO24-5531 (2.5 nmol/kg feed) before and during (initiation arm), or after AOM or vehicle administration (postinitiation arm). After 34 weeks of study, animals in each group were sacrificed, and their colons were removed and examined macroscopically and microscopically for the presence of tumors. At the time of sacrifice, the animals' serum calcium, phosphorus, 25-hydroxyvitamin D3 and 1 alpha,25(OH)2D3 levels were also analyzed. The results of these studies demonstrated that dietary RO24-5531 supplementation during the initiation arm of these experiments significantly reduced (by 70%) the incidence of AOM-induced colonic tumors compared to rats on the standard diet without RO24-5531. Moreover, this dietary regimen abolished the development of adenocarcinomas in this model. Although there was also a trend for dietary RO24-5531 supplementation during the postinitiation arm of this study to reduce the incidence of colon tumors, this did not reach statistical significance (P > 0.05). In addition, neither dietary RO24-5531 supplementation regimen significantly influenced the animals' rates of growth or their serum levels of calcium, phosphorus, or 25-hydroxyvitamin D3. These studies, therefore, demonstrate for the first time that supplemental dietary RO24-5531 is a chemopreventive agent in the AOM model of experimental colonic carcinogenesis. They also suggest that this agent may ultimately prove useful in clinical colon cancer chemopreventive trials.

Mechanism of action of chemoprotective ursodeoxycholate in the azoxymethane model of rat colonic carcinogenesis: potential roles of protein kinase C-alpha, -beta II, and -zeta.

Several lines of evidence from our laboratory and others indicate that epigenetic alterations in protein kinase C (PKC) are involved in colonic carcinogenesis in both man and experimental animals. Furthermore, bile salts, known activators of PKC, have also been implicated in colonic tumor development. Recently, however, our laboratory has demonstrated that, whereas dietary cholic acid increased the occurrence of azoxymethane (AOM)-induced rat colonic tumors, ursodeoxycholic acid was associated with a significant protective effect. In the present studies, we therefore examined changes in PKC isoforms that accompanied AOM-induced tumor formation and investigated whether the chemopromotional and/or chemopreventional actions of these supplemental dietary bile salts involved changes in specific isoforms of PKC. Rats treated with vehicle (saline) or AOM and maintained on bile salt unsupplemented or supplemented diets were used to isolate control colonocytes and carcinogen-induced tumors, which were then subjected to subcellular fractionation. The homogenates and subcellular fractions were then probed for individual PKC isoforms by quantitative Western blotting using isoform-specific antibodies. Normal rat colonocytes expressed PKC-alpha, -beta II, -delta, -epilson, and -zeta. AOM, in unsupplemented or cholate-supplemented groups, caused significant down-regulation of PKC-alpha, -delta and -zeta and up-regulation of PKC-beta II, while increasing particulate PKC-alpha, -beta II, and -zeta in carcinogen-induced tumors compared to normal colonocytes. Dietary supplementation with ursodeoxycholic acid, in marked contrast to these groups, prevented the changes in the subcellular distributions of PKC-alpha, -beta II, and -zeta, and preserved the expression of PKC-zeta in AOM-induced tumors. These studies suggest that changes in specific isoforms of PKC (particularly, PKC-alpha, -beta II, -delta, and/or -zeta) are involved in colonic malignant transformation in the AOM model but do not account for the chemopromotional actions of cholic acid in this model. Furthermore, the ability of ursodeoxycholic acid to block AOM-induced increases in particulate PKC-alpha, -beta II, and -zeta, and/or inhibit down-regulation of PKC-zeta, may contribute to the chemopreventive effects of this bile acid.

Dietary triacylglycerol modulates sodium-dependent D-glucose transport, fluidity and fatty acid composition of rat small intestinal brush-border membrane.

Rats were maintained on nutritionally complete diets enriched in unsaturated (menhaden fish oil) or saturated (butter fat) triacylglycerols. After 4 weeks, the animals were killed, proximal small intestinal brush-border membranes were prepared, and examined and compared with respect to their lipid composition, molecular species of phosphatidylcholine, lipid fluidity and sodium-dependent D-glucose transport. Membranes prepared from the two dietary groups were found to possess similar ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), and protein/lipid (w/w). In contrast to these findings, however, striking differences were noted in the total fatty acid compositions of these membranes. Plasma membranes prepared from animals fed the fish oil diet possessed higher percentages of saturated fatty acids as well as (n - 3) unsaturated fatty acids and lower percentages of monounsaturated and (n - 6) unsaturated fatty acids than those prepared from animals fed the butter fat diet. Analysis of the molecular species of phosphatidylcholine by HPLC, moreover, revealed that membranes from rats fed fish oil had higher levels of 16:0-20:5, 16:0-22:6 and 18:0-20:5 and lower levels of 18:0-18:2 and 16:0-18:1 than their butter fat counterparts. As assessed by steady-state fluorescence polarization, differential polarized phase fluorometric and excimer/monomer fluorescence intensity techniques using various fluorophores, the lipid fluidity of membranes from rats fed fish oil was also found to be significantly lower compared to membranes from rats fed butter fat. Finally, comparison of the kinetic parameters of Na+-dependent D-glucose transport revealed that fish oil-membrane vesicles had a higher maximum velocity (Vmax) than butter fat membrane vesicles but a similar Km for glucose.

Down-regulation of protein kinase C activity in 1,2-dimethylhydrazine-induced rat colonic tumors.

Recent studies by our laboratory have indicated that alterations in protein kinase C activity may be involved in the early stage(s) of malignant transformation in the 1,2-dimethylhydrazine model of colonic adenocarcinoma. In order to further evaluate the possible role of protein kinase C in this multistage process, rats were given subcutaneous weekly injections of this procarcinogen (20 mg/kg body weight) or diluent for 26 weeks. One week after receiving the last injection, animals were killed and control colonic tissue, tumor tissue and tissue at least 1 cm away from these tumors ('uninvolved mucosa') were harvested. The activity and distribution of protein kinase C in the cytosolic and membrane fractions of these preparations as well as their 1,2-diacylglycerol mass were then examined and compared. The results of these studies demonstrated that: (1) total protein kinase C activity was reduced by approximately 35% and 60%, respectively, in the 'uninvolved' colonic mucosa and tumors of carcinogen-treated rats compared to their control counterpart values; (2) in the 'uninvolved' mucosa, this decrease in total activity was secondary to a decrease solely in cytosolic protein kinase C, whereas, in tumors both membrane and cytosolic activities were reduced; and (3) 1,2-diacylglycerol mass was significantly increased in colonic tumors versus control values. Based on these findings, it would appear that alterations in the cellular distribution and total activity of protein kinase C, possibly secondary to increases in 1,2-diacylglycerol mass, may also play a role in the latter stage(s) of malignant transformation in this experimental model.

TPA causes divergent responses of Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC in the nuclei of Caco-2 cells.

The present studies were undertaken to examine the expression of PKC isoforms within the nucleus of Caco-2 cells, a cell line widely used to investigate intestinal cell growth and differentiation, in order to begin to explore their roles in modulating gene expression. Purified nuclei were, therefore, prepared from Caco-2 cells and found to contain PKC-zeta, but not -alpha. The phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) caused an acute redistribution of PKC-alpha to the nucleus, but did not change the distribution of PKC-zeta. Chronic treatment with TPA down-regulated total PKC-alpha, but not -zeta. Moreover, in contrast to acute TPA treatment, after chronic treatment, nuclear PKC-alpha was no longer detectable, whereas nuclear PKC-zeta was unchanged. These studies demonstrate for the first time the constitutive expression and divergent responses to TPA of the Ca(2+)-dependent and Ca(2+)-independent isoforms of PKC in the nuclei of Caco-2 cells and suggest that these specific isoforms may be involved in modulating gene expression.

Metabolic acidosis. Development in two patients receiving a potassium-sparing diuretic and total parenteral nutrition.

Two patients developed a metabolic acidosis associated with a normal undetermined anion concentration while receiving a potassium-sparing diuretic and total parenteral nutrition. In both cases the metabolic acidosis resolved within one week after discontinuing administration of the diuretic. The use of potassium-sparing diuretics in a patient receiving total parenteral nutrition requires caution and continued monitoring for this potential drug-nutrient interaction.

1,25-dihydroxyvitamin D3 inhibits Na(+)-H+ exchange by stimulating membrane phosphoinositide turnover and increasing cytosolic calcium in CaCo-2 cells.

We have examined the effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the phosphoinositol signal transduction pathway in the human colon cancer-derived cell line CaCo-2 and have studied the regulation of intracellular calcium ([Ca2+]i) and pH (pHi) by this secosteroid. CaCo-2 cells were prelabeled with [3H]myoinositol and treated with 10(-8) M 1,25-(OH)2D3 or vehicle for 90 sec. 1,25-(OH)2D3 caused a decrease in labeled phosphatidylinositol-4-5-bis-phosphate and an increase in labeled inositol 1,4,5-trisphosphate. Treatment with 10(-8) M 1,25-(OH)2D3 for 90 sec also raised the cellular content of diacylglycerol. In a dose-dependent manner, 1,25-(OH)2D3 caused the translocation of protein kinase-C activity from the cytosolic to the membrane fraction, which occurred after as little as 15 sec of exposure to the secosteroid, peaked at about 1-5 min, and then returned toward baseline values. In these CaCo-2 cells, baseline [Ca2+]i was 258 +/- 2 nM (mean +/- SE), as assessed using the fluorescent dye fura-2. After exposure to 10(-8) M 1,25-(OH)2D3, [Ca2+]i rapidly increased to 392 +/- 14 nM after 100 sec, fell, and then subsequently rose to a plateau of 350 +/- 3 nM after 400 sec. In Ca(2+)-free buffer, 1,25-(OH)2D3 caused only a transient rise in [Ca2+]i, indicating that 1,25-(OH)2D3 stimulated both the release of intracellular calcium stores and calcium influx. 1,25-(OH)2D3 caused a dose-dependent decrease in pHi in CaCo-2 cells, as assessed by the fluorescent dye BCECF, which was not observed in cells suspended in Na(+)-free buffer or pretreated with amiloride, indicating that the secosteroid inhibited Na(+)-H+ exchange. No effect of 1,25-(OH)2D3 on pHi was observed in cells in a Ca(2+)-free buffer or pretreated with the phospholipase-C inhibitor U-73,122, which also blocked the rise in [Ca2+]i, or in cells pretreated with the Ca2+/calmodulin inhibitor calmidazolium. Taken together, these studies indicate that 1,25-(OH)2D3 rapidly stimulates membrane phosphoinositide breakdown in CaCo-2 cells, generating the second messengers inositol 1,4,5-trisphosphate and diacylglycerol, causing translocation of protein kinase-C to the membrane, and increasing [Ca2+]i by both releasing calcium stores and promoting calcium influx. Secondary to the rise in [Ca2+]i, Na(+)-H+ exchange is inhibited by a calcium/calmodulin-dependent pathway.

1,25-Dihydroxycholecalciferol rapidly activates rat colonic particulate guanylate cyclase via a protein kinase C-dependent mechanism.

The present studies were performed to determine whether the major biologically active metabolite of vitamin D3, 1,25-dihydroxycholecalciferol [1,25(OH)2D3], could influence the activities of rat colonic particulate guanylate cyclase and adenylate cyclase. To address these issues, colonocytes were harvested from Sprague-Dawley rats and suspended in Krebs-Ringer bicarbonate buffer. The cells were then treated with 1,25(OH)2D3 or other agents (see below) and crude membranes were prepared and analyzed for particulate guanylate cyclase and adenylate cyclase activities. The results of these studies demonstrated that: 1) 1,25(OH)2D3, in a concentration-dependent manner, rapidly (within minutes) stimulated guanylate, but not adenylate cyclase activity; 2) preincubation of the cells with staurosporine, a protein kinase inhibitor, or U73122, an inhibitor of phosphoinositide-phospholipase C-dependent processes, blocked the increase in guanylate cyclase activity induced by 1,25(OH)2D3; and 3) 12-O-tetradecanoyl phorbol 13-acetate and 1,2-dioctanoyl-sn-glycerol, known activators of protein kinase C, also rapidly stimulated rat colonic particulate guanylate cyclase activity. Taken together, these results demonstrate that 1,25(OH)2D3 rapidly stimulates together, these results demonstrate that 1,25(OH)2D3 rapidly stimulates rat colonic particulate guanylate cyclase, at least in part, via a protein kinase C-dependent mechanism.

The role of protein kinase-C alpha in the activation of particulate guanylate cyclase by 1 alpha,25-dihydroxyvitamin D3 in CaCo-2 cells.

Recent studies have implicated protein kinase-C (PKC) in the regulation of guanylate cyclase in several cell types. In view of prior experiments by our laboratory which have demonstrated that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] can activate PKC in CaCo-2 cells, it was of interest to determine whether this secosteroid influenced particulate guanylate cyclase and, if so, to determine which isoforms of PKC were involved. To address these issues, CaCo-2 cells were treated with 1 alpha,25-(OH)2D3 or other agents (see below), and crude membranes prepared from these cells were assayed for guanylate cyclase activity. In several experiments, agents were added directly to isolated membranes, and guanylate cyclase activity was then assayed. These studies demonstrated that 1) the addition of 1 alpha,25-(OH)2D3 or 12-O-tetradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, to intact CaCo-2 cells stimulated particulate guanylate cyclase activity in a time- and concentration-dependent manner; 2) these agents induced the translocation of PKC alpha, but not PKC zeta, from the cytosolic to the membrane fraction of these cells; 3) preincubation of cells with staurosporine (50 nM), a PKC inhibitor, or U73122 (10 microM), an inhibitor of phospholipase-C-dependent processes, significantly reduced (P < 0.05) the stimulatory effect of 1 alpha,25-(OH)2D3 (3 nM) on guanylate cyclase; 4) preincubation of isolated membranes with TPA, calcium, and Mg(2+)-ATP increased guanylate cyclase activity, an affect that was augmented by purified rat brain PKC and inhibited by the PKC inhibitor peptide, PKC-(19-36); and 5) selective down-regulation of PKC alpha by treatment of cells with TPA (200 nM) for 24 h concomitantly abolished the activation of guanylate cyclase by 1 alpha,25-(OH)2D3. Taken together, these studies demonstrate that 1 alpha,25-(OH)2D3 activates particulate guanylate cyclase at least in part via a PKC alpha-dependent mechanism.

Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of Caco-2 cells.

The effect of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or calcitriol, on the proliferation and differentiation of Caco-2 cells was studied. Vitamin D receptor mRNA was detected in both pre- and postconfluent cells, and its abundance was unchanged with time and in response to calcitriol. 1,25-(OH)2D3-binding activity increased during differentiation, but there was no difference in binding between 1,25-(OH)2D3-treated and control cells. 1,25-(OH)2D3 caused a dose-dependent reduction in proliferation, as assessed by [3H]thymidine incorporation and DNA content. 1,25-(OH)2D3 significantly enhanced the normal rise in alkaline phosphatase activity during differentiation and increased alkaline phosphatase mRNA abundance. In contrast, 1,25-(OH)2D3 inhibited the normal rise in sucrase-isomaltase activity and the corresponding mRNA level, although the inhibition occurred after the initial period of cell differentiation (> 10 days postplating). Morphological analysis demonstrated that by day 12 postplating, 1,25-(OH)2D3 increased the mean dome diameter and microvillus length and density. Although 1,25-(OH)2D3 decreases the proliferation of Caco-2 cells and enhances certain parameters of differentiation, not all brush-border hydrolases respond in a similar fashion, making it necessary to interpret with caution their individual use as markers of differentiation.

Gastro-oesophageal reflux disease in obesity: pathophysiological and therapeutic considerations.

Gastro-oesophageal reflux disease (GERD) is common in obese patients. Apart from the physical discomfort and the economic burden, GERD may increase morbidity and mortality through its association with oesophageal carcinoma. The pathophysiology of GERD differs between obese and lean subjects. First, obese subjects are more sensitive to the presence of acid in the oesophagus. Second, hiatal hernia, capable of promoting GERD by several mechanisms, is more prevalent among the obese. Third, obese subjects have increased intra-abdominal pressure that displaces the lower oesophageal sphincter and increases the gastro-oesophageal gradient. Finally, vagal abnormalities associated with obesity may cause a higher output of bile and pancreatic enzymes, which makes the refluxate more toxic to the oesophageal mucosa. The altered body composition associated with obesity affects the pharmacokinetics of drugs. There are no data regarding the efficacy of any of the drugs used for GERD treatment. The dosages of cimetidine and ranitidine should be calculated according to the patient's ideal body weight, not their actual weight. Of the operative procedures used for weight loss, Roux-en-Y gastric bypass was found to be most effective for GERD, while gastric banding was associated with a high prevalence of reflux. This review outlines the pathophysiology and the treatment of GERD in obesity with emphasis on the therapeutic considerations in this population of patients.

Vitamin D status modulates rat colonic M3 muscarinic receptor characteristics and coupling to guanylate cyclase.

The present studies were conducted to determine whether [3H]quinuclidinyl benzilate binding in rat colonic membranes and/or carbachol-mediated stimulation of particulate guanylate cyclase were altered by changes in vitamin D status. EC50 values for the stimulation of colonic guanylate cyclase by carbachol were found to be significantly greater in vitamin D-deficient rats compared to their D-sufficient counterparts. Concomitantly, the density of receptors (Bmax) were significantly lower, and dissociation constants (Kd) were significantly higher in D-deficient colonic membranes. In vitamin D-repleted animals, moreover, all of these aforementioned alterations were at least partially corrected.

Intestinal absorption of cholecalciferol and 25-hydroxycholecalciferol in chronic cholestatic liver disease.

We compared the absorption of cholecalciferol and 25-hydroxycholecalciferol in normal subjects and in patients with mild and severe cholestatic liver disease. 3H-cholecalciferol and 3H-25-hydroxycholecalciferol were given orally and serial blood samples were drawn for measurement of the serum level of radiolabeled vitamin. Absorption of 25-hydroxycholecalciferol peaked earlier and was greater than absorption of cholecalciferol at all times in all three groups. Patients with mild cholestasis (normal bilirubin and fecal fat excretion) absorbed both forms of the vitamin normally. Those with severe cholestasis (jaundice and steatorrhea) had minimal absorption of cholecalciferol but relatively preserved absorption of 25-hydroxycholecalciferol. Absorption of cholecalciferol and 25-hydroxycholecalciferol was inversely related to fecal fat excretion. The superior absorption of 25-hydroxycholecalciferol may partly explain its greater efficacy in oral treatment of vitamin D deficiency in patients with severe cholestasis.