Estrogen regulation of duodenal bicarbonate secretion and sex-specific protection of human duodenum.

BACKGROUND & AIMS: The reason that women have a lower prevalence of duodenal ulcer is not clear. We investigated whether estrogen regulates human duodenal bicarbonate secretion (DBS) and whether this process accounts for sex differences in the prevalence of duodenal ulcer. METHODS: We performed an epidemiologic study to correlate duodenal ulcer prevalence with sex and age. Proximal DBS was measured from healthy subjects. Estrogen-receptor expression was examined in human duodenal mucosa by immunoblot and immunohistochemical analyses. RESULTS: Among women, the prevalence of duodenal ulcer was significantly lower than among men. The reduced prevalence was greatest among premenopausal women (20-49 y), who were 3.91- to 5.09-fold less likely to develop duodenal ulcers than age-matched men; the difference was reduced to 1.32-fold or less among subjects aged 60 years or older. Premenopausal (20-29 y), but not postmenopausal (60-69 y), women had significantly higher basal and acid-stimulated DBS than the age-matched men. Basal and acid-stimulated DBS in premenopausal women (20-29 y) were significantly higher than in postmenopausal women (60-69 y), whereas there were no significant differences in basal or acid-stimulated DBS between men who were aged 20-29 years or 60-69 years. Serum levels of estradiol changed in parallel with basal and acid-stimulated DBS during the physiological menstrual cycle in premenopausal women. 17ß-estradiol-stimulated DBS was independent of age or sex. Estrogen receptors α and ß were detected on plasma membranes and in the cytosol of human duodenal epithelial cells. CONCLUSIONS: Estrogen regulates human DBS, which could reduce the risk for duodenal ulcer in women and contribute to sex differences in the prevalence of duodenal ulcer.

Calcium-sensing receptor modulates extracellular Ca(2+) entry via TRPC-encoded receptor-operated channels in human aortic smooth muscle cells.

Ca-sensing receptor (CaSR), a member of the G protein-coupled receptor family, regulates the synthesis of parathyroid hormone in response to changes in serum Ca(2+) concentrations. The functions of CaSR in human vascular smooth muscle cells are largely unknown. Here we sought to study CaSR activation and the underlying molecular mechanisms in human aortic smooth muscle cells (HASMC). Extracellular Ca(2+) ([Ca(2+)](o)) dose-dependently increased free cytosolic Ca(2+) ([Ca(2+)](cyt)) in HASMC, with a half-maximal response (EC(50)) of 0.52 mM and a Hill coefficient of 5.50. CaSR was expressed in HASMC, and the [Ca(2+)](o)-induced [Ca(2+)](cyt) rise was abolished by dominant negative mutants of CaSR. The CaSR-mediated increase in [Ca(2+)](cyt) was also significantly inhibited by pertussis toxin, the phospholipase C inhibitor U-73122, or the general protein kinase C (PKC) inhibitor chelerythrine, but not by the conventional PKC inhibitor, Gö6976. Depletion of membrane cholesterol by pretreatment with methyl-ß-cyclodextrin markedly decreased CaSR-induced increase in [Ca(2+)](cyt). Blockade of TRPC channels with 2-aminoethoxydiphenyl borate, SKF-96365, or La(3) significantly inhibited [Ca(2+)](o) entry, whereas activation of TRPC6 channels with flufenamic acid potentiated [Ca(2+)](o) entry. Neither cyclopiazonic acid nor caffeine or ionomycin had any effect on [Ca(2+)](cyt) in [Ca(2+)](o)-free solutions. TRPC6 and PKCε mRNA and proteins were detected in HASMC, and [Ca(2+)](o) induced PKCε phosphorylation, which could be prevented by chelerythrine. Our data suggest that CaSR activation mediates [Ca(2+)](o) entry, likely through TRPC6-encoded receptor-operated channels that are regulated by a PLC/PKCε cascade. Our study therefore provides evidence not only for functional expression of CaSR, but also for a novel pathway whereby it regulates [Ca(2+)](o) entry in HASMC.

Molecular mechanisms underlying Ca2+-mediated motility of human pancreatic duct cells.

We recently reported that transforming growth factor-ß (TGF-ß) induces an increase in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pancreatic cancer cells, but the mechanisms by which TGF-ß mediates [Ca(2+)](cyt) homeostasis in these cells are currently unknown. Transient receptor potential (TRP) channels and Na(+)/Ca(2+) exchangers (NCX) are plasma membrane proteins that play prominent roles in controlling [Ca(2+)](cyt) homeostasis in normal mammalian cells, but little is known regarding their roles in the regulation of [Ca(2+)](cyt) in pancreatic cancer cells and pancreatic cancer development. Expression and function of NCX1 and TRPC1 proteins were characterized in BxPc3 pancreatic cancer cells. TGF-ß induced both intracellular Ca(2+) release and extracellular Ca(2+) entry in these cells; however, 2-aminoethoxydiphenyl borate [2-APB; a blocker for both inositol 1,4,5-trisphosphate (IP(3)) receptor and TRPC], LaCl(3) (a selective TRPC blocker), or KB-R7943 (a selective inhibitor for the Ca(2+) entry mode of NCX) markedly inhibited the TGF-ß-induced increase in [Ca(2+)](cyt). 2-APB or KB-R7943 treatment was able to dose-dependently reverse membrane translocation of PKCα induced by TGF-ß. Transfection with small interfering RNA (siRNA) against NCX1 almost completely abolished NCX1 expression in BxPc3 cells and also inhibited PKCα serine phosphorylation induced by TGF-ß. Knockdown of NCX1 or TRPC1 by specific siRNA transfection reversed TGF-ß-induced pancreatic cancer cell motility. Therefore, TGF-ß induces Ca(2+) entry likely via TRPC1 and NCX1 and raises [Ca(2+)](cyt) in pancreatic cancer cells, which is essential for PKCα activation and subsequent tumor cell invasion. Our data suggest that TRPC1 and NCX1 may be among the potential therapeutic targets for pancreatic cancer.

TGF-beta downregulates PTEN via activation of NF-kappaB in pancreatic cancer cells.

TGF-beta utilizes receptor-activated SMAD signaling to mediate growth suppression; however, non-SMAD signaling that modulates the TGF-beta response in epithelial cells become apparent when the SMAD signaling is abrogated, a common occurrence in pancreatic cancers. Here, we examined whether TGF-beta utilized NF-kappaB to downregulate PTEN, a gene that is rarely mutated in pancreatic cancers. SMAD4-null BxPc3 and CAPAN-1 pancreatic cancer cells were treated with TGF-beta (10 ng/ml) and lysed, and cellular proteins were analyzed by Western blots using p-IkappaB, p65, and PTEN antibodies. PTEN promoter and NF-kappaB activities were assessed by PTEN-luc and p-NF-luc constructs, respectively. Dominant negative p-IkappaB-alpha-M (NF-kappaB superrepressor) was used to block activation of NF-kappaB. Cell motility was assessed by Boyden chamber migration assay. TGF-beta induced IkappaB-alpha phosphorylation followed by NF-kappaB p65 subunit nuclear translocation and increased NF-kappaB activity. IkappaB-alpha-M blocked TGF-beta-induced NF-kappaB activity, reversed downregulated PTEN promoter activity and PTEN expression, and prevented augmentation of cell motility induced by TGF-beta. SMAD4 restoration, but not knockdown of SMAD2 and/or 3, reversed TGF-beta-induced NF-kappaB activity. Thus TGF-beta suppresses PTEN in pancreatic cancer cells through NF-kappaB activation and enhances cell motility and invasiveness in a SMAD4-independent manner that can be counteracted when TGF-beta-SMAD signaling is restored. The TGF-beta/NF-kappaB/PTEN cascade may be a critical pathway for pancreatic cancer cells to proliferate and metastasize.

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Calcium sensing receptor suppresses human pancreatic tumorigenesis through a novel NCX1/Ca(2+)/ß-catenin signaling pathway.

The calcium sensing receptor (CaSR) is functionally expressed in normal human pancreases, but its pathological role in pancreatic tumorigenesis is currently unknown. We sought to investigate the role of CaSR in pancreatic cancer (PC) and the underlying molecular mechanisms. We revealed that the expression of CaSR was consistently downregulated in the primary cancer tissues from PC patients, which was correlated with tumor size, differentiation and poor survival of the patients. CaSR activation markedly suppressed pancreatic tumorigenesis in vitro and in vivo likely through the Ca(2+) entry mode of Na(+)/Ca(2+) exchanger 1 (NCX1) to induce Ca(2+) entry into PC cells. Moreover, NCX1-mediated Ca(2+) entry resulted in Ca(2+)-dependent inhibition of ß-catenin signaling in PC cells, eventually leading to the inhibition of pancreatic tumorigenesis. Collectively, we demonstrate for the first time that CaSR exerts a suppressive function in pancreatic tumorigenesis through a novel NCX1/Ca(2+)/ß-catenin signaling pathway. Targeting this specific signaling pathway could be a potential therapeutic strategy for PC.

TGF-beta mediates PTEN suppression and cell motility through calcium-dependent PKC-alpha activation in pancreatic cancer cells.

Transforming growth factor-beta (TGF-beta) suppresses growth via the TGF-beta-SMAD pathway but promotes growth in cancer cells with disrupted SMAD signaling and corresponds to an invasive phenotype. TGF-beta also downregulates the tumor suppressor PTEN that is rarely mutated in sporadic pancreatic cancer; this downregulation may mediate cell proliferation and invasiveness, but the mechanism is unknown. Here, we examined whether TGF-beta modulation of PTEN was mediated by protein kinase C (PKC). We have previously demonstrated that SMAD4-null BxPc-3 pancreatic cancer cells treated with TGF-beta1 (10 ng/ml) suppressed PTEN expression and increased cell proliferation. TGF-beta-treated cells were examined for PKC activation and its coupling to PTEN expression, utilizing pharmacological and knockdown methods. Calcium mobilization and cell migration were also examined. In BxPc-3 cells, only two PKC isoforms were activated by TGF-beta, and PTEN downregulation by TGF-beta was specifically mediated by PKC-alpha. In parallel, TGF-beta rapidly induced an increase in cytoplasmic free calcium from intracellular stores, consistent with subsequent PKC-alpha activation. The TGF-beta-induced increase in cell migration was blocked by knockdown of PKC-alpha. Thus calcium-dependent PKC-alpha mediates TGF-beta-induced transcriptional downregulation of PTEN, and this pathway promotes cell migration in a SMAD4-null environment. The TGF-beta-PKC-alpha-PTEN cascade may be a key pathway for pancreatic cancer cells to proliferate and metastasize.

TGFbeta modulates PTEN expression independently of SMAD signaling for growth proliferation in colon cancer cells.

Signaling pathways enabling transforming growth factor-beta (TGFbeta)'s conversion from a tumor suppressor to a tumor promoter are not well characterized. TGFbeta utilizes intracellular SMADs to mediate growth suppression; however, TGFbeta-induced proliferative pathways may become more apparent when SMAD signaling is abrogated. Here, we determined regulation of the tumor suppressor PTEN by TGFbeta utilizing SMAD4-null colon cancer cells. TGFbeta downregulated PTEN mRNA and simultaneously induced growth proliferation. TGFbeta also induced both SMAD2 and SMAD3 nuclear translocation, but only triggered SMAD2-specific transcriptional activity in the absence of SMAD4. Interference of SMAD2 with DN-SMAD2 enhanced TGFbeta-induced cell proliferation, but downregulation of PTEN expression by TGFbeta was unaffected. TGFbeta increased PI3K tyrosine phosphorylation, and inhibition of PI3K pharmacologically or by DN-p85 transfection reversed both TGFbeta-induced PTEN suppression and TGFbeta-induced cell proliferation. Thus, TGFbeta activates PI3K to downregulate PTEN for enhancement of cell proliferation that is independent of SMAD proteins.

Role of protein phosphatase 2A in calcium-dependent chloride secretion by human colonic epithelial cells.

EGF inhibits carbachol-induced chloride secretion by regulating a basolateral potassium channel via phosphatidylinositol 3-kinase (PI 3-kinase) and PKCepsilon activation. Although both EGF and carbachol cause tyrosine phosphorylation of p85 of PI 3-kinase, only EGF activates the enzyme. Serine phosphorylation of p85 is thought to suppress the lipid kinase of PI 3-kinase. Our present study examined whether the differential effects of carbachol and EGF on PI 3-kinase activity correspond to varying phosphorylation of p85, and the mechanisms and consequences. T(84) colonic epithelial cells were treated with either EGF or carbachol. Cell lysates were immunoprecipitated with p85 antibody and blotted with either phosphotyrosine or phosphoserine antibodies. Protein phosphatase (PP) 1 and 2A activities were also measured. Both tyrosine and serine residues of p85 were phosphorylated by carbachol, whereas EGF induced only tyrosine phosphorylation. Moreover, EGF abolished carbachol-induced serine phosphorylation of p85 and activated PP2A without affecting PP1. Carbachol did not affect either phosphatase. Calyculin A or okadaic acid pretreatment reversed the inhibitory action of EGF on carbachol-induced chloride secretion and restored serine phosphorylation of p85. Although carbachol recruits p85, it phosphorylates both serine and tyrosine residues so that the lipid kinase of PI 3-kinase is inhibited. EGF results in p85 tyrosine phosphorylation as well as dephosphorylation of serine residues via the activation of PP2A. This explains the differential induction of PI 3-kinase enzyme activity in response to EGF and/or carbachol and has functional implications. Our data provide further insights into negative signals that regulate chloride secretion and into the molecular basis of signaling diversification in the intestinal epithelium.

Bone morphogenetic protein signaling and growth suppression in colon cancer.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily, which utilize BMP receptors and intracellular SMADs to transduce their signals to regulate cell differentiation, proliferation, and apoptosis. Because mutations in BMP receptor type IA (BMPRIA) and SMAD4 are found in the germline of patients with the colon cancer predisposition syndrome juvenile polyposis, and because the contribution of BMP in colon cancers is largely unknown, we examined colon cancer cells and tissues for evidence of BMP signaling and determined its growth effects. We determined the presence and functionality of BMPR1A by examining BMP-induced phosphorylation and nuclear translocation of SMAD1; transcriptional activity via a BMP-specific luciferase reporter; and growth characteristics by cell cycle analysis, cell growth, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolic assays. These assays were also performed after transfection with a dominant negative (DN) BMPR1A construct. In SMAD4-null SW480 cells, we examined BMP effects on cellular wound assays as well as BMP-induced transcription in the presence of transfected SMAD4. We also determined the expression of BMPR1A, BMP ligands, and phospho-SMAD1 in primary human colon cancer specimens. We found intact BMP signaling and modest growth suppression in HCT116 and two derivative cell lines and, surprisingly, growth suppression in SMAD4-null SW480 cells. BMP-induced SMAD signaling and BMPR1A-mediated growth suppression were reversed with DN BMPR1A transfection. BMP2 slowed wound closure, and transfection of SMAD4 into SW480 cells did not change BMP-specific transcriptional activity over controls due to receptor stimulation by endogenously produced ligand. We found no cell cycle alterations with BMP treatment in the HCT116 and derivative cell lines, but there was an increased G1 fraction in SW480 cells that was not due to increased p21 transcription. In human colon cancer specimens, BMP2 and BMP7 ligands, BMPRIA, and phospho-SMAD1 were expressed. In conclusion, BMP signaling is intact and growth suppressive in human colon cancer cells. In addition to SMADs, BMP may utilize SMAD4-independent pathways for growth suppression in colon cancers.

Na(+)/Ca(2+) exchange regulates Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion in mice.

Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca(2+), which regulates ion transport, including HCO(3)(-) secretion. However, the underlying Ca(2+) handling mechanisms are poorly understood. The aim of the present study was to determine whether Na(+)/Ca(2+) exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and HCO(3)(-) secretion by controlling Ca(2+) homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current (I(sc)), and HCO(3)(-) secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot, and cytosolic Ca(2+) in duodenocytes was measured by fura 2. Carbachol (100 muM) increased I(sc) in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced HCO(3)(-) secretion peaked at 10 min. 2-Aminoethoxydiphenylborate (2-APB, 100 muM) or LiCl (30 mM) significantly reduced the initial peak in I(sc) by 51 or 47%, respectively, and abolished the plateau phase of I(sc) without affecting HCO(3)(-) secretion induced by carbachol. Ryanodine (100 muM), caffeine (10 mM), and nifedipine (10 muM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10-30 muM) significantly inhibited carbachol-induced increases in duodenal mucosal I(sc) and HCO(3)(-) secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae, and functional NCX in duodenocytes was demonstrated in Ca(2+) imaging experiments where Na(+) depletion elicited Ca(2+) entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca(2+)-dependent duodenal mucosal ion transport and HCO(3)(-) secretion that results from stimulation of muscarinic receptors.

Heat-stable enterotoxin of Escherichia coli stimulates a non-CFTR-mediated duodenal bicarbonate secretory pathway.

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is an important pathway for duodenal mucosal bicarbonate secretion. Duodenal biopsies from CF patients secrete bicarbonate in response to heat-stable enterotoxin from Escherichia coli (STa) but not cAMP. To explore the mechanism of STa-induced bicarbonate secretion in CF more fully, we examined the role of CFTR in STa-stimulated duodenal bicarbonate secretion in mice. In vivo, the duodenum of CFTR (-/-) or control mice was perfused with forskolin (10(-4) M), STa (10(-7) M), uroguanylin (10(-7) M), 8-bromoguanosine 3',5'-cGMP (8-Br-cGMP) (10(-3) M), genistein (10(-6) M) plus STa, or herbimycin A (10(-6) M) plus STa. In vitro, duodenal mucosae were voltage-clamped in Ussing chambers, and bicarbonate secretion was measured by pH-stat. The effect of genistein, DIDS (10(-4) M), and chloride removal was also studied in vitro. Control, but not CF, mice produced a significant increase in duodenal bicarbonate secretion after perfusion with forskolin, uroguanylin, or 8-Br-cGMP. However, both control and CF animals responded to STa with significant increases in bicarbonate output. Genistein and herbimycin A abolished this response in CF mice but not in controls. In vitro, STa-stimulated bicarbonate secretion in CF tissues was inhibited by genistein, DIDS, and chloride-free conditions, whereas bicarbonate secretion persisted in control mice. In the CF duodenum, STa can stimulate bicarbonate secretion via tyrosine kinase activity resulting in apical Cl(-)/HCO(3)(-) exchange. Further studies elucidating the intracellular mechanisms responsible for such non-CFTR mediated bicarbonate secretion may lead to important therapies for CF.

Growth hormone reduces chloride secretion in human colonic epithelial cells via EGF receptor and extracellular regulated kinase.

BACKGROUND AND AIMS: Growth hormone (GH) has been shown to alleviate symptoms in patients with Crohn's disease. Chloride secretion is important in driving intestinal fluid secretion. We examined whether GH inhibits chloride secretion induced by carbachol (CCh, a calcium-dependent pathway), and the downstream effectors responsible. METHODS: T(84) cells were pretreated with GH at various concentrations followed by CCh (100 micromol/L). Chloride secretion was assessed as changes in short circuit current ( triangle up I(sc)) in Ussing chambers. Tyrphostins AG1478 (an epidermal growth factor receptor [EGFr] inhibitor) and AG490 (a Janus kinase 2 [JAK2] inhibitor), SB203580 (a p38 inhibitor), and PD98059 (a MEK1 inhibitor) were used. RESULTS: GH inhibited CCh-induced chloride secretion at up to 10 nmol/L, but higher concentrations were less effective. GH caused tyrosine phosphorylation of JAK2 and EGFr. AG490 suppressed activation of JAK2 and EGFr in response to GH. AG1478 prevented GH activation of EGFr and reversed its inhibitory effect on chloride secretion. GH also induced activation of both p38 and ERK1/2. AG490 reversed GH-induced tyrosine phosphorylation of both ERK1/2 and p38, but AG1478 reversed that of ERK1/2 only. PD98059, but not SB203580, reversed the inhibitory effect of GH on chloride secretion. CONCLUSIONS: GH inhibits CCh-induced chloride secretion via a JAK2-dependent mechanism involving transactivation of EGFr and consequent recruitment of ERK1/2. Although activated, p38 does not contribute to the inhibitory effect of GH on secretion. These data elucidate mechanisms of GH inhibition of chloride secretion in intestinal epithelia, which may be relevant to therapeutic benefits of GH in Crohn's disease or other diarrheal diseases.

Protein kinase C potentiates cAMP-stimulated mouse duodenal mucosal bicarbonate secretion in vitro.

PKC has been shown to regulate epithelial Cl(-) secretion in a variety of models. However, the role of PKC in duodenal mucosal bicarbonate secretion is less clear. We aimed to investigate the role of PKC in regulation of duodenal mucosal bicarbonate secretion. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers using a pH-stat technique. PKC isoform expression and activity were assessed by Western blotting and in vitro kinase assays, respectively. PMA (an activator of PKC) alone had no effect on duodenal bicarbonate secretion or short-circuit current (I(sc)). When PMA and dibutyryl-cAMP (db-cAMP) were added simultaneously, PMA failed to alter db-cAMP-stimulated duodenal bicarbonate secretion or I(sc) (P > 0.05). However, a 1-h preincubation with PMA potentiated db-cAMP-stimulated duodenal bicarbonate secretion and I(sc) in a concentration-dependent manner (from 10(-8) to 10(-5)M) (P < 0.05). PMA preincubation had no effects on carbachol- or heat-stable toxin-stimulated bicarbonate secretion. Western blot analysis revealed that PKCalpha, -gamma, -epsilon, -, -micro, and -iota/lambda were expressed in murine duodenal mucosa. Ro 31-8220 (an inhibitor active against PKCepsilon, -alpha, -beta, and -gamma), but not Gö 6983 (an inhibitor active against PKCalpha, -gamma, -beta, and -delta), reversed the potentiating effect of PMA on db-cAMP-stimulated bicarbonate secretion. PMA also time- and concentration-dependently increased the activity of PKCepsilon, an effect that was prevented by Ro 31-8220 but not Gö 6983. These results demonstrate that activation of PKC potentiates cAMP-stimulated duodenal bicarbonate secretion, whereas it does not modify basal secretion. The effect of PKC on cAMP-stimulated bicarbonate secretion is mediated by the PKCepsilon isoform.