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747 Cytosolic Phospholipase a2 Differentially Regulates Calcium and cAMP-Dependent Chloride Secretion in Colonic Epithelial Cells
AGA Abstracts
secretagogue, forskolin (FSK, 10 µM) induced phosphorylation of cPLA2, albeit with different kinetics. Surprisingly, in contrast to its effects on CCh-induced responses, AACOCF3 significantly inhibited Isc responses to FSK by 40.1 ± 0.1% (n = 6; p < 0.05). Conclusions: These data point to cPLA2 as an important regulator of intestinal secretory responses. Activation of cPLA2 limits the extent of Ca2+-dependent responses by a mechanism involving inhibition of basolateral K+ channels through activation of MAPKs. In contrast, by mechanisms yet to be determined, cPLA2 mediates the full extent of cAMP-dependent responses. These data point to cPLA2-dependent signalling mechanisms as possible targets for development of therapeutic agents that have the ability to differentially regulate Ca2+ and cAMP-dependent secretory mechanisms.
adenosine monophosphate-activated protein kinase (AMPK). Here, we investigated whether H2O2 inhibition of carbachol (CCh)-stimulated Ca2+-dependent Cl- secretion involves inhibition of the Na+,K+,2Cl- cotransporter-1, NKCC1, the dominant pathway for basolateral Cluptake, and whether this depends on an effect of AMPK on NKCC1 function in H2O2 treated epithelial cells. Methods: Protein phosphorylation was measured by Western blot, and ion transport was measured in Ussing chambers, or by measuring 86Rb+ influx as a surrogate for K+ transport. NKCC1 localization was determined by confocal microscopy. Results: H2O2 (500 µM) alone had no effect on baseline Rb+ influx, whereas H2O2 pretreatment inhibited CCh-stimulated Rb+ influx (56% ± 8%, p<0.001, n=8). In the presence of the NKCC1 inhibitor, bumetanide (10µM), CCh stimulated Rb+ influx was reduced (67% ± 9%, p<0.01, n=5) and H2O2 had no further inhibitory effect on CCh-stimulated Rb+ influx (n=5). To examine NKCC1 activity independent of CFTR-dependent Cl- efflux (a major activator of NKCC1 activity), T84 cells were incubated in Cl- free buffer to deplete cells of Cl-. When the cells were then placed in Cl- and Rb+ containing buffer, basal Rb+ influx was increased by approximately 2.3-fold (n=9). H2O2 pre-treatment for 30 min (in Cl- free buffer) significantly reduced the elevated baseline Rb+ influx (to 22% ± 10%, p<0.05, n= 9) and reduced CCh stimulated Rb+ influx to the same level (27% ± 8%, p<0.05, n=9). These data indicate that H2O2 inhibits NKCC1 activity independently of effects on [Cl-]cell or CFTR activity. The inhibitory effect of H2O2 was mediated by AMPK as the AMPK inhibitor, Compound C (50 µM), eliminated the inhibitory effect of H2O2 on CCh-stimulated Rb+ influx. Co-immunopreciptiation of AMPK with NKCC1 suggested that the inhibition of NKCC1 activity by AMPK involved a physical association between the two proteins (p<0.01; n=4). Confocal imaging indicated that H2O2 promoted internalization of NKCC1 from the basolateral membrane, and this was inhibited by Compound C. Conclusion: H2O2 inhibits CCh-stimulated Cl- secretion through AMPK-mediated effects on NKCC1 activity and/or internalization. These data demonstrate the role of energy sensing in the regulation of NKCC1 during inflammation and have implications for ion transport dysfunction in IBD. Supported by the Crohn's and Colitis Foundation of America and NIH.
748 Inhibition of Na-Glucose Co-Transport By Prostaglandin E2 Is Mediated By Calcium Calmodulin Kinase-II in Intestinal Epithelial Cells Jamilur R. Talukder, Steven Coon, Ramesh Kekuda, Uma Sundaram Background: Arachidonic acid metabolite prostaglandin E2 (PGE2) inhibits Na-dependent glucose co-transport (SGLT1) in intestinal epithelial cells by decreasing the number of cotransporters on the brush border membrane (BBM). However, the intracellular mechanism of PGE2 mediated inhibition of SGLT1 is unknown. Aim: Determine the intracellular mechanism of regulation of SGLT1 inhibition by PGE2 in enterocytes. Methods: Rat small intestinal epithelial cells (IEC-18) monolayers grown on transwell plates 10 days post confluence were utilized. Cells were treated with placebo or PGE2, and/or inhibitors of PKA, PKC and CamKII pathways for 24 hours. Na-dependent [3H]-O-methyl glucose uptake was measured to determine SGLT1 activity. cAMP levels were determined by EIA kit. Cytosolic Ca++ concentration was determined using Fluo-3AM. SGLT1 mRNA was quantitated by RTQPCR. Results: PGE2 decreased SGLT1 activity in IEC-18 (305±18 in control and 78±3 in PGE2 treated cells, n=4, p<0.01). PGE2 is known to mediate its effect via G-protein linked receptors thus, cells were treated with G-protein inhibitor suramin. PGE2 mediated inhibition of SGLT1 was reversed by suramin (216±11 pmol/mg protein/2 min in PGE2 +suramin treated cells, p<0.01, n=4). PGE2 treatment increased intracellular cAMP (436±18 pmol/ mg protein in control and 1439±54 in treated cells, n=6, p<0.01) and cytosolic Ca2+ (3fold) in IEC-18 cells. Inhibition of protein kinase A with Rp-8-Br-cAMPS did not fully reverse the PGE2 mediated inhibition of SGLT1 in IEC-18 cells. Further, PKC inhibitor (Calphostin C) also did not fully reverse PGE2 mediated inhibition of SGLT1 in IEC-18 cells. However, inhibition of CamK-II with KN93 reversed PGE2 mediated inhibition of SGLT1 in IEC-18 cells (275±12 pmol/mg protein/2 min in PGE2 + KN93 treated cells, n= 4, p <0.01). PGE2 inhibits SGLT1 secondary to a reduction in BBM co-transporter numbers. Kinetic studies demonstrated that the mechanism of reversal of PGE2 mediated inhibition of SGLT1 by KN93 in IEC-18 cells was secondary to the restoration of co-transporter numbers. Results of the RTQ-PCR studies demonstrated that diminished SGLT1 mRNA levels returned to near normal by KN93 in PGE2 treated cells. Conclusions: These studies demonstrated that while PGE2 increased intracellular cAMP and Ca++ levels, inhibition of SGLT1 appears to be mediated by intracellular Ca++ in IEC-18 cells. Specifically, Cadependent calmodulin kinase II rather than PKC mediates the inhibition of SGLT1 by PGE2 in intestinal epithelial cells.
746 Evidence Against Electrogenicity of Slc26a3 (Down-Regulated in Adenoma) Cl-/HCO3- Exchange in Murine Large Intestine Robert Z. Horack, Janet E. Simpson, Nancy M. Walker, Lane L. Clarke Down-regulated in adenoma (SLC26A3) is the major Cl-/HCO3- exchanger in the apical membrane of the human large intestine and loss of function mutations result in the genetic disease congenital chloride-losing diarrhea (CLD). Recent studies of recombinant SLC26A3 have proposed that its exchange activity is electrogenic with a 2Cl-:1HCO3- stoichiometry (J. Gen. Physiol. 127: 511-524, 2006). Mice with gene-targeted knockout of Slc26a3 (Dra KO) also show the CLD phenotype; however, preliminary studies of the Dra KO distal small intestine did not reveal major changes in transepithelial ionic currents, despite almost complete absence of net Cl- absorption. Therefore, we investigated the electrogenic properties of Dra using microscope-guided conventional microelectrodes to measure the apical membrane potential (Va) in the cecal surface epithelium of Dra wild-type (Dra WT) and Dra KO mice. Dra is expressed at very high levels in the murine cecum (Am.J.Physiol. 293: G923G934, 2007). Using BCECF microfluorimetry of intracellular pH, studies of the cecal surface epithelium showed robust Cl-/HCO3- exchange activity in the Dra WT (0.44 ± 0.03 pH units/min, n = 3) whereas activity was essentially absent in the Dra KO (0.01 ± 0.0 pH units/min, n = 3, p < 0.05). Microelectrode studies of the cecum involved the establishment of a stable impalement in 110 mM Clo Ringers, switch to 0 Clo Ringers to elicit Cl-out/HCO3in exchange and return to 110 mM Clo Ringers solution (22°C). Impalements were accepted if the signal returned to within ±2 mV of baseline upon retraction of the microelectrode. Initial Va after establishing impalement was -19.9 ± 2.2 mV in Dra WT and -23.5 ± 2.1 mV in Dra KO (n = 13-15 impalements, 3 mice each; ns). Switching to 0 Clo Ringers solution depolarized Va in all preparations, however, the ∆Va was not significantly different between Dra WT and KO (Dra WT = +5.2 ±0.7 vs. Dra KO = 4.8 ± 0.5 mV, ns). Preliminary microelectrode studies using the same protocol with solutions warmed to 37°C also did not reveal differences in the ∆Va induced by 0 Clo between Dra WT and KO. We conclude from these studies that 1) Dra (Slc26a3) is the dominant, perhaps sole, Cl-/HCO3- exchanger in the surface epithelium of the murine cecum, and 2) Dra Cl-/HCO3- exchange activity is characterized by electroneutral transport in the native surface epithelium of murine large intestine. Supported by NIDDK.
749 Nherf2 Is Necessary for Basal and All Second Messenger Regulation of NHE3 Activity in Intact Mouse Ileal Na Absorptive Cells Rakhilya Murtazina, Olga Kovbasnjuk, Yueping Chen, Boris M. Hogema, Ursula E. Seidler, Edward J. Weinman, Hugo de Jonge, Mark Donowitz Multi-PDZ-domains containing proteins of the NHERF family take part in regulation of NHE3 activity by providing scaffolding to form regulatory complexes. As an example, in PS120 fibroblasts, NHERF2 or NHERF1 were necessary for cAMP inhibition of NHE3 while only NHERF2 allowed cGMP and Ca2+ inhibition. That there were epithelial specific aspects of NHE3 regulation was demonstrated using NHERF1 null mice in which cAMP inhibition of NHE3 did not occur in proximal tubule, while there was normal inhibition in ileum. Therefore, these studies tested the hypothesis that NHERF2 was necessary for basal and all second messenger inhibition of NHE3 activity in intact mouse ileum. Methods: Initial rates of intact mouse ileal NHE3 activity were determined with two-photon confocal microscopy/ SNARF-4F comparing wild type (WT) and NHERF2 null littermates. Results: 1) Basal NHE3 activity in NHERF2 null ileum (0.17±0.02 ∆pH unit/min) was reduced compared to Wt (0.25±0.02 for WT ∆pH unit/min, p<0.05). Western blotting showed similar NHE3 expression in membrane preparations from both. 2) cAMP inhibited NHE3 activity in WT ileum but not in NHERF2-/- ileum. To examine the mechanism for this NHERF2 dependency of cAMP inhibition of NHE3, PKAII regulatory subunit amount and location were determined. IB showed a marked decrease in PKAII in NHERF2 null ileal membranes with IF demonstrating decrease in both ileal Na absorptive villus and Cl secretory crypt cells. The reduced level of PKA expression was not due to different amounts of mRNA (real time PCR). 3) Ca2+mediated inhibition of NHE3 activity was determined using 10 µM serotonin (5HT) which elevates ileal intracellular Ca2+. 5HT inhibited ileal NHE3 activity in WT ileum by 40±9% but had no effect in ileum from NHERF2 -/- mice. 4) cGMP inhibition of NHE3 activity was determined using 10 nM recombinant E. coli heat stable enterotoxin (STa). Sta decreased WT ileal NHE3 activity by 45±8% but had no effect in ileum from NEHRF2 -/- mice. Conclusions: These results demonstrate: 1) NHERF2 helps set basal ileal NHE3 activity, since NHE3 activity was decreased in ileum from NHERF2 null mice. 2) cAMP inhibition of NHE3 activity requires NHERF2. NHERF2 null ileum has decreased amount of the regulatory subunit of PKAII protein but normal amount of PKAII message. Whether this reduced amount of PKAII explains NHERF2 dependence of cAMP regulation of NHE3 activity is not known. 3) NHERF2 is necessary for Ca2+ -and cGMP-mediated inhibition of ileal NHE3. 4) NHERF2 is a major regulator of basal and second messenger inhibition of NHE3 activity in intact mouse ileum.
747 Cytosolic Phospholipase a2 Differentially Regulates Calcium and cAMPDependent Chloride Secretion in Colonic Epithelial Cells Fergal Donnellan, Frank E. Murray, Brian M. Harvey, Stephen J. Keely Background: Neuroimmune agonists promote intestinal Cl- and fluid secretion predominantly through Ca2+ and cAMP-dependent signalling mechanisms. Previous studies from our group have shown that ERK and JNK MAPKs are important in regulating the extent of secretory responses elicited by Ca2+ but not cAMP-dependent secretagogues. Cytosolic phospholipase A2 (cPLA2) has been shown to be an important constituent of MAPK-dependent signaling pathways in several cell types. Aim: To investigate potential relationships between MAPKs and cPLA2 in regulating colonic epithelial Cl- secretion. Methods: Monolayers of T84 colonic epithelial cells were grown on permeable supports. Protein phosphorylation was analyzed by western blotting. Cl- secretion was measured as changes in short-circuit current (Isc) across voltage-clamped T84 cells mounted in Ussing chambers. Results: Western blot analysis revealed that the Ca2+-dependent agonist, carbachol (CCh; 100 µM) rapidly stimulated cPLA2 phosphorylation (n = 6; p < 0.05) in T84 cells. Furthermore, pretreatment of voltage-clamped T84 cells with AACOCF3 (100 µM), a specific cPLA2 inhibitor, significantly potentiated Isc responses to CCh. Responses to CCh in AACOCF3-pretreated cells were 146.4 ± 2.5 % of those in paired controls (n = 6; p < 0.05). The ERK MAPK inhibitor, PD98059 (20 µM), did not alter CCh-induced cPLA2 phosphorylation, whereas AACOCF3, inhibited both CCh-stimulated ERK and JNK MAPK activation. In apically permeabilized T84 cell monolayers, AACOCF3, significantly potentiated CCh-stimulated K+ conductances, while it did not alter Na + /K + ATPase activity. Similar to CCh, the cAMP-dependent
AGA Abstracts
A-108
secretagogue, forskolin (FSK, 10 µM) induced phosphorylation of cPLA2, albeit with different kinetics. Surprisingly, in contrast to its effects on CCh-induced responses, AACOCF3 significantly inhibited Isc responses to FSK by 40.1 ± 0.1% (n = 6; p < 0.05). Conclusions: These data point to cPLA2 as an important regulator of intestinal secretory responses. Activation of cPLA2 limits the extent of Ca2+-dependent responses by a mechanism involving inhibition of basolateral K+ channels through activation of MAPKs. In contrast, by mechanisms yet to be determined, cPLA2 mediates the full extent of cAMP-dependent responses. These data point to cPLA2-dependent signalling mechanisms as possible targets for development of therapeutic agents that have the ability to differentially regulate Ca2+ and cAMP-dependent secretory mechanisms.
adenosine monophosphate-activated protein kinase (AMPK). Here, we investigated whether H2O2 inhibition of carbachol (CCh)-stimulated Ca2+-dependent Cl- secretion involves inhibition of the Na+,K+,2Cl- cotransporter-1, NKCC1, the dominant pathway for basolateral Cluptake, and whether this depends on an effect of AMPK on NKCC1 function in H2O2 treated epithelial cells. Methods: Protein phosphorylation was measured by Western blot, and ion transport was measured in Ussing chambers, or by measuring 86Rb+ influx as a surrogate for K+ transport. NKCC1 localization was determined by confocal microscopy. Results: H2O2 (500 µM) alone had no effect on baseline Rb+ influx, whereas H2O2 pretreatment inhibited CCh-stimulated Rb+ influx (56% ± 8%, p<0.001, n=8). In the presence of the NKCC1 inhibitor, bumetanide (10µM), CCh stimulated Rb+ influx was reduced (67% ± 9%, p<0.01, n=5) and H2O2 had no further inhibitory effect on CCh-stimulated Rb+ influx (n=5). To examine NKCC1 activity independent of CFTR-dependent Cl- efflux (a major activator of NKCC1 activity), T84 cells were incubated in Cl- free buffer to deplete cells of Cl-. When the cells were then placed in Cl- and Rb+ containing buffer, basal Rb+ influx was increased by approximately 2.3-fold (n=9). H2O2 pre-treatment for 30 min (in Cl- free buffer) significantly reduced the elevated baseline Rb+ influx (to 22% ± 10%, p<0.05, n= 9) and reduced CCh stimulated Rb+ influx to the same level (27% ± 8%, p<0.05, n=9). These data indicate that H2O2 inhibits NKCC1 activity independently of effects on [Cl-]cell or CFTR activity. The inhibitory effect of H2O2 was mediated by AMPK as the AMPK inhibitor, Compound C (50 µM), eliminated the inhibitory effect of H2O2 on CCh-stimulated Rb+ influx. Co-immunopreciptiation of AMPK with NKCC1 suggested that the inhibition of NKCC1 activity by AMPK involved a physical association between the two proteins (p<0.01; n=4). Confocal imaging indicated that H2O2 promoted internalization of NKCC1 from the basolateral membrane, and this was inhibited by Compound C. Conclusion: H2O2 inhibits CCh-stimulated Cl- secretion through AMPK-mediated effects on NKCC1 activity and/or internalization. These data demonstrate the role of energy sensing in the regulation of NKCC1 during inflammation and have implications for ion transport dysfunction in IBD. Supported by the Crohn's and Colitis Foundation of America and NIH.
748 Inhibition of Na-Glucose Co-Transport By Prostaglandin E2 Is Mediated By Calcium Calmodulin Kinase-II in Intestinal Epithelial Cells Jamilur R. Talukder, Steven Coon, Ramesh Kekuda, Uma Sundaram Background: Arachidonic acid metabolite prostaglandin E2 (PGE2) inhibits Na-dependent glucose co-transport (SGLT1) in intestinal epithelial cells by decreasing the number of cotransporters on the brush border membrane (BBM). However, the intracellular mechanism of PGE2 mediated inhibition of SGLT1 is unknown. Aim: Determine the intracellular mechanism of regulation of SGLT1 inhibition by PGE2 in enterocytes. Methods: Rat small intestinal epithelial cells (IEC-18) monolayers grown on transwell plates 10 days post confluence were utilized. Cells were treated with placebo or PGE2, and/or inhibitors of PKA, PKC and CamKII pathways for 24 hours. Na-dependent [3H]-O-methyl glucose uptake was measured to determine SGLT1 activity. cAMP levels were determined by EIA kit. Cytosolic Ca++ concentration was determined using Fluo-3AM. SGLT1 mRNA was quantitated by RTQPCR. Results: PGE2 decreased SGLT1 activity in IEC-18 (305±18 in control and 78±3 in PGE2 treated cells, n=4, p<0.01). PGE2 is known to mediate its effect via G-protein linked receptors thus, cells were treated with G-protein inhibitor suramin. PGE2 mediated inhibition of SGLT1 was reversed by suramin (216±11 pmol/mg protein/2 min in PGE2 +suramin treated cells, p<0.01, n=4). PGE2 treatment increased intracellular cAMP (436±18 pmol/ mg protein in control and 1439±54 in treated cells, n=6, p<0.01) and cytosolic Ca2+ (3fold) in IEC-18 cells. Inhibition of protein kinase A with Rp-8-Br-cAMPS did not fully reverse the PGE2 mediated inhibition of SGLT1 in IEC-18 cells. Further, PKC inhibitor (Calphostin C) also did not fully reverse PGE2 mediated inhibition of SGLT1 in IEC-18 cells. However, inhibition of CamK-II with KN93 reversed PGE2 mediated inhibition of SGLT1 in IEC-18 cells (275±12 pmol/mg protein/2 min in PGE2 + KN93 treated cells, n= 4, p <0.01). PGE2 inhibits SGLT1 secondary to a reduction in BBM co-transporter numbers. Kinetic studies demonstrated that the mechanism of reversal of PGE2 mediated inhibition of SGLT1 by KN93 in IEC-18 cells was secondary to the restoration of co-transporter numbers. Results of the RTQ-PCR studies demonstrated that diminished SGLT1 mRNA levels returned to near normal by KN93 in PGE2 treated cells. Conclusions: These studies demonstrated that while PGE2 increased intracellular cAMP and Ca++ levels, inhibition of SGLT1 appears to be mediated by intracellular Ca++ in IEC-18 cells. Specifically, Cadependent calmodulin kinase II rather than PKC mediates the inhibition of SGLT1 by PGE2 in intestinal epithelial cells.
746 Evidence Against Electrogenicity of Slc26a3 (Down-Regulated in Adenoma) Cl-/HCO3- Exchange in Murine Large Intestine Robert Z. Horack, Janet E. Simpson, Nancy M. Walker, Lane L. Clarke Down-regulated in adenoma (SLC26A3) is the major Cl-/HCO3- exchanger in the apical membrane of the human large intestine and loss of function mutations result in the genetic disease congenital chloride-losing diarrhea (CLD). Recent studies of recombinant SLC26A3 have proposed that its exchange activity is electrogenic with a 2Cl-:1HCO3- stoichiometry (J. Gen. Physiol. 127: 511-524, 2006). Mice with gene-targeted knockout of Slc26a3 (Dra KO) also show the CLD phenotype; however, preliminary studies of the Dra KO distal small intestine did not reveal major changes in transepithelial ionic currents, despite almost complete absence of net Cl- absorption. Therefore, we investigated the electrogenic properties of Dra using microscope-guided conventional microelectrodes to measure the apical membrane potential (Va) in the cecal surface epithelium of Dra wild-type (Dra WT) and Dra KO mice. Dra is expressed at very high levels in the murine cecum (Am.J.Physiol. 293: G923G934, 2007). Using BCECF microfluorimetry of intracellular pH, studies of the cecal surface epithelium showed robust Cl-/HCO3- exchange activity in the Dra WT (0.44 ± 0.03 pH units/min, n = 3) whereas activity was essentially absent in the Dra KO (0.01 ± 0.0 pH units/min, n = 3, p < 0.05). Microelectrode studies of the cecum involved the establishment of a stable impalement in 110 mM Clo Ringers, switch to 0 Clo Ringers to elicit Cl-out/HCO3in exchange and return to 110 mM Clo Ringers solution (22°C). Impalements were accepted if the signal returned to within ±2 mV of baseline upon retraction of the microelectrode. Initial Va after establishing impalement was -19.9 ± 2.2 mV in Dra WT and -23.5 ± 2.1 mV in Dra KO (n = 13-15 impalements, 3 mice each; ns). Switching to 0 Clo Ringers solution depolarized Va in all preparations, however, the ∆Va was not significantly different between Dra WT and KO (Dra WT = +5.2 ±0.7 vs. Dra KO = 4.8 ± 0.5 mV, ns). Preliminary microelectrode studies using the same protocol with solutions warmed to 37°C also did not reveal differences in the ∆Va induced by 0 Clo between Dra WT and KO. We conclude from these studies that 1) Dra (Slc26a3) is the dominant, perhaps sole, Cl-/HCO3- exchanger in the surface epithelium of the murine cecum, and 2) Dra Cl-/HCO3- exchange activity is characterized by electroneutral transport in the native surface epithelium of murine large intestine. Supported by NIDDK.
749 Nherf2 Is Necessary for Basal and All Second Messenger Regulation of NHE3 Activity in Intact Mouse Ileal Na Absorptive Cells Rakhilya Murtazina, Olga Kovbasnjuk, Yueping Chen, Boris M. Hogema, Ursula E. Seidler, Edward J. Weinman, Hugo de Jonge, Mark Donowitz Multi-PDZ-domains containing proteins of the NHERF family take part in regulation of NHE3 activity by providing scaffolding to form regulatory complexes. As an example, in PS120 fibroblasts, NHERF2 or NHERF1 were necessary for cAMP inhibition of NHE3 while only NHERF2 allowed cGMP and Ca2+ inhibition. That there were epithelial specific aspects of NHE3 regulation was demonstrated using NHERF1 null mice in which cAMP inhibition of NHE3 did not occur in proximal tubule, while there was normal inhibition in ileum. Therefore, these studies tested the hypothesis that NHERF2 was necessary for basal and all second messenger inhibition of NHE3 activity in intact mouse ileum. Methods: Initial rates of intact mouse ileal NHE3 activity were determined with two-photon confocal microscopy/ SNARF-4F comparing wild type (WT) and NHERF2 null littermates. Results: 1) Basal NHE3 activity in NHERF2 null ileum (0.17±0.02 ∆pH unit/min) was reduced compared to Wt (0.25±0.02 for WT ∆pH unit/min, p<0.05). Western blotting showed similar NHE3 expression in membrane preparations from both. 2) cAMP inhibited NHE3 activity in WT ileum but not in NHERF2-/- ileum. To examine the mechanism for this NHERF2 dependency of cAMP inhibition of NHE3, PKAII regulatory subunit amount and location were determined. IB showed a marked decrease in PKAII in NHERF2 null ileal membranes with IF demonstrating decrease in both ileal Na absorptive villus and Cl secretory crypt cells. The reduced level of PKA expression was not due to different amounts of mRNA (real time PCR). 3) Ca2+mediated inhibition of NHE3 activity was determined using 10 µM serotonin (5HT) which elevates ileal intracellular Ca2+. 5HT inhibited ileal NHE3 activity in WT ileum by 40±9% but had no effect in ileum from NHERF2 -/- mice. 4) cGMP inhibition of NHE3 activity was determined using 10 nM recombinant E. coli heat stable enterotoxin (STa). Sta decreased WT ileal NHE3 activity by 45±8% but had no effect in ileum from NEHRF2 -/- mice. Conclusions: These results demonstrate: 1) NHERF2 helps set basal ileal NHE3 activity, since NHE3 activity was decreased in ileum from NHERF2 null mice. 2) cAMP inhibition of NHE3 activity requires NHERF2. NHERF2 null ileum has decreased amount of the regulatory subunit of PKAII protein but normal amount of PKAII message. Whether this reduced amount of PKAII explains NHERF2 dependence of cAMP regulation of NHE3 activity is not known. 3) NHERF2 is necessary for Ca2+ -and cGMP-mediated inhibition of ileal NHE3. 4) NHERF2 is a major regulator of basal and second messenger inhibition of NHE3 activity in intact mouse ileum.
747 Cytosolic Phospholipase a2 Differentially Regulates Calcium and cAMPDependent Chloride Secretion in Colonic Epithelial Cells Fergal Donnellan, Frank E. Murray, Brian M. Harvey, Stephen J. Keely Background: Neuroimmune agonists promote intestinal Cl- and fluid secretion predominantly through Ca2+ and cAMP-dependent signalling mechanisms. Previous studies from our group have shown that ERK and JNK MAPKs are important in regulating the extent of secretory responses elicited by Ca2+ but not cAMP-dependent secretagogues. Cytosolic phospholipase A2 (cPLA2) has been shown to be an important constituent of MAPK-dependent signaling pathways in several cell types. Aim: To investigate potential relationships between MAPKs and cPLA2 in regulating colonic epithelial Cl- secretion. Methods: Monolayers of T84 colonic epithelial cells were grown on permeable supports. Protein phosphorylation was analyzed by western blotting. Cl- secretion was measured as changes in short-circuit current (Isc) across voltage-clamped T84 cells mounted in Ussing chambers. Results: Western blot analysis revealed that the Ca2+-dependent agonist, carbachol (CCh; 100 µM) rapidly stimulated cPLA2 phosphorylation (n = 6; p < 0.05) in T84 cells. Furthermore, pretreatment of voltage-clamped T84 cells with AACOCF3 (100 µM), a specific cPLA2 inhibitor, significantly potentiated Isc responses to CCh. Responses to CCh in AACOCF3-pretreated cells were 146.4 ± 2.5 % of those in paired controls (n = 6; p < 0.05). The ERK MAPK inhibitor, PD98059 (20 µM), did not alter CCh-induced cPLA2 phosphorylation, whereas AACOCF3, inhibited both CCh-stimulated ERK and JNK MAPK activation. In apically permeabilized T84 cell monolayers, AACOCF3, significantly potentiated CCh-stimulated K+ conductances, while it did not alter Na + /K + ATPase activity. Similar to CCh, the cAMP-dependent
AGA Abstracts
A-108