Roli Prasad | Benedictine University (original) (raw)

Papers by Roli Prasad

Research paper thumbnail of Galanin Contributes to the Excess Colonic Fluid Secretion Observed in Dextran Sulfate Sodium Murine Colitis

Inflammatory Bowel Diseases, 2004

Galanin is present in enteric nerves lining the gastrointestinal (GI) tract where it is normally ... more Galanin is present in enteric nerves lining the gastrointestinal (GI) tract where it is normally involved in regulating intestinal motility by binding to the galanin-1 receptor (Gal1R) subtype expressed by smooth muscle cells. In contrast, although epithelial cells lining the colon do not normally express Gal1R, this protein is upregulated by the inflammation-associated transcription factor NF-B. We previously showed that the murine colitis induced by dextran sulfate sodium (DSS) was associated with increased Gal1R expression as well as by increased colonic fluid secretion. Although Gal1R upregulation by colonic epithelial cells results in increased intestinal Cl − secretion, the relative contributions of galanin to this excess colonic fluid secretion could not be determined. We therefore created a mouse genetically incapable of synthesizing Gal1R (GAL1R −/− mice). We herein demonstrate that both wild-type and GAL1R −/− mice developed identical histologic lesions in response to DSS. This was characterized by a marked inflammatory infiltrate, activation of NF-B in both enterocytes and enteric nerves, and a threefold increase in neuronal galanin. Colonic fluid secretion, while increased, was approximately half that in GAL1R −/− mice as compared with their wild-type littermates. Overall, then, these findings strongly suggest that approximately half of the increase in colonic fluid secretion in DSS colitis is due to up-regulation of the Gal1R.

Research paper thumbnail of Neurotensin (NT) signaling in the rabbit proximal colon involves phospholipase C (PLC), Src Kinases, and ERK phosphorylation

Research paper thumbnail of Taurodeoxycholate (TDC) signaling in the human colon involves PLC translocation and tyrosine phosphorylation

Gastroenterology, 2001

The intracellular signalling pathways responsible for differentiation of intestinal cells remain ... more The intracellular signalling pathways responsible for differentiation of intestinal cells remain largely unknown. An important role for p38 Mitogen-Activated Protein Kinases (MAPKs) in various vertebrate cell differentiation processes has emerged recently. In the present study, we analyzed the involvement of p38 MAPKs in differentiation of human intestinal cells. Methods: Studies were done in the human colon cancer cell line Caco-2/15 which spontaneously differentiates into enterocytes several days after confluence, and in PCDE which are primary cultures of differentiated villus enterocytes. Immunofluorescence experiments were performed on cryostat sections of human fetal intestine. Results: 1-p38 MAPK activity dramatically increased as soon as Caco-2/15 cells reached confluence. 2-Addition of SB203580 (a specific p38 inhibitor) during differentiation of Caco-2/15 cells interfered with the sustained activation of p38 and strongly attenuated sucrase-isomaltase (SI) gene and protein expression as well as lactase and alkaline phosphatase activities. 3-Conversely, treatment of primary cultures of normal differentiated enterocytes with 20 uM SB203580 also significantly reduced expression of SI by 55% after 2 days. 4-Ectopic expression of Cdx2 resulted in a 13-fold increase in SI promoter activity and addition of $8203580 dose-dependenfiy inhibite~ the effect of Cdx2 with a maximal effect observed with 20 uM 5-To determine whether p38 increases the transcriptional activity of Cdx2, we used fusion proteins containing the transactivation domain of Cdx2 fused to Gal4(DBD). Caco-2/15 cells were cotransfected with a luciferase reporter gene containing five copies of a Gal4 binding site upstream of a minimal promoter and Gal4-Cdx2 expression plasmids. Inhibition of the p38 pathway reduced by 58% Cdx2dependent reporter gene expression. In contrast, inhibition of the p42/p44 MAPK pathway with the PD98059 inhibitor had no effect. 6-Pull-down GST and immunoprecipitation experiments demonstrated both a direct and specific interaction of Cdx2 with p38 and Cdx2 phosphorylafion in differentiating Caco-2/15 cells. 7-Although p38 was expressed in both villus and crypt cells, phosphorylated and active forms were detected primarily in the nuclei of differentiated villus cells. Conclusion: Our results indicate that p38 MAPK may be involved in the regulation of Cdx2 function and in the control of intestinal cell differentiation as well.

Research paper thumbnail of Taurodeoxycholate (TDC) signaling in rabbit distal colon involves inhibition of ERK phosphorylation but does not involve EGFR stimulation

Research paper thumbnail of Weanling, but not adult, rabbit colon absorbs bile acids: flux is linked to expression of putative bile acid transporters

Research paper thumbnail of transporters acids: flux is linked to expression of putative bile acid Weanling, but not adult, rabbit colon absorbs bile

Research paper thumbnail of Evidence for the presence of cGMP-dependent protein kinase-II in human distal colon and in T84, the colonic cell line

Biochimica Et Biophysica Acta-molecular Cell Research, 2000

Heat-stable enterotoxin (STa) stimulates intestinal Cl 3 secretion by activating guanylate cyclas... more Heat-stable enterotoxin (STa) stimulates intestinal Cl 3 secretion by activating guanylate cyclase C (GCC) to increase intracellular cyclic GMP (cGMP). In the colon, cGMP action could involve protein kinase (PK) G-II or PKA pathways, depending on the segment and species. In the human colon, both PKG and PKA pathways have been implicated, and, therefore, the present study examined the mechanism of cGMP-mediated Cl 3 transport in primary cultures of human distal colonocytes and in T84, the colonic cell line. Both cell preparations express mRNA for CFTR, Na -K -2Cl 3 cotransporter (NKCC1), GCC and PKG-II as detected by RT-PCR. The effects of STa and the PKG-specific cGMP analogues, 8Br-cGMP and 8pCPT-cGMP, on Cl 3 transport were measured using a halide-sensitive probe. In primary human colonocytes and T84 cells, STa, the cGMP analogues and the cAMP-dependent secretagogue, prostaglandin E 1 (PGE 1 ), enhanced Cl 3 transport. The effects of 8Br-cGMP and 8pCPT-cGMP suggested the involvement of PKG, and this was explored further in T84 cells. The effects of 8pCPT-cGMP were dose-dependent and sensitive to the PKG inhibitor, H8 (70 WM), but H8 had no effect on PGE 1 -induced Cl 3 secretion. In contrast, a PKA inhibitor, H7 (50 WM), blocked PGE 1 -mediated but not 8pCPT-cGMP-induced Cl 3 transport. 8pCPT-cGMP enhanced phosphorylation of the PKG-specific substrate, 2A3, by T84 membranes in vitro. This phosphorylation was inhibited by H8. These results strongly suggest that cGMP activates Cl 3 transport through a PKG-II pathway in primary cells and in the T84 cell line of the human colon. ß

Research paper thumbnail of Epidermal Growth Factor Stimulates Chloride Transport in Primary Cultures of Weanling and Adult Rabbit Colonocytes

Journal of Pediatric Gastroenterology and Nutrition, 2007

Objectives: We have shown that Ca 2þ -dependent regulation of Cl À secretion in the mammalian col... more Objectives: We have shown that Ca 2þ -dependent regulation of Cl À secretion in the mammalian colon exhibits age dependence. Because epidermal growth factor (EGF) has a well-established role in growth and can increase intracellular calcium [Ca 2þ ] i , it is conceivable that its developmental influence may extend to the regulation of intestinal ion transport. In this study, we examined the role of EGF in the regulation of Cl À transport in the developing rabbit distal colon. Materials and Methods: Because serum contains growth factors, which could have confounded our studies, we first established an optimal milieu for testing EGF in primary cultures of adult rabbit distal colonocytes by culturing them for 24 h in media containing 0%, 1%, 5%, and 20% serum. Chloride transport (millimoles per second) and [Ca 2þ ] i were measured with use of the fluorescent indicator N-(ethoxycarbonylmethyl ) -6 -methoxyquinolinium bromide (MQAE) and Fura-2AM, respectively. Results: Serum depletion had no effect on cell number, DNA content, or basal Cl À transport, but it significantly affected cell viability. In media with 0%, 1%, or 20% serum, bethanechol, 8BrcAMP, taurodeoxycholate, and EGF stimulated Cl À transport to a similar extent. EGF maximally stimulated Cl À transport at 16.3 nmol/L and 20 minutes. Bethanechol, but not EGF, increased [Ca 2þ ] i . EGF did not alter bethanecholstimulated Cl À transport or [Ca 2þ ] i . EGF acts via an EGFreceptor and mitogen activated protein kinase (MAPK) signaling pathway, since stimulation of Cl À transport was abolished by genistein, AG1478, and PD98059. Weanling and adult colonocytes, cultured in 1% serum, showed similar basal and EGF-stimulated Cl À transport. Conclusions: EGF stimulates rabbit colonic Cl À transport via a Ca 2þ -independent, tyrosine kinase-and MAPK-dependent pathway, and its effects are not age dependent. JPGN 44: 300-311, 2007.

Research paper thumbnail of Role of protein kinase C- in the age-dependent secretagogue action of bile acids in mammalian colon

American Journal of Physiology-cell Physiology, 2007

The role of specific PKC isoforms in the regulation of epithelial Clsecretion by Ca 2+dependent s... more The role of specific PKC isoforms in the regulation of epithelial Clsecretion by Ca 2+dependent secretagogues remains controversial. In the developing rabbit distal colon, the bile acid taurodeoxycholate (TDC) acts via [Ca 2+ ] i to stimulate Cltransport in adult, but not in young, animals, whereas the PKC activator, phorbol dibutyrate (PDB), stimulates Cltransport at all ages. We tested the hypothesis that specific PKC isoforms account for the age-specific effects of TDC. The effects of conventional and novel PKC-specific inhibitors on TDC-and PDBstimulated Cltransport in adult and weanling colonocytes were assessed using 6-methoxyquinolyl acetoethyl ester. In adult colonocytes, the cPKC inhibitor Gö6976 inhibited PDB, but not TDC action, whereas the cPKC and nPKC inhibitor Gö6850 blocked both TDC and PDB action. Additionally rottlerin and the PKC -specific inhibitor peptide ( V1-1) inhibited TDCand PDB-stimulated Cltransport in adult colonocytes. Rottlerin also decreased TDC-stimulated short-circuit current in intact colonic epithelia. Only Gö6976, but neither rottlerin nor V1-1 inhibited PDB-stimulated transport in weanling colonocytes. Colonic lysates express PKC ,and -protein equally at all ages but not PKC or -at any age. Expression of PKC and PKC protein was newborn>adult>weanling, whereas PKC was expressed in adult but not in weanling or newborn colonocytes. TDC (1.6-fold) and PDB (2.0-fold) stimulated PKC enzymatic activity in adult but failed to do so in weanling colonocytes. PKC mRNA expression showed age-dependence. Thus, PKC appears critical for the action of TDC in the adult colon, and its low expression in young animals may account for their inability to secrete in response to bile acids.

Research paper thumbnail of Phospholipase C and Src Tyrosine Kinases Mediate Neurotensin-Stimulated Cl− Secretion in Rabbit Proximal Colon

Digestive Diseases and Sciences, 2004

Calcium-dependent secretagogues, such as neurotensin, stimulate age-dependent chloride transport ... more Calcium-dependent secretagogues, such as neurotensin, stimulate age-dependent chloride transport in rabbit distal colonocytes, but their action in the proximal colon is unknown. This study examines the effect of neurotensin on chloride transport and its mechanism of action in rabbit proximal colonocytes. Our results show that neurotensin stimulates chloride transport only in adult, and not weanling or newborn, colonocytes. The calcium ionophore A23187 shows similar age dependence, while PGE2, which acts via cAMP, stimulates transport in all ages. The roles of phospholipase C, tyrosine kinases, and src tyrosine kinases were examined using specific inhibitors, i.e., U73122, genistein, and PP2, respectively. All three agents significantly inhibit neurotensin-stimulated chloride transport in adult colonocytes. In conclusion, this study reports for the first time that neurotensin stimulates chloride secretion in rabbit proximal colonocytes. This is also the first demonstration that neurotensin action exhibits age dependence and is dependent on phospholipase C and src tyrosine kinase activity.

Research paper thumbnail of Galanin Contributes to the Excess Colonic Fluid Secretion Observed in Dextran Sulfate Sodium Murine Colitis

Inflammatory Bowel Diseases, 2004

Galanin is present in enteric nerves lining the gastrointestinal (GI) tract where it is normally ... more Galanin is present in enteric nerves lining the gastrointestinal (GI) tract where it is normally involved in regulating intestinal motility by binding to the galanin-1 receptor (Gal1R) subtype expressed by smooth muscle cells. In contrast, although epithelial cells lining the colon do not normally express Gal1R, this protein is upregulated by the inflammation-associated transcription factor NF-B. We previously showed that the murine colitis induced by dextran sulfate sodium (DSS) was associated with increased Gal1R expression as well as by increased colonic fluid secretion. Although Gal1R upregulation by colonic epithelial cells results in increased intestinal Cl − secretion, the relative contributions of galanin to this excess colonic fluid secretion could not be determined. We therefore created a mouse genetically incapable of synthesizing Gal1R (GAL1R −/− mice). We herein demonstrate that both wild-type and GAL1R −/− mice developed identical histologic lesions in response to DSS. This was characterized by a marked inflammatory infiltrate, activation of NF-B in both enterocytes and enteric nerves, and a threefold increase in neuronal galanin. Colonic fluid secretion, while increased, was approximately half that in GAL1R −/− mice as compared with their wild-type littermates. Overall, then, these findings strongly suggest that approximately half of the increase in colonic fluid secretion in DSS colitis is due to up-regulation of the Gal1R.

Research paper thumbnail of Neurotensin (NT) signaling in the rabbit proximal colon involves phospholipase C (PLC), Src Kinases, and ERK phosphorylation

Research paper thumbnail of Taurodeoxycholate (TDC) signaling in the human colon involves PLC translocation and tyrosine phosphorylation

Gastroenterology, 2001

The intracellular signalling pathways responsible for differentiation of intestinal cells remain ... more The intracellular signalling pathways responsible for differentiation of intestinal cells remain largely unknown. An important role for p38 Mitogen-Activated Protein Kinases (MAPKs) in various vertebrate cell differentiation processes has emerged recently. In the present study, we analyzed the involvement of p38 MAPKs in differentiation of human intestinal cells. Methods: Studies were done in the human colon cancer cell line Caco-2/15 which spontaneously differentiates into enterocytes several days after confluence, and in PCDE which are primary cultures of differentiated villus enterocytes. Immunofluorescence experiments were performed on cryostat sections of human fetal intestine. Results: 1-p38 MAPK activity dramatically increased as soon as Caco-2/15 cells reached confluence. 2-Addition of SB203580 (a specific p38 inhibitor) during differentiation of Caco-2/15 cells interfered with the sustained activation of p38 and strongly attenuated sucrase-isomaltase (SI) gene and protein expression as well as lactase and alkaline phosphatase activities. 3-Conversely, treatment of primary cultures of normal differentiated enterocytes with 20 uM SB203580 also significantly reduced expression of SI by 55% after 2 days. 4-Ectopic expression of Cdx2 resulted in a 13-fold increase in SI promoter activity and addition of $8203580 dose-dependenfiy inhibite~ the effect of Cdx2 with a maximal effect observed with 20 uM 5-To determine whether p38 increases the transcriptional activity of Cdx2, we used fusion proteins containing the transactivation domain of Cdx2 fused to Gal4(DBD). Caco-2/15 cells were cotransfected with a luciferase reporter gene containing five copies of a Gal4 binding site upstream of a minimal promoter and Gal4-Cdx2 expression plasmids. Inhibition of the p38 pathway reduced by 58% Cdx2dependent reporter gene expression. In contrast, inhibition of the p42/p44 MAPK pathway with the PD98059 inhibitor had no effect. 6-Pull-down GST and immunoprecipitation experiments demonstrated both a direct and specific interaction of Cdx2 with p38 and Cdx2 phosphorylafion in differentiating Caco-2/15 cells. 7-Although p38 was expressed in both villus and crypt cells, phosphorylated and active forms were detected primarily in the nuclei of differentiated villus cells. Conclusion: Our results indicate that p38 MAPK may be involved in the regulation of Cdx2 function and in the control of intestinal cell differentiation as well.

Research paper thumbnail of Taurodeoxycholate (TDC) signaling in rabbit distal colon involves inhibition of ERK phosphorylation but does not involve EGFR stimulation

Research paper thumbnail of Weanling, but not adult, rabbit colon absorbs bile acids: flux is linked to expression of putative bile acid transporters

Research paper thumbnail of transporters acids: flux is linked to expression of putative bile acid Weanling, but not adult, rabbit colon absorbs bile

Research paper thumbnail of Evidence for the presence of cGMP-dependent protein kinase-II in human distal colon and in T84, the colonic cell line

Biochimica Et Biophysica Acta-molecular Cell Research, 2000

Heat-stable enterotoxin (STa) stimulates intestinal Cl 3 secretion by activating guanylate cyclas... more Heat-stable enterotoxin (STa) stimulates intestinal Cl 3 secretion by activating guanylate cyclase C (GCC) to increase intracellular cyclic GMP (cGMP). In the colon, cGMP action could involve protein kinase (PK) G-II or PKA pathways, depending on the segment and species. In the human colon, both PKG and PKA pathways have been implicated, and, therefore, the present study examined the mechanism of cGMP-mediated Cl 3 transport in primary cultures of human distal colonocytes and in T84, the colonic cell line. Both cell preparations express mRNA for CFTR, Na -K -2Cl 3 cotransporter (NKCC1), GCC and PKG-II as detected by RT-PCR. The effects of STa and the PKG-specific cGMP analogues, 8Br-cGMP and 8pCPT-cGMP, on Cl 3 transport were measured using a halide-sensitive probe. In primary human colonocytes and T84 cells, STa, the cGMP analogues and the cAMP-dependent secretagogue, prostaglandin E 1 (PGE 1 ), enhanced Cl 3 transport. The effects of 8Br-cGMP and 8pCPT-cGMP suggested the involvement of PKG, and this was explored further in T84 cells. The effects of 8pCPT-cGMP were dose-dependent and sensitive to the PKG inhibitor, H8 (70 WM), but H8 had no effect on PGE 1 -induced Cl 3 secretion. In contrast, a PKA inhibitor, H7 (50 WM), blocked PGE 1 -mediated but not 8pCPT-cGMP-induced Cl 3 transport. 8pCPT-cGMP enhanced phosphorylation of the PKG-specific substrate, 2A3, by T84 membranes in vitro. This phosphorylation was inhibited by H8. These results strongly suggest that cGMP activates Cl 3 transport through a PKG-II pathway in primary cells and in the T84 cell line of the human colon. ß

Research paper thumbnail of Epidermal Growth Factor Stimulates Chloride Transport in Primary Cultures of Weanling and Adult Rabbit Colonocytes

Journal of Pediatric Gastroenterology and Nutrition, 2007

Objectives: We have shown that Ca 2þ -dependent regulation of Cl À secretion in the mammalian col... more Objectives: We have shown that Ca 2þ -dependent regulation of Cl À secretion in the mammalian colon exhibits age dependence. Because epidermal growth factor (EGF) has a well-established role in growth and can increase intracellular calcium [Ca 2þ ] i , it is conceivable that its developmental influence may extend to the regulation of intestinal ion transport. In this study, we examined the role of EGF in the regulation of Cl À transport in the developing rabbit distal colon. Materials and Methods: Because serum contains growth factors, which could have confounded our studies, we first established an optimal milieu for testing EGF in primary cultures of adult rabbit distal colonocytes by culturing them for 24 h in media containing 0%, 1%, 5%, and 20% serum. Chloride transport (millimoles per second) and [Ca 2þ ] i were measured with use of the fluorescent indicator N-(ethoxycarbonylmethyl ) -6 -methoxyquinolinium bromide (MQAE) and Fura-2AM, respectively. Results: Serum depletion had no effect on cell number, DNA content, or basal Cl À transport, but it significantly affected cell viability. In media with 0%, 1%, or 20% serum, bethanechol, 8BrcAMP, taurodeoxycholate, and EGF stimulated Cl À transport to a similar extent. EGF maximally stimulated Cl À transport at 16.3 nmol/L and 20 minutes. Bethanechol, but not EGF, increased [Ca 2þ ] i . EGF did not alter bethanecholstimulated Cl À transport or [Ca 2þ ] i . EGF acts via an EGFreceptor and mitogen activated protein kinase (MAPK) signaling pathway, since stimulation of Cl À transport was abolished by genistein, AG1478, and PD98059. Weanling and adult colonocytes, cultured in 1% serum, showed similar basal and EGF-stimulated Cl À transport. Conclusions: EGF stimulates rabbit colonic Cl À transport via a Ca 2þ -independent, tyrosine kinase-and MAPK-dependent pathway, and its effects are not age dependent. JPGN 44: 300-311, 2007.

Research paper thumbnail of Role of protein kinase C- in the age-dependent secretagogue action of bile acids in mammalian colon

American Journal of Physiology-cell Physiology, 2007

The role of specific PKC isoforms in the regulation of epithelial Clsecretion by Ca 2+dependent s... more The role of specific PKC isoforms in the regulation of epithelial Clsecretion by Ca 2+dependent secretagogues remains controversial. In the developing rabbit distal colon, the bile acid taurodeoxycholate (TDC) acts via [Ca 2+ ] i to stimulate Cltransport in adult, but not in young, animals, whereas the PKC activator, phorbol dibutyrate (PDB), stimulates Cltransport at all ages. We tested the hypothesis that specific PKC isoforms account for the age-specific effects of TDC. The effects of conventional and novel PKC-specific inhibitors on TDC-and PDBstimulated Cltransport in adult and weanling colonocytes were assessed using 6-methoxyquinolyl acetoethyl ester. In adult colonocytes, the cPKC inhibitor Gö6976 inhibited PDB, but not TDC action, whereas the cPKC and nPKC inhibitor Gö6850 blocked both TDC and PDB action. Additionally rottlerin and the PKC -specific inhibitor peptide ( V1-1) inhibited TDCand PDB-stimulated Cltransport in adult colonocytes. Rottlerin also decreased TDC-stimulated short-circuit current in intact colonic epithelia. Only Gö6976, but neither rottlerin nor V1-1 inhibited PDB-stimulated transport in weanling colonocytes. Colonic lysates express PKC ,and -protein equally at all ages but not PKC or -at any age. Expression of PKC and PKC protein was newborn>adult>weanling, whereas PKC was expressed in adult but not in weanling or newborn colonocytes. TDC (1.6-fold) and PDB (2.0-fold) stimulated PKC enzymatic activity in adult but failed to do so in weanling colonocytes. PKC mRNA expression showed age-dependence. Thus, PKC appears critical for the action of TDC in the adult colon, and its low expression in young animals may account for their inability to secrete in response to bile acids.

Research paper thumbnail of Phospholipase C and Src Tyrosine Kinases Mediate Neurotensin-Stimulated Cl− Secretion in Rabbit Proximal Colon

Digestive Diseases and Sciences, 2004

Calcium-dependent secretagogues, such as neurotensin, stimulate age-dependent chloride transport ... more Calcium-dependent secretagogues, such as neurotensin, stimulate age-dependent chloride transport in rabbit distal colonocytes, but their action in the proximal colon is unknown. This study examines the effect of neurotensin on chloride transport and its mechanism of action in rabbit proximal colonocytes. Our results show that neurotensin stimulates chloride transport only in adult, and not weanling or newborn, colonocytes. The calcium ionophore A23187 shows similar age dependence, while PGE2, which acts via cAMP, stimulates transport in all ages. The roles of phospholipase C, tyrosine kinases, and src tyrosine kinases were examined using specific inhibitors, i.e., U73122, genistein, and PP2, respectively. All three agents significantly inhibit neurotensin-stimulated chloride transport in adult colonocytes. In conclusion, this study reports for the first time that neurotensin stimulates chloride secretion in rabbit proximal colonocytes. This is also the first demonstration that neurotensin action exhibits age dependence and is dependent on phospholipase C and src tyrosine kinase activity.