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Characterization of endothelin vasomotor responses in submucosal arterioles in the guinea pig intestine
April 1995
• THE ROLE OF DIFFERENTIATION AND GROWTH FACTORS IN T H E EXPRESSION OF NHE-2 AND NHE-3 mRNA IN CACO-2 C E L L S . K,E. Kim. L. Schmidt, P.K. Dudeja, T.J. Layden and K. Ramaswamy. Department of Medicine, University of Illinois at Chicago and Westside VA Medical Ceoler, Chicago, IL. A family of functionally and structurally related Na+/H + exchanger (NHE) isoforms NHE 1-4, have been identified in mammalian species. NHE-2 and 3 appear to be localized to the apical membrane 'and may be involved in luminal Na + absorption. We have been interested in determining the factors which regulate and determine the expression of the putative apical Na + absorptive isoforms, using the human epithelial cell line, Caco-2. The Caco-2 cell line has been used as a moclel for the study of enterocyte function and gene expression during differentiation, v-ras transfected Caco-2 cells provide a comparative model of terminal differentiation with the early induulion of differentiation markers such as TGF-oc and 13. Our previous studies using CaCo-2 cells using the qualitative, highly sensilive RT-PCR suggest that NHE-3 mRNA is expressed in confluenl, not preconfiuent cells. NHE-2 mRNA expression, in contrast, was present under both conditions. The aims of this study, therefore, are to investigate the role of differentiation and growth promoting agents in the quantitative expression of NHE 2 and 3 mRNA using Caco-2 cells in continuous culture. Specifically, Caco-2 cells were cultured with and without the addition of factors known to promote cell proliferation and differentiation (TGF-~, TGF-[~), while v-ras transfected Caco-2 cells were used to assess the effect of terminal differentiation on these isoforms. 32p labelled antisense riboprobes specific for human NHE 1-3 have been designed in our laboratory to quantitate mRNA levels by RNase protection assay (RPA). The abnndance of NHE-2 and 3 mRNA, normalized to G-3-PDH, does not change with differentiation (2-21 days post plating), after the intial onset of expression. TGF-c~ or TGF-~ at a concentration of 5ng/ml, stimulated NHE 2 and 3 expression in preconfluent conditions at a twolbld greater abundance than confluent conditions in the absence of growth factors, v-ras transfected Caco-2 cells, in contrast, express both NHE-2 and 3 mRNA by RT-PCR in pre and confluent conditions. These results indicate that NHE-2 may be constitutively expressed, upregulated in response to growth factors and therefore may share characteritics with the basolateral NHE-I isoform. In contrast, NHE-3 mRNA expression is regulated by conditions and growth factors that promote terminal differentiation. (Supported by the Stetler Foundation, NIDDK and the Dept. of Veterans Affairs))
OXIDANT-MEDIATED PHAGOCYTIC KILLING BY THE HEPATIC RETICULOENDOTHELIAL SYSTEM A. Klein, D. Wang, S. Takao, S. Kondo, G. Bulkley, Dept. Surgery, Johns Hopkins University, Baltimore, MD Introducton: We discriminately quantified hepatic phagocytic clearance (HPC) and hepatic killing of intravenously injected circulating E.coli in rats, measuring the contribution of reactive oxygen intermediates to HPC and hepatic killing. Methods: Intravenonsly injected E.coli labeled with Na~lCr and 5-[a~51]-deoxyutidine were cleared rapidly from the bloodstream by the liver. Hepatic 5~Crremained stable for 24h following injection and provided a reliable measurement of HPC. Hepatic x2sI decreased over time, accurately reflecting the number of viable bacteria remaining in the fiver (validated by quantitative culture of liver homoganates). In order to compare hepatic killing of bacteria among rats with different levds of HPC, hepatic killing efficiency was calculated (HIKE=[~ICr - a25I] / ~Cr). Four strains of E.coli were employed in this study: UM1 (cataiase-deficient); CSH7 (parental strain of UM1, catalase-suflicient); AB1157 (SOD-deficient); and JI132 (parental strain ofAB1157, SOD-sufficient). Xanthine Oxidase (XO) inhibition was produced either with allopurinol (50mg/kg) or a tungsten diet (3 wks). Superoxide dismutase (SOD) or cataiase were administered to a subgroup of rats at the time of bacteria inoculation(4mg/kg bolus, lmg/kg/min IV). Seven separate pairs of experiments were carried out in which rats were injecte d with one of the E.coli strains ± pretreatment with alloporinol,tungsten, catalase, or SOD (treatment group n-~5 and corresponding control group n=6 in each experiment). Results: HPC was similar in catalase-deficient, SOD-deficient, or wild-type enzymesufficient E.coli strains, and was not altered by pre-treatment with allopurinol, tungsten, catalase, nor SOD. HKE ofcatalase-deficent E.coli was significantly greater than that of the paired catalase-sufficient strain (55±7% vs 21±7%; p<0.01). Administration of exogenous catalase to rats ininbited KKE of UM1 (p<0.01 vs untreated UM1), as did inhibition of XO-dependent production of toxic oxidants by pre-treatrnent with alloputinol or administration of a tungsten diet (!0<0.05 and p<0.01 vs untreated UMI, respectively). SOD treaianent of rats did not alter HKE ofUM1. Allopurinol did not alter HKE of SOD-deficient or wild-type E.coli. Condusion: The increased susceptibility to hepatic killing of catatase-deficient UM1 compared to catalase-sufficient CSH7 or to catalase-treated UM1 indicates that hydrogen peroxide plays a role in bacterial killing by the hepatic RES. The suppression of UM1 killing by allopurinol pre-tteatment or by administration of a tungsten diet, suggests that XO is a source of this oxidant generation.
Intestinal Disorders
A295
• CHARACTERIZATION OF ENDOTHELIN VASOMOTOR RESPONSES IN S U B M U C O S A L ARTERIOLES IN T H E GUINEA PIG INTESTINE. A. Kirton., S. Vanner . GI Diseases Research Unit, Queen's University, Kingston, Canada. Endothelin (El'), one of the most potent vasoaetive peptides known, appears to be released during multiple pathological states but little is known about its mechanism of action in the intestine. This study characterized the actions of endothelin on in vitro submueosal arterioles (n=82) and examined the cellular properties involved, Videdomicroscopy was used to monitor changes in the outside diameter of arterioles (od = 4 0 - 9 2 #m) in submucosal preparations from the guinea pig ileum. All drugs were added by superfusion. ET-1 (200 pM20nM) evoked a dose-dependent vasoconstriction (EC~o=2nM)~ ET-3 was less potent ( E C 5 0 = 4 0 n M ) . Prepro-ET-1 (Big endothelin-l)(100nM) also evoked constrictions but was less potent (equipotent to 600 pM ET-1) and slower in onset (9 times longer vs. ET-1). Compared to vasoconstrictions evoked by phenylephrine, P G F 2 ~, and vasopressin, responses to ET-1 were dramatically sustained following washout of the drug (time to relaxation o f 50% of the resting diameter 8 times greater for ET-1). The ETA receptor antagonist BQ-123 (200nM) caused a parallel shift to the fight in the ET-1 vasoconstrictor dose-response curve. The ETa agonist IRL 1620 (200nM) ha~i no effect on the resting diameter. The possibility that endothelin dilated arterioles through activation of ET a receceptors was also examined. Arterioles were preconstricted with PGF2c~ (300nM) and sup~rfusion of E T - 3 , ET-1, or IRL 1620 failed to evoke a dilator response. Furthermore, constrictor responses to submaximal ET-1 (2nM) were not altered by the nitric oxide (NO) synthase antagonist L - N M M A (300/zM), suggesting there had not been co-incident activation of ET e receptors on endothelium causing NO release. Because ETc receptors have been found on neurons, intracellular recordings were made from submucosal neurons to determine if ET may activate submucsoal vasodilator neurons. ET-1 (50nM) had no effect on resting membrane potentials. In addition, constrictor responses to submaximal ET-1 (2nM) were not altered by TI'X. These findings suggest that ET acts as a potent and sustained vasoconstrictor in this intestinal vascular bed by activating arteriolar ETA receptors.
VASOCONSTRICTORS INDUCE H I G H FREQUENCY PANCREATIC ARTERIOLE VASOMOTION. R Kleinman, PH Guth, FC Brunicardi, RS Zhang, E~J_Livingston. Division of General Surgery, VAMC West Los Angeles, UCLA School of Medicine; Los Angeles, Calif. 90024-6904. Vasomotion is a phenomenon thought to occur at low frequency during stressful conditions. Our preliminary observations have been that for the pancreatic microcirculation, high frequency vasomotor events were inducible by norepinephrine. The purpose of the present study was to determine if non-adrenergic vasoconstrictors could reproduce this phenomenon. Because of the high frequency of the oscillations we hypothesized that nerves might mediate the contractions. Therefore, we performed experiments in a group of capsaicin denervated animals. Methods: The pancreas of anesthetized Sprague-Dawley rats (n = 6-12 per group) was exteriorized, maintained at 37°C and viewed under an Olympus microscope utilizing monochromatic (495 nm) light with a final magnification of 300x. Images were recorded on video tape and later analyzed with an image-splitting device to measure vessel diameter. Following equilibration, the pancreas was exposed to one of the following suffusions: C: Krebs buffer control, NE: graded doses of norepinephrine, CAP-NE: same as NE except in rats chronically denervated with capsaicin, Via: graded doses ofvasopressin Results: Animals perfused only with Krebs did not display vasomotion nor change in their arteriotar diameter for the duration of the experiment. Upon exposure to NE vasomotion was observed at a frequency of 16 + 1.0 cycles per second (cpm) and this rate was unaffected by capsaicin pretreatment. The frequency was significantly higher with VP at 23.5 _+6.0 cpm (p<.05, ANOVA with contrasts). NE resulted in a dose dependent decrease in mean arteriolar diameter which was also observed in the CAP-NE group. VP resulted in substantial decreases in mean vessel diameter (26.3+ 5.8 to 46.8 + 7.3 % of basal values, p<.05, ANOVA with contrasts) at all doses tested but there was no significant dose dependency. Conclusion: Pancreatic arterioles demonstrate high frequency vasomotion when exposed to vasoconsttictive agents that constrict by different mechanisms. This effect has not been observed in other splanchnic beds in our laboratory and is not affected by capsaicin sensitive nerves. The oscillations prevent sustained reductions in blood flow and might serve as a protective mechanism for the pancreas during stress.
• THE ROLE OF DIFFERENTIATION AND GROWTH FACTORS IN T H E EXPRESSION OF NHE-2 AND NHE-3 mRNA IN CACO-2 C E L L S . K,E. Kim. L. Schmidt, P.K. Dudeja, T.J. Layden and K. Ramaswamy. Department of Medicine, University of Illinois at Chicago and Westside VA Medical Ceoler, Chicago, IL. A family of functionally and structurally related Na+/H + exchanger (NHE) isoforms NHE 1-4, have been identified in mammalian species. NHE-2 and 3 appear to be localized to the apical membrane 'and may be involved in luminal Na + absorption. We have been interested in determining the factors which regulate and determine the expression of the putative apical Na + absorptive isoforms, using the human epithelial cell line, Caco-2. The Caco-2 cell line has been used as a moclel for the study of enterocyte function and gene expression during differentiation, v-ras transfected Caco-2 cells provide a comparative model of terminal differentiation with the early induulion of differentiation markers such as TGF-oc and 13. Our previous studies using CaCo-2 cells using the qualitative, highly sensilive RT-PCR suggest that NHE-3 mRNA is expressed in confluenl, not preconfiuent cells. NHE-2 mRNA expression, in contrast, was present under both conditions. The aims of this study, therefore, are to investigate the role of differentiation and growth promoting agents in the quantitative expression of NHE 2 and 3 mRNA using Caco-2 cells in continuous culture. Specifically, Caco-2 cells were cultured with and without the addition of factors known to promote cell proliferation and differentiation (TGF-~, TGF-[~), while v-ras transfected Caco-2 cells were used to assess the effect of terminal differentiation on these isoforms. 32p labelled antisense riboprobes specific for human NHE 1-3 have been designed in our laboratory to quantitate mRNA levels by RNase protection assay (RPA). The abnndance of NHE-2 and 3 mRNA, normalized to G-3-PDH, does not change with differentiation (2-21 days post plating), after the intial onset of expression. TGF-c~ or TGF-~ at a concentration of 5ng/ml, stimulated NHE 2 and 3 expression in preconfluent conditions at a twolbld greater abundance than confluent conditions in the absence of growth factors, v-ras transfected Caco-2 cells, in contrast, express both NHE-2 and 3 mRNA by RT-PCR in pre and confluent conditions. These results indicate that NHE-2 may be constitutively expressed, upregulated in response to growth factors and therefore may share characteritics with the basolateral NHE-I isoform. In contrast, NHE-3 mRNA expression is regulated by conditions and growth factors that promote terminal differentiation. (Supported by the Stetler Foundation, NIDDK and the Dept. of Veterans Affairs))
OXIDANT-MEDIATED PHAGOCYTIC KILLING BY THE HEPATIC RETICULOENDOTHELIAL SYSTEM A. Klein, D. Wang, S. Takao, S. Kondo, G. Bulkley, Dept. Surgery, Johns Hopkins University, Baltimore, MD Introducton: We discriminately quantified hepatic phagocytic clearance (HPC) and hepatic killing of intravenously injected circulating E.coli in rats, measuring the contribution of reactive oxygen intermediates to HPC and hepatic killing. Methods: Intravenonsly injected E.coli labeled with Na~lCr and 5-[a~51]-deoxyutidine were cleared rapidly from the bloodstream by the liver. Hepatic 5~Crremained stable for 24h following injection and provided a reliable measurement of HPC. Hepatic x2sI decreased over time, accurately reflecting the number of viable bacteria remaining in the fiver (validated by quantitative culture of liver homoganates). In order to compare hepatic killing of bacteria among rats with different levds of HPC, hepatic killing efficiency was calculated (HIKE=[~ICr - a25I] / ~Cr). Four strains of E.coli were employed in this study: UM1 (cataiase-deficient); CSH7 (parental strain of UM1, catalase-suflicient); AB1157 (SOD-deficient); and JI132 (parental strain ofAB1157, SOD-sufficient). Xanthine Oxidase (XO) inhibition was produced either with allopurinol (50mg/kg) or a tungsten diet (3 wks). Superoxide dismutase (SOD) or cataiase were administered to a subgroup of rats at the time of bacteria inoculation(4mg/kg bolus, lmg/kg/min IV). Seven separate pairs of experiments were carried out in which rats were injecte d with one of the E.coli strains ± pretreatment with alloporinol,tungsten, catalase, or SOD (treatment group n-~5 and corresponding control group n=6 in each experiment). Results: HPC was similar in catalase-deficient, SOD-deficient, or wild-type enzymesufficient E.coli strains, and was not altered by pre-treatment with allopurinol, tungsten, catalase, nor SOD. HKE ofcatalase-deficent E.coli was significantly greater than that of the paired catalase-sufficient strain (55±7% vs 21±7%; p<0.01). Administration of exogenous catalase to rats ininbited KKE of UM1 (p<0.01 vs untreated UM1), as did inhibition of XO-dependent production of toxic oxidants by pre-treatrnent with alloputinol or administration of a tungsten diet (!0<0.05 and p<0.01 vs untreated UMI, respectively). SOD treaianent of rats did not alter HKE ofUM1. Allopurinol did not alter HKE of SOD-deficient or wild-type E.coli. Condusion: The increased susceptibility to hepatic killing of catatase-deficient UM1 compared to catalase-sufficient CSH7 or to catalase-treated UM1 indicates that hydrogen peroxide plays a role in bacterial killing by the hepatic RES. The suppression of UM1 killing by allopurinol pre-tteatment or by administration of a tungsten diet, suggests that XO is a source of this oxidant generation.
Intestinal Disorders
A295
• CHARACTERIZATION OF ENDOTHELIN VASOMOTOR RESPONSES IN S U B M U C O S A L ARTERIOLES IN T H E GUINEA PIG INTESTINE. A. Kirton., S. Vanner . GI Diseases Research Unit, Queen's University, Kingston, Canada. Endothelin (El'), one of the most potent vasoaetive peptides known, appears to be released during multiple pathological states but little is known about its mechanism of action in the intestine. This study characterized the actions of endothelin on in vitro submueosal arterioles (n=82) and examined the cellular properties involved, Videdomicroscopy was used to monitor changes in the outside diameter of arterioles (od = 4 0 - 9 2 #m) in submucosal preparations from the guinea pig ileum. All drugs were added by superfusion. ET-1 (200 pM20nM) evoked a dose-dependent vasoconstriction (EC~o=2nM)~ ET-3 was less potent ( E C 5 0 = 4 0 n M ) . Prepro-ET-1 (Big endothelin-l)(100nM) also evoked constrictions but was less potent (equipotent to 600 pM ET-1) and slower in onset (9 times longer vs. ET-1). Compared to vasoconstrictions evoked by phenylephrine, P G F 2 ~, and vasopressin, responses to ET-1 were dramatically sustained following washout of the drug (time to relaxation o f 50% of the resting diameter 8 times greater for ET-1). The ETA receptor antagonist BQ-123 (200nM) caused a parallel shift to the fight in the ET-1 vasoconstrictor dose-response curve. The ETa agonist IRL 1620 (200nM) ha~i no effect on the resting diameter. The possibility that endothelin dilated arterioles through activation of ET a receceptors was also examined. Arterioles were preconstricted with PGF2c~ (300nM) and sup~rfusion of E T - 3 , ET-1, or IRL 1620 failed to evoke a dilator response. Furthermore, constrictor responses to submaximal ET-1 (2nM) were not altered by the nitric oxide (NO) synthase antagonist L - N M M A (300/zM), suggesting there had not been co-incident activation of ET e receptors on endothelium causing NO release. Because ETc receptors have been found on neurons, intracellular recordings were made from submucosal neurons to determine if ET may activate submucsoal vasodilator neurons. ET-1 (50nM) had no effect on resting membrane potentials. In addition, constrictor responses to submaximal ET-1 (2nM) were not altered by TI'X. These findings suggest that ET acts as a potent and sustained vasoconstrictor in this intestinal vascular bed by activating arteriolar ETA receptors.
VASOCONSTRICTORS INDUCE H I G H FREQUENCY PANCREATIC ARTERIOLE VASOMOTION. R Kleinman, PH Guth, FC Brunicardi, RS Zhang, E~J_Livingston. Division of General Surgery, VAMC West Los Angeles, UCLA School of Medicine; Los Angeles, Calif. 90024-6904. Vasomotion is a phenomenon thought to occur at low frequency during stressful conditions. Our preliminary observations have been that for the pancreatic microcirculation, high frequency vasomotor events were inducible by norepinephrine. The purpose of the present study was to determine if non-adrenergic vasoconstrictors could reproduce this phenomenon. Because of the high frequency of the oscillations we hypothesized that nerves might mediate the contractions. Therefore, we performed experiments in a group of capsaicin denervated animals. Methods: The pancreas of anesthetized Sprague-Dawley rats (n = 6-12 per group) was exteriorized, maintained at 37°C and viewed under an Olympus microscope utilizing monochromatic (495 nm) light with a final magnification of 300x. Images were recorded on video tape and later analyzed with an image-splitting device to measure vessel diameter. Following equilibration, the pancreas was exposed to one of the following suffusions: C: Krebs buffer control, NE: graded doses of norepinephrine, CAP-NE: same as NE except in rats chronically denervated with capsaicin, Via: graded doses ofvasopressin Results: Animals perfused only with Krebs did not display vasomotion nor change in their arteriotar diameter for the duration of the experiment. Upon exposure to NE vasomotion was observed at a frequency of 16 + 1.0 cycles per second (cpm) and this rate was unaffected by capsaicin pretreatment. The frequency was significantly higher with VP at 23.5 _+6.0 cpm (p<.05, ANOVA with contrasts). NE resulted in a dose dependent decrease in mean arteriolar diameter which was also observed in the CAP-NE group. VP resulted in substantial decreases in mean vessel diameter (26.3+ 5.8 to 46.8 + 7.3 % of basal values, p<.05, ANOVA with contrasts) at all doses tested but there was no significant dose dependency. Conclusion: Pancreatic arterioles demonstrate high frequency vasomotion when exposed to vasoconsttictive agents that constrict by different mechanisms. This effect has not been observed in other splanchnic beds in our laboratory and is not affected by capsaicin sensitive nerves. The oscillations prevent sustained reductions in blood flow and might serve as a protective mechanism for the pancreas during stress.