- Email: [email protected]
Modulation of vascular norepinephrine responsiveness by potassium channels in normal and portal hypertensive rats
A382 AGA ABSTRACTS
• G1557 MODULATION OF VASCULAR NOREPINEPHRINE RESPONSIVENESS BY POTASSIUM CHANNELS IN NORMAL AND PORTAL HYPERTENSIVE RATS. Z. Jiao, M.S. Taylor and J.N. Benoit. Department of Physiology, University of South Alabama College of Medicine. Mobile, AL. Studies in recent years have provided evidence that opening of potassium channels on the plasma membrane of smooth muscle hyperpolarizes the membrane and induces vasodilatation. To date, 4 types of K+ have been identified on vascular smooth muscle cells. These are voltage-dependent K+channels (K), Ca2+-activated K+-channels ( K ) , inward rectifying K+channels (Ka) and ATP-sensitive K+-channels (K~,~), In spite of the known existence of these various K+-channels on vascular smooth muscle, very little is known about the role of these channels as modulators of mesenteric vascular tone in normal and chronic portal hypertensive conditions. The present study was designed to test the role of K+-channels in the arterial norepinephrine (NE) hyporesponsiveness of chronic portal hypertension. Methods: Pre-hepatic portal hypertension (PH) was produced by portal vein stenosis. Sham operated animals served as controls (SHAM). Small mesenteric resistance arteries (A2) were dissected from the intestinalmesenteric border, cannulated with glass micropipettes and pressurized to 60 mm Hg. Vascular responses to incremental doses of norepinephrine (NE) were assessed after incubation with each of the following channel blockers: 4-Aminopyridine (4-AP, 3mM; Kv-blocker), Iberiotoxin (IBT, 100 nM; K -blocker), Barium Chloride (BaCI2, 50 laM; K~-blocker) or Glibenclamide (GLB., 10 laM; KAw-blocker). Physiological Salt Solution (PSS) was used as a vehicle for all experiments. Results: The table presents the pD2 values (-log[ECho]) for each of the experimental groups (*p < 0.05 from same group Vehicle; # p < 0.05 from same drug SHAM). Group Vehicle 4-AP IBT BaCI2 GLB SHAM 6.33 ~-0.04 6.02± 0.06* 6.64 ± 0.08* 6.47 ± 0.03* 6.51 ± 0.03* PH 6,05 ± 0.06# 5.78 ± 0.09*# 6.39 ± 0.07*# 6.31 ± 0.05*# 6.29 ± 0.05*#
The NE concentration-response relationship of Vehicle treated small mesenteric arteries from PH rats were significantly shifted to the right when compared with SHAM, Treatment of vessels with IBT, BaC12 and GLB increased the sensitivity of both SHAM and PH resistance arteries to NE. On the other hand, K~ channel inhibition reduced vascular NE sensitivity in both SHAM and PH vessels. The magnitude of the shift in sensitivity observed in response to K+-channel blockers was similar between SHAM and PH groups. Conclusions: The results of the present study suggest that potassium channels play a role as modulators of NE responsiveness in both SHAM and PH rats. The hyporesponsiveness to NE in portal hypertensive rats cannot be selectively reversed by K+-blockers. Supported by NIH/NIDDK-51430, • G1558 REDUCED MESENTERIC ARTERIAL NOREPINEPHRINE RESPONSIVENESS IN PORTAL HYPERTENSIVE RATS IS ENDOTHELIAL INDEPENDENT. Z. Jiao and J.N. Bend it. Department of Physiology, University of South Alabama College of Medicine. Mobile, AL.
It is well established that chronic portal hypertension leads to reductions in the responsiveness of splanchnic resistance vessels to vasoconstrictor stimuli. Furthermore, it has been suggested that overproduction of factors known to be synthesized and/or released from the endothelium (e.g., NO and Prustaglandins) could mediate the reduced vasoconstrictor effectiveness. However, the endothelial dependency of the portal hypertension induced vasoconstrictor dysfunction has not been tested. In the present study, we used a unique isolated vascular preparation to assess the role of endothelial dependent factors as mediators of reduced vasoconstrictor effectiveness in portal hypertension. Methods: Prehepatic portal hypertension was produced by portal vein stenosis. Sham operated animals served as controls. Small mesenteric resistance arteries from SHAM and PH rats were dissected from the intestinal-mesentario border, cannulated with glass micropipettes and pressurized to 60 mmHg. Vessels were divided into endothelium intact (E +) and endothelium denuded (E -) groups. The endothelium was removed by rubbing the lumen of the vessel with a human hair. Responses to cumulative doses of norepinephrine (NE) were assessed. Results: Baseline arterial diameter in E + was significantly larger in PH vessels (343 +_11 ~am)than in SHAM (294 + 9 ~am). Removal of the endothelium did not alter diameter of SHAM (302 _+6 pro) or PH (357 -+7 lain). The pD2 (-log [EC~0]) for NE was significantly lower (p < 0.05) in E + vessels from PH rats (6.15 +_0.05) than SHAM (6.37_+0,05), findings consistent with reduced responsiveness. Endothelial denudation increased vascular sensitivity of SHAM (pD2=6.67 _+0.06, p<0.05 from SHAM-E+) and PH (pDz=6.38 _+0.04, p < 0.05 from PH-E+). E- vessels from PH remained less sensitivity to NE than E- vessels from SHAM. The magnitude of the increase in vascular sensitivity induced by endothelial removal in SHAM was similar to that observed in PH. Conclusions: The results of the present study demonstrate a decreased responsiveness to NE in small mesenteric resistance arteries from PH rats. Endothelial denudation does not correct the hyporesponsiveness to NE. We conclude that portal hypertension induced reductions in vascular NE responsiveness are not related to overproduction of endothelial dependent vasodilators. Supported by NIH/NIDDK-51430.
GASTROENTEROLOGYVol. 114, No. 4 • G1559 IL-8 SECRETION BY ENTEROCYTES IN RESPONSE TO TNF-ct REQUIRES MAPK AND IS INDEPENDENT OF PKC. H.B. Jij6n and H.G. Parsons. Department of Medicine, University of Calgary, Calgary, AB. The chemokine interleukin 8 (IL-8) is a key molecule involved in the recruitment of inflammatory cells and its levels are elevated in various pathogenic states, including IBD. This is in accordance with observed exacerbated levels of various potent inducers of IL-8 in IBD mucosa, namely IL-1 and TNF-ct. In the present study we have partially elucidated the mechanism by which the proinflammatory agonist TNF-ct stimulates IL-8 production in the gut. TNF-ct (10 ng/ml) and phorbol esters (PMA, 100 nM) stimulate IL-8 secretion by HT-29 colonic epithelial cells 10 to 30 fold. Maximal IL-8 protein is found 2 hrs. and persists 6 hrs. post-stimulus. This increase is regulated at the transcriptional level as IL-8 production is almost completely inhibited by actinomycin D ( > 90%, 100 laM). RT-PCR analysis indicates that TNF treatment results in a rapid increase in IL-8 mRNA detectable 20 rains, after stimulation and peaking by 60 mins. Phorbol ester stimulation of IL-8 production implies a role for protein kinase C (PKC) in the transduction of signals to the nucleus. Experiments using the PKC inhibitor bisindolylmaleimide (10 laM) confirm PMA-stimulated IL-8 production is PKC-dependent. Further, PKC depletion following chronic PMA exposure (24 hrs., 500 nM) also resulted in an abrogated response to PMA. On the other hand, IL-8 secretion following TNF stimulation is unaffected by these conditions suggesting TNF-mediated IL-8 production is PKC-independant. However, both PMA and TNF treatments lead to tyrosine phosphorylation of MAPK. Western Blotting using polyclonal anti-MAPK sera indicates that both treatments result in the hyper shifting of two bands corresponding to the ERK-1 and ERK-2 isoforms of MAPK. Inhibition of IL-8 secretion using the MAPK-selective inhibitors olomoucine ( > 90%, 20 pM), apigenin ( > 90%, 20 laM) and PD98059 (MAPK-kinase inhibitor, > 70%, 100 ~tM) in response to both PMA and TNF suggests an absolute requirement for MAPK in the regulation of TNF- and PMA-dependent IL-8 production in this colonic cell line. This implies that the various signal transduction pathways which lead to IL-8 secretion (PKC-dependent and independent) may converge at the level of MAPK which may then relay information downstream terminating at nuclear signalling elements (eg. NFKB) resulting in IL-8 secretion. G1560 INTRACELLULAR OXIDANTS AND MAPK ARE REQUIRED FOR TNF-c~-STIMULATED IL-8 SECRETION IN ENTEROCYTES. HB Jiion and H.G. Parsons GI Research Group, University of Calgary, Calgary, AB.-
TNF-~x is a critical component of the inflammatory response in IBD. We have previously shown that TNF-ct stimulates Mitogen Activated Protein Kinase (MAPK), an event which is required for the expression and secretion of Interleukin 8 in HT-29 cells. Thus, at least some of the effects of TNF-o~ on enterocytes may be mediated via MAPK. We have looked at the events responsible for MAPK activation in HT-29 cells. Using TNF receptor blocking antibodies we have determined that MAPK activation following TNF-ct stimulation requires the type I and not the type II TNF-ct receptor. Using analogues of sphingomyelin metabolism we have observed very low activation of MAPK in response to C8- and C2- caramide analogues, a response which does not lead to IL-8 secretion (or apoptosis). The activity of the small GTP-binding protein Ras does not seem to be required as, IL-8 secretion and MAPK activation following TNF-ct treatment are insensitive to FarnesyI Transferase inhibitors. However, MAPK activation and IL-g secretion are profoundly inhibited ( > 80% for IL-8 secretion) by thiol-containing moieties and antioxidants (e.g. nordihydroguariaetic acid, NDGA (100uM)). This suggests that TNF-ct activation of MAPK requires the intracellular generation of reactive oxygen species (ROSs), leading to IL-8 expression and secretion. This is supported by the fact that treatment of HT-29 monolayers with 1 mM hydrogen peroxide, also leads to a significant increase in IL-8 secretion ( > 5 fold). Further, this response is also sensitive to MAPK inhibitors ( > 50%, PD98059). Initial characterization of the intracellular sources of ROIs has ruled out 5-1ipoxygenase, cycloxygenase, xanthine oxidase and mitochondrial respiration. This study demonstrates that MAPK activation following TNF-ct treatment does not require Ras, a ROS-sensitive protein, yet is dependent on ROS generation, an event which can be mimicked by H20 z. Intervention using antioxidants inhibits this event. Therefore, oxidative stress may stimulate the enterocyte to secrete IL-8 in the context of inflammation, leading to an exacerbation of mucosal damage. Further, benefits from antioxidant therapy may partly be mediated via inhibition of ROS-sensitive gene expression in the enterocyte. • G1561
EXTRACELLULAR MATRIX PROTEIN PROMOTES SURVIVAL OF HUMAN COLONIC SUB-EPITHELIAL MYOFIBROBLASTS BY BETAI-INTEGRIN DEPENDENT INHIBITION OF APOPTOSIS. T. M. Jobson, B. C. McKalg, J. Savill, Y. R. Mahida, L P. Hall. Dept of Medicine, Divisions of Therapeutics, Gastroenterology and Renal/Inflammatory Disease, University Hospital, Nottingham, U.K. We have previously shown that human colonic sub-epithelial myofibroblasts (SEMFs) proliferate in response to growth factors and inflammatory
• G1557 MODULATION OF VASCULAR NOREPINEPHRINE RESPONSIVENESS BY POTASSIUM CHANNELS IN NORMAL AND PORTAL HYPERTENSIVE RATS. Z. Jiao, M.S. Taylor and J.N. Benoit. Department of Physiology, University of South Alabama College of Medicine. Mobile, AL. Studies in recent years have provided evidence that opening of potassium channels on the plasma membrane of smooth muscle hyperpolarizes the membrane and induces vasodilatation. To date, 4 types of K+ have been identified on vascular smooth muscle cells. These are voltage-dependent K+channels (K), Ca2+-activated K+-channels ( K ) , inward rectifying K+channels (Ka) and ATP-sensitive K+-channels (K~,~), In spite of the known existence of these various K+-channels on vascular smooth muscle, very little is known about the role of these channels as modulators of mesenteric vascular tone in normal and chronic portal hypertensive conditions. The present study was designed to test the role of K+-channels in the arterial norepinephrine (NE) hyporesponsiveness of chronic portal hypertension. Methods: Pre-hepatic portal hypertension (PH) was produced by portal vein stenosis. Sham operated animals served as controls (SHAM). Small mesenteric resistance arteries (A2) were dissected from the intestinalmesenteric border, cannulated with glass micropipettes and pressurized to 60 mm Hg. Vascular responses to incremental doses of norepinephrine (NE) were assessed after incubation with each of the following channel blockers: 4-Aminopyridine (4-AP, 3mM; Kv-blocker), Iberiotoxin (IBT, 100 nM; K -blocker), Barium Chloride (BaCI2, 50 laM; K~-blocker) or Glibenclamide (GLB., 10 laM; KAw-blocker). Physiological Salt Solution (PSS) was used as a vehicle for all experiments. Results: The table presents the pD2 values (-log[ECho]) for each of the experimental groups (*p < 0.05 from same group Vehicle; # p < 0.05 from same drug SHAM). Group Vehicle 4-AP IBT BaCI2 GLB SHAM 6.33 ~-0.04 6.02± 0.06* 6.64 ± 0.08* 6.47 ± 0.03* 6.51 ± 0.03* PH 6,05 ± 0.06# 5.78 ± 0.09*# 6.39 ± 0.07*# 6.31 ± 0.05*# 6.29 ± 0.05*#
The NE concentration-response relationship of Vehicle treated small mesenteric arteries from PH rats were significantly shifted to the right when compared with SHAM, Treatment of vessels with IBT, BaC12 and GLB increased the sensitivity of both SHAM and PH resistance arteries to NE. On the other hand, K~ channel inhibition reduced vascular NE sensitivity in both SHAM and PH vessels. The magnitude of the shift in sensitivity observed in response to K+-channel blockers was similar between SHAM and PH groups. Conclusions: The results of the present study suggest that potassium channels play a role as modulators of NE responsiveness in both SHAM and PH rats. The hyporesponsiveness to NE in portal hypertensive rats cannot be selectively reversed by K+-blockers. Supported by NIH/NIDDK-51430, • G1558 REDUCED MESENTERIC ARTERIAL NOREPINEPHRINE RESPONSIVENESS IN PORTAL HYPERTENSIVE RATS IS ENDOTHELIAL INDEPENDENT. Z. Jiao and J.N. Bend it. Department of Physiology, University of South Alabama College of Medicine. Mobile, AL.
It is well established that chronic portal hypertension leads to reductions in the responsiveness of splanchnic resistance vessels to vasoconstrictor stimuli. Furthermore, it has been suggested that overproduction of factors known to be synthesized and/or released from the endothelium (e.g., NO and Prustaglandins) could mediate the reduced vasoconstrictor effectiveness. However, the endothelial dependency of the portal hypertension induced vasoconstrictor dysfunction has not been tested. In the present study, we used a unique isolated vascular preparation to assess the role of endothelial dependent factors as mediators of reduced vasoconstrictor effectiveness in portal hypertension. Methods: Prehepatic portal hypertension was produced by portal vein stenosis. Sham operated animals served as controls. Small mesenteric resistance arteries from SHAM and PH rats were dissected from the intestinal-mesentario border, cannulated with glass micropipettes and pressurized to 60 mmHg. Vessels were divided into endothelium intact (E +) and endothelium denuded (E -) groups. The endothelium was removed by rubbing the lumen of the vessel with a human hair. Responses to cumulative doses of norepinephrine (NE) were assessed. Results: Baseline arterial diameter in E + was significantly larger in PH vessels (343 +_11 ~am)than in SHAM (294 + 9 ~am). Removal of the endothelium did not alter diameter of SHAM (302 _+6 pro) or PH (357 -+7 lain). The pD2 (-log [EC~0]) for NE was significantly lower (p < 0.05) in E + vessels from PH rats (6.15 +_0.05) than SHAM (6.37_+0,05), findings consistent with reduced responsiveness. Endothelial denudation increased vascular sensitivity of SHAM (pD2=6.67 _+0.06, p<0.05 from SHAM-E+) and PH (pDz=6.38 _+0.04, p < 0.05 from PH-E+). E- vessels from PH remained less sensitivity to NE than E- vessels from SHAM. The magnitude of the increase in vascular sensitivity induced by endothelial removal in SHAM was similar to that observed in PH. Conclusions: The results of the present study demonstrate a decreased responsiveness to NE in small mesenteric resistance arteries from PH rats. Endothelial denudation does not correct the hyporesponsiveness to NE. We conclude that portal hypertension induced reductions in vascular NE responsiveness are not related to overproduction of endothelial dependent vasodilators. Supported by NIH/NIDDK-51430.
GASTROENTEROLOGYVol. 114, No. 4 • G1559 IL-8 SECRETION BY ENTEROCYTES IN RESPONSE TO TNF-ct REQUIRES MAPK AND IS INDEPENDENT OF PKC. H.B. Jij6n and H.G. Parsons. Department of Medicine, University of Calgary, Calgary, AB. The chemokine interleukin 8 (IL-8) is a key molecule involved in the recruitment of inflammatory cells and its levels are elevated in various pathogenic states, including IBD. This is in accordance with observed exacerbated levels of various potent inducers of IL-8 in IBD mucosa, namely IL-1 and TNF-ct. In the present study we have partially elucidated the mechanism by which the proinflammatory agonist TNF-ct stimulates IL-8 production in the gut. TNF-ct (10 ng/ml) and phorbol esters (PMA, 100 nM) stimulate IL-8 secretion by HT-29 colonic epithelial cells 10 to 30 fold. Maximal IL-8 protein is found 2 hrs. and persists 6 hrs. post-stimulus. This increase is regulated at the transcriptional level as IL-8 production is almost completely inhibited by actinomycin D ( > 90%, 100 laM). RT-PCR analysis indicates that TNF treatment results in a rapid increase in IL-8 mRNA detectable 20 rains, after stimulation and peaking by 60 mins. Phorbol ester stimulation of IL-8 production implies a role for protein kinase C (PKC) in the transduction of signals to the nucleus. Experiments using the PKC inhibitor bisindolylmaleimide (10 laM) confirm PMA-stimulated IL-8 production is PKC-dependent. Further, PKC depletion following chronic PMA exposure (24 hrs., 500 nM) also resulted in an abrogated response to PMA. On the other hand, IL-8 secretion following TNF stimulation is unaffected by these conditions suggesting TNF-mediated IL-8 production is PKC-independant. However, both PMA and TNF treatments lead to tyrosine phosphorylation of MAPK. Western Blotting using polyclonal anti-MAPK sera indicates that both treatments result in the hyper shifting of two bands corresponding to the ERK-1 and ERK-2 isoforms of MAPK. Inhibition of IL-8 secretion using the MAPK-selective inhibitors olomoucine ( > 90%, 20 pM), apigenin ( > 90%, 20 laM) and PD98059 (MAPK-kinase inhibitor, > 70%, 100 ~tM) in response to both PMA and TNF suggests an absolute requirement for MAPK in the regulation of TNF- and PMA-dependent IL-8 production in this colonic cell line. This implies that the various signal transduction pathways which lead to IL-8 secretion (PKC-dependent and independent) may converge at the level of MAPK which may then relay information downstream terminating at nuclear signalling elements (eg. NFKB) resulting in IL-8 secretion. G1560 INTRACELLULAR OXIDANTS AND MAPK ARE REQUIRED FOR TNF-c~-STIMULATED IL-8 SECRETION IN ENTEROCYTES. HB Jiion and H.G. Parsons GI Research Group, University of Calgary, Calgary, AB.-
TNF-~x is a critical component of the inflammatory response in IBD. We have previously shown that TNF-ct stimulates Mitogen Activated Protein Kinase (MAPK), an event which is required for the expression and secretion of Interleukin 8 in HT-29 cells. Thus, at least some of the effects of TNF-o~ on enterocytes may be mediated via MAPK. We have looked at the events responsible for MAPK activation in HT-29 cells. Using TNF receptor blocking antibodies we have determined that MAPK activation following TNF-ct stimulation requires the type I and not the type II TNF-ct receptor. Using analogues of sphingomyelin metabolism we have observed very low activation of MAPK in response to C8- and C2- caramide analogues, a response which does not lead to IL-8 secretion (or apoptosis). The activity of the small GTP-binding protein Ras does not seem to be required as, IL-8 secretion and MAPK activation following TNF-ct treatment are insensitive to FarnesyI Transferase inhibitors. However, MAPK activation and IL-g secretion are profoundly inhibited ( > 80% for IL-8 secretion) by thiol-containing moieties and antioxidants (e.g. nordihydroguariaetic acid, NDGA (100uM)). This suggests that TNF-ct activation of MAPK requires the intracellular generation of reactive oxygen species (ROSs), leading to IL-8 expression and secretion. This is supported by the fact that treatment of HT-29 monolayers with 1 mM hydrogen peroxide, also leads to a significant increase in IL-8 secretion ( > 5 fold). Further, this response is also sensitive to MAPK inhibitors ( > 50%, PD98059). Initial characterization of the intracellular sources of ROIs has ruled out 5-1ipoxygenase, cycloxygenase, xanthine oxidase and mitochondrial respiration. This study demonstrates that MAPK activation following TNF-ct treatment does not require Ras, a ROS-sensitive protein, yet is dependent on ROS generation, an event which can be mimicked by H20 z. Intervention using antioxidants inhibits this event. Therefore, oxidative stress may stimulate the enterocyte to secrete IL-8 in the context of inflammation, leading to an exacerbation of mucosal damage. Further, benefits from antioxidant therapy may partly be mediated via inhibition of ROS-sensitive gene expression in the enterocyte. • G1561
EXTRACELLULAR MATRIX PROTEIN PROMOTES SURVIVAL OF HUMAN COLONIC SUB-EPITHELIAL MYOFIBROBLASTS BY BETAI-INTEGRIN DEPENDENT INHIBITION OF APOPTOSIS. T. M. Jobson, B. C. McKalg, J. Savill, Y. R. Mahida, L P. Hall. Dept of Medicine, Divisions of Therapeutics, Gastroenterology and Renal/Inflammatory Disease, University Hospital, Nottingham, U.K. We have previously shown that human colonic sub-epithelial myofibroblasts (SEMFs) proliferate in response to growth factors and inflammatory