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An intracellular site of angiotensin action
AJH–April 2001–VOL. 14, NO. 4, PART 2
O-41 RENIN EXPRESSION AT SITES OF REPAIR IN THE INFARCTED RAT HEART Yao Sun, Jiakun Zhang, John Zhang, Karl T. Weber. 1 Medicine/Cardiology, University of Tennessee, Memphis, TN, United States,2Kenesiology and Health Program, University of Texas, San Antonio, TX, United States Angiotensin (Ang) II has paracrine and autocrine functions that contribute to structural cardiac remodeling by fibrous tissue following myocardial infarction (MI). The temporal and spatial recruitment of angiotensin converting enzyme (ACE) and AngII receptors by inflammatory and fibroblast-like cells involved in tissue repair of the infarcted heart is now well established. However,the cellular source and involvement of renin in Ang peptide generation in normal and infarcted hearts have not been fully elucidated. In the present study we sought to assess the cellular source, spatial distribution and temporal response of renin expression and synthesis in the rat heart following anterior transmural MI and its relationship to circulating renin activity. At day 3 and week 1, 2, 3 and 4 following left coronary artery ligation, the localization and optical density of cardiac renin mRNA was detected by quantitative in situ hybridization; cardiac and circulating renin activity was measured by radioimmunoassay; cells expressing cardiac renin were detected by immunolabeling; and cardiac injury/repair were assessed by hematoxylin/eosin and collagen specific picrosirius red staining. Unoperated rats served as normal controls. We found: 1) renin mRNA and activity were not detectable in either normal control or noninfarcted myocardium, but were expressed at the site of infarction and other sites of repair involving visceral pericardium and endocardium of interventricular septum at all time points; 2) cells expressing renin at day 3 and week 1, and 2 were predominantly macrophages, while at week 3 and 4, they were primarily myofibroblasts; 3) renin activity in the infarcted myocardium rose progressively over the course of 4 weeks; and 4) circulating renin activity was significantly increased at day 3 and week 1, reached a peak at week 2, declined at week 3 and returned to normal levels at week 4. Thus, renin expression and activity appear on day 3 at sites of repair in the infarcted rat heart and rise progressively thereafter over 4-weeks. Several types of cells are responsible for renin synthesis at sites of repair: primarily macrophages during inflammatory phase of repair; and myofibroblasts during the subsequent fibrogenic phase. Cardiac renin production following MI appears to be independent of circulating renin and to contribute to local AngII generation that regulates tissue repair and structural remodeling following MI. Key Words: Myocardial infarction, Tissue repair, Cardiac renin synthesis
O-42 ANG II ACTIVATES P38 MAP KINASE AND ERK1/2, BUT NOT JNK, IN VASCULAR SMOOTH MUSCLE CELLS FROM SHR R. M. Touyz, Q. N. Diep, G. He, M. El Mabrouk, V. Mardigyan, E. L. Schiffrin. 1Clinical Research Institute of Montreal, University of Montreal, QC, Canada Mitogen activated protein kinases (MAPK) comprise three major members, ERK1/2, p38MAPK and JNK, which influence vascular smooth muscle cell (VSMC) function. The aims of the present study were to determine whether Ang II differentially regulates these MAPKs in VSMCs from SHR. Vascular smooth muscle cells from mesenteric arteries of WKY and SHR were studied. Ang II-induced phosphorylation of ERK1/2, JNK and p38MAPK were assessed by Western blot analysis. mRNA expression of c-fos, a potential downstream target of MAPKs, was assessed by RT-PCR in the absence and presence of PD98059, selective inhibitor of ERK1/2-dependent pathways, and SB202190, selective p38MAPK inhibitor. Ang II increased phosphoryation of ERK1/2 and p38MARK, but not JNK. Responses were significantly increased in © 2001 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
ORALS: Is Angiotensin Doing Anything Inside the Heart
19A
SHR compared with WKY. PP2, selective Src inhibitor, decreased Ang II-mediated activation of MAPKs. Ang II increased c-fos mRNA expression in SHR and had a small stimulatory effect in WKY. These actions were inhibited by PD98059 but not by SB202190. Our data demonstrate that Ang II activates p38 MAPK and ERK1/2, but no JNK, in VSMCs. These effects are enhanced in SHR and are associated with ERK1/2dependent, p 38 MAPK-independent overexpression of c-fos mRNA. Differential activation of MAPKs by Ang II may be important in altered growth signaling in VSMCs from SHR. Key Words: MAP kinases, Angiotensin II, Vascular smooth muscle cells
O-43 INTRACRINE PRODUCTION OF ANGIOTENSIN II INITIATED BY INSULIN Farida Villette-Bounoua, Peter Eggena, Jack D. Barrett, Michael Nyby, Dalila B. Corry, Victoria Smutko, Anne M. Fredal, John Scordato, Yoshitoki Takagawa, Keiko Matsumoto, Michael L. Tuck. 1 Division of endocrinology, Veterans affairs, Greater los Angeles health Care System, UCLA School of Medicine, Sepulveda, CA, United States, 2Renin Biochemistry and Vascular Pharmacology, Veterans Affairs, Greater Los Angeles Health Care System, Sepulveda, CA, United States We have previously reported that exposure of vascular smooth muscle cells (VSMCs) to insulin results in stimulation of angiotensinogen gene expression. The objective of this investigation was to determine whether, after insulin stimulation, the angiotensin II (Ang II) generating cascade takes place intracellularly. We also evaluated the effect of insulin-induced production of AngII on the VSMCs growth, and the action of the angiotensin converting enzyme inhibitor (ACEI), lisinopril (Lis), or the vasopeptidase inhibitor (VPI), omapatrilat (OP), on the insulin-induced growth. Cultured VSMCs, from rat thoracic aorta, were deprived of serum for 24h. Insulin with OP or Lis was added. After 48 hours incubation, Ang II concentration in the cell homogenate was quantified by radioimmunoassay, a method highly specific and sensitive for Ang II. Proliferation was estimated by determination of the rate of DNA synthesis as assayed by pulse labeled 3H-methyl thymidine (3H-TdR) uptake, adjusted to the protein concentration of each sample. Exposure of VSMCs to 1000U/ml insulin for 48 hours resulted in a 37.4% increases in Ang II (p⬍0.01). OP (10-7M) or Lis (10-7M), prevented this insulinmediated increase of Ang II. Insulin increased the rate of 3H-TdR uptake by 31.3⫹/- 6.7% (p⬍0.01) compared to control. In comparison, OP and LIS reduced this action to basal values not significantly different from control. In conclusion, our results demonstrate that in VSMCs : 1) Insulin increases intracellular Ang II production, indicating the presence of an intracellular renin-angiotensin system; 2) Both inhibitors suppress insulin-induced intracellular production of Ang II, suggesting an intracellular action of these drugs. This may clarify the action of ACEI irrespective of the plasma renin-angiotensin system level; and 3) ACEI and VPI inhibit the insulin-mediated growth of VSMCs to the same extent. Key Words: renin angiotensin system, insulin, vascular smooth muscle cells
O-44 AN INTRACELLULAR SITE OF ANGIOTENSIN ACTION Julia L. Cook, Zhuo Zhang, Richard N. Re. 1Research Division, Alton Ochsner Medical Institution, New Orleans, LA, United States Background: This laboratory and others have demonstrated the existence of angiotensin II (AII) nuclear receptors and nuclear AII-mediated changes in transcription. Nevertheless, differentiation of AII effects mediated by cell surface versus nuclear receptors remains a challenge. 0895-7061/01/$20.00
20A
ORALS: Is Angiotensin Doing Anything Inside the Heart
Approach: To address this issue, we mutated the angiotensinogen (Aogen) cDNA by removal of the signal sequence (the AI-encoding region remains intact) in order to produce a non-secreted form of Aogen [Ang(-S)Exp]. A corresponding control [Ang(-S)Cntr] was designed to encode a mutant AI (minus the amino-terminal DR). The entire 3’untranslated region, possessing identified mRNA stabilizing sequences, is retained in both constructs. The modified Aogen-encoding cDNAs were ligated into the pSVL expression plasmid (Pharmacia, SV40 late promoter). Results: H4-II-E-C3 rat liver cells (which express renin and ACE mRNAs as determined by RT-PCR) were stably transfected with Ang(S)Exp/pSVL and Ang(-S)Cntr/pSVL. RNA was purified from expanded clonal isolates and tested by primer extension for presence of transcripts derived from the introduced plasmids. Control and experimental lines demonstrate a 123 bp band (consistent with anticipated size) not present in naive cells. Mitotic indices were measured for cell lines stably trans-
AJH–April 2001–VOL. 14, NO. 4, PART 2
fected with Ang(-S)Exp and Ang(-S)Cntr. Experimental clonal cell lines demonstrate an average of 39 ⫾ 4.8% (p ⬍ .01%) increase in labeled/ unlabeled DNA (BrdU) ratio as compared to control cell lines. The mitogenic effect is blocked by 10-6M losartan but not by 10-6M candesartan (CV 11974). Moreover, candesartan pretreatment blocks the antimitogenic effect of losartan for these transfected cell lines. In contrast, exogenous AII treatment increases BrdU-labeling of naive H4-II-E-C3 cells by 25%, the mitogenic effect of which is completely blocked by both candesartan and losartan (p ⬍ .005). Conclusion: Collectively, the results suggest that a nonsecreted form of Aogen is mitogenic for rat liver cells via production of an intracellularly acting angiotensin and that losartan, but not candesartan, blocks the relevant intracellular receptor after cell-surface receptor endocytosis. Key Words: Angiotensin Receptor Blockade, Intracrine, Angiotensin
O-41 RENIN EXPRESSION AT SITES OF REPAIR IN THE INFARCTED RAT HEART Yao Sun, Jiakun Zhang, John Zhang, Karl T. Weber. 1 Medicine/Cardiology, University of Tennessee, Memphis, TN, United States,2Kenesiology and Health Program, University of Texas, San Antonio, TX, United States Angiotensin (Ang) II has paracrine and autocrine functions that contribute to structural cardiac remodeling by fibrous tissue following myocardial infarction (MI). The temporal and spatial recruitment of angiotensin converting enzyme (ACE) and AngII receptors by inflammatory and fibroblast-like cells involved in tissue repair of the infarcted heart is now well established. However,the cellular source and involvement of renin in Ang peptide generation in normal and infarcted hearts have not been fully elucidated. In the present study we sought to assess the cellular source, spatial distribution and temporal response of renin expression and synthesis in the rat heart following anterior transmural MI and its relationship to circulating renin activity. At day 3 and week 1, 2, 3 and 4 following left coronary artery ligation, the localization and optical density of cardiac renin mRNA was detected by quantitative in situ hybridization; cardiac and circulating renin activity was measured by radioimmunoassay; cells expressing cardiac renin were detected by immunolabeling; and cardiac injury/repair were assessed by hematoxylin/eosin and collagen specific picrosirius red staining. Unoperated rats served as normal controls. We found: 1) renin mRNA and activity were not detectable in either normal control or noninfarcted myocardium, but were expressed at the site of infarction and other sites of repair involving visceral pericardium and endocardium of interventricular septum at all time points; 2) cells expressing renin at day 3 and week 1, and 2 were predominantly macrophages, while at week 3 and 4, they were primarily myofibroblasts; 3) renin activity in the infarcted myocardium rose progressively over the course of 4 weeks; and 4) circulating renin activity was significantly increased at day 3 and week 1, reached a peak at week 2, declined at week 3 and returned to normal levels at week 4. Thus, renin expression and activity appear on day 3 at sites of repair in the infarcted rat heart and rise progressively thereafter over 4-weeks. Several types of cells are responsible for renin synthesis at sites of repair: primarily macrophages during inflammatory phase of repair; and myofibroblasts during the subsequent fibrogenic phase. Cardiac renin production following MI appears to be independent of circulating renin and to contribute to local AngII generation that regulates tissue repair and structural remodeling following MI. Key Words: Myocardial infarction, Tissue repair, Cardiac renin synthesis
O-42 ANG II ACTIVATES P38 MAP KINASE AND ERK1/2, BUT NOT JNK, IN VASCULAR SMOOTH MUSCLE CELLS FROM SHR R. M. Touyz, Q. N. Diep, G. He, M. El Mabrouk, V. Mardigyan, E. L. Schiffrin. 1Clinical Research Institute of Montreal, University of Montreal, QC, Canada Mitogen activated protein kinases (MAPK) comprise three major members, ERK1/2, p38MAPK and JNK, which influence vascular smooth muscle cell (VSMC) function. The aims of the present study were to determine whether Ang II differentially regulates these MAPKs in VSMCs from SHR. Vascular smooth muscle cells from mesenteric arteries of WKY and SHR were studied. Ang II-induced phosphorylation of ERK1/2, JNK and p38MAPK were assessed by Western blot analysis. mRNA expression of c-fos, a potential downstream target of MAPKs, was assessed by RT-PCR in the absence and presence of PD98059, selective inhibitor of ERK1/2-dependent pathways, and SB202190, selective p38MAPK inhibitor. Ang II increased phosphoryation of ERK1/2 and p38MARK, but not JNK. Responses were significantly increased in © 2001 by the American Journal of Hypertension, Ltd. Published by Elsevier Science Inc.
ORALS: Is Angiotensin Doing Anything Inside the Heart
19A
SHR compared with WKY. PP2, selective Src inhibitor, decreased Ang II-mediated activation of MAPKs. Ang II increased c-fos mRNA expression in SHR and had a small stimulatory effect in WKY. These actions were inhibited by PD98059 but not by SB202190. Our data demonstrate that Ang II activates p38 MAPK and ERK1/2, but no JNK, in VSMCs. These effects are enhanced in SHR and are associated with ERK1/2dependent, p 38 MAPK-independent overexpression of c-fos mRNA. Differential activation of MAPKs by Ang II may be important in altered growth signaling in VSMCs from SHR. Key Words: MAP kinases, Angiotensin II, Vascular smooth muscle cells
O-43 INTRACRINE PRODUCTION OF ANGIOTENSIN II INITIATED BY INSULIN Farida Villette-Bounoua, Peter Eggena, Jack D. Barrett, Michael Nyby, Dalila B. Corry, Victoria Smutko, Anne M. Fredal, John Scordato, Yoshitoki Takagawa, Keiko Matsumoto, Michael L. Tuck. 1 Division of endocrinology, Veterans affairs, Greater los Angeles health Care System, UCLA School of Medicine, Sepulveda, CA, United States, 2Renin Biochemistry and Vascular Pharmacology, Veterans Affairs, Greater Los Angeles Health Care System, Sepulveda, CA, United States We have previously reported that exposure of vascular smooth muscle cells (VSMCs) to insulin results in stimulation of angiotensinogen gene expression. The objective of this investigation was to determine whether, after insulin stimulation, the angiotensin II (Ang II) generating cascade takes place intracellularly. We also evaluated the effect of insulin-induced production of AngII on the VSMCs growth, and the action of the angiotensin converting enzyme inhibitor (ACEI), lisinopril (Lis), or the vasopeptidase inhibitor (VPI), omapatrilat (OP), on the insulin-induced growth. Cultured VSMCs, from rat thoracic aorta, were deprived of serum for 24h. Insulin with OP or Lis was added. After 48 hours incubation, Ang II concentration in the cell homogenate was quantified by radioimmunoassay, a method highly specific and sensitive for Ang II. Proliferation was estimated by determination of the rate of DNA synthesis as assayed by pulse labeled 3H-methyl thymidine (3H-TdR) uptake, adjusted to the protein concentration of each sample. Exposure of VSMCs to 1000U/ml insulin for 48 hours resulted in a 37.4% increases in Ang II (p⬍0.01). OP (10-7M) or Lis (10-7M), prevented this insulinmediated increase of Ang II. Insulin increased the rate of 3H-TdR uptake by 31.3⫹/- 6.7% (p⬍0.01) compared to control. In comparison, OP and LIS reduced this action to basal values not significantly different from control. In conclusion, our results demonstrate that in VSMCs : 1) Insulin increases intracellular Ang II production, indicating the presence of an intracellular renin-angiotensin system; 2) Both inhibitors suppress insulin-induced intracellular production of Ang II, suggesting an intracellular action of these drugs. This may clarify the action of ACEI irrespective of the plasma renin-angiotensin system level; and 3) ACEI and VPI inhibit the insulin-mediated growth of VSMCs to the same extent. Key Words: renin angiotensin system, insulin, vascular smooth muscle cells
O-44 AN INTRACELLULAR SITE OF ANGIOTENSIN ACTION Julia L. Cook, Zhuo Zhang, Richard N. Re. 1Research Division, Alton Ochsner Medical Institution, New Orleans, LA, United States Background: This laboratory and others have demonstrated the existence of angiotensin II (AII) nuclear receptors and nuclear AII-mediated changes in transcription. Nevertheless, differentiation of AII effects mediated by cell surface versus nuclear receptors remains a challenge. 0895-7061/01/$20.00
20A
ORALS: Is Angiotensin Doing Anything Inside the Heart
Approach: To address this issue, we mutated the angiotensinogen (Aogen) cDNA by removal of the signal sequence (the AI-encoding region remains intact) in order to produce a non-secreted form of Aogen [Ang(-S)Exp]. A corresponding control [Ang(-S)Cntr] was designed to encode a mutant AI (minus the amino-terminal DR). The entire 3’untranslated region, possessing identified mRNA stabilizing sequences, is retained in both constructs. The modified Aogen-encoding cDNAs were ligated into the pSVL expression plasmid (Pharmacia, SV40 late promoter). Results: H4-II-E-C3 rat liver cells (which express renin and ACE mRNAs as determined by RT-PCR) were stably transfected with Ang(S)Exp/pSVL and Ang(-S)Cntr/pSVL. RNA was purified from expanded clonal isolates and tested by primer extension for presence of transcripts derived from the introduced plasmids. Control and experimental lines demonstrate a 123 bp band (consistent with anticipated size) not present in naive cells. Mitotic indices were measured for cell lines stably trans-
AJH–April 2001–VOL. 14, NO. 4, PART 2
fected with Ang(-S)Exp and Ang(-S)Cntr. Experimental clonal cell lines demonstrate an average of 39 ⫾ 4.8% (p ⬍ .01%) increase in labeled/ unlabeled DNA (BrdU) ratio as compared to control cell lines. The mitogenic effect is blocked by 10-6M losartan but not by 10-6M candesartan (CV 11974). Moreover, candesartan pretreatment blocks the antimitogenic effect of losartan for these transfected cell lines. In contrast, exogenous AII treatment increases BrdU-labeling of naive H4-II-E-C3 cells by 25%, the mitogenic effect of which is completely blocked by both candesartan and losartan (p ⬍ .005). Conclusion: Collectively, the results suggest that a nonsecreted form of Aogen is mitogenic for rat liver cells via production of an intracellularly acting angiotensin and that losartan, but not candesartan, blocks the relevant intracellular receptor after cell-surface receptor endocytosis. Key Words: Angiotensin Receptor Blockade, Intracrine, Angiotensin