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The endogenous ouabainlike factor in human plasma and MHS rat tissues: comparison of two independent assays
AJH 1996; 9:34A-35A
POSTERS: Cell Biology and Growth Factors
Cl
C2
GTP-BINDING (G) PROTEIN CARBOXYL METHYLATION CONTROLS AGONIST-INDUCED INTRACELLULAR Ca" RESPONSE IN RAT RESISTANCE ARTERIES AND AORTIC VASCULAR SMOOTH MUSCLE CELLS (VSMC). JB Roullet·, H Xue·, U Lull, CM Roullet, and DA McCarron. Oregon Health Sciences University, Portland, OR. Carboxyl methylation of the prenylcysteine terminus of G proteins controls receptor-mediated signal transduction in nonvascular tissues. Previous experiments by us with rat arteries have shown that the response to vasoconstnctors such as norepinephrine (NE) was significantly reduced by N-acetyl-Sgeranylgeranyl-L-cysteine (AGGC), a specific Inhibitor of carboxyl methyl transferase. We therefore studied the effect of AGGC on intracellular Ca" (iCa") signaling. [iCa'j was deterrOined (fura-2) in rat resistance arteries (RA, - 200 J.lm diam.) challenged with NE (10"M), and in vasopressin (AVP)stimulated rat aortic Al0 VSMC. Ethanol and N-acetyl-S-geranylL-cysteine (AGC) were used as negative controls. AGGC (5 J.lM, 30 min. incub.), but not AGC, significantly decreased the iCa"response of RA to NE, as well as contraction (m±SEM, Table). MARV (n = 10) Control AGC AGGC [iCa'*], nM 289±37 296±40 97:1:10·<0001 Contraction, mN/mm 4.6:1:0.2 4.5:1:0.2 1.77:1:0.2 p
THE EFFECT OF ENDOTHELlN-1 (ET·1) AND PROTEIN KINASE C (PKC) INHIBITOR· CHELERITHRIN ON DNA SYNTHESIS IN VASCULAR SMOOTH MUSCLE CELLS (VSMC) FROM YOUNG SHR AND WKY RATS. B Rosen, J Barg, D Gefel, R Zimllchman·. Wolfson Medical Center, Holan and Sackler Medical School, Tel Aviv, Israel. ET.1 Is I vasoactive peptide produced by endothelial cells. ET1 has a vasoconstrictive and mitogenic effect upon VSMC, and it may play I role In the pathogenesis of hypertension and atherosclerosis. Its mitogenic activity Is mediated by PKC. Oblective: To determine the responsiveness of VSMC's from young SHR and WI('( rats to the mitogenic effect of ET-1 and the effect of PKC inhibitor- Chelerithrin (Chel.). Methods: VSMC's from aortas of young SHR and WKY rats (1-3 weeks old) were grown in microplates for 24 and 48 hours. Then, they were Incubated In the presence of different concentrations of ET-1 (0.1- 100 nM) for 24 hours. VSMC's were Incubated for different times (1-4 hours) with ET-1 In the presence of different concentrations of chelerithrin (0-10 nM). The rate of DNA synthesis was examined by Incorporation of labeled thymidine to DNA of VSMC's. ~ Increasing concentrations of ET-1 dose dependently increased thymidine Incorporation to DNA. There was no difference In thymidine incorporation between SHR and WKY VSMC's Incubated for 24 and 48 hours. No difference was found between VSMS's from SHR and WKY rats Incubated with ET-1 for 1,2,3, and 4 hours In SHR and WKY rats. Incubation for 1 hour caused maximal mitogenic effect. Chel caused a dose dependent Inhibition of the stimulatory effect of ET-1 In VSMC's from SHR and WKY rats. VSMC's from SHR were significantly more susceptible to the Inhibitory effect of Chel in comparison with VSMC's from WI('( rats. Conclusions' ET-1 exerts a similar mitogenic effect on VSMC's from aortas from SHR and WI('( rats. Stimulation with ET-1 for 1 hour Is sufficient to cause an almost maximal stimulatory effect. PKC Inhibitor -Chel. causes a dose- dependent inhibition of the stimulatory effect of ET-1 In both strains, although VSMC's from SHR are more susceptible to inhibition.
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EXPRESSION OF TGF-B IS ELEVATED IN THE RENAL CORTEX OF SHR/N-CORPULENT RATS WITH DIABETIC NEPHROPATHY. ~, JS Striffler, AA Abraham, OE Michaelis IV, E Scalbert, N Thibault, and MT Velasquez•. George Washington University, Washington, DC,
AUTOCRINE/PARACRINE CONTROL OF VASCULAR SMOOTH MUSCLE CELL GROWTH BY MACROPHAGE COLONY-STIMULATING FACTOR. ~ ~, Gogusev J", Herembert T, Drueke T and Zhu DL". C~S URA 1482 and 'INSERM U 90, Necker Hosp, p~s" France and ,. Shanghai Institute of Hypertension, RUI-Jm Hosp, Shanghai, China. In several vascular diseases an abnormal of vascular smooth muscle cell (VSMC) proliferation is associated with the presence of macrophages. We therefore examined whether macrophage colony-stimulating factor (M-CSF) might playa role in the control of VSMC growth. VSMCs were isolated from rat aorta and maintained in culture. The influen~e of M-CSF on DNA s~nthesis and proto-oncogene expressIon was assessed by H-thymidine incorporation measurement and Northern analysis, respectively. Macrophage COlony-stimulating activity (M-CSA) was ascertained by a bioassay. Addition to quiescent VSMCs of various dilutions of L929 serum-free condition medium (S-FCM), as a source of M-CSF, concentration-dependently induced DNA synthesis; this effect could be blunted by the concomitant addition of a specific anti M-CSF anltbody. SFCM from L929 also induced and/or modulated the expression of c-fos, c-myc and jun B. Recombinant human M-CSF weakly induced DNA synthesis when added alone, but synergistically enhanced that induced by thrombin, PDGF and bFGF. M-CSA was clearly detected in VSMC-derived S-FCM. The presence of c-fms mRNA and fms oncoprotein, as revealed by Northern blot analysis and immunocytochemistry, respectively, indicated that VSMCs expressed M-CSF receptors. These results support the hypotheSIS of a paracrinelautocrine mechanism Whereby M-CSF could affect the growth of cultured rat VSMCs.
S
I : CI
~
[)
Beltsville Human Nutrition Research Center, USDA,
Beltsville, Maryland, and Institut de Recherches Internationales Servier, Par~s, France. D~abet~c nephropathy ~s characterized by glomerular mesangial expansion with accumulation of extracellular matrix
(ECM)
sclerosis, and interstitial factor
involved
in
ECM
proteins,
fibros~s.
production
glomerular
TGF-B, a key and
tissue
repair, has been implicated in the pathogenesis of diabetic renal lesions. We investigated TGF-B express~on in kidneys of SHR/N-corpulent (cp) rats, a genetic model of type 2 diabetes and hypertension with renal les~ons resembling human d~abetic nephropathy. Expression of TGF-B protein was assessed by immunohistochemistry using a polyclonal rabbit antibody which recognizes extracellular TGFB in kidneys of 6-8 month old obese male SHR/N-cp rats (n=5) and lean controls (n-5). Rats were fed 54' carbohydrate diet containing 18' sucrose and 36' starch. Renal morphology was evaluated for
glomerular
lesions
of
mesangial
expansion
and
sclerosis (MES). A tubulointerstitial score (TIS), an index of severity of tubulointerstitial lesions, e.g., tubular atrophy, cellular ~nf~ltration. and interstitial fibros~s was assigned. Morphologic analys~s showed that the number of MES and TIS were higher in kidneys of obese than in lean rats (MES, 31±5' vs 4±1'. pc.Ol, TIS, +2.2±0.4 vs +0.S±0.2, pc.Ol). A 4-fold increase of TGF-B immunostaining in the renal cortex (expressed as number of foci of
interstitial staining) was found in kidneys of obese rats compared to lean rats (pc. 02) . Treatment of obese rats (n-s) with the ACE inh~b~tor per~ndopril for 3 months reduced the glomerular les~ons,
TIS, and the amount of TGF-B
stain~ng
in
renal cortex. We conclude that 1) TGF-B protein ~s elevated in the renal parenchyma of obese SHR/N-cp rats with diabetic nephropathy, and 2) treatment of
hypertension with perindopril ameliorates the renal
and the increased TGF-B expression. Key Words:
les~ons
Diabetic nephropathy, perindopril, TGF-B, rat model
Key Words: Endothelin-1, Chelerithrin, Protein Kinase-C, SHR.
Key words: vascular smooth muscle cell, M-CSF, cell growth, macrophage, vascular hypertrophy.
POSTERS: Cell Biology and Growth Factors
Cl
C2
GTP-BINDING (G) PROTEIN CARBOXYL METHYLATION CONTROLS AGONIST-INDUCED INTRACELLULAR Ca" RESPONSE IN RAT RESISTANCE ARTERIES AND AORTIC VASCULAR SMOOTH MUSCLE CELLS (VSMC). JB Roullet·, H Xue·, U Lull, CM Roullet, and DA McCarron. Oregon Health Sciences University, Portland, OR. Carboxyl methylation of the prenylcysteine terminus of G proteins controls receptor-mediated signal transduction in nonvascular tissues. Previous experiments by us with rat arteries have shown that the response to vasoconstnctors such as norepinephrine (NE) was significantly reduced by N-acetyl-Sgeranylgeranyl-L-cysteine (AGGC), a specific Inhibitor of carboxyl methyl transferase. We therefore studied the effect of AGGC on intracellular Ca" (iCa") signaling. [iCa'j was deterrOined (fura-2) in rat resistance arteries (RA, - 200 J.lm diam.) challenged with NE (10"M), and in vasopressin (AVP)stimulated rat aortic Al0 VSMC. Ethanol and N-acetyl-S-geranylL-cysteine (AGC) were used as negative controls. AGGC (5 J.lM, 30 min. incub.), but not AGC, significantly decreased the iCa"response of RA to NE, as well as contraction (m±SEM, Table). MARV (n = 10) Control AGC AGGC [iCa'*], nM 289±37 296±40 97:1:10·<0001 Contraction, mN/mm 4.6:1:0.2 4.5:1:0.2 1.77:1:0.2 p
THE EFFECT OF ENDOTHELlN-1 (ET·1) AND PROTEIN KINASE C (PKC) INHIBITOR· CHELERITHRIN ON DNA SYNTHESIS IN VASCULAR SMOOTH MUSCLE CELLS (VSMC) FROM YOUNG SHR AND WKY RATS. B Rosen, J Barg, D Gefel, R Zimllchman·. Wolfson Medical Center, Holan and Sackler Medical School, Tel Aviv, Israel. ET.1 Is I vasoactive peptide produced by endothelial cells. ET1 has a vasoconstrictive and mitogenic effect upon VSMC, and it may play I role In the pathogenesis of hypertension and atherosclerosis. Its mitogenic activity Is mediated by PKC. Oblective: To determine the responsiveness of VSMC's from young SHR and WI('( rats to the mitogenic effect of ET-1 and the effect of PKC inhibitor- Chelerithrin (Chel.). Methods: VSMC's from aortas of young SHR and WKY rats (1-3 weeks old) were grown in microplates for 24 and 48 hours. Then, they were Incubated In the presence of different concentrations of ET-1 (0.1- 100 nM) for 24 hours. VSMC's were Incubated for different times (1-4 hours) with ET-1 In the presence of different concentrations of chelerithrin (0-10 nM). The rate of DNA synthesis was examined by Incorporation of labeled thymidine to DNA of VSMC's. ~ Increasing concentrations of ET-1 dose dependently increased thymidine Incorporation to DNA. There was no difference In thymidine incorporation between SHR and WKY VSMC's Incubated for 24 and 48 hours. No difference was found between VSMS's from SHR and WKY rats Incubated with ET-1 for 1,2,3, and 4 hours In SHR and WKY rats. Incubation for 1 hour caused maximal mitogenic effect. Chel caused a dose dependent Inhibition of the stimulatory effect of ET-1 In VSMC's from SHR and WKY rats. VSMC's from SHR were significantly more susceptible to the Inhibitory effect of Chel in comparison with VSMC's from WI('( rats. Conclusions' ET-1 exerts a similar mitogenic effect on VSMC's from aortas from SHR and WI('( rats. Stimulation with ET-1 for 1 hour Is sufficient to cause an almost maximal stimulatory effect. PKC Inhibitor -Chel. causes a dose- dependent inhibition of the stimulatory effect of ET-1 In both strains, although VSMC's from SHR are more susceptible to inhibition.
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C4
EXPRESSION OF TGF-B IS ELEVATED IN THE RENAL CORTEX OF SHR/N-CORPULENT RATS WITH DIABETIC NEPHROPATHY. ~, JS Striffler, AA Abraham, OE Michaelis IV, E Scalbert, N Thibault, and MT Velasquez•. George Washington University, Washington, DC,
AUTOCRINE/PARACRINE CONTROL OF VASCULAR SMOOTH MUSCLE CELL GROWTH BY MACROPHAGE COLONY-STIMULATING FACTOR. ~ ~, Gogusev J", Herembert T, Drueke T and Zhu DL". C~S URA 1482 and 'INSERM U 90, Necker Hosp, p~s" France and ,. Shanghai Institute of Hypertension, RUI-Jm Hosp, Shanghai, China. In several vascular diseases an abnormal of vascular smooth muscle cell (VSMC) proliferation is associated with the presence of macrophages. We therefore examined whether macrophage colony-stimulating factor (M-CSF) might playa role in the control of VSMC growth. VSMCs were isolated from rat aorta and maintained in culture. The influen~e of M-CSF on DNA s~nthesis and proto-oncogene expressIon was assessed by H-thymidine incorporation measurement and Northern analysis, respectively. Macrophage COlony-stimulating activity (M-CSA) was ascertained by a bioassay. Addition to quiescent VSMCs of various dilutions of L929 serum-free condition medium (S-FCM), as a source of M-CSF, concentration-dependently induced DNA synthesis; this effect could be blunted by the concomitant addition of a specific anti M-CSF anltbody. SFCM from L929 also induced and/or modulated the expression of c-fos, c-myc and jun B. Recombinant human M-CSF weakly induced DNA synthesis when added alone, but synergistically enhanced that induced by thrombin, PDGF and bFGF. M-CSA was clearly detected in VSMC-derived S-FCM. The presence of c-fms mRNA and fms oncoprotein, as revealed by Northern blot analysis and immunocytochemistry, respectively, indicated that VSMCs expressed M-CSF receptors. These results support the hypotheSIS of a paracrinelautocrine mechanism Whereby M-CSF could affect the growth of cultured rat VSMCs.
S
I : CI
~
[)
Beltsville Human Nutrition Research Center, USDA,
Beltsville, Maryland, and Institut de Recherches Internationales Servier, Par~s, France. D~abet~c nephropathy ~s characterized by glomerular mesangial expansion with accumulation of extracellular matrix
(ECM)
sclerosis, and interstitial factor
involved
in
ECM
proteins,
fibros~s.
production
glomerular
TGF-B, a key and
tissue
repair, has been implicated in the pathogenesis of diabetic renal lesions. We investigated TGF-B express~on in kidneys of SHR/N-corpulent (cp) rats, a genetic model of type 2 diabetes and hypertension with renal les~ons resembling human d~abetic nephropathy. Expression of TGF-B protein was assessed by immunohistochemistry using a polyclonal rabbit antibody which recognizes extracellular TGFB in kidneys of 6-8 month old obese male SHR/N-cp rats (n=5) and lean controls (n-5). Rats were fed 54' carbohydrate diet containing 18' sucrose and 36' starch. Renal morphology was evaluated for
glomerular
lesions
of
mesangial
expansion
and
sclerosis (MES). A tubulointerstitial score (TIS), an index of severity of tubulointerstitial lesions, e.g., tubular atrophy, cellular ~nf~ltration. and interstitial fibros~s was assigned. Morphologic analys~s showed that the number of MES and TIS were higher in kidneys of obese than in lean rats (MES, 31±5' vs 4±1'. pc.Ol, TIS, +2.2±0.4 vs +0.S±0.2, pc.Ol). A 4-fold increase of TGF-B immunostaining in the renal cortex (expressed as number of foci of
interstitial staining) was found in kidneys of obese rats compared to lean rats (pc. 02) . Treatment of obese rats (n-s) with the ACE inh~b~tor per~ndopril for 3 months reduced the glomerular les~ons,
TIS, and the amount of TGF-B
stain~ng
in
renal cortex. We conclude that 1) TGF-B protein ~s elevated in the renal parenchyma of obese SHR/N-cp rats with diabetic nephropathy, and 2) treatment of
hypertension with perindopril ameliorates the renal
and the increased TGF-B expression. Key Words:
les~ons
Diabetic nephropathy, perindopril, TGF-B, rat model
Key Words: Endothelin-1, Chelerithrin, Protein Kinase-C, SHR.
Key words: vascular smooth muscle cell, M-CSF, cell growth, macrophage, vascular hypertrophy.