Vol. 173, No. 3, 1990 December 31, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1137-1142
AORTIC SMOOTH MUSCLE CELLS ARE ABLE TO CONVERT ANGIOTENSIN I TO ANGIOTENSIN II Philippe ANDRE 1, Chrism SCHOTT, Herrade NEHLIG and Jean-Claude STOCLET Laboratoire de Pharmacologic Mol6culaire et Cellulaire CNRS URA600, Universit6 Louis Pasteur de Strasbourg, B.P. 24, 67401 Illkirch, France Received October 5, 1990
SUMMARY: The role of vascular smooth muscle cells (VSMC) in the intraparietal conversion of angiotensin I (AngI) to angiotensin II (AngII) was investigated in rat aortic tissue. The responses of rat aortic vascular smooth muscle cells to AngI and AnglI were assessed by studying contraction of endothelium-denuded aortic rings and by measuring intracellular Ca ++ ion concentration in primary cultures of VSMC free of endothelial cells. In both preparations, AngI and AnglI induced identical responses which were inhibited by saralasin, a blocker of AnglI receptors. In the presence of captopril, an inhibitor of the angiotensin converting enzyme, the increase in calcium caused by AngI was abolished in VSMC cultures and the contractile effect of this peptide in aortic rings was strongly decreased, whereas the responses to AngII remained unaffected. These results demonstrate that VSMC are able to convert AngI to AngH. © 1990 Academic Press, Inc.
Angiotensin converting enzyme (ACE) is a dipeptidyl carboxipeptidase (EC 3.4.15.11) which cleaves AngI to AngII, a potent vasoconstrictor agent. It is well established that circulating AngI, produced by renin, has no direct vasoconstrictor effect but it is converted into AngII, by circulating and endothelial ACE (1-2). This mechanism has a major importance in the regulation of vascular tone. However, evidence exists suggesting the presence of a renin-angiotensin system in the vessel wall: the presence of renin in smooth muscle and endothelial cells (3) and of angiotensinogen mRNA (4) in the rat aorta adventitia and periaortic brown adipose tissue have previously been reported. Futhermore, AngI induces contractions of isolated endotheliumdenuded vessels (5). It has been also suggested that the local production of AnglI could play an autocrine or paracrine role on VSMC (6). However, the localization of a functional extraendothelial ACE in the vascular wall is uncertain, due to the presence of various cell types. The aim of the present work was to examine whether vascular smooth muscle cells are able to convert AngI to AngII. An ACE activity-dependent effect of AngI on AngII receptors has been demonstrated on rat aortic smooth muscle cells in these experiments.
1To whom correspondence should be addressed. 0006-291X/90 $1.50 1137
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS
Cell cultures Primary cultures of aortic smooth muscle cells from 10 to 12 week-old Wistar rats were obtained as previously described (7). The aorta was treated with CLII collagenase at 70 U/ml in Hank's balanced salt solution for 30 rain at 37°C. The adventitia was stripped mechanically and the endothelium was removed with a fine paint brush after the vessel had been opened longitudinally. The aorta was incubated with an elastase-collagenase digestion medium. After 90 min, the tissue was homogenized and centrifuged. The pellet was resuspended in MEM with 2% Ultroser G and the cells were plated in 25 cm 2 flasks. After a 2 h-preplating, the supernatant was transferred to a new flask and seeded at 3x105 cells/ml on sterile glass coverslips. The medium was changed every two days. Endothelial cells were collected from fresh bovine aorta using collagenase digestion as described by Schini et al (8). The endothelial cells were cultivated on 25 cm 2 plastic flasks in MEM/HAM F12 (1:1) supplemented with 20% foetal calf serum and were used at the third passage. Fluorescence microscopy Endothelial cells and vascular smooth muscle cells were distinguished by the selective kinetic uptake of acetylated-low density lipoproteins (9). Briefly, confluent endothelial cells or VSMC were incubated for 4 hours at 37°C with 10 ~tg/ml of lipoproteins labelled with the fluorescent probe 1,1'dioctadecyl 3,3,3',3'tetramethyl-indocarbocyanine perchlorate (Di I-Ac-LDL). VSMC and endothelial cells were washed three times with a physiological solution for 10 min and fixed in 4% paraformaldehyde in 0.12M Na/K phosphate buffer saline.The samples were examined with a fluorescence microscope (Zeiss). Intracellular calcium measurement Intracellular free calcium concentration (Ca+÷i) was measured according to the method used by Cornwell and Lincoln (10). Fifteen day-old VSMC cultures, grown on glass coverslips, were washed 3 times with 2 ml of balanced salt solution (BSS). The glass coverslip was inserted in quartz spectrophotometric cuve. 3 ml of Fura-2/AM,5 p.M, were added to the cells and incubated 30 min at 37°C. The cells were washed 3 times with BSS without bovine serum albumin and incubated for an additional 20 min to ensure complete hydrolysis of the Fura-2/acetoxymethyl ester. C a ~ i measurements were made in a SPEX 1681 spectrometer Fluorescence measurements were made with two different excitation wavelengths, 340 and 380 nm. The emission was measured at 510 nm. Calculation of Ca++i was done as described previously by Grynkiewicz et al.(11) Contractile experiments Two mm-long aortic rings from 10-12 week old male Wistar rats were rubbed with fine forceps to remove the endothelium and were mounted under an initial tension of 2 g. The bath containing 20 mi of physiological solution (composition mM: NaC1 118; NaHCO3 25; glucose 10; KC1 4.7; CaC12 1.25; MgSO4 1.19; KH2PO4 1.14) was maintained at 37°C and bubbled with a mixture of 95% 02-5% CO2. After a 60 min equilibration, the tension was readjusted to 2 g. The absence of functional endothelium was assessed by the absence of a relaxing effect of acetylcholine 10-6M added when the maximal tension had been reached with 10-6M noradrenaline. Thereafter, tissues were washed every 15 min during 60 min. When indicated, saralasin or captopril were added 15 min before AngI or AngII addition. Isometric responses to AngI and AngII were recorded Statistical analysis Statistical analysis was performed using the Wilcoxon test, taking P
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1137-1142
AORTIC SMOOTH MUSCLE CELLS ARE ABLE TO CONVERT ANGIOTENSIN I TO ANGIOTENSIN II Philippe ANDRE 1, Chrism SCHOTT, Herrade NEHLIG and Jean-Claude STOCLET Laboratoire de Pharmacologic Mol6culaire et Cellulaire CNRS URA600, Universit6 Louis Pasteur de Strasbourg, B.P. 24, 67401 Illkirch, France Received October 5, 1990
SUMMARY: The role of vascular smooth muscle cells (VSMC) in the intraparietal conversion of angiotensin I (AngI) to angiotensin II (AngII) was investigated in rat aortic tissue. The responses of rat aortic vascular smooth muscle cells to AngI and AnglI were assessed by studying contraction of endothelium-denuded aortic rings and by measuring intracellular Ca ++ ion concentration in primary cultures of VSMC free of endothelial cells. In both preparations, AngI and AnglI induced identical responses which were inhibited by saralasin, a blocker of AnglI receptors. In the presence of captopril, an inhibitor of the angiotensin converting enzyme, the increase in calcium caused by AngI was abolished in VSMC cultures and the contractile effect of this peptide in aortic rings was strongly decreased, whereas the responses to AngII remained unaffected. These results demonstrate that VSMC are able to convert AngI to AngH. © 1990 Academic Press, Inc.
Angiotensin converting enzyme (ACE) is a dipeptidyl carboxipeptidase (EC 3.4.15.11) which cleaves AngI to AngII, a potent vasoconstrictor agent. It is well established that circulating AngI, produced by renin, has no direct vasoconstrictor effect but it is converted into AngII, by circulating and endothelial ACE (1-2). This mechanism has a major importance in the regulation of vascular tone. However, evidence exists suggesting the presence of a renin-angiotensin system in the vessel wall: the presence of renin in smooth muscle and endothelial cells (3) and of angiotensinogen mRNA (4) in the rat aorta adventitia and periaortic brown adipose tissue have previously been reported. Futhermore, AngI induces contractions of isolated endotheliumdenuded vessels (5). It has been also suggested that the local production of AnglI could play an autocrine or paracrine role on VSMC (6). However, the localization of a functional extraendothelial ACE in the vascular wall is uncertain, due to the presence of various cell types. The aim of the present work was to examine whether vascular smooth muscle cells are able to convert AngI to AngII. An ACE activity-dependent effect of AngI on AngII receptors has been demonstrated on rat aortic smooth muscle cells in these experiments.
1To whom correspondence should be addressed. 0006-291X/90 $1.50 1137
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS
Cell cultures Primary cultures of aortic smooth muscle cells from 10 to 12 week-old Wistar rats were obtained as previously described (7). The aorta was treated with CLII collagenase at 70 U/ml in Hank's balanced salt solution for 30 rain at 37°C. The adventitia was stripped mechanically and the endothelium was removed with a fine paint brush after the vessel had been opened longitudinally. The aorta was incubated with an elastase-collagenase digestion medium. After 90 min, the tissue was homogenized and centrifuged. The pellet was resuspended in MEM with 2% Ultroser G and the cells were plated in 25 cm 2 flasks. After a 2 h-preplating, the supernatant was transferred to a new flask and seeded at 3x105 cells/ml on sterile glass coverslips. The medium was changed every two days. Endothelial cells were collected from fresh bovine aorta using collagenase digestion as described by Schini et al (8). The endothelial cells were cultivated on 25 cm 2 plastic flasks in MEM/HAM F12 (1:1) supplemented with 20% foetal calf serum and were used at the third passage. Fluorescence microscopy Endothelial cells and vascular smooth muscle cells were distinguished by the selective kinetic uptake of acetylated-low density lipoproteins (9). Briefly, confluent endothelial cells or VSMC were incubated for 4 hours at 37°C with 10 ~tg/ml of lipoproteins labelled with the fluorescent probe 1,1'dioctadecyl 3,3,3',3'tetramethyl-indocarbocyanine perchlorate (Di I-Ac-LDL). VSMC and endothelial cells were washed three times with a physiological solution for 10 min and fixed in 4% paraformaldehyde in 0.12M Na/K phosphate buffer saline.The samples were examined with a fluorescence microscope (Zeiss). Intracellular calcium measurement Intracellular free calcium concentration (Ca+÷i) was measured according to the method used by Cornwell and Lincoln (10). Fifteen day-old VSMC cultures, grown on glass coverslips, were washed 3 times with 2 ml of balanced salt solution (BSS). The glass coverslip was inserted in quartz spectrophotometric cuve. 3 ml of Fura-2/AM,5 p.M, were added to the cells and incubated 30 min at 37°C. The cells were washed 3 times with BSS without bovine serum albumin and incubated for an additional 20 min to ensure complete hydrolysis of the Fura-2/acetoxymethyl ester. C a ~ i measurements were made in a SPEX 1681 spectrometer Fluorescence measurements were made with two different excitation wavelengths, 340 and 380 nm. The emission was measured at 510 nm. Calculation of Ca++i was done as described previously by Grynkiewicz et al.(11) Contractile experiments Two mm-long aortic rings from 10-12 week old male Wistar rats were rubbed with fine forceps to remove the endothelium and were mounted under an initial tension of 2 g. The bath containing 20 mi of physiological solution (composition mM: NaC1 118; NaHCO3 25; glucose 10; KC1 4.7; CaC12 1.25; MgSO4 1.19; KH2PO4 1.14) was maintained at 37°C and bubbled with a mixture of 95% 02-5% CO2. After a 60 min equilibration, the tension was readjusted to 2 g. The absence of functional endothelium was assessed by the absence of a relaxing effect of acetylcholine 10-6M added when the maximal tension had been reached with 10-6M noradrenaline. Thereafter, tissues were washed every 15 min during 60 min. When indicated, saralasin or captopril were added 15 min before AngI or AngII addition. Isometric responses to AngI and AngII were recorded Statistical analysis Statistical analysis was performed using the Wilcoxon test, taking P
