Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 907-912
October 31, 1991
Release of Nitric Oxide from Human Vascular Smooth Muscle Cells Jens Bernhardt 1, Marcel R. Tschudi 1, Yasuaki Dohi 1, Ivo Gut 2, Bernhard Urwyler 2, Fritz R. Bfihler 1 and Thomas F. Liischer 1,3 Department of 1Research, 2institut for Physical Chemistry and 3Department of Medicine, Divisions of Clinical Pharmacology and Cardiology, University of Basel, Switzerland Received September ii, 1991
Summary: It has recently been shown that nitric oxide (NO) or a labile NO-releasing compound is produced in endothelial cells. In the present study we measured the NOrelease from human vascular smooth muscle cells in culture. The cells released an average 2.2 x 10-9 moles nitric oxide per 108 cells in ten minutes with a large variation between different cell lines and passages without stimulators. The NO-release was markedly reduced by the inhibitor of NO-formation NG-momomethyl-L-arginine (L-NMMA, 10 -5 M) to 3 % of the control levels (p < 0.02, n = 4 ), but unaffected by acetylcholine, bradykinin or endothelin -1, -2 or -3. In a microperfusion system the compound released from human vascular smooth muscle cells caused profound relaxation of isolated rat mesenteric resistance arteries. Thus, human vascular smooth muscle cells in culture produce and release biologically active NO from L-arginine. ©1991 AcademicP . . . . . I n c .
Relaxation of smooth muscle cells can be induced by endothelium-derived relaxing factor (EDRF), which has been identified as nitric oxide (NO) or a closely related NO-releasing compound (1). Besides endothelial cells (2-4), NO also can be produced by macrophages (5) and brain tissues (6,7). NO most likely interferes with the heme group of the enzyme soluble guanylyl cyclase (8-12) and in turn activates the enzyme and increases the intracellular concentration of cGMP; cGMP leads to relaxation of smooth muscle cells (13,14) and, at least in higher cGMP concentrations, also appears to inhibit cell proliferation (15). Hence, there is increasing evidence that NO plays an important role as an inter- and intracellular messenger molecule. Immunhistochemical studies with antibodies against NO syntethase have shown that two types of enzymes appear to exist (6), one type with 150 kD present in endothelium cells and in brain cells, which is calmodulin-dependent, and an other form present in macrophages, which still has not been characterized. This study demonstrates basal release of NO from human vascular smooth muscle cells in culture. Since in endothelial cells the formation of NO can be stimulated with substances like acetylcholine and bradykinin and under certain conditions also by endothelin (16), we assessed the effect of these agonists on NO-production in human vascular smooth muscle cells. 0006-291X/91 $1.50 907
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Materials All tissue culture plastic materials were purchased from Falcon (Lincoln Park, N.J.), tissue culture media were obtained from Seromed (Berlin, Germany), microcarrier beads Cytodex 3 from Pharmacia (Uppsala, Sweden), NG-monomethyl-L-arginine (LNMMA) from Calbiochem Corp. (La Jolla, CA), and all other chemicals were purchased from Fluka AG (Buchs, Switzerland) or E. Merck (Darmstadt, Germany). Male Wistar Kyoto rats, 16 week old, were obtained from Charles River Wigam (Sulzfeld, Germany). Cell culture The isolation and characterization of human vascular smooth muscle cells (hVSMC) were performed as previously described (17). In brief, cells from the mesentery were obtained intraoperatively by enzymatic digestion of microarterioles prepared by microdissection. Cells were cultured in minimal essential medium (MEM) with Earles salts and addition of 10 % fetal bovine serum. In the present experiments four isolates of hVSMC between passage numbers 6-21 were used. Cells (1-3 x 108) were seeded onto 50-60 ml of preswollen Cytodex 3 beads and grown to confluence (2-3 days). Subsequently, cells were rendered quiescent by serum starvation for 36-48 h in medium supplemented with 0.1% (w/v) bovine serum albumin (MEM-Earle/BSA). Before the cell-carrying beads were packed into a column (40 ml bead-suspension in a 2.4cm x 9.0 cm column, dead volume ca. 30 ml), cell numbers were routinely determined with a Coulter Counter (Coulter Electronics Inc., Hialeah, FL) in each individual culture, to allow normalisation of NO-measurements according to cell numbers. The total amount of cells in the columns varied from 1.5 to 3.5 x 108 cells per column. Filled columns were washed twice before NO-measurement, with one column volume of either Krebs buffer or serum free medium (MEM-EARLES) containing 0.1% BSA. Nitric oxide-measurement NO was measured by chemoluminescence (1). The columns were perfused with serum free medium with or without addition of several substances. An aliquot of 5 ml of 50 ml column flow-through was injected into a glass chamber of the NO-measuring device (filled with 75 ml acetic acid (98%) and 25 ml 6% NaI (w/v)). The flow rate was 25 ml per minute. Developing gas was transported with a stream of nitrogen to a reaction chamber attached to a NO-chemoluminescence-analyser (Hammamatsu Photonics K.K., Hammamatsu City, Japan). For the calibration of the NO-analyser, NaNO2 (10 -11 to 2x10 -9 M) was used. The time of 10 minutes between injections was needed to allow the instrument to return to the baseline. Microt~erfusion assay In rats-anaesthetized with pentobarbital (50 mg/kg body weight i.p.) the entire mesentery was removed and placed in cold modified Krebs Ringer bicarbonate solution of the following composition (mM): NaC1 118.0, KC1 4.7, CaC12 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, EDTA calcium disodium 0.026, glucose 11.1 (Krebs solution). A 34 mm long segment of the third branch of the mesenteric artery was dissected and cleaned under a dissecting microscope and then transferred to an arteriograph chamber filled with oxygenated (95% 02 and 5% CO2) Krebs solution (37 °+ 0.5°C) (18). The chamber contained two glass microcannulae (efferent and afferent cannula). The proximal end of the artery was cannulated with the afferent cannula and secured with a suture. The distal end was attached to the inside of the efferent cannula. The artery was perfused with Krebs solution containing 1.0% albumin from bovine serum. A small catheter was located in the afferent cannula and was connected to a pressure transducer for measurement of transmural pressure. The column eluate was administered intraluminally through this catheter. Flow through the vascular segment was measured repeatly at the efferent cannula. Endothelium was removed by intraluminal perfusion with 0.5% 3-((3-Cholamidopropyl)diethylammonio)-l-propanesulfonate (CHAPS) for 30 seconds. The absence of the endothelium was confirmed by the absence of a relaxation to acetylcholine (10 -6 M). The arteriograph was placed on the stage of a microscope and intraluminal diameter and wall thickness were measured at a transmural pressure of 30 mm Hg (Living Systems Instrumentation, Burlington, Vt.) Calculation and Statistics Statistical evaluation was carded out by using the unpaired Student's t-test. Means were considered significantly different when the p-value was less than 0.05. Data are given as 908
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mean + SEM. The area under the curve (integral) drawn by the recorder was correlated with NaNO2 standard (standard not shown).
RESULTS A N D D I S C U S S I O N The average of the basal release of NO was 2.2 x 10 -9 +- 0.63 x 10 -9 mol per 108 cells (fig. 1, fig.2, N = 4) in MEM/Earles medium. Acetycholine, bradykinin (both 10 -6, fig.2, each N = 2), or endothelin-1, -2 or -3 did not affect the NO-release from the cells (all 10 -8 M, fig. 1, each N = 2). In the presence o f L - N M M A (10 -5 M) the NO-release was markedly diminished to 7.2 x 10 -11 + 4 . 9 x 10 -11 NO mol per 108 cells (p
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 907-912
October 31, 1991
Release of Nitric Oxide from Human Vascular Smooth Muscle Cells Jens Bernhardt 1, Marcel R. Tschudi 1, Yasuaki Dohi 1, Ivo Gut 2, Bernhard Urwyler 2, Fritz R. Bfihler 1 and Thomas F. Liischer 1,3 Department of 1Research, 2institut for Physical Chemistry and 3Department of Medicine, Divisions of Clinical Pharmacology and Cardiology, University of Basel, Switzerland Received September ii, 1991
Summary: It has recently been shown that nitric oxide (NO) or a labile NO-releasing compound is produced in endothelial cells. In the present study we measured the NOrelease from human vascular smooth muscle cells in culture. The cells released an average 2.2 x 10-9 moles nitric oxide per 108 cells in ten minutes with a large variation between different cell lines and passages without stimulators. The NO-release was markedly reduced by the inhibitor of NO-formation NG-momomethyl-L-arginine (L-NMMA, 10 -5 M) to 3 % of the control levels (p < 0.02, n = 4 ), but unaffected by acetylcholine, bradykinin or endothelin -1, -2 or -3. In a microperfusion system the compound released from human vascular smooth muscle cells caused profound relaxation of isolated rat mesenteric resistance arteries. Thus, human vascular smooth muscle cells in culture produce and release biologically active NO from L-arginine. ©1991 AcademicP . . . . . I n c .
Relaxation of smooth muscle cells can be induced by endothelium-derived relaxing factor (EDRF), which has been identified as nitric oxide (NO) or a closely related NO-releasing compound (1). Besides endothelial cells (2-4), NO also can be produced by macrophages (5) and brain tissues (6,7). NO most likely interferes with the heme group of the enzyme soluble guanylyl cyclase (8-12) and in turn activates the enzyme and increases the intracellular concentration of cGMP; cGMP leads to relaxation of smooth muscle cells (13,14) and, at least in higher cGMP concentrations, also appears to inhibit cell proliferation (15). Hence, there is increasing evidence that NO plays an important role as an inter- and intracellular messenger molecule. Immunhistochemical studies with antibodies against NO syntethase have shown that two types of enzymes appear to exist (6), one type with 150 kD present in endothelium cells and in brain cells, which is calmodulin-dependent, and an other form present in macrophages, which still has not been characterized. This study demonstrates basal release of NO from human vascular smooth muscle cells in culture. Since in endothelial cells the formation of NO can be stimulated with substances like acetylcholine and bradykinin and under certain conditions also by endothelin (16), we assessed the effect of these agonists on NO-production in human vascular smooth muscle cells. 0006-291X/91 $1.50 907
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Materials All tissue culture plastic materials were purchased from Falcon (Lincoln Park, N.J.), tissue culture media were obtained from Seromed (Berlin, Germany), microcarrier beads Cytodex 3 from Pharmacia (Uppsala, Sweden), NG-monomethyl-L-arginine (LNMMA) from Calbiochem Corp. (La Jolla, CA), and all other chemicals were purchased from Fluka AG (Buchs, Switzerland) or E. Merck (Darmstadt, Germany). Male Wistar Kyoto rats, 16 week old, were obtained from Charles River Wigam (Sulzfeld, Germany). Cell culture The isolation and characterization of human vascular smooth muscle cells (hVSMC) were performed as previously described (17). In brief, cells from the mesentery were obtained intraoperatively by enzymatic digestion of microarterioles prepared by microdissection. Cells were cultured in minimal essential medium (MEM) with Earles salts and addition of 10 % fetal bovine serum. In the present experiments four isolates of hVSMC between passage numbers 6-21 were used. Cells (1-3 x 108) were seeded onto 50-60 ml of preswollen Cytodex 3 beads and grown to confluence (2-3 days). Subsequently, cells were rendered quiescent by serum starvation for 36-48 h in medium supplemented with 0.1% (w/v) bovine serum albumin (MEM-Earle/BSA). Before the cell-carrying beads were packed into a column (40 ml bead-suspension in a 2.4cm x 9.0 cm column, dead volume ca. 30 ml), cell numbers were routinely determined with a Coulter Counter (Coulter Electronics Inc., Hialeah, FL) in each individual culture, to allow normalisation of NO-measurements according to cell numbers. The total amount of cells in the columns varied from 1.5 to 3.5 x 108 cells per column. Filled columns were washed twice before NO-measurement, with one column volume of either Krebs buffer or serum free medium (MEM-EARLES) containing 0.1% BSA. Nitric oxide-measurement NO was measured by chemoluminescence (1). The columns were perfused with serum free medium with or without addition of several substances. An aliquot of 5 ml of 50 ml column flow-through was injected into a glass chamber of the NO-measuring device (filled with 75 ml acetic acid (98%) and 25 ml 6% NaI (w/v)). The flow rate was 25 ml per minute. Developing gas was transported with a stream of nitrogen to a reaction chamber attached to a NO-chemoluminescence-analyser (Hammamatsu Photonics K.K., Hammamatsu City, Japan). For the calibration of the NO-analyser, NaNO2 (10 -11 to 2x10 -9 M) was used. The time of 10 minutes between injections was needed to allow the instrument to return to the baseline. Microt~erfusion assay In rats-anaesthetized with pentobarbital (50 mg/kg body weight i.p.) the entire mesentery was removed and placed in cold modified Krebs Ringer bicarbonate solution of the following composition (mM): NaC1 118.0, KC1 4.7, CaC12 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, EDTA calcium disodium 0.026, glucose 11.1 (Krebs solution). A 34 mm long segment of the third branch of the mesenteric artery was dissected and cleaned under a dissecting microscope and then transferred to an arteriograph chamber filled with oxygenated (95% 02 and 5% CO2) Krebs solution (37 °+ 0.5°C) (18). The chamber contained two glass microcannulae (efferent and afferent cannula). The proximal end of the artery was cannulated with the afferent cannula and secured with a suture. The distal end was attached to the inside of the efferent cannula. The artery was perfused with Krebs solution containing 1.0% albumin from bovine serum. A small catheter was located in the afferent cannula and was connected to a pressure transducer for measurement of transmural pressure. The column eluate was administered intraluminally through this catheter. Flow through the vascular segment was measured repeatly at the efferent cannula. Endothelium was removed by intraluminal perfusion with 0.5% 3-((3-Cholamidopropyl)diethylammonio)-l-propanesulfonate (CHAPS) for 30 seconds. The absence of the endothelium was confirmed by the absence of a relaxation to acetylcholine (10 -6 M). The arteriograph was placed on the stage of a microscope and intraluminal diameter and wall thickness were measured at a transmural pressure of 30 mm Hg (Living Systems Instrumentation, Burlington, Vt.) Calculation and Statistics Statistical evaluation was carded out by using the unpaired Student's t-test. Means were considered significantly different when the p-value was less than 0.05. Data are given as 908
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mean + SEM. The area under the curve (integral) drawn by the recorder was correlated with NaNO2 standard (standard not shown).
RESULTS A N D D I S C U S S I O N The average of the basal release of NO was 2.2 x 10 -9 +- 0.63 x 10 -9 mol per 108 cells (fig. 1, fig.2, N = 4) in MEM/Earles medium. Acetycholine, bradykinin (both 10 -6, fig.2, each N = 2), or endothelin-1, -2 or -3 did not affect the NO-release from the cells (all 10 -8 M, fig. 1, each N = 2). In the presence o f L - N M M A (10 -5 M) the NO-release was markedly diminished to 7.2 x 10 -11 + 4 . 9 x 10 -11 NO mol per 108 cells (p
