European Journal of Pharmacology, 215 (1992) 329-331
329
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 21054
Short communication
Transmurai pressure inhibits nitric oxide release from human endothelial cells Keiichi H i s h i k a w a a,b, T o s h i o N a k a k i b, H i r o m i c h i Suzuki a, T a k a o S a r u t a ~' a n d R y u i c h i K a t o b Departments of" Internal Medicine and b Pharmacology, Keio Unil~ersity School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Received 28 February 1992, accepted 10 March 1992
We examined the effect of transmural pressure on histamine-stimulated nitric oxide release from cultured endothelial cells prepared from human umbilical cord veins. PO: and pH were kept constant throughout the experiments. Various levels of transmural pressure and atmospheric pressure (40, 80, 120 and 160 mm Hg) were applied. Nitric oxide release was inhibited in a pressure-dependent manner. The inhibitory effects were reversible, and nitric oxide had no effect on the morphology of the cells. Our results suggest that transmural pressure-mediated inhibition of nitric oxide release contributes to pressure-induced vasoconstriction and reduced endothelium-dependent relaxation in patients with hypertension. EDRF (endothelium-derived relaxing factor); Nitric oxide (NO); Pressure; Endothelial cells; Mechanorcceptors; Hypertension
1. Introduction Increased intraluminal pressure has been reported to cause e n d o t h e l i u m - d e p e n d e n t vasoconstriction (Rubanyi, 1988; Harder et al., 1989). There have been no studies on the effect of transmural pressure alone on nitric oxide (NO) release from human endothelial cells. According to the law of Laplace, intraluminal pressure in a distensible hollow object such as a blood vessel causes both transmural pressure and wall tension. These changes appear to affect endothelial cell function, but it is impossible to examine the effect of transmural pressure alone on endothelial cells in situ. This study was designed to assess the effect of transmural pressure alone on NO release from human endothelial cells by exposing cells cultured on flat, solid flasks to elevated pressure and by measuring nitrite/nitrate, the oxidized stable products of NO. This is the first report that directly investigated the effect of transmural pressure alone on NO release from human endothelial cells.
2. Material and methods
2.1. Culture of human umbifical cord vein endothelial cells H u m a n umbilical vein endothelial cells were prepared from human umbilical cord veins according to
Correspondence to: T. Nakaki, Department of Pharmacology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. Fax 81.3.3359 8889.
the method described by Jaffe et al. (1973), with some minor modifications. Cells were grown to confluence (5-7 × 105 cells/flask) on 0.2% gelatin-coated 25-cm 2 flasks (Corning, NY, USA) containing 5.0 ml of a medium composed of Earle's M199 (GIBCO, NY, USA), 10% fetal calf serum (Mitsubishi-kasei, Tokyo, Japan), 30 /xg/ml endothelial cell growth supplement (Sigma, St. Louis, USA), and 6 U / m l heparin. Cells were used between passages 2 and 4.
2.2. Pressure-loading apparatus The pressure-loading apparatus was set up as follows. The flask was tightly sealed with a rubber cap and compressed helium (He) gas was pumped in to raise the internal pressure. The rubber cap was pierced by a needle connected to tubing attached to a three-way rotary valve (RT-N), sphygmomanometer and pressure valve. The connecting tubing (extension tube:61B) and the three-way rotary valve were obtained from Hakko Co., Tokyo, Japan. He gas and the pressure valve were obtained from Yamato-Sanki Co., Tokyo, Japan. The mercury sphygmomanometer was obtained from Sansei Medical Industry Co., Tokyo, Japan. While the He gas was being pumped in, no pre-packed room air was released, so that the partial pressures of the gases contained in the flask, such as oxygen, nitrogen oxide and carbon dioxide, were kept constant in accordance with Boyle-Charles' law. The flask was kept at 37°C on a hot plate. Different flasks were used for each pressure. To expose cells in a single flask to various pressures, it would be necessary to wash out histamine before changing pressures. As this was not possible to
330
do without giving shear stress, we examined the pressure effect in different flasks. 0.3
2.3. Measurement of nitrite / nitrate H u m a n umbilical vein endothelial cells were washed 3 times with 5 ml Hanks solution and then incubated at 37°C for 1 hour in 2 ml of Hanks solution before the experiments. The formation of NO from human umbilical vein endothelial cells was measured as the levels of n i t r i t e / n i t r a t e , oxidized products of nitric oxide, using an automated system based on the Griess reaction (Hishikawa et al., in press).
2.4. PO 2 and pH PO 2 and p H of the incubation medium were measured with an automated analyzer (pH gas analyzer 1306, Instrumental Laboratory, Tokyo, Japan).
2.5. Statistical analysis The data are expressed as the means and S.E.M. in the text and figures. Statistical significance was assessed with a t-test, and a P value less than 0.05 was considered significant.
3. Results
N i t r i t e / n i t r a t e at 30 min without histamine was 0.05 + 0.01 n m o l / f l a s k under atmospheric pressure (0
~. 0.8 0.7
0.6
=
0.5
c 0.4
o~ 0.3
"E 0.2 "~
"E 0.2 "~ 0.1
Z
0.0 0
i 5
,
i
10
15
J
20
i
i
25
30
Incubation Time (rain)
B
~
0.2
N ~
0.1
~
O.O 0
40 80 120 Pressure (mm Hg)
160
Fig. 2. Effect of transmural pressure on histamine-stimulated NO release from human umbilical vein endothelial cells. The concentration of histamine was 10 /~M. The indicated pressure is the exogenously applied pressure plus atmospheric pressure for 30 min. * P < 0.05 vs. 0 mm Hg. Experiments were done in quadruplicate. Means_+ S.E.M. are shown.
mm Hg). Histamine (10/~M) increased n i t r i t e / n i t r a t e in the medium in a time-dependent manner by more than 10-fold (fig. 1A), which is consistent with the results of Van de Voorde et al. (1987). NO release was inhibited by transmural pressure in a pressure-dependent manner (fig. 2). The PO 2 and p H of the incubation medium ranged from 154 + 4 to 158_+ 7 mm Hg and from 7.3 + 0.1 to 7.4_+ 0.1, respectively. There were no significant changes in these values throughout the experiments, as predicted by Boyle-Charles' law. The inhibitory effect of transmural pressure was reversible (fig. 1B). Cells that had been exposed to 160 m m Hg for 30 min were incubated under atmospheric pressure for 30 rain and were then stimulated with histamine. Pretreatment with a pressure of 160 mm Hg had no effect on N O release from the cells. The viability of human umbilical vein endothelial cells, as assessed with the trypan blue exclusion test, was over 99% throughout the experiments.
4. Discussion
~" 0.0 0
160
Pressure (mm Hg)
Fig. 1. Effect of histamine ( 1 0 / ~ M ) on nitric oxide ( N O ) release from human umbilical vein endothelial cells. ( A ) Histamine stimulated the
accumulation of nitrite/nitrate in a time-dependent manner. The concentration of nitrite/nitrate without histamine was about 0.05 + 0.01 nmol/flask per 30 rain. Experiments were done in quadruplicate. The standard errors were within the symbols. (B) Reversibility of the effects of treatment with 160 mm Hg on human umbilical vein endothelial cells. The cells were incubated under atmospheric pressure alone (designated 0 mm Hg) or 160 mm Hg plus atmospheric pressure (designated 160 mm Hg) for 30 min. After 30 min recovery time, the effect of histamine on NO release was examined under atmospheric pressure. There were no significant changes in NO release between pretreatment with 0 and 160 mm Hg. Means _+S.E.M. are shown. Experiments were done in quadruplicate.
Increased intraluminal pressure has been reported to cause vasoconstriction (Rubanyi, 1988; H a r d e r et al., 1989). The three best understood components of hemodynamic force are shear stress, wall tension, and transmural pressure. However, it is difficult to evaluate the effects of transmural pressure alone on vessels in situ. According to the law of Laplace, transmural pressure in a distensible hollow object causes wall tension, which may stretch endothelial cells. Both shear stress and stretch have been reported to enhance Ca 2 + infux and N O release (Rubanyi et al., 1986; Buga et al., 1991; Ohno et at., 1990; Lansman et al., 1987). The effect of transmural pressure on NO release from
331
human endothelial cells has not been directly investigated. This study was designed to assess the effects of transmural pressure alone on N O release from h u m a n umbilical vein endothelial cells by exposing cells cultured on flat, solid flasks to an elevated pressure. The pressure was raised by pumping compressed He gas into a flask packed with room air. While the He gas was being p u m p e d into the flask, no pre-packed room air was released and the temperature was kept at 37°C. In these systems, the partial pressure of 0 2 and CO 2 were kept constant, in accordance with the B o y l e Charles' law. Our results indicate that histamine-induced N O release was inhibited by increasing the transmural pressure. To examine whether the inhibition of N O release by transmural pressure was caused by cell damage, we also exposed cells to atmospheric pressure (0 m m Hg) and 160 m m Hg for 30 min, and N O release was assessed after 30 min recovery time (fig. 1B). There were no significant changes in N O release between the two experimental conditions. Cell viability was over 99% (trypan blue exclusion) and no morphological abnormalities were found by light microscopic observation at the end of the experiments. These results clearly show that the inhibition of N O release by transmural pressure is reversible and cannot be attributed to pressure-induced cell damage. Mean arterial pressure in healthy humans is in the range 80 to 100 m m Hg. The pressure in small arteries and arterioles generally ranges from 60 to 90 m m Hg in the small arteries to 40 to 60 m m Hg in the arterioles. Our results suggest that NO release may be suppressed, to some extent, under physiological conditions. It is interesting that NO release was better maintained in low-pressure vessels, such as capillaries than in high-pressure vessels, such as large arteries. Although the precise mechanism of the inhibitory effect of transmural pressure on NO release, especially
on basal (non-stimulated) N O release, remains to be elucidated, these results suggest that pressure-induced inhibition of N O release is one explanation for the pressure-induced vasoconstriction (Rubanyi, 1988; H a r d e r et al., 1989) and reduced endothelium-dependent relaxation seen in patients with hypertension (Panza et al., 1990).
References Buga, G.M., M.E. Gold, J.M. Fukuto and L.J. lgnarro, 1991, Shear stress-induced release of nitric oxide from endothelial cells grown on beads, Hypertension 17, 187. Harder, D.R., C. Sanchez-Ferrer, K. Kauser, W.J. Stekiel and G.M. Rubanyi, 1989, Pressure release a transferable endothelial contractile factor in cat cerebral arteries, Circ. Res. 65, 193. Hishikawa, K., T. Nakaki, M. Tsuda, H. Esumi, H. Ohshima, H. Suzuki, T. Saruta and R. Kato, Effect of systemic L-arginine administration on haemodynamics and nitric oxide release in man, Jap. Heart. J. (in press). Jaffe, E.A., R. Nachman, C. Becker and D. Minik, 1973, Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria, J. Clin. Invest. 52, 2754. Lansman, J.B., T.J. Hallam and T.J. Rink, 1987, Single stretchactivated ion channels in vascular endothelial cells as mechanotransducers?, Nature 325, 811. Ohno, M., M. Ochiai, J. Taguchi, K. Hara, N. Akatsuka and K. Kurokawa, 1990, Stretch may enhance the release of endothelium-derived relaxing factor in rabbit aorta, Biochem. Biophys. Res. Commun. 173, 1038. Panza, J.A., A.A. Quyyumi, J.E.Jr. Brush and S.E. Epstein, 1990, Abnormal endothelium-dependent relaxation in patients with essential hypertension, N. Engl. J. Med. 323, 22. Rubanyi, G.M., 1988, Endothelium-dependent pressure-induced contraction of isolated canine carotid arteries, Am. J. Physiol. 255, H783. Rubanyi, G.M., J.C. Romero and P.M. Vanhoutte, 1986, Flow-induced release of endothelium-derived relaxing factor, Am. J. Physiol. 250, H1145. Van de Voorde, J.E., H. Vanderstichele and I. Leusen, 1987, Release of endothelium-derived factor from human umbilical vessels, Circ. Res. 60, 517.
329
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 21054
Short communication
Transmurai pressure inhibits nitric oxide release from human endothelial cells Keiichi H i s h i k a w a a,b, T o s h i o N a k a k i b, H i r o m i c h i Suzuki a, T a k a o S a r u t a ~' a n d R y u i c h i K a t o b Departments of" Internal Medicine and b Pharmacology, Keio Unil~ersity School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan Received 28 February 1992, accepted 10 March 1992
We examined the effect of transmural pressure on histamine-stimulated nitric oxide release from cultured endothelial cells prepared from human umbilical cord veins. PO: and pH were kept constant throughout the experiments. Various levels of transmural pressure and atmospheric pressure (40, 80, 120 and 160 mm Hg) were applied. Nitric oxide release was inhibited in a pressure-dependent manner. The inhibitory effects were reversible, and nitric oxide had no effect on the morphology of the cells. Our results suggest that transmural pressure-mediated inhibition of nitric oxide release contributes to pressure-induced vasoconstriction and reduced endothelium-dependent relaxation in patients with hypertension. EDRF (endothelium-derived relaxing factor); Nitric oxide (NO); Pressure; Endothelial cells; Mechanorcceptors; Hypertension
1. Introduction Increased intraluminal pressure has been reported to cause e n d o t h e l i u m - d e p e n d e n t vasoconstriction (Rubanyi, 1988; Harder et al., 1989). There have been no studies on the effect of transmural pressure alone on nitric oxide (NO) release from human endothelial cells. According to the law of Laplace, intraluminal pressure in a distensible hollow object such as a blood vessel causes both transmural pressure and wall tension. These changes appear to affect endothelial cell function, but it is impossible to examine the effect of transmural pressure alone on endothelial cells in situ. This study was designed to assess the effect of transmural pressure alone on NO release from human endothelial cells by exposing cells cultured on flat, solid flasks to elevated pressure and by measuring nitrite/nitrate, the oxidized stable products of NO. This is the first report that directly investigated the effect of transmural pressure alone on NO release from human endothelial cells.
2. Material and methods
2.1. Culture of human umbifical cord vein endothelial cells H u m a n umbilical vein endothelial cells were prepared from human umbilical cord veins according to
Correspondence to: T. Nakaki, Department of Pharmacology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. Fax 81.3.3359 8889.
the method described by Jaffe et al. (1973), with some minor modifications. Cells were grown to confluence (5-7 × 105 cells/flask) on 0.2% gelatin-coated 25-cm 2 flasks (Corning, NY, USA) containing 5.0 ml of a medium composed of Earle's M199 (GIBCO, NY, USA), 10% fetal calf serum (Mitsubishi-kasei, Tokyo, Japan), 30 /xg/ml endothelial cell growth supplement (Sigma, St. Louis, USA), and 6 U / m l heparin. Cells were used between passages 2 and 4.
2.2. Pressure-loading apparatus The pressure-loading apparatus was set up as follows. The flask was tightly sealed with a rubber cap and compressed helium (He) gas was pumped in to raise the internal pressure. The rubber cap was pierced by a needle connected to tubing attached to a three-way rotary valve (RT-N), sphygmomanometer and pressure valve. The connecting tubing (extension tube:61B) and the three-way rotary valve were obtained from Hakko Co., Tokyo, Japan. He gas and the pressure valve were obtained from Yamato-Sanki Co., Tokyo, Japan. The mercury sphygmomanometer was obtained from Sansei Medical Industry Co., Tokyo, Japan. While the He gas was being pumped in, no pre-packed room air was released, so that the partial pressures of the gases contained in the flask, such as oxygen, nitrogen oxide and carbon dioxide, were kept constant in accordance with Boyle-Charles' law. The flask was kept at 37°C on a hot plate. Different flasks were used for each pressure. To expose cells in a single flask to various pressures, it would be necessary to wash out histamine before changing pressures. As this was not possible to
330
do without giving shear stress, we examined the pressure effect in different flasks. 0.3
2.3. Measurement of nitrite / nitrate H u m a n umbilical vein endothelial cells were washed 3 times with 5 ml Hanks solution and then incubated at 37°C for 1 hour in 2 ml of Hanks solution before the experiments. The formation of NO from human umbilical vein endothelial cells was measured as the levels of n i t r i t e / n i t r a t e , oxidized products of nitric oxide, using an automated system based on the Griess reaction (Hishikawa et al., in press).
2.4. PO 2 and pH PO 2 and p H of the incubation medium were measured with an automated analyzer (pH gas analyzer 1306, Instrumental Laboratory, Tokyo, Japan).
2.5. Statistical analysis The data are expressed as the means and S.E.M. in the text and figures. Statistical significance was assessed with a t-test, and a P value less than 0.05 was considered significant.
3. Results
N i t r i t e / n i t r a t e at 30 min without histamine was 0.05 + 0.01 n m o l / f l a s k under atmospheric pressure (0
~. 0.8 0.7
0.6
=
0.5
c 0.4
o~ 0.3
"E 0.2 "~
"E 0.2 "~ 0.1
Z
0.0 0
i 5
,
i
10
15
J
20
i
i
25
30
Incubation Time (rain)
B
~
0.2
N ~
0.1
~
O.O 0
40 80 120 Pressure (mm Hg)
160
Fig. 2. Effect of transmural pressure on histamine-stimulated NO release from human umbilical vein endothelial cells. The concentration of histamine was 10 /~M. The indicated pressure is the exogenously applied pressure plus atmospheric pressure for 30 min. * P < 0.05 vs. 0 mm Hg. Experiments were done in quadruplicate. Means_+ S.E.M. are shown.
mm Hg). Histamine (10/~M) increased n i t r i t e / n i t r a t e in the medium in a time-dependent manner by more than 10-fold (fig. 1A), which is consistent with the results of Van de Voorde et al. (1987). NO release was inhibited by transmural pressure in a pressure-dependent manner (fig. 2). The PO 2 and p H of the incubation medium ranged from 154 + 4 to 158_+ 7 mm Hg and from 7.3 + 0.1 to 7.4_+ 0.1, respectively. There were no significant changes in these values throughout the experiments, as predicted by Boyle-Charles' law. The inhibitory effect of transmural pressure was reversible (fig. 1B). Cells that had been exposed to 160 m m Hg for 30 min were incubated under atmospheric pressure for 30 rain and were then stimulated with histamine. Pretreatment with a pressure of 160 mm Hg had no effect on N O release from the cells. The viability of human umbilical vein endothelial cells, as assessed with the trypan blue exclusion test, was over 99% throughout the experiments.
4. Discussion
~" 0.0 0
160
Pressure (mm Hg)
Fig. 1. Effect of histamine ( 1 0 / ~ M ) on nitric oxide ( N O ) release from human umbilical vein endothelial cells. ( A ) Histamine stimulated the
accumulation of nitrite/nitrate in a time-dependent manner. The concentration of nitrite/nitrate without histamine was about 0.05 + 0.01 nmol/flask per 30 rain. Experiments were done in quadruplicate. The standard errors were within the symbols. (B) Reversibility of the effects of treatment with 160 mm Hg on human umbilical vein endothelial cells. The cells were incubated under atmospheric pressure alone (designated 0 mm Hg) or 160 mm Hg plus atmospheric pressure (designated 160 mm Hg) for 30 min. After 30 min recovery time, the effect of histamine on NO release was examined under atmospheric pressure. There were no significant changes in NO release between pretreatment with 0 and 160 mm Hg. Means _+S.E.M. are shown. Experiments were done in quadruplicate.
Increased intraluminal pressure has been reported to cause vasoconstriction (Rubanyi, 1988; H a r d e r et al., 1989). The three best understood components of hemodynamic force are shear stress, wall tension, and transmural pressure. However, it is difficult to evaluate the effects of transmural pressure alone on vessels in situ. According to the law of Laplace, transmural pressure in a distensible hollow object causes wall tension, which may stretch endothelial cells. Both shear stress and stretch have been reported to enhance Ca 2 + infux and N O release (Rubanyi et al., 1986; Buga et al., 1991; Ohno et at., 1990; Lansman et al., 1987). The effect of transmural pressure on NO release from
331
human endothelial cells has not been directly investigated. This study was designed to assess the effects of transmural pressure alone on N O release from h u m a n umbilical vein endothelial cells by exposing cells cultured on flat, solid flasks to an elevated pressure. The pressure was raised by pumping compressed He gas into a flask packed with room air. While the He gas was being p u m p e d into the flask, no pre-packed room air was released and the temperature was kept at 37°C. In these systems, the partial pressure of 0 2 and CO 2 were kept constant, in accordance with the B o y l e Charles' law. Our results indicate that histamine-induced N O release was inhibited by increasing the transmural pressure. To examine whether the inhibition of N O release by transmural pressure was caused by cell damage, we also exposed cells to atmospheric pressure (0 m m Hg) and 160 m m Hg for 30 min, and N O release was assessed after 30 min recovery time (fig. 1B). There were no significant changes in N O release between the two experimental conditions. Cell viability was over 99% (trypan blue exclusion) and no morphological abnormalities were found by light microscopic observation at the end of the experiments. These results clearly show that the inhibition of N O release by transmural pressure is reversible and cannot be attributed to pressure-induced cell damage. Mean arterial pressure in healthy humans is in the range 80 to 100 m m Hg. The pressure in small arteries and arterioles generally ranges from 60 to 90 m m Hg in the small arteries to 40 to 60 m m Hg in the arterioles. Our results suggest that NO release may be suppressed, to some extent, under physiological conditions. It is interesting that NO release was better maintained in low-pressure vessels, such as capillaries than in high-pressure vessels, such as large arteries. Although the precise mechanism of the inhibitory effect of transmural pressure on NO release, especially
on basal (non-stimulated) N O release, remains to be elucidated, these results suggest that pressure-induced inhibition of N O release is one explanation for the pressure-induced vasoconstriction (Rubanyi, 1988; H a r d e r et al., 1989) and reduced endothelium-dependent relaxation seen in patients with hypertension (Panza et al., 1990).
References Buga, G.M., M.E. Gold, J.M. Fukuto and L.J. lgnarro, 1991, Shear stress-induced release of nitric oxide from endothelial cells grown on beads, Hypertension 17, 187. Harder, D.R., C. Sanchez-Ferrer, K. Kauser, W.J. Stekiel and G.M. Rubanyi, 1989, Pressure release a transferable endothelial contractile factor in cat cerebral arteries, Circ. Res. 65, 193. Hishikawa, K., T. Nakaki, M. Tsuda, H. Esumi, H. Ohshima, H. Suzuki, T. Saruta and R. Kato, Effect of systemic L-arginine administration on haemodynamics and nitric oxide release in man, Jap. Heart. J. (in press). Jaffe, E.A., R. Nachman, C. Becker and D. Minik, 1973, Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria, J. Clin. Invest. 52, 2754. Lansman, J.B., T.J. Hallam and T.J. Rink, 1987, Single stretchactivated ion channels in vascular endothelial cells as mechanotransducers?, Nature 325, 811. Ohno, M., M. Ochiai, J. Taguchi, K. Hara, N. Akatsuka and K. Kurokawa, 1990, Stretch may enhance the release of endothelium-derived relaxing factor in rabbit aorta, Biochem. Biophys. Res. Commun. 173, 1038. Panza, J.A., A.A. Quyyumi, J.E.Jr. Brush and S.E. Epstein, 1990, Abnormal endothelium-dependent relaxation in patients with essential hypertension, N. Engl. J. Med. 323, 22. Rubanyi, G.M., 1988, Endothelium-dependent pressure-induced contraction of isolated canine carotid arteries, Am. J. Physiol. 255, H783. Rubanyi, G.M., J.C. Romero and P.M. Vanhoutte, 1986, Flow-induced release of endothelium-derived relaxing factor, Am. J. Physiol. 250, H1145. Van de Voorde, J.E., H. Vanderstichele and I. Leusen, 1987, Release of endothelium-derived factor from human umbilical vessels, Circ. Res. 60, 517.
