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salt hypertensive rats
Life Sciences, Vol. 48, pp. 465-468 Printed in the U.S.A.
Pergamon Press
AORTIC ORNITHINE DECARBOXYLASE ACTIVITY IN DEOXYCORTICOSTERONE/SALT HYPERTENSIVE RATS Mark Leitschuh, M.D. Vandana Hingorani, Ph.D. Peter Brecher, Ph.D. Aram V. Chobanian, M.D. Whitaker Cardiovascular Institute Boston University School of Medicine 80 East Concord Street Boston, Mass. 02118 (Received in final form November 27, 1990) Sum~_Ll
Previous studies from our own and other laboratories have shown that hypertension induces changes in the growth of arterial smooth muscle cells (SMC). The purpose of this study was to examine the role of ornithine decarboxylase (OrnDCase) in this process. OrnDCase, the rate limiting enzyme in polyamine biosynthesis, increases in activity early in the cell cycle, and has been used as a marker of cell growth or proliferation. Deoxycorticosterone (DOC)/salt hypertension was induced in male Wistar rats. At 1-3 day intervals of DOC/salt treatment, the aortas were removed and OrnDCase activity and DNA content were determined. The results indicated that OrnDCase activity increased as early as day 2 of DOC/salt administration, reached a peak at day 10, and fell to a baseline by day 16. DNA content increased after day 10 to levels approximately 25% greater than in controls. Significant increases in blood pressure were not observed until after day 8. The findings indicate that OrnDCase activity is stimulated by DOC/salt even before the rise in blood pressure and that factors other than blood pressure per se may be important in stimulating aortic smooth muscle cell growth in the development of hypertension. Hyperterlsion has been shown to increase aortic smooth muscle cell (SMC) mass in a number of experimental animal models (1,2,3). This increase is due to either hypertrophy, hyperplasia, or hyperploidy of the SMC, the extent of each of these changes dependent on the specific model of hypertension (4). We have previously shown, in the DOC/salt hypertensive rat, that hyperploidy is a major contributor to increased aortic SMC mass, with 15-30 percent of the cells becoming polyploid over a four- to six-week period (5). Although such changes in SMC growth have been well described, little is known regarding the exact mechanism involved. The current study was designed to determine whether arterial SMC growth might be initiated prior to any rise in blood pressure. We have used aortic ornithine decarboxylase activity as a marker of cell growth (6). This enzyme is rate-limiting in the biosynthesis of polyamines. Physiological concentrations of polyamines have been shown in vitro to increase DNA replication, transcription, and m R N A translation. Both polyamine levels and OrnDCase activity appear to be high in cells undergoing rapid growth. Furthermore, OrnDCase gene expression has been shown to increase as cells move from the resting or Go phase of the cell cycle through G 1 (7,8). With these considerations in mind, we have examined OrnDCase activity in the aorta of DOC/salt hypertensive rats and have correlated this activity with blood pressure and changes in aortic DNA content. Corresponding author: Aram V. Chobanian, M.D., Whitaker Cardiovascular Institute, Boston University School of Medicine. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
466
OrnDCase in DOC/Salt Hypertension
Vol. 48, No. 5, 1991
Methods Deoxycorticosterone (DOC)/salt hypertension was induced in 45 uninephrectomized male Wistar rats (Charles River Breeding Laboratory, Wilmington, Mass.) weighing between 175 and 200 gm by injection of deoxyeortieosterone pivalate (15 mg/kg subcutaneously, biweekly) and administration of 1% saline as drinking water. At two-day intervals, two to three animals were killed with a lethal dose of sodium pentobarbital after measurement of systolic blood pressure (BP) by indirect tail cuff technique (8). Aortic weight, OrnDCase activity, DNA content, and protein concentration were determined for each animal as described below. Ornithine Decarboxvlase Activity - - The aorta was removed from the arch to the bifurcation, stripped of adventitia, frozen in liquid nitrogen and stored at -70°C. Each aorta was homogenized individually in 0.5 ml of buffer containing 50 mM Tris HCI, pH 7.2, 5 mM dithiothreitol, 1 mM EDTA, and 0.5 mM pyridoxal-5'-phosphate using a polytron homogenizer (Brickman Instruments, Geneva, Switzerland). The homogenates were centrifuged at 26,000 x g for 30 minutes at 0-4°C. The supernatant (soluble fraction) was collected and used for determinations of OrnDCase activity. The pellet was saved for DNA measurement. OrnDCase activity was determined by measuring 14C_O2 release from L-[1-14C] ornithine HCI (Amersham, Arlington Heights, I11.) as described by Russell and Snyder (9). The incubation medium contained a final concentration of 0.5 mM dithiothreitol, 0.05 M pyridoxal phosphate, and 60 mM 14C-Ornithine as substrate. The reaction was allowed to proceed for three hours at 37°C in a closed vial, with 14CO~ collected on hyamine hydroxide-treated filter papers which were counted by a liquid scintillation counter. DNA Content and Protein Concentration - - Aortic DNA content was determined by the fluorometric assay of Labarca and Piegen (10). Aortic protein concentration was determined in the soluble fraction by the dye binding assay of Bradford (11) (Bio-Rad Laboratories, Richmond, Calif.). Results Ornithine Decarboxyl~se Activity and Svstolic Blood Pressure in Deoxvcorticoster0ne/s~lt Hyoerten~ion - - OrnDCase activity and systolic blood pressure (BP) are plotted against duration of deoxycorticosterone (DOC)/salt treatment in Figure 1. As shown,
~3
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~
a --.¢ 0 o E
10.0 9.0 8.0 7.0
• o ~/
.-~
o o ,, ~<>---'o'----
////
o
235 220
.¢ ~
0-205 190
I~. '
80
~/o 5.0 J o o /~°oo 4.0 o ~/ e 3.0 // 2.0 /D...o. _..oo S 1.0 ¢ ,
2
~
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i
10
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14
175
o • • ~e~'-e~
,
18
'
22
"~:Z 160 145 .2 130 115
,
26
30
100
Days of D O C - S a l t Treatment Fig. 1 Aortic ornithine decarboxylase activity and systolic blood pressure plotted against duration of DOC/salt treatment in the rat. Each point represents the average of determinations made in two to three animals.
Vol. 48, No. 5, 1991
OrnDCase in DOC/Salt Hypertension
467
OrnDCase activity clearly increases between 2 and 8 days of treatment, peaks at approximately day 8-10, returns to relatively low levels by day 16-18, and does not change appreciably despite a marked elevation of BP from 115 to greater than 200 mmHg over a two-week period. Note that OrnDCase activity appears to increase prior to significant elevation in blood pressure. Aortic DNA Content in Deoxvcorticosterone/salt Hypertension - - Figure 2 compares aortic DNA content and systolic blood pressure versus days of DOC/salt treatment. Increases in aortic DNA occur only after day 10 of treatment and were closely correlated to the rise in systolic blood pressure. The increase in DNA content occurred after the increase in OrnDCase activity shown in Figure 1.
.50 _
00
220
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-
.40
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!
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J180E
ioo __
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O
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<1:.30 Z
.20~r 0
t
I 4
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o°o
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I I I 12
140 120
.~_
°
(n
I I I I I I I ~100 16 20 24 28
D a y s of D O C - S a l t
Treatment
Fig. 2 Aortic DNA content and systolic blood pressure plotted against duration of DOC/salt treatment. Each point represents the average of determinations made in two to three animals. Discussion The main purpose of this study was to examine the temporal relationship between changes in aortic cell growth and the rise in blood pressure in DOC/salt hypertensive rats. The results indicate that OrnDCase activity, used as a marker of cell growth, increases early with DOC/salt treatment and prior to a significant rise in blood pressure. This increase in OrnDCase activity also precedes the increase in aortic DNA content, which more closely paralleled the blood pressure curve. The data suggest that factors other than blood pressure per se may be important stimulants to aortic cell growth in response to hypertension. The findings are consistent with those of Loeb et al. who found increased aortic DNA synthesis in two-kidney, one-clip renal hypertensive rats prior to significant increases in blood pressure (12). There are several mechanisms by which cell growth could be stimulated which involve increased OrnDCase activity. Johnson et al. (13) made rats hypertensive using chronic rate catecholamine infusion and found an early rise in aortic OrnDCase activity at a point of rapid cell growth in the aorta. Majesky et al. (14) observed a complex relationship between alphaand beta-adrenergic stimulation of OrnDCase activity in the cockerel aorta. The betaadrenerglc agonist isoproterenol reduced basal aortic OrnDCase activity and prevented induction of OrnDCase by the alpha-adrenergic agonist methoxamine. Further studies by Majesky (15) reported that alpha-adrenergic stimulation by phenylephrine and norepinephrine in Sprague-Dawley rats produced a dose-dependent stimulation of platelet-derived growth factor A-chaln (PDGF-A) gene expression by occupancy of alpha-adrenergic receptors.
468
OrnDCase in DOC/Salt Hypertension
Vol. 48, No. 5, 1991
There also was an increase in other growth-related genes including OrnDCase. PDGF also has been shown to stimulate OrnDCase activity in cultured arterial SMC (16). Rubin et al. (17) found PDGF-beta receptors in vascular SMC in atheroselerotic plaques and has suggested that induction of PDGF-type receptors may be involved in the etiology of proliferative vascular lesions. Thus, alpha-adrenergic receptor stimulation could mediate SMC growth by acting via PDGF which in turn may stimulate other growth-related genes such as OrnDCase. Owens (18) and others have suggested that neurohumoral factors known to be involved in the pathogenesis of hypertension, such as angiotension II, could act as growth stimulants. He reported that captopril treatment, which reduces angiotension II levels, has an effect over and above that predicted by its blood pressure-lowering action in reducing SMC polyploidy and hypertrophy in spontaneously hypertensive rats. His more recent studies (19) have suggested that contractile agonists, such as angiotension II, serve as "partial" growth factors for SMCs by initiating increases in cell mass and in some cases DNA replication, but not cell division. Because of the complex nature of the DOC/salt model of hypertension, which affects neurohumoral levels, pH, ion concentration and other metabolic factors, it is difficult to determine the exact factor or mechanism by which SMC growth is stimulated. Factors specific to this model of hypertension, such as renal mass reduction, sodium chloride or mineralocorticoid administration may be responsible for the changes seen. However, previous work from our laboratory (1) has shown that nephrectomy alone with and without salt or mineralocorticoid administration by itself will not bring about increases in cell mass or number seen in the aorta. In summary, we have shown that aortic OrnDCase activity, a marker of cell growth, increases early in the development of deoxycorticosterone/salt hypertension and prior to a rise in blood pressure. The findings indicate that factors other than blood pressure p~r se may be important in stimulating early aortic cell growth in the development of hypertension.
Acknowledgements Supported by NIH Grant HL 18318 (Hypertension SCOR) and HL 07224
Refergnees 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
A.H. LICHTENSTEIN, P. BRECHER, A. CHOBANIAN, Hypertension 8 II-50-II-54 (1986). G. OWENS, S. SCHWARTZ, Circ Res 51 280-289 (1982). G. OWENS, S. SCHWARTZ, Circ Res 56 525-536 (1985). M. LEITSCHUH, A. CHOBANIAN, Med Clin No Am 7...~1827-841 (1987). M. LEITSCHUH, A. CHOBANIAN, Hypertension 9 III-106 - III-109 0987). M. MAJESKY, M. CLOWES, S. SCHWARTZ, A. CLOWES, Circ Res 61 296-300 (1987). S. RITTLING, K. BROOKS, V. CRISTOFALO, R. BASENGA, PNAS 83 3316-3320 (1986). C.C. HAUDENSCHILD, M.F. PRESCOTT, A.V. CHOBANIAN, Hypertension 3 1-1481-153 (1981). D. RUSSELL, S. SNYDER, PNAS 60 1420-1427 (1968). C. LABARCA, K. PIEGEN, Anal Bioch 102 344-352 (1980). M. BRADFORD, Anal Bioch 7_22248-259 (1976). A. LOEB, G. MANDEL, J. STRAW, B. BEAN, Hypertension 8_ 754-761 (1986). M. JOHNSON, A. GRIGNOLO, C. KUHN, S. SCHANBERG, Life Sci 33 169-180 (1983). M. MAJESKY, L. HSUEH-YING, M. JACHAU, Life Sci 36153-159 (1985). M. MAJESKY, M. DAEMEN, S. SCHWARTZ, J Biol Chem 26_...551082-1088 (1990). J. THYBERG, B. FREDHOLM, Exp Cell Research 170160-169 (1987). K. RUBIN, G. HANSOON, L. RONNSTRAND, A. CLARESSON-WlLSH, B. FELLSTROM, A. TINGSTROM, E. LARSSON, L. KLARESKOG, C. HELDIN, L. TERRACIO, Lancet 11353-1356 (1988). G. OWENS, Hypertension 9178-187 (1987). G. OWENS, Am J Physiol 2___66HI755-H1765 (1989).
Pergamon Press
AORTIC ORNITHINE DECARBOXYLASE ACTIVITY IN DEOXYCORTICOSTERONE/SALT HYPERTENSIVE RATS Mark Leitschuh, M.D. Vandana Hingorani, Ph.D. Peter Brecher, Ph.D. Aram V. Chobanian, M.D. Whitaker Cardiovascular Institute Boston University School of Medicine 80 East Concord Street Boston, Mass. 02118 (Received in final form November 27, 1990) Sum~_Ll
Previous studies from our own and other laboratories have shown that hypertension induces changes in the growth of arterial smooth muscle cells (SMC). The purpose of this study was to examine the role of ornithine decarboxylase (OrnDCase) in this process. OrnDCase, the rate limiting enzyme in polyamine biosynthesis, increases in activity early in the cell cycle, and has been used as a marker of cell growth or proliferation. Deoxycorticosterone (DOC)/salt hypertension was induced in male Wistar rats. At 1-3 day intervals of DOC/salt treatment, the aortas were removed and OrnDCase activity and DNA content were determined. The results indicated that OrnDCase activity increased as early as day 2 of DOC/salt administration, reached a peak at day 10, and fell to a baseline by day 16. DNA content increased after day 10 to levels approximately 25% greater than in controls. Significant increases in blood pressure were not observed until after day 8. The findings indicate that OrnDCase activity is stimulated by DOC/salt even before the rise in blood pressure and that factors other than blood pressure per se may be important in stimulating aortic smooth muscle cell growth in the development of hypertension. Hyperterlsion has been shown to increase aortic smooth muscle cell (SMC) mass in a number of experimental animal models (1,2,3). This increase is due to either hypertrophy, hyperplasia, or hyperploidy of the SMC, the extent of each of these changes dependent on the specific model of hypertension (4). We have previously shown, in the DOC/salt hypertensive rat, that hyperploidy is a major contributor to increased aortic SMC mass, with 15-30 percent of the cells becoming polyploid over a four- to six-week period (5). Although such changes in SMC growth have been well described, little is known regarding the exact mechanism involved. The current study was designed to determine whether arterial SMC growth might be initiated prior to any rise in blood pressure. We have used aortic ornithine decarboxylase activity as a marker of cell growth (6). This enzyme is rate-limiting in the biosynthesis of polyamines. Physiological concentrations of polyamines have been shown in vitro to increase DNA replication, transcription, and m R N A translation. Both polyamine levels and OrnDCase activity appear to be high in cells undergoing rapid growth. Furthermore, OrnDCase gene expression has been shown to increase as cells move from the resting or Go phase of the cell cycle through G 1 (7,8). With these considerations in mind, we have examined OrnDCase activity in the aorta of DOC/salt hypertensive rats and have correlated this activity with blood pressure and changes in aortic DNA content. Corresponding author: Aram V. Chobanian, M.D., Whitaker Cardiovascular Institute, Boston University School of Medicine. 0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
466
OrnDCase in DOC/Salt Hypertension
Vol. 48, No. 5, 1991
Methods Deoxycorticosterone (DOC)/salt hypertension was induced in 45 uninephrectomized male Wistar rats (Charles River Breeding Laboratory, Wilmington, Mass.) weighing between 175 and 200 gm by injection of deoxyeortieosterone pivalate (15 mg/kg subcutaneously, biweekly) and administration of 1% saline as drinking water. At two-day intervals, two to three animals were killed with a lethal dose of sodium pentobarbital after measurement of systolic blood pressure (BP) by indirect tail cuff technique (8). Aortic weight, OrnDCase activity, DNA content, and protein concentration were determined for each animal as described below. Ornithine Decarboxvlase Activity - - The aorta was removed from the arch to the bifurcation, stripped of adventitia, frozen in liquid nitrogen and stored at -70°C. Each aorta was homogenized individually in 0.5 ml of buffer containing 50 mM Tris HCI, pH 7.2, 5 mM dithiothreitol, 1 mM EDTA, and 0.5 mM pyridoxal-5'-phosphate using a polytron homogenizer (Brickman Instruments, Geneva, Switzerland). The homogenates were centrifuged at 26,000 x g for 30 minutes at 0-4°C. The supernatant (soluble fraction) was collected and used for determinations of OrnDCase activity. The pellet was saved for DNA measurement. OrnDCase activity was determined by measuring 14C_O2 release from L-[1-14C] ornithine HCI (Amersham, Arlington Heights, I11.) as described by Russell and Snyder (9). The incubation medium contained a final concentration of 0.5 mM dithiothreitol, 0.05 M pyridoxal phosphate, and 60 mM 14C-Ornithine as substrate. The reaction was allowed to proceed for three hours at 37°C in a closed vial, with 14CO~ collected on hyamine hydroxide-treated filter papers which were counted by a liquid scintillation counter. DNA Content and Protein Concentration - - Aortic DNA content was determined by the fluorometric assay of Labarca and Piegen (10). Aortic protein concentration was determined in the soluble fraction by the dye binding assay of Bradford (11) (Bio-Rad Laboratories, Richmond, Calif.). Results Ornithine Decarboxyl~se Activity and Svstolic Blood Pressure in Deoxvcorticoster0ne/s~lt Hyoerten~ion - - OrnDCase activity and systolic blood pressure (BP) are plotted against duration of deoxycorticosterone (DOC)/salt treatment in Figure 1. As shown,
~3
I!
"F: :~ oO ~0
~
a --.¢ 0 o E
10.0 9.0 8.0 7.0
• o ~/
.-~
o o ,, ~<>---'o'----
////
o
235 220
.¢ ~
0-205 190
I~. '
80
~/o 5.0 J o o /~°oo 4.0 o ~/ e 3.0 // 2.0 /D...o. _..oo S 1.0 ¢ ,
2
~
6
i
10
'
14
175
o • • ~e~'-e~
,
18
'
22
"~:Z 160 145 .2 130 115
,
26
30
100
Days of D O C - S a l t Treatment Fig. 1 Aortic ornithine decarboxylase activity and systolic blood pressure plotted against duration of DOC/salt treatment in the rat. Each point represents the average of determinations made in two to three animals.
Vol. 48, No. 5, 1991
OrnDCase in DOC/Salt Hypertension
467
OrnDCase activity clearly increases between 2 and 8 days of treatment, peaks at approximately day 8-10, returns to relatively low levels by day 16-18, and does not change appreciably despite a marked elevation of BP from 115 to greater than 200 mmHg over a two-week period. Note that OrnDCase activity appears to increase prior to significant elevation in blood pressure. Aortic DNA Content in Deoxvcorticosterone/salt Hypertension - - Figure 2 compares aortic DNA content and systolic blood pressure versus days of DOC/salt treatment. Increases in aortic DNA occur only after day 10 of treatment and were closely correlated to the rise in systolic blood pressure. The increase in DNA content occurred after the increase in OrnDCase activity shown in Figure 1.
.50 _
00
220
//-.
-
.40
.,..
/
!
,oo; e
J180E
ioo __
•
O
_O., "/
<1:.30 Z
.20~r 0
t
I 4
t
o°o
//
t 8
t
I I I 12
140 120
.~_
°
(n
I I I I I I I ~100 16 20 24 28
D a y s of D O C - S a l t
Treatment
Fig. 2 Aortic DNA content and systolic blood pressure plotted against duration of DOC/salt treatment. Each point represents the average of determinations made in two to three animals. Discussion The main purpose of this study was to examine the temporal relationship between changes in aortic cell growth and the rise in blood pressure in DOC/salt hypertensive rats. The results indicate that OrnDCase activity, used as a marker of cell growth, increases early with DOC/salt treatment and prior to a significant rise in blood pressure. This increase in OrnDCase activity also precedes the increase in aortic DNA content, which more closely paralleled the blood pressure curve. The data suggest that factors other than blood pressure per se may be important stimulants to aortic cell growth in response to hypertension. The findings are consistent with those of Loeb et al. who found increased aortic DNA synthesis in two-kidney, one-clip renal hypertensive rats prior to significant increases in blood pressure (12). There are several mechanisms by which cell growth could be stimulated which involve increased OrnDCase activity. Johnson et al. (13) made rats hypertensive using chronic rate catecholamine infusion and found an early rise in aortic OrnDCase activity at a point of rapid cell growth in the aorta. Majesky et al. (14) observed a complex relationship between alphaand beta-adrenergic stimulation of OrnDCase activity in the cockerel aorta. The betaadrenerglc agonist isoproterenol reduced basal aortic OrnDCase activity and prevented induction of OrnDCase by the alpha-adrenergic agonist methoxamine. Further studies by Majesky (15) reported that alpha-adrenergic stimulation by phenylephrine and norepinephrine in Sprague-Dawley rats produced a dose-dependent stimulation of platelet-derived growth factor A-chaln (PDGF-A) gene expression by occupancy of alpha-adrenergic receptors.
468
OrnDCase in DOC/Salt Hypertension
Vol. 48, No. 5, 1991
There also was an increase in other growth-related genes including OrnDCase. PDGF also has been shown to stimulate OrnDCase activity in cultured arterial SMC (16). Rubin et al. (17) found PDGF-beta receptors in vascular SMC in atheroselerotic plaques and has suggested that induction of PDGF-type receptors may be involved in the etiology of proliferative vascular lesions. Thus, alpha-adrenergic receptor stimulation could mediate SMC growth by acting via PDGF which in turn may stimulate other growth-related genes such as OrnDCase. Owens (18) and others have suggested that neurohumoral factors known to be involved in the pathogenesis of hypertension, such as angiotension II, could act as growth stimulants. He reported that captopril treatment, which reduces angiotension II levels, has an effect over and above that predicted by its blood pressure-lowering action in reducing SMC polyploidy and hypertrophy in spontaneously hypertensive rats. His more recent studies (19) have suggested that contractile agonists, such as angiotension II, serve as "partial" growth factors for SMCs by initiating increases in cell mass and in some cases DNA replication, but not cell division. Because of the complex nature of the DOC/salt model of hypertension, which affects neurohumoral levels, pH, ion concentration and other metabolic factors, it is difficult to determine the exact factor or mechanism by which SMC growth is stimulated. Factors specific to this model of hypertension, such as renal mass reduction, sodium chloride or mineralocorticoid administration may be responsible for the changes seen. However, previous work from our laboratory (1) has shown that nephrectomy alone with and without salt or mineralocorticoid administration by itself will not bring about increases in cell mass or number seen in the aorta. In summary, we have shown that aortic OrnDCase activity, a marker of cell growth, increases early in the development of deoxycorticosterone/salt hypertension and prior to a rise in blood pressure. The findings indicate that factors other than blood pressure p~r se may be important in stimulating early aortic cell growth in the development of hypertension.
Acknowledgements Supported by NIH Grant HL 18318 (Hypertension SCOR) and HL 07224
Refergnees 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
A.H. LICHTENSTEIN, P. BRECHER, A. CHOBANIAN, Hypertension 8 II-50-II-54 (1986). G. OWENS, S. SCHWARTZ, Circ Res 51 280-289 (1982). G. OWENS, S. SCHWARTZ, Circ Res 56 525-536 (1985). M. LEITSCHUH, A. CHOBANIAN, Med Clin No Am 7...~1827-841 (1987). M. LEITSCHUH, A. CHOBANIAN, Hypertension 9 III-106 - III-109 0987). M. MAJESKY, M. CLOWES, S. SCHWARTZ, A. CLOWES, Circ Res 61 296-300 (1987). S. RITTLING, K. BROOKS, V. CRISTOFALO, R. BASENGA, PNAS 83 3316-3320 (1986). C.C. HAUDENSCHILD, M.F. PRESCOTT, A.V. CHOBANIAN, Hypertension 3 1-1481-153 (1981). D. RUSSELL, S. SNYDER, PNAS 60 1420-1427 (1968). C. LABARCA, K. PIEGEN, Anal Bioch 102 344-352 (1980). M. BRADFORD, Anal Bioch 7_22248-259 (1976). A. LOEB, G. MANDEL, J. STRAW, B. BEAN, Hypertension 8_ 754-761 (1986). M. JOHNSON, A. GRIGNOLO, C. KUHN, S. SCHANBERG, Life Sci 33 169-180 (1983). M. MAJESKY, L. HSUEH-YING, M. JACHAU, Life Sci 36153-159 (1985). M. MAJESKY, M. DAEMEN, S. SCHWARTZ, J Biol Chem 26_...551082-1088 (1990). J. THYBERG, B. FREDHOLM, Exp Cell Research 170160-169 (1987). K. RUBIN, G. HANSOON, L. RONNSTRAND, A. CLARESSON-WlLSH, B. FELLSTROM, A. TINGSTROM, E. LARSSON, L. KLARESKOG, C. HELDIN, L. TERRACIO, Lancet 11353-1356 (1988). G. OWENS, Hypertension 9178-187 (1987). G. OWENS, Am J Physiol 2___66HI755-H1765 (1989).