RELATION OF CATECHOLAMINES TO COLLAGEN SYNTHESIS S. Spector, F. M. Lai, A. Ooshima, G. Cardinale, and S. Udenfriend Depart men t of Phvsiological Ch ernis try Roche Instituteof Molecular Biology Nutley, N e w Jersey 071 10
Various laboratories have reported on the relationship between experimental arteriosclerosis and an increase in collagen biosynthesis.'.' One of the animal models employed to induce fibrous plaque formation within a few weeks is intravenous infusion of epinephrine to thyrotoxic rabbits. It is well known that alterations in thyroid activity result in profound changes in cardiovascular function. An increased heart rate, systolic blood pressure, and cardiac output a r e well-known signs not only of hyperthyroidism but also of excess sympathetic activity. Thus, it has been assumed that excess thyroid hormone potentiates the action of catecholamines. In a previous report, we have shown that the induction of hypertension in rats by the administration of desoxycorticosterone (DOCA) acetate and salt to uninephrectomized rats or that which naturally occurs in the genetically hypertensive rat (SHR), as developed by Ooshima et al. in Japan, is enough of an insult to the cardiovascular system to initiate collagen biosynthe~is.~ Wdenfriend4 showed that an increase in collagen synthesis seems to be related to the severity of the hypertension. We, therefore, generated the working hypothesis that collagen synthesis and deposition had to precede the formation of the fibrinous plaque. Because hypertension could stimulate collagen synthesis, our attention was focused on the possible interrelationship between collagen biosynthetis and the catecholamines. There are many reviews that discuss not only the role of catecholamines in the pathophysiology of hypertension but also the fact that many of the therapeutically efficacious antihypertensive drugs exert their action on the adrenergic nervous s y ~ t e m s . ~ This paper considers both peripheral blood vessels and cerebral microvessels, because we believe that the latter play a vital role. The deposition of collagen as a consequence of hypertension may compromise brain function. Cerebral microvessels, which are principally arterioles, venules, and capillaries, were isolated by the procedure of Brendel e f U I . . and ~ chemical analysis was performed to ascertain whether the synthetic machinery for catecholamine biosynthesis exists. TABLE1 compares the levels of the various enzymes involved in norepinephrine synthesis in the cerebral microvessels, whole brain, brain filtrate, which is brain tissue devoid of the cerebral microvessels, and a peripheral vascular artery. Although there a r e differences in enzymatic activities among these tissues, it should be noted that all of the enzymes required for synthesis of the adrenergic neurotransmitter are present and that the microvessels in some instances have greater specific activities than those seen in the whole brain. Tyrosine hydroxylase activity is lowest, however, in the microvessels. Nevertheless, the activity of this rate-limiting enzyme is sufficient, so that levels of norepinephrine in the cerebral microvessels are as great and possibly slightly higher than found in whole brain (TABLE2 ) . If one studies the catabolic enzymes of catecholamines, namely, catechol 0-methyltransferase ( C O M T ) and monoamine oxidase (MAO), the specific activities of these enzymes in the cerebral microvessels are greater than those found in whole brain tissue.
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TABLE1 NOREPINEPHRINE ANABOLIC ENZYMES IN BRAIN A N D IN BLOODVESSELS* Tissue Whole brain Brain filtrate Brain microvessels Mesentericartery
Tyrosine Hydroxylase Dopa Decarboxylase Dopamine j3-Hydroxylase (tyrosine oxidized) (CO, formed) (octopamine formed) 0.49 i 0.06 (3) 31.0 =t0.7 (3) 0.53 i 0.07 (5) 0.29 * 0.02 (3) 17.2 i 0.8 (3) 0.96 i 0.17 (5) 0.07 i 0.01 (3) 31.3 i 4.6 (3) 1.75 i 0.18 (5) 1.17 i 0.27 (3) 9.2 i 0.1 (3) 11.40 i 1.35 (5)
*Enzyme activities are expressed as nanomoles per nanograms of protein per hour with appropriate blanks substracted. Figures represent means =t SE. Numbers in parentheses refer to the number of experiments. Each experiment was performed on four to eight rats.
In rats made hypertensive by the administration of DOCA salt, collagen biosynthesis is stimulated, and this increase in collagen synthesis can be prevented if reserpine is given to these rats either simultaneously with DOCA or after a few days of DOCA treatment. Reserpine is an effective antihypertensive drug, and it has been postulated that its antihypertensive action is a consequence of its effect on biogenic amines. Because reserpine can deplete tissue norepinephrine content in brain and peripheral tissues, the possible relationship between the depletion of tissue norepinephrine and the reduction in collagen biosynthesis was studied. Reserpine at 0.75 mg/kg causes a slight reduction of tissue norepinephrine content prior to any effect on prolyl hydroxylase activity. However, levels and activity have to be considered with respect to turnover rates. Norepinephrine may have a faster turnover rate than prolyl hydroxylase, and one might therefore see an effect on one system prior to any observable effect on another. Reserpine, however, not only depletes biogenic amines but also can release hypothalamic hypophysiotropic factors, and there have been many studies that indicate modulation of pituitary function by brain transmitters.' After hypophysectomy, TABLE2 NOREPINEPHRINE CONTENT (Wg) Whole brain Brain filtrate Brain microvessels Mesenteric artery
0.45 i 0.06* -
0.64 i 0.09 2.76 i 0.34
*Means i SE.
TABLE3 CATECHOLAMINE DECRADATIVE ENZYMES Tissue Whole brain Brain filtrate Brain microvessels Mesenteric artery
Monoamine Oxidase (nmol/mn protein)
Catechol 0-Methyltransferase (nmol/mg protein)
179.7 i 8.4 134.8 & 4.7 425.4 i 23.1 76.3 =t 8.4
0.046 i 0.03 0.201 f 0.08 0.159 i 0.07
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Annals N e w York Academy of Sciences
1 I6
= =
20
E h
2
16
$ - c 2e
3 W
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CONTROL + RESERPINE HYPOPHYSECTOMY + RESERPINE
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* \ x m
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5 2 4 g
AORTA MES.ART.
HEART
KIDNEY
LIVER
LUNG
SKIN
FIGURE I . Effect of hypophysectomy and reserpine on prolyl hydroxylase in various rat tissues. Hypophysectomy was performed 2 weeks before the analysis of prolyl hydroxylase, while reserpine was administered daily at a dose of 0-75 mg/kg for 1 week. Values represent the means of rats.
there is also a decrease in collagen biosynthesis when prolyl hydroxylase is used as the indicator ( F I G U R EI), and this decrease in prolyl hydroxylase activity in the vasculature is even greater than that elicited by reserpine. In many tissues, particularly the aorta and rnesenteric artery, the effects of hypophysectomy and reserpine are additive, which indicates that reserpine has an effect on these tissues other than through action on the pituitary. These studies demonstrate that isolated brain microvessels posses the anabolic and catabolic enzymes necessary for catecholamine metabolism. The antihypertensive drug reserpine, which can deplete tissues of their norepinephrine content and decrease collagen biosynthesis, may modify collagen formation by affecting the pituitary as one of its many sites of action.
REFERENCES 1 . FULLER, G. C. & R. 0.LANGNOR. 1970. Science 168: 987. J . VITTO,P. HELIN & I. LORENZEN. 1972. Circulation Res. 30: 123. 2. LINDY,S., H. TURKO, 3. OOSHIMA, A., G . C. FULLER, G. S. CARDINALE, S. SPECTOR & S. UDENFRIEND. 1974. Proc. Nat. Acad. Sci. USA 71(8): 3019. 4. UDENFRIEND, S. This monograph. 1967. CirCUkitiOn Res. 5. CATECHOLAMINES IN CARDIOVASCULAR PHYSIOLOGY AND DISEASE. (Suppl. III)XXI(6). & E. C. CARLSON. 1974. Science 185: 953. 6. BRENDEL,K . , E. MEEZAN 7. D E WED, D. & W. DE JONG. 1974. Annu. Rev. Pharmacol. 14: 389.
Various laboratories have reported on the relationship between experimental arteriosclerosis and an increase in collagen biosynthesis.'.' One of the animal models employed to induce fibrous plaque formation within a few weeks is intravenous infusion of epinephrine to thyrotoxic rabbits. It is well known that alterations in thyroid activity result in profound changes in cardiovascular function. An increased heart rate, systolic blood pressure, and cardiac output a r e well-known signs not only of hyperthyroidism but also of excess sympathetic activity. Thus, it has been assumed that excess thyroid hormone potentiates the action of catecholamines. In a previous report, we have shown that the induction of hypertension in rats by the administration of desoxycorticosterone (DOCA) acetate and salt to uninephrectomized rats or that which naturally occurs in the genetically hypertensive rat (SHR), as developed by Ooshima et al. in Japan, is enough of an insult to the cardiovascular system to initiate collagen biosynthe~is.~ Wdenfriend4 showed that an increase in collagen synthesis seems to be related to the severity of the hypertension. We, therefore, generated the working hypothesis that collagen synthesis and deposition had to precede the formation of the fibrinous plaque. Because hypertension could stimulate collagen synthesis, our attention was focused on the possible interrelationship between collagen biosynthetis and the catecholamines. There are many reviews that discuss not only the role of catecholamines in the pathophysiology of hypertension but also the fact that many of the therapeutically efficacious antihypertensive drugs exert their action on the adrenergic nervous s y ~ t e m s . ~ This paper considers both peripheral blood vessels and cerebral microvessels, because we believe that the latter play a vital role. The deposition of collagen as a consequence of hypertension may compromise brain function. Cerebral microvessels, which are principally arterioles, venules, and capillaries, were isolated by the procedure of Brendel e f U I . . and ~ chemical analysis was performed to ascertain whether the synthetic machinery for catecholamine biosynthesis exists. TABLE1 compares the levels of the various enzymes involved in norepinephrine synthesis in the cerebral microvessels, whole brain, brain filtrate, which is brain tissue devoid of the cerebral microvessels, and a peripheral vascular artery. Although there a r e differences in enzymatic activities among these tissues, it should be noted that all of the enzymes required for synthesis of the adrenergic neurotransmitter are present and that the microvessels in some instances have greater specific activities than those seen in the whole brain. Tyrosine hydroxylase activity is lowest, however, in the microvessels. Nevertheless, the activity of this rate-limiting enzyme is sufficient, so that levels of norepinephrine in the cerebral microvessels are as great and possibly slightly higher than found in whole brain (TABLE2 ) . If one studies the catabolic enzymes of catecholamines, namely, catechol 0-methyltransferase ( C O M T ) and monoamine oxidase (MAO), the specific activities of these enzymes in the cerebral microvessels are greater than those found in whole brain tissue.
114
Spector et al. : Catecholamines & Collagen Synthesis
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TABLE1 NOREPINEPHRINE ANABOLIC ENZYMES IN BRAIN A N D IN BLOODVESSELS* Tissue Whole brain Brain filtrate Brain microvessels Mesentericartery
Tyrosine Hydroxylase Dopa Decarboxylase Dopamine j3-Hydroxylase (tyrosine oxidized) (CO, formed) (octopamine formed) 0.49 i 0.06 (3) 31.0 =t0.7 (3) 0.53 i 0.07 (5) 0.29 * 0.02 (3) 17.2 i 0.8 (3) 0.96 i 0.17 (5) 0.07 i 0.01 (3) 31.3 i 4.6 (3) 1.75 i 0.18 (5) 1.17 i 0.27 (3) 9.2 i 0.1 (3) 11.40 i 1.35 (5)
*Enzyme activities are expressed as nanomoles per nanograms of protein per hour with appropriate blanks substracted. Figures represent means =t SE. Numbers in parentheses refer to the number of experiments. Each experiment was performed on four to eight rats.
In rats made hypertensive by the administration of DOCA salt, collagen biosynthesis is stimulated, and this increase in collagen synthesis can be prevented if reserpine is given to these rats either simultaneously with DOCA or after a few days of DOCA treatment. Reserpine is an effective antihypertensive drug, and it has been postulated that its antihypertensive action is a consequence of its effect on biogenic amines. Because reserpine can deplete tissue norepinephrine content in brain and peripheral tissues, the possible relationship between the depletion of tissue norepinephrine and the reduction in collagen biosynthesis was studied. Reserpine at 0.75 mg/kg causes a slight reduction of tissue norepinephrine content prior to any effect on prolyl hydroxylase activity. However, levels and activity have to be considered with respect to turnover rates. Norepinephrine may have a faster turnover rate than prolyl hydroxylase, and one might therefore see an effect on one system prior to any observable effect on another. Reserpine, however, not only depletes biogenic amines but also can release hypothalamic hypophysiotropic factors, and there have been many studies that indicate modulation of pituitary function by brain transmitters.' After hypophysectomy, TABLE2 NOREPINEPHRINE CONTENT (Wg) Whole brain Brain filtrate Brain microvessels Mesenteric artery
0.45 i 0.06* -
0.64 i 0.09 2.76 i 0.34
*Means i SE.
TABLE3 CATECHOLAMINE DECRADATIVE ENZYMES Tissue Whole brain Brain filtrate Brain microvessels Mesenteric artery
Monoamine Oxidase (nmol/mn protein)
Catechol 0-Methyltransferase (nmol/mg protein)
179.7 i 8.4 134.8 & 4.7 425.4 i 23.1 76.3 =t 8.4
0.046 i 0.03 0.201 f 0.08 0.159 i 0.07
-
Annals N e w York Academy of Sciences
1 I6
= =
20
E h
2
16
$ - c 2e
3 W
n
CONTROL + RESERPINE HYPOPHYSECTOMY + RESERPINE
$2
* \ x m
k?g z x
0
-rE
5 2 4 g
AORTA MES.ART.
HEART
KIDNEY
LIVER
LUNG
SKIN
FIGURE I . Effect of hypophysectomy and reserpine on prolyl hydroxylase in various rat tissues. Hypophysectomy was performed 2 weeks before the analysis of prolyl hydroxylase, while reserpine was administered daily at a dose of 0-75 mg/kg for 1 week. Values represent the means of rats.
there is also a decrease in collagen biosynthesis when prolyl hydroxylase is used as the indicator ( F I G U R EI), and this decrease in prolyl hydroxylase activity in the vasculature is even greater than that elicited by reserpine. In many tissues, particularly the aorta and rnesenteric artery, the effects of hypophysectomy and reserpine are additive, which indicates that reserpine has an effect on these tissues other than through action on the pituitary. These studies demonstrate that isolated brain microvessels posses the anabolic and catabolic enzymes necessary for catecholamine metabolism. The antihypertensive drug reserpine, which can deplete tissues of their norepinephrine content and decrease collagen biosynthesis, may modify collagen formation by affecting the pituitary as one of its many sites of action.
REFERENCES 1 . FULLER, G. C. & R. 0.LANGNOR. 1970. Science 168: 987. J . VITTO,P. HELIN & I. LORENZEN. 1972. Circulation Res. 30: 123. 2. LINDY,S., H. TURKO, 3. OOSHIMA, A., G . C. FULLER, G. S. CARDINALE, S. SPECTOR & S. UDENFRIEND. 1974. Proc. Nat. Acad. Sci. USA 71(8): 3019. 4. UDENFRIEND, S. This monograph. 1967. CirCUkitiOn Res. 5. CATECHOLAMINES IN CARDIOVASCULAR PHYSIOLOGY AND DISEASE. (Suppl. III)XXI(6). & E. C. CARLSON. 1974. Science 185: 953. 6. BRENDEL,K . , E. MEEZAN 7. D E WED, D. & W. DE JONG. 1974. Annu. Rev. Pharmacol. 14: 389.
