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Altered norepinephrine synthesis of splanchnic vessels in neurogenic hypertension
EUROPEAN JOURNAL OF PHARMACOLOGY 26 (1974) 231-235. NORTH-HOLLAND PUBLSHING COMPANY
ALTERED
NOREPINEPHRINE SYNTHESIS OF SPLANCHNIC IN NEUROGENIC HYPERTENSION
VESSELS
Vincent DEQUATTRO and Natalie ALEXANDER Departments of Medicine, White Memorial Medical Center, and University of Southern California School of Medicine, Los Angeles, California 90033, U.S.A. Received 4 July 1973
Accepted 30 January 1974
V. DEQUATTRO and N. ALEXANDER, Altered norepinephrine synthesis of splanchnic vessels in neurogenic hypertension, European J. Pharmacol. 26 (1974) 231-235. Sino-aortic denervation (SAD) in the rabbit produced neurogenic hypertension which at first was characterized by increased cardiac output and later by increased peripheral vascular resistance. Tyrosine hydroxylase activity and catecholamine concentration of proximal mesenteric artery were greater than those of distal mesenteric 'vessels' in normal rabbits. 1 hr after SAD, NE synthesis, the activity of tyrosine hydroxylase assayed in vitro, was increased in proximal mesenteric artery and decreased in distal mesenteric "vessels'. 11 and 30 days after SAD, NE synthesis in vivo and the activity of tyrosine hydroxylase assayed in vitro was increased in distal mesenteric 'vessels' and decreased in proximal mesenteric artery. Sympatho-adrenal regulation of increased splanchnic vascular resistance is concluded to be an important factor in the initiation and maintenance of neurogenic hypertension in the rabbit. Neurogenic hypertension Noradrenaline synthesis
Splanchnic blood vessels Vascular resistance
Tyrosine hydroxylase
1. Introduction
2. Materials and methods
The sympathetic nervous system is important in the generation and maintenance o f hypertension in 3 animal models: the rat with doca salt hypertension (DeChamplain et al., 1968), the rat with renal artery stenosis hypertension (Grewal and Kaul, 1971), and the rabbit with neurogenic hypertension (DeQuattro et al., 1969). Further, sympathetic nerve dysfunction has been implicated as the cause o f the blood pressure elevation in some patients with primary hypertension (DeQuattro and Chan, 1972; Engelman et al., 1970; Nestel, 1970). Hypertension in the sino-aortic denervated (SAD) rabbit is associated with reductions of norepinephrine in the left ventricle and epinephrine in the adrenal glands despite increased catecholamine synthesis and turnover (DeQuattro et al., 1969). This paper reports our findings of alterations o f neurotransmitter metabolism and vascular resistance in splanchnic vessels o f SAD rabbits.
The acute and chronic effects o f sino-aortic denervation (SAD) or sham operation were studied in 46 female New Zealand rabbits weighing 2 . 5 - 3 . 5 kg. At 1 - 4 hr (acutely), hemodynamic measurements were made in 4 sham and 7 SAD rabbits, and assays o f tyrosine hydroxylase activity and o f catecholamine concentration of splanchnic and renal vessels were made in an additional 5 sham and 5 SAD rabbits. At 1 ! and 30 days (chronically), hemodynamic measurements were made in 12 sham and 1 1 SAD rabbits. Of these, 3 sham and 3 SAD at 11 days and 4 sham and 4 SAD at 30 days were used for assays o f norepinephrine synthesis in vivo and of tyrosine hydroxylase activity and catecholamine concentrations in vitro in splanchnic vessels. Blood pressure was detemfined after the cannulation o f the ear artery in resting, unanesthetized rabbits (DeQuattro et al., 1969). Cardiac o u t p u t was
v. De Quattro, N. Alexander, Vascularnorepinephrine synthesis in hypertension
232
determined from an arterial dilution curve of the isotope s 6 RbC1. Regionall flow was calculated as the product of the fractional isotope content of the region (or organ) and the cardiac output (Sapirstein, 1958). Vascular resistance was calculated as mean arterial pressure divided by blood flow. The proximal superior mesenteric artery (approximately 100 mg) was cleaned of all non-arterial tissue. The distal mesenteric 'vessels' consisted of remaining superior mesenteric artery, arteries and veins from coeliac and inferior mesenteric vessels, fat, and connective tissue; these 'vessels' were subdivided into proximal (I) and distal (II) parts for purposes of analysis. Tyrosine hydroxylase activity in fresh tissue homogenate was determined to estimate the norepinephrine synthesis rate in vitro (Nagatsu et al., 1964). Tissues (1 part) were homogenized in ice cold sucrose (15 parts). In vivo, NE synthesis was determined by infusion of L-tyrosine-3H and the subsequent recovery of norepinephrine-3H from tissues of the animals sacrificed 30 min later (De Quattro et al., 1969). The activity of the enzyme phenylethanolamine N-methyltransferase (PNMT)was determined in splanchnic vessels of normal rabbits (DeQuattro et al., 1969; Wurtman and Axelrod, 1965). Norepinephrine and protein were assayed by fluorometric and colorimetric methods, respectively (Crout, 1961; Lowry et al., 1951).
3. Results
3.1. Hemodynamic characterization of the hypertension after sino-aortic denervation Hypertension in SAD animals began immediately, was moderate, labile, and persisted for the duration of the study. 1 - 4 hr after SAD, the means of arterial pressure and cardiac output of SAD animals were 39 and 24% greater than values of sham rabbits, respectively (table 1). Splanchnic vascular resistance was increased 36% whereas total and renal vascular resistance were unchanged compared to values of sham animals. At 11 and 30 days post SAD, there was a 23% increase in mean arterial pressure and no significant change in the cardiac output; but total, splanchnic, and renal vascular resistance were all increased in the SAD animals compared to sham animals.
3.2. Tyrosine hydroxylase activities and catecholamine concentrations of blood vessels, heart and adrenal glands of normal rabbits Tyrosine hydroxylase activity per mg of protein of proximal mesenteric artery was less than adrenal #ands but greater than that of several other arteries and left ventricle; the mean values when expressed as percent of adrenal (2.2 _+0.2 m/lmoles/mg protein/hr)
Table 1 Alterations in arterial pressure, cardiac output and vascular resistance in the rabbit at 1-4 hr and 11-30 days after sino-aortic denervation. Time after SAD
1 - 4 hr Sham
SAD % increase p value 11-30 days Sham SAD % increase p value
4
7
12 11
* Values are means +_S.E.M.
Mean arterial pressure (mm Hg)
Cardiac output (ml/m/kg)
Vascular resistance Total (ram Hg/ml/ min/kg)
Splanchnic mm Hg/ml/ rain/100 kg)
Renal (ram Hg/ml/ min/100 kg)
69 _+2* 96 -+3 39 <0.001
212 _ 2 263 _+9 24 <0.01
0.33 - 0.02 0.37 -- 0.02 12 N.S.
0.89 _+0.04 1.21 _+0.08 36 <0.01
0.13 _+0.01 0.14 _+ 0.01 8 N.S.
79 + 1 97 _+2 23 <0.001
226 _+6 241 _+7 7 N.S.
0.35 _+ 0.01 0.43 _+ 0.03 23 <0.02
0.96 _+ 0.04 1.20 _+ 0.10 25 <0.05
0.16 _+ 0.01 0.19 __ 0.01 19 <0.01
V. De Quattro, N. Alexander, Vascular norepinephrine synthesis in hypertension
233
Table 2 Concentration of catecholamines and activities of tyrosine hydroxylase in mesenteric vessels of normal rabbits. Values are means + S.E.M. of 3 rabbits. 'Proximal' is proximal superior mesenteric artery. 'Distal l and Distal II' are the proximal and distal parts of the distal mesenteric vessels.
Proximal Distal I Distal II
Weight (g)
Norepinephrine (~g/g)
Epinephrine (~g/g)
Protein (mm Hg)
Total NE plus E (ng/mg protein)
Tyrosine hydroxylase (m~tmole/mg protein/hr)
0.10 ± 0.03 2.4 ± 0.3 1.8 ± 0.3
2.0 ± 0.2 0.27 ± 0.04 0.22 ± 0.04
1.1 ± 0.02 0.02 ± 0.01 0.02 ± 0.01
94 ± 5 22 ± 4 18 ± 3
33 ± 3 13 ± 2 13 ± 2
0.86 ± 0.1 0.22 ± 0.04 0.10 ± 0.02
200
(+ S.E.M.) are: adrenal 100 - 10, proximal mesenteric artery 45 + 13, renal artery 20 + 4.0, distal mesentetic 'vessels' 14 + 2.3, abdominal aorta 6.0 + 1.1, left ventricle 5.1 + 0.5, iliac artery 4.2 -+ 0.5, thoracic aorta 2.3 -+ 0.1. Tyrosine hydroxylase activity, catecholamine content per mg of protein, and protein concentration in proximal mesenteric artery were 3- to 4-fold greater than the respective values in distal 'vessels' (table 2). Epinephrine (E) accounted for 36% of the total catecholamine in proximal mesenteric artery, an unexpected finding. The PNMT activity of the pooled proximal mesenteric arteries of 4 control animals was 0.50 m/amoles/g/hr, but was not detectable in their distal mesenteric 'vessels'. The activity of the E-synthesizing enzyme in proximal mesenteric artery is approximately 1/300th that of rabbit adrenal gland (DeQuattro et al., 1969). The ratio of PNMT activity of proximal mesenteric artery to PNMT activity of adrenal is similar to the ratio of the respective concentrations of catecholamines.
3. 3. Catecholamine biosynthesis in blood vessels after sino-aortic denervation SAD changed catecholamine biosynthesis in splanchnic vasculature. Immediately (1 hr) tyrosine hydroxylase activity in vitro increased 53% in proximal mesenteric artery and decreased 50% in distal mesenteric 'vessels' ( p < 0 . 0 5 ) (fig. 1); later, both tyrosine hydroxylase activity in vitro and in vivo norepinephrine synthesis decreased in proximal mesenteric artery (p < 0.1) (60 and 50% respectively) at 30 days and increased in distal mesenteric 'vessels' (100 and 80% respectively) at 11 days compared with
PERCENT SHOF AM
r ~
r ~
100
--
i
--
RENAL MESENTERiC VESSELS ARTERY PROXIMAL DISTAL (10) (10) (10) p
<.02
<.05
<.01
Fig. 1. Tyrosine hydroxylase activities of splanchnic and renal vessels of SAD rabbits (compared with sham) 1 hr post SAD. Tyrosine hydroxylase activity in m#moles/mg protein/hr of sham tissues are: renal artery 0.22 ± 0.02; proximal mesenteric artery 0.63 ± 0.11; and distal mesenteric 'vessels' 0.30 ± 0.01 (means -+ S.E.M.). r - I TYROSINE HYDROXYLASEACTIVITY ~X,.~IN VIVO SYNTHESI: [ ] N.E CONTENT
200I PERCENT SHAM 100
I
I~OX. MESENTERICARTERY DISTALME~NTERICVESSELS N 8 8 pooled 6 6 6 <.05 <.02 <.05
Fig. 2. Norepinephrine synthesis and content of proximal inesenteric artery and distal mesenteric 'vessels' in SAD rabbits compared with sham at 30 and 11 days, respectively, post SAD. Values are ± S.E.M., except that in vivo synthesis and NE content of proximal mesenteric artery are values of pooled tissues. In vitro tyrosine hydroxylase activities in m#moles/mg protein/hr of sham animals were 0.96 -+0.17 for proximal mesenteric artery and 0.54 ± 0.10 for distal mesenteric 'vessels'. In vivo synthesis is represented by the specific activities of NE-3H 30 min after 0.3 mCi/kg tyrosine-aH i.v. The specific activities of the sham proximal mesenteric artery (pooled) and distal mesenteric 'vessels' (means ± S.E.M.) were 2,098 and 854 ± 31 cpm/~tg, respectively.
234
V. De Quattro, N. Alexander, Vascular norepinephrine synthesis in hypertension
sham values (p < 0.05) (fig. 2). NE concentration was increased 20% in proximal mesenteric artery at 30 days post SAD and was reduced 50% in distal mesenteric vessels 11 days after SAD. NE synthesis in vivo and catecholamine concentration of proximal mesenteric arteries at 30 days post SAD were measured in pooled tissues. NE synthesis in vitro of proximal mesenteric artery was determined as the means of the individual tissue values. Catecholamine biosynthesis was increased in renal artery after SAD; at 1 hr in vitro synthesis was increased 67% and at 30 days both in vitro and in vivo synthesis were increased 340 and 163%, respectively (p < 0.01, p < 0.01).
4. Discussion The high concentration of NE and tyrosine hydroxylase and the marked alterations of NE synthesis in proximal superior mesenteric artery and distal mesenteric 'vessels' of the SAD rabbit suggest that the neurogenic stimulus increasing splanchnic vascular resistance was a major factor causing hypertension in the SAD rabbit. There was considerable range in the amount of increase (acutely) or decrease (chronically) in the tyrosine hydroxylase activity of proximal superior mesenteric artery after SAD. The sham and SAD tissues were treated in pairs and the assay method was reliable and reproducible. Therefore, the wide range of change in blood vessel tyrosine hydroxylase activity may have been related to factors causing wide swings of blood pressure in these animals (DeQuattro et al., 1969). The increase in tyrosine hydroxylase activity in proximal superior mesenteric artery after SAD may indicate the increased synthesis of NE in vivo due to the increase in the enzyme protein. NE biosynthesis in this artery segment may be less sensitive to the inhibiting effects of the circulating catecholamines. There is some support for this reasoning from the findings of reduced neuronal uptake of NE in the rat mesenteric artery (Berkowitz et al., 1971), if an assumption is made that the entire rat mesenteric artery is analogous to the proximal part of the rabbit mesenteric artery. Mesenteric artery segments from 200 g rats weighed 15 mg (Berkowitz et al., 1971). In 2.5 kg rabbits the proximal part of the superior mesenteric artery alone weighed 100-150 mg. Thus, the
proximal part of the rabbit superior mesenteric artery may be similar functionally and anatomically to the entire arterial se~nent studied in the rats (Berkowitz et al., 1971); Tarver et al., 1971; Berkowitz et al., 1972). De la Lande et al. (1967) found that when NE was applied into the lumen of muscular arteries, it did not reach sympathetic nerves readily, perhaps due to the large distance between the lumen and the nerves. In rats, proximal mesenteric artery NE concentration was less than that of the distal segment (Berkowitz et al., 1971). In rabbits, we found less NE and tyrosine hydroxylase activity in distal mesenteric 'vessels' than in proximal mesenteric arteries. In our studies, rabbit distal 'vessels' included veins, fat, and traces of connective tissue. However, fat dissected from the vessels contained barely detectable amounts of tyrosine hydroxylase activity. Further, relatively more terminal arterioles were present in the distal vessels of rabbit than in distal mesenteric artery of the rat. We were surprised to find E in proximal mesenteric artery. Apparently, it can be synthesized in the arterial segment since small but significant amounts of PNMT also occur there. Preliminary microscopic studies of the proximal mesenteric artery wall (via the histochemical fluorescence method) indicated the presence of chromaffin cells. (We are grateful to Dr. D. Silva for his preparation of the tissue.) The possible role of these cells in regulatio n of mesenteric artery blood flow is unknown. The findings of reduced tyrosine hydroxylase activity in distal 'vessels' may indicate that an increased concentration of circulating catecholamines after SAD inhibited the enzyme by a feedback mechanism. Initially, after SAD, increased sympathetic outflow to proximal mesenteric artery may have increased splanchnic resistance and served to mitigate against the vasodilatory influences of circulating E. Later after SAD, the increased sympathetic nerve tonicity to distal mesenteric 'vessels' was important in maintaining increased splanchnic vascular resistance. Despite the rapid increase in NE synthesis of renal arteries, indicating increased sympathetic nerve tonicity to renal vessels, renal vascular resistance was not increased acutely. Similarly, renal vascular resistance was unchanged in the dog acutely after SAD (Passmore and Calhoon, 1972). In vivo NE synthesis in mesenteric arteries and 'vessels', as reflected by specific activity of NE after
V. De Quattro, N. Alexander, Vascular norepinephrine synthesis in hypertension
infusion of tritiated tyrosine, was altered in parallel with changes in tyrosine hydroxylase activity in vitro. We found earlier that tissue tyrosine specific activity after tyrosine-3H was unchanged in the heart and adrenals of SAD animals (DeQuattro et al., 1969). Further, the present study revealed that blood flow in splanchnic vasculature and therefore the distribution of infused tyrosine-3H was the same in sham and SAD rabbits. We have not determined whether these immediate changes were due to increased concentration of enzyme, cofactors, or activators. However, increased tyrosine hydroxylase activity assayed in vitro usually reflects increased enzyme concentrations because cofactors and conditions are optimized. The changes in cardiac output and vascular resistance in some patients with early primary or essential hypertension were similar to the changes occurring in the rabbit (Eich et al., 1966; Birkenhager et al., 1972). Increased splanchnic vascular resistance due to increased sympathetic nerve tonicity in these patients may be pathogenic in their hypertension. In studies of another animal model with hypertension, Tarver et al. (1971) found that tyrosine hydroxylase activity of mesenteric vessels of spontaneously hypertensive rats was decreased compared with controls. Earlier, Louis et al. (1968) found that NE synthesis is decreased in hearts of these animals, perhaps due to reflex changes, and they suggested that non-neurogenic factors caused the hypertension in that animal model.
Acknowledgement We wish to thank Mr. Howard Hammar and Mr. Daantje Meijer for their expert technical assistance.
References Berkowitz, B., J. Tarver and S. Spector, 1971, Norepinephrine in blood vessels: concentration, binding, uptake and depletion, J. Pharmacol. Exptl. Therap. 177, 119. Berkowitz, B., S. Spector and J. Tarver, 1972, Resistance of noradrenaline in blood vessels to depletion by 6-hydroxy-
235
dopamine or immunosympathectomy, Brit. J. Pharmacol. 44 (1), 10. Birkenhager, W.H., M. Schalenkamp, X. Krauss, G. Kolsters and G. Zaal, 1972, Consecutive haemodynamic patterns in essential hypertension, Lancet 1,560. Crout, J.R., 1961, Catecholamines in urine, in: Standard Methods of Clinical Chemistry, Vol. 3, ed. D. Seligson, (Academic Press, New York), p. 62. De Champlain, J.L., L. Krakoff, R. Lawrence and J. Axelrod, 1968, Relationship between sodium intake and norepinephrine storage during the development of experimental hypertension, Circulation Res. 23,479. De la Lande, I.S., D. Frewen and J.G. Waterson, 1967, The influence of sympathetic innervation on vascular sensitivity to noradrenaline, Brit. J. Pharmacol. 31, 82. DeQuattro, V. and S. Chan, 1972, Raised plasma-catechob amines in some patients with primary hypertension, Lancet 1,806. DeQuattro, V., T. Nagatsu, R. Maronde and N. Alexander, 1969, Catecholamine synthesis in rabbits with neurogenic hypertension, Circulation Res. 24,545. Eich, R.H., R.P. Cuddy, H. Smulyan and R.H. Lyons, 1966, Hemodynamics in labile hypertension: a follow-up study, Circulation 34,299. Engelman, K., B. Portnoy and A. Sjoerdsma, 1970, Plasma catecholamine concentrations in patients with hypertension, Circulation Res. 26, 27 (Suppl. 1), 141. Grewal, R.S. and C.L. Kaul, 1971, Importance of the sympathetic nervous system, in the development of renal hypertension in the rat, Brit. J. Pharmacol. 42, 497. Louis, W.J., S. Spector, R. Tabei and A. Sjoerdsma, 1968, Noradrenaline in the heart of the spontaneously hypertensive rat, Lancet I, 1013. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193,265. Nagatsu, T., M. Levitt and S. Udenfriend, 1964, Tyrosine hydroxylase - the initial step in norepinephrine biosynthesis, J, Biol. Chem. 239, 2910. Nestel, P.J. and M.D. Esler, 1970, Patterns of catecholamine excretion in urine in hypertension, Circulation Res. 27 (Suppl. 1I), 75. Passmore, J.C. and T. Calhoon, 1972, Renal blood flow distribution during hemorrhage in carotid sinus deprived dogs, Federation Proc. 31,318. Sapirstein, L.A., 1958, Regional blood flow by fractional distribution of indicators, Amer. J. Physiol. 193,161. Tarver, J., B. Berkowitz and S. Spector, 1971, Alterations in tyrosine hydroxylase and monoamine oxidase activity in blood vessels, Nature New Biol. 231,252. Wurtman, R.J. and J. Axelrod, 1965, Adrenaline synthesis: control by the pituitary gland and adrenal glucocorticolds, Science 150, 1464.
ALTERED
NOREPINEPHRINE SYNTHESIS OF SPLANCHNIC IN NEUROGENIC HYPERTENSION
VESSELS
Vincent DEQUATTRO and Natalie ALEXANDER Departments of Medicine, White Memorial Medical Center, and University of Southern California School of Medicine, Los Angeles, California 90033, U.S.A. Received 4 July 1973
Accepted 30 January 1974
V. DEQUATTRO and N. ALEXANDER, Altered norepinephrine synthesis of splanchnic vessels in neurogenic hypertension, European J. Pharmacol. 26 (1974) 231-235. Sino-aortic denervation (SAD) in the rabbit produced neurogenic hypertension which at first was characterized by increased cardiac output and later by increased peripheral vascular resistance. Tyrosine hydroxylase activity and catecholamine concentration of proximal mesenteric artery were greater than those of distal mesenteric 'vessels' in normal rabbits. 1 hr after SAD, NE synthesis, the activity of tyrosine hydroxylase assayed in vitro, was increased in proximal mesenteric artery and decreased in distal mesenteric "vessels'. 11 and 30 days after SAD, NE synthesis in vivo and the activity of tyrosine hydroxylase assayed in vitro was increased in distal mesenteric 'vessels' and decreased in proximal mesenteric artery. Sympatho-adrenal regulation of increased splanchnic vascular resistance is concluded to be an important factor in the initiation and maintenance of neurogenic hypertension in the rabbit. Neurogenic hypertension Noradrenaline synthesis
Splanchnic blood vessels Vascular resistance
Tyrosine hydroxylase
1. Introduction
2. Materials and methods
The sympathetic nervous system is important in the generation and maintenance o f hypertension in 3 animal models: the rat with doca salt hypertension (DeChamplain et al., 1968), the rat with renal artery stenosis hypertension (Grewal and Kaul, 1971), and the rabbit with neurogenic hypertension (DeQuattro et al., 1969). Further, sympathetic nerve dysfunction has been implicated as the cause o f the blood pressure elevation in some patients with primary hypertension (DeQuattro and Chan, 1972; Engelman et al., 1970; Nestel, 1970). Hypertension in the sino-aortic denervated (SAD) rabbit is associated with reductions of norepinephrine in the left ventricle and epinephrine in the adrenal glands despite increased catecholamine synthesis and turnover (DeQuattro et al., 1969). This paper reports our findings of alterations o f neurotransmitter metabolism and vascular resistance in splanchnic vessels o f SAD rabbits.
The acute and chronic effects o f sino-aortic denervation (SAD) or sham operation were studied in 46 female New Zealand rabbits weighing 2 . 5 - 3 . 5 kg. At 1 - 4 hr (acutely), hemodynamic measurements were made in 4 sham and 7 SAD rabbits, and assays o f tyrosine hydroxylase activity and o f catecholamine concentration of splanchnic and renal vessels were made in an additional 5 sham and 5 SAD rabbits. At 1 ! and 30 days (chronically), hemodynamic measurements were made in 12 sham and 1 1 SAD rabbits. Of these, 3 sham and 3 SAD at 11 days and 4 sham and 4 SAD at 30 days were used for assays o f norepinephrine synthesis in vivo and of tyrosine hydroxylase activity and catecholamine concentrations in vitro in splanchnic vessels. Blood pressure was detemfined after the cannulation o f the ear artery in resting, unanesthetized rabbits (DeQuattro et al., 1969). Cardiac o u t p u t was
v. De Quattro, N. Alexander, Vascularnorepinephrine synthesis in hypertension
232
determined from an arterial dilution curve of the isotope s 6 RbC1. Regionall flow was calculated as the product of the fractional isotope content of the region (or organ) and the cardiac output (Sapirstein, 1958). Vascular resistance was calculated as mean arterial pressure divided by blood flow. The proximal superior mesenteric artery (approximately 100 mg) was cleaned of all non-arterial tissue. The distal mesenteric 'vessels' consisted of remaining superior mesenteric artery, arteries and veins from coeliac and inferior mesenteric vessels, fat, and connective tissue; these 'vessels' were subdivided into proximal (I) and distal (II) parts for purposes of analysis. Tyrosine hydroxylase activity in fresh tissue homogenate was determined to estimate the norepinephrine synthesis rate in vitro (Nagatsu et al., 1964). Tissues (1 part) were homogenized in ice cold sucrose (15 parts). In vivo, NE synthesis was determined by infusion of L-tyrosine-3H and the subsequent recovery of norepinephrine-3H from tissues of the animals sacrificed 30 min later (De Quattro et al., 1969). The activity of the enzyme phenylethanolamine N-methyltransferase (PNMT)was determined in splanchnic vessels of normal rabbits (DeQuattro et al., 1969; Wurtman and Axelrod, 1965). Norepinephrine and protein were assayed by fluorometric and colorimetric methods, respectively (Crout, 1961; Lowry et al., 1951).
3. Results
3.1. Hemodynamic characterization of the hypertension after sino-aortic denervation Hypertension in SAD animals began immediately, was moderate, labile, and persisted for the duration of the study. 1 - 4 hr after SAD, the means of arterial pressure and cardiac output of SAD animals were 39 and 24% greater than values of sham rabbits, respectively (table 1). Splanchnic vascular resistance was increased 36% whereas total and renal vascular resistance were unchanged compared to values of sham animals. At 11 and 30 days post SAD, there was a 23% increase in mean arterial pressure and no significant change in the cardiac output; but total, splanchnic, and renal vascular resistance were all increased in the SAD animals compared to sham animals.
3.2. Tyrosine hydroxylase activities and catecholamine concentrations of blood vessels, heart and adrenal glands of normal rabbits Tyrosine hydroxylase activity per mg of protein of proximal mesenteric artery was less than adrenal #ands but greater than that of several other arteries and left ventricle; the mean values when expressed as percent of adrenal (2.2 _+0.2 m/lmoles/mg protein/hr)
Table 1 Alterations in arterial pressure, cardiac output and vascular resistance in the rabbit at 1-4 hr and 11-30 days after sino-aortic denervation. Time after SAD
1 - 4 hr Sham
SAD % increase p value 11-30 days Sham SAD % increase p value
4
7
12 11
* Values are means +_S.E.M.
Mean arterial pressure (mm Hg)
Cardiac output (ml/m/kg)
Vascular resistance Total (ram Hg/ml/ min/kg)
Splanchnic mm Hg/ml/ rain/100 kg)
Renal (ram Hg/ml/ min/100 kg)
69 _+2* 96 -+3 39 <0.001
212 _ 2 263 _+9 24 <0.01
0.33 - 0.02 0.37 -- 0.02 12 N.S.
0.89 _+0.04 1.21 _+0.08 36 <0.01
0.13 _+0.01 0.14 _+ 0.01 8 N.S.
79 + 1 97 _+2 23 <0.001
226 _+6 241 _+7 7 N.S.
0.35 _+ 0.01 0.43 _+ 0.03 23 <0.02
0.96 _+ 0.04 1.20 _+ 0.10 25 <0.05
0.16 _+ 0.01 0.19 __ 0.01 19 <0.01
V. De Quattro, N. Alexander, Vascular norepinephrine synthesis in hypertension
233
Table 2 Concentration of catecholamines and activities of tyrosine hydroxylase in mesenteric vessels of normal rabbits. Values are means + S.E.M. of 3 rabbits. 'Proximal' is proximal superior mesenteric artery. 'Distal l and Distal II' are the proximal and distal parts of the distal mesenteric vessels.
Proximal Distal I Distal II
Weight (g)
Norepinephrine (~g/g)
Epinephrine (~g/g)
Protein (mm Hg)
Total NE plus E (ng/mg protein)
Tyrosine hydroxylase (m~tmole/mg protein/hr)
0.10 ± 0.03 2.4 ± 0.3 1.8 ± 0.3
2.0 ± 0.2 0.27 ± 0.04 0.22 ± 0.04
1.1 ± 0.02 0.02 ± 0.01 0.02 ± 0.01
94 ± 5 22 ± 4 18 ± 3
33 ± 3 13 ± 2 13 ± 2
0.86 ± 0.1 0.22 ± 0.04 0.10 ± 0.02
200
(+ S.E.M.) are: adrenal 100 - 10, proximal mesenteric artery 45 + 13, renal artery 20 + 4.0, distal mesentetic 'vessels' 14 + 2.3, abdominal aorta 6.0 + 1.1, left ventricle 5.1 + 0.5, iliac artery 4.2 -+ 0.5, thoracic aorta 2.3 -+ 0.1. Tyrosine hydroxylase activity, catecholamine content per mg of protein, and protein concentration in proximal mesenteric artery were 3- to 4-fold greater than the respective values in distal 'vessels' (table 2). Epinephrine (E) accounted for 36% of the total catecholamine in proximal mesenteric artery, an unexpected finding. The PNMT activity of the pooled proximal mesenteric arteries of 4 control animals was 0.50 m/amoles/g/hr, but was not detectable in their distal mesenteric 'vessels'. The activity of the E-synthesizing enzyme in proximal mesenteric artery is approximately 1/300th that of rabbit adrenal gland (DeQuattro et al., 1969). The ratio of PNMT activity of proximal mesenteric artery to PNMT activity of adrenal is similar to the ratio of the respective concentrations of catecholamines.
3. 3. Catecholamine biosynthesis in blood vessels after sino-aortic denervation SAD changed catecholamine biosynthesis in splanchnic vasculature. Immediately (1 hr) tyrosine hydroxylase activity in vitro increased 53% in proximal mesenteric artery and decreased 50% in distal mesenteric 'vessels' ( p < 0 . 0 5 ) (fig. 1); later, both tyrosine hydroxylase activity in vitro and in vivo norepinephrine synthesis decreased in proximal mesenteric artery (p < 0.1) (60 and 50% respectively) at 30 days and increased in distal mesenteric 'vessels' (100 and 80% respectively) at 11 days compared with
PERCENT SHOF AM
r ~
r ~
100
--
i
--
RENAL MESENTERiC VESSELS ARTERY PROXIMAL DISTAL (10) (10) (10) p
<.02
<.05
<.01
Fig. 1. Tyrosine hydroxylase activities of splanchnic and renal vessels of SAD rabbits (compared with sham) 1 hr post SAD. Tyrosine hydroxylase activity in m#moles/mg protein/hr of sham tissues are: renal artery 0.22 ± 0.02; proximal mesenteric artery 0.63 ± 0.11; and distal mesenteric 'vessels' 0.30 ± 0.01 (means -+ S.E.M.). r - I TYROSINE HYDROXYLASEACTIVITY ~X,.~IN VIVO SYNTHESI: [ ] N.E CONTENT
200I PERCENT SHAM 100
I
I~OX. MESENTERICARTERY DISTALME~NTERICVESSELS N 8 8 pooled 6 6 6 <.05 <.02 <.05
Fig. 2. Norepinephrine synthesis and content of proximal inesenteric artery and distal mesenteric 'vessels' in SAD rabbits compared with sham at 30 and 11 days, respectively, post SAD. Values are ± S.E.M., except that in vivo synthesis and NE content of proximal mesenteric artery are values of pooled tissues. In vitro tyrosine hydroxylase activities in m#moles/mg protein/hr of sham animals were 0.96 -+0.17 for proximal mesenteric artery and 0.54 ± 0.10 for distal mesenteric 'vessels'. In vivo synthesis is represented by the specific activities of NE-3H 30 min after 0.3 mCi/kg tyrosine-aH i.v. The specific activities of the sham proximal mesenteric artery (pooled) and distal mesenteric 'vessels' (means ± S.E.M.) were 2,098 and 854 ± 31 cpm/~tg, respectively.
234
V. De Quattro, N. Alexander, Vascular norepinephrine synthesis in hypertension
sham values (p < 0.05) (fig. 2). NE concentration was increased 20% in proximal mesenteric artery at 30 days post SAD and was reduced 50% in distal mesenteric vessels 11 days after SAD. NE synthesis in vivo and catecholamine concentration of proximal mesenteric arteries at 30 days post SAD were measured in pooled tissues. NE synthesis in vitro of proximal mesenteric artery was determined as the means of the individual tissue values. Catecholamine biosynthesis was increased in renal artery after SAD; at 1 hr in vitro synthesis was increased 67% and at 30 days both in vitro and in vivo synthesis were increased 340 and 163%, respectively (p < 0.01, p < 0.01).
4. Discussion The high concentration of NE and tyrosine hydroxylase and the marked alterations of NE synthesis in proximal superior mesenteric artery and distal mesenteric 'vessels' of the SAD rabbit suggest that the neurogenic stimulus increasing splanchnic vascular resistance was a major factor causing hypertension in the SAD rabbit. There was considerable range in the amount of increase (acutely) or decrease (chronically) in the tyrosine hydroxylase activity of proximal superior mesenteric artery after SAD. The sham and SAD tissues were treated in pairs and the assay method was reliable and reproducible. Therefore, the wide range of change in blood vessel tyrosine hydroxylase activity may have been related to factors causing wide swings of blood pressure in these animals (DeQuattro et al., 1969). The increase in tyrosine hydroxylase activity in proximal superior mesenteric artery after SAD may indicate the increased synthesis of NE in vivo due to the increase in the enzyme protein. NE biosynthesis in this artery segment may be less sensitive to the inhibiting effects of the circulating catecholamines. There is some support for this reasoning from the findings of reduced neuronal uptake of NE in the rat mesenteric artery (Berkowitz et al., 1971), if an assumption is made that the entire rat mesenteric artery is analogous to the proximal part of the rabbit mesenteric artery. Mesenteric artery segments from 200 g rats weighed 15 mg (Berkowitz et al., 1971). In 2.5 kg rabbits the proximal part of the superior mesenteric artery alone weighed 100-150 mg. Thus, the
proximal part of the rabbit superior mesenteric artery may be similar functionally and anatomically to the entire arterial se~nent studied in the rats (Berkowitz et al., 1971); Tarver et al., 1971; Berkowitz et al., 1972). De la Lande et al. (1967) found that when NE was applied into the lumen of muscular arteries, it did not reach sympathetic nerves readily, perhaps due to the large distance between the lumen and the nerves. In rats, proximal mesenteric artery NE concentration was less than that of the distal segment (Berkowitz et al., 1971). In rabbits, we found less NE and tyrosine hydroxylase activity in distal mesenteric 'vessels' than in proximal mesenteric arteries. In our studies, rabbit distal 'vessels' included veins, fat, and traces of connective tissue. However, fat dissected from the vessels contained barely detectable amounts of tyrosine hydroxylase activity. Further, relatively more terminal arterioles were present in the distal vessels of rabbit than in distal mesenteric artery of the rat. We were surprised to find E in proximal mesenteric artery. Apparently, it can be synthesized in the arterial segment since small but significant amounts of PNMT also occur there. Preliminary microscopic studies of the proximal mesenteric artery wall (via the histochemical fluorescence method) indicated the presence of chromaffin cells. (We are grateful to Dr. D. Silva for his preparation of the tissue.) The possible role of these cells in regulatio n of mesenteric artery blood flow is unknown. The findings of reduced tyrosine hydroxylase activity in distal 'vessels' may indicate that an increased concentration of circulating catecholamines after SAD inhibited the enzyme by a feedback mechanism. Initially, after SAD, increased sympathetic outflow to proximal mesenteric artery may have increased splanchnic resistance and served to mitigate against the vasodilatory influences of circulating E. Later after SAD, the increased sympathetic nerve tonicity to distal mesenteric 'vessels' was important in maintaining increased splanchnic vascular resistance. Despite the rapid increase in NE synthesis of renal arteries, indicating increased sympathetic nerve tonicity to renal vessels, renal vascular resistance was not increased acutely. Similarly, renal vascular resistance was unchanged in the dog acutely after SAD (Passmore and Calhoon, 1972). In vivo NE synthesis in mesenteric arteries and 'vessels', as reflected by specific activity of NE after
V. De Quattro, N. Alexander, Vascular norepinephrine synthesis in hypertension
infusion of tritiated tyrosine, was altered in parallel with changes in tyrosine hydroxylase activity in vitro. We found earlier that tissue tyrosine specific activity after tyrosine-3H was unchanged in the heart and adrenals of SAD animals (DeQuattro et al., 1969). Further, the present study revealed that blood flow in splanchnic vasculature and therefore the distribution of infused tyrosine-3H was the same in sham and SAD rabbits. We have not determined whether these immediate changes were due to increased concentration of enzyme, cofactors, or activators. However, increased tyrosine hydroxylase activity assayed in vitro usually reflects increased enzyme concentrations because cofactors and conditions are optimized. The changes in cardiac output and vascular resistance in some patients with early primary or essential hypertension were similar to the changes occurring in the rabbit (Eich et al., 1966; Birkenhager et al., 1972). Increased splanchnic vascular resistance due to increased sympathetic nerve tonicity in these patients may be pathogenic in their hypertension. In studies of another animal model with hypertension, Tarver et al. (1971) found that tyrosine hydroxylase activity of mesenteric vessels of spontaneously hypertensive rats was decreased compared with controls. Earlier, Louis et al. (1968) found that NE synthesis is decreased in hearts of these animals, perhaps due to reflex changes, and they suggested that non-neurogenic factors caused the hypertension in that animal model.
Acknowledgement We wish to thank Mr. Howard Hammar and Mr. Daantje Meijer for their expert technical assistance.
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