European Journal of Pharmacology, 44 (1977) 131--142 © Elsevier/North-Holland Biomedical Press
131
RENIN-ANGIOTENSIN MEDIATION OF ADRENAL CATECHOLAMINE SECRETION I N D U C E D BY H A E M O R R H A G E * GIORA FEUERSTEIN, PUNYA BOONYAVIROJ and YEHUDA GUTMAN ** Department of Pharmacology, Hebrew University--Hadassah Medical School, Jerusalem, Israel Received 12 November 1976, revised MS received 27 January 1977, accepted 24 March 1977
G. FEUERSTEIN, P. BOONYAVIROJ and Y. GUTMAN, Renin--angiotensin mediation o f adrenal catecholamine secretion induced by haemorrhage, European J. Pharmacol. 44 (1977) 131--142. The mechanism involved in catecholamine (CA) release from eat adrenal gland, in response to haemorrhage was studied. In intact eats, in cats with bilateral cervical vagotomy or following bilateral ureteral ligation, haemorrhage induced an increased catecholamine release from the adrenal (with increased percentage of noradrenaline). Acute bilateral nephrectomy, chronic sodium loading with repeated administration of desoxyeorticosterone acetate (DOCA), or acute denervation of the adrenal gland, completely abolished the increased CA release from the adrenal gland following haemorrhage. Haemorrhage induced an increase of plasma renin concentration in intact cats and after ureteral ligation but there was no increase in plasma renin after haemorrhage in cats with bilateral nephrectomy or following pretreatment with DOCA and salt load. Following haemorrhage in intact cats, blood pressure showed an immediate fall followed by rapid recovery. The recovery of blood pressure after haemorrhage was abolished in cats with bilateral nephrectomy. It is concluded that the adreno-medullary response to haemorrhage in the eat, depends primarily on the intact renal renin angiotensin system. Angiotensin, generated peripherally, probably affects the CNS and activates the sympathetic nerves. Angiotensin
Catecholamines
Haemorrhage
1. I n t r o d u c t i o n A close interrelation b e t w e e n the r e n i n - a n g i o t e n s i n s y s t e m a n d t h e s y m p a t h e t i c nervous s y s t e m has b e e n s u g g e s t e d b y v a r i o u s types of experiments. Thus, sympathetic innervation of the juxtaglomerular apparatus has b e e n d e m o n s t r a t e d (Barajas, 1 9 6 4 ; Wagerm a k e t al., 1967} t o g e t h e r w i t h a d i r e c t e f f e c t of c a t e c h o l a m i n e s on r e n a l r e n i n r e l e a s e ( J o h n s o n e t al., 1 9 7 1 ; V a n d e r , 1 9 6 5 ) w h i c h * Parts of this paper were presented at the Jerusalem Satellite Symposia, 26th International Congress of Physiological Sciences, October 1974, at the International Congress on Central Actions of Angiotensin, Houston, Texas, January 1976 and at the 36th Meeting of the Israel Physiological & Pharmacological Society, Tel-Aviv, June 1976. ** Established Investigator, Chief Scientist's Bureau, Israeli Ministry of Health.
Adrenal medulla
c o u l d be b l o c k e d b y s p e c i f i c a d r e n e r g i c b l o c k ing a g e n t s ( B u h l e r , 1 9 7 4 ; L o e f f l e r e t al., 1 9 7 2 ) . On t h e o t h e r h a n d , t h e r e is e v i d e n c e s u g g e s t i n g a p r i m a r y r o l e o f a n g i o t e n s i n II in m o d u l a t i n g s y m p a t h e t i c tone and effects, through either central or peripheral mechanisms (Hughes and Roth, 1969; Robinson, 1 9 6 7 ; Yu a n d D i c k i n s o n 1 9 7 1 ) . Several i n v e s t i g a t i o n s have d e m o n s t r a t e d t h a t a n g i o t e n s i n II in small d o s e s e n h a n c e d v a s o m o t o r r e s p o n s e s t o c a t e c h o l a m i n e s in several v a s c u l a r b e d s ( D a y a n d M o o r e 1 9 7 6 ; Lias a n d Z i m m e r m a n , 1 9 7 2 ; N i c h o l a s , 1 9 7 0 ) . This e f f e c t has b e e n a t t r i b u t e d t o a d i r e c t s e n s i t i z i n g a c t i o n on t h e e f f e c t o r o r g a n s ( D a y a n d M o o r e , 1 9 7 6 ) , or t o an i n d i r e c t o n e i.e., facilitation of catecholamine release from nerve e n d i n g s ( H u g h e s a n d R o t h , 1 9 6 9 ) o r an i n h i b i t o r y e f f e c t on c a t e c h o l a m i n e r e - u p t a k e i n t o a d r e n e r g i c n e r v e t e r m i n a l s (Palaic a n d
132
Khairallah, 1971; Peach et al., 1969; Janovsky, 1972). Blockade of the re-uptake mechanism has been also postulated as the cause of the increased rate of catecholamine synthesis in several sympathetically innervated organs exposed to angiotensin (Davilla and Khairallah, 1971). Alternatively, a direct stimulatory effect of angiotensin, through induction of tyrosine hydroxylase synthesis, has also been suggested (Roth, 1972). Angiotensin II has also been found to have excitatory effects on sympathetic postganglionic neurons, through specific receptors, as demonstrated in the cat superior cervical ganglia (Lewis and Reit, 1966) and in adrenal medulla chromaffin cells of various species (Peach, 1971; Robinson, 1967; StaszeweskaBarczack and Vane, 1967). Some of these are conflicting, e.g. Hughes could not demonstrate inhibition of noradrenaline re-uptake by angiotensin II (Hughes, 1969) while Haefely et al. (1965) and Panniset et al. (1966) failed to demonstrate any excitatory effects of angiotensin II on sympathetic postganglionc neurons with "physiological" doses of angiotensin II. In addition to having these various peripheral sites of action, angiotensin was found to exert several central stimulatory properties, such as increase of water consumption (Epstein et al., 1970), vasopressin release (Mouw et al., 1971) and blood pressure elevation (Scroop and Whelan 1966; Yu and Dickinson, 1965). This latter effect has been shown to be mediated through the sympathetic nervous system (Ueda et al., 1969; Yu and Dickinson, 1971). These findings have suggested that angiotensin II may play an important role in blood pressure regulation and in the development of different types of hypertension. However, no clear relationship has been found between plasma renin activity, primary hypertension (Aurell et al., 1975) and several models of experimental renal hypertension (Brown et al., 1966; Fernandez et al., 1976), in which elevated blood pressure was sustained long after plasma angiotensin II concentration returned to control values. But in these vari-
G. F E U E R S T E I N E T AL.
ous types of hypertension, sympathetic overactivity has been suspected to play a major role in the maintenance of chronic high blood pressure (Dargie et al., 1976; Haeusler et al., 1972; Louis et al., 1973). In analyzing these conflicting data it should be realized that they derive mostly from in vitro experiments on isolated organs (using perfusion techniques), or tissue slices incubated in vitro. In many of the in vivo experiments pharmacological doses of angiotensin II were either implanted (as crystals) or injected through non-physiological routes; species differences and especially different anaesthetic procedures have probably also contributed to the differences. In order to asses the relationship between sympathetic responses and the renal renin-angiotensin system activity, it seemed of interest to us to use physiological stimuli in an in vivo situation and study whether the intrinsic renin system cm~ affect the sympathetic system under these conditions. The following experiments were carried out according to this outline.
2. Materials and methods Male cats weighing 2.5--4.5 kg were anaesthetized with pentobarbitone sodium (50 mg/kg, i.m.) and the right femoral artery and vein were cannulated. Cannulation of the left adrenolumbar vein and treatment of the adrenal vein blood samples were carried out as described previously (Feuerstein and Gutman, 1971). Blood from the adrenal vein was collected into tubes placed in ice. The samples were immediately centrifuged and the plasma was acidified with HC104 (final concentration 0.4 M). Haemorrhage was induced by withdrawal of 13.3 ml/kg of arterial blood over a period of 10 min. Catecholamines were assayed in adrenal vein blood collected for 30 rain before and 80 min after haemorrhage, at 10 min intervals. Blood pressure was recorded from the carotid artery on a F & M physiograph.
ANGIOTENSIN HAEMORRHAGE AND ADRENAL CATECHOLAMINES Bilateral cervical vagotomy was carried out through bilateral neck incision. Bilateral ureteral ligation and bilateral n e p h r e c t o m y were done through a mid abdominal incision. Left adrenal gland denervation was performed by carefully cutting the left splanchnic nerves. Sodium loading experiment: for 7 days cats were given a diet consisting of rice soaked in 1.2% NaC1 and the sole drinking fluid was 1.2% NaCt. Supplementary injections of deoxycortisone acetate (Sigma), 2 mg/kg/day, were given over the same period. On the 8th day, the experiment of haemorrhage and catecholamine release from the adrenal was carried out. 2.1. Assay o f plasma renin concentration (PRC) 1.5 ml arterial blood samples were collected into ice-cooled test tubes. Red blood cells were sedimented by centrifugation {6500 rpm, 5 rain, 4°C). The plasma was separated and kept at --20°C. PRC was determined with an [~2sI] radioimmunoassay kit (New England Nuclear) after generation of magiotensin I by incubation of the plasma samples with excess substrate (obtained from nephrectomized cats).
2.2. CatechoIamine assay 2.2.1. Isolation of CA by aluminium oxide and Bio-Rex 70 resin 2.2.1.1. Preparation o f alumina colums. (a) Activated (acid washed) aluminium oxide (0.7 g per column) was added to a 600 ml glass beaker containing 10 ml of 0.1 M a m m o n i u m acetate per column. The pH was adjusted to 8.4 with 1.0 M sodium hydroxide under constant mixing with a magnetic stirrer. (b) The supernatant liquid was discarded and the alumina was washed once with bidistilled water. (c) The alumina was then poured into a glass column plugged with glass wool. The column was washed with bidistilled water in order to settle the particles of alumina to the b o t t o m of the column.
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2.2.1.2. Preparation of Bio-Rex resin and column (Bio-Rex 70, 50--100 mesh). 3.0 g of the resin (sodium form) were washed 3 times with 10 ml of bidistilled water to remove the fine particles. The resin was then stirred with bidistilled water and allowed to settle. The supernatant suspension was decanted. After washing with bidistilled water, 10 ml of 0.4 M hydrochloric acid were added. The suspension was stirred and then allowed to settle. The supernatant was decanted. The resin was then washed 3 times with 10.0 ml bidistilled water, as described. After decanting the supernatant, the resin was stirred with 10.0 ml of 0.02 M sodium hydroxide and allowed to settle. The supernatant was decanted. The resin was washed 3 times with 10.0 ml of bidistilled water. The final supernatant had a pH of 6.0. 0.2 ml of the activated resin was transferred into each column (made from a 1.0 ml polyethylene syringe). A small a m o u n t of glass wool was packed into the lower tip of each column. A syringe needle (gauge 23) was fitted into the b o t t o m of the column to slow the flow rate. The flow rate for good recovery was 0.5 ml per min. After packing the resin, the column was washed 3 times with 1.0 ml of H20 in order to settle the particles of resin to the b o t t o m of the column. To adjust the pH of the column, 0.1 M potassium phosphate buffer, pH 6.5, was passed through {3 × I ml). The column was then washed 3 times with 1.0 ml H20 or until the pH of the final effluent
was 6.0-6.5.
2.2.2. Adsorption of catecholamines on columns (alumina and Bio-Rex resin) (a) The pH of the acidified plasma samples was adjusted to 5.5 with 5 M potassium carbonate. After pH adjustment, the potassium perchlorate formed was sedimented by centrifugation for 10 rain at 4000 r.p.m, in the cold. (b) The supernatant was collected. 1.0 ml of 10% disodium-EDTA was added to each sample (10 ml) and the pH of the sample was adjusted to 8.4 with a m m o n i u m hydroxide (0.3 M). (c) The samples were poured onto the columns. After adsorption of the sample,
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the alumina was washed with 10.0 ml of HzO and the catecholamines were then eluted with 6.0 ml of 0.05 M perchlorie acid and stored at 4°C. (d) 0.05 ml of 10.0% disodium-EDTA was added to each eluate from the alumina column. The pH was adjusted to 6.0 with 0.2 M sodium carbonate. The final volume was brought to 7.0 ml by adding HzO. Each sample was well mixed, then passed through the Bio-Rex column. The columns were washed with 2.5 ml of H20 and the catecholamines were then eluted with 1.5 ml of 2/3 M boric acid and placed in ice. Recovery through the complete procedure, using 50 ng to 1 yg of the catecholamine, was 70--90% for adrenaline and 75--95% for noradrenaline. Actual readings without correction for recovery are given in the results section.
2.2. 3. Fluorimetric analysis After elution from the Bio-Rex columns the catecholamines were immediately assayed. In the assay for adrenaline, 100 yl of eluate were buffered to pH 3.0 with 100 pl of 0.36 M sodium citrate buffer, pH 2.7. In this improved assay m e t h o d , 10 yl of 0.002% CuC12 were added to increase the fluorescence intensity of adrenaline. The adrenaline sample was then oxidized at room temperature by adding 10 yl of 0.1 M iodine (in 0.1 M KI). The optimal time for oxidation was 5 min. Oxidation was stopped by adding 10 pl of 0.1 M sodium thiosulfate. Then 10 yl of 2.5% ascorbie acid (freshly prepared) were added for stabilization of the fluorescent quinone products and to prevent the spontaneous oxidation of catecholamines in alkaline media. After the addition of ascorbic acid, the oxidized eateeholamines were tautomerized to the fluorescent trihydroxyindoles at pH 12.5--13.0 by adding 25 pl of 5 M sodium hydroxide; the optimal time for tautomerization was 15 min. Finally, 30 yl of saturated boric acid in glacial acetic acid were added and fluorescence was measured at neutral pH (6.8--7.0), excitation wavelength 420 m y and emission wavelength 494 my. The addition of
G. F E U E R S T E I N ET AL.
glacial acetic acid saturated with boric acid markedly increased the intensity and stability of the final fluorescent compounds at neutral pH. When the oxidation was carried out at pH 6.0, both adrenaline and noradrenaline were oxidized. To 100 yl of the eluate from the Bio-Rex column were added 100 gl of 0.1 M sodium acetate buffer, pH 6.0. Copper ions did not improve the sensitivity of the reaction at pH 6. Oxidation was started by the addition of 10 gl of 0.25% potassium ferricyanide, the optimal time for oxidation was 5 min. Oxidation was stopped by the addition of 15 pl of 20.0% sodium sulfite in the presence of 2.0% mercaptoethanol (freshly prepared). Mercaptoethanol stabilized the oxidized noradrenaline. The adrenochromes were then tautomerized with 25 pl of 5 M sodium hydroxide (the optimal time for tautomerization was 4 min) to the strongly fluorescent trihydroxyindoles, at pH 12.5--13.0. After tautomerization, the pH was brought to 6.5--7.0 with 3 0 / l l glacial acetic acid. In this procedure, the acidification of the alkaline reaction mixture with glacial acetic acid greatly increased the intensity of the maximal fluorescence. The fluorescence intensity was immediately measured with excitation at 400 mp and emission at 480 my.
2.2.4. Preparation o f blank In the fluorimetric assay of catecholamines in biological extracts it is essential to include sample blanks to correct for interfering compounds which might be present in the experimental samples. A blank was thus always included and was prepared by adding the reagents in reverse order to t h a t described above. After pH adjustement of the sample, cupric chloride was added followed by 5 M NaOH. Finally a mixture containing the other reagents was added. For the reaction at pH 3 (adrenaline oxidation) the mixture consisted of iodine, sodium thiosulfate, glacial acetic acid (saturated with boric acid) and ascorbic acid. For the reaction at pH 6.0 (noradrenaline and adrenaline) the mixture consisted of
A N G I O T E N S I N H A E M O R R H A G E AND A D R E N A L C A T E C H O L A M I N E S
potassium ferricyanide, glacial acetic acid, sodium sulfite and mercaptoethanol. The mixture of reagents for the blanks contained the same volumes of the various reagents as did the samples, but the order of addition prevented the production of fluorescent compounds from the catecholamines. Catecholamine secretion rate varied widely from cat to cat. Therefore, the mean secretion rate of CA during the last two periods before haemorrhage was taken as control value and the secretion rate of CA following haemorrhage is expressed as percent of control for each cat.
3. Results
3.1. Adrenal catecholamine secretion rate in cats after haemorrhage (fig. 1) In control experiments, adrenal catecholamine secretion rate and composition (percent adrenaline) in anaesthetized cats were found to be constant with a tendency to a
200 -
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m
.
t
• : ,/AT
0
100
i
.,¢:
0
I
i -20
I 0
I i 20 Time (rain)
L 60
decrease of secretion rate throughout the 90 min period of the experiment. A somewhat higher secretion rate was found in the first sample following surgery; this may be attributed to the stress induced by the surgical procedures. Although wide differences were found in CA secretion rate from animal to animal, the rate tended to be constant for each cat throughout the experiment. Following bleeding, there was an immediate increase of catecholamine secretion rate. The peak secretion rate following haemorrhage was 164.3 -+ 38.6 ng/kg/10 min in comparison to 52.8 -+ 7.8 ng/kg/10 min during the control period (p < 0.01, n = 8). Furthermore, there was a significant change in adrenaline/noradrenaline ratio: while adrenaline comprised 79.3 ± 5.5% of the total catecholamines in adrenal vein plasma during the control period, the percentage changed after haermorrhage to 55.4 ± 5.8% (t9 < 0.01). Thus haemorrhage elicited a relatively noradrenalinergic response. This corroborates our earlier report on the preferential secretion of noradrenaline from the cat adrenal following haemorrhage (Feuerstein and Gutman, 1971).
3.2. Effect o f bilateral cervical vagotomy on adrenal medulla response to haemorrhage (fig. 2)
•
•
'E 0
135
I 100
Fig. 1. C a t e c h o l a m i n e release f r o m t h e adrenal medulla o f t h e cat in r e s p o n s e t o h a e m o r r h a g e . O r d i n a t e : c a t e c h o l a m i n e s e c r e t i o n as p e r c e n t o f s e c r e t i o n rate during the c o n t r o l p e r i o d p r e c e e d i n g h a e m o r r h a g e . • ~: c o n t r o l cats, w i t h o u t h a e m o r r h a g e (n = 8). o o: cats e x p o s e d t o h a e m o r r h a g e (n = 8). H: Whenever used indicates t i m e o f s t a r t i n g h a e m o r r hage. Vertical bars: S.E. (in all figures). * p < 0.02, • * p < 0.01, * * * p < 0.001 for d i f f e r e n c e b e t w e e n c o n t r o l and e x p e r i m e n t a l g r o u p (in all figures).
The afferent fibres in the vagus nerve include most of the fibers originating from baroreceptors and volume receptors in the chest. The importance of these stimuli in mediating catecholamine secretion from the adrenal medulla following haemorrhage was studied in cats with bilateral cervical vagotomy. Following bilateral vagotomy but without haemorrhage (= control vagotomy), catecholamine secretion rate from the cat adrenal did not differ from that of control cats without vagotomy, as seen in fig. 2: there was no increase in CA secretion throughout the experiment. Induction of bleeding in vagotomized cats elicited a significant increase of CA secretion
136
G. F E U E R S T E I N ET AL.
T
200-
:
:
~ 100
•
H
131
RENIN-ANGIOTENSIN MEDIATION OF ADRENAL CATECHOLAMINE SECRETION I N D U C E D BY H A E M O R R H A G E * GIORA FEUERSTEIN, PUNYA BOONYAVIROJ and YEHUDA GUTMAN ** Department of Pharmacology, Hebrew University--Hadassah Medical School, Jerusalem, Israel Received 12 November 1976, revised MS received 27 January 1977, accepted 24 March 1977
G. FEUERSTEIN, P. BOONYAVIROJ and Y. GUTMAN, Renin--angiotensin mediation o f adrenal catecholamine secretion induced by haemorrhage, European J. Pharmacol. 44 (1977) 131--142. The mechanism involved in catecholamine (CA) release from eat adrenal gland, in response to haemorrhage was studied. In intact eats, in cats with bilateral cervical vagotomy or following bilateral ureteral ligation, haemorrhage induced an increased catecholamine release from the adrenal (with increased percentage of noradrenaline). Acute bilateral nephrectomy, chronic sodium loading with repeated administration of desoxyeorticosterone acetate (DOCA), or acute denervation of the adrenal gland, completely abolished the increased CA release from the adrenal gland following haemorrhage. Haemorrhage induced an increase of plasma renin concentration in intact cats and after ureteral ligation but there was no increase in plasma renin after haemorrhage in cats with bilateral nephrectomy or following pretreatment with DOCA and salt load. Following haemorrhage in intact cats, blood pressure showed an immediate fall followed by rapid recovery. The recovery of blood pressure after haemorrhage was abolished in cats with bilateral nephrectomy. It is concluded that the adreno-medullary response to haemorrhage in the eat, depends primarily on the intact renal renin angiotensin system. Angiotensin, generated peripherally, probably affects the CNS and activates the sympathetic nerves. Angiotensin
Catecholamines
Haemorrhage
1. I n t r o d u c t i o n A close interrelation b e t w e e n the r e n i n - a n g i o t e n s i n s y s t e m a n d t h e s y m p a t h e t i c nervous s y s t e m has b e e n s u g g e s t e d b y v a r i o u s types of experiments. Thus, sympathetic innervation of the juxtaglomerular apparatus has b e e n d e m o n s t r a t e d (Barajas, 1 9 6 4 ; Wagerm a k e t al., 1967} t o g e t h e r w i t h a d i r e c t e f f e c t of c a t e c h o l a m i n e s on r e n a l r e n i n r e l e a s e ( J o h n s o n e t al., 1 9 7 1 ; V a n d e r , 1 9 6 5 ) w h i c h * Parts of this paper were presented at the Jerusalem Satellite Symposia, 26th International Congress of Physiological Sciences, October 1974, at the International Congress on Central Actions of Angiotensin, Houston, Texas, January 1976 and at the 36th Meeting of the Israel Physiological & Pharmacological Society, Tel-Aviv, June 1976. ** Established Investigator, Chief Scientist's Bureau, Israeli Ministry of Health.
Adrenal medulla
c o u l d be b l o c k e d b y s p e c i f i c a d r e n e r g i c b l o c k ing a g e n t s ( B u h l e r , 1 9 7 4 ; L o e f f l e r e t al., 1 9 7 2 ) . On t h e o t h e r h a n d , t h e r e is e v i d e n c e s u g g e s t i n g a p r i m a r y r o l e o f a n g i o t e n s i n II in m o d u l a t i n g s y m p a t h e t i c tone and effects, through either central or peripheral mechanisms (Hughes and Roth, 1969; Robinson, 1 9 6 7 ; Yu a n d D i c k i n s o n 1 9 7 1 ) . Several i n v e s t i g a t i o n s have d e m o n s t r a t e d t h a t a n g i o t e n s i n II in small d o s e s e n h a n c e d v a s o m o t o r r e s p o n s e s t o c a t e c h o l a m i n e s in several v a s c u l a r b e d s ( D a y a n d M o o r e 1 9 7 6 ; Lias a n d Z i m m e r m a n , 1 9 7 2 ; N i c h o l a s , 1 9 7 0 ) . This e f f e c t has b e e n a t t r i b u t e d t o a d i r e c t s e n s i t i z i n g a c t i o n on t h e e f f e c t o r o r g a n s ( D a y a n d M o o r e , 1 9 7 6 ) , or t o an i n d i r e c t o n e i.e., facilitation of catecholamine release from nerve e n d i n g s ( H u g h e s a n d R o t h , 1 9 6 9 ) o r an i n h i b i t o r y e f f e c t on c a t e c h o l a m i n e r e - u p t a k e i n t o a d r e n e r g i c n e r v e t e r m i n a l s (Palaic a n d
132
Khairallah, 1971; Peach et al., 1969; Janovsky, 1972). Blockade of the re-uptake mechanism has been also postulated as the cause of the increased rate of catecholamine synthesis in several sympathetically innervated organs exposed to angiotensin (Davilla and Khairallah, 1971). Alternatively, a direct stimulatory effect of angiotensin, through induction of tyrosine hydroxylase synthesis, has also been suggested (Roth, 1972). Angiotensin II has also been found to have excitatory effects on sympathetic postganglionic neurons, through specific receptors, as demonstrated in the cat superior cervical ganglia (Lewis and Reit, 1966) and in adrenal medulla chromaffin cells of various species (Peach, 1971; Robinson, 1967; StaszeweskaBarczack and Vane, 1967). Some of these are conflicting, e.g. Hughes could not demonstrate inhibition of noradrenaline re-uptake by angiotensin II (Hughes, 1969) while Haefely et al. (1965) and Panniset et al. (1966) failed to demonstrate any excitatory effects of angiotensin II on sympathetic postganglionc neurons with "physiological" doses of angiotensin II. In addition to having these various peripheral sites of action, angiotensin was found to exert several central stimulatory properties, such as increase of water consumption (Epstein et al., 1970), vasopressin release (Mouw et al., 1971) and blood pressure elevation (Scroop and Whelan 1966; Yu and Dickinson, 1965). This latter effect has been shown to be mediated through the sympathetic nervous system (Ueda et al., 1969; Yu and Dickinson, 1971). These findings have suggested that angiotensin II may play an important role in blood pressure regulation and in the development of different types of hypertension. However, no clear relationship has been found between plasma renin activity, primary hypertension (Aurell et al., 1975) and several models of experimental renal hypertension (Brown et al., 1966; Fernandez et al., 1976), in which elevated blood pressure was sustained long after plasma angiotensin II concentration returned to control values. But in these vari-
G. F E U E R S T E I N E T AL.
ous types of hypertension, sympathetic overactivity has been suspected to play a major role in the maintenance of chronic high blood pressure (Dargie et al., 1976; Haeusler et al., 1972; Louis et al., 1973). In analyzing these conflicting data it should be realized that they derive mostly from in vitro experiments on isolated organs (using perfusion techniques), or tissue slices incubated in vitro. In many of the in vivo experiments pharmacological doses of angiotensin II were either implanted (as crystals) or injected through non-physiological routes; species differences and especially different anaesthetic procedures have probably also contributed to the differences. In order to asses the relationship between sympathetic responses and the renal renin-angiotensin system activity, it seemed of interest to us to use physiological stimuli in an in vivo situation and study whether the intrinsic renin system cm~ affect the sympathetic system under these conditions. The following experiments were carried out according to this outline.
2. Materials and methods Male cats weighing 2.5--4.5 kg were anaesthetized with pentobarbitone sodium (50 mg/kg, i.m.) and the right femoral artery and vein were cannulated. Cannulation of the left adrenolumbar vein and treatment of the adrenal vein blood samples were carried out as described previously (Feuerstein and Gutman, 1971). Blood from the adrenal vein was collected into tubes placed in ice. The samples were immediately centrifuged and the plasma was acidified with HC104 (final concentration 0.4 M). Haemorrhage was induced by withdrawal of 13.3 ml/kg of arterial blood over a period of 10 min. Catecholamines were assayed in adrenal vein blood collected for 30 rain before and 80 min after haemorrhage, at 10 min intervals. Blood pressure was recorded from the carotid artery on a F & M physiograph.
ANGIOTENSIN HAEMORRHAGE AND ADRENAL CATECHOLAMINES Bilateral cervical vagotomy was carried out through bilateral neck incision. Bilateral ureteral ligation and bilateral n e p h r e c t o m y were done through a mid abdominal incision. Left adrenal gland denervation was performed by carefully cutting the left splanchnic nerves. Sodium loading experiment: for 7 days cats were given a diet consisting of rice soaked in 1.2% NaC1 and the sole drinking fluid was 1.2% NaCt. Supplementary injections of deoxycortisone acetate (Sigma), 2 mg/kg/day, were given over the same period. On the 8th day, the experiment of haemorrhage and catecholamine release from the adrenal was carried out. 2.1. Assay o f plasma renin concentration (PRC) 1.5 ml arterial blood samples were collected into ice-cooled test tubes. Red blood cells were sedimented by centrifugation {6500 rpm, 5 rain, 4°C). The plasma was separated and kept at --20°C. PRC was determined with an [~2sI] radioimmunoassay kit (New England Nuclear) after generation of magiotensin I by incubation of the plasma samples with excess substrate (obtained from nephrectomized cats).
2.2. CatechoIamine assay 2.2.1. Isolation of CA by aluminium oxide and Bio-Rex 70 resin 2.2.1.1. Preparation o f alumina colums. (a) Activated (acid washed) aluminium oxide (0.7 g per column) was added to a 600 ml glass beaker containing 10 ml of 0.1 M a m m o n i u m acetate per column. The pH was adjusted to 8.4 with 1.0 M sodium hydroxide under constant mixing with a magnetic stirrer. (b) The supernatant liquid was discarded and the alumina was washed once with bidistilled water. (c) The alumina was then poured into a glass column plugged with glass wool. The column was washed with bidistilled water in order to settle the particles of alumina to the b o t t o m of the column.
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2.2.1.2. Preparation of Bio-Rex resin and column (Bio-Rex 70, 50--100 mesh). 3.0 g of the resin (sodium form) were washed 3 times with 10 ml of bidistilled water to remove the fine particles. The resin was then stirred with bidistilled water and allowed to settle. The supernatant suspension was decanted. After washing with bidistilled water, 10 ml of 0.4 M hydrochloric acid were added. The suspension was stirred and then allowed to settle. The supernatant was decanted. The resin was then washed 3 times with 10.0 ml bidistilled water, as described. After decanting the supernatant, the resin was stirred with 10.0 ml of 0.02 M sodium hydroxide and allowed to settle. The supernatant was decanted. The resin was washed 3 times with 10.0 ml of bidistilled water. The final supernatant had a pH of 6.0. 0.2 ml of the activated resin was transferred into each column (made from a 1.0 ml polyethylene syringe). A small a m o u n t of glass wool was packed into the lower tip of each column. A syringe needle (gauge 23) was fitted into the b o t t o m of the column to slow the flow rate. The flow rate for good recovery was 0.5 ml per min. After packing the resin, the column was washed 3 times with 1.0 ml of H20 in order to settle the particles of resin to the b o t t o m of the column. To adjust the pH of the column, 0.1 M potassium phosphate buffer, pH 6.5, was passed through {3 × I ml). The column was then washed 3 times with 1.0 ml H20 or until the pH of the final effluent
was 6.0-6.5.
2.2.2. Adsorption of catecholamines on columns (alumina and Bio-Rex resin) (a) The pH of the acidified plasma samples was adjusted to 5.5 with 5 M potassium carbonate. After pH adjustment, the potassium perchlorate formed was sedimented by centrifugation for 10 rain at 4000 r.p.m, in the cold. (b) The supernatant was collected. 1.0 ml of 10% disodium-EDTA was added to each sample (10 ml) and the pH of the sample was adjusted to 8.4 with a m m o n i u m hydroxide (0.3 M). (c) The samples were poured onto the columns. After adsorption of the sample,
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the alumina was washed with 10.0 ml of HzO and the catecholamines were then eluted with 6.0 ml of 0.05 M perchlorie acid and stored at 4°C. (d) 0.05 ml of 10.0% disodium-EDTA was added to each eluate from the alumina column. The pH was adjusted to 6.0 with 0.2 M sodium carbonate. The final volume was brought to 7.0 ml by adding HzO. Each sample was well mixed, then passed through the Bio-Rex column. The columns were washed with 2.5 ml of H20 and the catecholamines were then eluted with 1.5 ml of 2/3 M boric acid and placed in ice. Recovery through the complete procedure, using 50 ng to 1 yg of the catecholamine, was 70--90% for adrenaline and 75--95% for noradrenaline. Actual readings without correction for recovery are given in the results section.
2.2. 3. Fluorimetric analysis After elution from the Bio-Rex columns the catecholamines were immediately assayed. In the assay for adrenaline, 100 yl of eluate were buffered to pH 3.0 with 100 pl of 0.36 M sodium citrate buffer, pH 2.7. In this improved assay m e t h o d , 10 yl of 0.002% CuC12 were added to increase the fluorescence intensity of adrenaline. The adrenaline sample was then oxidized at room temperature by adding 10 yl of 0.1 M iodine (in 0.1 M KI). The optimal time for oxidation was 5 min. Oxidation was stopped by adding 10 pl of 0.1 M sodium thiosulfate. Then 10 yl of 2.5% ascorbie acid (freshly prepared) were added for stabilization of the fluorescent quinone products and to prevent the spontaneous oxidation of catecholamines in alkaline media. After the addition of ascorbic acid, the oxidized eateeholamines were tautomerized to the fluorescent trihydroxyindoles at pH 12.5--13.0 by adding 25 pl of 5 M sodium hydroxide; the optimal time for tautomerization was 15 min. Finally, 30 yl of saturated boric acid in glacial acetic acid were added and fluorescence was measured at neutral pH (6.8--7.0), excitation wavelength 420 m y and emission wavelength 494 my. The addition of
G. F E U E R S T E I N ET AL.
glacial acetic acid saturated with boric acid markedly increased the intensity and stability of the final fluorescent compounds at neutral pH. When the oxidation was carried out at pH 6.0, both adrenaline and noradrenaline were oxidized. To 100 yl of the eluate from the Bio-Rex column were added 100 gl of 0.1 M sodium acetate buffer, pH 6.0. Copper ions did not improve the sensitivity of the reaction at pH 6. Oxidation was started by the addition of 10 gl of 0.25% potassium ferricyanide, the optimal time for oxidation was 5 min. Oxidation was stopped by the addition of 15 pl of 20.0% sodium sulfite in the presence of 2.0% mercaptoethanol (freshly prepared). Mercaptoethanol stabilized the oxidized noradrenaline. The adrenochromes were then tautomerized with 25 pl of 5 M sodium hydroxide (the optimal time for tautomerization was 4 min) to the strongly fluorescent trihydroxyindoles, at pH 12.5--13.0. After tautomerization, the pH was brought to 6.5--7.0 with 3 0 / l l glacial acetic acid. In this procedure, the acidification of the alkaline reaction mixture with glacial acetic acid greatly increased the intensity of the maximal fluorescence. The fluorescence intensity was immediately measured with excitation at 400 mp and emission at 480 my.
2.2.4. Preparation o f blank In the fluorimetric assay of catecholamines in biological extracts it is essential to include sample blanks to correct for interfering compounds which might be present in the experimental samples. A blank was thus always included and was prepared by adding the reagents in reverse order to t h a t described above. After pH adjustement of the sample, cupric chloride was added followed by 5 M NaOH. Finally a mixture containing the other reagents was added. For the reaction at pH 3 (adrenaline oxidation) the mixture consisted of iodine, sodium thiosulfate, glacial acetic acid (saturated with boric acid) and ascorbic acid. For the reaction at pH 6.0 (noradrenaline and adrenaline) the mixture consisted of
A N G I O T E N S I N H A E M O R R H A G E AND A D R E N A L C A T E C H O L A M I N E S
potassium ferricyanide, glacial acetic acid, sodium sulfite and mercaptoethanol. The mixture of reagents for the blanks contained the same volumes of the various reagents as did the samples, but the order of addition prevented the production of fluorescent compounds from the catecholamines. Catecholamine secretion rate varied widely from cat to cat. Therefore, the mean secretion rate of CA during the last two periods before haemorrhage was taken as control value and the secretion rate of CA following haemorrhage is expressed as percent of control for each cat.
3. Results
3.1. Adrenal catecholamine secretion rate in cats after haemorrhage (fig. 1) In control experiments, adrenal catecholamine secretion rate and composition (percent adrenaline) in anaesthetized cats were found to be constant with a tendency to a
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decrease of secretion rate throughout the 90 min period of the experiment. A somewhat higher secretion rate was found in the first sample following surgery; this may be attributed to the stress induced by the surgical procedures. Although wide differences were found in CA secretion rate from animal to animal, the rate tended to be constant for each cat throughout the experiment. Following bleeding, there was an immediate increase of catecholamine secretion rate. The peak secretion rate following haemorrhage was 164.3 -+ 38.6 ng/kg/10 min in comparison to 52.8 -+ 7.8 ng/kg/10 min during the control period (p < 0.01, n = 8). Furthermore, there was a significant change in adrenaline/noradrenaline ratio: while adrenaline comprised 79.3 ± 5.5% of the total catecholamines in adrenal vein plasma during the control period, the percentage changed after haermorrhage to 55.4 ± 5.8% (t9 < 0.01). Thus haemorrhage elicited a relatively noradrenalinergic response. This corroborates our earlier report on the preferential secretion of noradrenaline from the cat adrenal following haemorrhage (Feuerstein and Gutman, 1971).
3.2. Effect o f bilateral cervical vagotomy on adrenal medulla response to haemorrhage (fig. 2)
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135
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Fig. 1. C a t e c h o l a m i n e release f r o m t h e adrenal medulla o f t h e cat in r e s p o n s e t o h a e m o r r h a g e . O r d i n a t e : c a t e c h o l a m i n e s e c r e t i o n as p e r c e n t o f s e c r e t i o n rate during the c o n t r o l p e r i o d p r e c e e d i n g h a e m o r r h a g e . • ~: c o n t r o l cats, w i t h o u t h a e m o r r h a g e (n = 8). o o: cats e x p o s e d t o h a e m o r r h a g e (n = 8). H: Whenever used indicates t i m e o f s t a r t i n g h a e m o r r hage. Vertical bars: S.E. (in all figures). * p < 0.02, • * p < 0.01, * * * p < 0.001 for d i f f e r e n c e b e t w e e n c o n t r o l and e x p e r i m e n t a l g r o u p (in all figures).
The afferent fibres in the vagus nerve include most of the fibers originating from baroreceptors and volume receptors in the chest. The importance of these stimuli in mediating catecholamine secretion from the adrenal medulla following haemorrhage was studied in cats with bilateral cervical vagotomy. Following bilateral vagotomy but without haemorrhage (= control vagotomy), catecholamine secretion rate from the cat adrenal did not differ from that of control cats without vagotomy, as seen in fig. 2: there was no increase in CA secretion throughout the experiment. Induction of bleeding in vagotomized cats elicited a significant increase of CA secretion
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G. F E U E R S T E I N ET AL.
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