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Effects of angiotensin on the autonomic nervous system
Fundamentals of clinioalcardiology
Effects of angiotensin
on the autonomic
nervous
system
R. D. Lowe, M.D. G. C. Scroo$, M.D. London, England
T
he physiological role and mechanism of action of angiotensin continue to excite the interest of a large number of workers. Angiotensin has at least three different effects: it causes contraction of a variety of smooth muscles, especially of vascular origin ; it increases the rate of secretion of aldosterone in a wide range of animal species; it has effects at various sites of the autonomic nervous system. The first of these actions to be described was the effect as a direct vasoconstrictor which occupied the attention of cardiovascular investigation until the late 1950’s. Since then its two other types of action have excited renewed interest among research workers but the results in the two fields of work provide an interesting contrast. The autonomic effects of angiotensin are complex and their physiological role is still obscure, whereas the effect on aldosterone secretion is well defined and appears to be of physiological importance in the control of blood volume. The first firm evidence that angiotensin could activate the autonomic nervous system in the intact animal was provided by the experiments of Renson and associates’ who in 1959 demonstrated that angiotensin could cause contraction of the nictitating membrane in cats (whose nerve supply is exclusively adrenergic) ; this action was blocked by the alpha-adrenergic blocking agent, phenoxybenzamine. Since then a From the Department
562
American
Heart
of Medicine.
Journal
St. Thomas’s
great deal of work has been done in this field, and it is therefore somewhat surprising that no clear picture has emerged as to the physiological role of these effects, i.e., what part they might play in the response to angiotensin generated in the blood stream by endogenous renin. Nevertheless, these effects are interesting and it is worth discussing them in some detail to illustrate how extremely complex the effect of angiotensin can be. The references cited are not necessarily the earliest ones, but have been selected as important ones which also give further references to much of the earlier literature. The effects which have been so far described fall into the following groups: (1) parasympathomimetic effects and potentiation of the effects of cholinergic nervous activity; (2) activation of sympathetic ganglia; (3) stimulation of catecholamine secretion from the adrenal medulla; (4) potentiation of the effects of adrenergic nerve stimulation ; (5) sympathomimetic effects due to an action on the brain; and (6) inhibition of parasympathetic nerves to the heart by an effect on the brain. In considering the possible physiological role of such effects, it is most important to bear in mind the concentration of angiotensin necessary to produce these effects and to compare this with those known to occur spontaneously in response to changes Hospital
Medical
School.
London.
S.E.I.,
England.
Afwil, 1970 Vol. 79, No. 4, pp. 562-567
Volumr Number
79 4
Effects of angiotensin
of renin secretion. The normal concentration of angiotensin in arterial blood in the dog and in man is probably in the region of 0.01 to 0.1 ng. per milliliter (i.e., 10 -11 to 10 -10 Gm. per milliliter). The concentration which occurs in response to severe circulatory stimuli (such as hemorrhage) is probably in the range 0.1 to 1 ng. per milliliter. In severe renal hypertension it may conceivably rise to much higher levels, but as a guide the “physiological” range is most likely to be 0.01 to 1 ng. per milliliter. Unfortunately, in many experiments in the intact animal the blood concentration is not measured, and only the dose is known. As a very rough guide, intravenous infusion rates of up to 100 ng. per kilogram per minute might produce concentrations within this physiological range. Effects on autonomic
nervous
system
Parasympathomimetic efects. Evidence of this action of angiotensin has come from studies of the response of intestinal smooth muscle which has a rich parasympathetic innervation by cholinergic fibers. When such tissues (e.g., guinea pig or rabbit ileum)2v3 are exposed to angiotensin in an organ bath, a contraction results which is increased by anticholinesterase and reduced by atropine or by botulinus toxin (which interferes with the synthesis and release of acetylcholine). Such results suggest that part of the effect of angiotensin on such tissues is mediated by cholinergic nerve endings in the tissue. This concept was supported by the experiments of Panisset* who studied the release of an acetylcholinelike substance into the organ bath when the tissue was stimulated via its nerve supply. In the presence of angiotensin (0.01 to 0.1 ng. per milliliter) stimulation of the nerve caused an increased release of acetylcholine. Since intestinal smooth muscle contains both pre- and postganglionic fibers as well as parasympathetic ganglia, this effect of angiotensin could be due to activation either of postganglionic cholinergic fibers or of preganglionic nerve terminals, or of the ganglia themselves. Angiotensin has not been found to potentiate the effects of cholinergic nerve stimulation in tissues where there are no ganglia, so that the most likely interpretation of these results
on autonomic nervous system
563
is that angiotensin can activate parasympathetic ganglia. Activation of sympathetic ganglia. Presumptive evidence of this effect was first provided by Lewis and Reit5 who demonstrated that intra-arterial injection of angiotensin close to the superior cervical ganglion of the cat caused contraction of the nictitating membrane, whereas similar injections to the membrane did not. The smallest effective dose was 100 ng. (by sudden injection) and the peak concentration was probably in the region of 100 ng. per milliliter. Panisset* studied the release of an acetylcholine-like substance into the effluent from the perfused superior cervical ganglion of the cat and found that angiotensin in a concentration of about 0.5 ng. per milliliter increased the amount of acetylcholine released in response to electrical stimulation of the preganglionic nerve. It is of interest that the effect of angiotensin on ganglionic transmission is not blocked by hexamethonium or pempidine5v6 (see below). Release of catecholamine from the adrenal medulla. Since the medulla is innervated by cholinergic “preganglionic” nerves and is in some respects the analogue of a ganglion, it might be expected that angiotensin would facilitate the release of catecholamines; such an effect has been described in several Feldberg and Lewis’ injected species. angiotensin into the aorta of eviscerated cats, assaying the released catecholamines by the response of the denervated nictitating membrane of the same animal; the smallest effective dose of angiotensin was 1 ng., and no effect was found after removal of the adrenal gland. Robinson8 perfused the isolated cat’s adrenal with an artificial perfusion fluid and measured the concentration of catecholamines in the venous effluent. Convincing increases of secretion were produced by injection of 0.1 ng. and some increase was seen with doses of 0.01 ng. or less; since the rate of perfusion was 0.5 to 5 ml. per minute it seems 1ikeIy that concentrations as low as 0.01 ng. per milliliter were effective. Sustained increases of catecholamine secretion in response to infusions of angiotensin in the dog were reported by Peach and associates9 who infused angiotensin
564
Lowe and &roop
intravenously in doses of 25, 50, and 100 ng. per kilogram per minute. The two higher doses caused a significant increase of catecholamine concentration in the inferior vena cava. Potentiation of the efects of adrenergic post-ganglionic nerve stimulation. The first evidence of this effect of angiotensin was produced by Benelli and co-workers’0 who demonstrated that the isolated guinea pig vas deferens (which is innervated only by adrenergic nerves) responded more strongly to stimulation of the hypogastric nerves when angiotensin was present in the organ bath, although angiotensin alone did not cause contraction. These results were not confirmed by Hughes” who stimulated the vas deferens transmurally; he suggested that the results of the Benelli group were due to the known effects of angiotensin at sympathetic ganglia which may have been present distal to the site of stimulation of the hypogastric nerve. Zimmerman and Gome+ also reported an effect which seemed likely to be due to some local interaction between sympathetic nerves and angiotensin. In the perfused paw of the dog, angiotensin infusion at 1,000 ng. per minute increased the constrictor effect of sympathetic nerve stimulation ; the concentration of angiotensin was approximately 30 ng. per milliliter. Similar effects were seen in the dog kidney with concentrations nearer 1 ng. per milliliter. It is possible that these effects were due to the known effects of angiotensin on sympathetic ganglia, because it is difficult to be certain that there were no ganglionic synapses distal to the site of stimulation of the nerve. An alternative approach to this problem is a pharmacological one, mimicking the effects of adrenergic nerve stimulation by the use of tyramine which is believed to act by releasing noradrenaline from symp’athetic nerve terminals. This drug has no known effects on ganglia, so that potentiation of the effects of tyramine by angiotensin cannot be due to facilitation of ganglionic transmission. Unfortunately, experiments with tyramine have usually involved injections or infusions into the whole animal and it is possible that it might cause release of catecholamines from
.‘lnwr.
Heart 1. .4pril, 1970
storage sites in the brain rather than from peripheral ones. In spite of these reservations it is nevertheless interesting that chronic renal hypertension13J4 or acute infusions of angiotensin at 30 ng. per kilogram per minute to the dog,15 or at 50 ng. per kilogram per minute into the rat,‘6 cause an increase in the pressor response to tyramine but not to noradrenaline. These results suggest that angiotensin may interact with factors causing release of noradrenaline at sympathetic nerve terminals to produce an increased response of the target organ. There is not yet sufficient evidence to be certain what is the nature of this interaction. Sympathomimetic effects due to an action on the brain. The first convincing experiments showing that angiotensin could produce such effects were those of Bickerton and Buckley,” who injected angiotensin into the arterial supply to the head of a dog, which was crossperfused from a donor. The only effective connection between the recipient’s head and body was via the nervous system, yet injection of angiotensin to the head caused a pressor response in the body which could be greatly reduced by an alpha-adrenergic blocking agent. The blood concentration producing these effects was probably above 100 ng. per milliliter Attempts to demonstrate this effect with more physiological concentrations have net been very successful. Infusions of angictensin into the carotid arteries of the intact animal do not apparently activate the syn.pathetic nervous system. Infusions into the vertebral artery of the rabbitl*Jg and the dogzO do have a specific pressor effect but it has not been shown that this is due to activation of the sympathetic nervous system. In the dog it is apparently due to inhibition of parasympathetic nerves to the heart, and a sympathomimetic effect can be demonstrated only in the vagotomized animal.*O Another method of demonstrating an effect of angiotensin on the brain is to inject or infuse it into the cerebrospinal fluid, but this is a very unphysiological route of administration and the concentrations required to demonstrate an effect are in the region of 100 ng. per milliliter. The resulting pressor effect seems likely to be
Volume h’umber
79 4
Effects of angiotensin
due to the sympathetic nervous system because it is reduced by the adrenergic blocking drugs, phenoxybenzamine and propranolol.21 A possible alternative explanation of this result is that adrenergic pathways in the brain were activated by angiotensin, and further evidence is required before it can be concluded that angiotensin can activate the sympathetic nervous system by an action on the brain in physiological concentrations in the intact animal. Inhibition of parasympathetic nerves to the heart due to an action on the brain. This effect can be demonstrated by infusions of angiotensin into the vertebral artery of the dog, which produces a rise of blood pressure due to an increase of cardiac output, which is unaffected by adrenergic blocking agents, but is almost abolished by atropine or vagotomy.20 Such effects can be demonstrated at local concentration of angiotensin in the region of 0.02 to 1 ng. per milliliter. Interaction of angiotensin with the sympathetic nervous system-site of action unknow. The preceding sections deal with the known sites of action of angiotensin on the autonomic nervous system, but there are a number of experiments which demonstrate a sympathomimetic action in which the site of action is unknown. It may subsequently transpire that the effects described fall into one or other of the categories already discussed, but until this has been shown they must be classified separately. The first evidence of such an action has already been mentioned, showing an effect on the nictitating membrane of cats. A similar effect on vascular smooth muscle was demonstrated by Laverty22; intravenous injection of angiotensin (doses of 1,000 ng. upward) caused a rise of resistance in the isolated perfused hind limb of the rat which communicated with the body only by the nerves. The peak concentrations of angiotensin were probably over 100 ng. per milliliter. Effects on the peripheral resistance have been demonstrated at lower concentration by McGiff and Fasyz3 and by Scroop and Whelan.24 McGiff and Fasy injected angiotensin (100 ng. per kilogram) intravenously in the anesthetized dog and recorded renal
on autonomic
nervous system
565
vasoconstriction which was abolished by guanethidine, bretylium, or renal denervation but not by the ganglion blocking agent, hexamethonium. The peak blood concentration in these experiments was probably about 3 ng. per milliliter. It is important to realize that the failure of hexamethonium to block this effect cannot be taken as evidence that the site of action of angiotensin was not on the sympathetic ganglia (see above). Scroop and Whelan infused angiotensin intravenously in man (dose 1,000 ng. per minute) and observed that the vasoconstrictor effect on the hand was greatly reduced by phenoxybenzamine or bretylium and did not occur in the sympathectomized limb or after denervation. The blood concentration of angiotensin in these experiments was probably in the region of 0.3 ng. per milliliter. Effects on the heart have been described by several workers. Farr and Grupp25 injected angiotensin (1,000 ng. per kilogram) intravenously in dogs, and noted a biphasic response of heart rate with a delayed rise above the control rate after the initial bradycardia; this delayed rise was accompanied by an increase of contractile force and was reduced by pronethalol or reserpine or by section of all nerves to the heart. It was not altered by adrenalectomy or by pentolinium or hexamethonium. Therefore, it cannot have been due to release of catecholamines from the adrenal medulla, but could have been due to an effect on ganglia (see above). The peak blood concentration was probably about 50 ng. per milliliter. Nishith and associates26 and Krasney and co-workers27 studied the effect on heart rate of rather low concentrations of angiotensin and concluded that there was activation of sympathetic nerves to the heart, but in their experiments interpretation is complicated by the fact that they stabilized arterial pressure during an angiotensin infusion and the effects on heart rate include the effects of controlled bleeding. Both groups of workers also studied the effects of angiotensin on heart rate after denervation of sinoaortic baroceptors (to prevent the reflex bradycardia in response to arterial hypertension) and they found a tachycardia. This was prevented by sympathetic blockade but not by ganglionic
566
Amer. Heart J. April. 1970
Lowe and Scroop
blockade or preganglionic neurectomy. These sympathomimetic effects could have been due either to a ganglionic or to a postganglionic action of angiotensin. Physiological role of effects on the autonomic nervous system. In many of the experiments described above both the absolute concentration of angiotensin and the shape of the concentration-line curve were very different from the physiological range. The question of the “physiological concentration” has already been discussed and a fairly broad range was accepted (0.0 to 1 ng. per milliliter), but it is also relevant to consider the way in which the “physiological concentration” changes with time, because endogenous changes of angiotensin are relatively slow, whereas injections produce a very sharp rise and decline. Infusions of angiotensin will mimic the effects of endogenous generation much better than will injections. Nevertheless, in spite of reservations as to the physiological relevance of some experiments, there is a sufficient body of evidence to suggest that generation of angiotensin in response to endogenous renin secretion might affect the autonomic nervous system in nearly all the ways which have been described. The fact that these autonomic effects can be expected to occur in many physiological circumstances does not enable us to draw any conclusions as to their relative importance compared to the direct vasoconstrictor effects. The net effect on arterial pressure must be the complex resultant of many conflicting mechanisms. In response to the direct vasoconstrictor effect the primary rise of blood pressure would be antagonized by baroceptor reflexes and perhaps by facilitation of transmission at parasympathetic ganglia supplying the heart. It would be enhanced by any central action of angiotensin on the brain, the adrenal medulla, the sympathetic ganglia, and the postganglionic sympathetic neurone. It is perhaps not surprising that no one has yet quantitated the relative importance of the various autonomic effects of angiotensin even in acute experiments, and the problem becomes even more complex in chronic experiments. It remains at least a possibility that the major cause of the sustained rise of arterial pressure in
chronic renal hypertension is due to the effects of angiotensin on the autonomic nervous system. REFERENCES 1. Renson, J., Barac, G., and Bacq, Z. M.: Effets de deux angiotensines synthetiques sur la pression arterielle et la membrane nictitante du chat, Comnt. rend. Sot. Biol. 153:1621. 1959. 2. Ross, 6. A., Sudden, C. T., and Stone, C. A.: Action of angiotensin on isolated guinea pig ileum, Proc. Sot. Exper. Biol. & Med. 105:558, 1960. 3. Robertson, P. A., and Rubin, D.: Stimulation of intestinal nervous elements by angiotensin, Brit. J. Pharmacol. 19:5, 1962. 4. Panisset, J. C.: Effect of angiotensin on the release of acetylcholine from pre-ganglionic and post-ganglion& nerve endings, Canad. J. Physiol. & Pharmacol. 45:313. 1967. 5. Lewis, G. P., and Reit, E.1 The action of angiotensin and bradykinin on the superior cervical ganglion of the cat, J. Physiol. 179:538, 1965. 6. Trendelenburg, U.: Observations on the ganglion stimulating action of angiotensin and bradykinin, J. Pharmacol. & Exper. Therap. 154:418, 1966. 7. Feldberg, W., and Lewis, G. P.: The action of peptides on the adrenal medulla. Release of adrenaline by bradykinin and angiotensin, J. Physiol. 171:98, 1964. of the cathe8. Robinson, R. L.: Stimulation cholamine output of the isolated perfused adrenal gland of the dog by angiotensin and bradvkinin. 1. Pharmacol. & Exaer. Therap. 156&2, 196i. 9. Peach, M. J., Cline, W. H., and Watts, D. J.: Release of adrenal catecholamines by angiotensin. II, Circulation Res. 19:571, 1966. 10. Benelli, G., Della-Bella, D., and Gandini, A.: Angiotensin and peripheral sympathetic nerve activity, Brit. J, Pharmacol. 22:211, 1964. 11. Hughes, I. E.: An investigation of the effects of angiotensin on the release of neurohumoral transmitters at the motor, adrenergic and cholineruic nerve terminals. T. Pharm. & Pharmacol. ib:116, 1969. 12. Zimmerman, B. G., and Gomez, J.: Increased response to sympathetic stimulation in the cutaneous vasculature in presence of angiotensin, Internat. J. Neuropharmacol. 4:185, 1965. 13. Verney, E. B., and Vogt, M.: An experimental investigation into hypertension of renal origin with some observations on convulsive “uraemia,” Quart. J. Exper. Physiol. !28:253, 1938. 14. McCubbin, J. W., and Page, I. H.: Renal pressor system and neurogenic control of arterial pressure, Circulation Res. 12:.553, 1963. 15. Louis, W. J., and Doyle, A. E.: The pressor response to noradrenaline and tyramine during angiotensin tachyphylaxis in the dog, Clin. SC. 31:247, 1966.
?u~ET’,“r ‘4’ 16.
17.
18.
19.
20.
21.
Efects
of angiotensin
Schmitt, H., and Schmitt, H.: Interrelation entre catecholamine et angiotensine, Compt. rend. Sot. Biol. 161:753, 1967. Bickerton, R. K,, and Buckley, J. P.: Evidence for a central mechanism in angiotensin-induced hvoertension. Proc. Sot. Exner. Biol. & Med. lbti:834, 1961. Dickinson, C. J., and Yu, R.: Mechanisms involved in the progressive pressor response to very small amounts of angiotensin in conscious rabbits, Circulation Res. (Supp. II) ZO-21:11 157, 1967. Cranston, W. I., Lavery, H. A., Lowe, R. D., and Rosendorff, C.: The central pressor action of angiotensin in the rabbit, J. Physiol. 198:3OP, 1968. Scroop, G. C., and Lowe, R. D.: A central pressor effect of angiotensin mediated by the parasympathetic nervous system, Nature 220: 1331, 1968. Severs, W. B., Daniels, A. E., Smookler, H. M., Kinnard, W. J., and Buckley, J. P.: Interrelationship between angiotensin II and the sympa-
on autonomic
nervous system
567
thetic nervous system, J. Pharmacol. & Exper. Therap. 153:530, 1966. 22. Laverty, R.: A nervously mediated action of angiotensin in anaesthetised rats, J. Pharm. & Pharmacol. 15:63, 1963. 23. McGiff, J. C., and Fasy, T. M.: The relationship of the renal vascular activity of angiotensin II to the autonomic nervous system, J. Clin. Invest. 44:1911, 1965. 24. Scroop, G. C., and Whelan, R. F.: A central vasomotor action of angiotensin in man, Clin. SC. 30:79, 1966. 2.5. Farr, W. C., and Grupp, G.: Sympathetically mediated effects of angiotensin on the dog heart in situ, J. Pharmacol. & Exper. Therap. 156: 528, 1967. S. D., Davis, L. D., and Youmans, 26. Nishith, W. B.: Cardioaccelerator action of angiotensin, Am. J. Physiol. 202:237,1962. 27. Krasney, J. A., Paudler, F. T., Smith, D. C., Davis, L. D., and Youmans, W. B.: Mechanisms of cardioaccelerator action of angiotensin, Am. J. Physiol. 209:539, 1965.
Effects of angiotensin
on the autonomic
nervous
system
R. D. Lowe, M.D. G. C. Scroo$, M.D. London, England
T
he physiological role and mechanism of action of angiotensin continue to excite the interest of a large number of workers. Angiotensin has at least three different effects: it causes contraction of a variety of smooth muscles, especially of vascular origin ; it increases the rate of secretion of aldosterone in a wide range of animal species; it has effects at various sites of the autonomic nervous system. The first of these actions to be described was the effect as a direct vasoconstrictor which occupied the attention of cardiovascular investigation until the late 1950’s. Since then its two other types of action have excited renewed interest among research workers but the results in the two fields of work provide an interesting contrast. The autonomic effects of angiotensin are complex and their physiological role is still obscure, whereas the effect on aldosterone secretion is well defined and appears to be of physiological importance in the control of blood volume. The first firm evidence that angiotensin could activate the autonomic nervous system in the intact animal was provided by the experiments of Renson and associates’ who in 1959 demonstrated that angiotensin could cause contraction of the nictitating membrane in cats (whose nerve supply is exclusively adrenergic) ; this action was blocked by the alpha-adrenergic blocking agent, phenoxybenzamine. Since then a From the Department
562
American
Heart
of Medicine.
Journal
St. Thomas’s
great deal of work has been done in this field, and it is therefore somewhat surprising that no clear picture has emerged as to the physiological role of these effects, i.e., what part they might play in the response to angiotensin generated in the blood stream by endogenous renin. Nevertheless, these effects are interesting and it is worth discussing them in some detail to illustrate how extremely complex the effect of angiotensin can be. The references cited are not necessarily the earliest ones, but have been selected as important ones which also give further references to much of the earlier literature. The effects which have been so far described fall into the following groups: (1) parasympathomimetic effects and potentiation of the effects of cholinergic nervous activity; (2) activation of sympathetic ganglia; (3) stimulation of catecholamine secretion from the adrenal medulla; (4) potentiation of the effects of adrenergic nerve stimulation ; (5) sympathomimetic effects due to an action on the brain; and (6) inhibition of parasympathetic nerves to the heart by an effect on the brain. In considering the possible physiological role of such effects, it is most important to bear in mind the concentration of angiotensin necessary to produce these effects and to compare this with those known to occur spontaneously in response to changes Hospital
Medical
School.
London.
S.E.I.,
England.
Afwil, 1970 Vol. 79, No. 4, pp. 562-567
Volumr Number
79 4
Effects of angiotensin
of renin secretion. The normal concentration of angiotensin in arterial blood in the dog and in man is probably in the region of 0.01 to 0.1 ng. per milliliter (i.e., 10 -11 to 10 -10 Gm. per milliliter). The concentration which occurs in response to severe circulatory stimuli (such as hemorrhage) is probably in the range 0.1 to 1 ng. per milliliter. In severe renal hypertension it may conceivably rise to much higher levels, but as a guide the “physiological” range is most likely to be 0.01 to 1 ng. per milliliter. Unfortunately, in many experiments in the intact animal the blood concentration is not measured, and only the dose is known. As a very rough guide, intravenous infusion rates of up to 100 ng. per kilogram per minute might produce concentrations within this physiological range. Effects on autonomic
nervous
system
Parasympathomimetic efects. Evidence of this action of angiotensin has come from studies of the response of intestinal smooth muscle which has a rich parasympathetic innervation by cholinergic fibers. When such tissues (e.g., guinea pig or rabbit ileum)2v3 are exposed to angiotensin in an organ bath, a contraction results which is increased by anticholinesterase and reduced by atropine or by botulinus toxin (which interferes with the synthesis and release of acetylcholine). Such results suggest that part of the effect of angiotensin on such tissues is mediated by cholinergic nerve endings in the tissue. This concept was supported by the experiments of Panisset* who studied the release of an acetylcholinelike substance into the organ bath when the tissue was stimulated via its nerve supply. In the presence of angiotensin (0.01 to 0.1 ng. per milliliter) stimulation of the nerve caused an increased release of acetylcholine. Since intestinal smooth muscle contains both pre- and postganglionic fibers as well as parasympathetic ganglia, this effect of angiotensin could be due to activation either of postganglionic cholinergic fibers or of preganglionic nerve terminals, or of the ganglia themselves. Angiotensin has not been found to potentiate the effects of cholinergic nerve stimulation in tissues where there are no ganglia, so that the most likely interpretation of these results
on autonomic nervous system
563
is that angiotensin can activate parasympathetic ganglia. Activation of sympathetic ganglia. Presumptive evidence of this effect was first provided by Lewis and Reit5 who demonstrated that intra-arterial injection of angiotensin close to the superior cervical ganglion of the cat caused contraction of the nictitating membrane, whereas similar injections to the membrane did not. The smallest effective dose was 100 ng. (by sudden injection) and the peak concentration was probably in the region of 100 ng. per milliliter. Panisset* studied the release of an acetylcholine-like substance into the effluent from the perfused superior cervical ganglion of the cat and found that angiotensin in a concentration of about 0.5 ng. per milliliter increased the amount of acetylcholine released in response to electrical stimulation of the preganglionic nerve. It is of interest that the effect of angiotensin on ganglionic transmission is not blocked by hexamethonium or pempidine5v6 (see below). Release of catecholamine from the adrenal medulla. Since the medulla is innervated by cholinergic “preganglionic” nerves and is in some respects the analogue of a ganglion, it might be expected that angiotensin would facilitate the release of catecholamines; such an effect has been described in several Feldberg and Lewis’ injected species. angiotensin into the aorta of eviscerated cats, assaying the released catecholamines by the response of the denervated nictitating membrane of the same animal; the smallest effective dose of angiotensin was 1 ng., and no effect was found after removal of the adrenal gland. Robinson8 perfused the isolated cat’s adrenal with an artificial perfusion fluid and measured the concentration of catecholamines in the venous effluent. Convincing increases of secretion were produced by injection of 0.1 ng. and some increase was seen with doses of 0.01 ng. or less; since the rate of perfusion was 0.5 to 5 ml. per minute it seems 1ikeIy that concentrations as low as 0.01 ng. per milliliter were effective. Sustained increases of catecholamine secretion in response to infusions of angiotensin in the dog were reported by Peach and associates9 who infused angiotensin
564
Lowe and &roop
intravenously in doses of 25, 50, and 100 ng. per kilogram per minute. The two higher doses caused a significant increase of catecholamine concentration in the inferior vena cava. Potentiation of the efects of adrenergic post-ganglionic nerve stimulation. The first evidence of this effect of angiotensin was produced by Benelli and co-workers’0 who demonstrated that the isolated guinea pig vas deferens (which is innervated only by adrenergic nerves) responded more strongly to stimulation of the hypogastric nerves when angiotensin was present in the organ bath, although angiotensin alone did not cause contraction. These results were not confirmed by Hughes” who stimulated the vas deferens transmurally; he suggested that the results of the Benelli group were due to the known effects of angiotensin at sympathetic ganglia which may have been present distal to the site of stimulation of the hypogastric nerve. Zimmerman and Gome+ also reported an effect which seemed likely to be due to some local interaction between sympathetic nerves and angiotensin. In the perfused paw of the dog, angiotensin infusion at 1,000 ng. per minute increased the constrictor effect of sympathetic nerve stimulation ; the concentration of angiotensin was approximately 30 ng. per milliliter. Similar effects were seen in the dog kidney with concentrations nearer 1 ng. per milliliter. It is possible that these effects were due to the known effects of angiotensin on sympathetic ganglia, because it is difficult to be certain that there were no ganglionic synapses distal to the site of stimulation of the nerve. An alternative approach to this problem is a pharmacological one, mimicking the effects of adrenergic nerve stimulation by the use of tyramine which is believed to act by releasing noradrenaline from symp’athetic nerve terminals. This drug has no known effects on ganglia, so that potentiation of the effects of tyramine by angiotensin cannot be due to facilitation of ganglionic transmission. Unfortunately, experiments with tyramine have usually involved injections or infusions into the whole animal and it is possible that it might cause release of catecholamines from
.‘lnwr.
Heart 1. .4pril, 1970
storage sites in the brain rather than from peripheral ones. In spite of these reservations it is nevertheless interesting that chronic renal hypertension13J4 or acute infusions of angiotensin at 30 ng. per kilogram per minute to the dog,15 or at 50 ng. per kilogram per minute into the rat,‘6 cause an increase in the pressor response to tyramine but not to noradrenaline. These results suggest that angiotensin may interact with factors causing release of noradrenaline at sympathetic nerve terminals to produce an increased response of the target organ. There is not yet sufficient evidence to be certain what is the nature of this interaction. Sympathomimetic effects due to an action on the brain. The first convincing experiments showing that angiotensin could produce such effects were those of Bickerton and Buckley,” who injected angiotensin into the arterial supply to the head of a dog, which was crossperfused from a donor. The only effective connection between the recipient’s head and body was via the nervous system, yet injection of angiotensin to the head caused a pressor response in the body which could be greatly reduced by an alpha-adrenergic blocking agent. The blood concentration producing these effects was probably above 100 ng. per milliliter Attempts to demonstrate this effect with more physiological concentrations have net been very successful. Infusions of angictensin into the carotid arteries of the intact animal do not apparently activate the syn.pathetic nervous system. Infusions into the vertebral artery of the rabbitl*Jg and the dogzO do have a specific pressor effect but it has not been shown that this is due to activation of the sympathetic nervous system. In the dog it is apparently due to inhibition of parasympathetic nerves to the heart, and a sympathomimetic effect can be demonstrated only in the vagotomized animal.*O Another method of demonstrating an effect of angiotensin on the brain is to inject or infuse it into the cerebrospinal fluid, but this is a very unphysiological route of administration and the concentrations required to demonstrate an effect are in the region of 100 ng. per milliliter. The resulting pressor effect seems likely to be
Volume h’umber
79 4
Effects of angiotensin
due to the sympathetic nervous system because it is reduced by the adrenergic blocking drugs, phenoxybenzamine and propranolol.21 A possible alternative explanation of this result is that adrenergic pathways in the brain were activated by angiotensin, and further evidence is required before it can be concluded that angiotensin can activate the sympathetic nervous system by an action on the brain in physiological concentrations in the intact animal. Inhibition of parasympathetic nerves to the heart due to an action on the brain. This effect can be demonstrated by infusions of angiotensin into the vertebral artery of the dog, which produces a rise of blood pressure due to an increase of cardiac output, which is unaffected by adrenergic blocking agents, but is almost abolished by atropine or vagotomy.20 Such effects can be demonstrated at local concentration of angiotensin in the region of 0.02 to 1 ng. per milliliter. Interaction of angiotensin with the sympathetic nervous system-site of action unknow. The preceding sections deal with the known sites of action of angiotensin on the autonomic nervous system, but there are a number of experiments which demonstrate a sympathomimetic action in which the site of action is unknown. It may subsequently transpire that the effects described fall into one or other of the categories already discussed, but until this has been shown they must be classified separately. The first evidence of such an action has already been mentioned, showing an effect on the nictitating membrane of cats. A similar effect on vascular smooth muscle was demonstrated by Laverty22; intravenous injection of angiotensin (doses of 1,000 ng. upward) caused a rise of resistance in the isolated perfused hind limb of the rat which communicated with the body only by the nerves. The peak concentrations of angiotensin were probably over 100 ng. per milliliter. Effects on the peripheral resistance have been demonstrated at lower concentration by McGiff and Fasyz3 and by Scroop and Whelan.24 McGiff and Fasy injected angiotensin (100 ng. per kilogram) intravenously in the anesthetized dog and recorded renal
on autonomic
nervous system
565
vasoconstriction which was abolished by guanethidine, bretylium, or renal denervation but not by the ganglion blocking agent, hexamethonium. The peak blood concentration in these experiments was probably about 3 ng. per milliliter. It is important to realize that the failure of hexamethonium to block this effect cannot be taken as evidence that the site of action of angiotensin was not on the sympathetic ganglia (see above). Scroop and Whelan infused angiotensin intravenously in man (dose 1,000 ng. per minute) and observed that the vasoconstrictor effect on the hand was greatly reduced by phenoxybenzamine or bretylium and did not occur in the sympathectomized limb or after denervation. The blood concentration of angiotensin in these experiments was probably in the region of 0.3 ng. per milliliter. Effects on the heart have been described by several workers. Farr and Grupp25 injected angiotensin (1,000 ng. per kilogram) intravenously in dogs, and noted a biphasic response of heart rate with a delayed rise above the control rate after the initial bradycardia; this delayed rise was accompanied by an increase of contractile force and was reduced by pronethalol or reserpine or by section of all nerves to the heart. It was not altered by adrenalectomy or by pentolinium or hexamethonium. Therefore, it cannot have been due to release of catecholamines from the adrenal medulla, but could have been due to an effect on ganglia (see above). The peak blood concentration was probably about 50 ng. per milliliter. Nishith and associates26 and Krasney and co-workers27 studied the effect on heart rate of rather low concentrations of angiotensin and concluded that there was activation of sympathetic nerves to the heart, but in their experiments interpretation is complicated by the fact that they stabilized arterial pressure during an angiotensin infusion and the effects on heart rate include the effects of controlled bleeding. Both groups of workers also studied the effects of angiotensin on heart rate after denervation of sinoaortic baroceptors (to prevent the reflex bradycardia in response to arterial hypertension) and they found a tachycardia. This was prevented by sympathetic blockade but not by ganglionic
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blockade or preganglionic neurectomy. These sympathomimetic effects could have been due either to a ganglionic or to a postganglionic action of angiotensin. Physiological role of effects on the autonomic nervous system. In many of the experiments described above both the absolute concentration of angiotensin and the shape of the concentration-line curve were very different from the physiological range. The question of the “physiological concentration” has already been discussed and a fairly broad range was accepted (0.0 to 1 ng. per milliliter), but it is also relevant to consider the way in which the “physiological concentration” changes with time, because endogenous changes of angiotensin are relatively slow, whereas injections produce a very sharp rise and decline. Infusions of angiotensin will mimic the effects of endogenous generation much better than will injections. Nevertheless, in spite of reservations as to the physiological relevance of some experiments, there is a sufficient body of evidence to suggest that generation of angiotensin in response to endogenous renin secretion might affect the autonomic nervous system in nearly all the ways which have been described. The fact that these autonomic effects can be expected to occur in many physiological circumstances does not enable us to draw any conclusions as to their relative importance compared to the direct vasoconstrictor effects. The net effect on arterial pressure must be the complex resultant of many conflicting mechanisms. In response to the direct vasoconstrictor effect the primary rise of blood pressure would be antagonized by baroceptor reflexes and perhaps by facilitation of transmission at parasympathetic ganglia supplying the heart. It would be enhanced by any central action of angiotensin on the brain, the adrenal medulla, the sympathetic ganglia, and the postganglionic sympathetic neurone. It is perhaps not surprising that no one has yet quantitated the relative importance of the various autonomic effects of angiotensin even in acute experiments, and the problem becomes even more complex in chronic experiments. It remains at least a possibility that the major cause of the sustained rise of arterial pressure in
chronic renal hypertension is due to the effects of angiotensin on the autonomic nervous system. REFERENCES 1. Renson, J., Barac, G., and Bacq, Z. M.: Effets de deux angiotensines synthetiques sur la pression arterielle et la membrane nictitante du chat, Comnt. rend. Sot. Biol. 153:1621. 1959. 2. Ross, 6. A., Sudden, C. T., and Stone, C. A.: Action of angiotensin on isolated guinea pig ileum, Proc. Sot. Exper. Biol. & Med. 105:558, 1960. 3. Robertson, P. A., and Rubin, D.: Stimulation of intestinal nervous elements by angiotensin, Brit. J. Pharmacol. 19:5, 1962. 4. Panisset, J. C.: Effect of angiotensin on the release of acetylcholine from pre-ganglionic and post-ganglion& nerve endings, Canad. J. Physiol. & Pharmacol. 45:313. 1967. 5. Lewis, G. P., and Reit, E.1 The action of angiotensin and bradykinin on the superior cervical ganglion of the cat, J. Physiol. 179:538, 1965. 6. Trendelenburg, U.: Observations on the ganglion stimulating action of angiotensin and bradykinin, J. Pharmacol. & Exper. Therap. 154:418, 1966. 7. Feldberg, W., and Lewis, G. P.: The action of peptides on the adrenal medulla. Release of adrenaline by bradykinin and angiotensin, J. Physiol. 171:98, 1964. of the cathe8. Robinson, R. L.: Stimulation cholamine output of the isolated perfused adrenal gland of the dog by angiotensin and bradvkinin. 1. Pharmacol. & Exaer. Therap. 156&2, 196i. 9. Peach, M. J., Cline, W. H., and Watts, D. J.: Release of adrenal catecholamines by angiotensin. II, Circulation Res. 19:571, 1966. 10. Benelli, G., Della-Bella, D., and Gandini, A.: Angiotensin and peripheral sympathetic nerve activity, Brit. J, Pharmacol. 22:211, 1964. 11. Hughes, I. E.: An investigation of the effects of angiotensin on the release of neurohumoral transmitters at the motor, adrenergic and cholineruic nerve terminals. T. Pharm. & Pharmacol. ib:116, 1969. 12. Zimmerman, B. G., and Gomez, J.: Increased response to sympathetic stimulation in the cutaneous vasculature in presence of angiotensin, Internat. J. Neuropharmacol. 4:185, 1965. 13. Verney, E. B., and Vogt, M.: An experimental investigation into hypertension of renal origin with some observations on convulsive “uraemia,” Quart. J. Exper. Physiol. !28:253, 1938. 14. McCubbin, J. W., and Page, I. H.: Renal pressor system and neurogenic control of arterial pressure, Circulation Res. 12:.553, 1963. 15. Louis, W. J., and Doyle, A. E.: The pressor response to noradrenaline and tyramine during angiotensin tachyphylaxis in the dog, Clin. SC. 31:247, 1966.
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thetic nervous system, J. Pharmacol. & Exper. Therap. 153:530, 1966. 22. Laverty, R.: A nervously mediated action of angiotensin in anaesthetised rats, J. Pharm. & Pharmacol. 15:63, 1963. 23. McGiff, J. C., and Fasy, T. M.: The relationship of the renal vascular activity of angiotensin II to the autonomic nervous system, J. Clin. Invest. 44:1911, 1965. 24. Scroop, G. C., and Whelan, R. F.: A central vasomotor action of angiotensin in man, Clin. SC. 30:79, 1966. 2.5. Farr, W. C., and Grupp, G.: Sympathetically mediated effects of angiotensin on the dog heart in situ, J. Pharmacol. & Exper. Therap. 156: 528, 1967. S. D., Davis, L. D., and Youmans, 26. Nishith, W. B.: Cardioaccelerator action of angiotensin, Am. J. Physiol. 202:237,1962. 27. Krasney, J. A., Paudler, F. T., Smith, D. C., Davis, L. D., and Youmans, W. B.: Mechanisms of cardioaccelerator action of angiotensin, Am. J. Physiol. 209:539, 1965.