Central Actions of Angiotensin and Related Hormones

55455

INITIAL STUDIES ON THE CENTRAL NERVOUS SYSTEM EFFECTS OF ANGIOTENSIN II

Joseph P. Buckley,     Cardiovascular Research Laboratories, College of Pharmacy, University of Houston and Norwich Pharmacal Company, Norwich, New York

Robert P. Halliday* and Robert K. Bickerton*,     *Norwich Pharmacal Company, Norwich, New York

Publisher Summary

This chapter presents the various effects induced by angiotensin I, II, III and renin-mediated effects via the central nervous system. It describes the initial studies, which suggest that angiotensin II produces a centrally mediated hypertensive response. It discusses the data obtained in the dog cross-circulation experiments. Angiotensin II, in sufficient dosage, is capable of stimulating structures within the central nervous system, producing an increase in peripheral blood pressure. The central hypertensive effect appears to be a result of an increase in sympathetic outflow from the central nervous system. The centrally induced pressor effects are not a result of an action of the peptide on baro- and/or chemo-receptors. The chapter also reviews that the central pressor effects of angiotensin II are not caused by hypoxia in the recipient’s central circulation as occlusion of the arterial inflow to the recipient’s head for 15 sec did not produce a pressor response, whereas the pressor response to angiotensin II occurred within this time period in an experiment described in the chapter.

This present Symposium is the result of conversations held between Drs. Ferrario, Epstein and myself during the meeting on Vasoactive Peptides and Hypertension held in Mendoza, Argentina, in August of 1974. Since there has been increasing interest in the various effects induced by angiotensin I, II, III and renin mediated via the central nervous system, the objective of this Symposium was to bring together scientists who are or who have investigated the centrally mediated effects of these peptides or renin on the cardiovascular system, dipsogenic activity, hormonal release, etc. The present report describes the initial studies undertaken at the University of Pittsburgh, which suggested that angiotensin II produced a centrally mediated hypertensive response.

Materials and Methods

Dog Cross-Circulation Studies

The dog cross-circulation preparation, modified after that described by Taylor and Page (Ref. 1) was used in these initial experiments. The recipient dog was anesthetized by an intravenous injection of 35 mg/kg of pentobarbital sodium. The internal jugular veins were doubly ligated and cut between the ligatures. The neck musculature was sectioned and removed using electrocautery to expose the vertebral column from C2 to C5. A dorsal laminectomy, which included removal of the spinous processes of the more caudad vertebrae and transverse processes of each of the two respective vertebrae was performed between C3 and C4. A length of 21-gauge stainless steel wire was inserted lengthwise through a soft rubber sponge (0.75 × 2.80 × 0.25 cm.) until the wire just protruded through the sponge. The wire and sponge were inserted under the spinal cord and the sponge positioned directly over the venous sinuses. The wire was then brought to the ventral surface, positioned in the intervertebral spaces and when tightened (using a Schiffren wire tightener) occluded the vertebral venous sinuses and vertebral arteries. The donor dog was then anesthetized with pentobarbital sodium, 35 mg/kg. Heparin, 500 U/kg, was administered intravenously to the donor and circulation established between the left common carotid artery of the anesthetized donor dog and two common carotid arteries of the recipient and the two jugular veins of the recipient and left jugular vein of the donor. Vascular isolation between the head and trunk of the recipient was determined by administering 10 µCi of ¹³¹I radioiodinated serum albumin into the arterial inflow to the recipient’s head and determining the radioactivity of the recipient’s femoral venous blood and the jugular outflow from the recipient’s head. Only those experiments in which there was no leakage between the head and trunk of the recipient were utilized in these studies. Arterial pressures were recorded from the femoral artery of each dog. In some of the experiments the carotid sinus-body areas of the recipient were bilaterally denervated by stripping the nervous tissue from the area or cutting the nerve of Hering. Synthetic angiotensin II was administered via the carotid inflow to the recipient’s head or via the femoral vein of either the recipient or the donor.

Cat Lateral Ventricle Preparation

The technique utilized in this series of experiments has been described by Bhattacharya and Feldberg (Ref. 2). Adult cats were anesthetized by an intravenous injection of alpha-chloralose, 60 mg/kg. The trachea was cannulated and the animal placed in a stereotaxic instrument and the left cerebrolateral ventricle cannulated with a 22-gauge unbeveled stainless steel needle 34 to 36 mm in length. A stainless steel screw was driven into the calvarium anterior to the cannula and both were fixed to the skull with dental acrylic. The cerebral ventricles were perfused with artificial cerebral spinal fluid maintained at 38 to 40°C. The inflow to the perfusion system was via the implanted cannula in the lateral ventricle and the outflow from the cisterna magna. The perfusion rate was maintained at 0.1 ml/min. After a stabilization period, angiotensin II (0.005-4 µg) was administered by intraventricular injection via a three-way stopcock placed into the ventricular cannula.

Results and Discussion

The administration of angiotensin II into the arterial inflow to the recipient’s head in doses varying from 0.2 to 4 µg/kg produced consistent pressor responses in both the recipient and donor animals (Ref. 3). The mean pressor response in the recipient animal was approximately 40% that obtained in the donor dog and the central hypertensive response was shorter in duration (0.5 to 5.0 min) than that obtained in the donor animal (2.0 to 5.0 min). The pressor responses to angiotensin II, 1 µg/kg, administered via the carotid inflow to the recipient’s head, could be blocked by the administration of 0.25 to 1.0 mg/kg of piperoxan, an alpha-adrenergic blocking agent, into the recipients femoral vein (Fig. 1)(Ref. 3, 4). The intravenous administration of angiotensin II into the peripheral circulation of the recipient produced marked hypertensive responses of relatively long duration (2.5 to 6.0 min) in the recipient animal only. Vasopressin, 0.1 U/kg, or 4-methyl-2-amino-pyridine, a potent pressor compound, administered into the arterial inflow to the recipient’s head in the dog cross-circulation preparations produced pressor effects in the donor animal only and did not demonstrate the central hypertensive properties of angiotensin II.

Fig. 1 Effect of Angiotensin II on donor and recipient blood pressures in the dog cross circulation preparation. (From Buckley et al., Ann. N.Y. Acad. Sci. 104, 299(1963). (Courtesy N.Y. Acad. Sci.)

Angiotensin II, 1 µg/kg, administered into the arterial inflow to the recipient’s head in which the buffer nerves arising from the carotid sinus-body areas of the recipient had been destroyed, produced consistent pressor responses in both the recipient and donor animals (Ref. 5). These results indicated that the centrally induced pressor responses of angiotensin II were not due to effects of the peptide on the baro- or chemo-receptors. Single doses of angiotensin II, 1.0 µg/kg, administered via the femoral vein of the donor, also produced pressor responses in the recipient’s trunk (Ref. 5). Infusion of angiotensin II, 0.1 µg/kg/min, via the femoral vein of the donor usually produced mild pressor responses in the recipient and marked pressor responses in the donor (Ref. 5). The data obtained in the dog cross-circulation experiments suggested the following:

1. Angiotensin II, in sufficient dosage, was capable of stimulating structures within the central nervous system, producing an increase in peripheral blood pressure.

2. The central hypertensive effect appeared to be due to an increase in sympathetic outflow from the central nervous system.

3. The centrally induced pressor effects were not due to an action of the peptide on baro- and/or chemo-receptors.

4. The central pressor effects of angiotensin II were not due to hypoxia in the recipient’s central circulation, since occlusion of the arterial inflow to the recipient’s head for 15 sec did not produce a pressor response, whereas the pressor response to angiotensin II occurred within this time period (Ref. 5).

The cat lateral ventricle preparation was utilized to further study the central actions of angiotensin II. Doses of angiotensin II ranging from 0.005 to 2.0 µg were administered via the lateral ventricles and dose-related hypertensive effects were produced (Ref. 6). The minimal effective dose producing an elevation in blood pressure was 0.01 µg, which produced a mean increase in systolic blood pressure of 19 + 5.3 mm Hg, with a duration of action of 9 + 1.6 min. (Ref. 6). The administration of 2 µg of angiotensin into the lateral ventricles of 28 anesthetized cats produced a mean rise in systolic and diastolic pressure of 45 + 4.3 mm Hg and 32 + 3.2 mm Hg, respectively, with a mean duration of action of 23 + 2.3 min. The onset of action was always less than 1 min following intraventricular injection and the pressor response was usually accompanied by a corresponding increase in heart rate and contraction of the nictitating membrane (Fig. 2). Maximal pressor effects in excess of 50 mm Hg were obtained with 4.0 µg of angiotensin II (Ref. 6). Section of the spinal cord at the C1 level essentially abolished the response to intraventricularly administered angiotensin II (Fig. 3). Additional injections of angiotensin II, administered intraventricularly 2 to 3 hours following C1 section also failed to induce pressor responses, whereas the intravenous administration of the peptide in doses of 0.4 to 0.5 µg/kg, following C1 section produced marked pressor effects. Phenoxybenzamine, 5 mg/kg, administered via the femoral vein, markedly attenuated the pressor response induced by intraventricularly administered angiotensin II; however, the degree of blockade was not as marked as that observed in the dog cross-circulation experiments (Ref. 7). These data further supported the hypothesis that the centrally induced pressor effects were due to an increase in sympathetic outflow from the central nervous system.

Fig. 2 Effects of Angiotensin II, administered into the cerebrolateral ventricle of the α-chloralose anesthetized cat. (From Smookler et al., J. Pharmacol. Exptl. Therap. 153, 485 (1966). Courtesy Williams & Wilkins Company)

Fig. 3 Effects of spinal section on the response to intraventricularly administered angiotensin II. (From Smookler et al., J. Pharmacol. Exptl. Therap. 153, 485 [1966].) (Courtesy Williams & Wilkins Company.)

Data obtained in dog cross-circulation studies indicate that angiotensin II in sufficient dosage does reach receptors within the central nervous system producing an increase in peripheral blood pressure. The centrally induced pressor effects do not appear to be due to an action of the peptide on reflexogenic receptors or due to hypoxia. Since alpha-adrenergic blocking agents administered via the femoral vein of the recipients in dog cross-circulation experiments and in cats in which angiotensin II was administered intraventricularly, blocked or markedly attenuated the centrally induced hypertension, we can conclude that these effects were due to stimulation of central sympathetic structures. Section of the spinal cord at the level of C1 abolished the central pressor effects of intraventricularly administered angiotensin in alpha-chloralose anesthetized cats, but did not block the effect of i.v. administered angiotensin. These data strengthened the hypothesis that the pressor response was of central origin and not due to escape of the compound into the peripheral vascular system or due to the release of a pressor substance from the central nervous system. The pressor effects induced by angiotensin II when administered via the cerebral lateral ventricles are not exclusive to the cat, since the peptide, in a dosage of 4 µg administered via the perfused lateral ventricles of dogs, produced a mean pressor response of 62 mm Hg, which was completely abolished by spinal cord section at the C1, level (Ref. 8).

References

1. Taylor, R.E., Page, I.H. Peripheral vasomotor effects of adrenaline and noradrenaline acting upon the isolated perfused central nervous system. Circulation. 1951; 4:564.

2. Bhattacharya, B.K., Feldberg, W. Perfusion of cerebral ventricles: Effects of drugs on outflow from the cisterna and the aqueduct. Brit. J. Pharmacol. 1958; 13:156.

3. Bickerton, R.K., Buckley, J.P. Evidence for a central mechanism in angiotensin induced hypertension. Proc. Soc. Exptl. Bio. Med. 1961; 106:834.

4. Buckley, J.P., Bickerton, R.K., Halliday, R.P., Kato, H. Central effects of peptides on the cardiovascular system. Ann. N.Y. Acad. Sci. 1963; 104:299.

5. Halliday, R.P., Buckley, J.P. Central hypertensive effects of angiotensin II. Int. J. Neuropharmacol. 1962; 1(1–3):43.

6. Smookler, H.H., Severs, W.B., Kinnard, W.J., Buckley, J.P. Centrally mediated cardiovascular effects of angiotensin II. J. Pharmacol. Expt. Ther. 1966; 153:485.

7. Severs, W.B., Daniels, A.E., Smookler, H.H., Kinnard, W.J., Buckley, J.P. Interrelationship between angiotensin II and the sympathetic nervous system. J. Pharmacol. Expt. Ther. 1966; 153:530.

8. Severs, W.B., Daniels, A.E., Buckley, J.P. On the central hypertensive effect of angiotensin II. Int. J. Neuropharmacol. 1967; 6:199.

ANGIOTENSIN RECEPTOR SITES

by

Philip A. Khairallah, M.D., Alan F. Moore, Ph.D. and Steve Gurchinoff, Ph.D.,     Research Division, Cleveland Clinic Foundation

Publisher Summary

This chapter discusses certain specialized receptor substances in tissues, with which drugs interact to produce a response. Cells are equipped with chemically reactive groups, with which drug molecules react as the first step in a biological response. It has been predicted that some sort of reciprocal relationship exists between the chemical structure of the drug molecule and the drug receptor. Angiotensinases are also present in plasma membranes, and the receptor sites and degrading sites are separate and distinguishable, although both bind peptide. The capacity to bind angiotensin and the capacity to activate biological responses are distinct processes and are dissociated. Thus, there is no direct relationship between hormone binding, receptor occupancy, and the magnitude of the response. In studying receptors, binding of a labeled hormone can be measured, which is difficult to interpret, or biological response, which may not be a direct one to one relation, can be measured. It also uses both binding and biological response data on angiotensin receptors.

The idea that there are in tissues certain specialized receptor substances with which drugs interact to produce a response, was first introduced by Langley in 1878 (1). This was further elaborated upon by the classical work of Ehrlich (2, 3) who suggested that cells were equipped with chemically reactive groups (called receptors), with which drug molecules can react as the first step in a biological response. Ehrlich postulated that some sort of reciprocal relationship exists between the chemical structure of the drug molecule and the drug receptor. Since then and especially during the past 10 years, work on receptors has increased logarithmically. In 1955, Hechter (4) proposed that hormonal responses required the concept that a single primary event, namely the binding of the hormone to a specific recognition site, initiates a sequence of steps terminating in a hormonal response. This concept was applied to peptide hormones insulin (5), ACTH (6), glucagon (7), and angiotensin (8). These have been very recently reviewed by Posner (9). A review of numerous publications dealing with receptors leads to the following working definition of an angiotensin receptor. It is a macromolecular binding site located primarily, but not always, on the plasma membrane of cells. This binding site is characterized by high affinity and high specificity to the peptide. The macromolecule may be a protein, but lipids and mucopolysaccharides are important. Angiotensinases (aminopeptidases) are also present in plasma membranes and the receptor sites and degrading sites are probably separate and distinguishable, although both bind peptide. Finally, the capacity to bind angiotensin and the capacity to activate biological responses (both being part of the more classical definition of receptors [4]) are distinct processes and can be dissociated. Thus, there is no direct relationship between hormone binding, receptor occupancy, and the magnitude of the response. However, in studying receptors, one can measure binding of a labelled hormone, which is difficult to interpret, or one can measure biological response, which may not be a direct one to one relation. In the present report on angiotensin receptors both binding and biological response data will be used.

How does one study angiotensin receptors biologically? Numerous studies can be classified into three areas; 1) studies on receptor desensitization and tachyphylaxis; 2) studies on structure-activity relations of angiotensin analogs on different tissues, and 3) studies on angiotensin antagonists.

Receptor Desensitization and Tachyphylaxis

Tachyphylaxis is the loss of response of tissues following repeated or continuous administration of a drug. Tachyphylaxis to angiotensin was first described by Page and Helmer (10), and has been shown to occur in most tissues that respond to the peptide. Several different mechanisms have been suggested to explain tachyphylaxis, increased destruction of agonist, exhaustion of a transmitter, changes in binding (increase) of agonist to its receptor, and receptor saturation. This latter mechanism although not perfect, explains most of the results observed when tachyphylaxis to angiotensin and its analogs is studied on isolated smooth muscle preparations (11, 12). Thus, tachyphylaxis to angiotensin can be defined as a saturation of angiotensin receptors with the peptide. Another molecule of angiotensin, not finding a free receptor, cannot produce a contractile response.

Carrying this definition farther, those analogs that react with the same receptor site will show cross-tachyphylaxis (13), while other agonists that react with different receptors, will still produce the expected response (14). Thus, it has been shown that the development of angiotensin tachyphylaxis cannot be blocked by alpha or beta adrenergic blocking agents, that strips tachyphylactic to angiotensin still respond to tyramine, norepinephrine, histamine and potassium, and that tachyphylaxis to angiotensin shows cross-tachyphylaxis to a number of angiotensin analogs (11, 12, 13, 14). From these studies we can conclude that there are discrete entities that can be defined as angiotensin receptor sites. They have high specificity, and are most likely present on the plasma membrane.

Structure-Activity Relation Studies

Since binding of a hormone to its receptor is thought to lead to a sequence of chemical events culminating in a response characteristic of the tissue, studies on structure-activity relationships of a series of analogs have been used to gain further knowledge of drug receptor interaction. All of these studies assume that with a series of closely related analogs, the sequence of chemical events leading to a response is the same, and thus changes in response necessarily indicate changes in binding of the hormone to its receptor. This has been done with angiotensin where over 400 structural analogs are now available. Results have been summarized recently (15, 16). Only a few results will be emphasized here. The carboxyl terminal hexapeptide, Val-Tyr-Ile-His-Pro-Phe, which is composed of alternate aliphatic and aromatic side chains carries the essential message of the hormone. A free carboxyl group on Phe is essential, as are the aromatic side groups 4-Tyr, 6-His and 8-Phe. Any changes in the above minimal requirements abolishes or greatly reduces biological activity, and changes binding characteristics (see later). Thus any postulated receptor must explain fully the necessity of these minimal structural requirements. Another result to be emphasized is the high biological activity retained by poly-O-acetyl-seryl angiotensin of around 50%. The poly-0-acetyl-serine residue is attached to the N-terminal end of angiotensin, increasing its molecular weight from around 1000 to over 28,000. If receptor sites to angiotensin coupled with smooth muscle contractile responses were any place else other than on the plasma membrane, the much larger molecule would not be able to bind, thus precluding a response.

Use of Antagonists in Studying Receptor Sites

During the study of structure-activity relations of angiotensin, a number of antagonists to the biological responses of the peptide were identified (17). Almost all of these antagonists have been classified as competitive antagonists, namely antagonists that reversibly combine with the same receptor site, with high affinity but with no intrinsic activity or efficacy. Thus, competitive antagonists must be close structural analogs, that bind to receptors without producing any biological response, and yet block the binding of other agonists. Looking at this from another point of view, when showing that competitive antagonism exists, this directly proves that both analogs react with the same specific receptor site. As a matter of fact, if an antagonist has the same pA2 value against the same agonist in two different tissues, then the likelihood is that we have the same receptor (18). Similarly, if a tissue still responds to an agonist after the addition of an antagonist, then there must be two different receptor sites (19). Again, when studying physiological responses to angiotensin, different antagonists have varying activities on separate tissues, then one has to conclude that although these tissues have angiotensin receptors, they are somewhat different. Thus, the angiotensin receptors on the adrenal medulla, mediating catecholamine release must be slightly different from those on vascular smooth muscle (20), and receptors in the adrenal zona glomerulosa cells, mediating aldosterone release must be slightly different (21).

Summarizing all the biological work, one can conclude that there is a great likelihood that angiotensin receptor sites occur on plasma membranes, that they have a high specificity for angiotensin and close analogs, and a high affinity for these peptides. However, to study these further, one has to isolate plasma membrane fragments, and study the binding of labelled hormone.

Binding of Tritiated Angiotensin

Tne specific binding of a hormone to its target cells should have the fundamental characteristics of reversibility, great affinity and high specificity (22). These parameters are used as the basis for all current research on binding of ³H angiotensin II to its receptors.

Smooth muscle.

Goodfriend and Lin (23) found specific binding of highly purified monoiodo-ang II to slices, particles and intact tissues of rat, rabbit and ox. However, monoiodo-ang II has only 80% of the full biological potency of ang II, and binding was reported to tissues such as the rat oesophagus which do not respond to angiotensin II.

Baudouin et al. (24) using ³H-ang II (with a biological potency of 100%) reported specific binding to slices of rabbit aorta. They found no specific binding in rat oesophagus. The following year (25) the same group reported binding of ³H-ang II to microsomal membranes prepared from homogenized rabbit aorta. They were able to correlate binding of ³H-ang II with a biological response – an increase in the release of membrane – incorporated calcium from rabbit aortic microsomes. Devynck et al. (26) reported the isolation of a membrane fraction derived from the plasma membrane fraction of rabbit aorta possessing specific binding sites for ang II. In 1974, Devynck et al. (27) reported an apparent solubilization of ang II receptors from the same preparation. The kinetic constants of ³H-angiotensin binding were similar to those reported in their previous studies on intact aorta and microsomal membranes. In all of these studies, specific ³H-ang II binding was reversible with angiotensin II, analog agonists and antagonists. Phe⁴-ang II and the 3-8 ang II fragment showed a higher affinity for the ang II receptor than would be expected from their contractile effect.

Further studies from this group (28) have shown a complex interaction of calcium with ³H-ang II binding. Concentrations of calcium from 10-⁶ to 10−4M increased binding in comparison with controls with zero calcium, but above 10−4M calcium binding was reduced.

Recently, Chevillotte et al. (29) observed specific binding of ³H-ang II to plasma membranes from myometrial homogenates of rat uterus. There was a significant increase in the number of receptor sites 15 hrs after bilateral nephrectomy. Long-term infusion of angiotensin II appeared to reduce the number of receptor sites.

Adrenal cortex.

Due to the known physiological importance of the action of angiotensin II on the adrenal cortex, interest in several laboratories, including our own, has centered on the binding of ³H-ang II to adrenal cortex cells.

Catt et al. (30) reported specific binding for ³H-ang II in purified homogenate of bovine and rat adrenal cortex. They found that the binding site for ang II was associated with vesicular membrane fragments and microsomal particles, and that the mitochondrial fraction displayed relatively low binding avidity for ang II. The relative potencies of angiotensin fragments and analogs in the adrenal receptor binding-inhibition system with ³H-ang II were closely related to their biological activities on smooth muscle.

Glossmann et al. (31) found an increased binding of ¹²⁵I-ang II to subcellular fractions of rat adrenal cortex in response to increasing concentrations of either K+ or Na+. In the presence of 140 mM Na+, increased uptake of ang II by adrenal receptors was associated with the appearance of a higher affinity binding site, which suggested, to the investigators, the presence of a cationic site close to the receptor site which may modify binding of ang II.

Recently, a report from our laboratory (32) demonstrated specific binding of ³H-ang II to zona glomerulosa cells from rabbit adrenal cortex. This binding was temperature dependent, and showed a complex interaction with ions. Binding of ³H-ang II increased from 0 to 40 mM Na+ then declined to the zero Na+ value at 150 mM Na+, Increasing concentrations of K+ decreased binding. Increasing concentrations of either MgCl2 or CaCl2 had no significant effect on the binding. In a further series of experiments the affinity of a series of angiotensin analogs for the receptor was examined by displacement studies, as seen in Table 1.

TABLE 1

Percent of bound tritiated angiotensin II displaced by approximately equimolar (10−8) concentrations of agonist and antagonist analogs.

Other tissues.

Sraer et al. (33) have recently reported that mono-iodinated ang II binds specifically to isolated rat glomeruli. Ang II, ang I, Ile⁸ ang II and Sar¹, Ile8 ang II all inhibited ¹²⁵I-ang II binding. They observed an excellent correlation between binding of ¹²⁵I-ang II and decrease of mean glomerular diameter.

In conclusion, one can summarize the results of binding studies as follows: 1) ³H-ang II appears to bind to cell membranes, both in smooth muscle and adrenal cortex; 2) there is a good, but not totally consistent, correlation between kinetic parameters of ³H-ang II binding and biological response; 3) the use of angiotensin fragments and analogs is a useful adjunct to kinetic data; 4) until more conclusive evidence exists, angiotensin binding sites should be referred to by the general term of acceptors rather than receptors.

In a symposium dealing with the central actions of angiotensin, one can ask whether there are receptor sites to angiotensin in specific areas of the brain. Many of the presentations to follow will study the biological actions of angiotensin on CNS function. Most of these can be interpreted as due to angiotensin and its analogs binding to specific receptor sites on specific neuronal cell membranes in specific regions of the brain. Very preliminary work from our laboratories have attempted to study binding of tritiated angiotensin to synaptosomes prepared from rat brain.

Synaptosomes

Homogenization of brain tissue in iso-osmotic sucrose under conditions of moderate shear force results in the formation of synaptosomes. These are central presynaptic nerve endings which have been torn away from their axons, and have sealed to form detached particles (34, 35, 36, 37). Due to the known interactions of angiotensin II with both cholinergic and adrenergic systems in the CNS, a study was undertaken to examine possible binding sites on brain synaptosomes.

Brains were removed from female rats (Sprague Dawley, 200-300 g), rapidly cooled in ice-cold 0.32 M sucrose (10:1 v/w), homogenized at 840 rpm in a smooth walled glass homogenizer, and centrifuged at 1000 g for 10 min. The supernatant was either diluted with Krebs solution and used in crude homogenate studies, or placed on a sucrose density gradient (0.8 M/1.2 M sucrose-equivolumes) and spun at 60,000 g for 90 min. The fraction collected at the 0.8 M/1.2 M interphase was collected, diluted with Tris buffer at pH 7.4 and used in the synaptosomal experiments.

Brain homogenates prepared as above were diluted 4:1 (v/v) with Ca²+-free Krebs solution containing 0.05% BSA and 2.5 mM Cleland’s reagent. ³H-angiotensin (10−8m) was added either with or without 10−5M cold ang II and 100 µl aliquots were withdrawn at the times shown in Fig. 1, filtered in a millipore filter apparatus, the filters dried, scintillation fluid added, and the samples counted. Our preliminary experiments indicate that crude homogenate of rat brain exhibits specific binding to ³H-ang II. A typical experiment is shown in Fig. 1, where it can be seen that at 22°C the homogenate exhibits specific binding which reaches equilibration between 10-20 mins.

Fig. 1 Binding of ³H-Ang II (10−8M) to rat brain homogenate

Experiments were performed to determine the active binding fraction. The three fractions obtained on differential centrifugation: myelin, synaptosomal fraction and mitochondrial fraction, were examined for binding activity. Specific, reversible binding of ³H-ang II was only observed in the synaptosomal fraction. A typical example is shown in Fig. 2, where a 100x excess of cold ang II induced a significant decrease in the amount of bound ³H-ang II (10-⁸M). It appears from our preliminary experiments that rat brains exhibit specific binding sites for angiotensin II, and that these sites are localized at the synaptosomal level.

Fig. 2 Binding of ³H-Ang II (10−8M) to rat brain synaptosomes

In conclusion, a good case can be made for angiotensin receptors on plasma membranes. Although some actions of this peptide may be mediated by other mechanisms, many responses follow binding of the octapeptide to specific sites.

Acknowledgements

This project was supported in part by NIH grants HL-6835 and HL-5126.

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ANGIOTENSIN ANTAGONISTS

by

F. Merlin Bumpus, Ph.D.,     Research Division, The Cleveland Clinic Foundation

Publisher Summary

The chapter presents an evidence for a direct action of angiotensin on ion transport, especially an effect on sodium and calcium. Various actions for angiotensin have been known and properties are found for this group of peptides. In addition to the ability to stimulate smooth muscle and to increase blood pressure, angiotensin has been reported to cause coronary vasoconstriction, reduce mesenteric blood flow, release catecholamines from the adrenal medulla, increase synthesis and release of aldosterone from adrenal cortex, stimulate central nervous system and affect the drinking phenomenon, increase sodium efflux from uterine and arterial strips, stimulate release of vasopressin, inhibit uptake of catecholamines by nerve endings, and to stimulate protein synthesis and possibly cardiac hypertrophy. The various stimuli are mediated by angiotensin. Evidence is lacking for the involvement of cyclic AMP except in situations where angiotensin stimulates the synthesis and release of a hormone, which is directly responsible for activation of adenyl cyclase.

Early studies relating structure of vasoactive peptide hormones to their biologic activity were limited to ability of these peptides to increase or reduce blood pressure or contract isolated smooth muscle preparations. Various actions for angiotensin have been known for several decades and in recent years additional properties have been found for this group of peptides. In addition to its ability to stimulate smooth muscle and to increase blood pressure, angiotensin has been reported to cause coronary vasoconstriction, reduce mesenteric blood flow, release catecholamines from the adrenal medulla, increase synthesis and release of aldosterone from adrenal cortex, stimulate central nervous system and affect the drinking phenomenon, increase sodium efflux from uterine and arterial strips, stimulate release of vasopressin, inhibit uptake of catecholamines by nerve endings and to stimulate protein synthesis and possibly cardiac hypertrophy.

For the most part, it is still unknown how the various stimuli are mediated by angiotensin. Evidence is lacking for the involvement of cyclic AMP except in situations where angiotensin stimulates the synthesis and release of a hormone which is directly responsible for activation of adenyl cyclase. Some evidence for a direct action of angiotensin on ion transport, especially an effect on sodium and calcium, has been given. More recently, involvement of the prostaglandin system has been demonstrated utilizing inhibitors of the biosynthetic pathway for this latter hormone.

In the late 1960s, observations by Peach, Khairallah and Bumpus (Ref. 1) led to the speculation that the angiotensin receptor mechanism was not identical in all organs. We first showed that the side group in position eight was unique. This became evident when we determined that removal of the aromatic side group in this position did not greatly reduce the potency of angiotensin in analogs to release catecholamines from the adrenal medulla nor its ability to inhibit uptake of these amines by nerve endings but almost completely abolished its response on contraction of smooth muscle. This led us to our original observation that modifications of the side group in position eight produced angiotensin antagonists (Ref. 2). Single substitutions of side groups in positions one through seven have not produced antagonists.

Since 1970 numerous eight substituted angiotensin analogs have been synthesized and tested for agonist and antagonist properties on various preparations (Ref. 3). Using aliphatic substitutions and testing for angiotensin II inhibition on smooth muscle, we have concluded that the angiotensin inhibition potency increases as the side chain increases in size and branching as follows:

Order of potencies of angiotensin antagonists for several eight position substitutions.

A small residual agonist property persists and also possibly increases with size and branching. [Ala⁸] ang II and [Ile⁸] ang II have been tested for their ability to release catecholamines and it was noted that [Ala⁸] peptide is the more potent agonist.

Substitution of cyclic or aromatic rings for the phenyl ring in position eight produced compounds with moderate agonist potency and high antagonist properties (Ref. 4). Dose response curves with these inhibitors suggest a non-competitive type of inhibition.

Introduction of sarcosine in position one of eight substituted angiotensin antagonists increased their inhibition potencies. Indeed, [Sar¹] ang II as well as [N-Me-β-AspNH2] ang II are all more potent agonists than the parent hormone (Ref. 5). We determined that the N-methylated, one substituted derivatives of angiotensin and analogs were more stable toward the proteolytic enzymes and also bound more strongly to receptor (Ref. 5).

Substitution of the more hydrophilic side group, threonine, into position eight has yielded an analog with a high inhibition index and with very low agonist property. It was surprising to us to observe that this peptide at levels tested show no catecholamine release when perfused through the adrenal medulla (Ref. 6).

Detailed comparisons have been made for [Sar¹, Thr⁸], [Sar¹, Ile⁸] and [Sar¹, Ala⁸] analogs. Angiotensin II inhibition on blood pressure increases in rats, if expressed as dose ratios and indicates that [Sar¹, Thr⁸] derivative has considerably more inhibitory activity than either of the other two compounds. All three peptides possess slight pressor activity and when tested in rats at 3, 10 and 30 minutes, the threonine derivative is considerably less agonistic than are the other two (Ref. 7). When these peptides were perfused in a retrograde manner through isolated cat adrenals [Sar¹, Ala⁸] ang II exhibited a rather high release of catecholamines. [Sar¹, Ile⁸] ang II is much less potent as an agonist, while the corresponding [Sar¹, Thr⁸] derivative did not release catecholamines even at 10−6 grams/ml infused (Ref. 6).

Insertion of 0-methylthreonine in position eight of [Sar¹] ang II increases in the inhibitory potency (in vivo) several fold over that of the threonine derivative but gives rise again to the catecholamine releasing