John A. Ackerly

Differentiation of neurogenic and myocardial angiotensin II receptors in isolated rabbit atria.

The effect of angiotensin on the action of tyramine was studied in isolated rabbit left atria paced by point and field stimulation to more clearly define the interaction of angiotensin with the sympathetic nervous system. Administration of angiotensin resulted in similar increases in contractility in both point- and field-stimulated atria. In point-stimulated preparations only the muscle is stimulated to contract, whereas in field-stimulated preparations both nerve and muscle are stimulated. l-Sar-8-Ala-angiotensin II completely blocked the direct inotropic effect of angiotensin in a molar dose ratio of 3:1 in both point- and field-stimulated preparations. However, angiotensin (0.05–10 ng/ml) potentiated the inotropic effect of tyramine in field-stimulated atria only. This facilitatory effect was not inhibited by l-Sar-8-Ala-angiotensin II at a molar dose ratio of 3:1; indeed, a ratio of 500:1 was necessary for complete blockade of this angiotensin-induced potentiation. This antagonist in doses of 0.1–1000 ng/ml was without contractile effect in any preparation, regardless of whether tyramine was present. The data suggest the presence of (1) a presynaptic angiotensin receptor that, in the presence of sympathetic nerve stimulation, modulates the release of norepinephrine and (2) a second angiotensin receptor in cardiac tissue that directly influences myocardial contractility.

The Renin–Angiotensin System during Controlled Hypotension with Sodium Nitroprusside

The role of the rennin–angiotensin system in blood pressure homeostasis during sodium nitroprusside (SNP) infusion in the rat was evaluated. Rats received infusions of SNP, 40 µg/kg/min, for one hour, and blood samples for renin determinations were drawn from the arterial cannula before and after infusion. Renin activity was measured by radioimmunoassay. At the termination of the SNP infusion, plasma renin activity had increased from 3.23 ± 1.53 to 13.25 ± 0.76 ng/ml/hr (P < 0.05). Control animals that received only vehicle showed no change in renin activity. When rats that had received SNP at 40 µg/kg/min for one hour were treated with a competitive inhibitor of angiotensin II (saralasin), there was a 7-torr decrease in blood pressure (P < 0.01). Control animals showed no hypotensive response to saralasin. The hypotensive response to the administration of SNP at 40 µg/kg/min in acutely nephrectomized rats exceeded that obtained in normal rats by 30 torr (P < 0.01). Enflurane anesthesia did not modify the renin response to SNP-induced hypotension. In conscious rats, larger doses of SNP (80 or 160 µg/kg/min) resulted in elevations of renin activity to 22.50 ± 0.90 and 19.84 ± 1.94 ng/ml/hr, respectively. When saralasin was infused into rats receiving 160 µg/kg/min, blood pressure decreased precipitously and the rats died. SNP-induced hypotension stimulates renin release and the subsequent production of angiotensin II helps maintain blood pressure.

The renin response to diuretic therapyl A limitation of antihypertensive potential.

We attempted to determine whether a diuretic-induced increase in renin secretion results in angiotensin II-mediated vasoconstriction which counteracts the antihypertensive action of diuretics. The angiotensui antagonist saralasin was administered to nine normal renin and five low renin essential hypertensives prior to and during stimulation of the renin-angiotendn system by 6 weeks of chronic diuretic therapy alone. Initial responses to saralasin infusion in all subjects on placebo varied, but were pressor overall. Following chronic polythiazide therapy, saralasin induced depressor responses of variable magnitude in seven of nine normal renin subjects. The combination of the diuretic and saralasin normalized blood pressure in four subjects. However, four normal renin subjects maintained a diastolic Mood pressure greater than 95 mm Hg despite simultaneous volume depletion and angiotensin blockade. One had a maximum antihypertensive effect with diuretic therapy alone. Pressor responses to saralasin persisted in four of five low renin subjects despite diuretic therapy. In general, the magnitude of the change hi plasma renin activity induced by diuretic therapy correlated with the difference in Mood pressure responses to saralasin. However, individual Mood pressure responses and absolute renin levels after diuretic therapy failed to predict consistently the ensuing responses to saralasin. Hence, in eight subjects (seven normal and one low renin), the compensatory increase in renin secretion hi response to diuretic-induced volume depletion limited diuretic antihypertensive efficacy. Yet diuretic-induced angiotensin dependency was not the sole mechanism supporting residual hypertension hi all subjects refractory to diuretic therapy.

Blood Pressure Support during General Anesthesia in a Renin-dependent State in the Rat

Previous work had shown that halothanc and enflurane at 1 MAC and ketamine, 125 mg/kg, did not increase plasma rcnin activity (PRA) in the normal sodium-replete rat. To investigate the renin-angiotensin system with increased PRA, 25 rats were fed a low-sodium diet for five to seven days and divided into four groups: awake; halothane, 1.26 vol per cent; enflurane, 1.75 vol per cent; ketaminc, 125 mg/kg, intramuscularly. The protocol consisted of a two-hour awake period, then an hour of stable anesthesia, followed by 30 min infusion of saralasin, an angiotensin II competitive inhibitor. An additional 18 rats had PRA measured by radioimmunoassay before and after an hour of stable anesthesia. Stable anesthesia decreased mean arterial pressure from 122 ± 2 to 69 ± 4 torr for the halothane group, 70 ± 3 torr for the enflurane group, and 103 ± 7 torr for the ketamine group. When saralasin was infused for 30 min, blood pressure decreased to 100 ± 3 torr for the awake groiip, 40 ± 1 torr for the halothane group, 44 ± 2 torr for the enflurane group, and 73 ± 3 torr for the ketamine group. PRA increased from 4.3 ± 0.5 ng/ml/hr for sodium-replete rats to 12.9 ± 1.7 ng/ml/hr for sodium- depleted rats. After an hour of stable anesthesia, PRA increased in all the anesthetized groups. The authors conclude that the anesthetic agents studied increase renin release in the sodium-depleted rat. The initial renin level may be important in. determining whether changes in rcnin release occur with anesthetic agents.

Des-Asp1-angiotensin I: a metabolite of angiotensin I in the perfused feline adrenal.

The administration of radioactive angiotensin I to the retrogradely perfused feline adrenal gland caused a brisk discharge of catecholamines. Recovery of the labelled decapeptide and metabolites in the adrenal effluent fluid was complete in 5 min. Radioimmunoassay of this perfusate revealed that most of the peptide remained as angiotensin I, but chromatographic and electrophoretic evaluation indicated that greater than 68% of the peptide had been metabolized to des-asp1 -angiotensin I. The absence of des-asp1 -angiotensin II, angiotensin II or his-3H-leu in adrenal effluent fluid suggested minimal dipeptidyl carboxypeptidase activity in this preparation. In addition, the profile of angiotensin I metabolites from the perfused adrenal was not altered by treatment with a converting enzyme inhibitor B. jararaca nonapeptide. The des-asp1-angiotensin I peptide was a very weak secretagogue in the adrenal medulla. If metabolism of the decapeptide to the nonapeptide occurs in the medulla, this may represent a pathway to limit the secretory action of angiotensin I. These results suggest a high degree of adrenal aminopeptidase activity which may be primarily localized in the adrenal cortex.

Peptide antagonists of angiotensin-induced adrenal catecholamine release.

The effect of analogs of angiotensin (modified with an Ile-substituted for Phe) was studied in the isolated, retrogradely perfused adrenal of the cat. Continuous differential analysis of norepinephrine and epinephrine output was quantified with an automated trihydroxyindole procedure. [Ile8]-angiotensin I and [Ile7]-angiotensin III exhibited negligible secretory activity, in contrast to the stimulatory effects of [Ile8]-angiotensin II (10-20% activity relative to angiotensin II). [Ile8]-angiotensin I blocked angiotensin II-induced catecholamine secretion and a pA2 value of 8.50 was obtained. [Ile7]-angiotensin III was an especially potent antagonist of angiotensin II and a pA2 value of 10.4 was calculated for this heptapeptide analog. The pA2 value for [Ile8]-angiotensin II, a partial agonist in the adrenal medulla was 9.33. These three analogs were equally effective against secretion induced by the corresponding unsubstituted homologs (Ang I and Ang III). These data suggest that all these angiotensin peptides interact with a common receptor. [Ile8]-angiotensin I and [Ile7]-angiotensin III had no effect on adrenal medullary responses induced by KCl, nicotine and bradykinin. These structural analogs of angiotensin are pure competitive antagonists of angiotensin in the cat adrenal chromaffin cell.

Angiotensin interactions with myocardial sympathetic neurons: enhanced release of dopamine-beta-hydroxylase during nerve stimulation.

In summary, the present study is further evidence for an interaction of angiotensin with adrenergic neurons in the myocardium. Concentrations of the peptides which do not display inotropic activity in point-stimulated atria result in marked facilitation of the release of neurotransmitter and DβH from adrenergic neurons in field-stimulated atrial preparations. The neuronal receptor for angiotensin is relatively resistant to blockade with an angiotensin antagonist, saralasin.

Inotropic cardiac and vascular actions of [Ala7]angiotensin analogs.

Summary Angiotensins I, II, and III are potent cardiac inotropic agents, but vasoconstrictor and steroidogenic activities compromise their use in conditions of cardiac dysfunction. This study examines the inotropic and vascular actions of [alanyl7]-substituted angiotensin analogs in field-stimulated rabbit left atria and isometrically contracting aortic strips. Angiotensin II increased cardiac contractility in a dose-dependent manner with an ED50 of 30 nm. Sarcosine substitution at the amino terminus of angiotensin II increased its potency 10-fold. Inhibition of converting enzyme with teprotide (10 μg/ml) had no effect on contractile responses to angiotensin II or [Sar1]angiotensin II. [Ala7]Angioten-sin II was a weaker cardiac stimulant than angiotensin II, but addition of teprotide enhanced its potency to that of angiotensin II. [Sar1, Ala7] Angiotensin II also was equipotent to [Sar1]angiotensin II in the presence of teprotide. The potency of angiotensin I as an inotropic agent (ED50 = 100 nm) was significantly less than that of angiotensin II, and the angiotensin I response was attenuated by teprotide. [Sar1, Ala7]Angiotensin I was a more effective inotropic agent following teprotide administration. These findings indicate that atrial converting enzyme metabolizes angiotensin I and the [Ala7] analogs of angiotensin I and II. Angiotensin II and [Sar1]angiotensin II had activities in aorta similar to those in atria. As previously reported for uterus, [Ala7]Angiotensin II was inactive in aorta whether teprotide was present or not. The decapeptides were equally potent in the aorta and atria. Sarcosine substitution at the NH2 terminus also was observed to attenuate cardiac selectivity. These data indicate that [Ala7]angiotensin analogs are cardioselective inotropic agents.

Converting-enzyme Activity and Pressor Responses to Angiotensin I and II in the Rat Awake and during Anesthesia

Plasma renin activity (rate of angiotensin I generation) does not increase during anesthesia with ketamine, fluroxene, halothane or enflurane in the sodium-repleted rat. However, blood pressure decreases when an angiotensin II antagonist, saralasin, is administered during halothane or enflurane anesthesia, but not during ketamine or fluroxene anesthesia. Differences in the rates of conversion of angiotensin I to angiotensin II induced by various anesthetic agents could help explain these previous findings. To determine the effects of anesthetic agents on angiotensin I conversion, experiments were performed in vitro and in vivo. The activities of rabbit pulmonary converting enzyme in the presence and absence of halothane or fluroxene were measured as rates of appearance of the dipeptide, histidyl-leucine, a product of angiotensin I hydrolysis to angiotensin II. Halothane and fluroxene did not alter conversion. Infusions of angiotensin I and angiotensin II were given to Wistar rats to construct dose-blood pressure response curves. The animals were then anesthetized with ketamine or halothane and infusions were repeated. Angiotensin I and angiotensin II induced similar blood pressure responses in awake and anesthetized rats. However, ketamine accentuated the pressor responses to angiotensin I and angiotensin II, whereas halothane depressed the responses. With the anesthetic agents studied, there is no significant effect on conversion of angiotensin I to angiotensin II either in vitro or in vivo.