Apparatus for solid-phase peptide synthesis.

The apparatus, designed for solid-phase peptide synthesis, consists of a round-bottom flask, rocked on a wrist-shaker, and fitted with a special dropping funnel and a fritted filter disc embedded within the flask. The dropping funnel is designed to wash down the polymer adhering to the neck of the flask. Solvents are removed through the fritted disc. Entire synthesis, and the removal of peptide from the polymer, are carried out without opening or removing the vessel from the shaker.

Efficacy of octa- and heptapeptide antagonists of angiotensin II as inhibitors of angiotensin III binding in the rat adrenal glomerulosa.

Angiotensin III (Ang III) is a carboxy-terminal 7-amino acid analog of angiotensin II (Ang II) with similar receptor binding affinity and biological activity in adrenal glomerulosa. Specific competitive antagonists have been synthesized for both compounds, and structure-activity studies have demonstrated that Ang II (octapeptide) antagonists compete better for Ang II receptors in adrenal glomerulosa than do Ang III (heptapeptide) antagonists. These differences were observed in spite of only 1 amino acid difference in chain length of antagonist analogs. These earlier observations by our group provided support for the current hypothesis that Ang III binding would be preferentially inhibited by Ang III antagonists compared to Ang II antagonists. To accomplish these studies, we used [125I]Ang III and [125I]Ang II as ligands and 5 pairs of heptapeptide and octapeptide antagonists with identical substituent amino acids in the carboxy-terminal position. [Sar1,Ile8]- and des Asp1 [Ile8]Ang II did not differ in potency as antagonists of Ang II binding, but with 4 other pairs of antagonists, the octapeptide antagonists were more potent than the corresponding hepatapeptide antagonist. Six of 10 antagonists exhibited similar potencies as antagonists of equimolar concentrations of Ang II and Ang III. One heptapeptide antagonist was twice as potent against Ang III, and 3 octapeptide antagonists were more potent against Ang II. In general, the order of potencies of the 10 antagonists as inhibitors of Ang III binding was linearly related to their potencies against Ang II. Hence, our hypothesis of preferential activity of Ang III antagonists (compared to Ang II antagonists) as inhibitors of Ang III binding to adrenal glomerulosa was not borne out by the present studies. When this observation was combined with the finding of similar receptor densities of Ang III and Ang II receptors, we concluded that Ang III and Ang II probably bind to the same receptor site in the adrenal glomerulosa.

Comparative studies of receptor binding and steroidogenic properties of angiotensins in the rat adrenal glomerulosa.

Angiotensin and analogs were tested in isolated rat adrenal zona glomerulosa cells to find if there was a correlation between receptor affinity and steroidogenic potency. Comparative receptor-binding affinities and corresponding aldosterone-releasing effects obtained for each analog were: [Asp1, Ile5]angiotensin II, 1.0 and 1.0: [Asp1, Val5]angiotensin II, 0.69 and 1.65; [Asn1, Val5]angiotensin II, 1.18 and 0.68; [Sar1]angiotensin II, 2.07 and 1.7; [Me2Gly1]angiotensin II, 0.63 and 0.72; [Ile5]angiotensin III, 0.72 and 0.59; [Val5]angiotensin III, 0.92 and 0.34; [Ile5]angiotensin I, 0.007 and 0.051; des-Asp1-[Ile5]-angiotensin I, 0.004 and 0.03; and [Val5, Ser9]angiotensin I, 0.03 and 0.098. Taken as a group, these agonist analogs demonstrated good correlations between these two variables (r = 0.76; P less than 0.001). There was parallelism between binding inhibition and aldosterone-releasing effect when position 5 was substituted with isoleucine, regardless of the substituent in position 1 of the angiotensins. This parallelism was lost when analogs of angiotensin II or III contained valine in position 5. In addition, angiotensin III was found to be less potent than angiotensin II, regardless of the substituent in position 5 (valine or isoleucine).

Intrarenal effects of [Ala-Pro-Gly-(Ile3-Val5)] angiotensin II in the conscious dog.

Amphibian skin has functional characteristics of epithelium in the mammalian distal nephron and plays an important role in sodium and water metabolism in these animals. A peptide extracted from the skin of the Australian frog Crinia georgiana has been purified, has been determined to have the amino acid sequence of [Ala-Pro-Gly-(Ile3-Val5)]angiotensin II, and recently has been synthesized. We studied the renal effect of synthetic frog skin angiotensin II (FSAII) infused via the renal artery in doses that were confined to the kidney. FSAII was infused intrarenally at 0.2, 2, and 4 pmol/kg.min in uninephrectomized conscious dogs (n = 5) in metabolic balance at a sodium intake of 80 meq/day. FSAII was confined to the kidney, as demonstrated by the absence of any systemic pressor response and/or any increase in plasma aldosterone concentrations during intrarenal FSAII infusion at rats of 0.2 and 2 pmol/kg.min. At 4 pmol/kg.min, FSAII traversed the kidney in amounts sufficient to stimulate aldosterone secretion (P less than 0.05). All three doses of FSAII caused significant antidiuresis and antinatriuresis, and decreased fractional excretion of sodium. There were no changes in the glomerular filtration rate (GFR) or renal plasma flow (RPF) during FSAII infusion at 0.2 and 2 pmol/kg.min. At 4 pmol/kg.min, FSAII engendered a significant decrease in GFR and RPF, while the filtration fraction increased. There were no significant changes in arterial blood pressure at any dose of FSAII. When confined to the kidney, FSAII caused antidiuresis and anti-natriuresis in the absence of a change in GFR and RPF. These results provide evidence that angiotensin acts directly at the renal tubular level to alter renal function.

Action of (1-des(aspartic acid), 8-isoleucine) angiotensin II upon the pressor and steroidogenic activity of angiotensin II.

The vascular and steroidogenic responses to (1-Sarcosine, 8-Isoleucine)-, and to (1-Des (Aspartic acid), 8-Isoleucine) angiotensin II were compared in bilaterally nephrectomized, ACTH-suppressed dogs receiving constant infusions of angiotensin II. Aldosterone secretion rate was significantly inhibited by pretreatment with 200 ng/Kg/min of the heptapeptide, (1-Des(Aspartic acid), 8-Isoleucine) ang II, but not by similar doses of the octapeptide, (1-Sarcosine, 8-Isoleucine) ang II. In contrast, the pressor action of ang II was unaffected by (1-Des (Aspartic acid), 8-Isoleucine) ang II though significant inhibition occurred with relatively small doses of (1-Sarcosine, 8-Isoleucine) ang II. This study suggests that: (a) angiotensin receptors in adrenal cortex and vascular smooth muscle are functionally different, and (b) (1-Des(Aspartic acid),8-Isoleucine) angiotensin II is a specific antagonist of steroidogenic effect of ang II.

Role of nitric oxide and angiotensin II in the regulation of sympathetic nerve activity in spontaneously hypertensive rats.

This study evaluated the actions of nitric oxide on the blood pressure and renal sympathetic nerve activity responses produced by angiotensin II (Ang II) blockade in conscious spontaneously hypertensive rats. Two days after implantation of electrodes, we measured mean arterial pressure, heart rate, and renal sympathetic nerve activity. Baroreceptor reflex function was assessed with a logistic function curve; the maximum slope of the curve estimated the baroreceptor reflex gain. Data were obtained in rats given acute intravenous administration of either vehicle, the Ang II type 1 receptor antagonist losartan, the type 2 antagonist CGP 42112A, or the converting enzyme inhibitor lisinopril. In comparison with vehicle (-1.1 +/- 0.2%/mm Hg), both losartan (-1.8 +/- 0.3%/mm Hg) and lisinopril (-2.4 +/- 0.2%/mm Hg) significantly increased the maximum gain of the baroreceptor reflex control of nerve activity (p < 0.05). In contrast, the type 2 receptor antagonist did not alter baroreceptor reflex function. Similar studies were performed in rats that received an intravenous injection of NG-monomethyl L-arginine (10 mg/kg). The nitric oxide synthase inhibitor increased baseline blood pressure and decreased renal sympathetic nerve activity. Subsequent administration of losartan or lisinopril returned blood pressure to initial hypertensive level, whereas sympathetic nerve activity was increased to a level above the initial control value. The maximum gain of the baroreceptor reflex control of renal nerve activity was increased after the nitric oxide inhibition. The present study demonstrates that blunted baroreceptor reflex function in conscious spontaneously hypertensive rats is mediated by an Ang II type 1 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of frog-skin angiotensin II in amphibians.

The role of frog-skin angiotensin II (AII) in amphibia was studied by comparing the sodium and water permeability effects of three angiotensins (AII): frog skin (Ala-Pro-Gly-[Ile3, Val5]-Ang II), human [( Asp1, Ile5]-AII), and Japanese goosefish [( Asn1-Val5]-AII). Frog-skin AII increased the short-circuit current (SCC) significantly after it was added to the dermal side of the isolated skin of the South American frogs, Leptodactylus chaquensis and ocellatus, and the toad, Bufo arenarum, in concentrations of 10(-6) M. In frogs, the effect was significant at 15 minutes and reached 45% over control after 2 1/2 hours. The effect cannot be achieved with concentrations lower than 10(-7) M. Since amiloride (10(-4) M) blocked the SCC response, and absence of chloride in the bathing fluid did not, the effect is probably dependent on sodium transport. Human AII (10(-6) M) produced a similar response in summer frogs that had been treated with 0.1% NaCl for 14 days. Goosefish AII was ineffective at similar concentrations, and none of the angiotensins modified SCC in the toad bladder. Hydrosmotic effects could be achieved with the three angiotensins, the response being dependent on seasonal and species factors but always considerably lower than that of the neurohypophyseal peptides. Vascular reactivity of the isolated frog hindlimbs was compared by dose-response curves. Potency ratios on a molar basis against frog-skin AII was 1.136 for human AII and 1.193 for goosefish AII. The results show that the effects of the angiotensins differ in both the response of SCC to frog-skin angiotensin and its higher vascular effects.

Angiotensin analogues that selectively augment the force of contraction of the isolated heart.

Angiotensin II (Ang II) produces a positive inotropic effect on the heart; however, its usefulness as an inotropic agent is limited because of its inherent vasoconstrictor action. We therefore designed Ang II analogues that are potent, positive inotropic agents with minimal myotropic properties. Replacement of the proline residue in position 7 with alanine reduced the pressor and vascular contractile response to less than 1% of Ang II. In spite of negligible vascular actions, however, [7-alanine]Ang II produced 50% of the inotropic activity of Ang II in the cat papillary muscle. The results of pharmacological evaluation of various position 7-substituted analogues were as follows: 1) Replacement of proline in position 7 of angiotensin I (Ang I) and Ang II with primary amino acids produced cardiac-specific, positive inotropic properties. 2) The selectivity of positive cardiac inotropic activity of position 7-substituted analogues of Ang II was dependent upon the nature of the amino acid in position 1. Replacement of aspartic acid in position 1 with sarcosine increased vasoconstrictor activity, thereby diminishing cardiac selectivity. However, this change did not affect cardiac selectivity in Ang I analogues. 3) Introduction of any type of steric hindrance in position 7 (e.g., replacement of alanine with N-methyl- or alpha-methylalanine) led to a considerable loss in inotropic activity. In conclusion, contrary to rigid, structural requirements (solution conformation) for the pressor action of Ang II, a less organized structure or a random conformation at the carboxyl terminus appears to favor cardiac-selective contractile response (or positive inotropic response).

Neurophysiological responses to angiotensin-(1-7).

The aim of this study was to investigate the action of the heptapeptide angiotensin-(1-7) on the spontaneous activity of paraventricular neurons using microiontophoresis. Recent immunocytochemical investigations have shown that this product of angiotensin I is predominantly located in cells and fibers of the forebrain and brain stem. Our results show that most neurons in the paraventricular nucleus are excited by angiotensin-(1-7) at a dose of 50-80 nA. In comparison with angiotensin II or angiotensin III, the onset of response and the occurrence of the maximal effect were significantly delayed. With higher doses of angiotensin-(1-7), there was a decrease in latency and a dose-dependent increase in firing frequency. Of all the angiotensin compounds tested, angiotensin III was the most potent. Preliminary results obtained with an angiotensin antagonist show that the action of angiotensin II, angiotensin III, and angiotensin-(1-7) is blocked by the angiotensin receptor subtype 2 antagonist CGP 42112A. Because the angiotensin-(1-7) system in the brain is associated with central vasopressinergic pathways, vasopressin was tested in a similar way. Neurons in the paraventricular nucleus that were excited by iontophoretically applied angiotensins showed a weak response to vasopressin. Occasionally, a small excitatory action was observed. Our results support the hypothesis that the heptapeptide angiotensin-(1-7) is a biologically active neuropeptide. The data also suggest that amino terminal fragments of angiotensin II are not inactive degradation products.

Subtype 2 angiotensin receptors mediate prostaglandin synthesis in human astrocytes.

We have identified two distinct cellular responses that occur in human astrocytes in the presence of angiotensin (Ang) peptides and are linked to specific receptor subtypes. Ang II and the N-terminal heptapeptide Ang-(1-7) stimulated release of prostaglandin (PG) E2 and PGI2 (measured as the stable metabolite 6-keto-PGF1 alpha). In contrast, only Ang II but not Ang-(1-7) activated phosphoinositide-specific phospholipase C, leading to mobilization of intracellular calcium. The Ang II-induced PGE2 and PGI2 syntheses were attenuated by [Sar1,Ile8]Ang II but not by [Sar1,Thr8]Ang II. Ang-(1-7)-induced PGE2 and PGI2 syntheses were not inhibited by either of these two classical antagonists. DuP 753, a subtype 1-selective Ang receptor antagonist, blocked the Ang II-induced release of PGE2 but not PGI2. In contrast, CGP 42112A, the subtype 2-selective antagonist, totally blocked the Ang II-induced PGI2 release and partially attenuated the PGE2 release. Ang-(1-7)-induced PGE2 and PGI2 release was not altered by DuP 753; however, CGP 42112A totally blocked the effects of Ang-(1-7) on PG stimulation. Calcium mobilization in response to Ang II was blocked by [Sar1,Thr8]Ang II, [Sar1,Ile8]Ang II, and DuP 753 but not by CGP 42112A. These data suggest that human astrocytes contain both Ang receptor subtypes. The subtype 1 Ang receptor participates both in the release of PGs and in the mobilization of calcium, whereas the subtype 2 receptor is coupled to the release of PGs only. In addition, PG release coupled to subtype 2 Ang II receptors occurs through a calcium-independent mechanism and responds uniquely to Ang-(1-7).

Identification and regulation of angiotensin II receptor subtypes on NG108-15 cells.

NG108-15 cells, a neurally derived clonal cell line, express various components of the renin-angiotensin system and thus serve as a model of the cellular action of angiotensin (Ang) II. NG108-15 cells contain a high-affinity binding site for Ang II, with a Kd of 1.1 nM and a Bmax of 6.5 fmol/mg protein. Ang peptides competed for 125I-Ang II binding with an order of potency of Ang II greater than Ang-(2-8) much greater than Ang-(1-7). The subtype 1 (or B)-selective Ang II receptor antagonist DuP 753 as well as [Sar1,Ile8]Ang II and [Sar1,Thr8]Ang II competed for Ang II binding with high affinity, whereas the subtype 2 (or A)-selective Ang receptor antagonist CGP 42112A was partially effective only at a 300-fold higher concentration. When NG108-15 cells were induced to differentiate by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate, the density of Ang II receptors increased dramatically, with little change in affinity (1.1 versus 4.2 nM) or competition by Ang peptides. In marked contrast to undifferentiated cells, CGP 42112A became a potent competitor (IC50, 1 nM) for the majority (90-95%) of Ang II binding, whereas DuP 753 competed for only 5-10% of the binding sites. Ang II caused a dose-dependent mobilization of cytosolic Ca2+ in undifferentiated NG108-15 cells through activation of phospholipase C and the production of inositol 1,4,5-trisphosphate. In these cells, Ca2+ mobilization was blocked by either DuP 753 or the sarcosine Ang II analogues, whereas CGP 42112A was ineffective. Ang II also mobilized intracellular Ca2+ in differentiated NG108-15 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin-(1-7). A member of circulating angiotensin peptides.

We measured the concentrations of three principal products of the renin-angiotensin system and seven of their metabolites in the plasma of anesthetized normal dogs and in dogs 24 hours after bilateral nephrectomy. The levels of the angiotensin peptides were measured by high-performance liquid chromatography combined with radioimmunoassay using three specific antibodies that recognized different epitotes in the sequences of angiotensin I, angiotensin II, and angiotensin-(1-7). The analysis revealed that angiotensin-(1-7) is present in the plasma of intact (4.9 +/- 2.2 fmol/ml) and nephrectomized (0.5 +/- 0.5 fmol/ml) dogs. An intravenous injection of purified hog renin (0.01 Goldblatt unit/kg) increased plasma levels of angiotensin I, angiotensin II, and angiotensin-(1-7) both before and after nephrectomy. These changes were associated with parallel increases in the concentrations of fragments of the three parent peptides. Administration of MK-422 led to the disappearance of circulating angiotensin II and its fragments both before and after a second injection of the same dose of renin. In contrast, MK-422 augmented the plasma levels of both angiotensin I and angiotensin-(1-7). The concentrations of these two peptides, but not the blood pressure, were again augmented by a second injection of renin given after blockade of converting enzyme. These effects were observed both before and after bilateral nephrectomy. These findings show that angiotensin-(1-7) circulates in the blood of normal and nephrectomized dogs. In addition, we found that angiotensin-(1-7) is generated in the blood from the cleavage of angiotensin I through a pathway independent of converting enzyme (EC 3.4.15.1).

Angiotensin-(1-7) potentiates the hypotensive effect of bradykinin in conscious rats.

Treatment with angiotensin-converting enzyme inhibitors increases the angiotensin-(1-7) [Ang-(1-7)] and bradykinin concentrations in plasma and tissue. In this study we evaluated the interaction between these peptides by determining the effect of Ang-(1-7) on the hypotensive action of bradykinin in conscious rats. Administration of Ang-(1-7) (5 nmol) did not change mean arterial pressure or heart rate. However, the hypotensive effect of bradykinin, produced by an intravenous or intra-arterial route, was potentiated by Ang-(1-7) in a dose-dependent manner. The Ang-(1-7) doses necessary to transform the effect of a single dose of bradykinin into that produced by a double dose (potentiating unit) were 2 nmol i.v. and 5 nmol IA. The Ang-(1-7) dose used did not change either the pressor effect of Ang II or the hypotensive effect of sodium nitroprusside. The bradykinin-potentiating Ang-(1-7) activity was significantly attenuated by pretreatment with indomethacin (5 mg/kg IM, n = 4). In an additional group the bradykinin-potentiating activity of Ang-(1-7) was evaluated 30 minutes after treatment with the angiotensin-converting enzyme inhibitor enalaprilat (10 mg/kg i.v., n = 9). Under this condition the bradykinin-potentiating activity of Ang-(1-7) was substantially increased, resulting in a potentiating unit of approximately 0.2 nmol IV. Pretreatment with indomethacin (5 mg/kg IM, n = 7) also attenuated the bradykinin-potentiating activity of Ang-(1-7) in enalaprilat-treated rats. These results show that Ang-(1-7) is a bradykinin-potentiating peptide in vivo. Furthermore, the data obtained with indomethacin suggest that prostaglandins participate in the mechanism of the bradykinin potentiation by Ang-(1-7). More importantly, these data suggest that the interaction between Ang-(1-7) and bradykinin can contribute to the pharmacological effects of angiotensin-converting enzyme inhibitors.

Potentiation of the hypotensive effect of bradykinin by short-term infusion of angiotensin-(1-7) in normotensive and hypertensive rats.

In this study we evaluated the effect of angiotensin-(1-7) on the hypotensive action of bradykinin (BK) in normotensive rats, renal hypertensive rats (RHR), and spontaneously hypertensive rats (SHR). In addition, we evaluated the effect of angiotensin-converting enzyme (ACE) inhibition with enalaprilat treatment (10 mg/kg I.V.) on the BK-potentiating activity of Ang-(1-7). Renal hypertension was produced by aorta coarctation between the origin of renal arteries. Ang-(1-7) (0.3 pmol/min) or saline (0.9% NaCl, 5 microL/min) was infused intravenously in conscious male Wistar rats, adult SHR, or RHR. Intravenous bolus injections of BK (0.1 to 1.6 nmol in RHR and SHR; 0.625 to 5 nmol in Wistar rats) were made before and within 30 and 60 minutes of Ang-(1-7) infusion. Ang-(1-7) infusion did not change mean arterial pressure (MAP) of Wistar rats (MAP=97+/-3 mm Hg), RHR (MAP=173+/-3 mm Hg), or SHR (MAP=177+/-5 mm Hg). In Wistar rats, Ang-(1-7) increased the BK hypotensive effect by 24+/-6% within 60 minutes of infusion. No significant changes were observed at 30 minutes of infusion. In additional groups of rats, Ang-(1-7) (5 pmol/min, n=5) was infused alone or combined with its selective antagonist D-Ala7-Ang-(1-7) (A-779) (5 pmol/min, n=6). The bradykinin-potentiating activity of Ang-(1-7) was completely abolished by A-779. In SHR and RHR, Ang-(1-7) significantly increased the hypotensive effect of BK by 59+/-8% and 57+/-9.8%, respectively, within 60 minutes of infusion. No significant changes were observed with saline infusion. In Wistar rats, enalaprilat treatment increased the BK-potentiating activity of Ang-(1-7) transforming the effect of 0.3 pmol/min into that observed with a rate 16-fold higher (5 pmol/min). On the other hand, in SHR enalaprilat did not change the Ang-(1-7) effect, while it abolished the BK potentiation in RHR. Our data show that the BK-potentiating activity of Ang-(1-7) is preserved and even augmented in hypertensive rats. The finding that the BK-potentiating activity of Ang-(1-7) could be demonstrated at a very low infusion rate suggests that this angiotensin can act as an endogenous modulator of the vascular actions of kinins. ACE inhibition can influence differently the BK-potentiating activity of Ang-(1-7) in normotensive and hypertensive rats.

Rat (Ile5) but not bovine (Val5) angiotensin raises plasma norepinephrine in rats.

A part of the vasoconstrictor activity of angiotensin II (AII) may result from its ability to enhance norepinephrine (NE) release from sympathetic noradrenergic nerve terminals. To investigate this proposed pressor mechanism of AII, the effects of intravenous (i.v.) infusion of AII on blood pressure and plasma catecholamines in pithed rats were determined. Two naturally occurring angiotensins, valine5 AII (bovine) and isoleucine5 AII (rat), were administered in equal (72 ng/min) doses. Valine5 AII caused an 80% increase in mean arterial pressure (MAP) from 54 +/- 4 to 97 +/- 19 mm Hg. Isoleucine5 AII caused an 82% increase in MAP from 49 +/- 5 to 89 +/- 18 mm Hg. Neither angiotensin caused a change in heart rate, suggesting that pithing completely destroyed the central baroreceptor reflex mechanism. Plasma catecholamines were differentially affected by the peptides:isoleucine5 AII significantly increased plasma NE concentration by 82% compared to saline-infused rats (p less than 0.01). Valine5 AII did not significantly affect plasma NE concentration. Plasma dopamine and epinephrine concentrations were not significantly altered by infusion of either analog. Despite the significant increases in plasma NE concentrations with isoleucine5 in AII-infusion rats, there was no correlation between plateau MAP or the percent increase in MAP and plasma NE concentrations of individual animals within this group. The ability of angiotensin to elevate MAP, increase NE release from sympathetic nerve terminals, as well as potential differences in the actions of angiotensins in different species, and angiotensin receptor heterogeneity, are discussed.

Synthesis of nonmammalian angiotensins and their comparative pressor properties in dogfish shark, domestic chicken, and rat.

To understand how vertebrates utilize angiotensins during evolutionary development, we undertook studies to synthesize and/or characterize angiotensin-like peptides from nonmammalian species. The present paper describes the synthesis of [Asp1,Val5,Asn9] angiotensin I (bull frog, Rana catesbeiana) (I), [Asn1,Val5,His9] angiotensin I (Japanese goosefish, Lophius litulon) (II), [Asn1, Val5,Asn9] angiotensin I (chum salmon, Oncorhynchus keta) (III), and [Asn1, Val5,Tyr9] angiotensin I (related to native angiotensin in snake, Elaphe climocophora (IV). Pressor properties of these peptides were compared with the peptides isolated from other species and related synthetic analogs in one representative species from three distinct classes of vertebrates: 1) elasmobranchs: spiny dogfish shark; 2) birds: domestic chicken; and 3) mammals: rat. The effect of angiotensins on short circuit current (to compare sodium and water permeability) was studied by adding these on the dermal side of the isolated frog skin. In the rat pressor bioassays, the above peptides possessed, respectively, I, 87.8%; II, 51.5%; III, 65.2%; and IV, 60.3% pressor activity of [Ile5] angiotensin II, which was blocked with a converting-enzyme inhibitor, captopril. In the conscious dogfish shark, the percentage increase of blood pressure based on preinjection level (= 100) in the dorsal aortic pressure was 35% to 60% for [Asp1,Ile5,His9] angiotensin I (human) (3 micrograms/kg), [Asp1,Val5,Ser9] angiotensin I (chicken) (3 micrograms/kg), [Asp1,Ile5] angiotensin II (3.6 micrograms/kg), and [Asn1,Val5] angiotensin II (6 micrograms/kg). Likewise, a 30% to 35% increase in blood pressure was obtained with angiotensin III (3 micrograms/kg), [Ile8] angiotensin II (4.4 micrograms/kg), and [Sar1,Ile8] angiotensin II (9.1 micrograms/kg). [Sar1,Thr8] angiotensin II and [Ile8] angiotensin I did not produce a significant pressor response even at high dose-level (8 micrograms/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of endothelial cell prostaglandin production by angiotensin peptides. Characterization of receptors.

Angiotensin II stimulates prostaglandin release in blood vessels via activation of angiotensin receptors present in endothelium, vascular smooth muscle cells, or both. We evaluated the response of angiotensin II, angiotensin I, and [des-Phe8] angiotensin II [angiotensin-(1-7)] on prostaglandin release in porcine aortic endothelial cells. Incubation of cell monolayers with angiotensin I and angiotensin-(1-7), but not angiotensin II, stimulated the release of prostaglandin E2 and prostaglandin I2 in a dose-dependent manner (10(-10) to 10(-6) M) with an EC50 of approximately 1 nM. In addition, we characterized the angiotensin receptor subtypes mediating prostaglandin synthesis by using subtype-selective antagonists. Angiotensin I-stimulated prostaglandin synthesis was not altered by either of the nonselective classical angiotensin receptor antagonists [Sar1,Thr8]angiotensin II or [Sar1,Ile8]angiotensin II. In contrast, either the angiotensin subtype 1 (AT1) antagonist DuP 753 or the subtype 2 (AT2) antagonist CGP42112A significantly attenuated the prostaglandin release in response to angiotensin I. However, PD123177, another AT2 antagonist, did not inhibit angiotensin I-stimulated prostaglandin release. Angiotensin-(1-7)-induced prostaglandin release was significantly attenuated by [Sar1,Thr8]angiotensin II (10(-6) M) and PD123177 (10(-6) M) but not by [Sar1,Ile8]angiotensin II, DuP 753, or CGP42112A. Higher doses (10(-5) M) of DuP 753 and CGP42112A attenuated the angiotensin-(1-7) response. These data suggest that in porcine aortic endothelial cells, angiotensin I and angiotensin-(1-7) but not angiotensin II are potent stimuli for prostaglandin synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathways of angiotensin formation and function in the brain.

New findings from this laboratory suggest that fragments of angiotensin derived from the amino (N-)terminus are biologically active end products of the renin-angiotensin system. In vitro and in vivo experiments revealed that the heptapeptide angiotensin-(1-7) [Ang-(1-7)] is a major endogenous product of the renin-angiotensin system cascade in the brains of rats and dogs. Additional studies with enzyme inhibitors showed that Ang-(1-7) is produced directly from angiotensin I by an enzyme other than the angiotensin converting enzyme. Immunocytochemical fibers within the hypothalamo-neurohypophyseal vasopressinergic system of the rat. Although Ang-(1-7) is as potent as angiotensin II (Ang II) in stimulating release of vasopressin from superperfused hypothalamo-neurohypophyseal explants, the heptapeptide has no dipsogenic or vasoconstrictor activity. In contrast, Ang-(1-7) mimics the effects of Ang II in augmenting the intrinsic discharge rate of neurons within the vagal-solitary complex and in causing monophasic depressor responses after microinjection into the medial region of the nucleus tractus solitarii. The evidence obtained in these experiments suggests novel mechanisms for the generation of angiotensin peptides in the brain. Additionally, the findings suggest that some of the biological actions ascribed to Ang II might be conveyed by the endogenous production of other angiotensin peptides that are generated by enzymatic pathways alternate to those described in the peripheral circulation.

Angiotensin-(1-7): a new hormone of the angiotensin system.

We provide a new foundation for an alternative interpretation of the biochemical physiology of the brain and other tissue angiotensin systems on the basis of research done in our laboratory. This perspective is prompted by the discovery that angiotensin-(1-7) has cellular functions that differ from those established for angiotensin II. Although angiotensin-(1-7) is not an agonist in terms of activating vasoconstriction, stimulating thirst, or promoting aldosterone release, the heptapeptide caused neuronal excitation and vasopressin release with a potency similar to that found with angiotensin II. Furthermore, angiotensin-(1-7) enhances the production of prostanoids by a receptor-mediated event that causes no associated rise in intracellular Ca2+. These actions of angiotensin-(1-7) provide a new understanding of the heterogeneous functions of angiotensin peptides as modulators of a wide range of regulatory functions in mammals.

Role of angiotensin-(1-7) in the modulation of the baroreflex in renovascular hypertensive rats.

In this study, we evaluated the effect produced by lateral ventricle (intracerebroventricular, I.C.V.) infusion of the selective angiotensin (Ang)-(1-7) antagonist, D-Ala7-Ang-(1-7) (A-779), in the modulation of the baroreflex control of heart rate in two-kidney, one clip renovascular hypertensive rats (2K1C) treated with the angiotensin-converting enzyme (ACE) inhibitor enalapril. Twenty days after the surgery to produce renovascular hypertension, I.C.V. cannulas were implanted in the rats with blood pressure (BP) greater than 145 mm Hg (n=33) and in sham-operated rats (n=32). Five days later, the rats were treated with enalapril (10 mg x kg(-1) x d(-1); 6 days, in the drinking water) or vehicle (tap water). On the sixth day of treatment, direct continuous BP recording and measurement of reflex changes in heart rate elicited by phenylephrine were made in conscious rats before and at 1 hour of I.C.V. infusion of saline (8 microL/h) or A-779 (4 microg/h). To evaluate the degree of ACE blockade produced by enalapril treatment, the pressor effect of Ang I (50 ng, I.V., and 100 ng, I.C.V.) and plasma ACE activity was determined. As expected, enalapril treatment in 2K1C produced a significant fall in BP, significant attenuation in the pressor response of Ang I (I.V.), and a reduction in plasma ACE activity. In addition, enalapril treatment increased the baroreflex sensitivity (0.76+/-0.04 versus 0.43+/-0.04 ms/mm Hg in 2K1C untreated rats). I.C.V. infusion of A-779 reverted the improvement in baroreflex sensitivity produced by enalapril treatment in 2K1C (from 0.80+/-0.07 to 0.42+/-0.08 ms/mm Hg) and also attenuated the baroreflex sensitivity in untreated 2K1C (0.36+/-0.05 versus 0.48+/-0.06 ms/mm Hg) and untreated sham-operated rats (1.21+/-0.05 versus 0.78+/-0.17 ms/mm Hg). These results suggest that central endogenous Ang-(1-7) is involved at least in part in the improvement of baroreflex sensitivity observed in 2K1C after peripheral chronic ACE inhibition.