Bioavailability of Orally Administered Des-Aspartate-Angiotensin I in Human Subjects.

In an earlier single-dose escalation study to evaluate the safety and pharmacokinetics of orally administered des-aspartate-angiotensin I (DAA-I) in healthy subjects, the plasma level of DAA-I could not be determined because DAA-I is rapidly degraded in the circulation. The present study investigated the oral bioavailability of DAA-I by measuring the prostaglandin E2 metabolite (PGEM) in the plasma samples of the same trial. PGEM is a stable derivative of PGE2, which has been shown to be a biomarker of DAA-I. The data show that plasma from two of the three subjects who were orally administered the efficacious preclinical dose of 0.70 mg/kg DAA-I exhibited a significant PGEM peak at 5-6 h postdose. Plasma of subjects who were administered 0.08 and 1.5 mg/kg DAA-I, the subefficacious and two-times efficacious dose, respectively, did not exhibit a similar PGEM peak. This observation is concordant with the known in vivo actions of DAA-I, especially its hypoglycemic action where maximum efficacy occurred at a dose of 0.7 mg/kg, and decreased to nil at the two-times efficacious dose. The onset of the PGEM peak at 5-6 h postdose was closed to the 4-h onset of absorption of [C14]DAA-I seen in preclinical rat studies, albeit the absorption kinetics between rodents and humans are not identical. The occurrence of polymorphism of enzymes involved in the formation and degradation of PGE2 is common, and this has been attributed to contributing to the variation in response, onset and peak PGEM observed among the three subjects who were administered the efficacious dose.

A Single Dose-Escalation Study to Evaluate the Safety and Pharmacokinetics of Orally Administered Des-Aspartate Angiotensin I in Healthy Subjects.

Des-aspartate-angiotensin I (DAA-I) is an endogenous angiotensin peptide and a prototype angiotensin receptor agonist (ARA). It acts on the angiotensin AT1 receptor and antagonises the deleterious actions of angiotensin II. DAA-I attenuates animal models of human disease in which angiotensin II has been implicated, such as cardiac hypertrophy, neointima formation, arteriosclerosis, renal failure, post-infarction injuries, diabetes, viral infection, chemical-induced inflammation, heat stroke, cancer, and gamma radiation lethality. DAA-I crosses Caco-2 cells and is effective at sub-nanomolar concentrations. These two properties are responsible for its oral efficacy. A single dose-escalation study was conducted to evaluate the safety, tolerability and pharmacokinetics of orally administered DAA-I in 18 healthy subjects. DAA-I was safe and well tolerated by the subjects, who were administered either 0.08, 0.70 or 1.50 mg/kg of the compound. The heart rate and systolic and diastolic blood pressures determined at each post-dose measurement remained within the clinically acceptable range. Across all cohorts, DAA-I had no substantial effect on blood pressures compared with placebo. Electrocardiographs (ECGs) were normal, and none of the subjects complained of chest discomfort. All clinical laboratory tests obtained before and after DAA-I and placebo treatment were normal. Pharmacokinetic analysis over a 12-h period following DAA-I administration did not show any increase of its level beyond basal concentration. This is in line with studies showing that intravenously administered DAA-I is rapidly metabolized and has a short half-life. We postulate that, during its short systemic sojourn, DAA-I exerts its actions via biased agonism on the angiotensin AT1 receptor.The ClinicalTrial.gov assignment number for this study is NCT02666196.

Des-aspartate-angiotensin I causes specific release of PGE2 and PGI2 in HUVEC via the angiotensin AT1 receptor and biased agonism.

DAA-I (des-aspartate-angiotensin I), an endogenous angiotensin, had been shown earlier to ameliorate animal models of cardiovascular diseases via the angiotensin AT1 receptor and prostaglandins. The present study investigated further the action of DAA-I on the release of PGE2, PGI2, PGF2α and TXA2 in HUVEC. 10(-11)-10(-8)M DAA-I and 15min incubation specifically released PGE2 and PGI2. The release was inhibited by losartan and indomethacin but not by PD123319 and NS398 indicating that the angiotensin AT1 receptor and COX-1 mediate the release. At concentrations higher than 10(-7)M, DAA-I mimics the action of angiotensin II by releasing TXA2 but had no effect on the production of PGF2α. At similar concentrations and 4h incubation, DAA-I increased the release of the 4 prostaglandins via the angiotensin AT1 receptor and COX-2, again mimicking the action of angiotensin II. HUVEC that were preincubated with DAA-I or angiotensin II, released similar profiles of prostaglandins when incubated with arachidonic acid after the angiotensin had been washed off. We postulate that the internalized DAA-I/receptor complex remains active and mediates the conversion of arachidonic acid to the respective prostaglandins. The release of PGE2 and PGI2 via the angiotensin AT1 receptor and COX-1 is a novel specific action of DAA-I and is likely responsible for its beneficial effects seen in earlier studies. This specific action is definable as a biased agonism of the angiotensin AT1 receptor, which identifies DAA-I as a novel biased agonist and potential therapeutic that is able to produce specific prostaglandins at nanomolar concentrations.

Des-Aspartate-Angiotensin I Attenuates Mortality of Mice Exposed to Gamma Radiation via a Novel Mechanism of Action.

ACE inhibitors and ARBs (angiotensin receptor blockers) have been shown to attenuate radiation injuries in animal models of lethal gamma irradiation. These two classes of drug act by curtailing the actions of angiotensin II-linked inflammatory pathways that are up-regulated during gamma radiation in organ systems such as the brain, lung, kidney, and bone marrow. ACE inhibitors inhibit ACE and attenuate the formation of angiotensin II from angiotensin I; ARBs block the angiotensin AT1 receptor and attenuate the actions of angiotensin II that are elicited through the receptor. DAA-I (des-aspartate-angiotensin I), an orally active angiotensin peptide, also attenuates the deleterious actions of angiotensin II. It acts as an agonist on the angiotensin AT1 receptor and elicits responses that oppose those of angiotensn II. Thus, DAA-I was investigated for its anticipated radioprotection in gamma irradiated mice. DAA-I administered orally at 800 nmole/kg/day for 30 days post exposure (6.4 Gy) attenuated the death of mice during the 30-day period. The attenuation was blocked by losartan (50 nmole/kg/day, i.p.) that was administered sequential to DAA-I administration. This shows that the radioprotection was mediated via the angiotensin AT1 receptor. Furthermore, the radioprotection correlated to an increase in circulating PGE2 of surviving animals, and this suggests that PGE2 is involved in the radioprotection in DAA-I-treated mice. At the hematopoietic level, DAA-I significantly improved two syndromes of myelosuppression (leucopenia and lymphocytopenia), and mice pre-treated with DAA-I prior to gamma irradiation showed significant improvement in the four myelodysplastic syndromes that were investigated, namely leucopenia, lymphocytopenia, monocytopenia and thrombocytopenia. Based on the known ability of PGE2 to attenuate the loss of functional hematopoietic stem and progenitor cells in radiation injury, we hypothesize that PGE2 mediated the action of DAA-I. DAA-I completely attenuated the increase in circulating level of two inflammatory cytokines, TNFα and IL-6, in irradiated mice; and this shows that DAA-I exerted additional anti-inflammatory actions, which could also have contributed to its radioprotection. These findings show that DAA-I acts via a novel mechanism of action on the angiotensin AT1 receptor to specifically release PGE2, which mediates radioprotection in the gamma irradiated mice.

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates.

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different ß-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.

Des-aspartate angiotensin I (DAA-I) reduces endothelial dysfunction in the aorta of the spontaneously hypertensive rat through inhibition of angiotensin II-induced oxidative stress.

Des-aspartate angiotensin I (DAA-I), an endogenous nonapeptide, counteracts several effects of angiotensin II on vascular tone. The aim of this study was to investigate the acute protective effect of DAA-I on endothelial function in the spontaneously hypertensive rat (SHR) as well as its effect on angiotensin II-induced contractions and oxidative stress. Aortic rings were incubated with DAA-I (0.1µM) for 30min prior to the assessment of angiotensin II-induced contractions (0.1nM-10µM) in WKY and SHR aortas. Total nitrate and nitrite levels were assessed using a colorimetric method and reactive oxygen species (ROS) were measured by dihydroethidium (DHE) fluorescence and lucigenin-enhanced chemiluminescence. The effect of DAA-I was also assessed against endothelium-dependent and -independent relaxations to acetylcholine and sodium nitroprusside, respectively. Angiotensin II-induced contractions were significantly reduced by DAA-I, losartan and tempol. Incubation with ODQ (soluble guanylyl cyclase inhibitor) and removal of the endothelium prevented the reduction of angiotensin II-induced contractions by DAA-I. Total nitrate and nitrite levels were increased in DAA-I, losartan and tempol treated-SHR tissues while ROS level was reduced by DAA-I and the latter inhibitors. In addition, DAA-I significantly improved the impaired acetylcholine-induced relaxation in SHR aortas whilst sodium nitroprusside-induced endothelium-independent relaxation remained unaffected. The present findings indicate that improvement of endothelial function by DAA-I in the SHR aorta is mediated through endothelium-dependent release of nitric oxide and inhibition of angiotensin II-induced oxidative stress.

Des-aspartate-angiotensin I, a novel angiotensin AT(1) receptor drug.

The review describes DAA-I (des-aspartate-angiotensin-I) as a prototype of a novel class of drugs that acts as agonists on the angiotensin AT1 receptor or ARAs (angiotensin receptor agonists). DAA-I is a component of the renin angiotensin system. Earlier studies showed that it was rapidly metabolized to angiotensin III. However, when administered at doses below the Km of enzymes, DAA-I produces specific actions that antagonize the deleterious actions of angiotensin II. DAA-I exerts protective actions in animal models of eight human pathologies in which angiotensin II is implicated. The pathologies include cardiac hypertrophy, neointima growth and cardiovascular hypertrophy, myocardial-ischemia reperfusion injury, hyperglycemia and insulin resistance, chemical induced inflammation, and exercise-induced skeletal muscle inflammation. Binding of DAA-I to the angiotensin AT1 receptors releases prostaglandins, which could either function as autocrines/paracrines or second messengers and attenuate the deleterious actions of angiotensin II. It is possible that in in vivo DAA-I functions as a physiological antagonist to angiotensin II, and exogenous DAA-I is a novel class of angiotensin receptor drug that could rival the angiotensin receptor blockers.

Des-aspartate-angiotensin I attenuates ICAM-1 formation in hydrogen peroxide-treated L6 skeletal muscle cells and soleus muscle of mice subjected to eccentric exercise.

L6 skeletal muscle cells overexpressed ICAM-1 when treated with H2O2. Maximum effect was observed at 200 µM H2O2. Des-aspartate-angiotensin I (DAA-I) concentration-dependently attenuated the overexpression. Maximum attenuation occurred at 10(-10) M DAA-I. H2O2 activated NFκB and its translocation into the nucleus of L6 muscle cells suggesting that NFκB mediates the H2O2-induced overexpression of ICAM-1. DAA-I inhibited the activation and translocation of NFκB. H2O2 is a major oxidant formed during skeletal muscle contraction and is implicated in oxidative stress and skeletal muscle damage in excessive unaccustomed exercise. The data show that DAA-I has antioxidant action, and its action was further investigated in the soleus muscle of mice subjected to 240 min of eccentric exercise on a rodent treadmill. The eccentric exercise induced superoxide formation and overexpression of ICAM-1 in the soleus muscle of the mice at 3 days post exercise. DAA-I (0.2 nmole/kg/day) administered orally on day 1 (pre-exercise) and 2 days post-exercise attenuated both the ROS formation and ICAM-1 overexpression. Earlier studies show that DAA-I acts as an agonist on the angiotensin AT1 receptor and elicits responses opposing those of angiotensin II. The present and earlier findings support the recent suggestion that angiotensin II is involved in skeletal muscle damage, and curtailment of its actions via ACE inhibitors and losartan protects and improves skeletal muscle damage. These findings open up new avenues for treatment and management of skeletal muscle damage via the interventions of the renin angiotensin system.

Des-aspartate-angiotensin I exerts antiviral effects and attenuates ICAM-1 formation in rhinovirus-infected epithelial cells.

The high frequency of rhinovirus (RV) infection and the lack of an effective treatment, underline the importance of research on novel anti-rhinoviral agents. The present study investigated the effects of des-aspartate-angiotensin I (DAA-I) on the survival of RV14-infected H1HeLa cells; and the early inflammatory processes in RV14-infected A549 lung epithelial cells. The study rationale was based on earlier findings showing that DAA-I is an effective anti-inflammatory agent, and that symptoms and severity of rhinoviral infection are related to the underling inflammation. RV14 concentration dependently caused the death of H1HeLa cells and DAA-I, at concentrations of 10⁻¹° to 10⁻¹² M, attenuated the lethal action of RV14 indicating that that DAA-I exerts antiviral action. Unlike its action on H1HeLa cells, RV14 did not cause apparent cytopathic effect on A549 cells, and these cells were used to study the antiviral action of DAA-I. RV14 induced overexpression of ICAM-1, E-selectin and overproduction of superoxide in A549 cells, and DAA-I attenuated the three increases to basal level at concentrations of 10⁻¹° to 10⁻¹² M. Losartan, an angiotensin AT1 receptor antagonist, blocked the inhibitory action of DAA-I on superoxide overproduction indicating that the AT1 receptor mediates the action of DAA-I. The present data represent a novel demonstration of the antiviral action of an angiotensin peptide, and a possible involvement of the renin angiotensin system in viral infection. Indeed the angiotensin AT1 receptor has been reported to be obligatory for the development of virus-induced myocardial injury through the proinflammatory action of angiotensin II via the NF-κB/cytokine pathway.

Des-aspartate-angiotensin-I and angiotensin IV improve glucose tolerance and insulin signalling in diet-induced hyperglycaemic mice.

Although clinical studies suggested that blockade of the renin-angiotensin system may prevent diabetes, the mechanism is uncertain. As a follow-up to an earlier study, we investigated how des-aspartate-angiotensin-1 (DAA-1) and its metabolite, angiotensin IV (Ang-IV) improved glucose tolerance in diet-induced hyperglycaemic mice. Male C57BL/6J mice were fed a high-fat-high-sucrose (HFD) or normal (ND) diet for 52 weeks. HFD animals were orally administered either DAA-I (600nmol/kg/day), Ang-IV (400nmol/kg/day) or distilled water. Body weight, blood glucose and insulin were measured fortnightly. Inflammatory and insulin signalling transducers that are implicated in hyperglycaemia were analyzed in skeletal muscles at 52 weeks. HFD animals developed hyperglycemia, hyperinsulinemia and obesity. DAA-I and Ang-IV improved glucose tolerance but had no effect on hyperinsulinemia and obesity. Skeletal muscles of HFD animals showed increased level of ROS, gp91 of NADPH oxidase, pJNK and AT(1)R-JAK-2-IRS-1 complex. Both DAA-I and Ang-IV attenuated these increases. Insulin-induced activation of IR, IRS-1, IRS-1-PI3K coupling, phosphorylation of Akt, and GLUT4 translocation were attenuated in skeletal muscles of HFD animals. The attenuation was significantly ameliorated in DAA-I-treated HFD animals. In corresponding Ang-IV treated animals, insulin induced IRAP and PI3K interaction, activation of pAkt and GLUT4 translocation, but no corresponding activation of IR, IRS-1 and IRS-1-PI3K coupling were observed. DAA-I and Ang-IV improved glucose tolerance, insulin signalling, and para-inflammatory processes linked to hyperglycaemia. DAA-I acts via the angiotensin AT(1) receptor and activates the insulin pathway. Ang-IV acts via IRAP, which couples PI3K and activates the later part of the insulin pathway.

Effects of des-aspartate-angiotensin I on myocardial ischemia-reperfusion injury in rats.

The present study investigated the actions of des-aspartate-angiotensin I (DAA-I) on infarct size and three early inflammatory events in acute myocardial ischemia-reperfusion injury in rats. The rationale was based on earlier findings showing that chronic daily administration of DAA-I attenuated infarct size of ischemic-reperfused rat heart, and cardiac hypertrophy in pressure overload rats. Anesthetized rats were subjected to 45 min of ischemia and 5h of reperfusion. Infarcted area, serum creatine kinase, and tissue myeloperoxidase activity were determined. The expression of intercellular adhesion molecule-1 (ICAM-1) was also investigated by immunohistochemistry and Western blotting. Intravenous administration of DAA-I at 5 min post reperfusion reduced myocardial infarct size by 45.5%, lowered serum creatine kinase activity, decreased myeloperoxidase activity in cardiac tissue, and inhibited the expression of ICAM-1 in cardiac capillary endothelium. The maximum effective dose was 1013 pmol/kg, and the cardioprotective actions of DAA-I were blocked by indomethacin. The data showed that the cardioprotection accorded by DAA-I was the result of its anti-inflammatory actions on early inflammatory processes in myocardial ischemia-reperfusion injury. The anti-inflammatory processes were indomethacin sensitive and probably mediated by prostaglandins.

Protective actions of des-aspartate-angiotensin I in mice model of CEES-induced lung intoxication.

The present study investigated the protective actions of des-aspartate-angiotensin I (DAA-I) in mice that were intranasally administered 2-chloroethyl ethyl sulfide (CEES), a half sulfur mustard. The protection was dose-dependent, and an oral dose of 75 mg kg⁻¹ per day administered 18 h post exposure and for the following 13 days, offered maximum protection that increased survival by a third. DAA-I attenuated the early processes of inflammation seen in the CEES-inoculated mice. DAA-I attenuated (i) elevated pulmonary ROS, and gp91-phox protein of NADPH oxidase, a non phagocytic enzyme that generates superoxide and subsequent ROS; (ii) intercellular adhesion molecule-1 (ICAM⁻¹) that is involved in the extravasation of circulating leucocytes; and (iii) myeloperoxidase activity, which is a surrogate enzymatic measurement of neutrophil infiltration. These actions led to improved histological lung structures, and survival of type-1 pneumocytes. The action of DAA-I on animal survival was blocked by losartan, a selective angiotensin AT1 receptor blocker, indicting that the AT1 receptor mediates the protection. The presence of elevated PGE2 and PGI2 in lung supernatants of DAA-I treated CEES-inoculated mice indicates that the two prostaglandins are involved in signaling the protective actions of DAA-I. This finding complements earlier studies showing that DAA-I acts on an indomethacin-sensitive angiotensin AT1 receptor. The findings of the present study are the first demonstration of an angiotensin peptide as an effective antidote for CEES intoxication. DAA-I is also an effective therapeutic intervention against CEES that was instituted at 18 h post exposure, and challenges conventional assumptions of limited efficacy with delayed action against alkylating agents.

Involvement of AT(1) angiotensin receptors in the vasomodulatory effect of des-aspartate-angiotensin I in the rat renal vasculature.

Angiotensin II is known to act primarily on the angiotensin AT(1) receptors to mediate its physiological and pathological actions. Des-aspartate-angiotensin I (DAA-I) is a bioactive angiotensin peptide and have been shown to have contrasting vascular actions to angiotensin II. Previous work in this laboratory has demonstrated an overwhelming vasodepressor modulation on angiotensin II-induced vasoconstriction by DAA-I. The present study investigated the involvement of the AT(1) receptor in the actions of DAA-I on angiotensin II-induced vascular actions in the renal vasculature of normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and streptozotocin (STZ)-induced diabetic rats. The findings revealed that the angiotensin receptor in rat kidney homogenate was mainly of the AT(1) subtype. The AT(1) receptor density was significantly higher in the kidney of the SHR. The increase in AT(1) receptor density was also confirmed by RT-PCR and Western blot analysis. In contrast, AT(1) receptor density was significantly reduced in the kidney of the streptozotocin-induced diabetic rat. Perfusion with 10(-9)M DAA-I reduced the AT(1) receptor density in the kidneys of WKY and SHR rats suggesting that the previously observed vasodepressor modulation of the nonapeptide could be due to down-regulation or internalization of AT(1) receptors. RT-PCR and Western blot analysis showed no significant changes in the content of AT(1) receptor mRNA and protein. This supports the suggestion that DAA-I causes internalization of AT(1) receptors. In the streptozotocin-induced diabetic rat, no significant changes in renal AT(1) receptor density and expression were seen when its kidneys were similarly perfused with DAA-I.

Des-aspartate-angiotensin I exerts hypoglycemic action via glucose transporter-4 translocation in type 2 diabetic KKAy mice and GK rats.

The present study investigated the hypoglycemic action of des-aspartate-angiotensin I (DAA-I), a metabolite of angiotensin I, in two animal models of type 2 diabetes. The rationale was based on our earlier studies demonstrating that DAA-I acts on the angiotensin AT(1) receptor and exerts responses opposing those of angiotensin II and on recent reports that curtailment of angiotensin II formation by angiotensin converting enzyme inhibitors and blockade of the AT(1) receptor attenuate hyperglycemia in type 2 diabetics and diabetic animals. Diabetic KKAy mice and GK rats were administered orally (by gavage) one of the following doses of DAA-I: 400, 600, or 800 nmol/kg.d for 4 and 6 wk, respectively. Control diabetic animals were similarly administered water. Blood glucose of each animal was determined fortnightly by oral glucose tolerance test and blood insulin on the last day of treatment. Animals were killed, and the levels of plasma membrane glucose transporter-4 and cytosolic tyrosine-phosphorylated insulin receptor substrate-1 in hind limb skeletal muscles were determined by Western blot in insulin-challenged and nonchallenged animals. Orally administered DAA-I had no effect on blood insulin level but exerted dose-dependent hypoglycemic action in KKAy mice and GK rats after 4 and 6 wk of treatment, respectively. At the maximal effective dose of 600 nmol/kg, insulin induced a significant increase in plasma membrane glucose transporter-4 and cytosolic tyrosine-phosphorylated insulin receptor substrate-1. These findings show that DAA-I is not an insulin secretagogue and exerts hypoglycemic action by attenuating insulin resistance, the first such demonstration indicating that the nonapeptide is involved in glycemic regulation.

Effector peptides of the renin-angiotensin system in the central mechanisms of acquired and innate behavior in thirst in rats.

We report here a comparative analysis of the involvement of a number of components of the renin-angiotensin system in the performance of simple and complex forms of drinking behavior and thirst-associated non-drinking types of behavior. On central (intracerebroventricular) microinjection, [des-Asp1]-angiotensin I at doses equieffective to those of angiotensins II and III was found to be involved only in the performance of simple (taking water from the bowl) and linked forms of activity (comfort behavior, stress grooming, orientational-investigative, and feeding behavior). Angiotensin II was involved in the central mechanisms of complex acquired drinking behavior, selectively modulating its key stages (initial, final), while angiotensin III was involved only in the mechanisms of reproduction of the complex skill. All three substances induced "innate patterns of behavior" specific for each compound, these occurring at fixed periods of time after intracerebral microinjection. The effects of these substances were selectively suppressed by the AT1 receptor blocker losartan potassium.

[Effector peptides of the brain renin-angiotensin system in central mechanisms of learned and natural forms of drinking behavior in rats].

Central mechanisms of angiotensin involvement in initiation and realization of operant forms of drinking behavior were investigated. It was suggested that intracerebroventricular microinjection of angiotensin-II and angiotensin-IIl specifically affected the learned forms of drinking behavior. The experiments demonstrated that [des-Asp1]-angiotensin-I produced only the natural forms of drinking behavior. Angiotensins modulated specific forms of thirst-associated behavior such as exploring, grooming, and ingestive behavior. Injections of AT1 receptor antagonist losartan were associated with acute water intake decrease and sharp operant behavior inactivation.

Effect of des-aspartate-angiotensin I on the actions of angiotensin II in the isolated renal and mesenteric vasculature of hypertensive and STZ-induced diabetic rats.

The present study investigated the action of des-aspartate-angiotensin I (DAA-I) on the pressor action of angiotensin II in the renal and mesenteric vasculature of WKY, SHR and streptozotocin (STZ)-induced diabetic rats. Angiotensin II-induced a dose-dependent pressor response in the renal vasculature. Compared to the WKY, the pressor response was enhanced in the SHR and reduced in the STZ-induced diabetic rat. DAA-I attenuated the angiotensin II pressor action in renal vasculature of WKY and SHR. The attenuation was observed for DAA-I concentration as low as 10(-18) M and was more prominent in SHR. However, the ability of DAA-I to reduce angiotensin II response was lost in the STZ-induced diabetic kidney. Instead, enhancement of angiotensin II pressor response was seen at the lower doses of the octapeptide. The effect of DAA-I was not inhibited by PD123319, an AT2 receptor antagonist, and indomethacin, a cyclo-oxygenase inhibitor in both WKY and SHR, indicating that its action was not mediated by angiotensin AT2 receptor and prostaglandins. The pressor responses to angiotensin II in mesenteric vascular bed were also dose-dependent but smaller in magnitude compared to the renal vasculature. The responses were significantly smaller in SHR but no significant difference was observed between STZ-induced diabetic and WKY rat. Similarly, PD123319 and indomethacin had no effect on the action of DAA-I. The findings reiterate a regulatory role for DAA-I in vascular bed of the kidney and mesentery. By being active at circulating level, DAA-I subserves a physiological role. This function appears to be present in animals with diseased state of hypertension and diabetes. It is likely that DAA-I functions are modified to accommodate the ongoing vascular remodeling.

Transport of angiotensin peptides across the Caco-2 monolayer.

The bidirectional transport of the angiotensin peptides--des-Asp-angiotensin I (DAAI), angiotensins III and IV--were studied using human intestinal Caco-2 monolayers. The peptides had low permeability rates but were relatively stable to enzymatic hydrolysis. DAAI was transported by diffusion while angiotensins III and IV were transported by an energy requiring, carrier-mediated process. The physicochemical properties and solution conformations of the peptides were investigated in an attempt to establish structure-transport correlations. Among the three peptides, DAAI was the most hydrophobic, had the highest hydrogen bonding potential and was the only peptide to have a random solution conformation, as determined from circular dichroism, two-dimensional (1)H NMR and molecular modelling. On the other hand, the more hydrophilic angiotensin IV had less hydrogen bonding potential and a solution conformation characterized by a beta turn. These factors may influence the transport characteristics of DAAI and angiotensin IV.