angiotensin-iii and amastatin

Endogenous angiotensin (Ang) II and/or an Ang II-derived peptide, acting on Ang type 1 (AT(1)) and Ang type 2 (AT(2)) receptors, can carry out part of the nociceptive control modulated by periaqueductal gray matter (PAG). However, neither the identity of this putative Ang-peptide, nor its relationship to Ang II antinociceptive activity was clarified. Therefore, we have used tail-flick and incision allodynia models combined with an HPLC time course of Ang metabolism, to study the Ang III antinociceptive effect in the rat ventrolateral (vl) PAG using peptidase inhibitors and receptor antagonists. Ang III injection into the vlPAG increased tail-flick latency, which was fully blocked by Losartan and CGP 42,112A, but not by divalinal-Ang IV, indicating that Ang III effect was mediated by AT(1) and AT(2) receptors, but not by the AT(4) receptor. Ang III injected into the vlPAG reduced incision allodynia. Incubation of Ang II with punches of vlPAG homogenate formed Ang III, Ang (1-7) and Ang IV. Amastatin (AM) inhibited the formation of Ang III from Ang II by homogenate, and blocked the antinociceptive activity of Ang II injection into vlPAG, suggesting that aminopeptidase A (APA) formed Ang III from Ang II. Ang III can also be formed from Ang I by a vlPAG alternative pathway. Therefore, the present work shows, for the first time, that: (i) Ang III, acting on AT(1) and AT(2) receptors, can elicit vlPAG-mediated antinociception, (ii) the conversion of Ang II to Ang III in the vlPAG is required to elicit antinociception, and (iii) the antinociceptive activity of endogenous Ang II in vlPAG can be ascribed preponderantly to Ang III.

Topics: Analgesics; Angiotensin II; Angiotensin III; Angiotensin Receptor Antagonists; Animals; Disease Models, Animal; Drug Interactions; Efferent Pathways; Glutamyl Aminopeptidase; Losartan; Male; Microinjections; Neural Inhibition; Nociceptors; Oligopeptides; Pain; Pain Measurement; Pain Threshold; Peptides; Periaqueductal Gray; Rats; Rats, Wistar; Receptors, Angiotensin

A previous study by our group has demonstrated that the selective AT1-receptor antagonist losartan behaves as a noncompetitive antagonist in rabbit isolated renal artery (RA). In the present investigation, the influence of losartan and irbesartan on the contractile effects of angiotensin II (AII) and its degradation products angiotensin III (AIII) and angiotensin IV (AIV) was determined in the rabbit isolated RA and femoral artery (FA). The arteries were set up in organ chambers and changes in isometric force were recorded. In both rabbit isolated RA and FA preparations, AII, AIII and AIV elicited significant contractile responses with a similar efficacy. These effects were impaired by the presence of functional endothelium in RA preparations but not in FA preparations. In both preparations studied, the effects of AII, AIII and AIV were influenced neither by the aminopeptidase-A and -M inhibitor amastatin (10 microM), nor by the aminopeptidase-B and -M inhibitor bestatin (10 microM). In endothelium-denuded FA preparations, preincubation with losartan (3-300 nM) antagonized AII-, AIII- and AIV-induced contractions in a competitive manner. However, in endothelium-denuded RA preparations, losartan depressed the maximal contractile responses induced by AII but not those induced by AIII and AIV. In the same preparations, preincubation of another selective AT1-receptor antagonist irbesartan (3-30 nM) concentration-dependently shifted AII and AIII curves to the right in an insurmountable manner. The reduction of the maximal response of AII is more potent when compared to that of AIII (47.7 +/- 1.51% vs. 66.7 +/- 1.88%, percentage of the initial maximal response; P < 0.05; n=5). The selective AT2-receptor antagonist PD123177 (1 microM) did not influence the responses to all three peptides in both RA and FA preparations. These heterogeneous antagonistic effects of the two AT1-receptor antagonists studied with respect to the contractile actions of AII, AIII and AIV suggest the possible existence of multiple, functionally relevant AT1-receptor subtypes in rabbit RA preparations.

Topics: Angiotensin II; Angiotensin III; Angiotensin Receptor Antagonists; Animals; Anti-Bacterial Agents; Antihypertensive Agents; Biphenyl Compounds; Endothelium, Vascular; Femoral Artery; Imidazoles; In Vitro Techniques; Irbesartan; Leucine; Losartan; Male; Peptides; Protease Inhibitors; Pyridines; Rabbits; Receptor, Angiotensin, Type 1; Renal Artery; Tetrazoles; Vasoconstrictor Agents

Elevated levels of angiotensin (Ang II) and its degradation products angiotensin III (Ang III) and angiotensin IV (Ang IV) may contribute to the regulation of vascular tone under various clinical circumstances. We investigated the contractile effects of Ang III and Ang IV in endothelium-denuded human saphenous vein (SV) preparations and compared them with those of Ang II. The veins were suspended in organ chambers, and changes in isometric force were recorded. Ang II (0.1-100 nM), Ang III (1 nM-3 microM), and Ang IV (0.3 microM-0.1 mM) caused concentration-dependent contractions with comparable maximal responses (Emax). Ang III was 16 times less active than Ang II, whereas Ang IV was approximately 2,700-fold less potent than Ang II. In the presence of the aminopeptidase-A and -M inhibitor amastatin (10 microM), the potencies of Ang III and Ang IV were increased by approximately 16 and 12 times, respectively, although no changes of Ang II potency were observed. The AT1-selective Ang II receptor antagonist losartan (10 and 100 nM) but not the AT2-selective antagonist PD123177 (1 microM), shifted the concentration-response curves (CRC) for the angiotensin peptides to the right in a parallel manner. Preincubation with indomethacin (10 microM), a cyclooxygenase inhibitor, did not influence the CRCs for any of the angiotensin peptides studied. Tachyphylaxis was investigated by constructing a second series of CRCs for the angiotensin peptides after an interval of 60 min. Ang II showed strong tachyphylaxis (the Emax value of the second Ang II CRC was approximately 50% of the first), whereas Ang III and Ang IV did not. Our results indicate that in endothelium-denuded human SV, both Ang III and Ang IV are less potent but similarly efficacious vasoconstrictor agents compared with Ang II. Endogenous aminopeptidase activity may counteract the effects of the angiotensin peptides. The contractile responses to all three peptides are mediated via AT1-receptors but not AT2-receptors.

Topics: Aged; Aged, 80 and over; Aminopeptidases; Angiotensin II; Angiotensin III; Angiotensin Receptor Antagonists; Anti-Bacterial Agents; Antihypertensive Agents; Biphenyl Compounds; Dose-Response Relationship, Drug; Drug Synergism; Female; Glutamyl Aminopeptidase; Humans; Imidazoles; In Vitro Techniques; Losartan; Male; Metalloendopeptidases; Methionyl Aminopeptidases; Middle Aged; Muscle Contraction; Muscle, Smooth, Vascular; Peptides; Protease Inhibitors; Pyridines; Saphenous Vein; Tachyphylaxis; Tetrazoles; Vasoconstrictor Agents

1. The direct positive chronotropic effects of angiotensin II (AII) and its degradation products angiotensin III (AIII) and angiotensin IV (AIV) were established in pithed rats and in rat spontaneously beating right atria. 2. In pithed rats, AII, AIII and AIV caused dose-dependent tachycardia with similar maximal responses (110 beats min-1). The beta-adrenoceptor antagonist propranolol (3.37 x 10(-6) mol kg-1) but not the alpha 1-adrenoceptor antagonist prazosin (2.38 x 10(-7) mol kg-1) significantly reduced these effects (P < 0.05; n = 7-8), but 20-25% of the responses could not be blocked by propranolol. 3. In isolated atria, AII, AIII and AIV caused concentration-dependent increases in beating rate with similar maximal responses to AII and AIII (34.3 +/- 0.4 and 34.7 +/- 0.4 beats min-1; n = 9-10), and a lower maximal response to AIV (26.8 +/- 0.6 beats min-1; P < 0.05; n = 8). AIII was about 9 times less potent than AII, whereas AIV proved approximately 3800 times less potent than AII. Neither propranolol (1 microM) nor prazosin (1 microM) could influence the effects of the angiotensin peptides. 4. In isolated atria, the selective AT1-receptor antagonist, losartan (10, 100 and 300 nM) caused parallel rightward shifts of the concentration-response curves for AII and AIII, whereas the selective AT2- receptor antagonist PD123177 (1 microM) did not influence the effects of AII and AIII. The aminopeptidase-A and -M inhibitor amastatin (10 microM), significantly steepened the slope of the AIII curves and increased the potency of AIII about 6 fold. Amastatin did not influence the responses to AII. 5. Our results indicate that both in vivo and in vitro, exogenous AII and AIII induced a direct dose-dependent chronotropic effect, which is independent of the adrenergic system. This chronotropic effect is mediated by AT1-subtype receptors.

Topics: Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Angiotensin II; Angiotensin III; Angiotensin Receptor Antagonists; Animals; Anti-Bacterial Agents; Decerebrate State; Heart Atria; Heart Rate; In Vitro Techniques; Injections, Intravenous; Male; Peptides; Prazosin; Propranolol; Rats; Rats, Wistar; Stimulation, Chemical; Vasoconstrictor Agents

In the present study the cardiovascular effects of intracerebroventricularly (i.c.v.) applied angiotensin II (AN II) and angiotensin III (AN III) were analysed in conscious Wistar rats. The baroreceptor heart reflex (BHR) was elicited by intravenous bolus injection of both phenylephrine (1 microgram) and sodium nitroprusside (5 micrograms) before and after i.c.v. administration (1.5 and 15 min) of the peptides. Administration of 20 ng and 200 ng AN II produced a short increase in inter-beat interval (IBI) and a long-lasting increase in mean blood pressure (MBP), inclusive of a drinking response. Only after the high dose of 200 ng AN II we found a continuous impairment in the BHR for reflex bradycardia. Inversely, the small doses of both 100 pg AN II and 100 pg AN III were without effects on IBI and MBP; they induced an enhancement in BHR for the reflex bradycardia and after 100 pg AN II it was also found for the reflex tachycardia. Pretreatment with 20 nmol amastatin (AM), a specified aminopeptidase A inhibitor, followed by 100 pg An II suppressed the enhancement in BHR. AM alone was without effects in this respect. These findings suggest that: 1) the influence of central angiotensin on the BHR could be dose-dependent in the opposite way and 2) AN III seems to be the active form and involved in the central blood pressure regulatory mechanism.

Topics: Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Baroreflex; Blood Pressure; Drinking; Heart Rate; Injections, Intraventricular; Male; Nitroprusside; Peptides; Phenylephrine; Rats; Rats, Wistar

Microinjection of angiotensin II and III into the rostral ventrolateral medulla of anesthetized barodenervated rabbits elicited in both cases pressor responses, which were of similar magnitude and time course. The responses to angiotensin II and III were either unchanged or increased in the presence of compounds which inhibit their degradation to shorter length peptides. The results indicate that both angiotensin peptides are independently capable of eliciting pressor responses in the rostral ventrolateral medulla.

Topics: Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Blood Pressure; Histocytochemistry; Horseradish Peroxidase; Leucine; Medulla Oblongata; Microinjections; Oligopeptides; Peptides; Rabbits; Sodium; Wheat Germ Agglutinin-Horseradish Peroxidase Conjugate; Wheat Germ Agglutinins

In homogenates of the endothelium and smooth muscle cum adventitia of the rat aorta, exogenous angiotensin (ANG) I was found to be degraded to des-aspartate-ANG I (des-Asp-ANG I) instead of ANG II. ANG II and ANG III were not detectable in either of the homogenates after 5, 10 and 30 min of incubation with the decapeptide. However, both the homogenates were able to catalyse hippuryl-L-histidyl-L-leucine (HHL) to hippuric acid and the catalysis was completely inhibited by 3 microM captopril. The data show that the angiotensin converting enzyme (ACE) present in the homogenates of rat aorta, prepared by normal laboratory procedures, is not able to hydrolyse ANG I to ANG II. This finding has important consequences in the study of vascular ACE as the assay of the enzyme is often carried out using crude homogenate and HHL or other artificial substrates. In addition, the aminopeptidase that degraded ANG I to des-Asp-ANG I was not inhibited by either amastatin or bestatin, indicating that it was not aminopeptidase A or B. Together with the recent findings of other investigators which show that the de novo production of ANG II in vascular tissues is stimulated and inhibited by beta- and alpha-agonists, respectively, our present data may suggest that production of vascular ANG II occurs only in intact tissues and is probably under adrenergic regulation.

Topics: Amino Acid Sequence; Aminopeptidases; Angiotensin I; Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Aorta; Endothelium, Vascular; Leucine; Male; Molecular Sequence Data; Muscle, Smooth, Vascular; Oligopeptides; Peptides; Rats; Rats, Sprague-Dawley

1. Experiments were performed with peptidase inhibitors on rabbit aortic strip preparations, to determine whether endogenous peptidase activity can influence the potency estimates for angiotensin receptor agonists and antagonists in this tissue. 2. Angiotensin II (A II) and angiotensin III (A III) both induced concentration-related contractions of rabbit aortic strip preparations. A III was approximately 38 fold less potent than A II, and the gradient of the A III concentration-response curve (1.00 +/- 0.04) was significantly more shallow than that (1.76 +/- 0.05) of the A II curve. 3. Neither the aminopeptidase-A and -M inhibitor, amastatin, nor the aminopeptidase-B and -M inhibitor, bestatin, affected the potency of, or the maximum response to, A II. In contrast, the potency of A III was increased by both amastatin and bestatin. Amastatin had the most marked effect and at 10 microM caused approximately a 12 fold increase in the potency of A III (EC50 values, 102 nM and 8.6 nM in the absence and presence of amastatin, respectively), and also significantly steepened the gradient of the A III concentration-response curve. Amastatin did not affect the position or shape of the concentration-response curve to the alpha 1-adrenoceptor agonist, phenylephrine. Finally, the carboxypeptidase-N inhibitor, D-L-mercaptomethyl-3-guanidine-ethylpropanoic acid (MERGETPA) did not change the position or shape of the concentration-response curves to either A II or A III.4. In the presence of amastatin, the potency of the peptide angiotensin receptor antagonist, Ile7-A III (100nM-l microM ), was increased approximately 13 fold (pA2, with A II as the agonist, 7.0 +/- 0.1 and 8.1 +/- 0.1, in the absence and presence of amastatin, respectively). However, the potency of the nonpeptide angiotensin receptor antagonist, DuP 753 (30-300 nM), was little affected by amastatin (pA2, 8.2 +/- 0.1 and 8.1 +/- 0.1 in the absence and presence of amastatin, respectively).5. The results of this study suggest that endogenous aminopeptidase activity in the rabbit thoracic aorta can profoundly affect estimates of the potency of peptide angiotensin receptor agonists and antagonists.A suitable aminopeptidase inhibitor should therefore be included in studies, using this tissue, which aim to classify angiotensin receptor subtype(s) based on the rank order of peptide angiotensin receptor agonist and/or antagonist potencies.

Topics: 3-Mercaptopropionic Acid; Adrenergic alpha-Agonists; Amino Acid Sequence; Aminopeptidases; Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Aorta, Thoracic; Dose-Response Relationship, Drug; In Vitro Techniques; Leucine; Male; Molecular Sequence Data; Muscle Contraction; Oligopeptides; Peptides; Phenylephrine; Rabbits; Receptors, Angiotensin; Vasoconstriction

1. The depolarizing responses to angiotensin II and angiotensin III of the rat superior cervical ganglion have been characterized in vitro, by the use of peptidase inhibitors, peptide and non-peptide antagonists and dithiothreitol (DTT). 2. Angiotensin II and III depolarized the ganglion in a concentration-related manner. Angiotensin II was approximately 30 fold more potent than angiotensin III. 3. The endopeptidase inhibitor, bacitracin, increased the potency of angiotensin II and III by approximately 4 and 20 fold respectively. The aminopeptidase inhibitor, amastatin, further increased the potency of angiotensin III (but not angiotensin II) by approximately 4 fold. In the presence of bacitracin and amastatin, angiotensin II and III were equipotent. 4. The peptide antagonist [Ile7]angiotensin III (0.01-0.3 microM) produced a non-parallel rightward displacement of the angiotensin II concentration-response curve, with a suppression of the maximum response. The potency of [Ile7]angiotensin III was increased by bacitracin and amastatin. 5. The AT1-selective non-peptide antagonist losartan (DuP 753; 0.03 and 0.1 microM) produced a parallel rightward displacement of the angiotensin II concentration-response curve, with an apparent pKB of 8.3 +/- 0.1. A higher concentration of losartan (0.3 microM) depressed the maximum agonist response by 32 +/- 6.5%, possibly reflecting non-competitive behaviour of the antagonist. The potency of losartan was not influenced by bacitracin. 6. The AT2-selective non-peptide antagonist, PD123177 (3 microM) failed to antagonize the angiotensin II-induced depolarizations. 7. DTT (1 mM) produced a 22% reduction of the maximum response to angiotensin II.8. We conclude that the angiotensin II-induced depolarizations of the rat superior cervical ganglion are mediated by angiotensin II receptors of the AT1 subclass. The ability of peptidase inhibitors to modify the potency of peptide agonists and antagonists highlights the difficulties associated with the use of peptide agents to characterize angiotensin II receptors in this preparation.

Topics: Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Bacitracin; Biphenyl Compounds; Dithiothreitol; Ganglia, Sympathetic; Imidazoles; In Vitro Techniques; Losartan; Male; Neuromuscular Depolarizing Agents; Oligopeptides; Peptides; Protease Inhibitors; Rats; Tetrazoles

Opioid peptides are present in human cerebrospinal fluid (CSF), and their levels are reported to change in some pathologic conditions. However, less is known about their degradation in CSF. In the present study, human CSF was found to contain aminopeptidase activity which hydrolyzed alanyl-, leucyl- and arginyl-naphthylamides in a ratio of 100:28:27. Twelve CSF samples hydrolyzed alanyl-2-naphthylamide and degraded Met5-enkephalin (N-terminal hydrolysis) at rates of 188 +/- 38 and 420 +/- 79 pmol/min/mL respectively. Further, the distribution of alanyl-naphthylamidase activity in individual samples (39-437 pmol/min/mL) was closely correlated with that of Met5-enkephalin degradation (37-833 pmol/min/mL). Both alanyl-naphthylamidase and enkephalin degradation were optimal at pH 7.0 to 7.5 and were inhibited by aminopeptidase inhibitors amastatin (IC50 = 20 nM), bestatin (4-7 microM) and puromycin (30-35 microM). Conversely, degradation was unaffected by inhibitors of neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin converting enzyme (captopril). The Km of Met5-enkephalin for the CSF aminopeptidase activity was 201 +/- 19 microM (N = 4). Rates of hydrolysis of the Tyr1-Gly2 bond of larger opioid peptides decreased with increasing peptide length. Pooled, concentrated CSF hydrolyzed Leu5-enkephalin, dynorphin A fragments [1-7], [1-10] and [1-13] and dynorphin A at rates of 2.05 +/- 0.27, 1.27 +/- 0.18, 0.94 +/- 0.06, 0.55 +/- 0.14 and 0.16 +/- 0.03 nmol/min/mL respectively. When analyzed by rocket-immunoelectrophoresis against antisera to aminopeptidase M (EC 3.4.11.2), the concentrated CSF formed an immunoprecipitate which could be stained histochemically for alanyl-naphthylamidase activity. These data are consistent with a significant role for aminopeptidase M activity in the degradation of low molecular weight opioid peptides in human CSF.

Topics: Aminopeptidases; Angiotensin III; Anti-Bacterial Agents; CD13 Antigens; Endorphins; Enkephalin, Methionine; Humans; Hydrogen-Ion Concentration; Hydrolysis; Immunoelectrophoresis; Molecular Weight; Oligopeptides; Peptides

Rats received the aminopeptidase inhibitors amastatin (AM) and bestatin (BE), and carboxypeptidase inhibitor Plummer's (PL) via intracerebroventricular infusion in various combinations, i.e. PL alone, AM + BE, and a cocktail consisting of AM + BE + PL. Blood pressure responses were recorded and a postinfusion sample of cerebrospinal fluid (CSF) was radioimmunoassayed for endogenous angiotensin levels. Results indicate that CSF angiotensin was increased approximately 1.5x over control levels when PL was infused; a 2.5x increase accompanied AM + BE administration; and a 10.3x elevation was measured when all 3 inhibitors were infused as a cocktail. Concomitant elevations in blood pressure accompanied increased concentrations of angiotensin. We conclude that endogenous levels of angiotensin can be significantly increased in the ventricular space when a combination of these inhibitors is utilized to protect both the amino and carboxyl terminals of the angiotensin molecule from enzymatic degradation.

Topics: 3-Mercaptopropionic Acid; Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Blood Pressure; Carboxypeptidases; Cerebral Ventricles; Injections, Intraventricular; Leucine; Male; Oligopeptides; Peptides; Protease Inhibitors; Radioimmunoassay; Rats; Rats, Inbred Strains

Two D-amino acid substitution angiotensin analogues were compared against native angiotensin II (AII) and angiotensin III (AIII) for their resistance to brain tissue-induced degradation and for pressor potency when intracerebroventricularly (i.c.v.) infused in Sprague-Dawley rats. The in vitro results indicate that [D-Asp1]AII was very resistant to degradation, AII and [D-Arg1]AIII were degraded at similar rates, while AIII was the most rapidly degraded. In vivo results revealed that AII, AIII and [D-Arg1]AIII produced greater pressor responses than [D-Asp1]AII. Intracerebroventricular pretreatment with the aminopeptidase A inhibitor, amastatin, significantly reduced the subsequent pressor response to i.c.v. infused [D-Asp1]AII presumably by inhibiting its conversion to AIII. In contrast, pretreatment with the aminopeptidase B inhibitor, bestatin, potentiated the subsequent pressor response to i.c.v. infused [D-Arg1]AIII, presumably by inhibiting the conversion of [D-Arg1]AIII to the less active hexapeptide AII(3-8). Next, i.c.v. pretreatment with the specific angiotensin receptor antagonist, [Sar1, Thr8]AII (Sarthran) was found to greatly diminish the subsequent pressor responses to i.c.v. infused [D-Asp1]AII and [D-Arg1]AIII, suggesting that these analogues are having their effect at the same brain angiotensin receptor site. These results support the hypothesis that AIII, or AIII-like ligands, may serve as the active form of brain angiotensin.

Topics: Aminopeptidases; Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Blood Pressure; Dose-Response Relationship, Drug; Enzyme Inhibitors; Injections, Intraventricular; Leucine; Male; Oligopeptides; Peptides; Rats; Rats, Inbred Strains

Recent iontophoretic data suggest that conversion of angiotensin II (AII) to angiotensin III (AIII) may be necessary before the peptide can activate central angiotensin-sensitive neurons. Furthermore, this conversion may be inhibited by the aminopeptidase A inhibitor, amastatin. In the present study we investigated the importance of aminopeptidase activity on central angiotensin-induced pressor responses. Intracerebroventricular (i.c.v.) pretreatment with amastatin, suppressed i.c.v. AII-induced pressor responses. Pretreatment with the aminopeptidase B inhibitor, bestatin, increased pressor responses to AIII. Pressor responses induced by the aminopeptidase-resistant analogue, [Sar1]angiotensin II, were not affected by pretreatment with angiotensin inhibitors. These results support the hypothesis that AII must be converted to AIII to be active in the brain.

Topics: Aminopeptidases; Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Blood Pressure; Cerebral Ventricles; Injections, Intraventricular; Leucine; Male; Oligopeptides; Peptides; Rats; Rats, Inbred Strains; Reference Values

Improved immunohistochemical and quantitative microiontophoretic methods were used to characterise angiotensinergic and angiotensin-sensitive neurones in the paraventricular nucleus (PVN) of the rat. The results can be summarised as follows: 1) Angiotensinogen was found in PVN neurones, astrocytes in the diencephalon which make putative contacts with microvessels, and in cells of the choroid plexus. 2) Affinity-purified angiotensin II/III antibodies were used to locate immunoreactive AII/III in large PVN neurones and their fibre tracts which project either caudally or ventrally to the neurohypophysis. 3) Quantitative microiontophoretic studies showed that PVN neurones are more sensitive to angiotensin II than to angiotensin II. 4) Iontophoretic co-application of the selective aminopeptidase inhibitors bestatin and amastatin, together with angiotensin II and angiotensin III produced results consistent with a central role for angiotensin III.

Topics: Aminopeptidases; Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Immunohistochemistry; Iontophoresis; Leucine; Male; Oligopeptides; Paraventricular Hypothalamic Nucleus; Peptides; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY

In the metabolism of angiotensin peptides by tissue angiotensinases, aminopeptidases A, B, M and leucine aminopeptidase have been identified as being particularly effective. Because the inhibitory actions of amastatin (AM) and bestatin (BE) are relatively specific for these aminopeptidases, we have examined the effects of these inhibitors on the binding, degradation and pressor activity of angiotensin II (AII) and angiotensin III (AIII). Within 30 min at 37 degrees C, significant metabolism of 125I-AII and 125I-AIII by homogenates of a block of tissue containing hypothalamus, thalamus, septum and anteroventral third ventricle regions of the brain was observed. A majority of 125I-AIII metabolism was due to soluble peptidases, whereas that of 125I-AII primarily resulted from membrane-bound peptidases. AM, BE and reduced incubation temperatures significantly decreased the metabolism of 125I-AII and 125I-AIII. After appropriate adjustments to reflect the proportion of intact radioligand bound, temperature- or inhibitor-induced decreases in metabolism were matched by corresponding increases in specific binding. Heat-treated bovine serum albumin, as a nonspecific peptidase inhibitor, had no effect on either the metabolism or binding of the ligands used. In accordance with their actions in vitro, i.c.v. administration of AM and BE prolonged the pressor activity of subsequently applied AII and AIII. Unexpectedly, the amplitude of the pressor response to AIII was increased by BE, whereas that to AII was decreased by AM. The results of this study indicate that the metabolism of AII and AIII by aminopeptidases is relatively specific and acts to modulate the actions of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aminopeptidases; Angiotensin II; Angiotensin III; Animals; Anti-Bacterial Agents; Blood Pressure; Iodine Radioisotopes; Leucine; Male; Oligopeptides; Peptides; Rats; Rats, Inbred Strains

In superfused anterior pituitary reaggregate cell cultures angiotensin II (AII) stimulated both spontaneous and dopamine-inhibited prolactin (PRL) release from subnanomolar concentrations. Angiotensin I (AI) and angiotensin III (AIII) also stimulated PRL release. The magnitude and rate of response to AI was equal to or only slightly lower than that to AII. However, the angiotensin converting enzyme (ACE) inhibitors captopril and teprotide (1 microM) completely abolished the PRL response to 0.1 nM AI and strongly reduced that to 1 nM AI. The intrinsic activity of AIII was lower than that of AII but could be enhanced by adding 2 microM of the aminopeptidase inhibitor amastatin to the superfusion medium. After withdrawal of AIII, PRL secretion rate rapidly returned to baseline levels, whereas after withdrawal of AI or AII, secretion fell to a level remaining significantly higher than basal release. The present findings indicate that stimulation of PRL release by AI is weak unless it is converted into AII by ACE and that aminopeptidase may be important in determining the magnitude and termination of the PRL response. Furthermore, the active peptides induce a different pattern of response.

Topics: Angiotensin I; Angiotensin II; Angiotensin III; Angiotensins; Animals; Anti-Bacterial Agents; Captopril; Cells, Cultured; Dopamine; Dose-Response Relationship, Drug; Male; Oligopeptides; Peptides; Pituitary Gland, Anterior; Prolactin; Radioimmunoassay; Rats; Rats, Inbred Strains