Bradykinin inactivation by perfused rat liver

Summary Previous data had suggested the presence of at least two enzymes in the perfused rat liver — a peptidyldipeptide hydrolase (EC 3.4.15.1) which inactivates bradykinin (BK) and converts angiotensin I (AI) to angiotensin II (AII), and an enzyme which inactivates AII. However, in the present studies the amides of BK and bradykinylglycine were inactivated by the perfused liver at the same rate as BK suggesting that the inactivation of BK was due not only to the presence of the peptidyldipeptide hydrolase which requires a free carboxyl group but to an additional, as yet unidentified kininase. A 10-min perfusion of rat liver with oxygenated Tyrode solution containing 0.05% (v/v) Triton X-100 liberated significant amounts of kininase, potassium, lactic dehydrogenase and acid phosphatase into the perfusion medium.DEAE-cellulose chromatography of this perfusate purified the kininase activity, which does not hydrolyze either AI or AII. BK hydrolysis at pH 7.0 by this enzyme was inhibited by 1×10^–3 M sodium tetrathionate but not by 3×10^–3 M EDTA or BPP_9a (10 g/ml), a BK-potentiating nonapeptide which inhibits the peptidyldipeptide hydrolase. The endopeptidase splits BK between the Phe⁵-Ser⁶ bond forming the peptides Arg¹-Phe⁵ and Ser⁶-Arg⁹ in stoichiometric amounts. It may be crucial for the BK inactivation by the perfused rat liver.Work supported by grants from FINEP, Rio de Janeiro and FAPESP, São PauloFrom Department of Medicine, EPMResearchers from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq), Rio de Janeiro.     

Prostacyclin (PGI2) release accompanying angiotensin conversion in rat mesenteric vasculature

The relationship between angiotensin conversion and release of prostaglandins (PGs) was studies in isolated, perfused, mesenteric vasculature of rats. PGs in the mesenteric effluent were detected by bovine coronary artery and rat stomach strip, and by radioimmunoassay of 6-oxo-PGF_1α and PGE₂. Angiotensin II (AII) was assayed simultaneously using rabbit aorta. Both angiotensin I (AI) and AII released a prostacyclin (PGI₂)-like substance, but little PGE₂ from the mesentery, AII being 5 times more potent. Indomethacin (2.8 μM) abolished angiotensin-induced release of PGI₂. Of the AI (0.2–1.5 nmol) injected into the perfusion medium, 3–8% was converted to AII in passage through the mesentery. Captopril (4 μM), infused through the mesentery, inhibited AI-induced PGI₂ release, but did not affect release induced by AII. Release of the PGI₂-like substance by higher doses of AI (2–20 nmol) in the presence of captopril was always accompanied by concomitant contraction of rabbit aorta, indicating residual conversion of AI. Infusion of (Sar¹, Ala⁸)AII (3–30 nM) through the mesentery abolished PGI₂ release by both peptides. By choosing doses of AI and AII which produced equivalent amounts of AII in the mesenteric effluent, it appeared that AII generated locally in the mesenteric vasculature was a more effective stimulus for PGI₂ generation than AII in the perfusion fluid. There is no evidence for intrinsic activity of AI, and release of the PGI₂-like substance appears to be mediated through an AII receptor.     

The effect of kinins on paw oedema and uterus in rats

Summary Bradykinin (BK) produced a dose-related increase in the paw volume of the rat. Responses to BK at all doses used were not affected by pretreating the rats with diphenhydramine, 1 mg kg^–1, or indomethacin, 2.5 and 5 mg kg^–1. Indomethacin, 10 mg kg^–1 produced a small but significant reduction in the responses to BK. Captopril 1 mg kg^–1 enhanced responses to low but not to high doses of BK.The rank order of potency of various kinin analogues to increase paw volume was found to be methionyl-lysyl-BK (met-lys-BK) > BK > kysyl-BK (Kallidin) des-Arg⁹-BK. The B₁-receptor antagonist des-Arg⁹-Leu⁸-BK did not affect responses to BK on paw volume.Two modified kinin fragments S2302 (H-D-Pro-Phe-Arg-p-Nitroaniline) and S2441 (H-D-Pro-Phe-Arg-NH-heptyl) produced dose-related increases in paw volume both having approximately half the potency of BK. These responses were not anagonised by diphenhydramine, 1 mg kg^–1 which reduced significantly the response to histamine.On the isolated rat uterus the rank order of potency of various kinins was BK > Kallidin > met-lys-BK > des-Arg⁹-BK. The two modified kinin fragments S2302 and S2441 (but not des-Arg⁹-Leu⁸-BK) antagonised BK induced contractions of the rat uterus.From the rank order of potency studies the receptor mediating contraction of the rat uterus in vitro and increase in rat paw volume to BK appear to be of the same type. Whether the differences in the action of S2302 and S2441 in the rat paw and rat uterus reflect a real difference in receptor type requires further study.     

Prejunctional modulation by angiotensins of noradrenaline release from sympathetic neurons in isolated rabbit aorta

The aims of the present work were to compare the modulating effect of angiotensins I, II, III, IV and (1–7) [AI, AII, AIII, AIV and A(1–7) respectively] on stimulation-evoked noradrenaline release from post-ganglionic sympathetic nerves in rabbit isolated aorta; to examine the influence of inhibiting the neuronal and extraneuronal uptake of noradrenaline on the modulating effect of AII and thirdly, to determine the role of angiotensin converting enzyme (ACE) in the modulating effects of AI and AII and the role of aminopeptidases A and M in the effects of AII and AIII. Rings of aorta were preloaded with (–)-³H-noradrenaline and then subjected to electrical field stimulation. Cumulative addition of AI (10^–8–10^–6M), AII (3×10^–11–10^–8M) and AIII (3×10^–10–10^–6M) enhanced the stimulation-evoked ³H-overflow up to 142, 165 and 188% respectively. The order of potency was AII > AIII > AI. AIV (10^–10–10^–7M) and A(1–7) (10^–10–10^–7M) caused no change. Single concentrations (10^–9–10^–7M) of AI, AII and AIII caused initial enhancement which subsequently decreased, i.e. development of tachyphylaxis. The effect of AII was independent of stimulation frequency at 1–10Hz, but absent at 30Hz. Cocaine (3×10^–5M) plus corticosterone (4×10^–5M) did not alter the enhancing effect of AII. CaNa₂EDTA (3×10^–5M) did not alter the enhancing effect of AI. Captopril (10^–6 and 10^–5M) and lisinopril (10^–6M) attenuated the enhancing effect of AI. Captopril and lisinopril (both 10^–6 and 10^–5M) did not alter the enhancing effect of AII. Captopril (10^–7– 10^–4M) and lisinopril (10^–7–10^–4M) themselves did not alter the stimulation-evoked ³H-overflow. Amastatin (10^–5M) increased the enhancement seen with AIII (3×10^–11–10^–9M) but did not alter the enhancing effect of AII (10^–9–10^–8M). Amastatin (10^–9–10^–5M) had no effect on the stimulation-evoked ³H-overflow. It is concluded that AI, AII and AIII facilitate the stimulation-evoked ³H-noradrenaline release to various degrees (relative order of potency: AII > AIII > AI and of efficacy: AIII > AII > AI). The estimates may be compromised by the development of tachyphylaxis. The facilitation by AII was independent of the neuronal and extraneuronal uptake mechanisms. The action of AI is in part due to its conversion to AII. The effect of AIII was probably underestimated due to its degradation to AIV. AII is apparently not a substrate for aminopeptidase M. Received: 10 September 1996 / Accepted: 18 August 1997     

Conversion of angiotensin I to angiotensin II and inactivation of bradykinin in canine peripheral vascular beds

We studied the effects of angiotensin I (AI), angiotensin II (AII), and bradykinin (BK) on systemic blood pressure and blood flow in the renal, mesenteric, coeliac, external iliac, and internal iliac vascular beds in the presence and absence of the converting enzyme inhibitor captopril in pentobarbital-anesthetized dogs. AI and AII caused dose-dependent pressor responses and blood flow reductions, and BK caused dose-related depressor responses and an increase in blood flow. The pressor response to AI was decreased and the depressor response to BK was potentiated by captopril. However, the effectiveness of captopril was greater when the peptides were injected intravenously than when they were injected intraaortically. The average conversion of AI to AII in the mesenteric, coeliac, external iliac, internal iliac, and renal vascular beds was 45.7, 31.6, 30.6, 28.0, and 8.9%, respectively. The dose ratio of equipotent BK-induced responses before and after captopril in the mesenteric, coeliac, external iliac, internal iliac, and renal vascular beds was 4.6, 3.5, 2.8, 2.5 and 1.4, respectively. This order was similar to that of the percentage conversion of AI to AII. These results suggest that the greatest proportion of angiotensin conversion and BK inactivation occurs in the pulmonary circulation and that the least proportion occurs in the kidney.     

The kinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin: an antagonist of the angiotensin AT1 receptor which also binds to the AT2 receptor.

1. Agonists and antagonists of kinin B1 and B2 receptors were evaluated in vitro for their effects against angiotensin II (AII)-induced contractile responses in the rabbit aorta and for their binding properties to angiotensin AT1 and AT2 receptors from purified membrane of rat liver and lamb uterus respectively. 2. In aortic rings, the kinin B1 receptor antagonist, des-Arg9-[Leu8]bradykinin (BK) (3-100 microM) caused a concentration-dependent decrease in sensitivity and a depression of the maximum response to AII. Des-Arg10-[Leu9]kallidin (KD), des-Arg9-BK, des-Arg10-KD, BK or KD at 3 microM had no effect against AII-induced contractions. 3. Des-Arg9-[Leu8]BK (3 or 100 microM) did not affect contractions of aortic rings to histamine, potassium chloride, endothelin-1, 5-hydroxytryptamine, noradrenaline and the thromboxane A2-mimetic, U46619. 4. Des-Arg9-[Leu8]BK displaced [125I]-Sar1-AII binding to the AT1 subtype in rat liver membranes with a Ki value of 1.1 +/- 0.4 microM. Values of Ki for des-Arg9-BK and KD were 45 +/- 13 microM and 25 +/- 22 microM, respectively. The other kinin derivatives des-Arg10-KD, BK and des-Arg10-[Leu9]KD at concentrations up to 100 microM did not bind to the AT1 receptor. 5. All the kinin derivatives except BK bound to AT2 receptors in lamb uterus membranes. Values of Ki for des-Arg9-[Leu8]BK, des-Arg10-[Leu9]KD, des-Arg9-BK, des-Arg10-KD and KD were 0.3 +/- 0.1, 0.7 +/- 0.1, 1.2 +/- 0.3, 1.5 +/- 0.3 and 7.0 +/- 1.6 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of bradykinin and angiotensin in the regulation of blood pressure in conscious rats

1. The interaction between bradykinin (BK) and the renin-angiotensin system was studied in conscious, catheterized rats. 2. Intravenous injection of BK induced dose-dependent decreases in blood pressure in normotensive Wistar and Wistar-Kyoto rats and spontaneously hypertensive rats. Pretreatment with the angiotensin-converting enzyme (ACE) inhibitor captopril markedly enhanced the effect of BK, such that the dose-response curve shifted significantly to the left in all three strains. 3. In a second series of experiments, captopril did not change basal blood pressure, but blocked the pressor response to angiotensin I (AI), but not angiotensin II (AII). 4. The partial agonist Sar1-Ala8-angiotensin II (SAR) increased blood pressure and blocked the pressor response to subsequent AII treatment. 5. After pretreatment with BK (50 micrograms/kg), captopril evoked a decrease in blood pressure, while still blocking the effect of AI. 6. After pretreatment with BK, SAR decreased blood pressure, while still antagonizing the action of AII. 7. These results suggest that ACE plays a role in the inactivation of circulating BK in normotensive and hypertensive rats. Conversely, BK can influence the activity of the renin-angiotensin system, probably by interacting with ACE.     

Potentiation of the pro-inflammatory effects of bradykinin by inhibition of angiotensin-converting enzyme and aminopeptidase P in rat paws

The influence of some peptidase inhibitors on oedema and plasma extravasation induced by bradykinin and carrageenan in rat paw was evaluated. Bradykinin-induced oedema in normal rats was increased by o-phenanthroline (3.10^–2 M), by captopril (10^–6 M to 10^–4 M), by lisinopril (10^–6 M to 10^–4 M), or by lisinopril (10^–5 M) in combination with apstatin (8.10^–5 M or 1.4 10^–4 M). It was not modified by phosphoramidon (10^–6 M to 10^–5 M) and by diprotin A (10^–3 M). It was increased by mergepta at high concentrations (2.10^–4 M). Mergepta did not increase the potentiating effect of captopril. Carrageenan-oedema in normal rats was increased by captopril (10^–5 M), lisinopril (10^–5 M) and apstatin (1.4 10 M). It was not modified by mergepta (10^–4 M), phosphoramidon (10^–5 M) and diprotin A (10^–3 M). Des-Argl-bradykinin and Des-Arg⁹-bradykinin have low oedema-promoting effects. Captopril (10^–5 M) increased the effects of bradykinin but not those of carrageenan in kininogen-deficient Brown Norway rats. Angiotensin-converting enzyme and amino-peptidase P appear to be main kinin-inactivating enzymes in rat paws. Carboxypeptidase N, neutral endopeptidase 24.11 and dipeptidyl(amino)peptidase IV do not play a significant role in this inactivation.     

Comparison of angiotensin converting enzyme inhibitors captopril and MK421-diacid in guinea pig atria

Angiotensin I (AI) and angiotensin II (AII) caused concentration-dependent increases in atrial rate in guinea pig isolated right atria. Converting enzyme inhibitors captopril and MK421-diacid did not alter the responses to AII but displaced the curves to AI to the right. The atrial response to generated AII from AI was used as a bioassay to estimate the dissociation constants of converting enzyme inhibitors (Kb) and test for kinetics of simple competition. MK421-diacid was 12-40 times more potent than captopril. However, estimations of Kb for captopril and MK421-diacid were unsatisfactory because at high concentrations of inhibitors the curves to AI were not displaced according to simple competition. We conclude that AI in high concentration can stimulate AII receptors accounting for the stationary displacement of curves to AI in the presence of converting enzyme inhibitors. MK421-diacid also potentiates responses to bradykinin in this assay.     

Sequential development of angiotensin receptors and angiotensin I converting enzyme during angiogenesis in the rat subcutaneous sponge granuloma

1. The vasoconstrictor peptide antiotensin II (AII) can stimulate angiogenesis, an important process in wound healing, tumour growth and chronic inflammation. To elucidate mechanisms underlying AII-enhanced angiogenesis, we have studied a subcutaneous sponge granuloma model in the rat by use of ¹³³Xe clearance, morphometry and quantitative in vitro autoradiography.
2. When injected directly into the sponge, AII (1 nmol day⁻¹) increased ¹³³Xe clearance from, and fibrovascular growth in sponge granulomas, indicating enhanced angiogenesis 6 to 12 days after implantation. This AII-enhanced angiogenesis was inhibited by daily doses (100 nmol/sponge) of the specific but subtype non-selective AII receptor antagonist (Sar¹, Ile⁸)AII, and by the selective non-peptide AT₁ receptor antagonists losartan and DuP 532. In contrast, AII-enhanced neovascularization was not inhibited by the AT₂ receptor antagonist PD123319, nor was it mimicked by the AT₂ receptor agonist CGP42112A (each at 100 nmol/sponge day⁻¹).
3. AI (1 nmol/sponge day⁻¹), the angiotensin converting enzyme (ACE) inhibitors captopril (up to 100 μg/sponge day⁻¹) and lisinopril (40 μg/sponge day⁻¹), or AII receptor antagonists did not affect angiogenesis in the absence of exogenous AII.
4. [¹²⁵I]-(Sar¹, Ile⁸)AII binding sites with characteristics of AT₁ receptors were localized to microvessels and to non-vascular cells within the sponge stroma from 4 days after implantation, and were at higher density than in skin throughout the study.
5. [¹²⁵I]-(Sar¹, Ile⁸)AII binding sites with characteristics of AT₂ receptors were localized to non-vascular stromal cells, were of lower density and appeared later than did AT₁ sites.
6. The ACE inhibitor [¹²⁵I]-351A bound to sites with characteristics of ACE, 14 days after sponge implantation. [¹²⁵I]-351A bound less densely to sponge stroma than to skin.
7. We propose that AII can stimulate angiogenesis, acting via AT₁ receptors within the sponge granuloma. AT₁ and AT₂ receptors and ACE develop sequentially during microvascular maturation, and the role of the endogenous angiotensin system in angiogenesis will depend on the balanced local expression of its various components. Pharmacological modulation of this balance may provide novel therapeutic approaches in angiogenesis-dependent diseases.

     

Effect of hypoxia on the conversion of angiotensin I to II in the isolated perfused rat lung

Acute hypoxia in the intact animal and in cultured endothelial cells has been shown to be associated with a decrease in conversion of angiotensin I (AI) to angiotensin II (AII). Alterations in capillary surface and in contact time resulting from hemodynamic changes have been shown to influence the rate of pulmonary AI conversion. The dependency of AI conversion on hemodynamics complicates the interpretation of experiments showing changes in AI conversion in intact animals. We studied the effect of acute hypoxia on AI conversion in the isolated rat lung perfused at constant flow without recirculation of perfusate. Three levels of oxygenation were produced by ventilating lungs and equilibrating perfusate with a range of hypoxic gas mixtures. AI (1 μg) was injected into the pulmonary artery, and the effluent was collected for measurement of AI and All. Instead of the expected hypoxic inhibition, percent conversion of AI to All increased slightly but significantly from 69.3 ± 3.1 (mean ± S.E.M.) at normal oxygenation to 74.4 ± 3.0 at moderate hypoxia (P < 0.005, paired t) and to 73.5 ± 3.9 at severe hypoxia (P < 0.01, paired t). Decreasing mean transit time of substrate through the lung (by increasing perfusate flow rate from 5 to 20 ml/min) resulted in a significant decrease in conversion of AI from 88.7 ± 2.9 to 73.4 ± 2.1% (P < 0.001, paired t). These data confirm the effect of contact time on the rate of AI conversion in the lungs. The isolated rat lung preparation does not exhibit the phenomenon of hypoxia-induced inhibition of AI conversion. The authors speculate that hypoxia-induced inhibition of AI conversion in vivo may be secondary to the effects of hypoxia on hemodynamics.     

Characterization of the angiotensin II-receptor subtype in the longitudinal smooth muscle of the rat portal vein

Summary The purpose of the present study was to identify the angiotensin II-receptor subtype involved in the enhancement of the amplitude of the phasic contractions by angiotensin II in the isolated rat portal vein preparation.At an extracellular Ca²⁺ concentration of 0.9 mmol/l and a K⁺ concentration of 4 mmol/l, angiotensin II induced concentration-dependent increases in the amplitude of the phasic contractions. The enhancement of phasic contraction amplitude caused by angiotensin II was not significantly altered by pretreatment of the rat portal vein with indomethacin 10^–5 mol/l or nitro-L-arginine 10^–4 mol/l, indicating that neither prostaglandins nor the endothelium derived-relaxing factor (NO) are involved. Losartan (DuP 753), a nonpeptide selective AT₁-receptor antagonist, concentration-dependently shifted the concentration-response curve for the effect of angiotensin II on the amplitude of the contractions to the right, without reducing the maximal response (pA₂ = 8.6, slope = 0.98), thus suggesting competitive antagonism at the level of AT₁-receptors. By contrast, PD 123177, a nonpeptide selective AT₂-receptor antagonist, even at 10^–5 mol/l, caused no significant change of the phasic myogenic response to angiotensin II, indicating the absence of AT₂-receptor involvement. Dithiothreitol, a disulfide-reducing agent which is known to inactivate AT₁-receptors in various tissues, markedly inhibited (3 mmol/l) or even abolished (5 mmol/l) the contractile response of the rat portal vein to angiotensin II, supporting the conclusion that these receptors can be classified as AT₁-receptors.In conclusion, the receptor subtype mediating the angiotensin II-induced potentiation of the spontaneous phasic contractions in the rat portal vein appears to belong to the AT₁-receptor subtype.Preliminary data were presented at the Spring Meeting of the German Pharmacological Society, Mainz, 1992Correspondence to J. S. Zhang at the above address     

Analysis of the mechanism of action of bradykinin on human basilar artery in vitro

Summary Bradykinin (BK) initially produced concentration-related relaxations of human basilar artery in vitro. Concentration-effect curves constructed at 2 h intervals to BK over an 8 h period were reproducible. The rank order of potency of three kinins on the human basilar artery was found to be BK > methionyl-lysyl-BK > des-Arg⁹-BK. The B₂-receptor antagonist Thi^5,8 d-Phe⁷-BK but not the B₁-receptor antagonist des-Arg⁹-Leu⁸-BK selectively blocked BK-induced relaxations of the human basilar artery.The relaxant effects of bradykinin and acetylcholine but not papaverine were attenuated after removal of the endothelium or treating the tissues with BW755C. Indomethacin was without effect. Concentration-effect curves to angiotensin I were markedly attenuated by captopril at a concentration which had no effect on BK, angiotensin II or 5-hydroxytryptamine responses. It is concluded that BK induced relaxations of the human basilar artery are mediated via activation of a B₂ receptor and the response is dependent upon the release of a factor present in the endothelium. Angiotensin converting enzyme is present in the human basilar artery and is important for the conversion of angiotensin I to angiotensin II but apparently not for the degradation of BK. It is likely that other kininases are present and active in the tissue. Send offprint requests to E. T. Whalley at the above address     

Investigation into atropine-induced antinociception.

1. The activity of angiotensin converting enzyme (ACE) has been studied on functional parameters of intact isolated preparations of extrapulmonary tissues. The conversion of angiotensin I (A I) to angiotensin II (A II) and the cleavage of bradykinin (BK) were used as indicators of ACE activity. Captopril was employed as a specific inhibitor of ACE. 2. Captopril augmented the BK-induced contractions of the rat isolated uterus, the BK- and substance P-induced contractions of the guinea-pig ileum, and the BK-induced venoconstriction in the isolated perfused ear of the rabbit. Degradation of BK by ACE was calculated to be 52% in the rat uterus and 75% in the rabbit perfused ear. 3. Captopril inhibited the A I-induced contractions of the rat isolated colon, the A I-induced vasoconstriction in the isolated perfused ear of the rabbit and the rise in blood pressure induced by i.a. injections of A I in pithed rats. Conversion of A I to A II was calculated to be 13% in the rat colon and 26% in the rabbit perfused ear. 4. From estimations of the A II activity (bioassay on the rat colon) in the effluent of the perfused ear of the rabbit after injections of A I into the arterial inflow cannula it was calculated that approximately one tenth of A I was converted to A II during a single passage through the ear (less than 15 s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Degradation of bradykinin, a cardioprotective substance, during a single passage through isolated rat-heart

Angiotensin converting enzyme (ACE) inhibitors have cardioprotective effects in different species including human. This cardioprotective effect is mainly due to the inhibition of bradykinin (BK) degradation rather than inhibition of the conversion of angiotensin I to angiotensin II. Bradykinin, a nonapeptide, has been considered to be the potential target for various enzymes including ACE, neutral endopeptidase 24.11, carboxypeptidase M, carboxypeptidase N, proline aminopeptidase, endopeptidase 24.15, and meprin. In the present study, the coronary vascular beds of Sprague Dawley rat isolated hearts were perfused (single passage) with Krebs solution alone or with different concentrations of BK i.e. 2.75x10(-10), 10(-7), 10(-6) and 10(-5) M solution. Percent degradation of BK was determined by radioimmunoassay. The degradation products of BK after passing through the isolated rat-hearts were determined using RP-HPLC and mass spectroscopy. All the four doses of BK significantly decreased the perfusion pressure during their passage through the hearts. The percentage degradation of all four doses was decreased as the concentration of drug was increased, implying saturation of a fixed number of active sites involved in BK degradation. Bradykinin during a single passage through the hearts degraded to give [1-7]-BK as the major metabolite, and [1-8]-BK as a minor metabolite, detected on HPLC. Mass spectroscopy not only confirmed the presence of these two metabolites but also detected traces of [1-5]-BK and arginine. These findings showed that primarily ACE is the major cardiac enzyme involved in the degradation of bradykinin during a single passage through the coronary vascular of bed the healthy rat heart, while carboxypeptidase M may have a minor role.     

METABOLISM OF TETRADECAPEPTIDE, ANGIOTENSINOGEN AND ANGIOTENSIN I AND II BY ISOLATED PERFUSED RAT HINDLIMBS

1. We investigated the mechanism of tetradecapeptide-induced vasoconstriction by studying the metabolism of tetradecapeptide (TDP), angiotensinogen, and angiotensin I (AI) and angiotensin II (AII) by isolated perfused rat hindlimbs. 2. Using HPLC and specific RIAs we have quantified six angiotensin peptides: pentapeptide(4-8), hexapeptide(3-8), heptapeptide(2-8), octapeptide(1-8), nonapeptide(2-10) and decapeptide(1-10) in hindlimb effluent. 3. TDP-induced vasoconstriction was associated with generation of predominantly AI and AII, with smaller amounts of the other peptides measured. 4. Captopril prevented vasoconstriction and inhibited AII production by 80%, indicating a dominant role for AI generation in the vascular response to TDP. 5. Evidence that renin is not the enzyme responsible for AI generation from TDP includes: first, the failure of angiotensinogen to cause vasoconstriction or angiotensin peptide generation despite very high amounts of AI and AII generation from TDP; second, the resistance of the TDP-induced vasoconstriction and angiotensin peptide generation to inhibition by pepstatin; and third, the failure of bilateral nephrectomy 24 h before the experiment to influence the vascular and angiotensin peptide response to TDP. 6. AII was cleared with 41% efficiency, with generation of penta-, hexa-, and heptapeptides. 7. AI was cleared with 59% efficiency; this was reduced to 24% by captopril, indicating a conversion of at least 35% of AI to AII by ACE. 8. These studies have identified vascular metabolism of AI and AII to be an efficient process, with both ACE and aminopeptidases playing an important role, and indicate that those peptidases which cleave TDP to generate AI are unlikely to play any role in AI generation in vivo.     

Antagonism of kinin-induced contraction of isolated rat uterus by the crude hydroalcoholic extract from Mandevilla illustris

1. The crude aqueous/alcoholic extract (CE) of Mandevilla illustris (Apocynaceae) rhizomes was analysed against contractile response elicited by bradykinin (BK), lysyl-bradykinin (L-BK), oxytocin(Ot), acetylcholine (Ach), angiotensin II (AII) and barium chloride (BaCl2) in the isolated uterus of the rat. 2. The CE of this plant (0.5-2.0 mg/ml) caused a parallel and concentration-related rightward displacement of BK and L-BK contractile responses. Schild plot revealed a linear relationship (r close to one) and yielded nominal PA2 values of 3.6 and 3.2 respectively, but the slopes were significantly different from unity. 3. However, the anti-BK action of the CE of M. illustris was not selective to kinin action, since in the same range concentration the CE also affected uterine contractile responses induced by Ot, Ach, AII and BaCl2.     

Ketoconazole impairs biliary excretory function in the isolated perfused rat liver

Summary The effects of ketoconazole (KT) on the hepatic excretory function was investigated in the isolated perfused rat liver. KT, at the concentrations of 5 × 10^–5 M or 10^–4 M caused dose-dependent decreases of the biliary bile acid concentration and excretion rate, with no significant effect on bile flow rates. Neither dose altered perfusate flow through the liver. Furthermore, at the same two concentrations, KT impaired the sulfobromophthalein transport in a dose-dependent manner. In contrast, the drug did not alter ¹⁴C-sucrose bile to perfusate ratio and did not cause enzyme release from the liver into the perfusate. The study demonstrates that KT possesses an intrinsic toxicity in the isolated perfused rat liver and suggests caution in the use of this drug in hepatopathic patients. Send offprint requests to G. B. Gaeta at the above address     

Alteration by kallikrein and bradykinin of the conversion of angiotensin I to angiotensin II in the isolated perfused rat lung

Pure kallikrein and bradykinin, when added to the perfusion medium of the isolated perfused rat lung, produced an equal inhibition in the conversion of angiotensin I to angiotensin II as measured in the venous return superfused over the rabbit aortic strips. Acetylsalicylic acid (ASA) prevented the inhibitory effect of kallikrein and bradykinin. Aprotinin, however, prevented the inhibitory effect of kallikrein without altering that of bradykinin. The recovery brought about by ASA of the bradykinin-produced inhibition of angiotensin I conversion was also prevented by prior addition of prostaglandin E2 (PGE2) into the perfusion medium. Neither kallikrein and bradykinin nor ASA altered the myotropic activity of angiotensin II. 5-Oxo-L-prolyl L-tryptophyl-L-prolyl-L-arginyl-L-prolyl-L-glutaminyl-L-isoleucyl-L-prolyl-L- proline (SQ 20 881), when added to the medium, greatly reduced the responses to angiotensin I but potentiated those of angiotensin II. The possible mechanisms of the inhibitory effects of kallikrein and bradykinin are discussed.     

Recovery and conversion of kinins in exsanguinated rat preparations

Summary A rat preparation in which the perfusion route bypassed the lungs, which were substituted by an artificial oxygenator, was exsanguinated and perfused with oxygenated 4% dextran (M.W. 70,000) in Tyrode's fluid; a peristaltic pump propelled the perfusing fluid at 20–25 ml/min through the aortic arch and the perfusion medium returned to the right ventricle. Known amounts of bradykinin (BK), kallidin (lysyl-bradykinin, LBK) and methionyllysylbradykinin (MLBK) were administered as single injections and samples of the perfusion fluid removed between 1.5 to 6 min following injection. Average total kinin activity remaining in the circulating fluid was calculated from assays on the isolated guinea pig ileum against respective kinin injected and found to be after 3 and 6 min respectively: 20 and 5% for BK, 54 and 21% for LBK, 60 and 30% for MLBK. When the lungs were introduced in the perfusion circuit BK recoveries decreased to 0.4% at 4 min and 0% at 6 min. In 2 experiments 1 mg MLBK and, in another two, 1 mg of LBK were recirculated for 3 to 3.5 min in the rat preparation with lung bypass; enzymic reactions were interrupted in the perfusates after removal by lowering the pH to 4.7 and placing them in a boiling water bath for 5 min. Following proper dilution, kinin activity left in the perfusates was separated on carboxymethyl-cellulose columns; in 3 experiments about 50% of the activity was identified as BK from its elution position and resistance to trypsin digestion.The average BK-inactivating potency of the perfusate obtained from the rat with lung bypass was 0.3 g BK/minxml compared to 16 g BK/minxml of rat plasma. The arylamidase activity on arginylnaphthylamide of the perfusate was 2 n moles NA/minxml and it was about 25-fold lower than that of rat plasma.Rat liver was exsanguinated and perfused in situ through the portal vein and inferior cava vein using the same conditions as for the whole animal. The perfusion rate was 12 ml/min. The recovery of injected BK in this preparation was 40% after 2 min of recirculation, declining progressively in the following minutes. When MLBK was perfused in this preparation for 3 min or glycylarginyllysylbradykinin (GALBK) for 3 and 5 min, significant amounts of BK were found in the perfusates.We conclude that LBK, MLBK and GALBK may be converted at a high rate into BK by tissue aminopeptidases found in the rat preparations used. BK inactivation in the whole rat is a fast reaction, even when the pulmonary tissue is not involved in the inactivation.Abbreviations BK Bradykinin - LBK Kallidin or lysylbradykinin - MLBK Methionyllysylbradykinin - GALBK Glycylarginyllysylbradykinin - AAMLBK Aspartylarginymethionyllysylbradikinin - NA Naphthlyamine This work was supported by grants from Convenio BIOQ/FAPESP (São Paulo) and FINEP (Rio)With a Fellowship from FAPESP