Mechanisms of hypoxic coronary vasodilatation in isolated perfused rat hearts.

We pharmacologically investigated the potential involvement of nitric oxide (NO), prostacyclin, adenosine, adenosine triphosphate (ATP)-sensitive K (K(ATP)) channel opening and Ca2+-activated K (K(Ca)) channel opening in coronary vasodilatation during 15 min of hypoxia in isolated rat hearts perfused at a constant pressure of 70 mm Hg. The coronary flow suppressed by 10(-4) M Nomega-nitro-L-arginine methyl ester (L-NAME), which corresponds to the NO-dependent flow, decreased to almost zero during hypoxia. In contrast, the NO-dependent coronary flow amounted to approximately 40% of the total coronary flow during normoxia. The suppression of coronary flow by 10(-5) M 8-phenyltheophylline (8-PT), which corresponds to the adenosine-dependent flow, was remarkable in the middle and the late phases of a 15-min hypoxia. The coronary flow suppressed by 2 x 10(-6) M glibenclamide, which corresponds to the K(ATP) channel opening-dependent flow, depended on the agents added to the perfusate. However, there was a marked increase in coronary flow in the early phase of hypoxia in the heart perfused with the combination of 8-PT, 10(-2) M tetraethylammonium (TEA) and L-NAME. During hypoxia, the coronary flow suppressed by TEA, which corresponds mainly to the K(Ca) channel opening-dependent flow, also depended on the agents added to the perfusate. However, during reoxygenation, there was a transient significant increase in any combination of the agents. Our study suggests that hypoxia almost completely inhibits NO production, and that K(ATP) channel opening immediately after hypoxia and subsequent enhanced adenosine production cause a marked hypoxic coronary vasodilatation. It also suggests that K(Ca) channel opening causes vasodilatation during reoxygenation.     

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.     

Angiotensin II activates NADPH oxidase in isolated rat hearts subjected to ischaemia-reperfusion

The role of angiotensin II in myocardial ischaemia-reperfusion is not clearly defined. In this respect, the involvement of NADPH oxidase remains to be determined. The aim of this study was 1) to evaluate the cardiac effects of angiotensin AT(1) receptor stimulation in non-ischaemic conditions of perfusion or during ischaemia-reperfusion, and 2) to measure the concomitant activation of NADPH oxidase in isolated rat hearts perfused with angiotensin II and/or Losartan. In non-ischaemic hearts, angiotensin II induced rapid and prolonged vasoconstrictive and negative inotropic effects. Ischaemia-reperfusion increased the mRNA expression of AT(1) and AT(2) receptors. During reperfusion, angiotensin II reduced the incidence of arrhythmias and the lactate dehydrogenase released, and increased NADPH oxidase mRNA expression and enzyme activity. Losartan co-administration totally antagonised the effects of angiotensin II. Our study demonstrates that ischaemia-reperfusion induces adaptative cardiac modifications, which allow exogenously added angiotensin II to stimulate myocardial NADPH oxidase through angiotensin AT(1) receptor activation.     

Metabolism of angiotensin I in isolated rat hearts: Effect of angiotensin converting enzyme inhibitors

In this study, the formation of biologically active angiotensins from angiotensin I (Ang I) in isolated rat hearts was evaluated. The role of angiotensin converting enzyme (ACE) in Ang I metabolism was also investigated. HPLC analysis of heart perfusate showed that ¹²⁵I-Ang I was metabolized extensively (single passage) in the rat coronary circulation in vitro leading to the formation of the biologically active angiotensins: angiotensin II (Ang II), Ang-(2–8), Ang-(3–8) and Ang-(l–7). Ang II was the major product identified in HPLC fractions, corresponding to 7.8 ± 0.89% of the total radioactivity recovered. A similar profile was observed when single-passage metabolism of non-isotopic Ang I was evaluated by HPLC, followed by radioimmunoassay of the eluate fractions. When ¹²⁵I-Ang I was perfused in the presence of ACE inhibitors (enalaprilat, ramiprilat) in concentrations up to 130 μM, the formation of Ang II was only partially inhibited (approximately 50%). A similar tendency was observed for Ang-(2–8), Ang-(3–8) and Ang-(2–7). The formation of Ang-(1–7) and its related fragments Ang-(3–7) and Ang-(4–7) was not changed significantly by ACE inhibitors, although a slight increase in formation of these fragments was observed. No significant changes were observed for the carboxyl-terminal fragments of Ang I: Ang-(2–10), Ang-(3–10), and Ang-(4–10). The fractional metabolism of Ang I was not modified by ACE inhibition. These findings suggest that biologically active angiotensins can be formed from Ang I in the rat coronary circulation. These locally generated peptides may contribute to the actions of the renin-angiotensin system in the heart.     

Angiotensin II type 2 receptor-mediated inhibition of norepinephrine release in isolated rat hearts

We investigated whether endogenous and exogenous angiotensin II (Ang II) regulates norepinephrine (NE) release from cardiac sympathetic nerves via both Ang II type 2 receptors (AT2Rs) and Ang II type 1 receptors (AT1Rs). Using isolated rat hearts, sympathetic nerves were electrically stimulated. Ang II with PD-123319 (AT2R antagonist) but not Ang II alone produced a significant increase in nerve stimulation-induced NE overflow, which was abolished by the addition of AT1R antagonist losartan. In contrast, NE overflow was markedly decreased by losartan with or without Ang II. This decrease was abolished by the combination with PD-123319, nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (NOARG), icatibant (bradykinin B2 receptor antagonist), or PKSI-527 (kininogenase inhibitor). CGP-42112A (AT2R agonist) suppressed nerve stimulation-induced NE overflow in the same way as the combination of Ang II and losartan, and this suppression was abolished by PD-123319, NOARG, icatibant, or PKSI-527. There were significant increases in NOx (NO2/NO3) contents in coronary effluent under conditions where NE overflow was suppressed. Ang II seems to function as an inhibitory modulator of cardiac noradrenergic neurotransmission via AT2Rs and well-known AT1R-mediated stimulatory actions. The inhibitory mechanism may involve local bradykinin production, its B2 receptor activation, and NO as a downstream effector.     

Angiotensin I as a dipsogen: efficacy in brain independent of conversion to angiotensin II

Angiotensin I or II injected into preoptic or anterior hypothalamic areas, or intraventricularly, evoked water drinking in rats. Neither vehicle controls nor SQ 20,881 (angiotensin-converting enzyme inhibitor) produced drinking. SQ 20,881, which inhibits the conversion of angiotensin I to II, did not suppress the drinking evoked by either angiotensin I or II even at inhibitor/angiotensin ratios of 100:1. The results indicate the possibility that brain receptor sites underlying the dipsogenic response to angiotensin II are responsive to angiotensin I as well, or that additional sites are responsive to angiotensin I.     

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.     

Angiotensin II receptor expression and inhibition in the chronically hypoxic rat lung.

1. Angiotensin II (AII) binding density and the effect of chronic AII receptor blockade were examined in the rat model of hypoxia-induced pulmonary hypertension. 2. [125I]-[Sar1,Ile2]AII binding capacity was increased in lung membranes from rats exposed to hypoxia (10% fractional inspired O2) for 7 days compared to normal rats (Bmax 108 +/- 12 vs 77 +/- 3 fmol mg-1 protein; P < 0.05), with no significant change in dissociation constant. Competition with specific AII receptor subtype antagonists demonstrated that AT1 is the predominant subtype in both normal and hypoxic lung. 3. Rats treated intravenously with the AT1 antagonist, GR138950C, 1 mg kg-1 day-1 rather than saline alone during 7 days of exposure to hypoxia developed less pulmonary hypertension (pulmonary arterial pressure: 21.3 +/- 1.7 vs 28.3 +/- 1.1 mmHg; P < 0.05), right ventricular hypertrophy (right/left ventricle weight ratio: 0.35 +/- 0.01 vs 0.45 +/- 0.01; P < 0.05) and pulmonary artery remodelling (abundance of thick-walled pulmonary vessels: 9.6 +/- 1.4% vs 20.1 +/- 0.9%; P < 0.05). 4. The reduction in cardiac hypertrophy and pulmonary remodelling with the AT1 antagonist was greater than that achieved by a dose of sodium nitroprusside (SNP) that produced a comparable attenuation of the rise in pulmonary arterial pressure during hypoxia. 5. The data suggest that AII, via the AT1 receptor, has a role in the early pathogenesis of hypoxia-induced pulmonary hypertension in the rat.     

Angiotensin II reduces infarct size and has no effect on post-ischaemic contractile dysfunction in isolated rat hearts

1. In order to test the hypothesis that angiotensin II exacerbates myocardial ischaemia-reperfusion (IR) injury, we examined the effects of graded angiotension II concentrations of angiotensin II on IR injury in both working and non-working (Langendorff) isolated rat hearts. 2. Non-working hearts were subjected to 30 min aerobic perfusion (baseline) then 25 min of global, no-flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=7) or presence of 1 (n=6) or 10 nM (n=5) angiotensin II). Recoveries of LV developed pressure and coronary flow after 30 min reperfusion in control hearts (58+/-9 and 40+/-8% of baseline levels, respectively) were no different from hearts treated with 1 or 10 nM angiotensin II. Infarct size (determined at the end of reperfusion by triphenyltetrazolium chloride staining) was reduced by angiotensin II in a concentration-dependent manner (from a control value of 27+/-3 to 18+/-4% and 9+/-3% of the LV, respectively). 3. Working hearts were subjected to 50 min pre-ischaemic (pre-I) aerobic perfusion then 30 min of global, no-flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=14) or presence of 1 (n=8), 10 (n=7) or 100 nM (n=7) angiotensin II). In controls, post-ischaemic (post-I) left ventricular (LV) work and efficiency of oxygen consumption were depressed (43+/-9 and 42+/-10% of pre-I levels, respectively). The presence of angiotensin II throughout IR had no effect on LV work compared with control. 4. Thus, angiotensin II reduces infarct size in a concentration-dependent manner but has no effect on contractile stunning associated with IR in isolated rat hearts.     

Selective blockade of neurotensin-induced coronary vessel constriction in perfused rat hearts by a neurotensin analogue

[D-Trp¹¹]-NT, an analogue of neurotensin (NT) in which Tyr¹¹ was replaced with D-Trp, was found to antagonize selectively NT-induced coronary vessel constriction in perfused rat hearts, in concentrations varying between 1.3 × 10^?7 and 1.1 × 10^?6 M. Higher concentrations of [D-Trp¹¹]-NT displayed NT-like activity. In rat stomach strips and guinea pig atria, [D-Trp¹¹]-NT exhibits full intrinsic activity, markedly reduced affinity for NT receptors, but no inhibitory effect against NT. These results suggest that the receptors mediating the constrictor action of NT in the coronary vessels of rat hearts are pharmacologically distinct from those subserving the stimulant effects of NT in rat stomach strips and guinea pig atria.     

Angiotensin II receptor internalization and signaling in isolated rat hepatocytes

Since angiotensin II (Ang II)-induced receptor internalization is required to maintain the production of certain intracellular signals in some target cells, we investigated the relationships between Ang II receptor endocytosis and the generation of second messengers in rat hepatocytes. The results of the present study demonstrate that in response to exposure of hepatocytes to Ang II, a decrease in surface Ang II receptors occurred, consistent with a rapid endocytosis of the receptor-bound hormone complex. Pretreatment of cells with okadaic acid (OA) did not have any effect on receptor-mediated internalization. In contrast, a marked reduction of the Ang II receptor endocytosis process occurred after treatment of hepatocytes with phenylarsine oxide (PAO), indicating that cysteine residues could be involved in receptor-mediated endocytosis. Stimulation of cells with Ang II blocked the generation of cyclic adenosine monophosphate (cAMP), which follows the stimulation of hepatocytes with forskolin. Moreover, Ang II increased both inositol 4,5-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) generation, and enhanced intracellular calcium concentration ([Ca2+]i). Exposure of cells to PAO did not alter the effect of Ang II on the accumulation of cAMP after forskolin stimulation, indicating that endocytosis of the agonist-receptor complex is not involved in adenylate cyclase inhibition. Conversely, PAO and OA markedly reduced IP2 and IP3 synthesis, and the plateau phase of Ang II-induced Ca2+ mobilization. The relationship between Ang II-induced endocytosis and the generation of phosphoinositols and increment in [Ca2+]i indicates that sequestration of the Ang II receptor is necessary to maintain the production of these intracellular signals in rat hepatocytes.     

Specific antagonists for the myotropic action of angiotensin II and angiotensin I on the isolated rat colon

Substitutions of 8 phenylalanine with L-alanine and D-phenylalanine abolish the myotropic action of the angiotensin II (AT(II)) analogues and confer inhibitory properties on the molecule. [8-L-Ala]-AT(II) and [8-D-Phe]-AT(II) antagonize specifically the myotropic action of AT(II) and angiotensin I (AT(I)) on the rat colon, while the action of other myotropic agents (acetylcholine, 5 hydroxytryptamine) is not modified.     

The angiotensin II AT1-receptor antagonist candesartan improves functional recovery and reduces the no-reflow area in reperfused ischemic rat hearts.

It is not yet clear if cardiac angiotensin II is involved in the pathophysiology of myocardial ischemia/ reperfusion injury. The aim of this study was to investigate the effect of the angiotensin II AT1-receptor antagonist candesartan on myocardial functional recovery in isolated rat hearts subjected to ischemia and reperfusion. Three groups of hearts perfused in the Langendorff mode with Krebs-Henseleit buffer under constant pressure received either vehicle (n = 7), candesartan, 1 nM (n = 6), or 100 nM (n = 7) at the start of 30 min of global ischemia. The recovery of the double product was significantly higher in the candesartan, 100 nM, group (75+/-9.2%) than in the vehicle group (40+/-5.1%; p < 0.05). At the end of 30 min of reperfusion, left ventricular end diastolic pressure was lower in rats given candesartan, 100 nM, than in rats given vehicle (10+/-4.3 vs. 38+/-4.8 mm Hg; p < 0.05). After ischemia and reperfusion, there was a large no-reflow area in the vehicle group (28+/-3.1% of the left ventricle), which was reduced by candesartan, 100 nM (12+/-1.3%; p < 0.05). In rats given candesartan, 1 nM, there was a trend toward a higher recovery of the double product (73+/-13.4%), a lower left ventricular end-diastolic pressure (29+/-6.6 mm Hg), and a smaller no-reflow area (19+/-3.5% of the left ventricle) compared with the rats receiving vehicle. These trends did, however, not reach statistical significance. Our results demonstrate that candesartan reduces myocardial ischemia/reperfusion injury, thus indicating that endogenous cardiac angiotensin II is involved in the tissue injury after myocardial ischemia and reperfusion.     

Developmental conversion of inotropism by endothelin I and angiotensin II from positive to negative in mice.

Inotropic effects on isolated neonatal and adult mouse myocardium of endothelin I and angiotensin II were examined. Endothelin I produced a sustained positive inotropic response in the neonate but a sustained negative response in the adult. Both were concentration-dependent and were inhibited by the endothelin ETA receptor antagonist, BQ-123 (Cyclo(D-a-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)). Angiotensin II produced a sustained positive inotropic response in the neonate while a sustained negative response in the adult. Both were concentration-dependent and were inhibited by the angiotensin AT1 receptor antagonist, YM358 (2,7-diethyl-5-((2'-(1 H-tetrazol-5-yl)biphenyl-4-yl)methyl-5H-pyrazolo(1,5-b)(1,2,4)tria zole potassium salt monohydrate). These results indicate that inotropic responses of the mouse heart to cardioactive peptides are unique among experimental animal species and may be reversed during development.     

Inhibition of neurotransmission by angiotensin I and II in rabbit isolated ear artery

Angiotensin I and II inhibited the nerve stimulation-induced pressure changes in isolated, perfused rabbit ear artery with an IC50 of 3.07 and 0.36 nM, respectively. Neither angiotensin I nor angiotensin II affected the basal pressure or the pressure changes elicited by exogenously administered norepinephrine (NE). The potency of angiotensin I was unaltered by 10(-5) M captopril, indicating that conversion by angiotensin converting enzyme (ACE) was not necessary and did not take place. [Sar1,Val5,Ala8]angiotensin II (3 x 10(-8) M) antagonized the effect of angiotensin I. These findings could have implications regarding ACE inhibitor therapy and the pathophysiology of migraine.     

Iloprost (ZK 36374) prevents angiotensin I conversion in the isolated perfused rat lung against anoxia

The tissue protective effect of iloprost against anoxia was studied in the isolated perfused rat lung. The change in angiotensin converting enzyme activity was taken as a sign of the biochemical activity of pulmonary vascular endothelium and was measured by bioassay of the vasoconstrictor effects of angiotensin I and angiotensin II. A significant decrease in angiotensin converting enzyme activity was observed in the lungs incubated with Krebs alone and exposed to anoxia for 2 h. The decrease in angiotensin converting enzyme activity following anoxia for 2 and 24 h was prevented by prior pretreatment with iloprost. The pulmonary vasoconstrictor effect of angiotensin II was significantly enhanced following anoxia and iloprost prevented this potentiation. The prevention by iloprost of the decrease in angiotensin converting enzyme activity and increase in the pressor response to angiotensin II was attributed to damage of pulmonary vascular endothelium during anoxia. Possible underlying mechanisms are discussed.     

Cross-talk related to insulin and angiotensin II binding on myocardial remodelling in diabetic rat hearts.

This study focused on the regulation and affinity modulation of angiotensin II (Ang II) binding to its receptor subtypes (AT(1)- and AT(2)-receptor) in the coronary endothelium (CE) and cardiomyocytes (CM) of Sprague-Dawley male rats in normal (N), normal treated with losartan (NL), streptozotocin-induced diabetic (D), insulin-treated diabetic (DI), losartan-treated diabetic (DL), and diabetic co-treated with insulin and losartan (DIL). Heart perfusion was used to estimate Ang II binding affinity (tau=1/k-(n)) to its receptor subtypes on CE and CM. Diabetes decreased tau value on CE and increased it on CM as compared to normal. In DL group, the tau value decreased on CE but was normalised on CM. Insulin treatment alone (DI) or with losartan (DIL) restored t to normal on both CE and CM. Western blot results for AT(1)-receptor density showed an increase in diabetics compared to normal with no normalising effect with insulin treatment. The AT(1)-receptor density was normalised in the diabetic groups treated with losartan +/- insulin. Results for AT(2)-receptor regulation revealed a significant difference between untreated (D) and losartan-treated (DL, DIL) diabetic groups. All of these data show the interrelated pathway and cross-talk between insulin and Ang II system indicating potentially negative effects on the diabetic heart.