Use of LC-MS/TOF, LC-MS(n), NMR and LC-NMR in characterization of stress degradation products: Application to cilazapril.

Forced degradation studies on cilazapril were carried out according to ICH and WHO guidelines. Significant degradation of the drug was observed in acid and base conditions, resulting primarily in cilazaprilat. In neutral condition, five degradation products were formed, while under oxidative condition, two degradation products were generated. In total, seven degradation products were formed, which were separated on an Inertsil C-18 column using a stability-indicating HPLC method. Structure elucidation of the degradation products was done by using sophisticated and hyphenated tools like, LC-MS/TOF, LC-MS(n), on-line H/D exchange, LC-NMR and NMR. Initially, comprehensive mass fragmentation pathway of the drug was laid down. Critical comparison of mass fragmentation pathways of the drug and its hydrolytic degradation products allowed structure characterization of the latter. 1D and 2D proton LC-NMR studies further confirmed the proposed structures of hydrolytic degradation products. The oxidative degradation products could not be characterized using LC-MS and LC-NMR tools. Hence, these degradation products were isolated using preparative HPLC and extensive 1D ((1)H, (13)C, DEPT) and 2D (COSY, TOCSY, HETCOR and HMBC) NMR studies were performed to ascertain their structures. Finally, degradation pathways and mechanisms of degradation of the drug were outlined.

Advantages of automation in plasma sample preparation prior to HPLC/MS/MS quantification: application to the determination of cilazapril and cilazaprilat in a bioequivalence study.

An HPLC/MS/MS method characterized by complete automation and high throughput was developed for the determination of cilazapril and its active metabolite cilazaprilat in human plasma. All sample preparation and analysis steps were performed by using 2.2 mL 96 deep-well plates, while robotic liquid handling workstations were utilized for all liquid transfer steps, including liquid-liquid extraction. The whole procedure was very fast compared to a manual procedure with vials and no automation. The method also had a very short chromatographic run time of 1.5 min. Sample analysis was performed by RP-HPLC/MS/MS with positive electrospray ionization using multiple reaction monitoring. The calibration curve was linear in the range of 0.500-300 and 0.250-150 ng/mL for cilazapril and cilazaprilat, respectively. The proposed method was fully validated and proved to be selective, accurate, precise, reproducible, and suitable for the determination of cilazapril and cilazaprilat in human plasma. Therefore, it was applied to a bioequivalence study after per os administration of 2.5 mg tablet formulations of cilazapril.

Triple pharmacological blockade of the renin-angiotensin-aldosterone system in nondiabetic CKD: an open-label crossover randomized controlled trial.

BACKGROUND: Agents inhibiting the renin-angiotensin-aldosterone (RAAS) system have an important role in slowing the progression of chronic kidney disease. We evaluated the hypothesis that the addition of an aldosterone receptor antagonist to an angiotensin-converting enzyme (ACE) inhibitor and angiotensin II type 1 (AT-1) receptor blocker (ARB) (triple RAAS blockade) may provide an additional benefit compared with an ACE inhibitor and ARB (double RAAS blockade). DESIGN: Randomized open controlled crossover study. SETTING & PARTICIPANTS: 18 whites (7 women, 11 men) from the Outpatient Department of Nephrology with chronic nondiabetic proteinuric kidney diseases, mean age 42.4 +/- 1.9 years (SEM). INTERVENTIONS: In the 8-week run-in period, all participants received the ACE inhibitor cilazapril (5 mg), the ARB telmisartan (80 mg), and the diuretic hydrochlorothiazide (12.5 mg) as double RAAS blockade to achieve the target blood pressure of less than 130/80 mm Hg. Participants were then randomly assigned to 2 treatment sequences, either the addition of spironolactone (25 mg) (triple RAAS blockade) through 8 weeks followed by double RAAS blockade through 8 weeks (sequence 1) or double RAAS blockade followed by triple RAAS blockade (sequence 2). MAIN OUTCOME MEASURES: 24-hour urine protein excretion (primary end point) and markers of tubular injury and fibrosis (secondary end points). Analysis was performed using analysis of variance for repeated measurements. RESULTS: At baseline, mean serum creatinine level was 1.16 +/- 0.09 mg/dL (103 +/- 8 micromol/L), estimated glomerular filtration rate was 107.8 mL/min (95% confidence interval, 93 to 140.9 [1.8 mL/s; 95% confidence interval, 1.55 to 2.35; Cockcroft-Gault formula), and 24-hour mean proteinuria was 0.97 +/- 0.18 g. Mean urine protein excretion was 0.7 g/24 h (95% confidence interval, 0.48 to 0.92) less after triple RAAS blockade than after double RAAS blockade (P = 0.01), without change in blood pressure. Urine excretion of N-acetyl-beta-d-glucosaminidase (P = 0.02) and amino-terminal propeptide of type III procollagen (P = 0.05) also significantly decreased. Potassium levels increased significantly after triple therapy (P = 0.02). However, no patient was withdrawn because of adverse effects. LIMITATIONS: Absence of blinding, small sample size, short treatment period, absence of histological assessment. CONCLUSIONS: Administration of an aldosterone receptor antagonist in addition to double RAAS blockade with an ACE inhibitor and ARB may slow the progression of chronic kidney disease. Additional studies are necessary to confirm this result.

Role of endothelium-derived hyperpolarizing factor in ACE inhibitor-induced renal vasodilation in vivo.

Although the angiotensin-converting enzyme (ACE) inhibitor-induced bradykinin enhances nitric oxide (NO) release, bradykinin may also stimulate the production of an additional vasodilator, endothelium-derived hyperpolarizing factor (EDHF). This study examined the role of EDHF in mediating the NO-independent action of ACE inhibitors in canine renal microcirculation in vivo. We used intravital CCD camera videomicroscopy that allowed direct visualization of renal microcirculation in superficial and juxtamedullary nephrons in an in vivo, in situ, and relatively intact setting. In the presence of E4177 (an angiotensin receptor blocker), cilazaprilat (30 microg/kg) had no effect on diameter of superficial afferent arterioles (Aff), but it increased renal contents of bradykinin and nitrate plus nitrite, and it elicited dilation of juxtamedullary Aff (from 24.0+/-0.2 to 28.2+/-0.8 microm), juxtamedullary efferent arterioles (Eff) (from 24.2+/-0.2 to 28.0+/-0.8 microm), and superficial Eff (from 18.2+/-0.2 to 19.7+/-0.2 microm). These changes in diameters were prevented by N(alpha)-adamantaneacetyl-d-Arg-[Hyp(3),Thi(5,8),D-Phe(7)]bradykinin, a bradykinin receptor antagonist. The pretreatment with nitro-l-arginine methylester (l-NAME) plus E4177 eliminated the dilator response of juxtamedullary/superficial Eff and the increase in renal nitrate plus nitrite levels induced by cilazaprilat. In contrast, in the presence of E4177+l-NAME, cilazaprilat still caused 8%+/-3% dilation of juxtamedullary Aff, which was completely eliminated by proadifen, a cytochrome-P450 and K(Ca) channel blocker. Collectively, the ACE inhibitor exerts multiple vasodilator mechanisms, including the inhibition of angiotensin II formation; blockade of angiotensin II activity appears to be a dominant mechanism in superficial Aff, whereas the bradykinin-induced NO acts on superficial Eff and juxtamedullary Aff/Eff. Furthermore, a putative EDHF is an additional mechanism for the ACE inhibitor-induced vasodilation of juxtamedullary Aff in vivo.

In vivo visualization of subendocardial arteriolar response in renovascular hypertensive hearts.

Time-sequential responses to endothelium-dependent and -independent vasodilators and angiotensin-converting enzyme (ACE) inhibitors were studied in the subendocardial arterioles (Endo) of canine renovascular hypertension (HT) compared with subepicardial arterioles (Epi; both <120 microm) by charge-coupled device intravital microscope. Vascular responses to acetylcholine, papaverine, and cilazaprilat were compared between normotensive (NT) and HT dogs [4 wk and 12 wk of HT (4wHT and 12wHT)]. The acetylcholine-induced vasodilation of Endo in both 4wHT and 12wHT was smaller than that of NT (both P < 0.01 vs. 4wHT and 12wHT), and that of Epi was smaller than that of NT only in 12wHT (P < 0.05). The papaverine-induced vasodilation of Endo, but not Epi, was impaired only in 12wHT (both P < 0.01 vs. NT and 4wHT). Vasodilation by cilazaprilat remained unchanged at 4wHT and 12wHT in both Epi and Endo. In conclusion, at the early stage, the endothelium-dependent response of Endo was impaired, whereas at the later stage, the endothelium-dependent and -independent responses of Endo and the endothelium-dependent response of Epi were impaired. However, the vasodilatory responses to the ACE inhibitor were maintained in both Endo and Epi of HT.

Differential regulation of elevated renal angiotensin II in chronic renal ischemia.

The present study was undertaken to clarify the role of intrarenal angiotensin (Ang) II and its generating pathways in clipped and nonclipped kidneys of 4-week unilateral renal artery stenosis in anesthetized dogs. After 4 weeks, renal plasma flow (RPF) decreased in clipped and nonclipped kidneys (baseline, 59+/-3; clipped, 16+/-1; nonclipped, 44+/-2 mL/min; P<0.01, n=22). Renal Ang I levels increased only in clipped, whereas intrarenal Ang II contents were elevated in both clipped (from 0.7+/-0.1 to 2.0+/-0.2 pg/mg tissue) and nonclipped kidneys (from 0.6+/-0.1 to 2.5+/-0.3 pg/mg tissue). Intrarenal ACE activity was increased in nonclipped kidneys but was unaltered in clipped kidneys. An angiotensin receptor antagonist (olmesartan medoxomil) given into the renal artery markedly restored RPF, and dilated both afferent and efferent arterioles (using intravital videomicroscopy). Furthermore, in clipped kidneys, the elevated Ang II was suppressed by a chymase inhibitor, chymostatin (from 2.1+/-0.6 to 0.8+/-0.1 pg/mg tissue; P<0.05), but not by cilazaprilat. In nonclipped kidneys, in contrast, cilazaprilat, but not chymostatin, potently inhibited the intrarenal Ang II generation (from 2.4+/-0.3 to 1.5+/-0.2 pg/mg tissue; P<0.05). Finally, [Pro11-D-Ala12]Ang I (an inactive precursor that yields Ang II by chymase but not by ACE; 1 to 50 nmol/kg) markedly elevated intrarenal Ang II in clipped, but not in nonclipped, kidneys. In conclusion, renal Ang II contents were elevated in both clipped and nonclipped kidneys, which contributed to the altered renal hemodynamics and microvascular tone. Furthermore, the mechanisms for intrarenal Ang II generation differ, and chymase activity is enhanced in clipped kidneys, whereas ACE-mediated Ang II generation is possibly responsible for elevated Ang II contents in nonclipped kidneys.

Determination of the antihypertensive drug cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine by solid-phase extraction and high-performance liquid chromatography with photometric detection.

A liquid chromatographic method with photometric detection for the determination of cilazapril and its active metabolite and degradation product cilazaprilat in urine and pharmaceuticals has been developed. The chromatographic method consisted of a microBondapak C18 column maintained at 30+/-0.2 degrees C, using a mixture of methanol-10 mM phosphoric acid (50:50 v/v) as mobile phase at a flow-rate of 1.0 ml/min. Enalapril maleate was used as internal standard. The detection was performed at a wavelength of 206 nm. A study of the retention of cilazapril and cilazaprilat using solid-liquid extraction has been carried out in order to optimise the clean-up procedure for urine samples, which consisted of a solid-liquid extraction using C(R) cartridges. Recoveries greater than 85% are obtained for both compounds. The method was sensitive, precise and accurate enough to be applied to the determination of urine samples obtained from three hypertensive patients up to 24 h after intake of a therapeutic dose (detection limit of 70 ng/ml for cilazapril and cilazaprilat in urine). A comparison of the method developed using photometric and amperometric detection has been carried out.

ACE-inhibition attenuates cardiac cell damage and preserves release of NO in the postischemic heart.

Cardioprotective effects of angiotensin-converting enzyme (ACE) inhibition have been demonstrated in postischemic reperfusion. This occurred via bradykinin and indirect evidence suggested mediation by nitric oxide (NO), which probably acts as a radical scavenger. To test this hypothesis, we measured release of lactate dehydrogenase (LDH) from isolated guinea pig hearts (constant flow perfusion, 37 degrees C) as a marker of cellular damage, before and after global ischemia (15 min), and we investigated the release of NO during reperfusion, both, without and with ACE inhibition. The main catabolites of NO, nitrate and nitrite, were also quantified. Coronary perfusion pressure (CPP) indicated coronary resistance changes. Cilazaprilat (CIL, 10 microM) was used for inhibition of ACE. Marked and protracted cellular damage occurred during reperfusion in the control group, myocardial LDH release rising nearly 10-fold from 1.5 mU/ml (basal level) to 14 mU/ml during acute reperfusion, then declining to 7 mU/ml after 5 min. ACE inhibition mitigated the acute rise of LDH (9 mU/ml), and reduced its release to preischemic values already after 3 min of reperfusion. Postischemic NO release in the 2nd min of reperfusion was about 40% of the preischemic value (approx. 200 nM) in untreated hearts, while there was 70% recovery after ACE inhibition. After 25 min, NO had recovered to 69% in controls vs. 100% with CIL. Coronary venous nitrate + nitrite was not infringed during early reperfusion (2nd min). After 25 min, nitrate + nitrite had decreased in controls (about 75% of preischemic values), but increased to 110% with CIL. In control hearts, CPP rose continuously from the 10th to the 25th min of reperfusion (from 39 to 55 mmHg), indicating progressive vasoconstriction. CIL significantly attenuated this effect. The results suggest that NO might be consumed during early reperfusion in the act of detoxifying radicals. In control hearts, "endothelial stunning" takes place. Concerning NO production and vasodilatory tone, ACE-inhibition augments postischemic NO release and mitigates disturbances caused by ischemia and reperfusion.

Zonal heterogeneity in action of angiotensin-converting enzyme inhibitor on renal microcirculation: role of intrarenal bradykinin.

The present study examined the role of intrarenal bradykinin in angiotensin-converting enzyme inhibitor (ACEI)-induced dilation of renal afferent (AFF) and efferent arterioles (EFF) in vivo, and further evaluated whether ACEI-stimulated bradykinin activity differed in superficial (SP) and juxtamedullary nephrons (JM). Arterioles of canine kidneys were visualized with an intravital charge-coupled device camera microscope. E4177 (an angiotensin receptor antagonist, 30 microg/kg) dilated AFF and EFF in SP (15 +/- 3% and 19 +/- 5%) and JM (15 +/- 3% and 18 +/- 4%). Subsequently, cilazaprilat (30 microg/kg) caused further dilation of both AFF (29 +/- 4%) and EFF (36 +/- 4%) in JM, whereas in SP it dilated only EFF (29 +/-3%). Similarly, in the presence of E4177, cilazaprilat caused further increases in sodium excretion. This cilazaprilat-induced vasodilation and natriuresis was abolished by a bradykinin antagonist (N(alpha)-adamantaneacetyl-D-Arg-[Hyp3,Thi5,8,D-Phe7]b radykinin). In parallel with these results, cilazaprilat increased renal bradykinin content, more greatly in the medulla than in the cortex (5.7 +/- 0.4 versus 4.6 +/- 0.1 ng/g). Similarly, cilazaprilat elicited greater bradykinin-dependent increases of nitrite/nitrate in the medulla. In conclusion, zonal heterogeneity in renal bradykinin/nitric oxide levels and segmental differences in reactivity to bradykinin contribute to the diverse responsiveness of renal AFF and EFF to ACEI. ACEI-enhanced kinin action would participate in the amelioration of glomerular hemodynamics and renal sodium excretion by ACEI.

Cardioprotective effect of angiotensin-converting enzyme inhibition against hypoxia/reoxygenation injury in cultured rat cardiac myocytes.

BACKGROUND: Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury. METHODS AND RESULTS: Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine. CONCLUSIONS: The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.

Quantitative determination of the angiotensin-converting enzyme inhibitor cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine by high-performance liquid chromatography with amperometric detection.

A rapid and simple high-performance liquid chromatographic method with amperometric detection has been developed for the quantitation of cilazapril and its active metabolite and degradation product cilazaprilat in urine and tablets. The chromatographic system consisted of a microBondapak C18 column, using a mixture of methanol-5 mM phosphoric acid (50:50, v/v) as mobile phase, which was pumped at a flow-rate of 1.0 ml/min. The column was kept at a constant temperature of (40+/-0.2) degrees C. Detection was performed using a glassy carbon electrode at a potential of 1350 mV. Sample preparation for urine consisted of a solid-phase extraction using C8 cartridges. This procedure allowed recoveries greater than 85% for both compounds. The method proved to be accurate, precise and sensitive enough to be applied to pharmacokinetic studies and it has been applied to urine samples obtained from four hypertensive patients (detection limit of 50 ng/ml for cilazapril and 40 ng/ml for cilazaprilat in urine). Results were in good agreement with pharmacokinetic data.

Role of Ca2+-activated K+ channels in the protective effect of ACE inhibition against ischemic myocardial injury.

Angiotensin-converting enzyme (ACE) inhibitors increase the production of nitric oxide (NO) and prostacyclin and open Ca2+-activated K+ channels. The effects of these actions of ACE inhibitors on infarct size were investigated in open-chest dogs subjected to myocardial ischemia and reperfusion. Infarct size was assessed 6 hours after the onset of reperfusion, subsequent to 90 minutes of occlusion of the left anterior descending coronary artery. The ACE inhibitor cilazaprilat was administered into the coronary artery 10 minutes before coronary occlusion, and infusion was continued until 1 hour after reperfusion. The bradykinin and NO concentrations in coronary venous blood 10 minutes after the onset of reperfusion were significantly higher in dogs treated with cilazaprilat (3 microg x kg(-1) x min(-1)) than in control animals. Although there were no significant differences in collateral flow during ischemia, infarct size in the cilazaprilat group was smaller than that in the control group (15.1+/-3.0% versus 46.7+/-4.2% of the area at risk, P<0.0001). The infarct size-limiting effect of cilazaprilat was partially reduced by either N(G)-nitro-L-arginine methyl ester (an inhibitor of NO synthase) or iberiotoxin (a blocker of Ca2+-activated K+ channels) and was abolished by N(G)-nitro-L-arginine methyl ester plus iberiotoxin. Indomethacin (an inhibitor of cyclooxygenase) had no effect on the beneficial action of cilazaprilat. Inhibition of ACE thus reduced myocardial infarct size, an effect that was mediated by NO and the opening of Ca2+-activated K+ channels in canine hearts.

Biphasic response to bradykinin in isolated porcine iliac arteries is mediated by bradykinin B1 and B2 receptors.

Bradykinin-induced responses were studied in isolated porcine iliac arteries. Relaxation was endothelium dependent and seen at low concentrations (10(-10)-10(-8) M) of bradykinin. It was inhibited by the bradykinin B2-receptor antagonist icatibant (HOE-140) and by the nitric oxide synthase inhibitor Nomega-nitro-L-arginine. Bradykinin-induced relaxation was significantly potentiated by the kininase I carboxypeptidase inhibitor mergepta (10(-6) M). Bradykinin (>10(-7) M) elicited contraction of preparations with or without endothelium. The contraction was abolished by indomethacin but was not affected by the thromboxane A2/prostaglandin H2-receptor antagonist SQ 29,548. Icatibant and the bradykinin B1-receptor antagonist desArg9[Leu8]bradykinin significantly decreased bradykinin-induced contraction regardless of endothelial function. The contraction also was decreased by treatment with mergepta. The bradykinin B1-receptor agonist desArg9-bradykinin contracted endothelium-denuded arterial strips. This contraction was significantly decreased by desArg9[Leu8]bradykinin but not by icatibant. The desArg9-bradykinin-induced contraction also was inhibited by the protein-synthesis inhibitor cycloheximide. Neither bradykinin-induced relaxation nor contraction was affected by the ACE inhibitors enalaprilat or cilazaprilat. In conclusion, bradykinin-induced relaxation of isolated porcine iliac arteries was mediated by endothelial bradykinin B2 receptors and mainly nitric oxide. Bradykinin-induced contraction was endothelium independent, indomethacin sensitive, and probably mediated by bradykinin B1 (inducible) and B2 receptors located in the vascular smooth-muscle layer. Kininase I carboxypeptidase, and not ACE, is the main enzyme responsible for bradykinin degradation in these vessels.

Inhibition of angiotensin-converting enzyme increases the nitric oxide levels in canine ischemic myocardium.

Since angiotensin-converting enzyme (ACE) produces angiotensin II in the heart, ACE inhibitors may prevent coronary vasoconstriction and increase coronary blood flow. On the other hand, since ACE inhibitors also inhibit kininase II which results in reduced degradation of bradykinin, ACE inhibitors may increase cardiac nitric oxide (NO) levels via stimulation of bradykinin receptors. This study was undertaken to test whether ACE inhibitors increase the cardiac NO levels and coronary blood flow in the ischemic myocardium. In 34 open-chest dogs, the left anterior descending coronary artery was perfused through an extracorporeal bypass tube from the left carotid artery. When either imidaprilat or cilazaprilat of 3 microg/kg/min was infused into the bypass tube for 10 min after reduction of coronary blood flow due to partial occlusion of the bypass tube, coronary blood flow increased from 31 +/- 1 to either 45 +/- 5 or 43 +/- 4 ml/100 g/min despite no changes in coronary perfusion pressure (43 +/- 2 mmHg). During an infusion of either imidaprilat or cilazaprilat, bradykinin and the end-products of NO (nitrate + nitrite) concentrations of coronary venous blood were markedly increased, which were attenuated by either HOE-140 (an inhibitor of bradykinin receptors) or by N(omega)-nitro-L-arginine methyl ester (an inhibitor of NO synthase). We also observed increases in cardiac bradykinin and NO levels due to either imidaprilat or cilazaprilat in the low constant coronary blood flow condition. It is concluded that ACE inhibitors can increase cardiac NO levels via the accumulation of bradykinin in the ischemic myocardium.

ACE-inhibition prevents postischemic coronary leukocyte adhesion and leukocyte-dependent reperfusion injury.

OBJECTIVE: Polymorphonuclear leukocytes (PMN), retained in the microvascular bed, can contribute to postischemic myocardial reperfusion injury. Since a beneficial effect of ACE-inhibition on reperfusion injury has been reported, we investigated the impact of cilazaprilat on PMN dependent reperfusion injury in isolated guinea pig hearts. METHODS: Hearts (n = 5 per group) were subjected to 15 min of ischemia. Immediately thereafter, a bolus of PMN was injected into the coronary system. External heart work (EHW) and total cardiac nitric oxide release were measured. For microscopic evaluation, hearts received rhodamine 6G labelled PMN after ischemia, were arrested 5 min later and further perfused with FITC dextran (0.1%). Localization of retained PMN was assessed by fluorescence microscopy. Leukocyte activation was studied by FACS analysis of the adhesion molecule CD11b before and after coronary passage of the PMN. The ACE-inhibitor cilazaprilat (Cila, 2 microM) and the NO-synthase inhibitor nitro-L-arginine (NOLAG, 10 microM) were used to modulate nitric oxide formation of the heart. RESULTS: Postischemic EHW recovered to 67 +/- 5% (controls) and 64 +/- 6% (Cila) of the preischemic value. Addition of PMN severely depressed recovery of EHW (39 +/- 2%) and NO release (39 +/- 6% of the preischemic value). Simultaneously, ischemia led to a substantial increase in postcapillary PMN adhesion (from 21 +/- 5 to 172 +/- 27 PMN/mm2 surface) and CD11b-expression of the recovered PMN (3-fold). Cila attenuated postischemic PMN adhesion (83 +/- 52 PMN/mm2) and activation of PMN, whereas it improved recovery of work performance (64 +/- 4%) and NO release (65 +/- 4%) in the presence of PMN. Conversely, NOLAG increased PMN adhesion (284 +/- 40 PMN/mm2) and myocardial injury. We conclude that ACE-inhibition prevents leukocyte dependent reperfusion injury mainly by inhibition of postcapillary leukocyte adhesion. The effect may be mediated by NO, given the proadhesive effect of NOLAG.

Effects of ACE-inhibition on redox status and expression of P-selectin of endothelial cells subjected to oxidative stress.

Redox stress during post-ischemic reperfusion may be the prime signal for processes leading to myocardial remodelling and hypertrophy. Nitric oxide (NO) is antioxidative, antiadhesive for neutrophils (PMN) and antiproliferative. Thus, enhancing endothelial production of NO, e.g. by inhibiting breakdown of endogenous bradykinin via angiotensin converting enzyme (ACE), could be beneficial. The effect of cilazaprilat (CILA, 10 micro M), an ACE inhibitor, on redox status, expression of the adhesion molecule P-selectin, and PMN adhesion under conditions of oxidative stress was investigated in cultured human umbilical vein endothelial cells (HUVECs). Incubation of the cells with H2O2 (0.1 and 1 mm) for 15 min served as oxidative stimulus. The intra- and extracellular concentrations of reduced and oxidized glutathione (GSH and GSSG) were measured by HPLC as indicators of endothelial redox status. Expression of P-selectin was measured by flow cytometry. Furthermore, firm leukocyte adhesion to HUVECs was assessed. In controls, the intracellular ratio GSH/GSSG averaged 47 and dropped to 30 after incubation with 0.1 mm H2O2. The ratio declined to 6.5 with 1 mm H2O2. CILA blocked the effects of 0.1 mm H2O2, but was ineffective against 1 mm peroxide. The extracellular ratio did not discriminate between 0.1 and 1 mm H2O2, falling from 18 to 1 in both situations. P-selectin expression rose from 100% (control) to 146% after 1 mm H2O2 without CILA, but only to 114% in the presence of CILA. PMN adhesion was enhanced from about 1600 PMN per microwell (control) to 4300/well by 1 mm H2O2. CILA had no significant effect on adhesion (3900 PMN/well). Exposure of HUVECs to 0.1 mm H2O2 affected neither P-selectin expression nor PMN adhesion. Consequently, ACE inhibition can mitigate mild (0.1 mm H2O2) but not more severe redox stress in HUVECs. Irrespectively, CILA reduced the upregulation of P-selectin at the higher H2O2 concentration, indicating that this process is regulated independently of the cellular redox status. The firm adhesion of PMN to HUVECs was independent of P-selectin expression.

Beneficial effects of inhibition of angiotensin-converting enzyme on ischemic myocardium during coronary hypoperfusion in dogs.

BACKGROUND: Angiotensin-converting enzyme (ACE) produces angiotensin II, causing vasoconstriction of coronary arteries and reduction of coronary blood flow. The present study was undertaken to test the hypothesis that an ACE inhibitor increases coronary blood flow and improves myocardial metabolic and contractile functions of ischemic myocardium. METHODS AND RESULTS: In 65 open-chest dogs, the left anterior descending coronary artery was perfused through an extracorporeal bypass tube from the left carotid artery. When cilazaprilat (3 micrograms/kg per minute) was infused into the bypass tube for 10 minutes after reduction of coronary blood flow due to partial occlusion of the bypass tube, coronary blood flow increased from 30 +/- 1 to 43 +/- 2 mL/100 g per minute despite there being no changes in coronary perfusion pressure (43 +/- 1 mm Hg). The ratio of myocardial endocardial flow to epicardial flow increased during an infusion of cilazaprilat. Both fractional shortening and lactate extraction ratio of the perfused area were increased (fractional shortening: 4.1 +/- 0.6% to 8.9 +/- 0.6%, P < .001; lactate extraction ratio: -55.7 +/- 3.3% to -36.7 +/- 3.9%, P < .001). During an infusion of cilazaprilat, the bradykinin concentration of coronary venous blood was markedly increased. The increased coronary blood flow due to cilazaprilat was attenuated by HOE-140 (an inhibitor of bradykinin receptors; coronary blood flow: 35 +/- 2 mL/100 g per minute), and by N omega-nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase; coronary blood flow: 34 +/- 2 mL/100 g per minute). Intracoronary administration of bradykinin mimicked the beneficial effects of cilazaprilat. Cyclic GMP content of the coronary artery was increased by cilazaprilat compared with the untreated condition in the ischemic myocardium. In the denervated hearts, the increased coronary blood flow due to cilazaprilat was not attenuated. On the other hand, CV11974, an inhibitor of angiotensin II receptors, slightly increased coronary blood flow to 34 +/- 2 from 30 +/- 1 mL/100 g per minute. CONCLUSIONS: We conclude that an inhibitor of ACE can increase coronary blood flow and ameliorate myocardial ischemia, primarily due to accumulation of bradykinin and production of nitric oxide from the ischemic myocardium. Inhibition of angiotensin II production due to inhibition of ACE partially contributes to coronary vasodilation in the ischemic myocardium.

Endothelium-modulated proliferation of medial smooth muscle cells: influence of angiotensin II and converting enzyme inhibition.

This study investigated the role of the endothelium and angiotensin II (Ang II) in regulating medial smooth muscle cell (SMC) proliferation. [3H]-thymidine incorporation into medial SMC of rat arteries was examined in vivo, using ballooned rat carotid arteries, as well as in vitro, using cultures of aortic tissue rings (organoids). In vivo, maximal medial [3H]-thymidine incorporation occurred within 3 days post-ballooning. In endothelium-denuded organoids, maximum medial DNA synthesis was achieved after 7 days of culture. [3H]-thymidine-labelling of SMC in intact organoids (with endothelium) increased minimally during culture, indicating that the endothelium provided protection with respect to medial proliferation under basal conditions (culture in the presence of 1% plasma-derived serum). Inclusion of 10(-7) M Ang II significantly elevated medial [3H]-thymidine incorporation above that in control cultures. The stimulatory effect of Ang II was much more pronounced in intact organoids that in endothelium-denuded organoids, indicating synergistic growth regulation by Ang II and endothelium-derived factors. When organoids were cultured in the combined presence of Ang II and the ACE inhibitor cilazaprilat, labelling indices of intact organoids were also significantly increased above control, but to a lower level than those obtained in the presence of Ang II alone. However, for endothelium-denuded organoids, medial [3H]-thymidine incorporation in the combined presence of Ang II and cilazaprilat was not significantly different from that in untreated controls. Thus, cilazaprilat exerts both endothelium-dependent and endothelium-independent negative regulatory effects on medial SMC proliferation.

Nitric oxide release from rat aortic smooth muscle cells is not attenuated by angiotensin converting enzyme inhibitors.

We investigated the effects of angiotensin converting enzyme inhibitors on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. We measured the production of nitrite, a stable metabolite of NO, and the expression of inducible NO synthase mRNA by vascular smooth muscle cells. Incubation of the culture with interleukin-1 beta (10 ng/ml) for 24 h caused a significant increase in nitrite levels. The basal and interleukin-1 beta-induced nitrite production by vascular smooth muscle cells were not affected by the presence of angiotensin converting enzyme inhibitors (0.1 approximately 10 microM), enalaprilat, cilazaprilat or captopril. Unstimulated vascular smooth muscle cells expressed no inducible NO synthase transcripts, whereas incubation with interleukin-1 beta for 24 h induced marked inducible NO synthase mRNA expression. The angiotensin converting enzyme inhibitors, however, had no effects on the interleukin-1 beta-induced inducible NO synthase mRNA expression. These results indicate that angiotensin converting enzyme inhibitors do not attenuate NO synthesis by vascular smooth muscle cells under basal and interleukin-1 beta-stimulated conditions.