Effects of dual angiotensin-converting enzyme and neutral endopeptidase 24-11 chronic inhibition by mixanpril on insulin sensitivity in lean and obese Zucker rats

The aim of this study was to examine the effects of chronic (8-day) oral treatment with the dual angiotensin-converting enzyme (ACE) and neutral endopeptidase 24-11 (NEP) inhibitor mixanpril (25 mg/kg twice a day), compared with the ACE inhibitor captopril (25 mg/kg twice a day), on whole body insulin-mediated glucose disposal in young (10-week) and old (19-week) obese Zucker rats (ZOs). Moreover, the effects of chronic mixanpril administration on femoral blood flow at rest and during an insulin infusion were assessed. In the young ZOs, mixanpril decreased the glucose response during an IV glucose tolerance test more effectively than did captopril (-49 and -30%, respectively, p < 0.05). Incremental glucose area under the curve in mixanpril-treated ZOs was then no longer different from that observed in vehicle-treated lean rats (1,592 +/- 175 and 1, 470 +/- 104 mg/dl x min, respectively). The beneficial effects resulting from mixanpril or captopril administration were observed in ZOs but not in lean littermates. In the old ZOs, mixanpril induced higher glucose infusion rates to maintain euglycemia than did captopril during a hyperinsulinemic euglycemic clamp test (+92 and +35%, respectively, p < 0.001). However, the glucose infusion rates in mixanpril-treated ZOs remained much lower than that observed in vehicle-treated lean rats (9.4 +/- 0.7 mg/kg/min vs 28.6 +/- 1.0 mg/kg/min, p < 0.001). Mixanpril did not affect resting femoral vascular bed hemodynamics but restored the femoral blood flow response to insulin infusion. In conclusion, in ZOs, chronic dual ACE/NEP inhibition improves whole body insulin-mediated glucose disposal more effectively than does ACE inhibition alone. This beneficial effect seems to be restricted to conditions of insulin resistance and not directly linked to the improvement in the femoral blood flow response to insulin.     

Role of the effect of inhibition of neutral endopeptidase on vascular and neural complications in streptozotocin-induced diabetic rats

We have previously shown that treating streptozotocin-induced diabetic rats, an animal model of type 1 diabetes, with Ilepatril (an inhibitor of neutral endopeptidase and angiotensin converting enzyme (ACE)) improves vascular and neural function. In this study we sought to determine the individual effect of inhibition of neutral endopeptidase and ACE on diabetes-induced vascular and neural dysfunction. After 4 weeks of untreated diabetes, rats were treated for 12 weeks with Ilepatril, Enalapril (ACE inhibitor) or Candoxatril (neutral endopeptidase inhibitor) followed by analysis of neural and vascular function. Diabetes caused slowing of motor and sensory nerve conduction, thermal hypoalgesia, reduction in intraepidermal nerve fiber density in the hindpaw and impairment in vascular relaxation to acetylcholine and calcitonin gene-related peptide in epineural arterioles of the sciatic nerve and to atrial natriuretic peptide and calcitonin gene-related peptide in renal arteries. Inhibition of neutral endopeptidase or ACE improved neural function; however, dual inhibition of neutral endopeptidase and ACE with Ilepatril tended to have the greatest efficacy. Ilepatril and Candoxatril treatment of diabetic rats was more efficacious in improving vascular responsiveness in epineurial arterioles than treatment with Enalapril. Ilepatril, Enalapril or Candoxatril treatment of diabetic rats were all efficacious in renal arteries. These studies suggest that combination therapy may be the most effective approach for treatment of diabetic neural and vascular complications.     

Acute effect of the dual angiotensin-converting enzyme and neutral endopeptidase 24-11 inhibitor mixanpril on insulin sensitivity in obese Zucker rat

The aim of this study was to determine whether acute dual angiotensin-converting enzyme (ACE)/neutral endopeptidase 24-11 (NEP) inhibition could improve whole body insulin-mediated glucose disposal (IMGD) more than ACE inhibition alone and whether this effect was mediated by the kinin-nitric oxide (NO) pathway activation. We therefore compared in anaesthetized obese (fa/fa) Zucker rats (ZOs) the effects of captopril (2 mg kg(-1), i.v.+2 mg kg(-1) h(-1)), retrothiorphan (25 mg kg(-1), i.v. +25 mg kg(-1) h(-1)), a selective NEP inhibitor, and mixanpril (25 mg kg(-1), i.v. +25 mg kg(-1) h(-1)), a dual ACE/NEP inhibitor, on IMGD using hyperinsulinaemic euglycaemic clamp technique. The role of the kinin-NO pathway in the effects of mixanpril was tested using a bradykinin B2 receptor antagonist (Hoe-140, 300 microg kg(-1)) and a NO-synthase inhibitor (N(omega)-nitro-L-arginine methyl ester, L-NAME, 10 mg kg(-1) i.v. +10 mg kg(-1) h(-1)) as pretreatments. Insulin sensitivity index (ISI) was lower in ZO controls than in lean littermates. Increases in ISI were observed in captopril- and retrothiorphan-treated ZOs. In mixanpril-treated ZOs, ISI was further increased, compared to captopril- and retrothiorphan-treated ZOs. In ZOs, Hoe-140 and L-NAME alone did not significantly alter and slightly reduced the ISI respectively. Hoe-140 and L-NAME markedly inhibited the ISI improvement induced by mixanpril. These results show that in obese insulin-resistant Zucker rats, under acute conditions, NEP or ACE inhibition can improve IMGD and that dual ACE/NEP inhibition improves IMGD more effectively than does either single inhibition. This effect is linked to an increased activation of the kinin-NO pathway.     

Effects of aerosolised substance P on lung resistance in guinea-pigs: a comparison between inhibition of neutral endopeptidase and angiotensin-converting enzyme.

1. We have examined in guinea-pigs, in vivo, the effects of inhibition of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) on the airway response to aerosolised substance P (SP). We aerosolised captopril (4.6 mM, 60 breaths; 210 nmol) to inhibit ACE and acetorphan (0.3, 1 and 3 mM, 60 breaths; 9 nmol, 33 nmol and 110 nmol respectively) to inhibit NEP. We also examined the effect of the highest dose of acetorphan (110 nmol) on the response to aerosolised acetylcholine (ACh). 2. Responsiveness to SP (or ACh) was measured as the change in lung resistance (RL) induced by nebulisation of increasing concentrations of SP (or ACh) before and after treatment with the inhibitor. PC200, defined as the provocative concentration inducing an increase in RL of 200% above baseline was calculated for each challenge. 3. Administration of acetorphan before the second SP-challenge induced a dose-dependent decrease in PC200 for SP amounting to 1.8 (+/- 0.3) log units after treatment with 11 nmol acetorphan. Treatment with vehicle before the second SP-challenge or with 3 mM acetorphan before the second ACh-challenge had no significant effect on PC200. 4. Treatment with captopril (21 nmol) induced only a small, nonsignificant leftward shift of PC200 to SP (0.3 +/- 0.2 log units). 5. We conclude that a NEP-like enzyme, but not ACE, regulates the response to aerosolised SP. We suggest that the same is true for SP released endogenously from sensory nerve endings in the airway epithelial layer.     

Triple vasopeptidase inhibition of angiotensin-converting enzyme/neutral endopeptidase/endothelin-converting enzyme activities on the hemodynamic profile of chronically instrumented unrestrained conscious spontaneously hypertensive rats

Inhibition of the renin-angiotensin system with an angiotensin-converting enzyme inhibitor (ACEi) is an effective therapy in hypertension. Vasopeptidase inhibition was initially proposed with compounds inhibiting both angiotensin-converting enzyme and neutral endopeptidase (omapatrilat), but clinical trials revealed that reducing angiotensin II while blocking the degradation of vasodilatory peptides was not without concerns. We have previously investigated the combination of an ACEi with an endothelin-converting enzyme inhibitor (ECEi); now we add a neutral endopeptidase inhibitor (NEPi) toward triple vasopeptidase inhibition. Male spontaneously hypertensive rats were surgically implanted with a vascular catheter and treated with an ACEi (benazepril), a NEPi (CGS 24592) and an ECEi (CGS 35066) (continuous intra-arterial infusion at 1 or 5 mg/kg/day x 5 days each). After 15 days, drugs administration was stopped for 3 days. ACEi (1 mg/kg per day) reduced the mean arterial blood pressure by 8.4%. The addition of a NEPi and an ECEi at the same dose did not shown any added benefit. The mean arterial blood pressure came back to baseline upon cessation of treatment. ACEi (5 mg/kg per day) reduced the mean arterial blood pressure by 28%. The mean arterial blood pressure remained attenuated by 21% and 19% with the addition of the NEPi and the ECEi. Again, the mean arterial blood pressure rose back to 148 +/- 4 mmHg following cessation of treatment. Daily biochemical and hematological analysis of plasma did not reveal any signs of toxicity, except for a rapid elevation in K (40%) after 1 day of ACEi. Thus, angiotensin II inhibition plays a primary role in controlling the blood pressure of spontaneously hypertensive whereas additional NEPi and ECEi did not provide further benefits under the present dose combinations. The normalizing effect of the higher dose of ACEi by itself made it impossible to discriminate the role of neutral endopeptidase and endothelin-converting enzyme-modulated peptides and to further define the paradigm of triple vasopeptidase inhibition toward better control of vascular hemodynamics. Additional studies are underway.     

Effects of SA7060, a novel dual inhibitor of neutral endopeptidase and angiotensin-converting enzyme, on deoxycorticosterone acetate-salt-induced hypertension in rats

We evaluated whether a novel dual inhibitor of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), SA7060, (S)-2-[3-[(S)-2-(butoxycarbonyl)-2-hydroxyethyl]-3-isobutylureido] -3-(2-naphtyl) propionic acid, prevents deoxycorticosterone acetate (DOCA)-salt-induced hypertension and related organ damage, such as cardiovascular hypertrophy, renal dysfunction and renal tissue injury in rats. The effectiveness was compared with candoxatril and enalapril, which are a selective NEP and ACE inhibitor, respectively. During DOCA-salt treatment for 4 weeks, the rats were given SA7060, candoxatril, enalapril or vehicle, once daily by gavage. The 4-weeks treatment with DOCA and salt produced progressive increases in systolic blood pressure. Daily administration of SA7060, candoxatril or enalapril significantly suppressed the development of hypertension induced by DOCA and salt, although the effect of enalapril was less potent at 4-weeks of the treatment period. In vehicle-treated DOCA-salt rats, decreases in creatinine clearance and increases in urinary excretion of protein and blood urea nitrogen were observed. This functional damage was improved most efficiently by the treatment with SA7060. There were significant increases in urinary excretions of atrial natriuretic peptide and cyclic GMP in SA7060- or candoxatril-treated animals. Histopathological examination of the kidney in DOCA-salt rats revealed tubular, glomerular and vascular lesions, all of which were improved in animals given SA7060 or candoxatril. When the vascular hypertrophy of the aorta was evaluated, there were significant increases in wall thickness, wall area and the wall-to-lumen ratio in vehicle-treated DOCA-salt rats compared with the sham rats. The development of vascular hypertrophy was suppressed by the treatment with SA7060, candoxatril or enalapril. Our findings indicate that SA7060 efficiently prevents DOCA-salt-induced hypertension and related tissue injury, mainly by inhibiting NEP. Thus, SA7060 may be useful for treatment of both renin-dependent and renin-independent hypertensive subjects, although further studies examining efficiency in a renin-dependent hypertensive model are needed.     

Antihypertensive activity during inhibition of neutral endopeptidase and angiotensin converting enzyme.

The specific neutral endopeptidase (NEP) inhibitor, SQ 29,072 (7-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid), was studied in conscious spontaneously hypertensive rats (SHRs) and in DOCA/salt hypertensive rats during inhibition of angiotensin-converting enzyme (ACE) activity with captopril or SQ 27,519 (the free acid of fosinopril). In the SHR, the maximal depressor responses to the combination of SQ 29,072 and SQ 27,519 (-44 +/- 4 mm Hg) were greater than the responses to any of the inhibitors given alone (-26 +/- 5, -40 +/- 10, and -28 +/- 6 mm Hg for SQ 29,072, captopril, and SQ 27,519, respectively). In contrast, the maximal antihypertensive activities of SQ 29,072 were the same in conscious DOCA/salt hypertensive rats infused with saline, captopril, or SQ 27,519 (-54 +/- 10, -51 +/- 8, and -58 +/- 11 mm Hg, respectively), indicating a lack of synergism in this model. In agreement, SQ 28,133 [N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-L-leucine], a compound that inhibits both NEP and ACE, elicited significant depressor activities in both SHR and DOCA/salt hypertensive rats. In conclusion, a selective NEP inhibitor enhanced the depressor activity of ACE inhibitors in the conscious SHR, indicating that these agents may be effectively combined for treatment of some types of hypertension.     

Effect of inhibition of angiotensin converting enzyme and/or neutral endopeptidase on vascular and neural complications in high fat fed/low dose streptozotocin-diabetic rats

Treating high fat fed/low dose streptozotocin-diabetic rats; model of type 2 diabetes, with ilepatril (vasopeptidase inhibitor, blocks neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE)) improved vascular and neural functions. Next, studies were performed to determine the individual effect of inhibition of NEP and ACE on diabetes-induced vascular and neural dysfunctions. High fat fed rats (8 weeks) were treated with 30 mg/kg streptozotocin (i.p.) and after 4 additional weeks, were treated for 12 weeks with ilepatril, enalapril (ACE inhibitor) or candoxatril (NEP inhibitor) followed by analysis of vascular and neural functions. Glucose clearance was impaired in diabetic rats and was not improved with treatment although treatment with ilepatril or candoxatril partially improved insulin stimulated glucose uptake by isolated soleus muscle. Diabetes caused slowing of motor and sensory nerve conduction, thermal hypoalgesia, reduction in intraepidermal nerve fiber (IENF) profiles and impairment in vascular relaxation to acetylcholine and calcitonin gene-related peptide (CGRP) in epineurial arterioles of the sciatic nerve. Inhibition of NEP improved nerve conduction velocity and inhibition of NEP or ACE improved thermal sensitivity and protected IENF density. Ilepatril and candoxatril treatments of diabetic rats were efficacious in improving vascular responsiveness to acetylcholine in epineurial arterioles; whereas all three treatments improved vascular response to CGRP. These studies suggest that inhibition of NEP and ACE activity is an effective approach for treatment of type 2 diabetes neural and vascular complications.     

Induction of neutral endopeptidase and angiotensin-converting enzyme activity of SK-N-SH cells in vitro by quercetin and resveratrol

Quercetin and resveratrol are weak inhibitors of neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) activity of the neuroblastoma cell line SK-N-SH. The long term incubation of the cells for 4 days with quercetin, resveratrol and a combination of both substances in concentrations lower than necessary for inhibition of NEP and ACE activity induced the cellular enzyme activity of NEP and ACE associated with an inhibition of cellular proliferation. The long term treatment of neuroblastoma cells with quercetin and resveratrol enhanced the differentiation state of the cells. Taking into account the significance of NEP and ACE for the degradation of amyloid beta peptides, the effect of quercetin and resveratrol as constituents of red wine for a neuroprotective activity is discussed.     

Effects of lisinopril on streptozotocin-induced diabetic neuropathy in rats

Streptozotocin (STZ)-induced diabetic neuropathy in rats was monitored by measuring the motor nerve conduction velocity (MNCV) and histopathology of the tibial nerve. Pretreatment with lisinopril (2 mg/kg p.o., 5 days prior to STZ and continued for 10 weeks) significantly (p < 0.01) prevented deterioration of MNCV as compared to STZ-diabetic animals. Nerve sections from the lisinopril pretreated group revealed less structural damage as compared to STZ-diabetic rats. However lisinopril had no effect on blood sugar, i.e., it did not alter the diabetic state. It is concluded that lisinopril prevents the development of experimental diabetic neuropathy in STZ-induced diabetic rats.     

High dietary sodium blunts affects of angiotensin-converting enzyme inhibition on vascular angiotensin I-to-angiotensin II conversion in rats

High sodium intake blunts the efficacy of angiotensin (Ang)-converting enzyme (ACE) inhibition (ACEi), but the underlying mechanism is incompletely characterized. High sodium has been reported to increase vascular expression and vascular activity of ACE. To investigate whether high-dietary sodium-induced effects on vascular conversion of Ang I might be involved in the sodium-induced blunting of the response to ACEi, the authors studied the vasoconstrictor responses to Ang I and Ang II of isolated aortic rings from healthy rats on low dietary sodium (LS: 0.05% NaCl) and high dietary sodium (HS: 2.0% NaCl) after 3 weeks of ACEi (lisinopril 75 mg/L) or vehicle (CON). Blood pressure was similar in LS and HS in CON, but HS blunted the blood pressure response to ACEi. Functional conversion of Ang I was assessed as the difference in dose-response curves to Ang I and Ang II in parallel aortic rings. Sodium intake did not affect the dose-response curves to Ang I and Ang II in CON. In the ACEi groups, a significant difference was present between the curves for Ang I and Ang II on LS (deltaEC50, 6.7 nM; range, 2.2-13 nM; P < 0.01) but not on HS (deltaEC50: 1.3 nM; range, 0.0-4.1 nM, median [interquartile range], NS). Thus, HS blunts the ACEi-induced reduction of functional vascular Ang I conversion compared with LS. Whether the blunted functional vascular conversion is causally related to the blunted blood pressure response remains to be elucidated.     

Endothelial angiotensin-converting enzyme and neutral endopeptidase in isolated human umbilical vein: an effective bradykinin inactivation pathway

Kinins are metabolized by metallopeptidases present in different tissues. The aim of this study was to evaluate, employing functional studies in isolated human umbilical vein, the possible participation of angiotensin-converting enzyme, neutral endopeptidase and aminopeptidase P as an inactivation pathway of bradykinin, as well as assess if the endothelial layer is involved in this process. Concentration-response curves to bradykinin were constructed after 120 min incubation period on human umbilical vein rings with and without endothelium and enzymatic inhibitors were applied 30 min before construction of concentration-response curves. The presence of endothelium was confirmed by histological studies. Bradykinin-induced contractile responses were potentiated in human umbilical vein without endothelium when compared to intact tissues. Application of captopril 1 μM (angiotensin-converting enzyme inhibitor) or phosphoramidon 10 μM (neutral endopeptidase inhibitor) induced a leftward shift of bradykinin-elicited responses in human umbilical vein with endothelium while no effect was observed in tissues denuded of endothelium under the same treatment. Exposure to apstatin 10 μM (aminopeptidase P inhibitor) did not potentiate bradykinin-induced effects in intact human umbilical vein. When angiotensin-converting enzyme and neutral endopeptidase were concomitantly inhibited, there was a higher potentiation of bradykinin-elicited responses compared to the effects observed under individual inhibition of either enzyme. Moreover, concentration-response curves to FR190997, a non-peptidic bradykinin B(2) receptor agonist, were not modified under dual enzymatic inhibition. In conclusion, our results demonstrate for the first time the functional relevance of angiotensin-converting enzyme and neutral endopeptidase, localized on the endothelial layer, acting concurrently as a bradykinin inactivating pathway in isolated human umbilical vein.     

Inhibition of both neutral endopeptidase and endothelin-converting enzyme by SLV306 reduces proteinuria and urinary albumin excretion in diabetic rats

Diabetic nephropathy is a serious complication of diabetes associated with a poor prognosis which deteriorates to end-stage renal disease. Increased urinary excretion of protein and albumin are early clinical markers for diabetic renal disease and increased risk of cardiovascular disease. Diabetes causes activation of the renal endothelin system inducing renal damage. We analyzed the effects of SLV306, an inhibitor of both neutral endopeptidase and endothelin-converting enzyme, on diabetes-induced alterations of kidney function and morphology in rats with streptozotocin-induced diabetes. The effects of SLV306 (30 mg/kg per day), captopril (10 mg/kg per day), and placebo on urinary protein and albumin excretion as well as on blood pressure were studied in diabetic rats in comparison to non-diabetic control rats. The rats were treated for 20 weeks. At the end of the study kidney morphology was also analyzed using computer-aided image analysis systems. Serum glucose and blood pressure were similar in all diabetic groups. No side-effects were observed with SLV306 and captopril treatment. Protein excretion was 17.3 +/- 3.0 mg/24 hours in untreated diabetic rats. Protein excretion decreased significantly in the SLV306 (4.8 +/- 0.9 mg/24 hours; P = 0.03 vs untreated diabetic rats) as well as in the captopril (5.1 +/- 1.0 mg/24 hours; P = 0.03 vs untreated diabetic rats) -treated diabetic rats. Albumin excretion was 0.51 +/- 0.12 mg/24 hours in the untreated diabetic group and decreased likewise in the SLV306-treated diabetic rats (0.09 +/- 0.03 mg/24 hours; P = 0.04 vs untreated diabetic rats). The captopril-treated diabetic rats showed a strong trend towards reduced albumin excretion (0.12 +/- 0.04 mg/24 hours; P = 0.06 vs untreated diabetic rats). Computer-aided image analysis revealed that renal interstitial matrix content was significantly decreased in diabetic rats treated with either the angiotensin-converting enzyme inhibitor or the neutral endopeptidase/endothelin-converting enzyme inhibitor as compared to untreated diabetic rats. It was found that SLV306 decreases renal matrix protein content as well as protein and albumin excretion in diabetic rats independent of blood pressure. These effects are comparable to those of angiotensinconverting enzyme inhibition.     

Cardiovascular status following combined angiotensin-converting enzyme and AT1 receptor inhibition in conscious spontaneously hypertensive rats

1. Combined treatment of spontaneously hypertensive rats (SHR) with AT1 receptor antagonists and angiotensin-converting enzyme (ACE) inhibitors has been shown to reduce mean arterial pressure (MAP) more than monotherapy with either agent. The aims of the present study were to investigate the effects of chronic dual renin-angiotensin system (RAS) inhibition using non-hypotensive doses of the AT1 receptor antagonist candesartan cilexetil and the ACE inhibitor perindopril on cardiovascular function and structure. 2. Adult male SHR, aged 15 weeks, were divided into four groups: (i) candesartan cilexetil (0.5 mg/kg per day in drinking water); (ii) perindopril (0.3 mg/kg per day in drinking water); (iii) combined treatment (dual RAS inhibition); or (iv) the appropriate vehicle (0.1% ethanol/0.1% polyethylene glycol/1.5 mmol/l sodium bicarbonate dissolved in water for candesartan cilexetil; distilled water for perindopril). Systolic blood pressure was measured weekly using the tail-cuff method and urinary microalbuminuria was measured fortnightly. 3. After 4 weeks, rats were instrumented for intravenous drug administration and measurement of MAP. At this time, the cardiovascular effects of angiotensin (Ang) I and AngII (5-20 ng) and sodium nitroprusside (SNP) and acetylcholine (ACh; 1-5 micro g) were assessed. In addition, left ventricular : bodyweight and media : lumen ratios were determined as indices of cardiac and vascular hypertrophy, respectively. 4. Candesartan cilexetil and perindopril alone had minimal effect on MAP when measured both directly and indirectly, whereas direct MAP was significantly decreased in the combined treatment group (131 +/- 6 mmHg; P < 0.05) compared with the vehicle group (156 +/- 9 mmHg). Pressor responses to AngI were significantly decreased in all groups compared with the vehicle-treated group and pressor responses to AngII were significantly decreased in the candesartan cilexetil-treated (P < 0.01) and combined treatment groups (P < 0.01) compared with the vehicle-treated group. Depressor responses to ACh and SNP were not significantly affected by any of the antihypertensive therapies compared with vehicle-treated SHR. 5. Vascular hypertrophy was significantly decreased in the candesartan cilexetil and combined groups compared with the vehicle-treated group, whereas cardiac hypertrophy was reduced, with the rank order of effect being: dual RAS inhibition > perindopril > candesartan cilexetil. Urinary albumin tended to decrease with dual RAS inhibition, but was not significantly affected by this short-term treatment. 6. These results demonstrate the efficacy of low-dose dual RAS inhibition as an antihypertensive modality, at least in SHR, not only in reducing arterial pressure, but also in improving cardiovascular structure.     

Comparison of a vasopeptidase inhibitor with neutral endopeptidase and angiotensin-converting enzyme inhibitors on bradykinin metabolism in the rat coronary bed

The in vitro effects of omapatrilat, a dual vasopeptidase inhibitor that simultaneously inhibits neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), on exogenous bradykinin metabolism after a single passage through the coronary bed were compared with that of a NEP inhibitor (retrothiorphan, 25 nM), an ACE inhibitor (enalaprilat, 130 nM), and omapatrilat (25 nM). Bradykinin and inhibitors were infused into isolated Langendorff rat hearts perfused at 1 ml/min followed by reperfusion at 10 ml/min. Residual bradykinin was quantified in the coronary effluent by enzyme-linked immunosorbent assay to calculate bradykinin recovery and its kinetic parameters (Vmax/Km). Bradykinin degradation rate at 1 ml/min was 4.56 +/- 0.39 1/min per gram without inhibitors and was significantly reduced to 2.57 +/- 0.19 1/min per gram in the presence of enalaprilat, to 2.97 +/- 0.38 1/min per gram with retrothiorphan, to 1.82 +/- 0.17 1/min per gram with both enalaprilat and retrothiorphan, and to 1.14 +/- 0.35 1/min per gram with omapatrilat. In a second set of experiments, the effect of a 14-day treatment of rats with either ACE inhibitors (enalapril, quinapril, and ramipril), a NEP inhibitor (candoxatril), or omapatrilat on exogenous bradykinin metabolism was studied in Langendorff perfused hearts isolated from these long-term treated rats. In untreated rats, bradykinin degradation at a coronary perfusion of 1 ml/min was 4.35 +/- 0.41 1/min per gram. This value was reduced by 30% for the NEP inhibitor, by 50% for all ACE inhibitors, and by 75% for omapatrilat. All inhibitors administered either short term or long term significantly reduced bradykinin degradation during a single passage through the coronary bed. However, omapatrilat administration resulted in the greatest protection from bradykinin breakdown than ACE or NEP inhibitors alone.     

Effects of gastroprokinetic agents on gastroparesis in streptozotocin-induced diabetic rats

The influence of diabetic hyperglycemia on solid gastric emptying in rats was examined. Diabetes was produced by streptozotocin (STZ, 40mg/kg i.v.), and diabetic hyperglycemia was observed from 1 day after the STZ injection. The gastric emptying of glass beads in the diabetic rats was significantly delayed compared with that in age-matched control rats at 1, 3 and 7 days after diabetes induction. A slight decrease in gastric emptying was observed in the diabetic rats from 2 to 52 weeks after the diabetes induction. We also investigated the influence of gastroprokinetic agents on STZ-induced diabetic gastroparesis and subdiaphragmatic vagotomy-induced gastroparesis in rats. The selective 5-HT₃ receptor antagonists ramosetron (YM060), YM114 (KAE-393), granisetron and ondansetron, and the substituted benzamides (5-HT₄ receptor agonist/5-HT₃ receptor antagonists) cisapride mosapride and SC-53116 dose-dependently enhanced gastric emptying in normal rats. These compounds also reversed the impairment of diabetic gastroparesis rats at 7 days after the STZ injection, but higher doses were required. The solid gastric emptying in subdiaphragmatic vagotomized rats was also delayed. Ramosetron and the substituted benzamides cisapride and zacopride partially reversed the gastroparesis in the vagotomized rats. These results suggest that acute hyperglycemia is important mechanism for the delay of solid gastric emptying in diabetic rats. It is also suggested that selective 5-HT₃ receptor antagonists and substituted benzamides enhance gastric emptying not only in normal rats but also in diabetic and vagotomized rats. Received : 9 December 1996 / Accepted: 25 Februar 1997     

Effects of arctiin on streptozotocin-induced diabetic retinopathy in sprague-dawley rats

Diabetic retinopathy is one of the most common and severe complications of diabetes mellitus. Arctiin, a bioactive compound isolated from the dry seeds of Arctium lappa L., has been reported to have antidiabetic activity. In this study, we investigated the effect of arctiin on the serum glucose and HBA1c levels, the blood viscosity, and VEGF expression in the retinal tissues of rats with diabetic retinopathy. We first extracted arctiin from Fructus Arctii and then investigated its chemopreventive effect on streptozotocin-induced diabetic retinopathy in male Sprague-Dawley rats. After the induction of diabetes using streptozotocin (30 mg/kg, i. p.), the rats were randomly divided into five groups (n = 20 per group) and treated with intragastric doses of 30, 90, or 270 mg/kg/d wt of arctiin, 100 mg/kg/d wt of calcium dobesilate, or 0.5 % CMC-Na. Twenty nondiabetic sham-treated rats were treated with 0.5 % CMC-Na. The occurrence of diabetic retinopathy did not differ dramatically among the groups. However, at week 16, the glycosylated haemoglobin (HBA1c) level was significantly decreased in all of the arctiin-treated groups when compared with the control group, and the serum glucose level was also decreased in the rats treated with the highest dose of arctiin. In addition, treatment with arctiin ameliorated retinal oedema, detachment of the retina, and VEGF expression in the retina, as detected using histological and immunochemical examinations. Finally, arctiin increased the viability of retinal microvascular endothelial cells in vitro. Together, these findings demonstrate that arctiin decreases the severity of diabetic complications, demonstrating the importance of this compound as an inhibitor of diabetic retinopathy.     

Dual inhibition of angiotensin converting enzyme and neutral endopeptidase by omapatrilat in rat in vivo.

The pharmacological profile of a vasopeptidase inhibitor is dependent on the ratio of neutral endopeptidase (NEP)vs angiotensin converting enzyme (ACE) inhibition of the particular drug. We used in vitro autoradiography to determine the local renal and cardiac NEP and ACE inhibition after oral treatment with the dual NEP/ACE inhibitor omapatrilat in rat. Maximal inhibition of both renal NEP and ACE was achieved at the omapatrilat dose of 40 mg kg(-1)day(-1). Effective local ACE inhibition was detected also in the myocardium. The haemodynamic effects were similar to captopril, but omapatrilat treatment produced more favorable effect on natriuretic peptide levels. In conclusion, good tissue penetration of omapatrilat and balanced NEP/ACE inhibition may prove to be useful in the treatment of hypertension and heart failure.     

Effects of neutral endopeptidase 24.11 inhibition on myocardial infarct size and ischemic preconditioning in rabbits

This study was designed to determine whether inhibition of neutral endopeptidase 24.11 (NEP) reduces infarct size and enhances protection afforded by ischemic preconditioning (PC) by elevation of the tissue bradykinin (BK) level in the heart in situ. In experiments to determine a dose of thiorphan (Thio) that inhibits NEP activity in the rabbit, infusion of Thio at a rate of 15 micro g/kg per min was found to be NEP-selective, since it increased the extent and duration of hypotension after BK injection (50 ng/kg and 100 ng/kg, i.v.) but did not inhibit pressor response to angiotensin I (100 ng/kg and 500 ng/kg, i.v.). Infusion of Thio at a rate of 25 micro g/kg per min blunted pressor response to angiotensin I by 30%, suggesting this dose partially inhibits angiotensin-converting enzyme activity. In the second series of experiments, myocardial infarction was induced by 30-min coronary occlusion and 3-h reperfusion in rabbits. In untreated controls, infarct size as a percentage of area at risk (%IS/AR) was 50.1+/-4.1%, and infusion of Thio at 15 micro g/kg per min and 25 micro g/kg per min failed to limit infarct size (54.3+/-4.0% and 50.1+/-2.8%, respectively). However, these doses of Thio significantly reduced %IS/AR when combined with PC with 2-min ischemia to 25.7+/-3.3% and 19.7+/-3.1%, respectively, although this submaximal PC protocol alone did not achieve significant cardioprotection (%IS/AR=35.6+/-4.0%). This effect of Thio on PC was abolished by pretreatment with icatibant (2 micro g/kg), a BK B(2) receptor blocker. The results of the present study suggest that NEP inhibition does not increase anti-infarct tolerance of the myocardium but significantly enhances cardioprotection of PC via a B(2) receptor-mediated mechanism.