Investigation of Dipicolinic Acid Isosteres for the Inhibition of Metallo-ß-Lactamases.

New Delhi metallo-ß-lactamase-1 (NDM-1) poses an immediate threat to our most effective and widely prescribed drugs, the ß-lactam-containing class of antibiotics. There are no clinically relevant inhibitors to combat NDM-1, despite significant efforts toward their development. Inhibitors that use a carboxylic acid motif for binding the ZnII ions in the active site of NDM-1 make up a large portion of the >500 inhibitors reported to date. New and structurally diverse scaffolds for inhibitor development are needed urgently. Herein we report the isosteric replacement of one carboxylate group of dipicolinic acid (DPA) to obtain DPA isosteres with good inhibitory activity against NDM-1 (and related metallo-ß-lactamases, IMP-1 and VIM-2). It was determined that the choice of carboxylate isostere influences both the potency of NDM-1 inhibition and the mechanism of action. Additionally, we show that an isostere with a metal-stripping mechanism can be re-engineered into an inhibitor that favors ternary complex formation. This work provides a roadmap for future isosteric replacement of routinely used metal binding motifs (i.e., carboxylic acids) for the generation of new entities in NDM-1 inhibitor design and development.

Effects of ACE inhibition and angiotensin II type 1 receptor blockade on cardiac function and G proteins in rats with chronic heart failure.

1. Inhibition of the renin-angiotensin system (RAS) improves symptoms and prognosis in heart failure. The experimental basis for these benefits remains unclear. We examined the effects of inhibition of ACE or blockade of angiotensin II type 1 (AT1) receptor on the haemodynamics, cardiac G-proteins, and collagen synthesis of rats with coronary artery ligation (CAL), a model in which chronic heart failure (CHF) is induced. 2. Rats were orally treated with the ACE inhibitor trandolapril (3 mg kg(-1) day(-1)) or the AT1 receptor blocker L-158809 (1 mg kg(-1) day(-1)) from the 2nd to 8th week after CAL. CAL resulted in decreases in the left ventricular systolic pressure and its positive and negative dP/dt, an increase in the left ventricular end-diastolic pressure, and the rightward shift of the left ventricular pressure-volume curve. Long-term treatment with either drug improved these signs of CHF to a similar degree. 3. Cardiac Gsalpha and Gqalpha protein levels decreased, whereas the level of Gialpha protein increased in the animals with CHF. Long-term treatment with trandolapril or L-158809 attenuated the increase in the level of cardiac Gialpha protein of the animals with CHF without affecting Gsalpha and Gqalpha protein levels. Cardiac collagen content of the failing heart increased, whose increase was blocked by treatment with either drug. 4. Exogenous angiotensin I stimulated collagen synthesis in cultured cardiac fibroblasts, whose stimulation was attenuated by either drug. 5. These results suggest that blockade of the RAS, at either the receptor level or the synthetic enzyme level, may attenuate the cardiac fibrosis that occurs after CAL and thus affect the remodelling of the failing heart.

Long-term treatment with neutral endopeptidase inhibitor improves cardiac function and reduces natriuretic peptides in rats with chronic heart failure.

OBJECTIVE: Increased secretion of atrial and brain natriuretic peptide (ANP and BNP) from hearts is known to exhibit favorable effects in patients and animals with heart failure, and inhibition of neutral endopeptidase (NEP), an enzyme that degrades ANP and BNP, may further increase these peptide levels. However, it is still unknown whether such elevation of the ANP and BNP may offer a therapeutic benefit to the progression of chronic heart failure (CHF). We examined the effects of ONO-9902, a novel NEP inhibitor, on changes in hemodynamic parameters, NEP activity and neurohumoral factors in rats with CHF induced by left coronary artery ligation (CAL). METHODS: Male Wistar rats (220-240 g) were subjected to induction of acute myocardial infarction by CAL. Rats were orally treated with ONO-9902 (300 mg/kg/day) from the 1st to 6th week after the operation. Hemodynamic and/or biochemical assessments were performed at the 1st and 6th weeks after the operation. RESULTS: A single administration of ONO-9902 inhibited the plasma and kidney NEP activities and thereby further augmented the elevation of plasma ANP concentration in rats with CAL at the 1st week after the operation. In rats with CAL at the 6th week after the operation, the left ventricular end-diastolic pressure (LVEDP) increased and cardiac output index (COI) decreased as compared with those of sham-operated rats. These changes were accompanied by marked increases in the plasma ANP, BNP and endothelin-1 (ET-1). Chronic treatment with ONO-9902 attenuated the increase in LVEDP and the decrease in COI. These changes were associated with a decrease in plasma ANP, BNP and ET-1 concentrations. CONCLUSIONS: The results suggest that chronic treatment with NEP inhibitor improves depressed cardiac function in rats with CHF. ONO-9902 may offer a new and possible therapeutic approach in patients with CHF.

Role of cardiac renin-angiotensin system in sarcoplasmic reticulum function and gene expression in the ischemic-reperfused heart.

The aim of this study was to explore the possible participation of cardiac renin-angiotensin system (RAS) in the ischemia-reperfusion induced changes in heart function as well as Ca2+-handling activities and gene expression of cardiac sarcoplasmic reticulum (SR) proteins. The isolated rat hearts, treated for 10 min without and with 30 microM captopril or 100 microM losartan, were subjected to 30 min ischemia followed by reperfusion for 60 min and processed for the measurement of SR function and gene expression. Attenuated recovery of the left ventricular developed pressure (LVDP) upon reperfusion of the ischemic heart was accompanied by a marked reduction in SR Ca2+-pump ATPase, Ca2+-uptake and Ca2+-release activities. Northern blot analysis revealed that mRNA levels for SR Ca2+-handling proteins such as Ca2+-pump ATPase (SERCA2a), ryanodine receptor, calsequestrin and phospholamban were decreased in the ischemia-reperfused heart as compared with the non-ischemic control. Treatment with captopril improved the recovery of LVDP as well as SR Ca2+-pump ATPase and Ca2+-uptake activities in the postischemic hearts but had no effect on changes in Ca2+-release activity due to ischemic-reperfusion. Losartan neither affected the changes in contractile function nor modified alterations in SR Ca2+-handling activities. The ischemia-reperfusion induced decrease in mRNA levels for SR Ca2+-handling proteins were not affected by treatment with captopril or losartan. The results suggest that the improvement of cardiac function in the ischemic-reperfused heart by captopril is associated with the preservation of SR Ca2+-pump activities; however, it is unlikely that this action of captopril is mediated through the modification of cardiac RAS. Furthermore, cardiac RAS does not appear to contribute towards the ischemia-reperfusion induced changes in gene expression for SR Ca2+-handling proteins.

Role of energy metabolism in the preconditioned heart--a possible contribution of mitochondria.

A brief period of ischemia and reperfusion has been shown to protect the myocardium against subsequent sustained ischemia and reperfusion injury, which is called "preconditioning". A great number of investigators have explored the mechanisms underlying this preconditioning-induced cardioprotection. This article dealt with possible mechanisms of energy metabolism and mitochondrial activity for preconditioning-induced cardioprotection. Particularly, the contribution of energy metabolites produced during a brief period of ischemia and reperfusion injury, as well as mitochondrial function that is modified by changes in mitochondrial ATPase activity, opening of mitochondrial ATP-dependent potassium channels and production of free radicals in mitochondria, to ischemic preconditioning is discussed.

Improvement of exercise capacity of rats with chronic heart failure by long-term treatment with trandolapril.

1. The effects of long-term treatment with trandolapril, an angiotensin I-converting enzyme inhibitor, on exercise capacity of rats with chronic heart failure (CHF) following coronary artery ligation were examined. CHF was developed by 8 weeks after the coronary artery ligation. 2. The running time of rats with CHF in the treadmill test was shortened to approximately 65% of that of sham-operated rats (16.3+/-1.2 vs. 25.1+/-1.6 min, n = 7; P<0.05). ATP, creatine phosphate (CP), and lactate contents of the gracilis muscle of rats with CHF were similar to those of sham-operated rats before running. After running, ATP and CP were decreased and lactate was increased in both rats with CHF and sham-operated rats. There were no significant differences in the levels of energy metabolites between rats with CHF and sham-operated rats. The rates of decrease in ATP and CP and rate of increase in lactate in the gracilis muscle of rats with CHF during exercise were greater than those of sham operated rats (2.5, 2.0 and 1.5 fold high, respectively), suggesting wastage of energy during exercise in the animals with CHF. 3. Myofibrillar Ca2+ -stimulated ATPase (Ca-ATPase) activity of skeletal muscle of rats with CHF was increased over that of the sham-operated control (62.03+/-1.88 vs. 52.34+/-1.19 micromol Pi mg(-1) protein h(-1) n = 7; P<0.05). The compositions of myosin heavy chain (MHC) isoforms of gracilis muscle were altered by CHF; decreases in MHC types I and IIb and an increase in MHC type IIa were found (P<0.05). 4. Rats with CHF were treated with 1 mg kg(-1) day(-1) trandolapril from the 2nd to 8th week after surgery. Treatment with trandolapril prolonged the running time, reversed the rates of decrease in ATP and CP and the rate of increase in lactate, and restored the Ca-ATPase activity (51.11+/-0.56 micromol Pi mg(-1) protein h(-1), n = 7; P<0.05) and composition ratio of MHC isoforms in the gracilis muscle. 5. The results suggest that long-term trandolapril treatment of rats with CHF may restore their ability to utilize energy without wastage and thus improve exercise capacity.

Effects of long-term treatment with eicosapentaenoic acid on the heart subjected to ischemia/reperfusion and hypoxia/reoxygenation in rats.

The effects of eicosapentaenoic acid (EPA) and long-term treatment with EPA-ethylester (EPA-E) were examined in perfused rat hearts subjected to ischemia/reperfusion and adult rat cardiomyocytes subjected to hypoxia/reoxygenation. EPA (0.1 microM) improved postischemic contractile dysfunction of the ischemic/reperfused heart. EPA (10 microM) attenuated hypoxia/reoxygenation-induced morphological deterioration of cardiomyocytes. The results suggest the presence of direct cardioprotective effects of EPA. Rats were orally treated for 4 weeks with 1 g/kg/day of EPA-E to elucidate ex vivo effects of EPA, and the fatty acid composition of cardiac phospholipids was determined. The percent ratio of EPA in total fatty acids of cardiac phospholipids increased whereas that of arachidonic acid decreased. The percent ratio of n-3/n-6 fatty acid did not increase. Treatment with EPA-E did not improve the post-ischemic contractile function, but attenuated the ischemia/reperfusion-induced release of prostaglandins during reperfusion. Treatment with EPA-E preserved a better morphological appearance of the cardiomyocytes subjected to hypoxia/reoxygenation. The results suggest that the mechanisms responsible for cytoprotective effects of hypoxic/reoxygenated cardiomyocytes or inhibition of metabolic alterations of the ischemic/reperfused heart by long-term EPA-E treatment did not contribute substantially to recovery of post-ischemic contractile dysfunction. The direct in vitro effects of EPA may play a role in the protection of the heart from ischemia/reperfusion or hypoxia/reoxygenation injury.

Modulation of cAMP-mediated vasorelaxation by endothelial nitric oxide and basal cGMP in vascular smooth muscle.

Recent in vitro evidence shows a role of endothelial nitric oxide (NO) in the modulation of isoproterenol-induced vasorelaxation. To elucidate roles of endothelial cells and NO in cyclic adenosine monophosphate (cAMP)-mediated vasodilators we examined the effects of removal of endothelium and a NO synthase (NOS) inhibitor on relaxant responses in vitro of rat aortic strips to beta-adrenoceptor stimulants and colforsin dapropate, a water-soluble forskolin, and changes in cAMP and cyclic guanosine monophosphate (cGMP) contents. Relaxant responses of rat aorta to isoproterenol, denopamine, salbutamol, colforsin, and dibutyryl cAMP (dbcAMP) were blunted by removal of endothelial cells or treatment with NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Relaxant response of endothelium-intact segments to isoproterenol was associated with increases in tissue cAMP and cGMP contents. Removal of endothelium or treatment with L-NAME markedly reduced basal cGMP and abolished the isoproterenol-induced increase in cGMP but not cAMP content. In endothelium-removed segments, pretreatment with sodium nitroprusside (SNP) restored the diminished relaxant response to isoproterenol and increased basal cGMP (from 0.08 +/- 0.01 to 0.16 +/- 0.02 pmol/mg protein), whereas it did not affect the isoproterenol-induced increase in cAMP. The diminished relaxant response of endothelium-removed segments to dbcAMP was not restored by SNP pretreatment. The results suggest that relaxant response of rat aorta to cAMP-mediated vasodilators is mediated, in part, by NO production in endothelium and subsequent increase in cGMP in vascular smooth-muscle cells.

Long-term supplementation with eicosapentaenoic acid salvages cardiomyocytes from hypoxia/reoxygenation-induced injury in rats fed with fish-oil-deprived diet.

Dietary supplementation of fish oil containing eicosapentaenoic acid (C20:5 n-3, EPA) and docosahexaenoic acid (C22:6 n-3, DHA) has been shown to exert protective effects on ischemic/reperfused hearts. We determined whether deprivation of fish oil from the diet paradoxically enhances susceptibility of cardiomyocytes to hypoxia/reoxygenation-induced injury and whether supplementation with either EPA or DHA overcomes such alterations. Rats were fed with fish-oil-rich (FOR) diet, fish-oil-deprived (FOD) diet alone, FOD diet with EPA (1 g/kg/day), or FOD diet with DHA (1 g/kg/day) for 4 weeks. The FOD diet reduced n-3 polyunsaturated fatty acids (PUFAs) and increased n-6 PUFAs such as linoleic (C18:2) and arachidonic acids (C20:4) in myocardial phospholipids. EPA or DHA supplementation increased its incorporation into phospholipid pools. Cardiomyocytes isolated by treatment with collagenase were subjected to 150 min of hypoxia and subsequent reoxygenation for 15 min. In the FOD diet group, the number of surviving rod-shaped cells after hypoxia and reoxygenation was smaller than that of the FOR group. Supplementation with EPA did not affect the number of rod-shaped cells, but attenuated reoxygenation-induced reduction in the number of square-shaped cells. In contrast, DHA supplementation did not afford any protection. The results suggest that deprivation of fish oil from dietary intake enhances the susceptibility of cardiomyocytes to hypoxic injury, and EPA, but not DHA, is capable of salvaging cardiomyocytes from hypoxia/reoxygenation-induced damage.

The effect of chronic treatment with trandolapril on cyclic AMP-and cyclic GMP-dependent relaxations in aortic segments of rats with chronic heart failure.

1 Characteristics of cyclic GMP- and cyclic AMP-mediated relaxation in aortic segments of rats with chronic heart failure (CHF) and the effects of chronic treatment with an angiotensin I converting enzyme (ACE) inhibitor, trandolapril, were examined 8 weeks after coronary artery ligation. 2 Cardiac output indices of coronary artery-ligated and sham-operated rats were 125+/-8 and 189+/-10 ml min(-1) kg(-1), respectively (P<0.05), indicating the development of CHF at this period. 3 The maximal relaxant response of aortic segments to 10 microM acetylcholine in rats with CHF and sham-operated rats was 64.0+/-5.7 and 86.9+/-1.9%, respectively (P<0.05), whereas the relaxant response to sodium nitroprusside (SNP) remained unchanged. Tissue cyclic GMP content in rats with CHF was lower than that of sham-operated rats. 4 In endothelium-intact segments of rats with CHF, the maximal relaxant response to 10 microM isoprenaline (44.5+/-6.7%) was lower that sham-operated rats (81.3+/-2.5%, P<0.05) and the concentration-response curve for NKH477, a water-soluble forskolin, was shifted to the right without a reduction in the maximal response. Isoprenaline-induced relaxation of aortic segments was attenuated by NG-nitro-L-arginine methyl ester (L-NAME) in sham-operated rats, but not in rats with CHF. Relaxation to 30 microM dibutyryl cyclic AMP in rats with CHF (26.8+/-2.7%) was lower than that in sham-operated rats (63.4+/-11.8%, P<0.05). 5 Trandolapril (3 mg kg(-1) day(-1)) was orally administered from the 2nd to 8th week after the operation. Aortic blood flow of rats with CHF (38.5+/-3.6 ml min(-1)) was lower than that of sham-operated rats (55.0+/-3.0 ml min(-1)), and this reduction was reversed (54.1+/-3.4 ml min(-1)) by treatment with trandolapril. The diminished responsiveness described above was normalized in the trandolapril-treated rat with CHF (i.e., the maximal relaxation to acetylcholine, 94.7+/-1.0%; that to isoprenaline, 80.5+/-2.8%; that to dibutyryl cyclic AMP, 54.7+/-6.2%). However, aortic segments of trandolapril-treated rats with CHF, L-NAME did not attenuate isoprenaline-induced relaxation and the tissue cyclic GMP level was not fully restored, suggesting that the ability of the endothelium to produce NO was still partially damaged. 6 The results suggest that vasorelaxation in CHF, diminished mainly due to dysfunction in endothelial nitric oxide (NO) production and cyclic AMP-mediated signal transduction, was partially restored by long-term treatment with trandolapril. The mechanism underlying the restoration may be attributed in part to prevention of CHF-induced endothelial dysfunction.

Effects of the antihypertensive agent, cicletanine, on noradrenaline release and vasoconstriction in perfused mesenteric artery of SHR.

1. The mechanism by which cicletanine (CIC) exerts its antihypertensive effects has not been fully elucidated. The present study was undertaken to examine the effects of in vivo and in vitro treatment with CIC on the pressor response and noradrenaline (NA) overflow during periarterial nerve stimulation (PNS) in perfused mesenteric arterial beds isolated from spontaneously hypertensive rats (SHR). 2. CIC at a dose of 50 mg kg(-1) day(-1) was administered orally to both SHR and normotensive Wistar-Kyoto rats (WKY) from the 6th to 10th week of age. At the 10th week, the isolated mesenteric arterial bed was perfused with Krebs-Henseleit buffer and changes in perfusion pressure and NA overflow during PNS were measured. 3. Chronic treatment with CIC suppressed the age-related elevation of systemic blood pressure in SHR but not in WKY. 4. The PNS (20 Hz)-induced mesenteric vasoconstrictor response and NA overflow were greater in SHR than in WKY. In the vasculature of SHR chronic treatment with CIC resulted in a significant attenuation of the vasoconstriction and the NA overflow during PNS, whereas it did not alter vasoconstrictor responses to bolus injections of KCl and phenylephrine. 5. Treatment with 30 microM CIC in vitro diminished the PNS-induced vasoconstriction and NA overflow but not the NA- and KCl-induced vasoconstriction in the vasculature of untreated SHR. 6. In the vasculature of SHR PNS-induced NA overflow was attenuated by prostaglandin E2 (0.05 microM), whereas it was augmented by the cyclo-oxygenase inhibitor diclofenac-Na (30 microM). In the presence of diclofenac, in vitro treatment with CIC did not attenuate the NA overflow during PNS. 7. The results suggest that the antihypertensive effect of CIC in SHR is partially due to the presynaptic inhibition of NA release during sympathetic nerve activation. Transjunctional inhibition of NA release by prostaglandins may contribute to the inhibitory action of CIC on NA release in the vasculature of SHR.

Changes in fatty acid compositions of myocardial lipids in rats with heart failure following myocardial infarction.

Changes in fatty acid composition of myocardial lipids were examined in rats with heart failure following myocardial infarction. Left ventricular systolic pressure (LVSP) was decreased and left ventricular end-diastolic pressure (LVEDP) was elevated 24 h, 1 and 12 weeks after left coronary artery ligation (CAL), suggesting the development of heart failure at these periods in this model. Hearts were isolated 24 h, 1 week and 12 weeks after the operation. Myocardial lipids in the infarcted scar tissue, non-infarcted remaining left ventricle including interseptum and right ventricle were separated into phospholipid (PL), triacylglycerol (TG), diacylglycerol (DAG) and free fatty acid (FFA) fractions. In the scar tissue PL content markedly decreased whereas TG, DAG and FFA contents increased 24 h after CAL. Despite a marked decrease in constituted fatty acids of PL fraction in the scar tissue the percentage of arachidonic acid in PL was elevated 12 weeks after CAL, suggesting that release of arachidonic acid during PL degradation was suppressed. In the non-infarcted viable left ventricle PL content remained unchanged throughout the experiment whereas TG, DAG and FFA contents were elevated 24 h after CAL. Despite no changes in PL and other lipid contents in the non-infarcted tissue the percentage of linoleic acid in PL was reduced and that of docosahexaenoic acid in PL was elevated 12 weeks after CAL. Our findings showed that myocardial lipid composition of the non-infarcted left ventricle was altered only in an early stage of the development of heart failure and fatty acid compositions of PL was exchanged in a late stage of the development of heart failure. The exchange may be related to cardiac dysfunction or myocardial remodelling in the rat with heart failure.

Protective effect of amiloride against reperfusion damage as evidenced by inhibition of accumulation of free fatty acids in working rat hearts.

To examine whether amiloride protects against ischemia-induced or reperfusion-induced damage to the heart, mechanical and metabolic studies were performed in the isolated, working rat heart. Ischemia decreased both mechanical function and the tissue levels of high-energy phosphates and increased the tissue levels of free fatty acids (FFAs). Reperfusion restored the levels of high-energy phosphates but further increased FFA accumulation. For this reason, accumulation of FFAs was used as an indicator of both ischemia-induced and reperfusion-induced damage. Drugs were added to the perfusion solution 5 min before ischemia until the end of ischemia (pre) or until 10 min after reperfusion (pre + post). Diltiazem (1 or 5 mumol/L pre) decreased the mechanical function of the non-ischemic heart and attenuated both ischemia-induced and reperfusion-induced accumulation of FFAs. Amiloride (50 mumol/L pre) did not affect the mechanical function of the non-ischemic heart or attenuate ischemia-induced or reperfusion-induced FFA accumulation effectively. However, amiloride (50 mumol/L pre + post) did markedly attenuate the reperfusion-induced accumulation of FFAs. In conclusion, diltiazem attenuates both ischemia-induced and reperfusion-induced myocardial damage, probably through its energy-sparing effect as a result of a decrease in mechanical function before ischemia. In contrast, amiloride attenuates only the reperfusion-induced myocardial damage through mechanisms other than the energy-sparing effect.

Beta-adrenoceptor stimulation-mediated preconditioning-like cardioprotection in perfused rat hearts.

To determine whether adrenergic stimulation induces preconditioning-like cardioprotection, rat hearts were perfused for 2 min with either norepinephrine, phenylephrine, or isoproterenol followed by 10-min drug-free perfusion. Then the hearts were subjected to 40-min ischemia and 30-min reperfusion. Little recovery of left ventricular developed pressure (LVDP) and loss of the myocardial creatine kinase (CK) during reperfusion were observed in the drug-untreated heart. Preperfusion with norepinephrine (0.25 microM) or isoproterenol (0.25 microM), but not phenylephrine (10 microM), resulted in a better recovery of LVDP in the postischemic reperfused heart and a reduction in CK release during reperfusion. A similar improvement of postischemic cardiac contractile dysfunction and CK loss was seen in the heart subjected to 5-min ischemia followed by 5-min reperfusion (ischemic preconditioning) before the prolonged period of ischemia/reperfusion. Pretreatment with timolol, a beta-adrenoceptor blocker, abolished the protective effect of norepinephrine, whereas pretreatment with bunazosin, an alpha 1-adrenoceptor blocker, did not affect the protective effect of isoproterenol. The results suggest that a brief period of stimulation of cardiac beta-adrenoceptor exerts the preconditioning-mimetic protective effect against postischemic contractile dysfunction in perfused rat hearts.

Preconditioning preserves mitochondrial function and glycolytic flux during an early period of reperfusion in perfused rat hearts.

OBJECTIVE: The purpose of the present study was to examine the effects of preconditioning on glycolysis and oxidative phosphorylation during reperfusion in perfused rat hearts. METHODS: Preconditioning was induced by 5 min of ischemia and 5 min of reperfusion before 40 min of sustained ischemia and subsequent 30 min of reperfusion. Tissue energy metabolite levels, mitochondrial oxygen consumption capacity and adenine nucleotide translocator content of the perfused hearts were assessed at 40 min of ischemia, 5 and 30 min of reperfusion. RESULTS: Preconditioning improved the postischemic recovery of rate x pressure product (92.5 +/- 8.7 vs. 24.9 +/- 1.2% for non-preconditioned group) and high-energy phosphate content (ATP and CrP; 39.5 +/- 2.0 and 96.2 +/- 4.9% of initial vs. 24.1 +/- 0.9 and 56.1 +/- 4.3% of initial for the non-preconditioned group). The mitochondrial oxygen consumption capacity and the adenine nucleotide translocator content of the non-preconditioned heart were decreased by sustained ischemia and remained decreased throughout reperfusion. Preconditioning prevented these decreases. The tissue lactate level of the non-preconditioned heart was high throughout reperfusion (16.5-fold vs. basal), whereas in the preconditioned heart it returned to the basal level within a few minutes of reperfusion. Furthermore, the ratios of [fructose 1,6-bisphosphate]/([glucose 6-phosphate] + [fructose 6-phosphate]) at 5-min reperfusion were higher (2.2-fold) than those of the non-preconditioned heart. CONCLUSIONS: The results suggest that preconditioning preserves the capacity for normal mitochondrial function and the facilitation of glycolysis during reperfusion, which may play an important role in the improvement of postischemic contractile function and high-energy phosphate content.

beta-Adrenoreceptor antagonistic actions and mutagenicities of R(+)- and S(-)-enantiomers of N-desisopropylpropranolol and its N-acetyl conjugate.

Enantiospecific acetyl conjugation was examined in the rat liver 105000 x g supernatant (cytosol) system using racemic 1-amino-3-(1-naphthyloxy)-2-propanol (NDP), a N-desisopropyl metabolite of propranolol. From the results of chiral separative determination of the samples by HPLC using a Chiralcel OD-R column, more remarkable enantiospecificity was observed in the R(+)-enantiomer on NDP elimination and N-acetyl conjugate (AcNDP) formation. Next, the strength of beta-adrenoceptor antagonistic actions and mutagenicities was compared between R(+)- and S(-)-enantiomers of NDP and AcNDP, respectively. In the case of NDP, both enantiomers possessed weak beta1-adrenoceptor antagonistic effects on isoproterenol-induced positive inotropic and chronotropic actions in the left and right atria isolated from a guinea pig. These actions of R(+)- and S(-)-NDP were 1700-times and 100-times less potent, respectively, than those of propranolol. beta2-Adrenoceptor antagonistic actions of R(+)- and S(-)-NDP in the trachea were 1600-times and 200-times less potent, respectively, than those of propranolol. Enantiospecificity was observed in the beta-adrenoceptor antagonistic action of S(-)-NDP, while R(+)-NDP and both enantiomers of AcNDP appeared to be negligible in this action. On the other hand, the mutagenicities of each enantiomer were examined by the Ames method using 13 kinds of Salmonella typhimurium strains. In the case of AcNDP, the numbers of colonies increased according to the substrate concentration only when rat liver 9000 x g supernatant fraction (S-9 mixture) was added to the plates containing TA100, YG1029, TA104 and YG3003, and then enantiospecificity was observed in the mutagenicity of S(-)-AcNDP. Thus, the ultimate mutagen might be an active metabolite formed mainly from S(-)-AcNDP. Despite of the addition of rat liver S-9 mixture, R(+)-AcNDP and both enantiomers of NDP did not indicate mutagenicity.

Activation of cardiac muscarinic receptor and ischemic preconditioning effects in in situ rat heart.

Activation of cardiac muscarinic receptors by vagal stimulation decreases cardiac work, which may have a protective effect against ischemic injury. To determine whether cardiac muscarinic receptors contribute to the mechanisms of preconditioning effects, we examined the effect of carbachol on ischemia/reperfusion damage and the effect of vagotomy on cardioprotection induced by ischemic preconditioning. Rats were subjected to 30 min of left coronary artery occlusion followed by 30-min reperfusion in situ. Pre-conditioning was induced by three cycles of 2-min coronary artery occlusion and, subsequently by 5 min of reperfusion. The incidence of ischemic arrhythmias, such as ventricular tachycardia (VT) and ventricular fibrillation (VF), and the development of myocardial infarction were markedly reduced by the preconditioning. Carbachol infusion (4 micrograms/kg per min) delayed the occurrence of VT and VF during ischemia and reduced the infarct size. Compared with non-ischemic left ventricle, the cyclic guanosine monophosphate (GMP) content in the ischemic region of the left ventricle was decreased by ischemia/reperfusion, whereas the cyclic adenosine monophosphate (AMP) content of this region was increased. These changes were reversed by preconditioning. Similar changes in cyclic GMP and AMP content in the ischemic region were seen in rats undergoing carbachol treatment. These results suggest the possible contribution of muscarinic receptor stimulation to preconditioning. Vagotomy prior to preconditioning diminished the antiarrhythmic effects, whereas it did not block the anti-infarct effect afforded by pre-conditioning. Vagotomy abolished the preconditioning effect on the tissue cyclic GMP, but it did not attenuate the decrease in tissue cyclic AMP. The results suggest that muscarinic stimulation exerts preconditioning-mimetic protective effects in ischemic/reperfused hearts, but that a contribution of reflective vagal activity to the mechanism for preconditioning is unlikely.

Acetylcholine-induced vasoconstrictor response of coronary vessels in rats: a possible contribution of M2 muscarinic receptor activation.

A mechanism by which acetylcholine (ACh) may elicit vasoconstrictor response in coronary vessels was studied in rat hearts perfused at a constant flow rate. In spontaneously beating hearts, bolus injections of ACh and carbachol (CCh) produced biphasic changes in coronary perfusion pressure (CPP): a transient increase at the initial period followed by a sustained decrease. In KCl-arrested hearts, ACh and CCh produced a monophasic increase in CPP, which was attenuated by either removal of endothelial cells by saponin or cyclooxygenase inhibition by diclofenac sodium. In the spontaneously beating heart, ACh-induced vasoconstriction was almost abolished by atropine (0.1 microM) and was markedly attenuated by an M2 antagonist, methoctramine (0.1 microM), but not by an M1 antagonist, pirenzepine (1 microM). Arecaidine propargyl ester (APE), an M2 agonist, produced coronary artery constriction which was attenuated by methoctramine (0.1 microM) but not by pirenzepine (0.1 microM) in both spontaneously beating and KCl-arrested hearts. McN-A-343, an M1 agonist, increased CPP in both beating and KCl-arrested hearts, but to a lesser degree than APE. These results suggest that the release of vasoconstrictor prostaglandins from endothelial cells contributes to the vasoconstrictor response to ACh in perfused rat coronary vessels, and the response to ACh appears to be mediated, in part, via the M2 subtype of muscarinic receptors.

Effects of long-term treatment with trandolapril on augmented vasoconstriction in rats with chronic heart failure.

BACKGROUND: Despite the clinical relevance of angiotensin I-converting enzyme (ACE) inhibitors, their effects on impaired vascular function in patients and animals with chronic heart failure (CHF) have not been fully understood. This study was undertaken to determine whether long-term treatment with an ACE inhibitor improved the altered contractile properties of vessels from rats with CHF. METHODS AND RESULTS: Twelve weeks after coronary artery ligation, the rats were sacrificed and the isometric tension development of thoracic aorta, pulmonary artery, and mesenteric artery with and without endothelium was examined. Contractile responses to norepinephrine and prostaglandin F2 alpha were augmented in endothelium-intact, but not in endothelium-denuded, thoracic aorta and pulmonary artery segments of the rat with CHF. The contractile response to angiotensin II was augmented in endothelium-denuded mesenteric artery segments of the rat with CHF, which was attenuated by indomethacin or diclofenac sodium but not by bunazosin. Trandolapril (3 mg/kg/d) was administered orally from the 2nd to 12th week after the operation. Treatment with trandolapril reversed the augmented contractile response of the rat with CHF to norepinephrine, prostaglandin F2 alpha, and angiotensin II almost to the levels in the sham-operated rat. CONCLUSIONS: The results demonstrate that an ACE inhibitor is capable of reversing altered vascular function in the rat with CHF, suggesting that vascular beds are possible sites of action for ACE inhibitors in the therapy for CHF.

Impairment of cGMP- and cAMP-mediated vasorelaxations in rats with chronic heart failure.

To elucidate pathophysiological alterations in vascular relaxation in rats with chronic heart failure (CHF), guanosine 3',5'-cyclic monophosphate (cGMP)- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated vasorelaxations in pulmonary artery (PA) and thoracic aorta (TA) of rats were examined 12 wk after coronary artery ligation. Acetylcholine (ACh)-induced relaxation was attenuated in endothelium-intact segments of both arteries, whereas sodium nitroprusside-induced relaxation was attenuated only in endothelium-intact TA segments of rats with CHF. Vasorelaxations elicited by isoproterenol and NKH-477, a water-soluble forskolin analogue, were diminished mainly in PA segments of the CHF rat. NG-nitro-L-arginine methyl ester (L-NAME)-induced decrease in cGMP level was less in endothelium-intact TA segments of the rat with CHF (0.20 +/- 0.06 vs. 0.99 +/- 0.26 pmol/mg protein in control), suggesting that basal nitric oxide (NO) production is reduced in CHF. Treatment with L-NAME attenuated the isoproterenol-induced relaxation only in endothelium-intact TA segments in control rats but not in CHF rats. The results suggest that both cGMP- and cAMP-mediated relaxations are impaired in CHF, and a reduction of NO synthesis, presumably in endothelial cells, plays a significant role in pathophysiological alterations in vessels of rats with CHF.