Effect of bradykinin metabolism inhibitors on evoked hypotension in rats: rank efficacy of enzymes associated with bradykinin-mediated angioedema.

BACKGROUND AND PURPOSE: Inhibition of bradykinin metabolizing enzymes (BMEs) can cause acute angioedema, as demonstrated in a recent clinical trial in patients administered the antihypertensive, omapatrilat. However, the relative contribution of specific BMEs to this effect is unclear and confounded by the lack of a predictive pre-clinical model of angioedema. EXPERIMENTAL APPROACH: Rats were instrumented to record blood pressure and heart rate; inhibitors were infused for 35 min and bradykinin was infused during the last 5 min to elicit hypotension, as a functional marker of circulating bradykinin and relative angioedema risk. KEY RESULTS: In the presence of omapatrilat bradykinin produced dose-dependent hypotension, an effect abolished by B(2) blockade. In the presence of lisinopril (ACE inhibitor), but not candoxatril (NEP inhibitor) or apstatin (APP inhibitor), bradykinin also elicited hypotension. Lisinopril-mediated hypotension was unchanged with concomitant blockade of NEP or NEP/DPPIV (candoxatril+A-899301). However, hypotension was enhanced upon concomitant blockade of APP and further intensified in the presence of NEP inhibition to values not different from omapatrilat alone. CONCLUSIONS AND IMPLICATIONS: We demonstrated that bradykinin is degraded in vivo with an enzyme rank-efficacy of ACE>APP>>NEP or DPPIV. These results suggest the effects of omapatrilat are mediated by inhibition of three BMEs, ACE/APP/NEP. However, dual inhibition of ACE/NEP or ACE/NEP/DPPIV elicits no increased risk of angioedema compared to ACE inhibition alone. Thus, novel BME inhibitors must display no activity against APP to avoid angioedema risk due to high prevalence of ACE inhibitor therapy in patients with diabetes and cardiovascular disease.

Bcl-2 family proteins are essential for platelet survival.

Platelets are relatively short-lived, anucleated cells that are essential for proper hemostasis. The regulation of platelet survival in the circulation remains poorly understood. The process of platelet activation and senescence in vivo is associated with processes similar to those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, and cell shrinkage. ABT-737, a potent antagonist of Bcl-2, Bcl-X(L), and Bcl-w, induces apoptosis in nucleated cells dependent on these proteins for survival. In vivo, ABT-737 induces a reduction of circulating platelets that is maintained during drug therapy, followed by recovery to normal levels within several days after treatment cessation. Whole body scintography utilizing ([111])Indium-labeled platelets in dogs shows that ABT-737-induced platelet clearance is primarily mediated by the liver. In vitro, ABT-737 treatment leads to activation of key apoptotic processes including cytochrome c release, caspase-3 activation, and PS externalization in isolated platelets. Despite these changes, ABT-737 is ineffective in promoting platelet activation as measured by granule release markers and platelet aggregation. Taken together, these data suggest that ABT-737 induces an apoptosis-like response in platelets that is distinct from platelet activation and results in enhanced clearance in vivo by the reticuloendothelial system.

Dependence of delta1-opioid receptor-induced cardioprotection on a tyrosine kinase-dependent but not a Src-dependent pathway.

We investigated the possibility that opioids activate a tyrosine kinase (TK) that mediates cardioprotection in an in vivo rat model of myocardial infarction. All animals underwent 30 min of regional ischemia and 2 h of reperfusion. Infarct size was expressed as a percentage of the area at risk (IS/AAR). Control animals had an IS/AAR of 58.2 +/- 0.6. Cardioprotection was induced with the delta1- or delta1/delta2-selective opioid agonists, TAN-67, or D-Ala D-Leu enkephalin (DADLE). Both significantly reduced IS/AAR (28.8 +/- 3.6 and 34.8 +/- 3.8, respectively). The general TK inhibitor, genistein, abolished cardioprotection produced by TAN-67 or DADLE (59.1 +/- 3.2 and 61.5 +/- 3.4, respectively), whereas the structural analog, daidzein, lacking TK inhibitory activity, did not. Interestingly, the selective Src/epidermal growth factor (EGF) receptor TK inhibitor, lavendustin A, did not abolish TAN-67-induced cardioprotection (22.1 +/- 6.8). Similarly, the Src-selective TK antagonist, PP2, had no effect on DADLE-induced cardioprotection (31.1 +/- 7.3). These unexpected findings suggest that Src and EGF receptor TKs are not important in the genesis of cardioprotection produced by TAN-67. Finally, we demonstrate that genistein did not affect protein kinase C (PKC) translocation induced by TAN-67. These data suggest that a TK, but most likely not an Src/EGF receptor TK, is important in cardioprotection via opioid receptor stimulation and that the pathway for TK activation is downstream from or parallel to PKC activation in the in situ rat heart since genistein could not affect PKC translocation of selective isoforms induced by TAN-67 and assessed by immunohistochemistry.

Production and metabolism of ceramide in normal and ischemic-reperfused myocardium of rats.

Ceramide has been shown to be a key signaling molecule involved in the apoptotic effect of tumor necrosis factor alpha (TNF-alpha) and other cytokines. Given the importance of cytokines such as TNF-alpha in myocardial ischemia-reperfusion injury, we hypothesize that ceramide is increased during ischemia or reperfusion, and that the activity of enzymes responsible for its production or breakdown should be increased and/or decreased, respectively. Therefore, in the present study, we characterized the enzymatic activities responsible for ceramide production and metabolism in the myocardium of rats, and determined the contribution of these enzymes to altered ceramide levels during myocardial ischemia and reperfusion. The basal ceramide concentration in the myocardium of rats was 34.0 pmol/mg tissue. As determined by the conversion of 14C-sphingomyelin into ceramide and 14C-choline phosphate, both neutral (N-) and acidic (A-) SMase were detected in the myocardium, with a conversion rate of 0.09 +/- 0.008 and 0.32 +/- 0.05 nmol/min per mg protein, respectively. The activity of A-SMase (78 % of total cellular activity) was significantly higher in microsomes than in cytosol, while the activity of N-SMase was similar in both fractions. Ceramidase, a ceramide-metabolizing enzyme, was also detected in the myocardium of rats. It metabolized ceramide into sphingosine at a rate of 9.94 +/- 0.42 pmol/min per mg protein. In anesthetized rats, 30 min of ischemia had no apparent effect on ceramide concentrations in the myocardium, while 30 min of ischemia followed by 3 h of reperfusion resulted in a significant increase in ceramide by 48 %. The activities of both N- and A-SMase were reduced by 44 % and 32 %, respectively, in the myocardium subjected to ischemia followed by reperfusion, but unaltered in the ischemic myocardium. It was also found that myocardial ischemia followed by reperfusion produced a marked inhibition of ceramidase (by 29 %). These results demonstrate that the myocardium of rats expresses N- and A-SMase and ceramidase, which contribute to the production and metabolism of ceramide, respectively. Tissue ceramide concentrations increased in reperfused myocardium. These increases in ceramide were not associated with enhanced SMase activity, but rather with reduced ceramidase activity.

Mitochondrial K(ATP) channel opening is important during index ischemia and following myocardial reperfusion in ischemic preconditioned rat hearts.

We have previously demonstrated that K(ATP)channel openers administered just prior to and throughout reperfusion induce cardioprotection in the blood-perfused canine heart. However, a recent report suggests that the mitochondrial K(ATP)channel is only a trigger of ischemic preconditioning (IPC). These recent data are, however, in contrast to most previous investigations that suggested that activation of the mitochondrial K(ATP)channel is an important downstream mediator of cardioprotection. Therefore, we examined the role of the mitochondrial K(ATP)channel as a downstream mediator of IPC in a rat model by administering the selective mitochondrial K(ATP)channel antagonist, 5-hydroxydecanoate (5-HD), at several points during IPC. Infarct size (IS) was determined by tetrazolium chloride staining and expressed as a percent of the area at risk (AAR). Control animals had an IS/AAR of 58.4+/-0.6 and IS/AAR was reduced to 6.2+/-1.7 following IPC. 5-HD (10 mg/kg), attenuated cardioprotection when administered either 5 min prior to the IPC stimulus (40.4+/-1.4), during the reperfusion phase of the IPC stimulus (39.7+/-5.9), or 5 min prior to reperfusion during prolonged ischemia (34.3+/-6.9). Additionally, when 5-HD was administered at 5 mg/kg during the reperfusion phase of index ischemia plus 5 min prior to IPC or plus during the reperfusion phase of IPC, cardioprotection was also attenuated (36.3+/-5.5 and 43.8+/-6.9, respectively). These data suggest that activation of the mitochondrial K(ATP) channel is an important downstream regulator of myocardial protection with effects lasting into the reperfusion period following prolonged ischemia.

Opioid-induced cardioprotection against myocardial infarction and arrhythmias: mitochondrial versus sarcolemmal ATP-sensitive potassium channels.

We examined the role of the sarcolemmal and mitochondrial ATP-sensitive potassium (K(ATP)) channel in a rat model of myocardial infarction after stimulation with the selective delta(1)-opioid receptor agonist TAN-67. Hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Infarct size was expressed as a percentage of the area at risk. TAN-67 significantly reduced infarct size/area at risk (29.6 +/- 3.3) versus control (63. 1 +/- 2.3). The sarcolemmal-selective K(ATP) channel antagonist HMR 1098, administered 10 min before TAN-67, did not significantly attenuate cardioprotection (26.0 +/- 7.3) at a dose (3 mg/kg) that had no effect in the absence of TAN-67 (56.3 +/- 4.3). Pretreatment with the mitochondrial selective antagonist 5-hydroxydecanoic acid (5-HD) 5 min before the 30-min occlusion completely abolished TAN-67-induced cardioprotection (54.3 +/- 2.7), but had no effect in the absence of TAN-67 (62.6 +/- 4.1), suggesting the involvement of the mitochondrial K(ATP) channel. Additionally, we examined the antiarrhythmic effects of TAN-67 in the presence or absence of 5-HD and HMR 1098 during 30 min of ischemia. Control animals had an average arrhythmia score of 10.40 +/- 2.41. TAN-67 significantly reduced the arrhythmia score during 30 min of ischemia (2.38 +/- 0. 85). 5-HD and HMR 1098 in the absence of TAN-67 produced an insignificant decrease in the arrhythmia score (8.80 +/- 2.56 and 4. 20 +/- 1.07, respectively). 5-HD administration before TAN-67 treatment abolished its antiarrhythmic effect (4.71 +/- 1.11). However, HMR 1098 did not abolish TAN-67-induced protection against arrhythmias (1.67 +/- 0.80). These data suggest that delta(1)-opioid receptor stimulation is cardioprotective against myocardial ischemia and sublethal arrhythmias and suggest a role for the mitochondrial K(ATP) channel in mediating these cardioprotective effects.

Ischemic preconditioning in rats: role of mitochondrial K(ATP) channel in preservation of mitochondrial function.

We examined the role of the sarcolemmal and mitochondrial K(ATP) channels in a rat model of ischemic preconditioning (IPC). Infarct size was expressed as a percentage of the area at risk (IS/AAR). IPC significantly reduced infarct size (7 +/- 1%) versus control (56 +/- 1%). The sarcolemmal K(ATP) channel-selective antagonist HMR-1098 administered before IPC did not significantly attenuate cardioprotection. However, pretreatment with the mitochondrial K(ATP) channel-selective antagonist 5-hydroxydecanoic acid (5-HD) 5 min before IPC partially abolished cardioprotection (40 +/- 1%). Diazoxide (10 mg/kg iv) also reduced IS/AAR (36.2 +/- 4.8%), but this effect was abolished by 5-HD. As an index of mitochondrial bioenergetic function, the rate of ATP synthesis in the AAR was examined. Untreated animals synthesized ATP at 2.12 +/- 0.30 micromol x min(-1) x mg mitochondrial protein(-1). Rats subjected to ischemia-reperfusion synthesized ATP at 0.67 +/- 0.06 micromol x min(-1) x mg mitochondrial protein(-1). IPC significantly increased ATP synthesis to 1.86 +/- 0.23 micromol x min(-1) x mg mitochondrial protein(-1). However, when 5-HD was administered before IPC, the preservation of ATP synthesis was attenuated (1.18 +/- 0.15 micromol x min(-1) x mg mitochondrial protein(-1)). These data are consistent with the notion that inhibition of mitochondrial K(ATP) channels attenuates IPC by reducing IPC-induced protection of mitochondrial function.

Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning.

Ischemic preconditioning (IPC) is a phenomenon in which single or multiple brief periods of ischemia have been shown to protect the heart against a more prolonged ischemic insult, the result of which is a marked reduction in myocardial infarct size, severity of stunning, or incidence of cardiac arrhythmias. Although a number of substances and signaling pathways have been proposed to be involved in mediating the cardioprotective effect of IPC, the overwhelming majority of evidence suggests that the ATP-sensitive potassium channel (KATP channel) is an important component of this phenomenon and may serve as the end effector in this process. Initially, it was hypothesized that the surface or sarcolemmal KATP (sarc KATP) channel mediated protection observed after IPC; however, subsequent evidence suggested that the recently identified mitochondrial KATP channel (mito KATP) may be the potassium channel mediating IPC-induced cardioprotection. In this review, evidence will be presented supporting a role for either the sarc KATP or the mito KATP in IPC and potential mechanisms by which opening these channels may produce cardioprotection; additionally, we will address important questions that still need to be investigated to define the role of the sarc or mito KATP channel, or both, in cardiac pathophysiology.