Kevin M. Mullane

Myeloperoxidase activity as a quantitative assessment of neutrophil infiltration into ischemie myocardium

The infiltration of neutrophils into ischemic myocardium exacerbates myocardial damage upon reperfusion, whereas drugs that inhibit neutrophil activity or function reduce infarct size. Consequently, it is important to accurately assess the myocardial neutrophil content. Histologic sections and radiolabeled cells have been used, but have major limitations. We have developed a method to measure the neutrophils present in cardiac tissue by utilizing a spectrophotometric assay for the neutrophil-specific myeloperoxidase enzyme (MPO) (Bradley et al., 1982a). Coronary artery occlusion and reperfusion in the anesthetized dog induces neutrophil accumulation into the ischemic heart, which shows a linear relationship with time. An increase in activity from 0.014 +/- 0.001 units (u) MPO/100 mg tissue to 0.091 +/- 0.02 u MPO/100 mg is already apparent at the end of the 90-min occlusion period. This activity increases over 5 hr reperfusion to 0.32 +/- 0.018 u MPO/100 mg tissue. Histologic analyses confirmed the temporal association of neutrophil accumulation. Moreover, there is a correlation between infarct size and tissue MPO activity. Measuring the MPO content in preparations of canine neutrophils, which is directly correlated with cell number, allows units of MPO activity to be converted into a tissue neutrophil content. This assay is simple, sensitive, and provides a quantitative index of myocardial neutrophil accumulation that can be used to study the relationship between leukocyte infiltration and myocardial injury.

Adenosine kinase inhibitors

Adenosine (ADO) is an endogenous modulator of intercellular signaling that provides homeostatic reductions in cell excitability during tissue stress and trauma. The inhibitory actions of ADO are mediated by interactions with specific cell-surface G-protein coupled receptors regulating membrane cation flux, polarization, and the release of excitatory neurotransmitters. ADO kinase (AK; EC 2.7.1.20) is the key intracellular enzyme regulating intra- and extracellular ADO concentrations. Inhibition of AK produces marked increases in extracellular ADO levels that are localized to cells and tissues undergoing accelerated ADO release. Thus AK inhibition represents a mechanism to selectively enhance the protective actions of ADO during tissue trauma without producing the nonspecific effects associated with the systemic administration of ADO receptor agonists. During the last 10 years, specific inhibitors of AK based on the endogenous purine nucleoside substrate, ADO, have been developed. Potent AK inhibitors have recently been synthesized that demonstrate high specificity for this enzyme as compared to other ADO metabolic enzymes, transporters, and receptors. In both in vitro and in vivo models, AK inhibitors have been shown to potently increase ADO concentrations in a tissue and event specific fashion and to demonstrate potential clinical utility in animal models of epilepsy, ischemia, pain, and inflammation. AK inhibitors have demonstrated superior efficacy in these models as compared to other mechanisms of modulating ADO availability, and these agents exhibit reduced side-effect liabilities compared to direct acting ADO receptor agonists. The preclinical profile of AK inhibitors indicate that these agents may have therapeutic utility in a variety of central and peripheral diseases associated with cellular trauma and inflammation. Clinical trials are currently underway to evaluate the efficacy of AK inhibitors in seizure disorders and pain.

Harnessing an Endogenous Cardioprotective Mechanism: Cellular Sources and Sites of Action of Adenosine

Endogenous adenosine is produced by the heart during ischemia-reperfusion as a natural cardioprotectant. The benefits of this local protective mechanism can be harnessed by ischemic preconditioning and amplified by drugs such as acadesine, that augment extracellular adenosine levels specifically during an ischemic event. Classically, adenosine production by cardiomyocytes, and measured in the interstitial fluid, is considered the relevant source of this mediator. In contrast, it is proposed that there are two independent sites of adenosine formation in the heart--the myocytes and the endothelial cells, that are differentially regulated. Recent evidence implicates the vascular endothelium as a potentially important site of both adenosine formation and action that subserves the cardioprotective effects of the nucleoside. The mechanisms by which endogenous adenosine protects the heart from ischemia-reperfusion injury require clarification, and may involve different adenosine receptors (A1, A2, and A3) acting through various second messenger systems that contribute to the overall response. Additional studies are required to define the source of adenosine, the mechanisms by which its levels are regulated, and the effector pathways responsible for the myocardial protection observed.

Neutrophil depletion suppresses 111In-labeled platelet accumulation in infarcted myocardium.

Summary: Platelets and neutrophils accumulate rapidly in infarcted myocardium. Although antineutrophil agents reduce the size of the infarcted area, this is not observed with antiplatelet drugs. The possibility that myocardial ischemia–induced platelet deposition was secondary to a neutrophil-mediated event was assessed by injecting prostacyclin-washed autologous 111In-labeled platelets and measuring the amount of radioactivity in different regions of the heart following 90-min occlusion of the left anterior descending coronary artery followed by reperfusion for periods up to 5 h. Platelet deposition during the reperfusion phase was linear with time and similar to the time course of neutrophil accumulation. There was a transmural distribution of radioactivity across the myocardium where the “zone” between infarcted and risk regions, called the “interface,” > infarct > risk > normal. Neutropenia (21 ± 2% control levels), induced with specific sheep anti-dog neutrophil antiserum, had minimal effects on platelet aggregation ex vivo, but significantly reduced platelet accumulation in the ischemic myocardium following 5-h reperfusion and abolished the transmural platelet distribution. These results suggest that myocardial platelet deposition is secondary to a neutrophil-mediated event in this occlusion–reperfusion model of myocardial injury. Interactions between platelets and neutrophils at the site of tissue damage may influence the process of myocardial ischemic injury.

Acadesine extends the window of protection afforded by ischaemic preconditioning in conscious rabbits

OBJECTIVE Ischaemic preconditioning protects myocardium from infarction if the reperfusion interval between the brief and prolonged ischaemic intervals is less than 1 h. In anaesthetised rabbits acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine enhancer which increases tissue adenosine during ischaemia, prolongs the window of protection to 2 h. The aim of this study was to try to determine the maximum extension of this window of protection, using chronically instrumented, unsedated rabbits. METHODS Rabbits were instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion. Five to seven days after surgery all animals underwent a 30 min coronary occlusion. Animals were randomised to one of seven groups: (1) No additional treatment (control); (2) Ischaemic preconditioning with 5 min regional ischaemia followed by 10 min reperfusion before the 30 min coronary occlusion; (3) and (4) Ischaemic preconditioning followed by 2 or 4 h of reperfusion before the 30 min occlusion, respectively; (5) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min and then 0.5 mg.kg-1.min-1 beginning 45 min before and continuing until 30 min after release of the 30 min occlusion) without ischaemic preconditioning; (6) and (7) Treatment with the higher dose of acadesine for 5 min beginning 35 min before the 5 min ischaemic period, and then the lower dose continuing until 30 min after release of the 30 min coronary occlusion in rabbits with 4 or 6 h reperfusion intervals, respectively. RESULTS Rabbits with ischaemic preconditioning with 10 min reperfusion preceding the 30 min coronary occlusion (group 2) had only 5.6(SEM 1.1)% infarction of the ischaemic zone. Ischaemic preconditioning followed by 2 h reperfusion (group 3) offered continued protection [18.2(2.2)% infarction] as compared to control animals [37.7(2.6)% infarction]. However, protection waned if ischaemic preconditioning was followed by 4 h reperfusion (group 4) [36.7(3.0)% infarction]. Additionally, treatment with acadesine alone did not modify infarct size (group 7) [39.5(4.0)%], but acadesine largely restored the protection of ischaemic preconditioning despite a 4 h reperfusion interval (group 5) [20.4(3.0)% infarction, P < 0.01 v control]. However, when reperfusion was extended to 6 h (group 6) acadesine could no longer restore protection [36.2(0.9)% infarction]. CONCLUSIONS The protection afforded by a 5 min ischaemic preconditioning period lasts from 2 to 4 h in the awake, unsedated rabbit, and acadesine can extend the duration of this window of protection to at least 4 h but not to 6 h.

Impaired endothelium-dependent relaxations in rabbits subjected to aortic coarctation hypertension.

Rabbits were rendered hypertensive by suprarenal coarctation of the abdominal aorta. Seven days later, endothelium-dependent and endothelium-independent vascular relaxations were examined in vascular rings taken from hypertensive (thoracic aorta, carotid artery) and normotensive (abdominal aorta) regions. Relaxation of phenylephrine-contracted rings in response to endothelium-dependent agonists (acetylcholine, A23187) was impaired, compared with that in sham-operated and intact controls, in regions exposed to the elevated blood pressure (i.e., above the coarctation). Responses to acetylcholine and A23187 in the abdominal aorta, below the coarctation, were not altered. The diminished endothelium-dependent responses in the thoracic aorta were not affected by pretreatment with the cyclooxygenase inhibitor indomethacin. In contrast to acetylcholine and A23187, responses to the endothelium-independent agonist nitroprusside were not attenuated in vessels from hypertensive regions, indicating that the defect occurred in the endothelium. The EC50 for acetylcholine-induced relaxations of thoracic aorta correlated significantly with mean arterial pressure above the coarctation, indicating that the extent to which endothelium-dependent relaxation is impaired is in proportion to the degree of blood pressure elevation. This study suggests that the diminished relaxations by endothelium-dependent agonists is a local response to the elevation of blood pressure and is not due to a circulating factor.

Prostacyclin mediates the potentiated hypotensive effect of bradykinin following captopril treatment

The effect of angiotensin-converting enzyme inhibition by captopril on the release of a prostacyclin-like substance by bradykinin, angiotensin I and angiotensin II was studied by means of the blood-bathed bioassay technique of Vane. Administration of captopril abolished the release of prostacyclin-like substance induced by angiotensin I, potentiated the release provoked by bradykinin and did not alter that due to angiotensin II. Potentiation of the bradykinin-induced renal vasodilatation with captopril could be completely reversed by indomethacin, which also abolished the kinin-induced release of prostacyclin-like substance. Potentiation of the bradykinin-induced hypotension was markedly attenuated but not completely reversed by cyclo-oxygenase inhibition. It is suggested that following converting inhibition increased production of prostacyclin by elevated kinin levels may contribute to the antihypertensive action of angiotensin-converting enzyme inhibitors.

Presence of cytochrome P-450-dependent monooxygenase in intimal cells of the hog aorta.

Cytochrome P-450-dependent mixed function oxidase activity is present in vascular tissue; however, as far as we could determine, the distribution of monooxygenase activity across the blood vessel wall has not previously been assessed. The aryl-hydrocarbon hydroxylase activity was examined by metabolism of benzo[a]pyrene in microsomes prepared from intimal and smooth muscle cell scrapings of the hog thoracic aorta. Microsomes of intimal cells comprising 95% endothelial cells showed an approximately 2.5-fold increase in aryl-hydrocarbon hydroxylase activity compared with that in microsomes prepared from medial smooth muscle cells. Michaelis-Mentin kinetics for the intimal enzyme yielded an apparent Km value of 11.11 microM and an apparent Vmax of 3-OH benzo[a]pyrene of 40 pmol/mg protein/10 min. Aryl-hydrocarbon hydroxylase activity was dependent on nicotinamide adenine dinucleotide phosphate and was inhibited by 7,8 benzoflavone, SKF 525A, and carbon monoxide. The localization of cytochrome P-450-dependent mixed function oxidase primarily to the intimal surface of the aorta may indicate a role for this enzyme system in vasoregulation and the pathogenesis of atherosclerosis.

Biotransformation and cardiovascular effects of arachidonic acid in the dog.

The biotransformation and cardiovascular effects of arachidonic acid (AA) were studied in the circulation of anaesthetized dogs. Arterial blood was continuously bioassayed for arachidonate metabolites using the blood-bathed organ technique of Vane. AA (5-10 microgram/ml) infused into an incubation coil of flowing blood was converted into a labile substance which contracted the vascular tissues (rabbit aorta, RbA; rabbit coeliac and mesenteric arteries, RbCA and RbMA; bovine coronary artery, BCA) and the gastrointestinal smooth muscle strips (rat stomach strip, RSS; rat colon, RC). These effects could be mimicked by exogenously generated thromboxane A2 (TXA2). Conversion of AA was inhibited by indomethacin and the selective thromboxane synthetase inhibitor, imidazole (100 microgram/ml). The half-life of TXA2 in blood was 30-47 sec, a similar value to that found in aqueous solutions at 37 degrees C. PGH2 was also converted in blood to other product(s) which contracted RSS and RC, relaxed RbCA and RbMA but had little effect on RbA. Intravenous infusion of AA (50-800 microgram kg-1 min-1) caused effects on the bioassay tissues which could be mimicked by prostacyclin. The AA infusion also induced falls in pulmonary and systemic arterial pressures and bradycardia. All effects were abolished by indomethacin (5 mg/kg) or aspirin (200 mg/kg). Radioimmunoassay confirmed that the major product of intravenously infused AA was 6-oxo-PGF1alpha, the chemical degradation product of prostacyclin. Thus, although AA is transformed to the vasoconstrictor TXA2 when incubated for sufficient time with blood alone, on rapid pulmonary transit it is transformed into a prostacyclin-like substance.

Myocardial salvage induced by REV-5901: an inhibitor and antagonist of the leukotrienes

Summary: Lipoxygenase metabolites of arachidonic acid have been implicated in myocardial injury induced by coronary artery occlusion and reperfusion, as dual inhibitors of the lipoxygenase and cyclooxygenase enzymes, but not selective cyclooxygenase inhibitors, reduce infarct size. However, interpretation of these studies has been clouded by the lack of specificity of the drugs previously used. A specific 5-lipoxygenase inhibitor, REV-5901, has recently been developed. This drug inhibits A23187-induced immunoreactive leukotriene B4 generation by canine neutrophils (IC50 ∼2.5 μM), and when given intravenously, attenuates the formation of a leukotriene D4-like material in blood ex vivo. The release of bioassayable leukotriene-like material from rabbit hearts infarcted in vivo and subsequently perfused in vitro is prevented by REV-5901. Moreover, the inhibitor also acts as an end-organ antagonist to prevent the spasmogenic effects of the peptide-containing leukotrienes in vitro with an IC50 ∼0.1 μM. REV-5901 (10 + 2 mg/kg i.v.) reduces infarct size produced by coronary artery occlusion and reperfusion in the anesthetized dog from 56.6 ± 2 to 28.6 ± 3.7% of the hypoperfused zone. Salvage of the ischemic myocardium occurs independently of any apparent hemodynamic effect of the drug, but is accompanied by a diminution in neutrophil accumulation in the ischemic heart. It is proposed that inhibition of leukotriene B4 formation by REV-5901 suppresses the accumulation of neutrophils, thereby attenuating neutro-phil-mediated cardiac damage.