Nitric oxide mediated effects of nebivolol in myocardial infarction: the source of nitric oxide.

OBJECTIVE: After MI pathological LV remodeling is one of the major causes of death. We previously showed the NO mediated beneficial effects of nebivolol in rat MI model, in this study we aimed to evaluate the NOS related mechanisms in this phenomenon. MATERIALS AND METHODS: Rats were divided into four groups: sham operated (sham-control), MI-induced (MI-control), immediate nebivolol loaded (MI-neb1), orally nebivolol treated (MI-neb2). MI was induced by the ligation of the LAD. Loading dose of nebivolol (0.1 mg/kg) was administrated i.v. during reperfusion and continuation dose was administrated orally (2 mg/kg) by gastric gavages once daily. NOS related mechanisms were assessed either in acute (2nd day) and sub-acute (28th day) period of MI by histologic, hemodynamic and biologic studies. RESULTS: Compared to MI-control rats, physiological functions of LV (LVEDP, Δ±dp/dt) were prevented in both nebivolol treated groups. Improvements in anatomical parameters (LEV, HW, LVW/HW) were consistent with functional improvement too. Moreover, oxidative (characterized by decreased MDA and increased SOD levels) and nitrosative (characterized by decreased ONOO- levels) damage were limited in these groups. Compared to MI-control rats, most marked change was seen in the nNOS labelling in the nebivolol treated groups. The decrease in iNOS labelling was also prominent in these groups too. CONCLUSIONS: NOS mediated mechanisms of nebivolol can be summarized as: 1) diminishing iNOS expression together with restoration of MI induced eNOS activation both in vascular bed and myocytes at the acute period of MI, and 2) prevention of deterioration in nNOS expression in myocardial cells at the sub-acute period of MI.

Chronic emotional stress exposure increases infarct size in rats: the role of oxidative and nitrosative damage in response to sympathetic hyperactivity.

We investigated the level of sympathetic hyperactivity in response to stress exposure in an acute myocardial infarction (AMI) model and the contribution of oxidative and nitrosative damage to this phenomenon. Stress was induced by 20-day administration of different emotional stress factors: daylight/darkness exposure, overcrowding, isolation, new hierarchy, tilting the cage and restriction of water or food. AMI was induced surgically. Heart rate (HR) and heart rate variability (HRV) measurements were done before and after AMI. Oxidant parameters were measured in heart tissue and cortisol levels were measured in plasma specimens. Compared with the nonstressed group, stress-exposed rats showed sympathetic hyperactivity characterized by increased HR together with decreased HRV. In the stressed group serum corticosterone levels were high both before and after AMI. Mean infarct size in the stressed group was significantly larger (44.6+/-3.23% and 53.1+/-4.52%, respectively; P<0.05). Increased tissue malondialdehyde (MDA) levels (0.63+/-0.59 and 1.60+/-0.31 nmol/mg protein, respectively; P<0.05) and decreased superoxide dismutase (SOD) activity and glutathione (GSH) content were seen in stress-exposed rats. Likewise, heart peroxynitrite levels were also high in stress-exposed rats (141.8+/-18 nmol/g tissue vs. 164.2+/-21 nmol/g tissue). Chronic emotional stress is a deteriorating factor for the induction and prognosis of MI. Exaggerated sympathetic activity may be the major contributing factor. Oxidative and nitrosative damage in response to this sympathetic hyperactivity is the key mechanism.

Cardioprotective effects of adenosine A1 and A3 receptor activation during hypoxia in isolated rat cardiac myocytes.

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A1 and A3 receptors (A1R and A3R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4-5 days in vitro), to an atmosphere of a N2 (95%) and CO2 (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A1 and A3 receptor antagonists abolished the protective effects of adenosine. A1R and A3R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A1R agonist, CCPA, was abolished by an A1R antagonist, DPCPX, and was not affected by an A3R antagonist, MRS 1523. Cardioprotection caused by the A3R agonist, Cl-IB-MECA, was antagonized completely by MRS 1523 and only partially by DPCPX. Activation of both A1R and A3R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.

Selective neurotoxicity induced by the ionophore lasalocid in rat dissociated cerebral cultures, involvement of the NMDA receptor/channel.

An in vitro model of dissociated cerebral cultures, prepared from prenatal 15-16-days rat fetuses, was used to further characterize the neurotoxic effects caused by the antibiotic ionophore lasalocid-X-537A. The damage caused by lasalocid (1-2 microM, 2-4 hr) included swelling of perikarya, followed by cytolysis of most neurons present in the cultures. The neuronal damage was dose-dependent, noticeable at concentrations above 0.5 microM, and was more pronounced in established cultures (14 days in vitro-DIV) than in younger ones (7 DIV). Unlike neurons, no damage was observed in glia and other non-neuronal cells present in the cultures by exposure to 2 microM lasalocid. Moreover, the drug was not toxic for cultures of rat astrocytes and C6 glioma cells. Another calcium ionophore A-23187 (calcimycin, 1 microM), destroyed both neuronal and non-neuronal cells within 1 hr. Ca2+ influx was increased by 140% in cultures exposed to lasalocid (1.5 microM). The lasalocid neurotoxic effects were neither inhibited by 10 microM nimodipine (a calcium channel antagonist) nor by 10 microM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX)(a non-N-methyl-D-aspartic acid (NMDA) receptor antagonist), but were exclusively blocked by 10 microM MK-801 (a non-competitive NMDA receptor/channel antagonist). The neurotoxicity induced by lasalocid was further confirmed by measurements of lactate dehydrogenase (LDH) released into the media. Lasalocid (1.5 microM) induced the release of both LDH and arachidonic acid (AA) (by 8 and 4 fold of control values, respectively), and this was blocked by MK-801 but not by CNQX. These results are in according with the observations that activation of calcium influx through the NMDA receptor leads to activation of phospholipase A2 (PLA2) and release of AA. In contrast, MK-801 did not block the release of either LDH or AA mediated by the calcium ionophore A-23187 (1 microM) in these cultures. [3H]-MK-801 binding to washed rat cortical membranes, a measure of direct interaction with the NMDA receptor/channel complex, was not affected by lasalocid either alone or in the presence of glutamate and glycine. [3H]-D-aspartate release, a measure of excitatory amino acid (EAA) secretion mediated by NMDA receptor activation, was increased by lasalocid and could be blocked by MK-801. These observations suggest that lasalocid induces selective neurotoxicity, which involves the NMDA receptor/channel complex, possibly indirectly, resulted in elevated intracellular Ca2+ levels and the subsequent glutamate or aspartate release.

Paralytic syndrome attributed to lasalocid residues in a commercial ration fed to dogs.

Lasalocid, accidently introduced into a commercial dog food, was found to be the cause of neuromuscular toxicosis in 10 dogs. Toxicosis was confirmed in 4 principal dogs. The history, clinical signs, and pattern of onset of lasalocid-induced toxicosis in the 14 dogs were similar to those reported for botulism. The signs, which were related to a generalized lower motor neuron deficit, were generally different from ionophore-induced toxicosis reported in other species. Supportive therapy and close monitoring were sufficient to bring about a gradual improvement in all of the dogs, despite the severity of clinical signs.

Selective neurotoxicity induced by lasalocid in dissociated cerebral cultures.

Dispersed cerebral cells prepared from 15-16-day mouse foetuses were cultured for 7 days and exposed to lasalocid for periods of 4, 18 or 48 hr. Cultures were examined by phase contrast microscopy and processed for scanning electron microscopy. Lasalocid (1 and 2 mum) induced neurotoxic effects that were evident already at 4 hr, including swelling of perikarya, followed by cytolysis of most neurons present in the cultures. During the following 18 hr virtually the entire neuronal population degenerated completely. Cultures exposed to a lower concentration of lasalocid (0.5 mum) for 48 hr showed only mild damage to some neurons, and at 0.2 mum no damage could be observed, even after several days of continuous exposure. Notably, glial and other non-neuronal cells were not damaged by exposure to 2 mum-lasalocid, and cell division resumed on returning the cultures to regular growth media. Similarly, lasalocid (2 mum) was not toxic to cultured rat astrocytes. These morphological observations were followed by measurements of (45)Ca(2+) influx in the dissociated cerebral cultures. Lasalocid (1 mum) induced (45)Ca(2+) influx of 40% above dimethyl sulphoxide control values. The voltage-sensitive calcium channel antagonists nimodipine (10 mum) and D-600 (50 mum) did not inhibit lasalocid-mediated (45)Ca(2+) influx, whereas MK-801 [10 mum; a non-competitive N-methyl-d-aspartate (NMDA) receptor/channel antagonist] exclusively blocked this influx and prevented cytotoxic damage. Conversely, NMDA and glutamate, which by themselves mediated (45)Ca(2+) influx in these cultures, both potentiated lasalocid-induced (45)Ca(2+) influx and cellular damage. These observations clearly demonstrate the selective neurotoxicity of lasalocid to cultured cerebral neurons, and may imply involvement of the NMDA receptor/channel in lasalocid-mediated neurotoxicity.

Isolation and preliminary characterisation of a novel retrovirus isolated from a leukaemic dog.

A novel canine retrovirus was isolated from mononuclear cells of the peripheral blood of a leukaemic dog. The main clinical and pathological findings in this dog were lethargy, anorexia, weakness, dyspnoea, severe anaemia, thrombocytopenia and a high white blood cell count, practically all of which were lymphoblasts. The virus was isolated from mononuclear cells obtained from the blood, cocultivated with indicator cells. The virus particles encode a reverse transcriptase with Mg++ preference, have a density in sucrose gradients of 1.16 g ml-1, and induce syncytia in permissive cell cultures such as Himalayan tahr ovary and canine fetal thymus lines. This agent replicates to high titres. The virus exhibits a morphogenesis and morphology typical of lentiviruses. Immunoblotting and competitive radioimmunoassays failed to detect immunological crossreactivity with other representative lentiviruses and oncoviruses of the retrovirus family.

Propagation and characterization of novel canine lentivirus isolated from a dog.

We have recently isolated a novel canine lentivirus (canine immunodeficiency virus, [CIV]) from a leukemic dog. The virus was isolated from buffy coat cells obtained from the leukemic dog co-cultivated with indicator cells. The virus particles encode a reverse transcriptase with a preference for magnesium, have a density of 1.16 g/ml in sucrose, and induce syncytia in permissive cell lines such as Himalayan tahr ovary and canine fetal thymus. CIV replicates to high titer and highly purified virus can readily be prepared. The ultrastructure and morphogenesis of CIV is strikingly similar to that displayed by other lentiviruses, while immunoblot analysis failed to demonstrate close immunological relatedness to any other lentivirus or oncovirus. These findings suggest that this canine virus, representing the first isolation of a canine retrovirus, belongs to the lentivirus subfamily but is not closely related to other known members.

Transforming potential of a retrovirus isolated from lung carcinoma of sheep.

A retrovirus isolated from experimentally induced sheep lung carcinoma (SPCTV) was propagated in chronically infected Himalayan tahr ovarian cells and in normal sheep lung cells. Follow-up of infection of the cells with SPCTV showed the appearance of syncytium, plaque formation, partial recovery and the establishment of a chronic infection. Virus-associated reverse transcriptase activity in the medium fluctuated but remained at a constantly high level at the stage of chronic infection. Stages of type-C virus morphogenesis were demonstrated by electron microscopy. The viral genome was detected in both the nucleus and cytoplasm by in situ hybridization. Chronically infected cells formed colonies when plated in soft agar. Following subcutaneous inoculation of chronically infected cells (of fibroblast origin) into nude mice, lymphoid tumors developed at the site of inoculation and in vital organs.