Chemoprophylaxis of influenza A virus infections, with single doses of zanamivir, demonstrates that zanamivir is cleared slowly from the respiratory tract.
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Zanamivir (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid; Relenza; GG167) is a potent and highly specific neuraminidase (sialidase) inhibitor with inhibitory activity in vivo against both influenza A and B viruses. This compound has been extensively tested in both mouse and ferret models of influenza and has recently been approved for the treatment of influenza in Europe and Australasia. The compound markedly reduces the clinical course of disease in humans when given therapeutically by inhalation directly into the respiratory tract. In addition, experimental influenza infections in phase I clinical trials have shown the benefit of giving a single prophylactic dose of zanamivir in addition to a therapeutic regime. The studies reported here were designed to determine the persistence of zanamivir, as assessed by its antiviral activity in vivo, in the respiratory tracts of infected animals. We have shown that the prophylactic administration of zanamivir, when the drug is given in a single dose by the intranasal route, can significantly reduce lung virus titers in the mouse and can reduce both viral titers and symptoms in the ferret. Whole-body autoradiographical analyses of mice have indicated a long retention time for this compound in respiratory tract tissues when it is given in a single dose by the intranasal route. These results indicate that zanamivir may have clinical value as a prophylactic agent in protecting at-risk groups from influenza virus infection. In addition, these data may be useful in the design of prophylactic protocols for humans, in that the dosing schedule may only need to be intermittent to provide protection. (+info)
Genetic analysis reveals that both haemagglutinin and neuraminidase determine the sensitivity of naturally occurring avian influenza viruses to zanamivir in vitro.
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The basis of differential sensitivity of replication of influenza viruses to the neuraminidase-specific inhibitor zanamivir was examined using four avian influenza viruses and reassortants produced between them. IC(50) values for inhibition of neuraminidase activity by zanamivir were similar for each of the four viruses, whereas the haemagglutinating activity of each of the viruses was relatively insensitive to zanamivir. However, the four viruses showed distinct zanamivir-sensitivity profiles in tissue culture. Analysis of the reassortant viruses showed that sensitivity was determined by the haemagglutinin gene (segment 4) and the neuraminidase gene (segment 6) and was independent of the remaining six RNA segments. Decreased sensitivity to zanamivir was associated with possession of a haemagglutinin that is released from cells with decreased dependence on neuraminidase and with possession of a neuraminidase that has a short stalk region. (+info)
Inhibition of Na+-H+ exchange impairs receptor-mediated albumin endocytosis in renal proximal tubule-derived epithelial cells from opossum.
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1. Receptor-mediated endocytosis is an important mechanism for transport of macromolecules and regulation of cell-surface receptor expression. In renal proximal tubules, receptor-mediated endocytosis mediates the reabsorption of filtered albumin. Acidification of the endocytic compartments is essential because it interferes with ligand-receptor dissociation, vesicle trafficking, fusion events and coat formation. 2. Here we show that the activity of Na+-H+ exchanger isoform 3 (NHE3) is important for proper receptor-mediated endocytosis of albumin and endosomal pH homeostasis in a renal proximal tubular cell line (opossum kidney cells) which expresses NHE3 only. 3. Depending on their inhibitory potency with respect to NHE3 and their lipophilicity, the NHE inhibitors EIPA, amiloride and HOE694 differentially reduced albumin endocytosis. The hydrophilic inhibitor HOE642 had no effect. 4. Inhibition of NHE3 led to an alkalinization of early endosomes and to an acidification of the cytoplasm, indicating that Na+-H+ exchange contributes to the acidification of the early endosomal compartment due to the existence of a sufficient Na+ gradient across the endosomal membrane. 5. Exclusive acidification of the cytoplasm with propionic acid or by removal of Na+ induced a significantly smaller reduction in endocytosis than that induced by inhibition of Na+-H+ exchange. 6. Analysis of the inhibitory profiles indicates that in early endosomes and endocytic vesicles NHE3 is of major importance, whereas plasma membrane NHE3 plays a minor role. 7. Thus, NHE3-mediated acidification along the first part of the endocytic pathway plays an important role in receptor-mediated endocytosis. Furthermore, the involvement of NHE3 offers new ways to explain the regulation of receptor-mediated endocytosis. (+info)
Cardioprotective effects of propofol and sevoflurane in ischemic and reperfused rat hearts: role of K(ATP) channels and interaction with the sodium-hydrogen exchange inhibitor HOE 642 (cariporide).
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BACKGROUND: Sodium ion-hydrogen ion (Na(+)-H(+)) exchange inhibitors are effective cardioprotective agents. The N(+)-H(+) exchange inhibitor HOE 642 (cariporide) has undergone clinical trials in acute coronary syndromes, including bypass surgery. Propofol and sevoflurane are also cardioprotective via unknown mechanisms. The authors investigated the interaction between propofol and HOE 642 in the ischemic reperfused rat heart and studied the role of adenosine triphosphate-sensitive potassium (K(ATP)) channels in the myocardial protection associated with propofol and sevoflurane. METHODS: Isolated rat hearts were perfused by the Langendorff method at a constant flow rate, and left ventricular function and coronary pressures were assessed using standard methods. Energy metabolites were also determined. To assess the role of K(ATP) channels, hearts were pretreated with the K(ATP) blocker glyburide (10 microM). Hearts were then exposed to either control buffer or buffer containing HOE 642 (5 microM), propofol (35 microM), sevoflurane (2.15 vol%), the K(ATP) opener pinacidil (1 microM), or the combination of propofol and HOE 642. Each heart was then subjected to 1 h of global ischemia followed by 1 h of reperfusion. RESULTS: Hearts treated with propofol, sevoflurane, pinacidil, or HOE 642 showed significantly higher recovery of left ventricular developed pressure and reduced end-diastolic pressures compared with controls. The combination of propofol and HOE 642 provided superior protection toward the end of the reperfusion period. Propofol, sevoflurane, and HOE 642 also attenuated the onset and magnitude of ischemic contracture and preserved high-energy phosphates (HEPs) compared with controls. Glyburide attenuated the cardioprotective effects of sevoflurane and abolished the protection observed with pinacidil. In contrast, glyburide had no effect on the cardioprotection associated with propofol treatment. CONCLUSION: HOE 642, propofol, and sevoflurane provide cardioprotection via different mechanisms. These distinct mechanisms may allow for the additive and superior protection observed with the combination of these anesthetics and HOE 642. (+info)
Structure of a soluble super-active insulin is revealed by the nature of the complex between cyanogen-bromide-activated sepharose and amines.
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Insulin-like material with elevated insulin specific acitivity is released from insulin-Sepharose in the presence of bovine-serum albumin. The mechanism of release and the chemical nature of this insulin-like material are revealed by the finding that amine-Sepharose is O-Sepharose-N-substituted isourea. Nucleophilic attack by amino groups releases N-1-N-2-disubstituted guanidines. Correspondingly, it is shown that the super-active insulin-like material is an N-1-N-2-disubstituted guanidine in which insulin and bovine-serum albumin are the substituents. (+info)
Cardioprotective effects of N-hydroxyguanidine PR5 in myocardial ischaemia and reperfusion in rats.
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1. The potential for the N-hydroxyguanidine compound PR5 (N-(3, 4-dimethoxy-2-chlorobenzylideneamino)-N'-hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models. 2. Administration of 1-10 mg kg-1 of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg-1 of PR5, respectively). 3. Administration of 3 mg kg-1 of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion. 4. Coronary occlusion/reperfusion (10 - 20 min) increased malondialdehyde (MDA) of rat hearts (0.88+/-0.13 for sham vs 1.45+/-0.12 nmol mg-1 protein for ischaemic; P<0.05). In rats where 3 mg kg-1 PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04+/-0.12; P<0.05 vs ischaemic). 5. PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326+/-32 mg for controls vs 137+/-21 mg for animals treated with 3x3 mg kg-1 of PR5 (P<0.01). 6. PR5 reduced the elevation of the ST-segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion. 7 We conclude that the N-hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life-threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N-hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme. (+info)
Stoichiometry of potassium channel opener action.
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Potassium channel openers (KCOs; e.g., P1075, pinacidil) exert their effects on excitable cells by opening ATP-sensitive potassium channels. These channels are heteromultimers composed with a 4:4 stoichiometry of an inwardly rectifying K(+) channel subunit plus a regulatory subunit comprising the receptor sites for hypoglycemic sulfonylureas and KCOs (a sulfonylurea receptor). To elucidate stoichiometry of KCO action, we analyzed P1075 sensitivity of channels coassembled from sulfonylurea receptor isoforms with high or low P1075 affinity. Concentration activation curves for cDNA ratios of 1:1 or 1:10 resembled those for channel opening resulting from interaction with a single site, whereas models for activation requiring occupation of two, three, or four sites were incongruous. We conclude KCO-induced channel activation to be mediated by interaction with a single binding site per tetradimeric complex. (+info)
Creatine accumulation and exchange by HEK293 cells stably expressing high levels of a creatine transporter.
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We have generated a stable HEK293 cell line expressing high levels of a creatine transporter (CREAT). This cell line (HEK293-CREAT) was used to study the properties of CREAT in terms of the accumulation and release of creatine. HEK293-CREAT cells accumulated high steady state levels of creatine under saturating creatine levels (approx. 25-fold higher intracellular creatine levels than seen in control cells). The accumulation of high levels of creatine affected [3H]creatine uptake by decreasing the Vmax for transport. High intracellular creatine levels were maintained in the absence of extracellular creatine. External creatine stimulated the release of stored creatine by an exchange mechanism dependent on extracellular Na+. These studies have shown that cellular creatine levels can be affected by the amount of creatine transporter in the membrane and exchange through the creatine transporter. These findings highlight the importance of the creatine transporter in the maintenance of intracellular creatine levels. (+info)