s-nitro-n-acetylpenicillamine and Disease-Models--Animal

Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as V(L)12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which V(L)12.3 does not change. In contrast, expression of V(L)12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells.

Topics: Animals; Antibodies; Antibody Specificity; Brain; Cell Line, Transformed; Disease Models, Animal; Epitope Mapping; Humans; Huntingtin Protein; Immunoglobulin Variable Region; In Vitro Techniques; Luminescent Proteins; Mutation; Nerve Tissue Proteins; Neurotoxicity Syndromes; Nuclear Proteins; Penicillamine; Peptides; Proline; Protein Binding; Protein Structure, Tertiary; Transfection

This study investigated the involvement of the L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in the antidepressant-like effect of an acute administration of memantine in the forced swimming test (FST) in mice, since this signaling pathway is supposed to play a significant role in depression. The antidepressant-like effect of memantine (3 mg/kg, i.p.) was prevented by pretreatment with L-arginine (750 mg/kg, i.p.) or S-nitroso-N-acetyl-penicillamine (SNAP, 25 microg/site, i.c.v.), but not with d-arginine (750 mg/kg, i.p.).The treatment of mice with NG-nitro-L-arginine (L-NNA, 0.03 and 0.3 mg/kg, i.p.) potentiated the effect of a subeffective dose of memantine (0.3 mg/kg, i.p.) in the FST. Moreover, the pretreatment of mice with (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one) (ODQ, 30 pmol/site, i.c.v.) produced a synergistic antidepressant-like effect with subeffective doses of memantine (0.3 and 1 mg/kg, i.p.) in the FST. Furthermore, the reduction in the immobility time elicited by memantine (3 mg/kg, i.p.) in the FST was prevented by pretreatment with sildenafil (5 mg/kg, i.p.). Taken together, the results demonstrate that memantine produced an antidepressant-like effect in the FST that seems to be mediated through an interaction with the L-arginine-NO-cGMP pathway.

Topics: Analysis of Variance; Animals; Antidepressive Agents; Arginine; Cyclic GMP; Depression; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Female; Immobility Response, Tonic; Male; Memantine; Mice; Nitric Oxide; Nitric Oxide Donors; Nitroarginine; Penicillamine; Piperazines; Purines; Sildenafil Citrate; Sulfones; Swimming

Visceral organs display differential sensitivity to ischemia and reperfusion injury, but the cellular mechanisms underlying these differential responses are not completely understood. A significant response to ischemia identified in brain is stress to the endoplasmic reticulum (ER), as indicated by PKR-like endoplasmic reticulum eIF2alpha kinase (PERK)-mediated phosphorylation of eIF2alpha. To determine the generality of this response, we evaluated the PERK pathway in brain, GI tract, heart, liver, lung, kidney, pancreas and skeletal muscle following a clinically relevant, 10 min cardiac arrest-induced whole body ischemia and either 10 or 90 min reperfusion. The potential role of nitric oxide (NO) on PERK activation was investigated by conducting ischemia and reperfusion in the presence and absence of the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME). Organ stress could be ranked with respect to the degree of eIF2alpha phosphorylation at 10 min reperfusion. Brain, kidney and GI tract were reactive organs, showing 15 to 20-fold increases in eIF2alpha(P) compared to controls. Moderately reactive organs included liver and heart, showing <10-fold increases in eIF2alpha(P). Pancreas, lung and skeletal muscle were nonreactive. Although treatment of cultured neuroblastoma 104 cells with the NO-donor S-nitroso-N-acetyl-penicillamine (SNAP) activated PERK, administration of L-NAME had no effect on PERK activation or eIF2alpha phosphorylation in organs following ischemia and reperfusion. Thus, PERK is activated differentially in reperfused organs independent of NO. These results suggest that ER stress may play a role in differential responses of viscera to ischemia and reperfusion. ER stress in viscera may contribute to the pathophysiology of resuscitation from cardiac arrest and during organ transplantation procedures.

Topics: Animals; Brain; Cardiopulmonary Resuscitation; Disease Models, Animal; eIF-2 Kinase; Eukaryotic Initiation Factor-2; Gastrointestinal Tract; Heart Arrest; Kidney; Liver; Lung; Male; Muscle, Skeletal; Nitric Oxide Donors; Pancreas; Penicillamine; Rats; Rats, Long-Evans; Reperfusion Injury; Tumor Cells, Cultured

Guanosine 3', 5'-cyclic monophosphate (cGMP) acts as a relaxant second messenger in the cerebral vessels. cGMP-specific phosphodiesterase type 5 (PDE5) inhibitor increases intracellular cGMP levels. This study investigated the effect of the PDE5 inhibitor on the ischemic brain.. Regional cerebral blood flow (rCBF), cGMP concentration, and infarction volume were measured in the rat middle cerebral artery occlusion model. Ten minutes after ischemia, the animals received an intravenous (i.v.) infusion of vehicle (phosphate-buffered saline), PDE5 inhibitor, zaprinast (10 mg/kg), or nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP, 100 microg/kg). rCBF was measured continuously by laser-Doppler flowmetry in the ischemic penumbra of the ischemic and contralateral sides under continuous blood pressure monitoring. cGMP concentrations were determined using the enzyme immunoassay and infarct volumes were estimated by 2,3,5-triphenyltetrazolium chloride staining.. The administration of zaprinast significantly increased rCBF in the ischemic brain compared with the pre-drug control value despite the decreased mean blood pressure, whereas it did not affect rCBF in the contralateral side. The cGMP concentration was significantly higher in the ischemic cortex compared with the contralateral side. SNAP infusion increased the cGMP concentration in the bilateral cortices to a similar extent. The volume of cerebral infarction was significantly decreased by zaprinast administration.. The PDE5 inhibitor zaprinast may selectively increase CBF in the ischemic brain via increased cGMP levels, thus providing a new strategy against acute cerebral infarction.

Topics: Analysis of Variance; Animals; Blood Circulation Time; Blood Pressure; Brain Ischemia; Cerebrovascular Circulation; Cyclic GMP; Disease Models, Animal; Functional Laterality; Immunoenzyme Techniques; Infarction, Middle Cerebral Artery; Laser-Doppler Flowmetry; Male; Nitric Oxide Donors; Penicillamine; Phosphodiesterase Inhibitors; Purinones; Rats; Rats, Wistar; Regional Blood Flow; Tetrazolium Salts; Time Factors

Several reports propose that apoptosis of pancreatic beta cells may play a central role in the pathogenesis of both spontaneous and induced insulin-dependent diabetes mellitus (IDDM) in animal models. Whether apoptosis is a major cell death pathway during diabetes development, however, is highly controversial. The aim of this study was to examine the mode of islet cell death in prediabetic diabetes-prone (dp) BB rats, which spontaneously develop diabetes and serve as an animal model for human IDDM. In addition we investigated the cell death pathway of islet cells treated with the widely used diabetogenic compound streptozotocin or with nitric oxide (NO), which during IDDM development has been found to be present in inflamed islets in high concentrations because of the expression of inducible NO synthase. Islets of prediabetic BBdp rats were analyzed for DNA strand breaks and screened by electron microscopy. The mode of islet cell death in vitro after treatment with cytotoxic concentrations of streptozotocin or of NO was investigated using different methods including morphologic analysis by electron microscopy, detection of DNA strand breaks, poly(ADP-ribose) polymerase cleavage, and annexin V staining. Although cells with DNA stand breaks-often accepted as a proof for apoptosis-could be identified, we did not find apoptosis-specific features during islet cell death. Instead we observed massive necrosis as evidenced by disrupted plasma membranes and spilled-out cellular constituents in vitro as well as during disease manifestation in BBdp rats. These results may have serious consequences with regard to the treatment of humans to prevent the development of IDDM.

Topics: Animals; Annexin A5; Apoptosis; Blotting, Western; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; DNA; DNA Fragmentation; Flow Cytometry; In Situ Nick-End Labeling; Islets of Langerhans; Male; Mice; Mice, Inbred C57BL; Microscopy, Electron; Necrosis; Nitric Oxide; Penicillamine; Poly(ADP-ribose) Polymerases; Rats; Rats, Inbred BB; Rats, Wistar; Streptozocin

Traumatic brain injury (TBI) attenuates the cerebral vasodilation to hypercapnia. Cortical spreading depression (CSD) also transiently reduces hypercapnic vasodilation. The authors sought to determine whether the CSD elicited by a controlled cortical impact (CCI) injury masks the true effect of TBI on hypercapnic vasodilation, and whether a nitric oxide (NO) donor can reverse the attenuation of hypercapnic vasodilation following CCI.. Anesthetized rats underwent moderate CCI. Cerebral blood flow was monitored with laser Doppler flowmetry and the response to hypercapnia was determined for injured and sham-injured animals. The effect of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), on this response was also assessed. At an uninjured cortical site ipsilateral to the CCI, a single wave of CSD was recorded and the CO2 response at this location was significantly attenuated for up to 30 minutes (seven rats, p < 0.05). At the injured cortex, hypercapnic vasodilation continued to be attenuated for 7 hours. The cerebral vasodilation to CO2 was 37 +/- 5% in injured rats (six) compared with 84 +/- 10% in the sham-injured group (five rats, p < 0.05). After 30 minutes of topical superfusion with SNAP, hypercapnic vasodilation was restored to 74 +/- 7% (nine rats, p > 0.1 compared with that in the sham-injured group). In contrast, papaverine, an NO-independent vasodilator, failed to reverse the attenuation of the CO2 response to CCI.. The authors conclude that CSD elicited by CCI can mask the true effect of TBI on hypercapnic vasodilation for at least 30 minutes. Exogenous NO, but not papaverine, can reverse the attenuation of cerebrovascular reactivity to CO2 caused by TBI. This result supports the hypothesis that NO production is reduced after TBI and that the NO donor has a potential beneficial role in the clinical management of head injury.

Topics: Animals; Blood Pressure; Brain Injuries; Carbon Dioxide; Cerebrovascular Circulation; Cortical Spreading Depression; Disease Models, Animal; Hypercapnia; Male; Nitric Oxide; Nitric Oxide Donors; Oxygen; Papaverine; Penicillamine; Rats; Rats, Sprague-Dawley; Vasodilator Agents

The effects of local application of a cream containing nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine (SNAP) or isosorbide dinitrate were studied in a rat model of incision pain. An incision was made in the plantar aspect of a hind paw and the cream was applied inside the surgical wound. SNAP (1-10%) or isosorbide (2.5-5%) reduced the incision allodynia as measured with von Frey filaments. Higher concentrations produced a smaller or no effect, but SNAP (30%) intensified the allodynia. Allodynia was also intensified by SNAP (5% or 30%) in rats pretreated with intraplantar 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, 4 microg), a guanylate cyclase inhibitor. The effect of isosorbide (5%) was prevented by ODQ. The cream containing SNAP released 10- to 20-fold more nitrite than did isosorbide from a macrophage culture. We conclude that local application of drugs generating a low NO concentration reduces incision pain through activation of guanylate cyclase. Drugs generating high NO concentrations, however, intensify pain via a guanylate cyclase-independent mechanism.

Topics: Animals; Disease Models, Animal; Diuretics, Osmotic; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guanylate Cyclase; Hindlimb; Hyperalgesia; Isosorbide; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Donors; Nitrites; Ointments; Oxadiazoles; Pain Threshold; Pain, Postoperative; Penicillamine; Quinoxalines; Rats; Rats, Wistar; Time Factors

In a rat model of the ischemia-reperfusion with pylorus ligation, gastric ulcer was formed, although gastric acid secretion was reduced. When the polymorphonuclear leukocytes were inactivated in advance, gastric ulcer was not formed, but acid secretion was increased, indicating that gastric acid is not a cause of the ulcer formation in this model. The mechanism of gastric acid suppression accompanied by ischemia-reperfusion was examined in relation to the role of oxygen-free radicals in this rat model. Prior administration of superoxide dismutase did not modulate acid secretion, but N-nitro-L-arginine methyl ester (L-NAME) increased acid secretion. The action of L-NAME was antagonized specifically by L-arginine, but not by D-arginine. S-nitroso-N-acetylpenicillamine did not inhibit basal acid secretion but antagonized the action of L-NAME. Aminoguanidine increased significantly the gastric acid output that was suppressed by ischemia-reperfusion. When polymorphonuclear leukocytes were inactivated by treatment with their antibody, the gastric acid output recovered to the level in the pylorus-ligated rat without ischemia-reperfusion. These results suggested that nitric oxide (NO) produced by the infiltrated polymorphonuclear leukocytes plays an important role in the suppression of acid secretion induced by ischemia-reperfusion.

Topics: Animals; Arginine; Disease Models, Animal; Enzyme Inhibitors; Free Radical Scavengers; Gastric Acid; Gastric Mucosa; Ligation; Male; Neutrophils; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Penicillamine; Pylorus; Rats; Rats, Inbred Strains; Reperfusion Injury; Stomach; Stomach Ulcer; Superoxide Dismutase; Time Factors

Lipopolysaccharide (LPS) has been proposed to act as one of the pathogens in endotoxemia-induced gastric lesions, but its action on mucosal integrity has not been fully clarified.. We compared the effects of LPS originating from Escherichia coli and the chemical donor of nitric oxide (NO), S-nitroso-acetylpenicillamine (SNAP), on acute gastric lesions induced by 100% ethanol, mucosal blood flow (GBF), and mucosal generation of prostaglandin E2 (PGE2) and examined the expression of constitutive NO synthase (cNOS) and inducible NO synthase (iNOS) mRNA in the gastric mucosa of rats treated with LPS, by using reverse transcription polymerase chain reaction (RT-PCR).. LPS (0.01-1.0 mg/kg) or SNAP (0.37-3.0 mg/kg) given intraperitoneally, dose-dependently prevented ethanol-induced mucosal lesions, and these protective effects were accompanied by a significant increase in the GBF and excessive mucosal release of NO. Suppression of NOS activity by NG-nitro-L-arginine methyl ester (L-NAME) (20mg/kg intravenously) or L-NG-(1-iminoethyl)-lysine (L-NIL) (30mg/kg intraperitoneally) and NOS induction by treatment with dexamethasone (2 mg/kg intraperitoneally) reversed the protective and hyperemic effects of LPS, and this reversal by L-NAME was significantly antagonized by addition of the substrate for NOS, L-arginine, but not D-arginine. Both LPS and SNAP increased PGE2 generation significantly, and this effect was reduced by pretreatment with L-NAME, L-NIL, or dexamethasone. Expression of cNOS was detected by RT-PCR in the intact mucosa, but intense signals for expression of both cNOS and iNOS were detected in the mucosa of LPS-treated rats.. Parenteral LPS, similarly to the chemical NO donor, SNAP, protects the gastric mucosa against ethanol-induced damage via an increase in GBF mediated by NO due to the activation of arginine-NO system and possibly also enhanced generation of PGE2.

Topics: Analysis of Variance; Animals; Arginine; Base Sequence; Dexamethasone; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Escherichia coli; Ethanol; Gastric Acid; Gastric Mucosa; Gene Expression; Lipopolysaccharides; Male; Molecular Sequence Data; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Penicillamine; Pepsin A; Polymerase Chain Reaction; Rats; Rats, Wistar; Regional Blood Flow; RNA, Messenger; Stomach; Stomach Diseases

The mechanisms associated with dysfunction of the cerebral vasculature following head trauma have not yet been fully elucidated. In an attempt to shed more light on the matter, we investigated the endothelial-mediated dilations in the rat middle cerebral artery (MCA) following severe traumatic brain injury (TBI). Rats were subjected to severe controlled cortical impact injury (CCI; 5 m/s, 130 ms duration, 3 mm deformation) over the right parietal cortex. At 24 h postinjury, ipsilateral segments of MCA and corresponding contralateral segments were isolated, mounted in a vessel chamber, and pressurized. The responses to 2 methylthio-ATP (2MeSATP), a selective agonist for the P2Y1 purinoceptors, N(omega)-nitro-L-arginine (L-NAME), an NO synthase inhibitor, and S-nitroso-N-acetylpenicillamine (SNAP), an exogenous NO donor, were determined. 2MeSATP elicited concentration dependent dilations in all MCAs studied. Ipsilateral MCAs harvested following TBI or sham-TBI, showed similar maximum dilations to 2MeSATP [70 +/- 4% (n = 17) and 72 +/- 6% (n = 13), respectively]. However, TBI reduced the concentration of 2MeSATP necessary to elicit one-half of the maximum dilation (EC50) from 15 to 9 nM (p < 0.05). Inhibition of NO synthase with 10(-5) M L-NAME abolished the dilation to 2MeSATP in both TBI and sham-TBI MCAs. The constriction to L-NAME was significantly reduced in TBI MCAs compared to sham vessels. Dilations to SNAP, an NO donor, were not altered by TBI indicating that the mechanisms of dilation involving NO in the vascular smooth muscle were not affected. Unlike other pathological conditions which often diminish endothelial-mediated responses, severe TBI enhanced the sensitivity to 2MeSATP without altering the maximum response.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Brain Injuries; Cerebral Arteries; Cerebrovascular Circulation; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Inhibitors; Linear Models; Male; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Penicillamine; Rats; Rats, Long-Evans; Receptors, Purinergic P2; Thionucleotides; Vasodilation