P2 purinergic receptor activation of neuronal nitric oxide synthase and guanylyl cyclase in the dorsal facial area of the medulla increases blood flow in the common carotid arteries of cats.

In the dorsal facial area (DFA) of the medulla, an activation of either P2 purinergic receptor or nitric oxide synthase (NOS) results in the release of glutamate, leading to an increase in blood flow of the common carotid artery (CCA). It is not known whether activation of the P2 receptor by ATP may mediate activation of NOS/guanylyl cyclase to cause glutamate release and/or whether L-Arg (nitric oxide (NO) precursor) may also cause ATP release from any other neuron, to cause an increase in CCA flow. We demonstrated that microinjections of P2 receptor agonists (ATP, α,ß-methylene ATP) or NO precursor (L-arginine) into the DFA increased CCA blood flow. The P2-induced CCA blood flow increase was dose-dependently reduced by pretreatment with NG-nitro-arginine methyl ester (L-NAME, a non-specific NOS inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NOS inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with D-NAME (an isomer of L-NAME) or N5-(1-iminoethyl)-L-ornithine (L-NIO, a potent endothelial NOS inhibitor). Involvement of glutamate release in these responses were substantiated by microdialysis studies, in which perfusions of ATP into the DFA increased the glutamate concentration in dialysates, but co-perfusion of ATP with L-NAME or 7-NI did not. Nevertheless, the arginine-induced CCA blood flow increase was abolished by combined pretreatment of L-NAME and MB, but not affected by pretreatment with a selective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). In conclusion, ATP activation of the P2 receptor in the DFA induced activation of neuronal NOS/guanylyl cyclase, which causes glutamate release leading to an increase in CCA blood flow. However, arginine activation of neuronal NOS/guanylyl cyclase, which also caused glutamate release and CCA blood flow increase, did not induce activation of P2 receptors. These findings provide important information for drug design and/or developing therapeutic strategies for the diseases associated with CCA blood flow that supplies intra- and extra-cranial tissues.

Mycophenolate mofetil alleviates lupus nephritis through urokinase receptor signaling in a mice model.

Lupus nephritis (LN) is usually associated with widespread effacement of the podocytes' foot processes leading to proteinuria. Induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via promoting podocytes' motility and kidney permeability in the glomerulus. Very little is known about uPAR signaling in LN. Mycophenolate mofetil (MMF), an immunosuppressive agent, efficiently modulates the development of LN in humans and mice, but there are no data concerning the direct uPAR involvement on podocytes in LN. The MMF efficiency and uPAR involvement signaling in NZB×NZW F1 lupus-prone mice were examined by proteinuria, renal function and pathology, immune complex deposits, and uPAR expression of podocytes by immunofluorescence staining and quantitative RT-PCR. After MMF treatment, the proteinuria (p < 0.01), BUN level (p < 0.05) and immunodeposition in glomeruli (p < 0.001) were significantly improved. Most important, the renal uPAR mRNA levels (p < 0.001) and uPAR protein level of podocytes (p < 0.001) were significantly reduced. The beneficial effect of MMF on LN could be attributed, at least in part, to the inhibition of uPAR expression in podocytes. These findings demonstrated uPAR could have potential as a predictive index for response to LN therapeutics.

Glutamate release upon purinergic action in the dorsal facial area of the medulla increases blood flow in the common carotid artery in cats.

Interactions of glutamatergic and purinergic actions in the medulla regulate important cardiovascular functions. The glutamatergic action in dorsal facial area (DFA) of the medulla increases blood flow of common carotid artery (CCA) in cats. We hypothesized that interactions of glutamatergic and purinergic actions in the DFA may regulate the CCA blood flow. Purinergic and glutamatergic agonists and antagonists were microinjected into the DFA through a four-barrel tubing in anesthetized cats. Drug effects were evaluated by changes in the CCA blood flow. Microinjection with 20 nmol ATP or alpha,beta-methyleneATP (alpha,beta-MeATP, a P2 purinergic receptor agonist) induced an increase of the CCA blood flow. This increase was dose-dependently reduced by prior administration with 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX, a specific P1 purinergic receptor antagonist), or pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, a selective P2 purinergic receptor antagonist) as well as with MK-801 (a non-competitive NMDA receptor antagonist) or glutamate diethyl ester (GDEE, a competitive AMPA/kainate receptor antagonist). It was almost completely blocked by administrations with combined maximal doses of P1 and P2 receptor antagonists as well as NMDA and AMPA receptor antagonists. Nevertheless, P1 receptor agonist induced only mild and poorly reproducible increase in the CCA blood flow. In conclusion, prominent P2 and minor P1 purinergic receptors appear to be present in the DFA; the purinergic activation can mediate a release of glutamate that stimulates NMDA and AMPA to induce the increase of the CCA blood flows. These findings may provide important information for developing therapeutic strategy for diseases involving the CCA blood flow, such as hypertensive disease and cerebral ischemia.

Regulation of the common carotid arterial blood flow by nicotinic receptors in the medulla of cats.

BACKGROUND AND PURPOSE: Actions of glutamate and serotonin on their respective receptors in the dorsal facial area (DFA) of the medulla are known to regulate common carotid arterial (CCA) blood flow in cats. Less is known about acetylcholine action on its nicotinic receptor (nAChR) subtypes in the DFA for regulation of CCA blood flow and this aspect was investigated. EXPERIMENTAL APPROACH: Nicotinic and muscarinic agonists and antagonists were microinjected into the DFA through a three-barrel tubing in anesthetized cats. RESULTS: CCA blood flow was dose-dependently increased by nicotine (a non-selective nAChR agonist) and choline (a selective alpha7-nAChR agonist). These effects of nicotine were attenuated by alpha-bungarotoxin (an alpha7-nAChR antagonist), methyllycaconitine (an alpha7-nAChR antagonist), mecamylamine (a relatively selective alpha3beta4-nAChR antagonist) and dihydro-beta-erythroidine (a relatively selective alpha4beta2-nAChR antagonist). The choline-induced flow increase was attenuated by alpha-bungarotoxin and mecamylamine, but not by dihydro-beta-erythroidine. Muscarinic agonists (muscarine and methacholine) and antagonist (atropine) affected neither the basal nor the nicotine-induced increase in the CCA blood flow. CONCLUSIONS AND IMPLICATIONS: Functional alpha7, alpha4beta2, and alpha3beta4 subunits of the nAChR appear to be present on the DFA neurons. Activations of these receptors increase the CCA blood flow. The present findings do not preclude the presence of other nAChRs subunits. Muscarinic receptors, if any, on the DFA are not involved in regulation of the CCA blood flow. Various subtypes of nAChRs in the DFA may mediate regulation of the CCA and cerebral blood flows.

Comparison of heart failure in children with enterovirus 71 rhombencephalitis and cats with norepinephrine cardiotoxicity.

The mechanism of heart failure in patients with enterovirus 71 rhombencephalitis (brain stem encephalitis) remains unknown. Our previous reports hypothesized that a catecholamine storm induced by rhombencephalitis may account for the heart failure. The aim of this study was to develop a novel feline model of norepinephrine cardiotoxicity and compare the resulting heart failure to that in children with enterovirus 71 rhombencephalitis. Nine of 75 children (12%) with enterovirus 71 rhombencephalitis (5 boys and 4 girls; age, 4-28 months; median age, 16 months) were complicated with left ventricular hypokinesia (ejection fraction, 31 +/- 9%). Six cats (weight, 3.03 +/- 0.64 kg) were administered intravenous norepinephrine 30 microg/kg/min for 3 hours. Echocardiography assessed the left ventricular diameter and function before and after the administration of norepinephrine. Pathology studies included hematoxylin and eosin stain and in situ terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling assay. In the feline model, norepinephrine induced significant left ventricular dilatation (end diastolic diameter from 1.18 +/- 0.19 to 1.62 +/- 0.22 cm, p = 0.001; endsystolic diameter from 0.54 +/- 0.09 to 1.36 +/- 0.32 cm, p = < 0.001) and hypokinesia (ejection fraction from 87.5 +/- 4.1 to 35.2 +/- 16.3%, p = 0.001). Heart specimens from 4 patients and six cats showed similar pathology findings, including myocardial hemorrhage, cardiomyocyte apoptosis, and coagulative myocytolysis, which is characterized by sarcoplasmic coagulation, granulation, vacuolization, myofibrillar waving, and disruption. Both groups showed no significant inflammatory reaction. In conclusion, heart failure in patients with enterovirus 71 rhombencephalitis is similar to that in cats with norepinephrine cardiotoxicity. Norepinephrine cardiotoxicity may play a role in the pathogenesis of heart failure in enterovirus 71 rhombencephalitis.

Diethylmaleate decreased ascorbic acid release induced by cerebral ischemia in cerebral cortex of the anesthetized rat.

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.

Effects of naloxone on lactate, pyruvate metabolism and antioxidant enzyme activity in rat cerebral ischemia/reperfusion.

Whether naloxone may modulate energy metabolism and endogenous antioxidant enzyme activities in ischemic cortex was studied. Cerebral ischemia/reperfusion (I/R) was produced by occluding two common carotid arteries and the right middle cerebral artery for 90 min followed by reperfusion in anesthetized Sprague-Dawley rats. Both pre-treatment (0.03 or 0.3 mg) and post-treatment (0.3 mg) of naloxone by intracerebroventricular infusion significantly reduced cortical infarct volumes. Pre-treatment with 0.03 mg reduced ischemia-induced suppression of extracellular pyruvate level and enhancement of lactate/pyruvate ratio as well as cerebral I/R-induced increases of endogenous catalase, glutathione peroxidase, and manganese superoxide dismutase activities. In conclusion, neuroprotective effects of naloxone in terms of reducing brain infarction involve attenuation of the disturbance of cellular functions following cerebral I/R via restoration of mitochondrial activities or energy metabolism.

Dorsal facial area of cat medulla; 5-HT2 action on glutamate release in regulating common carotid blood flow.

Serotonin (5-HT) may inhibit glutamate release in the dorsal facial area (DFA) of the medulla and decrease common carotid arterial (CCA) blood flow. We attempted to clarify which subtype(s) of 5-HT receptor was involved. A microdialysis probe was inserted in DFA. The concentration of glutamate in dialysates were determined by chromatography. Glutamate concentration was dose-dependently decreased by perfusion of 5-HT or DOI, a 5-HT2 agonist, but not by 5-CT, a 5-HT1 agonist. The 5-HT-induced decrease in glutamate was reversed by co-perfusion of ketanserin, a 5-HT2 antagonist, but not by propranolol, a 5-HT1 antagonist. CCA blood flow was decreased when 5-HT or DOI was perfused, and was reversed by co-perfusing ketanserin. In conclusion, 5-HT may inhibit glutamate release via 5-HT2 receptor in DFA, resulting in the reduction of CCA blood flow.

Elevated extracellular glutamate concentrations increased malondialdehyde production in anesthetized rat brain cortex.

Oxidative stress is believed to be involved in the damaging mechanism of excitotoxic insult. Thus, we investigated the effect of elevated extracellular glutamate levels on malondialdehyde production, a common index of lipid peroxidation, in anesthetized rat brain cortex. Elevation of extracellular glutamate levels was achieved either by exogenously perfusing glutamate solutions, or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a competitive inhibitor of glutamate uptake transporter, through an implanted microdialysis probe. Malondialdehyde levels in the microdialysates, which were reacted with thiobarbituric acid, were analyzed by a high performance liquid chromatography system equipped with a fluorescence detector. Perfusion of glutamate (1.5 and 15 mM) resulted in dose-dependent increases in extracellular malondialdehyde production (as high as a 6-fold increase in malondialdehyde production following perfusion of 15 mM glutamate solution). PDC (3.14 and 31.4 mM), not only significantly increased the extracellular glutamate levels in a dose-dependent manner, but also dramatically increased malondialdehyde production (as high as 20-fold increase). These results suggest that excitotoxicity induces oxidative stress in anesthetized rat brain cortex, as evidenced by the glutamate-induced increase in malondialdehyde production.

Evaluation of extracellular lipid peroxidation in brain cortex of anaesthetized rats by microdialysis perfusion and high-performance liquid chromatography with fluorimetric detection.

A method for in vivo evaluation of lipid peroxidation in the extracellular space of anaesthetized rat brain cortex was developed. This method involved the use of microdialysis perfusion and high-performance liquid chromatography. The microdialysates, eluted from implanted probes, were reacted with thiobarbituric acid (TBA) prior to analysis by an HPLC system equipped with a fluorescence detector (excitation and emission wavelengths were 515 and 550 nm, respectively). Lipid peroxidation in the extracellular space was evaluated as the concentration of malondialdehyde, a lipid peroxidation end product which reacts with TBA to form a fluorescent conjugate. Significantly increased production of malondialdehyde following hydrogen peroxide perfusion (0.03%, 0.3% at a flow-rate of 1 microl/min) was observed in the brain cortex of anaesthetized rats.

In vivo, continuous and automatic monitoring of extracellular ascorbic acid by microdialysis and on-line liquid chromatography.

A system for in vivo, automatic, continuous monitoring of organ extracellular ascorbic acid in anesthetized rat is described. This system involves microdialysis perfusion and a LC system equipped with an electrochemical detector. Microdialysate, eluted from a microdialysis probe implanted in the brain cortex or in the left ventricular myocardium of anesthetized rats was collected in the sample loop of an on-line injector for direct injection onto the LC system. This automated method provides a shortened sample processing time. This system was utilized to investigate the effect of cerebral ischemia on cortex extracellular ascorbic acid and the effect of myocardial ischemia on left ventricular myocardium extracellular ascorbic acid in anesthetized rats. Basal ascorbic acid concentrations in the cortex and left ventricular myocardium ranged from 9.7 to 15.4 microM (mean +/- S.D., 12.7 +/- 2.5 microM from the results of eight rats) and from 9.3 to 36.0 microM (mean +/- S.D., 24.3 +/- 8.9 microM from the results of twelve rats), respectively. Cerebral ischemia significantly elevated ascorbic acid levels in the cortex extracellular space, while myocardial ischemia did not significantly alter ascorbic acid levels in the left ventricular myocardium extracellular space.

In vivo evidence of hydroxyl radical formation induced by elevation of extracellular glutamate after cerebral ischemia in the cortex of anesthetized rats.

The in vivo interrelation between excitotoxicity and oxidative stress following cerebral ischemia in the cortex of anesthetized rats was investigated. Cerebral ischemia was induced by ligation of the bilateral common carotid arteries and the unilateral middle cerebral artery. Microdialysis perfusion with on-line high-performance liquid chromatography was used to monitor the hydroxyl radical levels. Extracellular hydroxyl radical levels were quantitated as the increased formation of 2.3 and 2.5 dihydroxybenzoic acid (DHBA), the hydroxylative products of salicylic acid contained in the microdialysis perfusion solutions. Elevated cortex extracellular glutamate content, resulting from the cerebral ischemia, caused an increase in the formation of hydroxyl radicals. Exogenous perfusion of authentic glutamate solutions through implanted microdialysis probes also resulted in increased hydroxyl radical formation in the cortex. The 2.3 and 2.5 DHBA levels remained elevated for an entire 80-min ischemic period. These results suggest that, after cerebral ischemia, increased oxidative stress did occur in anesthetized rats, and the oxidative stress may result from increased excitotoxicity.

Lowered brain glutathione by diethylmaleate decreased the glutamate release induced by cerebral ischemia in anesthetized rats.

The effect of lowered brain glutathione content on the glutamate release following cerebral ischemia was investigated. Diethylmaleate (4 mmol/kg, i.p.), a commonly used chemical reagent for tissue glutathione depletion, significantly reduced the ischemia-induced glutamate release. The release of another excitatory amino acid aspartate was not affected by the diethylmaleate administration. These results suggested that part of the elevated glutamate content induced by ischemia might result from the cellular GSH.

Elevated extracellular glutamate levels increased the formation of hydroxyl radical in the striatum of anesthetized rat.

Results from various in vitro experiments have indicated that excitotoxicity and oxidative stress are two interrelated major mechanisms in causing neuronal damage in brain disorders such as cerebral ischemia/reperfusion. Thus, we have conducted experiments to investigate whether in the striatum of anesthetized rats the elevated brain extracellular concentrations of glutamate could increase the formation of hydroxyl radical. Elevation of glutamate levels and trapping of hydroxyl radical were accomplished by perfusing Ringer solutions containing both glutamate and salicylic acid through microdialysis probes implanted in rat striatum. The formation of hydroxyl radical was quantitated as the increased amounts of 2,3 and 2,5 dihydroxybenzoic acid (DHBA) which were the hydroxylative products of salicylic acid. Eluted microdialysates were directly injected onto high performance liquid chromatography (HPLC) with electrochemical detector via an on-line automatic injector. This method was authenticated by in vitro studies employing Fenton-type hydroxyl radicals generation system. Our results indicated that elevated glutamate concentrations (15 mM, 1.5 mM, and 150 microM glutamate in perfusing solutions) would significantly increased both the concentrations of 2,3 and 2,5 DHBA. In conclusion, we have obtained direct evidence showing that the elevated glutamate concentrations in brain extracellular space would increase the formation of hydroxyl radical, and these results implied that oxidative stress occurring in brain disorders might be induced by excitotoxicity.

Determination of extracellular glutathione in rat brain by microdialysis and high-performance liquid chromatography with fluorescence detection.

A method for the continuous monitoring of extracellular glutathione (GSH) concentrations in rat brain has been developed. This method involved the in vivo sampling of brain extracellular fluid by microdialysis perfusion and the subsequent analysis by high-performance liquid chromatography (HPLC) with fluorescence detection. Perfusates from the microdialysis probes were directly derivatized with methanolic monobromobimane which acted as the fluorescence tag. Separation of the derivatized perfusate was achieved on narrow-bore reversed-phase C18 columns. Recoveries of GSH from the microdialysis probes ranged from 1.5% to 4%. The basal extracellular GSH concentration in rat (Sprague-Dawley) brain cortex was found to be 2.10 +/- 1.78 microM (mean +/- S.D.) (results of 18 rats). Fluorescence detection and separation on narrow-bore columns provided adequate sensitivity for accurate determination of brain extracellular GSH concentrations in rats. With this method, the extracellular GSH concentrations in the cerebral cortex were found to be significantly elevated upon the onset of cerebral ischemia induced by the ligation of bilateral common carotid arteries.

Pulmonary edema and rapid transfusion: the comparison between rapid intravenous and intraarterial infusion in the severely hemorrhagic anesthesized pigs.

BACKGROUND: Patients with shock often develop pulmonary edema (PE) after rapid and massive fluid supplement and intravenous infusion. Rapid intraarterial infusion (RIA) is often used for fluid supplement in cardiac surgery, but has not yet been applied to treatment of hemorrhagic shock. However, by perfusing the ischemic peripheral organs through RIA, the fluid should flow first through the venous system to the heart and lung in less volume at lower speed. Therefore, the probability of developing PE should be less than that in rapid intravenous infusion (RIV) to the heart and lung in the same condition regarding volume and speed. Accordingly, we compared RIV and RIA in the treatment of hemorrhagic shock (HS) to determine if RIA provides any beneficial effect in reducing the development of PE. METHODS: Eleven male mini-pigs weighing 17.5-32 kg were randomly divided into two groups to have RIV and RIA. Under general anesthesia, HS was induced by shedding blood (about 35 ml/kg) through the femoral artery until the mean arterial blood pressure (MAP) fell to 50 mm Hg. This condition was maintained for three hours. Then, lactated Ringer's solution (LRS) was infused thrice by force through a femoral artery (RIA) or an external jugular vein (RIV) at a speed of 25 ml/kg/min for 3 min. Data include hemodynamics, arterial blood gases, urine output, total extravascular lung water index (ETVI), and total amount of infused LRS used to induce gross PE (endotracheal release of pinkish foamy sputum). Serum concentrations of catecholamines, platelet activating factor (PAF) and thromboxane B2 (TxB2) were measured. RESULTS: The total amount of LRS needed to induce gross PE was significantly greater in RIA than in RIV group. ETVI after rapid transfusion with a total of 225 ml/kg LRS was significantly less in RIA than in RIV group. Also, TxB2 concentrations in serum were less in RIA group. However, there was no difference in changes of hemodynamics, blood gases, acid-base, pulmonary shunting, urine output, serum concentrations of PAF or catecholamines between these two groups. CONCLUSIONS: RIA may be a better choice for fluid replacement in HS in terms of decreasing the development of PE and lessening the release of ETVI and TxB2 in severely hemorrhagic anesthetized pigs. Further human investigation is warranted.

Effects of reducing sympathetic activities on acute myocardial ischemia in cats.

The importance of the sympathetic nervous system in the genesis of ventricular arrhythmias associated with acute myocardial ischemia (AMI) was assessed in urethane-chloralose anesthetized cats. AMI was created by a ligation of the left anterior descending coronary artery. All control cats developed ventricular tachyarrhythmia (VT), ventricular fibrillation (VF), bradycardia and hypotension, resulting in 50% mortality. In other groups the paramedian reticular nucleus (PRN) was intermittently stimulated to inhibit the sympathetic nervous system. Both sides of the stellate ganglia (SG) or adrenal glands (AG) were removed to exclude direct effects of cardiac sympathetic nerve or indirect effects of circulatory catecholamines from the adrenal medulla on the heart, respectively. AMI did not affect plasma norepinephrine (NE) and epinephrine (EPI) concentrations in all groups. However, NE concentration was higher in SG removed but lower in AG removed group. Removal of SG and stimulation of PRN significantly reduced arrhythmia score and the incidence of VT, VF and mortality. Removal of AG appeared to be less effective. These findings suggest that cardiac sympathetic innervation is more important than neural and adrenal catecholamine secretions in the plasma.

Effects of paweiwan on streptozotocin-induced hyperglycemia in rats.

The ancient Chinese remedy of Paweiwan was used by patients with polyuria, polydipsia, and polyphagia. The present study investigated the hypoglycemic effects of Paweiwan using streptozotocin-induced hyperglycemic rats. The effects on serum glucose in a 4-day course, in a 7-week course, on the standard oral glucose tolerance test, and on the liver glycogen content were studied. In the glucose tolerance test, chlorpropamide and insulin were used as the positive controls and 0.5% CMC (Carboxymethylcellulose) was used as the negative control. We found that Paweiwan decreased the baseline glucose concentration, ameliorated the blood glucose elevation after glucose challenge, and increased liver glycogen content. The results may imply that Paweiwan increases glucose entrance into cells.