Andrew Yau-Chik Shum

Oxidative neurotoxicity in rat cerebral cortex neurons: synergistic effects of H2O2 and NO on apoptosis involving activation of p38 mitogen-activated protein kinase and caspase-3.

Oxidative stress in the brain has been increasingly associated with the development of numerous human neurological diseases. Microglia, activated upon neuronal injury or inflammatory stimulation, are known to release superoxide anion (·O  2− ), hydrogen peroxide (H2O2), and nitric oxide (NO), thereby further contributing to oxidative neurotoxicity. The reaction of NO and ·O  2− , forming the toxic peroxynitrite (ONOO−), has been proposed to play a pathogenic role in neuronal injury. However, the interactions between H2O2 and NO during oxidative stress, which may promote or diminish cell death, is less clear. In this study, we explored oxidative neurotoxicity induced by H2O2 plus NO in primary cultures of rat cerebral cortex neurons. As the mechanisms may involve reactions between H2O2 and NO, we monitored the production of ONOO−and reactive oxygen species (ROS) throughout the experiments. Results indicated that the NO donor S‐nitroso‐N‐acetyl‐D, L‐penicillamine (SNAP) and H2O2 by themselves elicited neuronal death in a concentration‐ and time‐dependent manner. Sublytic concentrations of H2O2 plus SNAP were sufficient to induce neuronal apoptosis as determined by DNA laddering and fluorescent staining of apoptotic nuclei. Transient ONOO−increase was accompanied by rapid H2O2 decay and NO production, whereas ROS slowly decreased following treatment. Furthermore, p38 mitogen‐activated protein kinase (MAPK) activation and the cleavage of caspase‐3 were observed. Conversely, inhibition of p38 MAPK and caspase‐3 significantly reduced apoptotic death induced by H2O2 plus SNAP. These data suggest that H2O2 and NO act synergistically to induce neuronal death through apoptosis in which activation of p38 MAPK and caspase‐3 is involved.

The vasorelaxant effect of evodiamine in rat isolated mesenteric arteries: mode of action

The roles of the endothelium, Ca2+ and K+ fluxes in the evodiamine-induced attenuation of vascular contractile responses to vasoactive agents were examined. The results showed that: (1) in rat mesenteric artery rings, evodiamine elicited a concentration-dependent attenuation in the contractile response generated by phenylephrine. The inhibitory potency was greater for intact than for endothelium-denuded preparations. Thus, the vasodilator action of evodiamine appeared to be partially endothelium-interactive (dependent). (2) Evodiamine pretreatment had a greater inhibitory effect on the phenylephrine-induced tonic contraction (via Ca2+ influx) than on the phasic contraction (via Ca2+ release). In addition, evodiamine was more potent to inhibit the restoration by CaCl2 of contractile responses to phenylephrine than a potassium depolarizing solution in media that had been kept calcium-free. These results suggest that block of the Ca2+ influx through receptor-mediated Ca2+ channels may be the major mechanism underlying the vasodilator effect of evodiamine. (3) A K+ channel blocker, tetraethylammonium, almost completely abolished the vasodilatation induced by minoxidil (a known K+ channel opener) but not evodiamine. The possible involvement of K+ channel activation of the vasodilator effect produced by evodiamine was therefore excluded.

Production of macrophage inflammatory protein‐2 following hypoxia/reoxygenation in glial cells

Polymorphonuclear neutrophils (PMNs) are known to mediate brain inflammation following hypoxia/reoxygenation (H/R), but the precise mechanisms leading to PMN recruitment are undefined. The α‐chemokine macrophage inflammatory protein‐2 (MIP‐2) has specificity for the recruitment of PMNs. In this study, we found that 8 or 12 h of hypoxia followed by 24‐h reoxygenation (H8/R24 or H12/R24) induced MIP‐2 secretion in cultures of enriched microglia or mixed glia, respectively. Microglia, however, could not survive longer duration (>12 h) of hypoxia. Astrocytes did not produce any significant amount of MIP‐2 even though astrocytes maintained 98–99% viability following H12/R24. We also found that microglia survived the H/R treatment better (following H24) in the presence of astrocytes (mixed glial culture) than in microglia‐enriched culture. Reoxygenation for prolonged periods (3 and 5 days) following H24 resulted in progressively larger increases in MIP‐2 production (20‐ and 60‐fold, respectively) in mixed glial cultures. Immunocytochemical staining revealed that the cells expressing MIP‐2 in response to H/R were microglia rather than astrocytes in mixed glial cultures. Examination of MIP‐2 mRNA expression showed that H/R upregulated MIP‐2 gene expression. Taken together, our data suggest that microglial cells are an important source of MIP‐2 production and suggest a potential injury mechanism involving brain‐derived production of MIP‐2 in H/R. GLIA 32:155–164, 2000.

Determination of chlorogenic acid in rat blood by microdialysis coupled with microbore liquid chromatography and its application to pharmacokinetic studies.

To investigate the pharmacokinetics of unbound chlorogenic acid, a sensitive microbore liquid chromatographic method for the determination of chlorogenic acid in rat blood by microdialysis has been developed. A microdialysis probe was inserted into the jugular vein of male Sprague-Dawley rats, to which chlorogenic acid (20, 40, 60 or 80 mg/kg, i.v.) had been administered. On-line microdialysate was directly injected into a microbore column using a methanol-100 mM sodium dihydrogenphosphate (30:70, v/v, pH 2.5 adjusted with orthophosphoric acid) as the mobile phase and ultraviolet detection at 325 nm. The method is rapid, easily reproduced, selective and sensitive. The limit of detection for chlorogenic acid was 0.01 microg/ml and the limit of quantification was 0.05 microg/ml. The in vivo recovery of the chlorogenic acid of the microdialysis probe, based on a 5 microg/ml standard, was approximately 49-65% (n=6). The disposition of chlorogenic acid at each dose was best fitted to a two-compartment pharmacokinetic model. The area under the concentration curve increased greater than in direct proportion with the dose and terminal disposition become much slower as the dose was increased. The results indicated that the pharmacokinetics of unbound chlorogenic acid in rat blood is non-linear.

Hypoxia/reoxygenation induces cell injury via different mechanisms in cultured rat cortical neurons and glial cells.

Hypoxia/reoxygenation (H/R) causes cell injury/death. We examined the protection by drugs intervening at various stages of the injury cascade in cultured neurons and glia. Primary cultures of rat cortical neurons and mixed glia were subjected to H/R. Measurements of cell death (by lactate dehydrogenase release into the medium) and viability (by MTT reduction) indicated that H/R led to time-dependent injury in both neuronal and mixed glial cultures. The extent of cell injury in neurons was significantly greater than in glia cells. Pretreatment with (+)-MK-801 hydrogen maleate (MK-801) (an N-methyl-D-aspartate antagonist), N(omega)-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of nitric oxide synthase) or free radical scavengers reduced the extent of the H/R-elicited neuronal damage. MK-801, in contrast, was without effect on glial cells while L-NAME was effective. Our results suggest differential mechanism(s) and susceptibility to injury caused by H/R for neurons and mixed glia.

Enterohepatic circulation of chloramphenicol and its glucuronide in the rat by microdialysis using a hepato-duodenal shunt.

A system consisting of a hepato-duodenal shunt in which the bile of a drug-treated donor rat was diverted to the duodenum of an untreated recipient rat via a bile cannula was used to assess the role of hepatic metabolism and enterohepatic circulation in the pharmacokinetics of chloramphenicol. Blood concentrations of unbound chloramphenicol and its glucuronide were measured by on-line microdialysis coupled to a microbore liquid chromatographic system. Results indicated that chloramphenicol and its glucuronide were detected in the blood of both donor and recipient rats following an intravenous 100 mg/kg dose of chloramphenicol succinate to the donor rat. Our finding suggests that although enterohepatic circulation contributed only to a minor extent (approximately 1.8%) was involved in the disposition of unbound chloramphenicol in the rat on-line microdialysis techniques were applicable for such studies.

SIMULTANEOUS BLOOD AND BILIARY SAMPLING OF ESCULETIN BY MICRODIALYSIS IN THE RAT

Biliary excretion and intestinal reabsorption in enterohepatic circulation play major dispositional roles for some drugs. To circumvent multiple blood sampling and interruption of enterohepatic circulation in conventional biliary cannulation, the present study utilized the minimally invasive sampling technique of microdialysis in pharmacokinetics and biliary excretion studies. Microdialysis probes were inserted into the jugular vein and bile duct in the anesthetized rat for simultaneous and continuous sampling following intravenous administration of esculetin, a bioactive coumarin derivative. Placements of the microdialysis probes were designed to minimize obstruction to normal flows of the body fluids. Separation and quantitation of esculetin in the dialysates were achieved using high performance liquid chromatography (HPLC) coupled to UV detection. The results indicated higher drug concentrations in the bile than in the blood, suggesting active biliary excretion. The study also provided an example of successful application of in vivo microdialysis as an interesting and feasible alternative for pharmacokinetics and biliary drug excretion studies.

Studies of the cellular mechanisms underlying the vasorelaxant effects of rutaecarpine, a bioactive component extracted from an herbal drug

We conducted studies to investigate the nature and underlying mechanisms of the vascular effects of rutaecarpine (Rut), an alkaloid isolated from the Chinese herbal drug Evodia rutaecarpa. By using largely the effects on phenylephrine (PE)-induced contraction in the isolated rat aorta as the experimental index and by comparison with several known vascular muscle relaxants such as acetylcholine (ACh), histamine, and A23187, Rut relaxed PE-precontracted aorta in concentration-(10(-7)-10(-4) M) and endothelium-dependent manners. Studies with appropriate antagonists indicated that this was coupled to nitric oxide (NO) and guanylyl cyclase. Extracellular Ca2+ removal and treatment with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), suggested that influx of extracellular Ca2+ was the major factor contributing to the action of Rut. Pertussis toxin suppressed the relaxation potency of histamine but had no effects on the actions of Rut. NaF, the G proteins activator, attenuated the actions of ACh, but only minimally affected Na-NP, A23187, and Rut. 1-[6-{[17 beta-3-methoxyestra-1,2,3(10)-trien-17-yl]amino} hexyl]-1H-pyrrole-2,5-dione (U73122), the phospholipase C inhibitor, again suppressed the actions of ACh but had few effects on A23187 and Rut. Taken together, these results suggest that these vasorelaxants had different cellular mechanisms and that neither pertussis toxin-sensitive Gi protein, other G proteins, nor phospholipase C activation was involved in the cellular response to rutaecarpine.

Mechanisms of vasorelaxant effect of dehydroevodiamine: a bioactive isoquinazolinocarboline alkaloid of plant origin.

We examined the mechanisms underlying the vasorelaxant effect of dehydroevodiamine (DeHE), one of the bioactive components of the Chinese herbal drug Evodia rutaecarpa that has been shown to produce vasorelaxant and hypotension. DeHE (10(-7)-10(-4) M) concentration-dependently relaxed isolated rat mesenteric arteries precontracted with phenylephrine (PE). This vasorelaxant potency was diminished by 15% by endothelial removal, L-NG-nitro arginine, or methylene blue (MB), but not indomethacin treatment, indicating that the vasorelaxant effect of DeHE was partially endothelium dependent and mediated by nitric oxide (NO) and the cyclic GMP pathway. In endothelium-denuded preparations, DeHE caused a rightward shift of the contractile concentration-response curve (CRC) to PE in a dose-dependent manner with a pA2 value of 6.15. Maximal response was unaffected. Receptor binding assay indicated that DeHE competed with alpha 1-adrenoceptor ligand prazosin with a Ki value of 3.57 microM. Potassium channel activity-attenuating conditions such as increased level of extracellular K+ (20 mM) and treatment with the antagonist tetraethylammonium (TEA) significantly inhibited DeHEs effect, suggesting a mode of action similar to that of a potassium channel activator. In addition, high concentrations of DeHE (3 x 10(-5) and 10(-4) M) relaxed high K+ (80 mM)-evoked contraction, indicating that DeHE might possess K+ channel blocking properties. Multiple-action mechanisms, including endothelium dependence, alpha 1-adrenoceptor blockade, K+ channel activation, and Ca2+ channel blockade were probably involved in the vasorelaxant effects of DeHE.

Increase of plasma neuropeptide Y-like immunoreactivity following chronic hypoxia in the rat

In an attempt to understand the changes of circulating neuropeptide Y (NPY) during hypoxia, the plasma level of NPY was investigated by radioimmunoassay. Exposure of rats to hypobaric hypoxia at an altitude of 18,000 ft for 4 weeks causes an increase of pulmonary pressure and an elevation of plasma NPY-like immunoreactivity (NPY-LI). However, the systemic blood pressure was not elevated by this chronic hypoxia. Also, plasma noradrenaline (NA) estimated by chromatographic analysis (HPLC-ECD) was not markedly raised. Failure of bretylium and guanethidine, sympathetic neuron blockers, in reducing the plasma NPY-LI level of these rats ruled out the participation of adrenergic nervous terminals. Adrenal medulla seems responsible for this elevation of plasma NPY-LI because this magnitude disappeared in adrenalectomized rats. These data suggest that chronic hypoxia induced an elevation of circulating NPY from the adrenal gland of rats.