Involvement of the bed nucleus of the stria terminalis in hippocampal cholinergic system-mediated activation of the hypothalamo--pituitary--adrenocortical axis in rats

We tested the hypothesis that activation of Na(+)/H(+) exchanger is involved in dilator responses of the basilar artery to endothelium-dependent vasodilators in vivo. Using a cranial window in anesthetized rats, we examined responses of the basilar artery to acetylcholine and bradykinin. Topical application of acetylcholine and bradykinin increased diameter of the basilar artery in a concentration-related manner. Because N(G)-nitro-L-arginine, an inhibitor of nitric oxide synthase, almost abolished vasodilator responses to acetylcholine and bradykinin, vasodilatation produced by the agonists appears to be mediated primarily by nitric oxide. 5-N,N-Hexamethyleneamiloride, an inhibitor of Na(+)/H(+) exchanger, did not affect baseline diameter of the basilar artery, but inhibited vasodilatation in response to acetylcholine and bradykinin, without affecting vasodilatation produced by sodium nitroprusside. FR183998, another inhibitor of Na(+)/H(+) exchanger, also attenuated acetylcholine-induced dilatation of the basilar artery without affecting vasodilatation in response to sodium nitroprusside. Monomethylamine hydrochloride, which produces intracellular alkalinization, enhanced acetylcholine-induced dilatation of the basilar artery in the presence of 5-N,N-hexamethyleneamiloride. These results suggest that intracellular alkalinization produced by activation of Na(+)/H(+) exchanger may enhance nitric oxide production in the basilar arterial endothelium and thereby contribute to dilator responses of the artery in vivo.     

NADPH-oxidase activity is elevated in penumbral and non-ischemic cerebral arteries following stroke

Reactive oxygen species play a role in neuronal damage following cerebral ischemia-reperfusion. We tested whether activity of the superoxide-generating enzyme, NADPH-oxidase, is enhanced in cerebral arteries within, adjacent and distant from the ischemic core. The right middle cerebral artery (MCA) of conscious rats was temporarily occluded by perivascular injection of endothelin-1 to induce stroke (ET-1; n=19). Control rats were injected with saline (n=9). At 24 h or 72 h post-administration of ET-1, the MCA and its branches within the ipsilateral penumbra and infarcted core, corresponding arteries in the contralateral hemisphere, and basilar artery were excised. Anatomically similar arteries were excised from saline-injected rats. At 24 h after stroke, NADPH-stimulated superoxide production by arteries from the infarcted core did not differ from levels generated by arteries from control rats, whereas levels were significantly lower 72 h after stroke. However, at both time points after stroke, superoxide production by arteries from the ischemic penumbra was 8-fold greater than levels generated by arteries from control rats. Surprisingly, even in the non-ischemic arteries from the contralateral hemisphere and in the basilar artery, superoxide production was increased approximately 4- to 6-fold at 24 h, but had returned to normal 72 h after stroke. The NADPH-oxidase inhibitor, diphenyleneiodonium, virtually abolished superoxide production by all arteries. Thus, the activity of NADPH-oxidase is enhanced in cerebral arteries from the ischemic penumbra at 24 h and 72 h following cerebral ischemia. Additionally, NADPH-oxidase activity is temporarily enhanced after cerebral ischemia within arteries from non-ischemic parts of the brain.     

Superoxide dismutase ameliorates impaired nitric oxide synthase-dependent dilatation of the basilar artery during chronic alcohol consumption

The goals of this study were to determine whether chronic alcohol consumption impairs nitric oxide synthase-dependent reactivity of the basilar artery and to determine a potential mechanism which might account for the effects of alcohol on the basilar artery. Sprague-Dawley rats were fed liquid diets with or without alcohol for 8 weeks. Using intravital microscopy, we measured the diameter of basilar artery in response to nitric oxide synthase-dependent agonists (acetylcholine and bradykinin) and a nitric oxide synthase-independent agonist (nitroglycerin). Topical application of acetylcholine (0.1 and 1 microM) and bradykinin (1 and 10 nM) produced dose-related dilatation of the basilar artery in non-alcohol-fed and alcohol-fed rats. However, the magnitude of vasodilatation in response to acetylcholine and bradykinin was significantly less in alcohol-fed rats compared to non-alcohol-fed rats. Dilatation of the basilar artery in response to nitroglycerin was similar in non-alcohol-fed and alcohol-fed rats. Next, we examined whether impaired responses of the basilar artery in alcohol-fed rats in response to acetylcholine and bradykinin may be related to the production of oxygen radicals. We found that topical application of superoxide dismutase (150 U/ml) significantly improved impaired receptor-mediated nitric oxide synthase-dependent dilatation of basilar artery in alcohol-fed rats. However, superoxide dismutase did not alter responses of the basilar artery to nitroglycerin in alcohol-fed rats, and did not alter responses of the basilar artery to nitric oxide synthase-dependent or -independent agonists in non-alcohol-fed rats. Our findings suggest that chronic consumption of alcohol impairs nitric oxide synthase-dependent dilatation of a large cerebral artery which may be related to the receptor-mediated release of oxygen radicals to inactivate nitric oxide.     

Influence of gender on dilatation of the basilar artery during diabetes mellitus

The first goal of the present study was to examine the influence of gender on reactivity of the basilar artery. The second goal of this study was to examine the effect of diabetes mellitus on reactivity of the basilar artery in male and female rats. We examined in vivo responses of the basilar artery in male and female nondiabetic and diabetic rats in response to a nitric oxide synthase (NOS)-dependent (acetylcholine) and -independent (nitroglycerin) agonist. In nondiabetic male and female rats, acetylcholine and nitroglycerin produced dose-related dilatation of the basilar artery. However, the magnitude of vasodilatation in response to acetylcholine and a high concentration of nitroglycerin was significantly greater in female than in male rats. Acetylcholine (1.0 microM) dilated the basilar artery by 11 +/- 2% in nondiabetic males versus 25 +/- 4% in nondiabetic females (P<0.05). Nitroglycerin (1.0 microM) dilated the basilar artery by 37 +/- 8% in nondiabetic males versus 62 +/- 5% in nondiabetic females (P<0.05). Thus, there is a significant effect of gender on reactivity of the basilar artery during physiologic conditions. Dilatation of the basilar artery in response to acetylcholine, but not nitroglycerin, was impaired in diabetic male and female rats compared to their nondiabetic counterparts. Acetylcholine (1.0 microM) dilated the basilar artery by only 5 +/- 1% in diabetic males and by only 4 +/- 1% in diabetic females. In summary, dilatation of the basilar artery in response to NOS-dependent agonist was significantly greater in nondiabetic female than in nondiabetic male rats. In addition, diabetes mellitus impaired NOS-dependent dilatation of the basilar artery not only in male rats, but also in female rats. We suggest that the results of these studies provide insight into the pathogenesis of cerebrovascular abnormalities observed in postmenopausal women.     

氨甲酰化促红细胞生成素经PI3-K/Akt信号通路抗脑缺血损伤

目的观察脑缺血后氨甲酰化促红细胞生成素(CEPO)的神经保护作用并探讨其可能机制。方法健康雄性SD大鼠随机分为6组(n=10):(1)假手术组;(2)缺血组;(3)EPO组;(4)CEPO组;(5)LY(LY294002)组;(6)CEPO+LY组。应用大脑中动脉线栓法(MCAO)制作大鼠局灶性脑缺血模型,评定大鼠神经功能并计算脑梗死体积,Western blot方法检测PI3-K/Akt活性变化。结果 EPO组与CEPO组脑梗死体积均明显缩小,神经功能显著改善,磷酸化Akt(p Akt)水平明显增高,且两组之间无明显差异,但CEPO的神经保护作用及对Akt磷酸化的诱导效应均可被PI3-K抑制剂LY294002部分抵消。结论 CEPO具有与EPO相当的缺血后脑保护作用,其机制可能与PI3-K/Akt信号通路激活有关。     

Increased reactive oxygen species in familial amyotrophic lateral sclerosis with mutations in SOD1

Amyotrophic lateral sclerosis (ALS) is a paralytic disorder characterized by degeneration of large motor neurons of the brain and spinal cord. A subset of ALS is inherited (familial ALS, FALS) and is associated with more than 70 different mutations in the SOD1 gene. Here we report that lymphoblast cell lines derived from FALS patients with 16 different mutations in SOD1 gene exhibit significant increase of intracellular reactive oxygen species (ROS) compared with sporadic ALS (SALS) and normal controls (spouses of ALS patients). The ROS generation did not correlate with SOD1 activity. Further, cells incubated with vitamin C, catalase or the flavinoid quercetin significantly reduced ROS in all groups. The catalase inhibitor 3-amino-1,2,4-triazole resulted in a ten-fold increase of ROS in all groups. Neither L-nitroarginine, a nitric oxide synthase inhibitor or vitamin E altered the ROS levels. Thus, these studies suggest that hydrogen peroxide (H(2)O(2)) is a major ROS elevated in FALS lymphoblasts and it may contribute to the degeneration of susceptible cells. Further, we postulate a mechanism by which increased H(2)O(2) could be generated by mutant SOD1.     

Cerebral blood flow changes in a patient recovered from hypocarbia

The changes of cerebral blood flow velocity (CBFV) and pulsatility index (PI) in anterior cerebral artery were examined by cranial color Doppler ultrasonography while a patient recovered from hypocarbia. As arterial PCO2 was elevated, PI was decreased and color imaging of CBF from basilar artery to anterior cerebral artery were improved in sagittal view. Hypocarbia is considered to induce the constriction of the cerebral vessels and the decrease of CBF in premature infants.     

Nicotine exposure, mimicked smoking, directly and indirectly enhanced protein kinase C activity in isolated canine basilar artery, resulting in enhancement of arterial contraction.

Cigarette smoking is a significant risk factor in the incidence of cerebrovascular disorders. Among the many compounds in cigarette smoke, nicotine is considered to most significantly affect cerebral arterial tone. The purpose of this study is to investigate precise pharmacological effects of nicotine on the regulation of cerebral arterial tone. To mimic smoking, a low concentration of nicotine (10(-6) mol/L), which is equivalent to the serum level of habitual smokers, was treated for 1 hour in an isometric tension study and for 24 hours in a study using cultured vascular endothelial cells (VECs). Using the canine basilar artery, the effect of nicotine on uridine 5'-triphosphate (UTP)-induced vasoconstriction was examined in the isometric tension study. Protein kinase C (PKC) activity in the canine basilar artery was measured by enzyme immunoassay. Endothelial function was assessed by endothelium-dependent vasodilatation and endogenous nitric oxide (NO) synthesis in VECs using a fluorescent indicator, diaminofluorescein-FM diacetate (DAF-FM/DA). Nicotine significantly enhanced UTP-induced contraction and PKC activity in the artery, and attenuated endothelium-dependent vasodilatation and NO synthesis in VECs. Because PKC activity was increased by de-endothelialization itself, endothelial dysfunction by nicotine enhances PKC activity. Because PKC was further activated by nicotine even in the de-endothelialized artery, nicotine directly affects PKC activities in smooth muscle. These results indicate that nicotine potentiates contractile response through direct and indirect PKC activation in the canine basilar artery.     

Bad as a converging signaling molecule between survival PI3-K/Akt and death JNK in neurons after transient focal cerebral ischemia in rats.

Bad, a proapoptotic Bcl-2 family protein, plays a critical role in determining cell death/survival. The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the c-Jun N-terminal kinase (JNK) pathway are thought to be involved in regulation of Bad. Therefore, the present study was performed to clarify the role of Bad as a common target of the PI3-K/Akt and JNK pathways after transient focal cerebral ischemia (tFCI) in rats. We found that Akt activity increased at 3 h and then decreased, whereas JNK activity increased 7 to 24 h in the peripheral area after tFCI. Administration of LY294002, a PI3-K-specific inhibitor, exacerbated DNA fragmentation, whereas administration of SP600125, a JNK-specific inhibitor, attenuated it. Inhibited by LY294002, phospho-Bad (Ser136) expression increased in the peripheral area 3 h after tFCI, with suppression of Akt activity. Furthermore, phospho-Bad (Ser136) and phospho-Akt (Ser473) were colocalized. Decreases in phospho-Bad (Ser136) and Bad/14-3-3 dimerization and increases in Bcl-X(L)/Bad or Bcl-2/Bad dimerization observed 7 to 24 h after tFCI, were prevented by SP600125 administration, with inhibition of JNK activity. The present study indicates that signal predominance varies from PI3-K/Akt-mediated survival signaling to JNK-mediated death signaling with the development of neuronal damage in the peripheral area after tFCI. This study also suggests that PI3-K/Akt has a role in Bad inactivation, whereas the JNK pathway is involved in Bad activation. We conclude that Bad may be an integrated checkpoint of PI3-K/Akt-mediated survival signaling and JNK-mediated death signaling and that it contributes to cell fate in the peripheral area after cerebral ischemia.     

Responses of rat basilar artery to acetylcholine and platelet products in vivo

Studies in vitro suggest that the basilar artery has distinctive responses to endothelium-dependent stimuli. Our first goal was to examine the effects of acetylcholine on diameter of the basilar artery in vivo. Because aggregating platelets may have important effects on cerebral arteries, our second goal was to examine the effects on the basilar artery of products that are released by platelets (thromboxane, serotonin, and adenosine 5'-diphosphate). Diameter of the basilar artery was measured through a cranial window in anesthetized rats (n = 25). Baseline diameter of the basilar artery was 247 +/- 10 microns mean +/- SEM. Topical application of acetylcholine at 10(-6) and 10(-5) M dilated the basilar artery by 13 +/- 2% and 19 +/- 2%, respectively. The thromboxane analogue U46619 at 10(-8) and 10(-7) M reduced the diameter of the basilar artery by 18 +/- 5% and 29 +/- 4%, respectively. At 10(-8) and 10(-7) M, serotonin had little effect on pial arterioles on the cerebrum but constricted the basilar artery by 18 +/- 2% and 29 +/- 4%, respectively. At 10(-6) and 10(-5) M, adenosine 5'-diphosphate produced marked dilatation of pial arterioles on the cerebrum (9 +/- 2% and 20 +/- 3%, respectively) but had little effect on the basilar artery (increased diameter by 4 +/- 2% and 6 +/- 2%, respectively). Thus, in contrast to some studies of the basilar artery in vitro, acetylcholine produces dilatation of the basilar artery in vivo. Potent constrictor responses to thromboxane and serotonin, in combination with the minimal dilator effect of adenosine 5'-diphosphate, suggest that release of these products during platelet aggregation would favor constriction of the basilar artery.     

Effect of diabetes mellitus on flow-mediated and endothelium-dependent dilatation of the rat basilar artery.

BACKGROUND AND PURPOSE: Diabetes mellitus may impair endothelium-dependent responses in cerebral arterioles. The basilar artery dilates in response to increases in blood flow. The goal of this study was to examine effects of diabetes mellitus on "flow-mediated" and endothelium-dependent dilatation of the basilar artery. METHODS: Diabetes was induced in rats with 50 mg/kg streptozotocin. Six months later, vessel diameter and velocity of blood flow through the basilar artery were measured using a cranial window in anesthetized rats under baseline conditions and during occlusion of the carotid arteries. Changes in vessel diameter were also measured during topical application of acetylcholine and sodium nitroprusside. RESULTS: With aortic pressure maintained at baseline levels, blood flow velocity through the basilar artery increased similarly in control and diabetic rats during unilateral common carotid artery occlusion and during bilateral occlusion. In control and diabetic rats, diameter of the basilar artery increased by 10 +/- 2% and 10 +/- 4% during unilateral occlusion and by 27 +/- 5% and 31 +/- 4% during bilateral occlusion, respectively. Thus, diabetes did not impair flow-mediated dilatation of the basilar artery. In contrast, dilatation in response to 10(-5) M topical acetylcholine was less in diabetic rats (13 +/- 2%) than in control rats (45 +/- 8%) (p less than 0.05). Dilator responses to nitroprusside were not impaired by diabetes. CONCLUSIONS: The findings suggest that diabetes produces impairment of endothelium-dependent responses to acetylcholine, but not flow-mediated dilatation, in the basilar artery.     

Impairment of endothelium-dependent dilatation of the basilar artery during diabetes mellitus

The goal of this study was to determine whether responses of the basilar artery are altered during diabetes mellitus. We measured the diameter of the basilar artery in vivo in non-diabetic and diabetic rats (streptozotocin; 50–60 mg/kg i.p.). Responses of the basilar artery to agonists, which presumably produce dilatation by releasing endothelium-derived relaxing factor (EDRF), were impaired in diabetic rats compared to non-diabetic rats. Acetylcholine (1.0 and 10 μM) dilated the basilar artery by 13 ± 2 and 26 ± 4% (means±S.E.M.), respectively, in non-diabetic rats, but by only 13 ± 1 and 9 ± 2%, respectively, in diabetic rats (P < 0.05). Bradykinin (1.0 and 10 μM) dilated the basilar artery by14 ± 2 and 35 ± 6 % (means±S.E.M.), respectively, in non-diabetic rats, but by only5 ± 1 and 6± 2%, respectively, in diabetic rats (P < 0.05). The response to nitroglycerin was similar in non-diabetic and diabetic rats. Thus, impairment of vasodilatation in diabetic rats in response to acetylcholine and bradykinin is not related to non-specific impaired of vasodilation than impaired dilator responses of the basilar artery in response to acetylcholine and bradykinin in diabetic rats may be related to the activation of the throm☐ane A₂-prostaglandin H₂ receptor. SQ 29548 (a specific throm☐ane A₂-prostaglandin H₂ receptor antagonist) did not alter responses of the basilar artery to acetylcholine and bradykinin. These findings suggest that diabetes mellitus impairs endothelium-dependent dilation of the basilar artery. In addition, the mechanism of impaired responses of the basilar artery during diabetes mellitus does not appear to be related to the activation of the throm☐ane A₂-prostaglandin H₂ receptor.     

Goat cerebrovascular reactivity to ADP after ischemia-reperfusion. Role of nitric oxide, prostanoids and reactive oxygen species

To analyze the cerebrovascular effects of ischemia-reperfusion, cerebrovascular reactivity to ADP was studied after inducing 60-min occlusion followed by 60-min reperfusion of the left middle cerebral artery (MCA) in anesthetized goats. In 12 goats, at the end of reperfusion, left MCA resistance was decreased by 19%, and reactive hyperemia to 5- and 10-s occlusions as well as the cerebral vasodilatation to ADP (0.03-0.3 microg) but not to sodium nitroprusside (0.3-3 microg) was decreased. In 28 animals, killed at the end of reperfusion, segments 3-mm long were obtained from the left (ischemic) and right (control) MCA, prepared for isometric tension recording, and precontracted with the thromboxane A2 analogue U46619. The relaxation to ADP (10(-8) to 10(-5) M) but not to sodium nitroprusside (10(-8) to 10(-4) M) was lower in ischemic arteries. L-NAME (inhibitor of nitric oxide synthesis, 10(-4) M), charybdotoxin (10(-7) M)+apamin (10(-6) M) (blockers of KCa), or catalase (1000 U/ml) reduced the relaxation to ADP only in control arteries. Charybdotoxin+apamin further augmented the L-NAME-induced reduction in the relaxation to ADP in control arteries. The inhibitor of cyclooxygenase meclofenamate (10(-5) M) increased the relaxation to ADP only in ischemic arteries. The superoxide dismutase mimetic tiron (10(-2) M) increased the ADP-induced relaxation only in ischemic arteries. Therefore, it is suggested that ischemia-reperfusion produces cerebrovascular endothelial dysfunction, which may be associated with decreased nitric oxide bioavailability, decreased release of an EDHF, and increased production of vasoconstrictor prostanoids. All these alterations may be related in part with an increased production of superoxide anion.     

Experimental alterations in cyclic adenosine monophosphate concentrations in the cat basilar artery.

Levels of cyclic adenosine monophosphate (AMP) in the basilar artery and in circulating blood of cats were determined after the production of spasm by topical application of blood to the vessel and following treatment with agents known to alter cyclic AMP. Isoproterenol, known to stimulate adenyl cyclase, and aminophylline, a phosphodiesterase inhibitor, were studied alone and in combination. Cyclic AMP of the basilar artery fell from a mean control value of 43 to 26 pmoles per milligram of protein following the production of vasospasm. Intravenous administration of isoproterenol alone and in combination with aminophylline produced dilatation of the basilar artery, which was associated with a marked rise in the cyclic AMP concentration in the vessel. The finding that cerebral vasospasm is associated with a fall and vasodilation with a rise in cyclic AMP concentration supports the hypothesis of an active role for cyclic nucleotides in the regulation of cerebrovascular smooth muscle tone.     

Superoxide dismutase partially restores impaired dilatation of the basilar artery during diabetes mellitus

The goal of this study was to test the hypothesis that administration of superoxide dismutase restores nitric oxide synthase-mediated dilatation of the basilar artery during diabetes mellitus. We measured the diameter of the basilar artery in vivo in nondiabetic and diabetic rats (streptozotocin; 50–60 mg/kg i.p.) in response to nitric oxide synthase-dependent agonists (acetylcholine and bradykinin) and a nitric oxide synthase-independent agonist (nitroglycerin) before and during application of superoxide dismutase. Topical application of acetylcholine (1.0 and 10 μM) and bradykinin (1.0 and 10 μM) produced dose-related dilatation of the basilar artery in nondiabetic and diabetic rats. However, the magnitude of vasodilation produced by acetylcholine and bradykinin was significantly less in diabetic rats. Topical application of nitroglycerin (0.1 and 1.0 μM) produced similar dose-related dilatation of the basilar artery in nondiabetic and diabetic rats. Treatment with superoxide dismutase (150 U/ml) did not alter baseline diameter of the basilar artery in nondiabetic and diabetic rats. However, topical application of superoxide dismutase partially restored nitric oxide synthase-dependent dilatation of the basilar artery in diabetic rats towards that observed in nondiabetic rats. Superoxide dismutase did not alter dilatation of the basilar artery in nondiabetic rats. These findings suggest that impaired nitric oxide synthase-dependent dilatation of the basilar artery during diabetes mellitus may be related, in part, to enhanced release of oxygen-derived free radicals.     

Insulin reverses impaired acetylcholine-induced dilatation of the rat basilar artery during diabetes mellitus

The goal of this study was to determine whether treatment of diabetic rats with insulin reverses impaired nitric oxide synthase-dependent reactivity of the basilar artery in vivo. We measured the diameter of the basilar artery in non-diabetic rats, diabetic (streptozotocin; 50-60 mg/kg i.p.) rats, and diabetic rats treated with insulin implants in response to acetylcholine and nitroglycerin before and following topical application of N(G)-monomethyl-L-arginine (L-NMMA). Reactivity of the basilar artery was measured 2-3 months after injection of streptozotocin. We found that topical application of acetylcholine (1.0 and 10 microM) produced dose-related dilatation of the basilar artery in non-diabetic rats, which was inhibited (>80%) by topical application of L-NMMA (10 microM). In diabetic rats, the magnitude of vasodilation produced by acetylcholine was significantly less than in non-diabetic rats. Further, topical application of L-NMMA did not affect dilatation of the basilar artery in diabetic rats in response to acetylcholine. In insulin treated diabetic rats, dilatation of the basilar artery in response to acetylcholine was significantly greater than that observed in either non-diabetic or diabetic rats. In contrast, dilatation of the basilar artery in response to nitroglycerin was similar in insulin treated diabetic rats, non-diabetic rats and diabetic rats. Topical application of L-NMMA only partially inhibited dilatation of the basilar artery in response to acetylcholine in diabetic rats treated with insulin. Thus, the findings of this study suggest that impaired acetylcholine-induced dilatation of the basilar artery during diabetes mellitus can be restored by treatment with insulin. Further, it appears that the contribution of cellular pathways, in addition to nitric oxide synthase, may contribute to dilatation of the basilar artery in response to acetylcholine in rats treated with insulin.     

Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells

Previous in vitro studies in our laboratory have shown that mancozeb (MZ) and maneb (MB), both widely used EBDC fungicides, are equipotent neurotoxicants that produce cell loss in mesencephalic dopaminergic and GABAergic cells after an acute 24h exposure. Mitochondrial uncoupling and inhibition were associated with fungicide exposure. Inhibition of mitochondrial respiration is known to increase free radical production. Here the mechanism(s) of neuronal damage associated with MZ exposure was further explored by determining the role that reactive oxygen species (ROS) played in toxicity. Damage to mesencephalic dopamine and GABA cell populations were significantly attenuated when carried out in the presence of ascorbate or SOD, indicative of a free radical-mediated contribution to toxicity. ROS generation monitored by hydrogen peroxide (H(2)O(2)) production using Amplex Red increased in a dose-dependent manner in response to MZ. Inhibition of intracellular catalase with aminotriazole had little effect on H(2)O(2) generation, whereas exogenously added catalase significantly reduced H(2)O(2) production, demonstrating a large extracellular contribution to ROS generation. Conversely, cells preloaded with the ROS indicator dye DCF showed significant MZ-induced ROS production, demonstrating an increase in intracellular ROS. Both the organic backbone of MZ as well as its associated Mn ion, but not Zn ion, were responsible and required for H(2)O(2) generation. The functionally diverse NADPH oxidase inhibitors, diphenylene iodonium chloride, apocynin, and 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride significantly attenuated H(2)O(2) production by MZ. In growth medium lacking cells, MZ produced little H(2)O(2), but enhanced H(2)O(2) generation when added with xanthine plus xanthine oxidase whereas, in cultured cells, allopurinol partially attenuated H(2)O(2) production by MZ. Minocycline, an inhibitor of microglial activation, modestly reduced H(2)O(2) formation in mesencephalic cells. In contrast, neuronal-enriched cultures or cultures treated with MAC-1-SAP to kill microglia, did not show an attenuation of ROS production. These findings demonstrate that Mn-containing EBDC fungicides such as MZ and MB can produce robust ROS generation that likely occurs via redox cycling with extracellular and intracellular oxidases. The findings further show that microglia may contribute to but are not required for ROS production by MZ.     

Isolated human and rat cerebral arteries constrict to increases in flow: role of 20-HETE and TP receptors

Elevation of intraluminal pressure increases vasomotor tone, which thought to have a substantial role in regulation of cerebral blood flow (CBF). Interestingly, responses of cerebral vessels to increases in flow varied and have not been studied in human cerebral arteries. We hypothesized that increases in flow elicit constrictions of isolated human and rat cerebral arteries and aimed to elucidate the underlying mechanisms. Human cerebral arteries and rat middle cerebral arteries constricted to increases in flow (P<0.05). Simultaneous increase in intraluminal flow+pressure further reduced the diameter compared with pressure-induced changes (P<0.05), leading to constant estimated CBF. Flow-induced constrictions were abolished by HET0016 (inhibitor of synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) or inhibition of COXs or blocking TP (thromboxane A₂/prostaglandin H₂, receptors and attenuated by scavenging reactive oxygen species (ROS). Flow-enhanced ROS formation was significantly reduced by HET0016. In conclusion, in human and rat cerebral arteries (1) increases in flow elicit constrictions, (2) signaling mechanism of flow-induced constriction of cerebral arteries involves enhanced production of ROS, COX activity, and mediated by 20-HETE via TP receptors, and (3) we propose that simultaneous operation of pressure- and flow-induced constrictions is necessary to provide an effective autoregulation of CBF.     

Activation of the Akt/GSK3beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats.

Recent studies have revealed that the phosphatidylinositol 3-kinase (PI3-K) pathway is involved in apoptotic cell death after experimental cerebral ischemia. The serine-threonine kinase, Akt, functions in the PI3-K pathway and prevents apoptosis by phosphorylation at Ser473 after a variety of cell death stimuli. After phosphorylation, activated Akt inactivates other apoptogenic factors, including glycogen synthase kinase-3beta (GSK3beta), thereby inhibiting cell death. However, the role of Akt/GSK3beta signaling in the delayed death of hippocampal neurons in the CA1 subregion after transient global cerebral ischemia (tGCI) has not been clarified. Transient global cerebral ischemia for 5 mins was induced by bilateral common carotid artery occlusion combined with hypotension. Western blot analysis showed a significant increase in phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) in the hippocampal CA1 subregion after tGCI. Immunohistochemistry showed that expression of phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) was markedly increased in the vulnerable CA1 subregion, but not in the ischemic-tolerant CA3 subregion. Double staining with phospho-GSK3beta (Ser9) and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed different cellular distributions in the CA1 subregion 3 days after tGCI. Phosphorylation of Akt and GSK3beta was prevented by LY294002, a PI3-K inhibitor, which facilitated subsequent DNA fragmentation 3 days after tGCI. Moreover, transgenic rats that overexpress copper/zinc-superoxide dismutase, which is known to be neuroprotective against delayed hippocampal CA1 injury after tGCI, had enhanced and persistent phosphorylation of both Akt and GSK3beta after tGCI. These findings suggest that activation of the Akt/GSK3beta signaling pathway may mediate survival of vulnerable hippocampal CA1 neurons after tGCI.     

Catalase prevents elevation of [Ca(2+)](i) induced by alcohol in cultured canine cerebral vascular smooth muscle cells: Possible relationship to alcohol-induced stroke and brain pathology

Several studies have suggested that alcohol-induced brain injury is associated with generation of reactive oxygen species (ROS). The recent findings, that antioxidants (Vitamin E and pyrrolidine dithiocarbamate (PDTC)) prevent intracellular Ca(2+) ([Ca(2+)](i)) overload in cerebral vascular smooth muscle cells, induced by alcohol, demonstrate indirectly that ROS formation is related to cerebral vascular injury. The present experiments were designed to test the hypothesis that catalase, an hydrogen peroxide (H(2)O(2)) scavenging enzyme, can prevent or ameliorate alcohol-induced elevation of [Ca(2+)](i). Preincubation of cultured canine cerebral vascular smooth muscle cells with catalase (20-1000 units/ml) didn't produce any apparent changes from controls in resting levels of [Ca(2+)](i) after 1-3 days. Exposure of the cerebral vascular cells to culture media containing 10-100mM ethanol resulted in significant rises in [Ca(2+)](i) (p<0.01). Although exposure of these cells to a low concentration of catalase (20 units/ml) failed to prevent the increased level of [Ca(2+)](i) induced by ethanol, concomitant addition of higher concentrations of catalase (100-1000 units/ml) and ethanol (10-100mM) inhibited or ameliorated the rises of [Ca(2+)](i) induced by ethanol either at 24h or at 3 days, in a concentration-dependent manner. Catalase, in the range of 100-200 units/ml, inhibited approximately 50% of the [Ca(2+)](i) increases caused by ethanol in the first 24h. Catalase at a concentration of 1000 units/ml inhibited completely excessive [Ca(2+)](i) accumulation. The present results when viewed in light of other recently published data suggest that H(2)O(2) generation may be one of the earliest events triggered by alcohol in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.