Cerebroprotective actions of hydrogen sulfide in the epileptic brain in newborn pigs.

BACKGROUND: Neonatal epileptic seizures cause postictal dysregulation of cerebral blood flow. Hydrogen sulfide (H2S), a mediator with vasodilator and antioxidant properties, is produced in the brain by astrocyte cystathionine ß-synthase (CBS). This study investigated whether H2S improves the cerebral vascular outcome of seizures. METHODS: Epileptic seizures were induced in newborn pigs using bicuculline. The effects of the CBS inhibitor aminooxyacetate (AOA) and the H2S donor NaHS on cerebral vascular outcome of seizures were examined in live pigs, cerebral endothelial cells, and cortical astrocytes. RESULTS: Brain H2S was elevated during seizures. AOA blocked H2S and reduced functional hyperemia in the epileptic brain. The endothelium- and astrocyte-dependent vasodilation of pial arterioles was impaired 48 h after seizures suggesting cerebral vascular dysfunction. Systemic NaHS elevated brain H2S and blocked reactive oxygen species in the epileptic brain and in primary endothelial cells and astrocytes during inflammatory and excitotoxic conditions. Postictal cerebrovascular dysfunction was exaggerated in H2S-inhibited pigs and minimized in NaHS-treated pigs. CONCLUSIONS: H2S elevation in the epileptic brain via activation of CBS contributes to functional hyperemia and exhibits cerebroprotective properties. The H2S donor NaHS enhances brain antioxidant defense and provides a therapeutic approach for preventing adverse cerebral vascular outcome of neonatal epileptic seizures. IMPACT: Epileptic seizures in neonates lead to prolonged postictal cerebral vascular dysregulation. The role of hydrogen sulfide (H2S), a mediator with vasodilator and antioxidant properties, in the epileptic brain has been explored. Astrocytes are major sites of enzymatic H2S production in the epileptic brain. Postictal cerebral vascular dysfunction is exaggerated when astrocyte H2S production is pharmacologically inhibited during seizures. Postictal cerebral vascular dysfunction is minimized when the brain H2S is elevated by systemic administration of NaHS during seizures. NaHS provides a therapeutic approach for improving cerebrovascular outcome of epileptic seizures via a mechanism that involves the antioxidant potential of H2S.

H2S mediates the vasodilator effect of endothelin-1 in the cerebral circulation.

H2S is an endogenous gasotransmitter that increases cerebral blood flow. In the cerebral vascular endothelium, H2S is produced by cystathionine δ-lyase (CSE). Endothelin-1 (ET-1) has constrictor and dilator influences on the cerebral circulation. The mechanism of the vasodilation caused by ET-1 may involve endothelium-derived factors. We hypothesize that ET-1-elicited dilation of pial arterioles requires an elevation of H2S production in the cerebral vascular endothelium. We investigated the effects of ET-1 on CSE-catalyzed brain H2S production and pial arteriolar diameter using cranial windows in newborn pigs in vivo. H2S was measured in periarachnoid cerebrospinal fluid. ET-1 (10-12-10-8 M) caused an elevation of H2S that was reduced by the CSE inhibitors propargylglycine (PPG) and ß-cyano-l-alanine (BCA). Low doses of ET-1 (10-12-10-11 M) produced vasodilation of pial arterioles that was blocked PPG and BCA, suggesting the importance of H2S influences. The vasodilator effects of H2S may require activation of smooth muscle cell membrane ATP-sensitive K+ (KATP) channels and large-conductance Ca2+-activated K+ (BK) channels. The KATP inhibitor glibenclamide and the BK inhibitor paxilline blocked CSE/H2S-dependent dilation of pial arterioles to ET-1. In contrast, the vasoconstrictor response of pial arterioles to 10-8 M ET-1 was not modulated by PPG, BCA, glibenclamide, or paxilline and, therefore, was independent of CSE/H2S influences. Pial arteriolar constriction response to higher levels of ET-1 was independent of CSE/H2S and KATP/BKCa channel activation. These data suggest that H2S is an endothelium-derived factor that mediates the vasodilator effects of ET-1 in the cerebral circulation via a mechanism that involves activation of KATP and BK channels in vascular smooth muscle. NEW & NOTEWORTHY Disorders of the cerebral circulation in newborn infants may lead to lifelong neurological disabilities. We report that vasoactive peptide endothelin-1 exhibits vasodilator properties in the neonatal cerebral circulation by stimulating production of H2S, an endothelium-derived messenger with vasodilator properties. The ability of endothelin-1 to stimulate brain production of H2S may counteract the reduction in cerebral blood flow and prevent the cerebral vascular dysfunction caused by stroke, asphyxia, cerebral hypoxia, ischemia, and vasospasm.

Selective head cooling during neonatal seizures prevents postictal cerebral vascular dysfunction without reducing epileptiform activity.

Epileptic seizures in neonates cause cerebrovascular injury and impairment of cerebral blood flow (CBF) regulation. In the bicuculline model of seizures in newborn pigs, we tested the hypothesis that selective head cooling prevents deleterious effects of seizures on cerebral vascular functions. Preventive or therapeutic ictal head cooling was achieved by placing two head ice packs during the preictal and/or ictal states, respectively, for the ∼2-h period of seizures. Head cooling lowered the brain and core temperatures to 25.6 ± 0.3 and 33.5 ± 0.1°C, respectively. Head cooling had no anticonvulsant effects, as it did not affect the bicuculline-evoked electroencephalogram parameters, including amplitude, duration, spectral power, and spike frequency distribution. Acute and long-term cerebral vascular effects of seizures in the normothermic and head-cooled groups were tested during the immediate (2-4 h) and delayed (48 h) postictal periods. Seizure-induced cerebral vascular injury during the immediate postictal period was detected as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive staining of cerebral arterioles and a surge of brain-derived circulating endothelial cells in peripheral blood in the normothermic group, but not in the head-cooled groups. During the delayed postictal period, endothelium-dependent cerebral vasodilator responses were greatly reduced in the normothermic group, indicating impaired CBF regulation. Preventive or therapeutic ictal head cooling mitigated the endothelial injury and greatly reduced loss of postictal cerebral vasodilator functions. Overall, head cooling during seizures is a clinically relevant approach to protecting the neonatal brain by preventing cerebrovascular injury and the loss of the endothelium-dependent control of CBF without reducing epileptiform activity.

Endothelial Nitric Oxide Mediates Caffeine Antagonism of Alcohol-Induced Cerebral Artery Constriction.

Despite preventive education, the combined consumption of alcohol and caffeine (particularly from "energy drinks") continues to rise. Physiologic perturbations by separate intake of ethanol and caffeine have been widely documented. However, the biologic actions of the alcohol-caffeine combination and their underlying subcellular mechanisms have been scarcely studied. Using intravital microscopy on a closed-cranial window and isolated, pressurized vessels, we investigated the in vivo and in vitro action of ethanol-caffeine mixtures on cerebral arteries from rats and mice, widely recognized models to address cerebrovascular pathophysiology and pharmacology. Caffeine at concentrations found in human circulation after ingestion of one to two cups of coffee (10 µM) antagonized the endothelium-independent constriction of cerebral arteries evoked by ethanol concentrations found in blood during moderate-heavy alcohol intoxication (40-70 mM). Caffeine antagonism against alcohol was similar whether evaluated in vivo or in vitro, suggesting independence of systemic factors and drug metabolism, but required a functional endothelium. Moreover, caffeine protection against alcohol increased nitric oxide (NO•) levels over those found in the presence of ethanol alone, disappeared upon blocking NO• synthase, and could not be detected in pressurized cerebral arteries from endothelial nitric-oxide synthase knockout (eNOS(-/-)) mice. Finally, incubation of de-endothelialized cerebral arteries with the NO• donor sodium nitroprusside (10 µM) fully restored the protective effect of caffeine. This study demonstrates for the first time that caffeine antagonizes ethanol-induced cerebral artery constriction and identifies endothelial NO• as the critical caffeine effector on smooth muscle targets. Conceivably, situations that perturb endothelial function and/or NO• availability will critically alter caffeine antagonism of alcohol-induced cerebrovascular constriction without significantly disrupting endothelium-independent, alcohol-induced cerebral artery constriction itself.

Enteral supplements of a carbon monoxide donor CORM-A1 protect against cerebrovascular dysfunction caused by neonatal seizures.

Cerebral blood flow dysregulation caused by oxidative stress contributes to adverse neurologic outcome of seizures. A carbon monoxide (CO) donor CORM-A1 has antioxidant and cytoprotective properties. We investigated whether enteral supplements of CORM-A1 can improve cerebrovascular outcome of bicuculline-induced seizures in newborn piglets. CORM-A1 (2 mg/kg) was given to piglets via an oral gastric tube 10 minutes before or 20 minutes after seizure onset. Enteral CORM-A1 elevated CO in periarachnoid cerebrospinal fluid and produced a dilation of pial arterioles. Postictal cerebral vascular responses to endothelium-, astrocyte-, and vascular smooth muscle-dependent vasodilators were tested 48 hours after seizures by intravital microscopy. The postictal responses of pial arterioles to bradykinin, glutamate, the AMPA receptor agonist quisqualic acid, ADP, and heme were greatly reduced, suggesting that seizures cause injury to endothelial and astrocyte components of the neurovascular unit. In contrast, in the two groups of piglets receiving enteral CORM-A1, the postictal cerebral vascular responsiveness to these dilators was improved. Overall, enteral supplements of CORM-A1 before or during seizures offer a novel effective therapeutic option to deliver cytoprotective mediator CO to the brain, reduce injury to endothelial and astrocyte components of cerebral blood flow regulation and to improve the cerebrovascular outcome of neonatal seizures.

Contributions of KATP and KCa channels to cerebral arteriolar dilation to hypercapnia in neonatal brain.

Mechanisms by which Pco2 controls cerebral vascular tone remain uncertain. We hypothesize that potassium channel activation contributes to the neonatal cerebrovascular dilation in response to increases in Paco2. To test this hypothesis, experiments were performed on newborn pigs with surgically implanted, closed cranial windows. Hypercapnia was induced by ventilation with elevated Pco2 gas in the absence and presence of the KATP channel inhibitor, glibenclamide and/or the KCa channel inhibitor, paxillin. Dilations to pinacidil, a selective KATP channel activator, without and with glibenclamide, were used to evaluate the efficacy of KATP channel inhibition. Dilations to NS1619, a selective KCa channel activator, without and with paxillin, were used to evaluate the efficacy of KCa channel inhibition. Cerebrovascular responses to the KATP and KCa channel activators, pinacidil and NS1619, respectively, cAMP-dependent dilator, isoproterenol, and cGMP-dependent dilator, sodium nitroprusside (SNP), were used to evaluate the selectivity of glibenclamide and paxillin. Glibenclamide blocked dilation to pinacidil, but did not inhibit dilations to NS1619, isoproterenol, or SNP. Glibenclamide prior to hypercapnia decreased mean pial arteriole dilation ~60%. Glibenclamide treatment during hypercapnia constricted arterioles ~35%. The level of hypercapnia, Paco2 between 50 and 75 mmHg, did not appear to be involved in efficacy of glibenclamide in blocking dilation to Paco2. Similarly to glibenclamide and KATP channel inhibition, paxillin blocked dilation to the KCa channel agonist, NS1619, and attenuated, but did not block, arteriolar dilation to hypercapnia. Treatment with both glibenclamide and paxillin abolished dilation to hypercapnia. Therefore, either glibenclamide or paxillin that block dilation to their channel agonists, pinacidil or NS1619, respectively, only partially inhibit dilation to hypercapnia. Block of both KATP and KCa channels completely prevent dilation hypercapnia. These data suggest hypercapnia activates both KATP and KCa channels leading to cerebral arteriolar dilation in newborn pigs.

Cerebrovascular dilation via selective targeting of the cholane steroid-recognition site in the BK channel ß1-subunit by a novel nonsteroidal agent.

The Ca(2+)/voltage-gated K(+) large conductance (BK) channel ß1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK ß1 allows the BK channels to reduce myogenic tone, facilitating vasodilation. The endogenous steroid lithocholic acid (LCA) dilates cerebral arteries via BK channel activation, which requires recognition by a BK ß1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate ß1-containing BK channels and evoke vasodilation remain unknown. We performed a chemical structure database similarity search using LCA as a template, along with a two-step reaction to generate sodium 3-hydroxyolean-12-en-30-oate (HENA). HENA activated the BK (cbv1 + ß1) channels cloned from rat cerebral artery myocytes with a potency (EC50 = 53 µM) similar to and an efficacy (×2.5 potentiation) significantly greater than that of LCA. This HENA action was replicated on native channels in rat cerebral artery myocytes. HENA failed to activate the channels made of cbv1 + ß2, ß3, ß4, or ß1T169A, indicating that this drug selectively targets ß1-containing BK channels via the BK ß1 steroid-sensing site. HENA (3-45 µM) dilated the rat and C57BL/6 mouse pressurized cerebral arteries. Consistent with the electrophysiologic results, this effect was larger than that of LCA. HENA failed to dilate the arteries from the KCNMB1 knockout mouse, underscoring BK ß1's role in HENA action. Finally, carotid artery-infusion of HENA (45 µM) dilated the pial cerebral arterioles via selective BK-channel targeting. In conclusion, we have identified for the first time a nonsteroidal agent that selectively activates ß1-containing BK channels by targeting the steroid-sensing site in BK ß1, rendering vasodilation.

Functional role of astrocyte glutamate receptors and carbon monoxide in cerebral vasodilation response to glutamate.

In newborn pigs, vasodilation of pial arterioles in response to glutamate is mediated via carbon monoxide (CO), a gaseous messenger endogenously produced from heme degradation by a heme oxygenase (HO)-catalyzed reaction. We addressed the hypothesis that ionotropic glutamate receptors (iGluRs), including N-methyl-D-aspartic acid (NMDA)- and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid (AMPA)/kainate-type receptors, expressed in cortical astrocytes mediate glutamate-induced astrocyte HO activation that leads to cerebral vasodilation. Acute vasoactive effects of topical iGluR agonists were determined by intravital microscopy using closed cranial windows in anesthetized newborn pigs. iGluR agonists, including NMDA, (±)1-aminocyclopentane-cis-1,3-dicarboxylic acid (cis-ACPD), AMPA, and kainate, produced pial arteriolar dilation. Topical L-2-aminoadipic acid, a gliotoxin that selectively disrupts glia limitans, reduced vasodilation caused by iGluR agonists, but not by hypercapnia, bradykinin, or sodium nitroprusside. In freshly isolated and cultured cortical astrocytes constitutively expressing HO-2, iGluR agonists NMDA, cis-ACPD, AMPA, and kainate rapidly increased CO production two- to threefold. Astrocytes overexpressing inducible HO-1 had high baseline CO but were less sensitive to glutamate stimulation of CO production when compared with HO-2-expressing astrocytes. Glutamate-induced astrocyte HO-2-mediated CO production was inhibited by either the NMDA receptor antagonist (R)-3C4HPG or the AMPA/kainate receptor antagonist DNQX. Accordingly, either antagonist abolished pial arteriolar dilation in response to glutamate, NMDA, and AMPA, indicating functional interaction among various subtypes of astrocytic iGluRs in response to glutamate stimulation. Overall, these data indicate that the astrocyte component of the neurovascular unit is responsible for the vasodilation response of pial arterioles to topically applied glutamate via iGluRs that are functionally linked to activation of constitutive HO in newborn piglets.

Antioxidant roles of heme oxygenase, carbon monoxide, and bilirubin in cerebral circulation during seizures.

Postictal cerebrovascular dysfunction is an adverse effect of seizures in newborn piglets. The brain heme oxygenase (HO) provides protection against cerebrovascular dysfunction. We investigated the contribution of reactive oxygen species (ROS) to seizure-induced vascular damage and the mechanism of HO vasoprotection. In a bicuculline model of seizures, we addressed the hypotheses: (1) seizures increase brain ROS; (2) ROS contribute to cerebral vascular dysfunction; (3) ROS initiate a vasoprotective mechanisms by activating endogenous HO; and (4) HO products have antioxidant properties. As assessed by dihydroethidium oxidation (ox-DHE), seizures increased ROS in cerebral vessels and cortical astrocytes; ox-DHE elevation was prevented by tiron and apocynin. An HO inhibitor, tin protoporphyrin, potentiated, whereas an HO-1 inducer, cobalt protoporphyrin, blocked seizure-induced increase in DHE oxidation. Heme oxygenase products carbon monoxide (CO) (CORM-A1) and bilirubin attenuated ox-DHE elevation during seizures. Antioxidants tiron and bilirubin prevented the loss of postictal cerebrovascular dilations to bradykinin, glutamate, and sodium nitroprusside. Tiron and apocynin abrogated activation of the brain HO during seizures. Overall, these data suggest that long-term adverse cerebrovascular effects of seizures are attributed to oxidative stress. On the other hand, seizure-induced ROS are required for activation of the endogenous antioxidant HO/CO/bilirubin system that alleviates oxidative stress-induced loss of postictal cerebrovascular function in piglets.

Hydrogen sulfide and cerebral microvascular tone in newborn pigs.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that appears to be involved in numerous biological processes, including regulation of blood pressure and vascular tone. The present study is designed to address the hypothesis that H2S is a functionally significant, endogenous dilator in the newborn cerebrovascular circulation. In vivo experiments were conducted using newborn pigs with surgically implanted, closed, cranial windows. Topical application of H2S concentration-dependently (10(-6) to 2×10(-4) M) dilated pial arterioles. This dilation was blocked by glibenclamide (10(-6) M). L-cysteine, the substrate of the H2S-producing enzymes cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS), also dilated pial arterioles. The dilation to L-cysteine was blocked by the CSE inhibitor d,l-propargylglycine (PPG, 10 mM) but was unaffected by the CBS inhibitor amino-oxyacetate (AOA, 1 mM). Western blots detected CSE, but not CBS, in cerebral microvessels, whereas CBS is detected in brain parenchyma. Immunohistological CSE expression is predominantly vascular while CBS is expressed mainly in neurons and astrocytes. L-cysteine (5 mM) increased H2S concentration in cerebrospinal fluid (CSF), measured by GC-MS, from 561±205 to 2,783±818 nM before but not during treatment with PPG (1,030±70 to 622±78 nM). Dilation to hypercapnia was inhibited by PPG but not AOA. Hypercapnia increased CSF H2S concentration from 763±243 to 4,337±1789 nM before but not during PPG treatment (357±178 vs. 425±217 nM). These data show that H2S is a dilator of the newborn cerebral circulation and that endogenous CSE can produce sufficient H2S to decrease vascular tone. H2S appears to be a physiologically significant dilator in the cerebral circulation.

Time-dependent action of carbon monoxide on the newborn cerebrovascular circulation.

Carbon monoxide (CO) causes cerebral arteriolar dilation in newborn pigs by the activation of large-conductance Ca(2+)-activated K(+) channels. In adult rat cerebral and skeletal muscle arterioles, CO has been reported to produce constriction caused by the inhibition of nitric oxide (NO) synthase (NOS). We hypothesized that, in contrast to dilation to acute CO, more prolonged exposure of newborn cerebral arterioles to elevated CO produces constriction by reducing NO. In piglets with closed cranial windows, pial arteriolar responses to isoproterenol (10(-6) M), sodium nitroprusside (SNP; 10(-7) and 3 x 10(-7) M), and L-arginine ethyl ester (L-Arg; 10(-5) and 10(-4) M) were determined before and after 2 h of treatment with CO. CO (10(-7) M) caused transient dilation and had no further effects. CO (2 x 10(-7) and 10(-6) M) initially caused vasodilation, but over the 2-h exposure, pial arterioles constricted and removal of the CO caused dilation. Exposure to elevated CO (2 h) did not alter dilation to SNP or isoproterenol. Conversely, the NOS substrate L-Arg caused dilation before CO that was progressively lost over 90 min of elevated CO. If NO was held constant, CO caused dilation that was sustained for 2 h. We conclude that in neonates, cerebral arteriole responses to CO are biphasic: dilation to acute elevation with subsequent constriction from NOS inhibition after more prolonged exposure. As a result, short episodic production of CO allows function as a dilator gasotransmitter, whereas prolonged elevation can reduce NO to elevate cerebrovascular tone. The interaction between heme oxygenase/CO and NOS/NO could form a negative feedback system in the control of cerebral vascular tone.

Age and species dependence of pial arteriolar responses to topical carbon monoxide in vivo.

In newborn pigs, carbon monoxide (CO) contributes to regulation of cerebrovascular circulation. Results from isolated adult cerebral arteries suggest CO may have less dilatory potential in mature animals. However, few data are available on the direct effects of CO on cerebrovascular circulation in vivo except for those from newborn pigs. Therefore, we tested the hypothesis that i) rat cerebral arterioles dilate to CO in vivo and ii) CO-induced cerebrovascular dilatory responses are age dependent in pigs. Also, we examined whether the permissive role of nitric oxide in CO-induced dilation observed in piglets is present in older pigs and rats. Experiments used anesthetized newborn, 7-week-old, and juvenile (3- to 4-month-old) pigs and 3- to 4-month-old rats with closed cranial windows and topical applications of CO and sodium nitroprusside (SNP). Dilations to SNP were not different at different ages in pigs or between pigs and rats. CO produced pial arteriolar dilations in all groups. Dilation to 10(-5) M CO was reduced in juvenile pigs as compared to newborn and 7-week-old pigs, and tended to less at 10(-6) M CO. Dilations of rat pial arterioles to all concentrations were less than those of newborn and 7-week-old pigs, but not different from those of juvenile pig pial arterioles. In newborn and 7-week-old pigs, l-nitro-arginine (LNA) inhibited the dilation to CO, an effect reversed by a constant background of SNP. In contrast, LNA did not reduce dilation to CO in juvenile pigs or rats. In conclusion, rat pial arterioles like those in piglets dilate to CO in vivo, but there are age and species differences with regard to reactivity and interaction with NO.

Cerebroprotective effects of the CO-releasing molecule CORM-A1 against seizure-induced neonatal vascular injury.

Endogenous CO, a product of heme oxygenase activity, has vasodilator and cytoprotective effects in the cerebral circulation of newborn pigs. CO-releasing molecule (CORM)-A1 (sodium boranocarbonate) is a novel, water-soluble, CO-releasing compound. We addressed the hypotheses that CORM-A1 1) can deliver CO to the brain and exert effects of CO on the cerebral microvasculature and 2) is cerebroprotective. Acute and delayed effects of topically and systemically administered CORM-A1 on cerebrovascular and systemic circulatory parameters were determined in anesthetized newborn pigs with implanted closed cranial windows. Topical application of CORM-A1 (10(-7)-10(-5) M) to the brain produced concentration-dependent CO release and pial arteriolar dilation. Systemically administered CORM-A1 (2 mg/kg ip or iv) caused pial arteriolar dilation and increased cortical cerebrospinal fluid CO concentration. Systemic CORM-A1 did not have acute or delayed effects on blood pressure, heart rate, or blood gases. Potential cerebroprotective vascular effects of CORM-A1 (2 mg/kg ip, 30 min before seizures) were tested 2 days after bicuculline-induced epileptic seizures (late postictal period). In control piglets, seizures reduced postictal cerebrovascular responsiveness to selective physiologically relevant vasodilators (bradykinin, hemin, and isoproterenol) indicative of cerebrovascular injury. In contrast, in CORM-A1-pretreated animals, no loss of postictal cerebrovascular reactivity was observed. We conclude that systemically administered CORM-A1 delivers CO to the brain, elicits the vasodilator and cytoprotective effects of CO in the cerebral circulation, and protects the neonatal brain from cerebrovascular injury caused by epileptic seizures.

Contributions of astrocytes and CO to pial arteriolar dilation to glutamate in newborn pigs.

Astrocytes can act as intermediaries between neurons and cerebral arterioles to regulate vascular tone in response to neuronal activity. Release of glutamate from presynaptic neurons increases blood flow to match metabolic demands. CO is a gasotransmitter that can be related to neural function and blood flow regulation in the brain. The present study addresses the hypothesis that glutamatergic stimulation promotes perivascular astrocyte CO production and pial arteriolar dilation in the newborn brain. Experiments used anesthetized newborn pigs with closed cranial windows, piglet astrocytes, and cerebrovascular endothelial cells in primary culture and immunocytochemical visualization of astrocytic markers. Pial arterioles and arteries of newborn pigs are ensheathed by astrocytes visualized by glial fibrillary acidic protein staining. Treatment (2 h) of astrocytes in culture with L-2-alpha-aminoadipic acid (L-AAA), followed by 14 h in toxin free medium, dose-dependently increased cell detachment, suggesting injury. Conversely, 16 h of continuous exposure to L-AAA caused no decrease in endothelial cell attachment. In vivo, topical L-AAA (2 mM, 5 h) disrupted the cortical glia limitans histologically. Such treatment also eliminated pial arteriolar dilation to the astrocyte-dependent dilator ADP and to glutamate but not to isoproterenol or CO. Glutamate stimulated CO production by the brain surface that also was abolished following L-AAA. In contrast, tetrodotoxin blocked dilation to N-methyl-D-aspartate but not to glutamate, isoproterenol, or CO or the glutamate-induced increase in CO. The concurrent loss of CO production and pial arteriolar dilation to glutamate following astrocyte injury suggests astrocytes may employ CO as a gasotransmitter for glutamatergic cerebrovascular dilation.

Cyclooxygenase products stimulate carbon monoxide production by piglet cerebral microvessels.

Products of arachidonic acid (AA) metabolism by cyclooxygenase (Cox) are important in regulation of neonatal cerebral circulation. The brain and cerebral microvessels also express heme oxygenase (HO) that metabolizes heme to carbon monoxide (CO), biliverdin, and iron. The purpose of this study in newborn pig cerebral microvessels was to address the hypothesis that Cox products affect HO activity and HO products affect Cox activity. AA (2.0-20 microM) increased prostaglandin E2 (PGE2) measured by radioimmunoassay (RIA) and also CO measured by gas chromatography/mass spectrometry (GC/MS). Further, 10(-4) M indomethacin, which inhibited Cox, reduced both AA and heme-induced CO production. Conversely, neither exogenous 2 x 10(-6) M heme, which markedly increased CO production, nor the inhibitor of HO, chromium mesoporphyrin, altered PGE2 synthesis. Because AA metabolism by Cox generates both prostanoids and superoxides, we determined the effects of the predominant prostanoid and superoxide on CO production. Although PGE2 caused a small increase in CO production, xanthine oxidase plus hypoxanthine, which produces superoxide, strongly stimulated the production of CO by cerebral microvessels. This increase was mildly attenuated by catalase. These data suggest that Cox-catalyzed AA metabolites, most likely superoxide and/or a subsequent reactive oxygen species, increase cerebrovascular CO production. This increase seems to be caused, at least in part, by the elevation of HO-2 catalytic activity. Conversely, Cox activity is not affected by HO-catalyzed heme metabolites. These data suggest that some cerebrovascular functions attributable to Cox activity could be mediated by CO.

Nitric oxide increases carbon monoxide production by piglet cerebral microvessels.

Carbon monoxide (CO) and nitric oxide (NO) can be involved in the regulation of cerebral circulation. Inhibition of production of either one of these gaseous intercellular messengers inhibits newborn pig cerebral arteriolar dilation to the excitatory amino acid glutamate. Glutamate can increase NO production. Therefore, the present study tests the hypothesis that NO, which is increased by glutamate, stimulates the production of CO by cerebral microvessels. Experiments used freshly isolated cerebral microvessels from piglets that express only heme oxygenase-2 (HO-2). CO production was measured by gas chromatography-mass spectrometry. Although inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-l-arginine (l-NNA) did not alter basal HO-2 catalytic activity or CO production, l-NNA blocked glutamate stimulation of HO-2 activity and CO production. Furthermore, the NO donor sodium nitroprusside mimicked the actions of glutamate on HO-2 and CO production. The action of NO appears to be via cGMP because 8-bromo-cGMP mimics and 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) blocks glutamate stimulation of CO production and HO-2 catalytic activity. Inhibitors of neither casein kinase nor phosphotidylinositol 3-kinase altered HO-2 catalytic activity. Conversely, inhibition of calmodulin with calmidazolium chloride blocked glutamate stimulation of CO production and reduced HO-2 catalytic activity. These data suggest that glutamate may activate NOS producing NO that leads to CO synthesis via a cGMP-dependent elevation of HO-2 catalytic activity. These results are consistent with the findings in vivo that either HO or NOS inhibition blocks cerebrovascular dilation to glutamate in piglets.

Permissive contributions of NO and prostacyclin in CO-induced cerebrovascular dilation in piglets.

Endogenously produced CO is an important dilator in newborn cerebrovascular circulation. CO dilates cerebral arterioles by activating Ca2+-activated K+ channels, but modulatory actions of other effectors and second messenger inputs are unclear. Specifically, the mechanisms behind the obligatory permissive roles of prostacyclin and NO are uncertain. Therefore, the present study was performed using acutely implanted, closed cranial windows in newborn pigs to address the hypothesis that the permissive roles of NO and prostacyclin in cerebrovascular dilation in response to CO involve a common mechanism. The NO donor sodium nitroprusside restored dilation in response to CO after inhibition of that dilation with the prostaglandin cyclooxygenase inhibitor indomethacin. The stable prostacyclin analog iloprost restored CO-induced dilation blocked by the NO synthase inhibitor Nomega-nitro-L-arginine. Restoration of dilation in response to CO by the cGMP-dependent phosphodiesterase inhibitor zaprinast and blockade of CO dilation by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) suggests involvement of the cGMP/PKG pathway. Iloprost or the cAMP-dependent dilator isoproterenol restored dilation in response to CO after ODQ administration. However, CO-induced dilation blocked by the cGMP-dependent PKG inhibitor Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine could not be reversed by administration of sodium nitroprusside, iloprost, or isoproterenol. Conversely, PKA inhibition did not block dilation in response to CO. Overall, data indicate that activation of PKG is the predominant mechanism of the permissive actions of NO and prostacyclin for CO-induced pial arteriolar dilation.

Mechanism of glutamate stimulation of CO production in cerebral microvessels.

Dilation of piglet pial arterioles to glutamate involves carbon monoxide (CO) produced from heme by heme oxygenase-2 (HO-2). Piglet cerebral microvessels and endothelial and smooth muscle cells grown on microcarrier beads were used to address the hypothesis that glutamate increases endothelial CO production by increasing HO-2 catalytic activity. CO was measured by gas chromatography/mass spectrometry. Glutamate increased CO production from endogenous heme by cerebral microvessels, endothelial cells, and smooth muscle cells. Glutamate increased the conversion of exogenous heme to CO. Protein tyrosine kinase inhibition blocked glutamate stimulation of CO production. Inhibition of protein tyrosine phosphatases stimulated CO production. Conversely, neither phorbol myristate acetate nor H-7 changed glutamate stimulation of CO production. The mechanism of HO-2 stimulation by glutamate appears to be independent of cytosolic Ca, because stimulation of CO production by glutamate was the same in Careplete medium, Ca-free medium with ionomycin, and Careplete medium with ionomycin. Therefore, glutamate appears to increase HO-2 catalytic activity in cerebral microvessels via a tyrosine kinase mediated pathway.

Regulation of CO production in cerebral microvessels of newborn pigs.

Carbon monoxide (CO) is produced from heme by heme oxygenase-2 (HO-2) in cerebral blood vessels. Gas chromatography-mass spectrometry was used on piglet cerebral microvessels to address the hypothesis that CO production is regulated by heme delivery and HO-2 catalytic activity. CO production appears to be substrate limited because heme and its precursor aminolevulinate increase CO production. Ionomycin also increases CO production. However, CO production from exogenous heme was the same in Ca-replete medium, Ca-free medium with ionomycin, and Ca-replete medium with ionomycin. Phorbol myristate acetate increases CO production but does not change the catalytic activity of HO-2. Also, the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine has no effect on the HO-2 catalytic activity. Protein tyrosine kinase inhibition reduces HO-2 catalytic activity. Inhibition of protein tyrosine phosphatases increased HO-2 catalytic activity. Therefore, regulation of CO production by cerebral microvessels can include changing heme availability and HO-2 catalytic activity. HO-2 catalytic activity is stimulated by tyrosine phosphorylation.

Role of carbon monoxide in glutamate receptor-induced dilation of newborn pig pial arterioles.

The hypothesis that glutamate dilates pial arterioles of newborn pigs through the production of carbon monoxide (CO) was addressed. Anesthesized newborn pigs were equipped with cranial windows to measure pial arteriolar responses to stimuli. Heme oxygenase (HO) inhibitors added topically inhibited dilation to glutamate and to specific glutamate receptor agonists. The initial dilation to glutamate (10(-5) M) was 22% from baseline without an inhibitor and decreased to 9% with the HO inhibitor chromium mesoporphyrin (CrMP). Inhibition of dilation upon HO inhibition was similar when specific glutamate receptor agonists were employed. RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid caused 24% dilation from the baseline without an inhibitor, and the dilation was decreased to 1% with tin protoporphyrin (SnPP). (RS)-2-amino-3-(3-hydroxy-5-t-butylisoxazol-4-yl)propionic acid (kainate receptors) caused dilation of 18% from baseline without an inhibitor, but only 2% when tin mesoporphyrin was applied. 1-Aminocyclopropanecarboxylic acid (N-methyl-D-aspartate receptors) dilated pial arterioles 33% from baseline in control, but only to 2% in the presence of SnPP. Neither copper mesoporphyrin, which does not inhibit HO, nor light-inactivated CrMP affected the dilations. Furthermore, cerebral microvessels removed from the brain produced CO (stable isotope dilution gas chromatography-mass spectrometry), and this production was dose dependently increased by glutamate and inhibited by metal porphyrin HO inhibitors. These data suggest that dilation of newborn pig pial arterioles to glutamate and specific glutamate receptor agonists involves vascular production of CO. Additional cerebral sources of CO also could be stimulated by glutamate and contribute to the dilation.