Alexander L. Fedinec

Carbon monoxide and cerebral microvascular tone in newborn pigs.

The present study addresses the hypothesis that CO produced from endogenous heme oxygenase (HO) can dilate newborn cerebral arterioles. HO-2 protein was highly expressed in large and small blood vessels, as well as parenchyma, of newborn pig cerebrum. Topically applied CO dose-dependently dilated piglet pial arterioles in vivo over the range 10(-11)-10(-9) M (maximal response). CO-induced cerebrovascular dilation was abolished by treatment with the Ca2+-activated K+ channel inhibitors tetraethylammonium chloride and iberiotoxin. The HO substrate heme-L-lysinate also produced tetraethylammonium-inhibitable, dose-dependent dilation from 5 x 10(-8) to 5 x 10(-7) M (maximal). The HO inhibitor chromium mesoporphyrin blocked dilation of pial arterioles in response to heme-L-lysinate. In addition to inhibiting dilation to heme-L-lysinate, chromium mesoporphyrin also blocked pial arteriolar dilations in response to hypoxia but did not alter responses to hypercapnia or isoproterenol. We conclude that CO dilates pial arterioles via activation of Ca2+-activated K+ channels and that endogenous HO-2 potentially can produce sufficient CO to produce the dilation.The present study addresses the hypothesis that CO produced from endogenous heme oxygenase (HO) can dilate newborn cerebral arterioles. HO-2 protein was highly expressed in large and small blood vessels, as well as parenchyma, of newborn pig cerebrum. Topically applied CO dose-dependently dilated piglet pial arterioles in vivo over the range 10-11-10-9M (maximal response). CO-induced cerebrovascular dilation was abolished by treatment with the Ca2+-activated K+ channel inhibitors tetraethylammonium chloride and iberiotoxin. The HO substrate heme-l-lysinate also produced tetraethylammonium-inhibitable, dose-dependent dilation from 5 × 10-8 to 5 × 10-7 M (maximal). The HO inhibitor chromium mesoporphyrin blocked dilation of pial arterioles in response to heme-l-lysinate. In addition to inhibiting dilation to heme-l-lysinate, chromium mesoporphyrin also blocked pial arteriolar dilations in response to hypoxia but did not alter responses to hypercapnia or isoproterenol. We conclude that CO dilates pial arterioles via activation of Ca2+-activated K+ channels and that endogenous HO-2 potentially can produce sufficient CO to produce the dilation.

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 β-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.

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.

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.

Cerebrovascular Dilation via Selective Targeting of the Cholane Steroid-Recognition Site in the BK Channel β1-Subunit by a Novel Nonsteroidal Agent

The Ca2+/voltage-gated K+ large conductance (BK) channel β1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK β1 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 β1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate β1-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 + β1) 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 + β2, β3, β4, or β1T169A, indicating that this drug selectively targets β1-containing BK channels via the BK β1 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 β1’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 β1-containing BK channels by targeting the steroid-sensing site in BK β1, 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.

A BRIEF COMMUNICATION: 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.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.

Prostacyclin Receptor Activation and Pial Arteriolar Dilation After Endothelial Injury in Piglets

BACKGROUND AND PURPOSE Both light/dye endothelial injury and indomethacin treatment inhibit hypercapnia-induced cerebral prostacyclin synthesis and pial arteriolar dilation in newborn pigs. Topical iloprost can allow hypercapnia-induced dilation of pial arterioles to occur in piglets treated with indomethacin. We addressed the hypothesis that prostacyclin receptor activation with iloprost can return the ability of pial arterioles with endothelial injury to respond to hypercapnia. We also examined whether the endothelial dependence and the permissive role of prostacyclin extended to histamine-induced dilation or are specific for hypercapnia. METHODS Experiments used chloralose-anesthetized piglets equipped with closed cranial windows. Hypercapnia (PaCO2 approximately 80 mm Hg) and topically applied histamine (10(-6) and 10(-5) mol/L) dilated pial arterioles. Dilations in response to both stimuli were abolished by light/dye treatment. RESULTS Simultaneous topical treatment with iloprost (10(-12) mol/L, which caused no residual dilation, returned dilation of pial arterioles to both hypercapnia and histamine. On removal of iloprost, responses were again absent and returned with readdition of iloprost to the cortical cerebrospinal fluid. Neither isoproterenol nor sodium nitroprusside returned responses to hypercapnia after light/dye treatment. CONCLUSIONS These data add further support to the hypothesis that prostacyclin represents an important endothelial-derived signal in the newborn pig cerebral circulation that can permit appropriate responses by adjacent smooth muscle in response to specific stimuli.

Dietary Cholesterol Protects Against Alcohol-Induced Cerebral Artery Constriction

BACKGROUND Binge drinking represents the major form of excessive alcohol (ethanol [EtOH]) consumption in the United States. Episodic (such as binge) drinking results in blood alcohol levels (BAL) of 18 to 80 mM and leads to alcohol-induced cerebral artery constriction (AICAC). AICAC was shown to arise from EtOH-induced inhibition of large-conductance, calcium/voltage-gated potassium (BK) channels in the vascular smooth muscle. Factors that modulate BK channel-mediated AICAC remain largely unknown. METHODS Male Sprague Dawley rats were placed on high-cholesterol (2% of cholesterol) diet for 18 to 23 weeks. Their littermates were placed on control iso-caloric diet. AICAC was evaluated both in vivo and in vitro, by means of pial arteriole diameter monitoring through a closed cranial window and diameter measurements of isolated, pressurized cerebral arteries. Cholesterol level in the cerebral artery tissue was manipulated by methyl-β-cyclodextrin to reverse dietary-induced accumulation of cholesterol. BK channel surface presence on the plasma membrane of cerebral artery myocytes was evaluated by immunofluorescence staining. BK channel function in pressurized cerebral artery was assessed using selective BK channel blocker paxilline. RESULTS Within 5 minutes of 50 mM EtOH injection into carotid artery in vivo, arteriole diameter decreased by 20% in control group. Pial arteriole constriction was significantly reduced in rats on high-cholesterol diet, resulting in only 10% reduction in diameter. BAL in both groups, however, remained the same. Significant reduction in AICAC in group on high-cholesterol diet compared to control was also observed after middle cerebral artery dissection and in vitro pressurization at 60 mmHg, this reduction remaining after endothelium removal. Cholesterol level in de-endothelialized cerebral arteries was significantly increased in rats on high-cholesterol diet. Removal of excessive cholesterol content restored AICAC to the level observed in cerebral arteries of rats on normal diet. Immunofluorescence staining of BK channel-forming and accessory, smooth muscle-specific β1 subunit in freshly isolated cerebral artery myocyte showed that high-cholesterol diet did not down-regulate surface presence of BK protein. However, paxilline-induced cerebral artery constriction was diminished in arteries from rats on high-cholesterol diet. CONCLUSIONS Our data indicate that dietary cholesterol protects against AICAC. This protection is caused by cholesterol buildup in the arterial tissue and diminished function (but not surface presence) of EtOH target-BK channel.

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.