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.

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-␣-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 astrocytedependent 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. glia limitans; cerebrovascular circulation; gasotransmitter; glia toxin IN THE CEREBROVASCULAR circulation, signals to vascular smooth muscle can come from endothelium, nerves, astrocytes, or pericytes, which interact to form a neurovascular unit (15). L-Glutamic acid (glutamate) is the principal excitatory neurotransmitter in the central nervous system (38). It is a dilator of cerebral arterioles in vivo (8, 12), although direct effects of glutamate on the vascular smooth muscle are uncertain. Release of glutamate from presynaptic neurons dilates cerebral arterioles, causing blood flow to increase to match metabolic demands (37).Astrocytes are the most abundant cell type in the higher mammalian brain. They function as intermediaries between neurons and cerebral arterioles in regulation of cerebral vascular tone in response to neuronal activity, thereby adjusting cerebral blood flow to metabolism (15,25). Whether astrocytes are involved in dilation in response to glutamate released at excitatory synapses and the nature of astrocyte-derived vasodilator(s) employed remain uncertain.The gas CO is produced physiologically by catabolism of heme to CO, iron, and biliverdin (36). This reaction is catalyzed by heme oxygenase (HO) with oxidation of...

drogen 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 Ϫ6 to 2 ϫ 10 Ϫ4 M) dilated pial arterioles. This dilation was blocked by glibenclamide (10 Ϫ6 M). L-Cysteine, the substrate of the H2S-producing enzymes cystathionine ␥-lyase (CSE) and cystathionine ␤-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 Ϯ 205 to 2,783 Ϯ 818 nM before but not during treatment with PPG (1,030 Ϯ 70 to 622 Ϯ 78 nM). Dilation to hypercapnia was inhibited by PPG but not AOA. Hypercapnia increased CSF H2S concentration from 763 Ϯ 243 to 4,337 Ϯ 1789 nM before but not during PPG treatment (357 Ϯ 178 vs. 425 Ϯ 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.

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.

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.

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.

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral microcirculation. Other dilators include prostanoids and nitric oxide (NO), and interactions among the systems are likely. Experiments on anesthetized piglets with cranial windows address the hypothesis that CO-induced dilation of pial arterioles involves interaction with the prostanoid and NO systems. Topical application of CO or the heme oxygenase substrate heme-L-lysinate (HLL) produced dilation. Indomethacin, N(omega)-nitro-L-arginine (L-NNA), and either iberiotoxin or tetraethylammonium chloride (TEA) were used to inhibit prostanoids, NO, and Ca(2+)-activated K(+) (K(Ca)) channels, respectively. Indomethacin, L-NNA, iberiotoxin, or TEA blocked cerebral vasodilation to CO and HLL. Vasodilations to both CO and HLL were returned to indomethacin-treated piglets by topical application of iloprost. Vasodilations to both CO and HLL were returned to L-NNA-treated piglets by sodium nitroprusside but not iloprost. In iberiotoxin- or TEA-treated piglets, dilations to CO and HLL could not be restored by either iloprost or sodium nitroprusside. The dilator actions of CO involve prostacyclin and NO as permissive enablers. The permissive actions of prostacyclin and NO may alter the K(Ca) channel response to CO because neither iloprost nor sodium nitroprusside could restore dilation to CO when these channels were blocked.

The Ca 21 /voltage-gated K 1 large conductance (BK) channel b1 subunit is particularly abundant in vascular smooth muscle. By determining their phenotype, BK b1 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 b1 site that includes Thr169. Whether exogenous nonsteroidal agents can access this site to selectively activate b1-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 1 b1) channels cloned from rat cerebral artery myocytes with a potency (EC 50 5 53 mM) similar to and an efficacy (Â2.5 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 1 b2, b3, b4, or b1T169A, indicating that this drug selectively targets b1-containing BK channels via the BK b1 steroid-sensing site. HENA (3-45 mM) 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 b1's role in HENA action. Finally, carotid artery-infusion of HENA (45 mM) 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 b1-containing BK channels by targeting the steroid-sensing site in BK b1, rendering vasodilation.