Carbon monoxide mediates vasodilator effects of glutamate in isolated pressurized cerebral arterioles of newborn pigs

The excitatory neurotransmitter glutamate causes dilation of newborn pig cerebral arterioles in vivo that is blocked by inhibition of carbon monoxide (CO) production. CO, a potent dilator in cerebral circulation in vivo, is produced endogenously in cerebral microvessels via heme oxygenase (HO). In isolated pressurized cerebral arterioles (approximately 200 microm) from newborn pigs, we investigated the involvement of CO and the endothelium in response to glutamate. A CO-releasing molecule, dimanganese decacarbonyl (10(-8)-10(-6) M), dilated cerebral arterioles. Glutamate (10(-6)-10(-4) M) and 1-aminocyclopentane-cis-1,3-dicarboxylic acid (cis-ACPD; 10(-6)-10(-5) M), a N-methyl-D-aspartate (NMDA) receptor agonist, caused cerebral vascular dilation. Dilation of cerebral arterioles to glutamate and cis-ACPD was abolished by chromium mesoporphyrin (CrMP; 10(-6) M), a HO inhibitor. In contrast, CrMP did not alter dilation to isoproterenol, a -adrenergic receptor agonist. Endothelium-denuded cerebral arterioles did not dilate to glutamate or bradykinin (endothelium-dependent dilator), whereas responses to isoproterenol were preserved. These data indicate that cerebral arterioles from newborn pigs may directly respond to glutamate and the NMDA receptor agonists by endothelium-dependent dilation that involves stimulation of CO production via the HO pathway in the endothelium.     

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.     

Contributions of prostacyclin and nitric oxide to carbon monoxide-induced cerebrovascular dilation in piglets

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.     

Carbon monoxide as an attenuator of vasoconstriction in piglet cerebral arterioles

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral circulation. The present study addressed the hypothesis that endogenous CO attenuates pial arteriolar vasoconstriction caused by hypocapnia, platelet activating factor, and elevated blood pressure. Experiments used anesthetized piglets with implanted, closed cranial windows. Topical application of a metal porphyrin inhibitor of heme oxygenase was used to inhibit production of CO. Chromium mesopophyrin increased vasoconstriction in response to hypocapnia. The constrictor response to a topical stimulus, platelet activating factor, was also increased by application of chromium mesoporphyrin. Inhibition of heme oxygenase did not constrict pial arterioles in normotensive newborn pigs (mean arterial pressure of about 70 mmHg), but did constrict pial arterioles of piglets with experimentally induced increases in arterial pressure (mean arterial pressure greater than 90 mmHg). In fact, pial arterioles of normotensive piglets transiently dilated to chromium mesoporphyrin, whereas those of hypertensive piglets progressively constricted during 10 min of chromium mesoporphyrin treatment. Therefore, inhibition of heme oxygenase augments cerebral vasoconstriction in response to several very different constrictor stimuli. These data suggest endogenous CO attenuates vasoconstrictor responses in the newborn cerebral circulation.     

Effects of carbon monoxide and heme oxygenase inhibitors in cerebral vessels of rats and mice

Carbon monoxide (CO) has been postulated to be a signaling molecule in many tissues, including the vasculature. We examined vasomotor responses of adult rat and mouse cerebral arteries to both exogenously applied and endogenously produced CO. The diameter of isolated, pressurized, and perfused rat middle cerebral arteries (MCAs) was not altered by authentic CO (10(-6) to 10(-4) M). Mouse MCAs, however, dilated by 21 +/- 10% at 10(-4) M CO. Authentic nitric oxide (NO., 10(-10) to 10(-7) M) dilated both rat and mouse MCAs. At 10(-8) M NO., rat vessels dilated by 84 +/- 4%, and at 10(-7) M NO., mouse vessels dilated by 59 +/- 9%. Stimulation of endogenous CO production through heme oxygenase (HO) with the heme precursor delta-aminolevulinic acid (10(-10) to 10(-4) M) did not dilate the MCAs of either species. The metalloporphyrin HO inhibitor chromium mesoporphyrin IX (CrMP) caused profound constriction of the rat MCA (44 +/- 2% at 3 x 10(-5) M). Importantly, this constriction was unaltered by exogenous CO (10(-4) M) or CO plus 10(-5) M biliverdine (both HO products). In contrast, exogenous CO (10(-4) M) reversed CrMP-induced constriction in rat gracilis arterioles. Control mouse MCAs constricted by only 3 +/- 1% in response to 10(-5) M CrMP. Magnesium protoporphyrin IX (10(-5) M), a weak HO inhibitor used to control for nonspecific effects of metalloporphyrins, also constricted the rat MCA to a similar extent as CrMP. We conclude that, at physiological concentrations, CO is not a dilator of adult rodent cerebral arteries and that metalloporphyrin HO inhibitors have nonspecific constrictor effects in rat cerebral arteries.     

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.     

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.     

Carbon monoxide as an endogenous vascular modulator

Carbon monoxide (CO) is produced by heme oxygenase (HO)-catalyzed heme degradation to CO, iron, and biliverdin. HO has two active isoforms, HO-1 (inducible) and HO-2 (constitutive). HO-2, but not HO-1, is highly expressed in endothelial and smooth muscle cells and in adjacent astrocytes in the brain. HO-1 is expressed basally only in the spleen and liver but can be induced to a varying extent in most tissues. Elevating heme, protein phosphorylation, Ca(2+) influx, and Ca(2+)/calmodulin-dependent processes increase HO-2 activity. CO dilates cerebral arterioles and may constrict or dilate skeletal muscle and renal arterioles. Selected vasodilatory stimuli, including seizures, glutamatergic stimulation, hypoxia, hypotension, and ADP, increase CO, and the inhibition of HO attenuates the dilation to these stimuli. Astrocytic HO-2-derived CO causes glutamatergic dilation of pial arterioles. CO dilates by activating smooth muscle cell large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels. CO binds to BK(Ca) channel-bound heme, leading to an increase in Ca(2+) sparks-to-BK(Ca) channel coupling. Also, CO may bind directly to the BK(Ca) channel at several locations. Endothelial nitric oxide and prostacyclin interact with HO/CO in circulatory regulation. In cerebral arterioles in vivo, in contrast to dilation to acute CO, a prolonged exposure of cerebral arterioles to elevated CO produces progressive constriction by inhibiting nitric oxide synthase. The HO/CO system is highly protective to the vasculature. CO suppresses apoptosis and inhibits components of endogenous oxidant-generating pathways. Bilirubin is a potent reactive oxygen species scavenger. Still many questions remain about the physiology and biochemistry of HO/CO in the circulatory system and about the function and dysfunction of this gaseous mediator system.     

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.     

Interaction between endogenously produced carbon monoxide and nitric oxide in regulation of renal afferent arterioles

Heme oxygenases (HO-1 and HO-2) catalyze the conversion of heme to carbon monoxide (CO), iron, and biliverdin. CO causes vasorelaxation via stimulation of soluble guanylate cyclase (sGC) and/or activation of calcium-activated potassium channels. Because nitric oxide (NO) exerts effects via the same pathways, we tested the interaction between CO and NO on rat afferent arterioles (AAs) using the blood-perfused juxtamedullary nephron preparation. AAs were superfused with either tricarbonyldichlororuthenium (II) dimer, known as CO releasing molecule (CORM-2), 10 micromol/l CO solution, or 15 micromol/l chromium mesoporphyrin (CrMP, HO inhibitor). AAs were also superfused with 1 mmol/l N(omega)-nitro-L-arginine (L-NNA) to inhibit NO synthase (NOS) or 10 micromol/l 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one to inhibit sGC, and then CrMP was superfused during NOS inhibition or sGC inhibition. Treatment with 150 and 300 micromol/l CORM-2 or with CO (10 micromol/l) significantly dilated AAs (22.0 +/- 0.9 and 22.8 +/- 0.9 vs. 18.3 +/- 0.9 microm, n = 5, P < 0.05; and 26.0 +/- 1.4 vs. 18.8 +/- 0.7 microm, n = 5, P < 0.05). In untreated vessels, HO inhibition did not alter AA diameter (17.5 +/- 0.7 vs. 17.2 +/- 0.6 microm, n = 7, P > 0.05); however, during inhibition of NO production, which constricted arterioles to 14.6 +/- 1.2 microm, n = 6, P < 0.05, concurrent HO inhibition led to further vasoconstriction (11.7 +/- 1.6 microm, n = 6, P < 0.05). CORM-2 attenuated the L-NNA-induced vasoconstriction. Inhibition of sGC caused significant constriction (15.7 +/- 0.4 vs. 18.8 +/- 0.4 microm, n = 6, P < 0.05). HO inhibition during sGC inhibition did not cause further change in AAs (15.5 +/- 0.7 microm, n = 6). We conclude that endogenously produced CO does not exert a perceptible influence on AA diameter in the presence of intact NO system; however, when NO production is inhibited, CO serves as an important renoprotective reserve mechanism to prevent excess afferent arteriolar constriction.     

Carbon monoxide and Ca2+-activated K+ channels in cerebral arteriolar responses to glutamate and hypoxia in newborn pigs

Large-conductance calcium-activated potassium (K(Ca)) channels regulate the physiological functions of many tissues, including cerebrovascular smooth muscle. l-Glutamic acid (glutamate) is the principal excitatory neurotransmitter in the central nervous system, and oxygen tension is a dominant local regulator of vascular tone. In vivo, glutamate and hypoxia dilate newborn pig cerebral arterioles, and both dilations are blocked by inhibition of carbon monoxide (CO) production. CO dilates cerebral arterioles by activating K(Ca) channels. Therefore, the present study was designed to investigate the effects of glutamate and hypoxia on cerebral CO production and the role of K(Ca) channels in the cerebral arteriolar dilations to glutamate and hypoxia. In the presence of iberiotoxin or paxilline that block dilation to the K(Ca) channel opener, NS-1619, neither CO nor glutamate dilated pial arterioles. Conversely, neither paxilline nor iberiotoxin inhibited dilation to acute severe or moderate prolonged hypoxia. Both glutamate and hypoxia increased cerebrospinal fluid (CSF) CO concentration. Iberiotoxin that blocked dilation to glutamate did not attenuate the increase in CSF CO. The guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), which blocked dilation to sodium nitroprusside, did not inhibit dilation to hypoxia. These data suggest that dilation of newborn pig pial arterioles to glutamate is mediated by activation of K(Ca) channels, consistent with the intermediary signal being CO. Surprisingly, although 1) heme oxygenase (HO) inhibition attenuates dilation to hypoxia, 2) hypoxia increases CSF CO concentration, and 3) K(Ca) channel antagonists block dilation to CO, neither K(Ca) channel blockers nor ODQ altered dilation to hypoxia, suggesting the contribution of the HO/CO system to hypoxia-induced dilation is not by stimulating vascular smooth muscle K(Ca) channels or guanylyl cyclase.     

Dependence of acetylcholine and ADP dilation of pial arterioles on heme oxygenase after transfusion of cell-free polymeric hemoglobin

Polymers of cell-free hemoglobin have been designed for clinical use as oxygen carriers, but limited information is available regarding their effects on vascular regulation. We tested the hypothesis that the contribution of heme oxygenase (HO) to acetylcholine-evoked dilation of pial arterioles is upregulated 2 days after polymeric hemoglobin transfusion. Dilator responses to acetylcholine measured by intravital microscopy in anesthetized cats were blocked by superfusion of the HO inhibitor tin protoporphyrin-IX (SnPPIX) in a group that had undergone exchange transfusion with hemoglobin 2 days earlier but not in surgical sham and albumin-transfused groups. However, immunoblots from cortical brain homogenates did not reveal changes in expression of the inducible isoform HO1 or the constitutive isoform HO2 in the hemoglobin-transfused group. To test whether the inhibitory effect of SnPPIX was present acutely after hemoglobin transfusion, responses were measured within an hour of completion of the exchange transfusion. In control and albumin-transfused groups, acetylcholine responses were unaffected by SnPPIX but were blocked by addition of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine (l-NNA) to the superfusate. In hemoglobin-transfused groups, the acetylcholine response was blocked by either SnPPIX or l-NNA alone. The effect of another HO inhibitor, chromium mesoporphyrin (CrMP), was tested on ADP, another endothelial-dependent dilator, in anesthetized rats. Pial arteriolar dilation to ADP was unaffected by CrMP in controls but was attenuated 62% by CrMP in rats transfused with hemoglobin. It is concluded that 1) polymeric hemoglobin transfusion acutely upregulates the contribution of HO to acetylcholine-induced dilation of pial arterioles in cats, 2) this upregulation persists 2 days after transfusion when 95% of the hemoglobin is cleared from the circulation, and 3) this acute upregulation of HO signaling is ubiquitous in that similar effects were observed with a different endothelial-dependent agonist (i.e., ADP) in a another species (rat).     

Heme oxygenase-derived carbon monoxide promotes arteriolar endothelial dysfunction and contributes to salt-induced hypertension in Dahl salt-sensitive rats

Vascular tissues express heme oxygenase (HO), which metabolizes heme to form carbon monoxide (CO). Heme-derived CO inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. After 4 wk of high-salt diet, Dahl salt-sensitive (Dahl-S) rats display hypertension, increased vascular HO-1 expression, and attenuated vasodilator responses to ACh that can be completely restored by acute treatment with an inhibitor of HO. In this study, we examined the temporal development of HO-mediated endothelial dysfunction in isolated pressurized first-order gracilis muscle arterioles, identified the HO product responsible, and studied the blood pressure effects of HO inhibition in Dahl-S rats on a high-salt diet. Male Dahl-S rats (5-6 wk) were placed on high-salt (8% NaCl) or low-salt (0.3% NaCl) diets for 0-4 wk. Blood pressure increased gradually, and responses to an endothelium-dependent vasodilator, ACh, decreased gradually with the length of high-salt diet. Flow-induced dilation was abolished in hypertensive Dahl-S rats. Acute in vitro pretreatment with an inhibitor of HO, chromium mesoporphyrin (CrMP), restored endothelium-dependent vasodilation and abolished the differences between groups. The HO product CO prevented the restoration of endothelium-dependent dilation by CrMP. Furthermore, administration of an HO inhibitor lowered blood pressure in Dahl-S rats with salt-induced hypertension but did not do so in low-salt control rats. These results suggest that hypertension and HO-mediated endothelial dysfunction develop gradually and simultaneously in Dahl-S rats on high-salt diets. They also suggest that HO-derived CO underlies the impaired endothelial dysfunction and contributes to hypertension in Dahl-S rats on high-salt diets.     

Role of cGMP in carbon monoxide-induced cerebral vasodilation in piglets

The hypothesis was addressed that CO-induced cerebral vasodilation requires a permissive cGMP signal that can be produced by nitric oxide (NO). Anesthetized piglets were implanted with cranial windows for measurement of pial arteriolar responses to stimuli. Pial arterioles dilated in response to isoproterenol (Iso), sodium nitroprusside (SNP), and CO or the CO-releasing molecule Mn2(CO)10 [dimanganese decacarbonyl (DMDC)]. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, decreased cerebrospinal fluid (CSF) cGMP and selectively inhibited dilations to SNP and DMDC without affecting the dilation to Iso. However, DMDC did not cause an increase in cortical periarachnoid CSF cGMP concentration. cGMP clamp with a threshold dilator level of 8-bromo-cGMP (10(-4) M) and ODQ restored the dilation to DMDC that had been blocked by ODQ alone. Under these conditions, cGMP was present but could not increase. Inhibition of the pial arteriolar dilation to glutamate by N-nitro-l-arginine, which blocks NO synthase, was similar to that by heme oxygenase inhibitors, which block endogenous CO production. The dilation to glutamate, similar to dilation to DMDC, was partially restored by 8-bromo-cGMP and completely restored by SNP (5 x 10(-7) M). These data suggest that the permissive role of NO in CO- and glutamate-induced vasodilation involves maintaining the minimum necessary cellular level of cGMP to allow CO to cause dilation independently of increasing cGMP.     

The permissive role of endothelial NO in CO-induced cerebrovascular dilation

Carbon monoxide (CO) and nitric oxide (NO) are important paracrine messengers in the newborn cerebrovasculature that may act as comessengers. Here, we investigated the role of NO in CO-mediated dilations in the newborn cerebrovasculature. Arteriolar branches of the middle cerebral artery (100-200 microm) were isolated from 3- to 7-day-old piglets and cannulated at each end in a superfusion chamber, and intravascular pressure was elevated to 30 mmHg, which resulted in the development of myogenic tone. Endothelium removal abolished dilations of pressurized pial arterioles to bradykinin and to the CO-releasing molecule Mn(2)(CO)(10) [dimanganese decacarbonyl (DMDC)] but not dilations to isoproterenol. With endothelium intact, N(omega)-nitro-l-arginine (l-NNA), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), or tetraethylammonium chloride (TEA(+)), inhibitors of NO synthase (NOS), guanylyl cyclase, and large-conductance Ca(2+)-activated K(+) (K(Ca)) channels, respectively, also blocked dilation induced by DMDC. After inhibition of NOS, a constant concentration of sodium nitroprusside (SNP), a NO donor that only dilated the vessel 6%, returned dilation to DMDC. The stable cGMP analog 8-bromo-cGMP also restored dilation to DMDC in endothelium-intact, l-NNA-treated, or endothelium-denuded arterioles, and this effect was blocked by TEA(+). Similarly, in the continued presence of ODQ, 8-bromo-cGMP restored DMDC-induced dilations. These findings suggest that endothelium-derived NO stimulates guanylyl cyclase in vascular smooth muscle cells and, thereby, permits CO to cause dilation by activating K(Ca) channels. Such a requirement for NO could explain the endothelium dependency of CO-induced dilation in piglet pial arterioles.     

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.     

Carbon monoxide and hydrogen sulfide: gaseous messengers in cerebrovascular circulation.

This review focuses on two gaseous cellular messenger molecules, CO and H2S, that are involved in cerebrovascular flow regulation. CO is a dilatory mediator in active hyperemia, autoregulation, hypoxic dilation, and counteracting vasoconstriction. It is produced from heme by a constitutively expressed enzyme [heme oxygenase (HO)-2] expressed highly in the brain and by an inducible enzyme (HO-1). CO production is regulated by controlling substrate availability, HO-2 catalytic activity, and HO-1 expression. CO dilates arterioles by binding to heme that is bound to large-conductance Ca2+-activated K+ channels. This binding elevates channel Ca2+ sensitivity, that increases coupling of Ca2+ sparks to large-conductance Ca2+-activated K+ channel openings and, thereby, hyperpolarizes the vascular smooth muscle. In addition to dilating blood vessels, CO can either inhibit or accentuate vascular cell proliferation and apoptosis, depending on conditions. H2S may also function as a cerebrovascular dilator. It is produced in vascular smooth muscle cells by hydrolysis of l-cysteine catalyzed by cystathione gamma-lyase (CSE). H2S dilates arterioles at physiologically relevant concentrations via activation of ATP-sensitive K+ channels. In addition to dilating blood vessels, H2S promotes apoptosis of vascular smooth muscle cells and inhibits proliferation-associated vascular remodeling. Thus both CO and H2S modulate the function and the structure of circulatory system. Both the HO-CO and CSE-H2S systems have potential to interact with NO and prostanoids in the cerebral circulation. Much of the physiology and biochemistry of HO-CO and CSE-H2S in the cerebral circulation remains open for exploration.     

Chronic administration of indomethacin increases role of nitric oxide in hypercapnic cerebrovasodilation in piglets.

Hypercapnia-induced cerebral vasodilation is associated with prostanoids in the piglet, but is a primarily nitric oxide (NO) associated response in many adult models. Hypercapnia-induced cerebral vasodilation is both NO and prostanoid associated in the juvenile pig. We hypothesized that with chronic administration of indomethacin the piglet would advance the role of the NO system in cerebrovascular responses. The closed cranial window technique was used in piglets to determine pial arteriolar response. Chronically indomethacin treated newborn animals dilated in response to CO2 similarly to control newborns (40.9+/-4.4% vs 48.4+/-4.1%). Topical n-nitro L-arginine (L-NA, 10(-3) M), attenuated CO2 induced dilation in the chronically indomethacin treated animals (11.7+/-3.3% vs 40.9+/-4.4%; p < 0.001), but had no effect on the response to hypercapnia of piglets not treated with indomethacin. Neither indomethacin nor L-NA altered response to topical isoproterenol (10(-6) M). We conclude that with chronic indomethacin administration there develops a significant hypercapnia-induced cerebral vasodilation in which NO has an important role. The chronic inhibition of the newborn's principal dilator system appears to increase the role of NO in newborn cerebral hemodynamics.     

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.     

Metabolic syndrome increases endogenous carbon monoxide production to promote hypertension and endothelial dysfunction in obese Zucker rats

Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide (CO). Increased heme-derived CO inhibits nitric oxide synthase and can contribute to hypertension via endothelial dysfunction in Dahl salt-sensitive rats. Obese Zucker rats (ZR) are models of metabolic syndrome. This study tests the hypothesis that endogenous CO formation is increased and contributes to hypertension and endothelial dysfunction in obese ZR. Awake obese ZR showed increased respiratory CO excretion, which was lowered by HO inhibitor administration [zinc deuteroporphyrin 2,4-bis glycol (ZnDPBG) 25 micromol.kg(-1).24 h(-1) ip]. In awake obese ZR, chronically instrumented with femoral arterial catheters, blood pressure was elevated but was decreased by the HO inhibitor ZnDPBG. Body weight, blood glucose, glycated hemoglobin, plasma insulin, total and LDL cholesterol, oxidized LDL, and triglyceride levels were elevated in obese ZR, and, except for LDL cholesterol, were unchanged by HO inhibition. Total HO-1 protein levels were not different between lean and obese ZR aortas. In vitro experiments used isolated skeletal muscle arterioles with constant pressure and no flow, or constant midpoint, but altered endpoint pressures to establish graded levels of luminal flow. In obese ZR arterioles, responses to ACh and flow were attenuated. Acute in vitro pretreatment with an HO inhibitor, chromium mesoporphyrin, enhanced ACh and flow-induced dilation and abolished the differences between groups. Furthermore, exogenous CO prevented the restoration of flow-induced dilation by the HO inhibitor in obese ZR arterioles. These results suggest that HO-derived CO production is increased and promotes hypertension and arteriolar endothelial dysfunction in obese ZR with metabolic syndrome independent of affecting metabolic parameters.