Cortical spreading depression confounds concentration-dependent pial arteriolar dilation during N-methyl-D-aspartate superfusion

Pial arterioles do not express N-methyl-D-aspartate (NMDA) receptors but dilate in response to topical NMDA application. We explored the mechanism underlying NMDA-mediated responses in murine pial arterioles (11-31 microm), using a closed cranial window preparation, and found that arteriolar dilation was not concentration dependent. Pial arteriolar diameter abruptly increased within 3 min of superfusing 50 or 100 microM NMDA. Dilation reached a peak within 1 min (46 +/- 14%) and then declined to a plateau (28 +/- 13%) for the duration of superfusion. Whereas a higher concentration (200 microM) did not produce further dilation, lower concentrations (1-10 microM) did not dilate the arterioles at all. MK-801 (10 microM) abrogated the dilation response, whereas Nomega-nitro-L-arginine (1 mM) attenuated the peak and abolished the sustained dilation during NMDA superfusion. We determined that NMDA-induced pial arteriolar responses were evoked by cortical spreading depression, because abrupt vasodilation during 50 or 100 microM NMDA superfusion was associated with a large negative slow potential shift and electrocorticogram suppression that spread from the superfusion window to distant cortical areas. Our data suggest that the responses of pial arterioles to NMDA are caused in part by neurovascular coupling due to cortical spreading depression.     

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.     

N-methyl-D-aspartate-induced vasodilation is mediated by endothelium-independent nitric oxide release in piglets

N-methyl-D-aspartate (NMDA) elicits pial arteriolar dilation that has been associated with neuronal nitric oxide (NO) production. However, endothelial factors or glial P-450 epoxygenase products may play a role. We tested whether NMDA-induced pial vasodilation 1) primarily involves NO diffusion from the parenchyma to the surface arterioles, 2) involves intact endothelial function, and 3) involves a miconazole-sensitive component. Arteriolar diameters were determined using closed cranial window-intravital microscopy in anesthetized piglets. NMDA (10-100 microM) elicited virtually identical dose-dependent dilations in paired arterioles (r = 0.94, n = 15). However, NMDA- but not bradykinin (BK)-induced dilations of arteriolar sections over large veins were reduced by 31 +/- 1% (means +/- SE, P < 0.05, n = 4) compared with adjacent sections on the cortical surface. Also, 100 microM NMDA increased cerebrospinal fluid levels of NO metabolites from 3.7 +/- 1.0 to 5.3 +/- 1.2 microM (P < 0.05, n = 6). Endothelial stunning by intracarotid injection of phorbol 12,13-dibutyrate did not affect NMDA-induced vasodilation but attenuated vascular responses to hypercapnia and BK by approximately 70% (n = 7). Finally, miconazole (n = 6, 20 microM) pretreatment and coapplication with NMDA did not alter vascular responses to NMDA. In conclusion, NMDA appears to dilate pial arterioles exclusively through release and diffusion of NO from neurons to the pial surface in piglets.     

Astrocytes are a key conduit for upstream signaling of vasodilation during cerebral cortical neuronal activation in vivo

Astrocytes play an important role in the coupling between neuronal activity and brain blood flow via their capacity to "sense" neuronal activity and transmit that information to parenchymal arterioles. Here we show another role for astrocytes in neurovascular coupling: the ability to act as a signaling conduit for the vitally important process of upstream vasodilation (represented by pial arterioles) during both excessive (seizure) and physiological (sciatic nerve stimulation) increases in cerebral cortical neuronal activity. The predominance of an astrocytic rather than a vascular route was indicated by data showing that pial arteriolar-dilating responses to neuronal activation were completely blocked following selective disruption of the superficial glia limitans, whereas interference with interendothelial signaling was without effect. Results also revealed contributions from connexin 43, implying a role for gap junctions and/or hemichannels in the signaling process and that signaling from the glia limitans to pial arterioles may involve a diffusible mediator.     

Purinergic mechanisms in gliovascular coupling

Regional elevations in cerebral blood flow (CBF) often occur in response to localized increases in cerebral neuronal activity. An ever expanding literature has linked this neurovascular coupling process to specific signaling pathways involving neuronal synapses, astrocytes and cerebral arteries and arterioles. Collectively, these structures are termed the "neurovascular unit" (NVU). Astrocytes are thought to be the cornerstone of the NVU. Thus, not only do astrocytes "detect" increased synaptic activity, they can transmit that information to proximal and remote astrocytic sites often through a Ca(2+)- and ATP-related signaling process. At the vascular end of the NVU, a Ca(2+)-dependent formation and release of vasodilators, or substances linked to vasodilation, can occur. The latter category includes ATP, which upon its appearance in the extracellular compartment, can be rapidly converted to the potent vasodilator, adenosine, via the action of ecto-nucleotidases. In the present review, we give consideration to experimental model-specific variations in purinergic influences on gliovascular signaling mechanisms, focusing on the cerebral cortex. In that discussion, we compare findings obtained using in vitro (rodent brain slice) models and multiple in vivo models (2-photon imaging; somatosensory stimulation-evoked cortical hyperemia; and sciatic nerve stimulation-evoked pial arteriolar dilation). Additional attention is given to the importance of upstream (remote) vasodilation; the key role played by extracellular ATP hydrolysis (via ecto-nucleotidases) in gliovascular coupling; and interactions among multiple signaling pathways.     

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.     

Effects of trigeminal neurotransmitters on piglet pial arterioles.

We investigated effects of calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) on pial arterioles in newborn pigs. Pial arteriolar diameter was determined using a closed cranial window and intravital microscopy. Initial diameters were approximately 100 microns. Calcitonin-gene related peptide dilated pial arterioles by 22 +/- 8% at 10(-9)M and by 34 +/- 6% at 10(-8)M (n = 8), and this response was not significantly altered by prior administration of indomethacin (5mg/kg, iv) (n = 6) or administration of NG-methyl-L-arginine (5mg/kg, iv, and 10(-3)M in CSF) (n = 10). Substance P dilated arterioles at 10(-10)M through 10(-5)M (maximal response = 23 +/- 3%) (n = 6), and this response was unaffected by indomethacin administration (n = 6). In contrast, NG-methyl-L-arginine blocked much of the pial arteriolar dilation to SP. Unlike the other two peptides, NKA did not change pial arteriolar diameter. Radioimmunoassay determinations indicated that cerebrospinal fluid levels of 6-keto-prostaglandin F1 and prostaglandin E2 did not change appreciably during application of CGRP or SP. We conclude that CGRP and SP but not NKA are dilator stimuli in the piglet pial circulation. Dilation by CGRP probably involves direct activation of receptors on vascular smooth muscle, while SP probably partially dilates pial arterioles via release of an endothelium-dependent relaxing factor.     

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.     

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.     

Exercise training normalizes impaired NOS-dependent responses of cerebral arterioles in type 1 diabetic rats

Our goal was to examine whether exercise training (ExT) could normalize impaired nitric oxide synthase (NOS)-dependent dilation of cerebral (pial) arterioles during type 1 diabetes (T1D). We measured the in vivo diameter of pial arterioles in sedentary and exercised nondiabetic and diabetic rats in response to an endothelial NOS (eNOS)-dependent (ADP), an neuronal NOS (nNOS)-dependent [N-methyl-D-aspartate (NMDA)], and a NOS-independent (nitroglycerin) agonist. In addition, we measured superoxide anion levels in brain tissue under basal conditions in sedentary and exercised nondiabetic and diabetic rats. Furthermore, we used Western blot analysis to determine eNOS and nNOS protein levels in cerebral vessels/brain tissue in sedentary and exercised nondiabetic and diabetic rats. We found that ADP and NMDA produced a dilation of pial arterioles that was similar in sedentary and exercised nondiabetic rats. In contrast, ADP and NMDA produced only minimal vasodilation in sedentary diabetic rats. ExT restored impaired ADP- and NMDA-induced vasodilation observed in diabetic rats to that observed in nondiabetics. Nitroglycerin produced a dilation of pial arterioles that was similar in sedentary and exercised nondiabetic and diabetic rats. Superoxide levels in cortex tissue were similar in sedentary and exercised nondiabetic rats, were increased in sedentary diabetic rats, and were normalized by ExT in diabetic rats. Finally, we found that eNOS protein was increased in diabetic rats and further increased by ExT and that nNOS protein was not influenced by T1D but was increased by ExT. We conclude that ExT can alleviate impaired eNOS- and nNOS-dependent responses of pial arterioles during T1D.     

Elevation in resting blood flow attenuates exercise hyperemia.

These experiments tested the hypothesis that elevating muscle blood flow before exercise would wash out vasoactive substances produced by muscle contraction and reduce the magnitude of exercise hyperemia and/or delay the response. In chronically instrumented dogs (n = 7), hindlimb blood flow was measured with chronically implanted flow probes during mild treadmill exercise. In an anesthetized preparation (n = 8), arterial and venous blood flows of a single hindlimb were obtained during 1-s tetanic contractions evoked by electrical stimulation of the cut sciatic nerve. Elevation of blood flow by intra-arterial infusion of adenosine attenuated the increase in flow during exercise and tetanic contraction by 48 and 47%, respectively. No delay was observed in the latency to peak flow. The attenuated hyperemic response to exercise or contraction is best explained by washout of vasoactive substance(s) produced by contracting muscle, but the residual response suggests that a metabolic mediator may not be the sole explanation for exercise hyperemia.     

Effect of adenosine receptor blockade on pial arteriolar dilation during sciatic nerve stimulation

In the present study, we report the effects of adenosine receptor antagonists on pial vasodilatation during contralateral sciatic nerve stimulation (SNS). The pial circulation was observed through a closed cranial window in alpha-chloralose-anesthetized rats. In artificial cerebrospinal fluid (CSF), SNS resulted in a 30.5 +/- 13.2% increase in pial arteriolar diameter in the hindlimb somatosensory cortex. Systemic administration of the selective adenosine A2A receptor antagonist, 4-(2-[7-amino-2-[2-furyl][3,2,4]triazolol[2,3-a][1,3,5]triazin-5-yl-amino] ethyl)phenol (ZM-241385), significantly (P < 0.05, n = 6) attenuated the SNS-induced vasodilatation. Systemic administration of 8-(p-sulfophenyl)theophylline (8SPT), a nonselective antagonist that is blood-brain barrier (BBB) impermeable, had no effect on vasodilatation to SNS. In contrast, systemic theophylline, which readily penetrates the BBB, nearly abolished the SNS-induced vasodilatation (P < 0.01; n = 7). Topical superfusion of 8SPT significantly (P < 0.01; n = 6) attenuated vasodilatation during SNS. Topical superfusion of 8- cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective adenosine A1 receptor antagonist, significantly potentiated SNS-induced vasodilatation (P < 0.01; n > or = 5). Hypercarbic vasodilatation and somatosensory-evoked potentials were not affected by any of the compounds tested. Our findings suggest that luminal endothelial adenosine receptors are not involved in the arteriolar response to SNS, as demonstrated by a lack of effect with systemic 8SPT. Furthermore, the adenosine A2A receptor subtype appears to be involved in the dilator response to SNS. Finally, the neuromodulatory action of adenosine, via the A1 receptor subtype, significantly influences SNS-induced vasodilatation. Thus the present study provides further evidence for a role of adenosine in the regulation of CBF during somatosensory stimulation.     

Rapid vasodilation in response to a brief tetanic muscle contraction.

To test the hypothesis that vasodilation occurs because of the release of a vasoactive substance after a brief muscle contraction and to determine whether acetylcholine spillover from the motor nerve is involved in contraction-induced hyperemia, tetanic muscle contractions were produced by sciatic nerve stimulation in anesthetized dogs (n = 16), instrumented with flow probes on both external iliac arteries. A 1-s stimulation of the sciatic nerve at 1. 5, 3, and 10 times motor threshold increased blood flow above baseline (P < 0.01) for 20, 25, and 30 s, respectively. Blood flow was significantly greater 1 s after the contraction ended for 3 and 10 x motor threshold (P < 0.01) and did not peak until 6-7 s after the contraction. The elevations in blood flow to a 1-s stimulation of the sciatic nerve and a 30-s train of stimulations were abolished by neuromuscular blockade (vecuronium). The delayed peak blood flow response and the prolonged hyperemia suggest that a vasoactive substance is rapidly released from the contracting skeletal muscle and can affect blood flow with removal of the mechanical constraint imposed by the contraction. In addition, acetylcholine spillover from the motor nerve is not responsible for the increase in blood flow in response to muscle contraction.     

Slowing of Motor Nerve Conduction Velocity in Streptozotocin-induced Diabetic Rats is Preceded by Impaired Vasodilation in Arterioles that Overlie the Sciatic Nerve

Diabetes mellitus produces marked abnormalities in motor nerve conduction, but the mechanism is not clear. In the present study we hypothesized that in the streptozotocin (STZ)-induced diabetic rat impaired vasodilator function in arterioles that provide circulation to the region of the sciatic nerve is associated with reduced endoneural blood flow (EBF) and that these defects precede slowing of motor nerve conduction velocity, and thereby may contribute to nerve dysfunction. As early as three days after the induction of diabetes endoneural blood flow was reduced in the STZ-induced diabetic rat. Furthermore, after 1 week of diabetes acetylcholine- induced vasodilation was found to be impaired. This was accompanied by an increase in the superoxide level in arterioles that provide circulation to the region of the sciatic nerve as well as changes in the level of other markers of oxidative stress including an increase in serum levels of thiobarbituric acid reactive substances and a decrease in lens glutathione level. In contrast to the vascular related changes that occur within 1 week of diabetes, motor nerve conduction velocity and sciatic nerve Na⁺/k⁺ ATPase activity were significantly reduced following 2 and 4 weeks of diabetes, respectively. These studies demonstrate that changes in vascular function in the STZ-induced diabetic rat precede the slowing of motor nerve conduction velocity (MNCV) and are accompanied by an increase in superoxide levels in arterioles that provide circulation to the region of the sciatic nerve.     

Effect of pikamilon on the cortical blood supply and microcirculation in the pial arteriole system

Pikamilon was shown to increase blood supply in cerebral cortex in conscious rats and rabbits. The increase in blood flow has been revealed under intravenous, intraperitoneal and systemic administration of the drug. There is a pronounced dilatation of pial arterioles under pikamilon action while applied locally. Most dilation occurs in arterioles with the initial diameter of 10-20 microns. With the increase of pial arterioles diameter, dilatory effect of pikamilon, is reduced.     

Role of 20-HETE in the pial arteriolar constrictor response to decreased hematocrit after exchange transfusion of cell-free polymeric hemoglobin.

The cerebrovascular response to decreases in hematocrit and viscosity depends on accompanying changes in arterial O2 content. This study examines whether 1) the arteriolar dilation seen after exchange transfusion with a 5% albumin solution can be reduced by the K(ATP) channel antagonist glibenclamide (known to inhibit hypoxic dilation), and 2) the arteriolar constriction seen after exchange transfusion with a cell-free hemoglobin polymer to improve O2-carrying capacity can be blocked by inhibitors of the synthesis or vasoconstrictor actions of 20-HETE. In anesthetized rats, decreasing hematocrit by one-third with albumin exchange transfusion dilated pial arterioles (14 +/- 2%; SD), whereas superfusion of the surface of the brain with 10 muM glibenclamide blocked this response (-10 +/- 7%). Exchange transfusion with polymeric hemoglobin decreased the diameter of pial arterioles by 20 +/- 3% without altering arterial pressure. This constrictor response was attenuated by superfusing the surface of the brain with a 20-HETE antagonist, WIT-002 (10 microM; -5 +/- 1%), and was blocked by two chemically dissimilar selective inhibitors of the synthesis of 20-HETE, DDMS (50 microM; 0 +/- 4%) and HET-0016 (1 microM; +6 +/- 4%). The constrictor response to hemoglobin transfusion was not blocked by an inhibitor of nitric oxide (NO) synthase, and the inhibition of the constrictor response by DDMS was not altered by coadministration of the NO synthase inhibitor. We conclude 1) that activation of K(ATP) channels contributes to pial arteriolar dilation during anemia, whereas 2) constriction to polymeric hemoglobin transfusion at reduced hematocrit represents a regulatory response that limits increased O2 transport and that is mediated by increased formation of 20-HETE, rather than by NO scavenging.     

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.     

Carbon monoxide contributes to hypotension-induced cerebrovascular vasodilation in piglets

The gaseous compound carbon monoxide (CO) has been identified as an important endogenous biological messenger in brain and is a major component in regulation of cerebrovascular circulation in newborns. CO is produced endogenously by catabolism of heme to CO, free iron, and biliverdin during enzymatic degradation of heme by heme oxygenase (HO). The present study was designed to test the hypothesis that endogenously produced CO contributes to hypotension-induced vasodilation of cerebral arterioles. Experiments used anesthetized piglets with implanted, closed cranial windows. Topical application of the HO substrate heme-l-lysinate caused dilation of pial arterioles that was blocked by a metal porphyrin inhibitor of HO, chromium mesoporphyrin (CrMP). In normotensive piglets (arterial pressure 64 +/- 4 mmHg), CrMP did not cause vasoconstriction of pial arterioles but rather a transient dilation. Hypotension (50% of basal blood pressure) increased cerebral CO production and dilated pial arterioles from 66 +/- 2 to 92 +/- 7 microm. In hypotensive piglets, topical CrMP or intravenous tin protoporphyrin decreased cerebral CO production and produced pial arteriolar constriction to normotensive diameters. In additional experiments, because prostacyclin and nitric oxide (NO) are also key dilators that can contribute to cerebrovascular dilation, we held their levels constant. NO/prostacyclin clamp was accomplished with continuous, simultaneous application of indomethacin, N(omega)-nitro-l-arginine, and minimal dilatory concentrations of iloprost and sodium nitroprusside. With constant NO and prostacyclin, the transient dilator and prolonged constrictor responses to CrMP of normotensive and hypotensive piglets, respectively, were the same as when NO and prostaglandins were not held constant. These data suggest that endogenously produced CO contributes to cerebrovascular dilation in response to reduced perfusion pressure.     

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.     

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.