NOC/oFQ contributes to age-dependent impairment of NMDA-induced cerebrovasodilation after brain injury

This study characterized the effects of fluid percussion brain injury (FPI) on N-methyl-D-aspartate (NMDA)-induced vasodilation and determined the role of nociceptin/orphanin FQ (NOC/oFQ) in such changes as a function of age and time postinsult. FPI elevated cerebrospinal fluid (CSF) NOC/oFQ from 70 +/- 3 to 444 +/- 56 pg/ml ( approximately 10(-10) M) within 1 h and to 1,931 +/- 112 pg/ml within 8 h, whereas values returned to control levels within 168 h in the newborn pig. In contrast, FPI elevated CSF NOC/oFQ from 77 +/- 4 to 202 +/- 16 pg/ml within 1 h and values returned to control levels within 8 h in the juvenile pig. Topical NOC/oFQ (10(-10) M) had no effect on pial artery diameter but attenuated NMDA (10(-8), 10(-6) M)-induced dilation (9 +/- 1 and 16 +/- 1 vs. 5 +/- 1 and 10 +/- 1%) in both age groups. In the newborn, NMDA-induced pial artery dilation was reversed to vasoconstriction within 1 h post-FPI and responses remained impaired for 72 h, but such vasoconstriction was attenuated by pretreatment with [F/G]NOC/oFQ(1-13)-NH(2) (10(-6) M, 1 mg/kg iv), an NOC/oFQ antagonist (9 +/- 1 and 16 +/- 1 vs. -7 +/- 1 and -12 +/- 1 vs -2 +/- 1 and -3 +/- 1% for control, FPI, and FPI pretreated with the NOC/oFQ antagonist). In contrast, in the juvenile, NMDA-induced vasodilation was only attenuated within 1 h post-FPI and returned to control within 8 h. Such dilation was also partially restored by the NOC/oFQ antagonist. These data indicate that NOC/oFQ contributes to impaired NMDA pial artery dilation after FPI. These data suggest that the greater NOC/oFQ release in the newborn versus the juvenile may contribute to age-related differences in FPI effects on excitatory amino acid-induced pial dilation.     

NOC/oFQ PKC-dependent superoxide generation contributes to hypoxic-ischemic impairment of NMDA cerebrovasodilation

This study determined whether nociceptin/orphanin FQ (NOC/oFQ) generates superoxide anion (O(2)(-)) in a protein kinase C (PKC)-dependent manner and whether such production contributes to hypoxic-ischemic (H-I) impairment of N-methyl-D-aspartate (NMDA)-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was an index of O(2)(-) generation. Under non-H-I conditions, topical NOC/oFQ (10(-10) M, concentration present in cerebrospinal fluid after I or H-I) increased SOD-inhibitable NBT reduction from 1 +/- 1 to 20 +/- 3 pmol/mm(2). PKC inhibitors staurosporine and chelerythrine (10(-7) M) blunted NBT reduction (1 +/- 1 to 7 +/- 2 pmol/mm(2) for chelerythrine), whereas the NOC/oFQ receptor antagonist [F/G]NOC/oFQ (1-13)-NH(2) (10(-6) M) blocked NBT reduction. [F/G]NOC/oFQ(1-13)-NH(2) and staurosporine also blunted the NBT reduction observed after I or H-I. NMDA (10(-8), 10(-6) M)-induced pial artery dilation was reversed to vasoconstriction after H-I. The NOC/oFQ antagonist staurosporine and free radical scavengers partially prevented this impaired dilation (sham: 9 +/- 1 and 16 +/- 1; H-I: -5 and -10 +/- 1; H-I staurosporine pretreated: 3 +/- 1 and 6 +/- 1%). These data show that NOC/oFQ increased O(2)(-) production in a PKC-dependent manner and contributed to this production after insult and that NOC/oFQ contributed to impaired NMDA-induced pial artery dilation after H-I, suggesting, therefore, that PKC-dependent O(2)(-) generation by NOC/oFQ links NOC/oFQ release to impaired NMDA dilation after H-I.     

Relationship between nociceptin/orphanin FQ and cerebral hemodynamics after hypoxia-ischemia in piglets

This study was designed to characterize the role of the newly described endogenous opioid nociceptin/orphanin FQ (NOC/oFQ) in reduced cerebral blood flow (CBF) observed after ischemia-reperfusion (I/R) and combined hypoxia and ischemia-reperfusion (H-I/R), as a function of time after onset of reperfusion in newborn pigs equipped with a closed cranial window. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, whereas hypoxia (10 min) decreased PO(2) to 35 +/- 3 mmHg with unchanged PCO(2). I/R elevated cerebrospinal fluid (CSF) NOC/oFQ from 67 +/- 4 to 266 +/- 29 pg/ml within 1 h, whereas values returned to control level within 4 h of reperfusion. H-I/R elevated CSF NOC/oFQ to 483 +/- 67 pg/ml within 1 h, and such values returned slowly to control level within 12 h of reperfusion. Topical NOC/oFQ (10(-8) M, 10(-6) M)-induced vasodilation was attenuated by I/R and reversed to vasoconstriction by H-I/R at 1 h of reperfusion (control, 9 +/- 1 and 16 +/- 1%; I/R, 3 +/- 1 and 6 +/- 1%; H-I/R, -6 +/- 1 and -11 +/- 1%). Such altered dilation returned to control values within 4 h in I/R animals and within 12 h in H-I/R animals. Blood flow in the cerebrum was reduced from 58 +/- 4 to 33 +/- 2 ml x min(-1) x 100 g(-1) within 1 h and returned to control value within 4 h in I/R animals. In animals pretreated with [F/G]NOC/oFQ(1-13)-NH(2) (1 mg/kg iv), an NOC/oFQ antagonist, however, CBF only fell to 43 +/- 3 ml x min(-1) x 100 g(-1) at 1 h of reperfusion. Similar observations were made in H-I/R animals. These data suggest that an elevated CSF NOC/oFQ concentration and altered vascular responsiveness to this opioid contribute to reductions in CBF observed after either I/R or H-I/R.     

PTK,MAPK, and NOC/oFQ impair hypercapnic cerebrovasodilation after hypoxia/ischemia

This study characterized the contributions of protein tyrosine kinase (PTK) and mitogen-activated protein kinase (MAPK) in nociceptin/orphanin FQ (NOC/oFQ)-induced impairment of hypercapnic pial artery dilation (PAD) after hypoxia/ischemia (H/I) in piglets equipped with a closed cranial window. NOC/oFQ (10(-10) M cerebrospinal fluid H/I concentration) impaired hypercapnic PAD (21 +/- 2% vs. 13 +/- 1%). Coadministration of either of the PTK inhibitors genistein or tyrphostin A23 or the MAPK inhibitors U-0126 or PD-98059 with NOC/oFQ (10(-10) M) partially prevented the inhibition of hypercapnic PAD compared with that observed in their absence (21 +/- 2% vs. 17 +/- 1% for genistein). After exposure to H/I, PAD in response to hypercapnia was impaired, but pretreatment with either genistein, tyrphostin A23, U-0126, or PD-98059 partially protected such impairment (17 +/- 1% vs. 4 +/- 1% vs. 9 +/- 1% for sham control, H/I, and H/I + genistein pretreatment, respectively). These data show that PTK and MAPK activation contribute to NOC/oFQ-induced impairment of hypercapnic PAD. These data suggest that activation of PTK and MAPK is also involved in the mechanism by which NOC/oFQ impairs hypercapnic PAD after H/I.     

Heat shock protein modulation of KATP and KCa channel cerebrovasodilation after brain injury

Fluid percussion brain injury (FPI) impairs pial artery dilation to activators of the ATP-sensitive (K(ATP)) and calcium-activated (K(Ca)) K(+) channels. This study investigated the role of heat shock protein (HSP) in the modulation of K(+) channel-induced pial artery dilation after FPI in newborn pigs equipped with a closed cranial window. Under nonbrain injury conditions, topical coadministration of exogenous HSP-27 (1 mug/ml) blunted dilation to cromakalim, CGRP, and NS-1619 (10(-8) and 10(-6) M; cromakalim and CGRP are K(ATP) agonists and NS-1619 is a K(Ca) agonist). In contrast, coadministration of exogenous HSP-70 (1 mug/ml) potentiated dilation to cromakalim, CGRP, and NS-1619. FPI increased the cerebrospinal fluid (CSF) concentration of HSP-27 from 0.051 +/- 0.012 to 0.113 +/- 0.035 ng/ml but decreased the CSF concentration of HSP-70 from 50.42 +/- 8.96 to 30.9 +/- 9.9 ng/ml at 1 h postinsult. Pretreatment with topical exogenous HSP-70 (1 mug/ml) before FPI fully blocked injury-induced impairment of cromakalim and CGRP dilation and partially blocked injury-induced impairment of dilation to NS-1619. These data indicate that HSP-27 and HSP-70 contribute to modulation of K(+) channel-induced pial artery dilation. These data suggest that HSP-70 is an endogenous protectant of which its actions may be unmasked and/or potentiated with exogenous administration before brain injury.     

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.     

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.     

Adenosine receptors mediate glutamate-evoked arteriolar dilation in the rat cerebral cortex

We tested the hypothesis that adenosine (Ado) mediates glutamate-induced vasodilation in the cerebral cortex by monitoring pial arteriole diameter in chloralose-anesthetized rats equipped with closed cranial windows. Topical application of 100 microM glutamate and 100 microM N-methyl-d-aspartate (NMDA) dilated pial arterioles (baseline diameter 25 +/- 2 microm) by 17 +/- 1% and 18 +/- 4%, respectively. Coapplication of the nonselective Ado receptor antagonist theophylline (Theo; 10 microM) significantly reduced glutamate- and NMDA-induced vasodilation to 4 +/- 2% (P < 0.01) and 6 +/- 2% (P < 0.05), whereas the Ado A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM) had no effect. Moreover, application of the Ado A(2A) receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5)triazin-5-ylamino]ethyl)phenol (ZM-241385), either by superfusion (0.1 microM, 1 microM) or intravenously (1 mg/kg), significantly inhibited the pial arteriole dilation response to glutamate. Neither Theo nor ZM-241385 affected vascular reactivity to mild hypercapnia induced by 5% CO(2) inhalation. These results suggest that Ado contributes to the dilation of rat cerebral arterioles induced by exogenous glutamate, and that the Ado A(2A) receptor subtype may be involved in this dilation response.     

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.     

Enhanced pial arteriolar sensitivity to bioactive agents following exposure to endothelin-1

Effects of prior exposure of pial arterioles to endothelin-1 (ET-1) (10(-9) M) on the constriction induced by the by-products of hemolyzed blood (5-HT, LTC4, LPA, and thromboxane analog U-46619) were examined. Piglets (age: 1-3 d) anesthetized with a mixture of ketamine hydrochloride and acepromazine were implanted with cranial windows, and anesthesia was maintained with alpha-chloralose. Topical applications of the by-products of hemolyzed blood mildly constricted pial arterioles. Following prior exposure of the microvessels to ET-1, application of the by-products of hemolyzed blood produced significantly potentiated and long-lasting constrictions compared to the controls. In another experiment, pretreatment of pial arterioles with U-46619 (10(-8) M) also potentiated the constriction induced by ET-1. The constriction produced was fast and longer-lasting. Thus, these data show that by-products of hemolyzed blood, though not potent vasoconstrictors per se, potently constricted pial arterioles in the presence of ET-1. The same agents in the CSF can also potentiate constriction induced by ET-1. Hence, by-products of hemolyzed blood may play a significant role in the initiation and maintenance of cerebral arterial narrowing observed following intracranial bleeding.     

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.     

Leukotrienes increase levels of prostanoids in cerebrospinal fluid in piglets

We investigated effects of exogenous leukotrienes (C4, D4, or E4) on levels of prostanoids in cerebrospinal fluid in newborn pigs (1-5 days). A "closed" cranial window was placed over the parietal cortex. Pial arterial diameter was measured with a microscope and electronic micrometer system. Levels in cerebrospinal fluid (CSF) of 6-keto-Prostaglandin F1 alpha (6-keto-PGF1 alpha), Thromboxane B2 (TXB2), and Prostaglandin E2 (PGE2) were measured by radioimmunoassay. Topical application of leukotrienes C4, D4, or E4 (5,000 ng/ml) similarly constricted pial arteries by 15 +/- 2% (n = 14) (mean +/- SEM). In addition, leukotrienes increased levels of 6-keto-PGF1 alpha from 806 +/- 136 to 1,612 +/- 304 pg/ml (n = 13), TXB2 from 161 +/- 31 to 392 +/- 81 pg/ml (n = 10), and PGE2 from 2,271 +/- 342 to 4,636 +/- 740 pg/ml (n = 13). Each type of leukotriene had similar effects on prostanoid synthesis. In other experiments (n = 5), we found that 2.0 ng/ml PGE2 in CSF dilated pial arteries by 24 +/- 8% and that 1.0 ng/ml PGI2 dilated pial arteries by 15 +/- 6%. These results indicate that leukotrienes are able to increase levels of prostanoids in cerebral cortex.     

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.     

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.     

Endothelin-1 and endothelin-3 stimulate prostaglandin E2 secretion from the rat median eminence.

The in vitro effects of endothelin-1 (ET-1) and endothelin-3 (ET-3) on the release of prostaglandin (PG)E2 from the rat median eminence were investigated. The addition of ET-1 from 10(-9) M to 10(-6) M stimulated PGE2 release in a dose-dependent manner (from 10.5 +/- 2.1 to 54.4 +/- 5.6 pg/ME fragment/30 min; mean +/- SEM, p less than 0.001). ET-3 also stimulated the release of PGE2 from 10(-7) M to 10(-5) M dose dependently (from 18.1 +/- 0.7 to 60.9 +/- 17.4 pg/ME fragment/30 min p less than 0.05). The time course effect of ET-3 (10(-6) M) showed that PGE2 release was stimulated within five minutes (control, 1.5 +/- 0.5; ET-3, 15.8 +/- 3.0 pg/ME fragment/5 min, p less than 0.01). These results suggest that ET-1 and ET-3 have some physiological effects on the rat median eminence.     

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.     

Pial arteriole dilation during somatosensory stimulation is not mediated by an increase in CSF metabolites

Pial arterioles supplying the hindlimb somatosensory cortex dilate in response to contralateral sciatic nerve stimulation. The mechanism of this pial vasodilation is not well understood. One possibility is that vasoactive metabolites released during brain activation may diffuse to subarachnoid cerebrospinal fluid (CSF) to dilate pial vessels. To test this hypothesis, we implanted closed cranial windows in rats and measured pial arteriolar dilation to sciatic nerve stimulation during constant rate superfusion of the pial surface with artificial CSF. We reason that flushing the pial surface with CSF should quickly dissipate vasoactive substances and prevent these substances from dilating pial arterioles. CSF flow (1 and 1.5 ml/min) significantly reduced pial arteriole dilation induced by 5% CO2 inhalation, but the same flow rates did not affect dilator responses to sciatic nerve stimulation. We conclude that brain-to-CSF diffusion of vasoactive metabolites does not play a significant role in the dilation of pial arterioles during somatosensory activity.     

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.     

Effects of hypothermia on neuronal-vascular function after cerebral ischemia in piglets

We determined whether cerebral arteriolar dilation to N-methyl-d-aspartate (NMDA), a response dependent on stimulation of cortical neurons and inhibited by anoxic stress, would be preserved by hypothermia during and following ischemia. Pial arteriolar diameters in anesthetized piglets were determined via intravital microscopy. Arteriolar responses to NMDA (10, 50, and 100 micromol/l) were measured before and 1 h after 10 min of global ischemia. Piglets were exposed to either total body or selective brain cooling (33-34 degrees C). Arteriolar dilation to lower doses or to 100 micromol/l NMDA was not affected by hypothermia alone (51 +/- 3 vs. 46 +/- 7%, normothermia vs. hypothermia; n = 7) in nonischemic animals. However, arteriolar responses to 100 micromol/l NMDA were clearly attenuated after ischemia despite body cooling during ischemia (53 +/- 3 vs. 32 +/- 6%; n = 8), hypothermia during ischemia and early reperfusion (49 +/- 10 vs. 20 +/- 3%; n = 8), or selective brain cooling (48 +/- 5 vs. 20 +/- 5%; n = 10). In contrast, pretreatment with indomethacin resulted in complete preservation of NMDA-induced vasodilation after ischemia. Thus, hypothermia fails to protect against neuronal dysfunction during ischemia.