NOC/oFQ contributes to hypoxic-ischemic impairment of N-methyl-D-aspartate-induced cerebral vasodilation

Previous studies in piglets show that either hypoxia, ischemia-reperfusion (I+R) or combined hypoxia-ischemia-reperfusion (H+I+R) attenuated N-methyl-D-aspartate (NMDA)-induced pial artery dilation. This study was designed to determine the contribution of the newly described opioid nociceptin orphanin FQ (NOC/oFQ) to hypoxic-ischemic impairment of NMDA induced cerebral vasodilation in piglets equipped with a closed cranial window. Global cerebral ischemia was produced via elevated intracranial pressure. Hypoxia decreased P(O(2)) to 35+/-3 mmHg with unchanged P(CO(2)). I+R elevated CSF NOC/oFQ from 67+/-4 to 266+/-29 pg/ml ( approximately 10(-10) M) while H+I+R elevated CSF NOC/oFQ to 483+/-67 pg/ml within 1 h of reperfusion. Such elevated NOC/oFQ levels returned to control within 4 h in I+R animals and within 12 h in H+I+R animals. Topical NOC/oFQ (10(-10) M) had no effect on pial artery diameter by itself but attenuated NMDA (10(-8), 10(-6) M) induced pial dilation (control, 9+/-1 and 16+/-1; coadministered NOC/oFQ, 5+/-1 and 10+/-1%). NMDA induced pial artery dilation was attenuated by I+R or H+I+R; but such dilation was partially restored by pretreatment with the putative NOC/oFQ antagonist [F/G] NOC/oFQ (1-13) NH(2) (10(-6) M) (control, 9+/-1 and 16+/-1; I+R, 3+/-1 and 5+/-1; I+R+NOC/oFQ antagonist, 6+/-1 and 11+/-1%) Similar results were obtained for glutamate. These data suggest that NOC/oFQ release contributes to impaired NMDA and glutamate-induced cerebrovasodilation following I+R or H+I+R.     

Altered release of prostaglandins contributes to hypoxic/ischemic impairment of NOC/oFQ cerebrovasodilation

This study was designed to determine if altered release of prostaglandins contributes to impaired pial artery dilation to the newly described opioid, nociceptin/orphanin FQ (NOC/oFQ), following hypoxia/ischemia in newborn pigs equipped with a closed cranial window. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, while hypoxia (10 min) decreased P(O(2)) to 35+/-3 mmHg with unchanged P(CO(2)). NOC/oFQ (10(-8) and 10(-6) M) modestly increased cerebrospinal fluid (CSF) 6-Keto PGF(1alpha) and TXB(2), the stable breakdown products of PGI(2) and TXA(2), in sham animals (1199+/-39 to 1704+/-104 and 299+/-9 to 409+/-12 pg/ml for control and 10(-6) M NOC/oFQ 6-Keto PGF(1alpha) and TXB(2), respectively). In 1 h post ischemia/reperfusion (I+R) animals, basal levels of 6-Keto PGF(1alpha) and TXB(2) were elevated. NOC/oFQ-stimulated release of 6-Keto PGF(1alpha) was blocked while such release of TXB(2) was enhanced (526+/-15 to 822+/-36 pg/ml for control and 10(-6) M NOC/oFQ CSF TXB(2)). Similar, though more pronounced, changes were observed in hypoxia/ischemia/reperfusion (H+I+R) animals. Pretreatment with indomethacin (5 mg/kg i.v.) or SQ 29,548 (10(-4) M), cyclooxygenase and PGH(2)/TXA(2) receptor antagonists, partially restored attenuated NOC/oFQ pial artery dilation 1 h after I+R (9+/-1 and 18+/-1 vs. 3+/-1 and 6+/-1 vs. 8+/-1 and 13+/-1% for 10(-8) and 10(-6) M NOC/oFQ in sham, I+R, and I+R - SQ 29,548 pretreated animals). In contrast, NOC/oFQ-induced vasodilation was reversed to vasoconstriction in H+I+R animals and indomethacin or SQ 29,548 similarly partially restored such pial vasodilation. These data indicate that altered stimulated prostaglandin release contributes to hypoxic/ischemic impairment of NOC/oFQ-mediated pial artery dilation.     

Role of NOC/oFQ in impaired opioid-induced pial artery dilation following brain injury

Previous studies in piglets show that opioid-induced pial artery dilation was impaired following fluid percussion brain injury (FPI). This study was designed to determine the role of the newly described opioid nociceptin orphanin FQ (NOC/oFQ) in such impaired dilation to other opioids after FPI. CSF NOC/oFQ concentration was elevated from 70+/-6 to 444+/-56 pg/ml ( approximately 10(-10) M) within 1 h of FPI. Coadministration of NOC/oFQ (10(-10) M) with methionine enkephalin (10(-10), 10(-8), 10(-6) M) attenuated pial dilation induced by this opioid (7+/-1, 13+/-2, and 19+/-2 vs. 2+/-1, 6+/-1, and 7+/-2%) under non-brain injury conditions. Similar inhibition by NOC/oFQ was observed for leucine enkephalin and dynorphin. Methionine enkephalin (10(-10), 10(-8), 10(-6) M)-induced pial artery dilation was also inhibited within 1 h of FPI, but such responses were partially restored in animals pretreated with the NOC/oFQ receptor antagonist [F/G] NOC/oFQ (1-13) NH(2) (10(-6) M) (8+/-1, 14+/-1, and 21+/-1 vs. 1+/-1, 3+/-1, and 4+/-1 vs. 7+/-1, 11+/-1, and 17+/-1% for sham control, FPI and FPI pretreated with the NOC/oFQ receptor antagonist). Leucine enkephalin and dynorphin-induced pial artery dilation were similarly altered by FPI and partially restored by [F/G] NOC/oFQ (1-13) NH(2). These data indicate that the NOC/oFQ released by FPI contributes to impaired dilation to other opioids observed following this insult.     

Role of cAMP and K(+) channel-dependent mechanisms in piglet hypoxic/ischemic impaired nociceptin/orphanin FQ-induced cerebrovasodilation

This study was designed to determine the role of altered cAMP and K(+) channel-dependent mechanisms in impaired pial artery dilation to the newly described opioid, nociceptin/orphanin FQ (NOC/oFQ) following hypoxia/ischemia in newborn pigs equipped with a closed cranial window. Recent studies have observed that NOC/oFQ elicits pial dilation via release of cAMP, which, in turn, activates the calcium sensitive (K(ca)) and the ATP-dependent K(+) (K(ATP)) channel. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, while hypoxia (10 min) decreased pO(2) to 35+/-3 mm Hg with unchanged pCO(2). Topical NOC/oFQ (10(-8), 10(-6) M) induced vasodilation was attenuated by ischemia/reperfusion (I+R) and reversed to vasoconstriction by hypoxia/ischemia/reperfusion (H+I+R) at 1 h of reperfusion (control, 9+/-1 and 16+/-1%; I+R, 3+/-1 and 6+/-1%; H+I+R, -7+/-1 and -12+/-1%). Such altered dilation returned to control values within 4 h in I+R animals and within 12 h in H+I+R animals. NOC/oFQ dilation was associated with elevated CSF cAMP in control animals but such biochemical changes were attenuated in I+R animals and reversed to decreases in cAMP concentration in H+I+R animals (control, 1037+/-58 and 1919+/-209 fmol/ml; I+R, 1068+/-33 and 1289+/-30 fmol/ml; H+I+R, 976+/-36 and 772+/-27 fmol/ml for absence and presence of NOC/oFQ 10(-6) M, respectively). Topical 8-Bromo cAMP (10(-8), 10(-6) M) pial dilation was unchanged by I+R but blunted by H+I+R (control, 10+/-1 and 20+/-1%; I+R, 11+/-1 and 20+/-2%; H+I+R, 0+/-1 and 0+/-2%). Pituitary adenylate cyclase activating polypeptide and cromakalim, adenylate cyclase and K(ATP) channel activators, respectively, elicited dilation that was blunted by both I+R and H+I+R while NS1619, a K(ca) channel activator, elicited dilation that was unchanged by I+R but blunted by H+I+R. These data indicate that impaired NOC/oFQ dilation following I+R results form altered adenylate cyclase and K(ATP) channel-dependent mechanisms. These data further indicate that impaired NOC/oFQ dilation following H+I+R results not only from altered adenylate cyclase and K(ATP) channel but also from altered cAMP and K(ca) channel-dependent mechanisms.     

Superoxide generation links nociceptin/orphanin FQ (NOC/oFQ) release to impaired N-methyl-D-aspartate cerebrovasodilation after brain injury

BACKGROUND AND PURPOSE: Although activation of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to altered cerebrovascular regulation after traumatic brain injury, the effects of such injury on the vascular response to NMDA itself has been less well appreciated. The newly described opioid nociceptin/orphanin FQ (NOC/oFQ) elicits pial artery dilation, at least in part, in a prostaglandin-dependent manner and is released into cerebrospinal fluid after fluid percussion brain injury (FPI). Generation of superoxide anion (O(2)(-)) occurs after FPI, and a byproduct of cyclooxygenase metabolism is the generation of O(2)(-). This study was designed to determine whether NOC/oFQ generates O(2)(-), which in turn could link NOC/oFQ release to impaired NMDA-induced pial artery dilation after FPI. METHODS: Injury of moderate severity (1.9 to 2.1 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase-inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O(2)(-) generation. RESULTS: Under non-brain injury conditions, topical NOC/oFQ (10(-)(10) mol/L, the concentration present in cerebrospinal fluid after FPI) increased superoxide dismutase-inhibitable NBT reduction from 1+/-1 to 20+/-3 pmol/mm(2) but had no effect itself on pial artery diameter. Indomethacin (5 mg/kg IV) blunted such NBT reduction (1+/-1 to 6+/-2 pmol/mm(2)), whereas the NOC/oFQ receptor antagonist [F/G] NOC/oFQ (1-13) NH(2) (10(-)(6) mol/L) blocked NBT reduction. [F/G] NOC/oFQ (1-13) NH(2) and indomethacin also blunted the NBT reduction observed after FPI (1+/-1 to 15+/-1 versus 1+/-1 to 4+/-1 versus 1+/-1 to 4+/-1 pmol/mm(2) for sham, NOC/oFQ antagonist, and indomethacin-treated animals, respectively). NMDA (10(-)(8) and 10(-)(6) mol/L)-induced pial artery dilation was reversed to vasoconstriction after FPI, and [F/G] NOC/oFQ (1-13) NH(2) attenuated such vasoconstriction (sham 9+/-1% and 16+/-1% versus FPI -7+/-1% and -12+/-1% versus FPI-[F/G] NOC/oFQ (1-13) NH(2)-pretreated animals -2+/-1% and -3+/-1%). Indomethacin and the free radical scavengers polyethylene glycol superoxide dismutase and catalase also partially restored NMDA-induced vasodilation. CONCLUSIONS: These data show that NOC/oFQ, in concentrations present in cerebrospinal fluid after FPI, increased O(2)(-) production in a cyclooxygenase-dependent manner and contributes to such production after FPI. These data show that NOC/oFQ contributes to impaired NMDA-induced pial artery dilation after FPI. Therefore, these data suggest that cyclooxygenase-dependent O(2)(-) generation links NOC/oFQ release to impaired NMDA-induced cerebrovasodilation after brain injury.     

NOC/oFQ activates ERK and JNK but not p38 MAPK to impair prostaglandin cerebrovasodilation after brain injury

Fluid percussion brain injury (FPI) elevates the CSF concentration of the opioid nociceptin/orphanin FQ (NOC/oFQ), which contributes to impairment of pial artery dilation to the prostaglandins (PG) PGE2 and PGI2. This study investigated the role of the ERK, p38, and JNK isoforms of mitogen-activated protein kinase (MAPK) in impaired PG cerebrovasodilation after FPI, and the relationship of brain injury induced release of NOC/oFQ to MAPK in such vascular impairment in newborn pigs equipped with a closed cranial window. FPI blunted PGE2 pial artery dilation, but U 0126 and SP 600125 (10(-6) M) (ERK and JNK MAPK inhibitors, respectively) partially prevented such impairment (7 +/- 1, 12 +/- 1, and 17 +/- 1 vs. 2 +/- 1, 3 +/- 1, and 5 +/- 1 vs. 4 +/- 1, 7 +/- 1, and 12 +/- 1% for 1, 10, and 100 ng/ml PGE2 in control, FPI, and FPI + U 0126 pretreated animals, respectively). In contrast, administration of SB 203580 (10(-5) M) (p38 MAPK inhibitor) did not prevent FPI impairment of PGE2 dilation. Co-administration of NOC/oFQ at the dose of 10(-10) M, the cerebrospinal fluid concentration observed after FPI, with PGE2 under non-brain injury conditions blunted PG dilation, but U 0126 or SP 600125 partially prevented such impairment (7 +/- 1, 11 +/- 1, and 16 +/- 2 vs. 0 +/- 1, 1 +/- 1, and 2 +/- 1, vs. 5 +/- 1, 9 +/- 1, and 13 +/- 2 for responses to PGE2 in control, NOC/oFQ, and NOC/oFQ + U 0126 treated animals, respectively). Administration of SB 203580 did not prevent impairment of PG pial artery dilation by NOC/oFQ. These data show that activation of ERK and JNK but not p38 MAPK contributes to impairment of PG cerebrovasodilation after FPI. These data suggest that NOC/oFQ induced ERK and JNK but not p38 MAPK activation contributes to impaired cerebrovasodilation to PG after FPI.     

Role of altered cyclooxygenase metabolism in impaired cerebrovasodilation to nociceptin/orphanin FQ following brain injury

This study was designed to determine the role of altered cyclooxygenase metabolism in impaired pial artery dilation to the newly described opioid, nociceptin orphanin FQ (NOC/oFQ), following fluid percussion brain injury (FPI) in newborn pigs equipped with a closed cranial window. Recent studies show that NOC/oFQ contributes to oxygen free radical generation observed post FPI in a cyclooxygenase dependent manner. FPI was produced by using a pendulum to strike a piston on a saline filled cylinder that was fluid coupled to the brain via a hollow screw inserted through the cranium. NOC/oFQ (10(-8), 10(-6) M) modestly increased cerebrospinal fluid (CSF) 6-keto-PGF(1alpha), and thromboxane B(2) (TXB(2)), the stable breakdown products of PGI(2) and TXA(2), in sham animals (1148 +/- 83 to 1681 +/- 114 and 308 +/- 16 to 424 +/- 21 pg/ml for control and 10(-6) M NOC/oFQ 6-keto-PGF(1alpha), and TXB(2), respectively). In 1-h post FPI animals, basal levels of 6-keto-PGF(1alpha), and TXB(2) were elevated. NOC/oFQ stimulated release of 6-keto-PGF(1alpha), was blocked while such release of TXB(2) was enhanced (720 +/- 63 to 1446 +/- 117 pg/ml for control and 10(-6) M NOC/oFQ CSF TXB(2)). NOC/oFQ (10(-8), 10(-6) M) induced pial artery dilation that was reversed to vasoconstriction by FPI while the cyclooxygenase inhibitor indomethacin (5 mg/kg, intravenous) partially restored such vascular responses (8 +/- 1 and 15 +/- 1 vs. -7 +/- 1 and -12 +/- 1 vs. 7 +/- 1 and 12 +/- 1% for 10(-8), 10(-6) M NOC/oFQ in sham, FPI and FPI-Indo pretreated animals). Similar observations were made in FPI animals pretreated with the thromboxane receptor antagonist SQ 29,548 or the free radical scavenger polyethylene glycol superoxide dismutase and catalase. These data indicate that altered NOC/oFQ induced cyclooxygenase metabolism contributes to impairment of dilation to this opioid following FPI.     

Nociceptin/orphanin FQ dilates pial arteries by KATP and Kca channel activation

Nociceptin/orphanin FQ (NOC/oFQ) is a recently discovered endogenous ligand for the opioid like receptor, ORL-1. In the piglet, cGMP activates the ATP sensitive (K(ATP)) while cAMP activates both the K(ATP) and the calcium sensitive (K(ca)) K(+) channel to elicit vasodilation. The present study was designed to characterize the role of cGMP, cAMP, K(ATP), and K(ca) channel activation in NOC/oFQ-induced pial artery dilation in newborn pigs equipped with a closed cranial window. NOC/oFQ (10(-8), 10(-6) M) induced pial arteriole dilation was decreased by the protein kinase A inhibitor Rp 8-Br cAMPs (16+/-1 and 30+/-1 vs. 5+/-1 and 10+/-1%). NOC/oFQ dilation was associated with elevated CSF cAMP (1037+/-58 vs. 1919+/-209 fmol/ml for control and 10(-6) M NOC/oFQ). Glibenclamide and iberiotoxin, K(ATP) and K(ca) channel antagonists, attenuated NOC/oFQ induced dilation (15+/-1 and 28+/-1 vs. 10+/-1 and 19+/-1% before and after iberiotoxin). In contrast, the nitric oxide synthase inhibitor, L-NNA, and the protein kinase G inhibitor, Rp 8-Br cGMPs had no effect on NOC/oFQ dilation while such dilation was not associated with a change in CSF cGMP. The putative ORL-1 receptor antagonist [F/G] NOC/oFQ (1-13)-NH(2) blocked NOC/oFQ dilation while responses were unchanged after naloxone (17+/-1 and 30+/-2 vs. 3+/-1 and 5+/-1%, before and after [F/G] NOC/oFQ (1-13)-NH(2)). Dilation to other opioids (e.g., methionine enkephalin) was unchanged by [F/G] NOC/oFQ (1-13)-NH(2). These data show that NOC/oFQ elicits pial artery dilation, at least in part, via cAMP, K(ATP), and K(ca) channel dependent mechanisms. These data suggest that such a mechanism involves the sequential release of cAMP and subsequent K(ATP) and K(ca) channel activation.     

Role of cAMP and K channel-dependent mechanisms in piglet hypoxic/ischemic impaired nociceptin/orphanin FQ-induced cerebrovasodilation

This study was designed to determine the role of altered cAMP and K⁺ channel-dependent mechanisms in impaired pial artery dilation to the newly described opioid, nociceptin/orphanin FQ (NOC/oFQ) following hypoxia/ischemia in newborn pigs equipped with a closed cranial window. Recent studies have observed that NOC/oFQ elicits pial dilation via release of cAMP, which, in turn, activates the calcium sensitive (K_ca) and the ATP-dependent K⁺ (K_ATP) channel. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, while hypoxia (10 min) decreased pO₂ to 35±3 mmHg with unchanged pCO₂. Topical NOC/oFQ (10⁻⁸, 10⁻⁶ M) induced vasodilation was attenuated by ischemia/reperfusion (I+R) and reversed to vasoconstriction by hypoxia/ischemia/reperfusion (H+I+R) at 1 h of reperfusion (control, 9±1 and 16±1%; I+R, 3±1 and 6±1%; H+I+R, −7±1 and −12±1%). Such altered dilation returned to control values within 4 h in I+R animals and within 12 h in H+I+R animals. NOC/oFQ dilation was associated with elevated CSF cAMP in control animals but such biochemical changes were attenuated in I+R animals and reversed to decreases in cAMP concentration in H+I+R animals (control, 1037±58 and 1919±209 fmol/ml; I+R, 1068±33 and 1289±30 fmol/ml; H+I+R, 976±36 and 772±27 fmol/ml for absence and presence of NOC/oFQ 10⁻⁶ M, respectively). Topical 8-Bromo cAMP (10⁻⁸, 10⁻⁶ M) pial dilation was unchanged by I+R but blunted by H+I+R (control, 10±1 and 20±1%; I+R, 11±1 and 20±2%; H+I+R, 0±1 and 0±2%). Pituitary adenylate cyclase activating polypeptide and cromakalim, adenylate cyclase and K_ATP channel activators, respectively, elicited dilation that was blunted by both I+R and H+I+R while NS1619, a K_ca channel activator, elicited dilation that was unchanged by I+R but blunted by H+I+R. These data indicate that impaired NOC/oFQ dilation following I+R results form altered adenylate cyclase and K_ATP channel-dependent mechanisms. These data further indicate that impaired NOC/oFQ dilation following H+I+R results not only from altered adenylate cyclase and K_ATP channel but also from altered cAMP and K_ca channel-dependent mechanisms.     

Stimulus duration modulates the interaction between opioids and nitric oxide in hypoxic pial artery dilation

Since recent studies show that pial artery dilation during a 20 or 40 min hypoxic exposure was less than that observed during a 5 or 10 min exposure, stimulus duration determines the nature of the vascular response to hypoxia. Decremented hypoxic pial dilation during longer exposure periods results, at least in part, from decreased release of methionine enkephalin (Met), an opioid known to contribute to dilation during hypoxia. Nitric oxide and cGMP contribute to both release and the vascular response to this opioid. The present study was designed to determine if the stimulus duration modulates the interaction between opioids and NO in hypoxic pial dilation using newborn pigs equipped with a closed cranial window. Elevation of CSF cGMP during hypoxia (Po2 approximately 35 mmHg) was dependent on stimulus duration (435+/-31, 934+/-46, 747+/-25, and 623+/-17 fmol/ml cGMP during normoxia and after 10, 20, and 40 min of hypoxia). Met-induced pial dilation during hypoxia was also stimulus duration dependent (7+/-1, 10+/-1, and 15+/-1, vs. 4+/-1, 6+/-1, and 8+/-2 vs. 2+/-1, 3+/-1, and 5+/-1% for 10(-10), 10(-8), 10(-6) M Met during normox and after 20, and 40 min of hypoxia). Additionally, the release of cGMP by Met during hypoxia was also stimulus duration dependent (1.8+/-0.1 vs. 1.6+/-0.1 vs. 1.3+/-0.1 fold change in CSF cGMP for 10(-8) M Met during normoxia and after 20 and 40 min of hypoxia). These data indicate that the diminished role of Met in pial dilation during longer hypoxic exposure periods results from a diminished capacity of this opioid to elicit dilation. Such impaired dilation is correlated with diminished stimulated cGMP release. These data also suggest that diminished CSF cGMP release during prolonged hypoxia contributes to decreased release of Met during longer hypoxic periods. Therefore, stimulus duration modulates the interaction between opioids and NO in hypoxic pial artery dilation.     

Differential role of PTK,ERK and p38 MAPK in superoxide impairment of NMDA cerebrovasodilation

Previous studies in piglets have shown that the generation of oxygen free radicals (O(-)(2)) following traumatic brain injury and hypoxia/ischemia contribute to the reversal of N-methyl-D-aspartate (NMDA)-induced pial artery dilation to vasoconstriction. This study determined the contribution of protein tyrosine kinase (PTK) and mitogen-activated protein (MAPK) activation to impairment of NMDA cerebrovasodilation by O(-)(2) in piglets equipped with a closed window. Exposure of the cerebral cortex to a xanthine oxidase O(-)(2) generating system (OX) reversed NMDA (10(-8), 10(-6) M) dilation to vasoconstriction but such impairment was partially prevented by the PTK inhibitor, genistein, the MAPK (ERK isoform) inhibitor, U0126, and the MAPK (p38 isoform) inhibitor, SB203580 (9+/-1 and 15+/-1 vs. -1+/-1 and -1+/-1 vs. 5+/-1 and 9+/-1% for sham control, OX and OX in the presence of genistein, respectively). However, the p38 MAPK inhibitor, SB203580, prevented NMDA dilator impairment significantly less than the ERK MAPK inhibitor, U0126. Similar results were obtained for glutamate. These data show that PTK and MAPK activation by the presence of O(-)(2) contributes to the impairment of NMDA dilation. Furthermore, these data indicate a differential role for ERK and p38 MAPK activation in impairment of NMDA dilation by O(-)(2) in the brain.     

Protein kinase C activation generates superoxide and contributes to impairment of cerebrovasodilation induced by G protein activation after brain injury

Previous studies have observed that activation of protein kinase C (PKC) contributes to generation of superoxide anion (O(-)(2)) after fluid percussion brain injury (FPI). This study was designed to characterize the effects of FPI on the vascular activity of two activators of a pertussis toxin sensitive G protein, mastoparan and mastoparan-7, and the role of PKC dependent O(-)(2) generation in such effects in newborn pigs equipped with a closed cranial window. Mastoparan (10(-8), 10(-6) M) elicited pial artery dilation that was blunted by FPI and partially restored by the PKC inhibitor chelerythrine (10(-7) M) or the O(-)(2) free radical scavengers polyethylene glycol superoxide dismutase and catalase (SODCAT) (9+/-1 and 16+/-1, sham control; 3+/-1 and 5+/-1, FPI; and 7+/-1 and 11+/-1%, FPI SODCAT pretreated). Similar results were observed for mastoparan-7 but the inactive analogue mastoparan-17 had no effect on pial artery diameter. Exposure of the cerebral cortex to a xanthine oxidase O(-)(2) generating system blunted mastoparan induced pial artery dilation similar to FPI (10+/-1 and 17+/-1 vs. 2+/-1 and 3+/-1%). Pertussis toxin (1 microg/ml) exposure blocked mastoparan and mastoparan-7 vasodilation. These data show that pertussis toxin sensitive G protein activation elicits cerebrovasodilation that is blunted following FPI in a PKC dependent manner. These data also show that O(-)(2) generation similarly blunts G protein mediated cerebrovasodilation. These data suggest that PKC dependent O(-)(2) generation contributes to impaired G protein mediated cerebrovasodilation after FPI.     

Protein tyrosine kinase and mitogen-activated protein kinase activation contribute to K(ATP) and K(ca) channel impairment after brain injury

Previous studies have observed that pial artery dilation to activators of the ATP sensitive K (K(ATP)) and calcium sensitive K (K(ca)) channel was blunted following fluid percussion brain injury (FPI) in the piglet. In recent studies in the rat, protein tyrosine kinase (PTK) activation was observed to contribute to K(ATP) channel impairment after FPI, but such a role in K(ca) channel impairment was unclear. This study investigated the role of PTK and mitogen activated protein kinase (MAPK) activation in blunted pial dilation to K(ATP) and K(ca) channel agonists following FPI in piglets equipped with a closed cranial window. Cromakalim and NS1619 (10(-8), 10(-6) M) induced pial artery dilation was blunted after FPI, but partially restored by the PTK inhibitors genistein (10(-6) M) and tyrphostin A23 (10(-5) M) (10+/-1 and 19+/-1%, sham control; 2+/-1 and 4+/-1%, FPI; and 7+/-1 and 11+/-1% FPI-genistein pretreated for NS1619 10(-8), 10(-6) M, respectively). Cromakalim- and NS1619-induced pial dilation was also partially restored after FPI by pretreatment with the MAPK inhibitors U0126 (10(-6) M) and PD98059 (10(-5) M) (12+/-1 and 21+/-1%, sham control; 2+/-1 and 4+/-1%, FPI; and 6+/-1 and 10+/-2%, FPI-U0126 pretreated for NS1619 10(-8), 10(-6) M, respectively). These data suggest that PTK and MAPK activation contribute to K(ATP) and K(ca) channel impairment following FPI.     

Stimulus duration-dependent contribution of k(ca) channel activation and cAMP to hypoxic cerebrovasodilation

Activation of calcium sensitive (K(ca)) K channels and cAMP contribute to pial artery dilation observed during a 10-min exposure to hypoxia. Recent studies show that pial dilation during a 20- or 40-min hypoxic exposure was less than that observed during a 5- or 10-min exposure indicating that stimulus duration determines the nature of the vascular response to hypoxia. The present study was designed to determine if the stimulus duration modulates the contribution of K(ca) channel activation and cAMP-dependent mechanisms to hypoxic pial artery dilation in piglets equipped with a closed cranial window. The K(ca) channel antagonist iberiotoxin had no influence on pial dilation during 5 min of hypoxia (pO(2) approximately 25 mmHg), decremented the dilation during 10- and 20-min exposure, but had no effect on the dilation during a 40-min exposure (33+/-1% vs. 32+/-3%, 33+/-1% vs. 25+/-1%, 23+/-1% vs. 19+/-1%, and 21+/-2% vs. 17+/-2% for 5-, 10-, 20-, and 40-min hypoxic dilations before and after iberiotoxin). NS1619, a K(ca) channel agonist, induced pial dilation during hypoxia that was attenuated by 20- and 40-min but not by 5- and 10-min exposure durations. Similarly, the cAMP antagonist Rp 8-Bromo cAMPs had no influence on pial dilation during 5 min of hypoxia, decremented the dilation during a 10-min exposure, but had no effect on the dilation during a 20- or 40-min exposure (36+/-1% vs. 34+/-2%, 34+/-1% vs. 22+/-1%, 24+/-2% vs. 21+/-2%, and 21+/-2% vs. 19+/-2% for 5-, 10-, 20-, and 40-min hypoxic dilations before and after Rp 8-Bromo cAMPs). Additionally, CSF cAMP was unchanged during 5 min, elevated during 10 min, but such elevations were attenuated during 20- and 40-min hypoxic exposure. Pial vasodilation to a cAMP analogue during hypoxia was attenuated by 20- and 40-min but not by 5- and 10-min hypoxic exposure durations. These data show that K(ca) channel activation and cAMP contribute to hypoxic pial artery dilation in a stimulus duration-dependent manner. These data suggest that diminished pial artery dilation during longer hypoxic exposure results from attenuated K(ca) channel and cAMP-dependent mechanisms.     

Stimulus duration-dependent contribution of Kca channel activation and cAMP to hypoxic cerebrovasodilation

Activation of calcium sensitive (K_ca) K channels and cAMP contribute to pial artery dilation observed during a 10-min exposure to hypoxia. Recent studies show that pial dilation during a 20- or 40-min hypoxic exposure was less than that observed during a 5- or 10-min exposure indicating that stimulus duration determines the nature of the vascular response to hypoxia. The present study was designed to determine if the stimulus duration modulates the contribution of K_ca channel activation and cAMP-dependent mechanisms to hypoxic pial artery dilation in piglets equipped with a closed cranial window. The K_ca channel antagonist iberiotoxin had no influence on pial dilation during 5 min of hypoxia (pO₂≈25 mmHg), decremented the dilation during 10- and 20-min exposure, but had no effect on the dilation during a 40-min exposure (33±1% vs. 32±3%, 33±1% vs. 25±1%, 23±1% vs. 19±1%, and 21±2% vs. 17±2% for 5-, 10-, 20-, and 40-min hypoxic dilations before and after iberiotoxin). NS1619, a K_ca channel agonist, induced pial dilation during hypoxia that was attenuated by 20- and 40-min but not by 5- and 10-min exposure durations. Similarly, the cAMP antagonist Rp 8-Bromo cAMPs had no influence on pial dilation during 5 min of hypoxia, decremented the dilation during a 10-min exposure, but had no effect on the dilation during a 20- or 40-min exposure (36±1% vs. 34±2%, 34±1% vs. 22±1%, 24±2% vs. 21±2%, and 21±2% vs. 19±2% for 5-, 10-, 20-, and 40-min hypoxic dilations before and after Rp 8-Bromo cAMPs). Additionally, CSF cAMP was unchanged during 5 min, elevated during 10 min, but such elevations were attenuated during 20- and 40-min hypoxic exposure. Pial vasodilation to a cAMP analogue during hypoxia was attenuated by 20- and 40-min but not by 5- and 10-min hypoxic exposure durations. These data show that K_ca channel activation and cAMP contribute to hypoxic pial artery dilation in a stimulus duration-dependent manner. These data suggest that diminished pial artery dilation during longer hypoxic exposure results from attenuated K_ca channel and cAMP-dependent mechanisms.     

Cerebral vasoconstriction in response to hypocapnia is maintained after ischemia/reperfusion injury in newborn pigs.

BACKGROUND AND PURPOSE: Hypocapnic cerebral vasoconstriction is used therapeutically to reduce elevated intracranial pressure caused by cerebral edema. Because cerebral ischemia/reperfusion injury causes a selective loss of prostanoid-dependent responses, including vasodilation to hypercapnia, we designed these experiments to examine the effect of ischemia/reperfusion on hypocapnic cerebral vasoconstriction. METHODS: Microvascular responses were studied in 10 newborn pigs (closed cranial window) in response to hyperventilation-induced hypocapnia (PaCO2, 22 +/- 2 mm Hg) both before and 45 minutes after 20 minutes of global cerebral ischemia. Responses to hypercapnia (PaCO2, 63 +/- 3 mm Hg), topical isoproterenol (10(-7) M), and norepinephrine (10(-4) M) were also studied before and after ischemia in the same animals for comparison. RESULTS: Before ischemia/reperfusion, pial arterioles vasoconstricted to hypocapnia (-17 +/- 2%) and norepinephrine (-35 +/- 4%) and vasodilated to CO2 (37 +/- 7%) and isoproterenol (25 +/- 2%). After ischemia/reperfusion, the constriction of pial arterioles to hypocapnia (-19 +/- 2%) was similar to that before ischemia. This is in contrast to the loss of dilation to hypercapnia. Dilation to isoproterenol and constriction to norepinephrine were not affected by ischemia. CONCLUSIONS: Hypocapnic cerebral vasoconstriction is maintained after ischemia/reperfusion. Since prostanoid-dependent responses, such as hypercapnic dilation, are lost following cerebral ischemia, these data suggest that hypocapnic constriction is not dependent on an intact prostanoid system and that cerebral vascular responses to CO2 involve multiple mechanisms, depending on whether CO2 is increasing or decreasing from baseline.     

Adrenomedullin reduces gender-dependent loss of hypotensive cerebrovasodilation after newborn brain injury through activation of ATP-dependent K channels.

Cerebrovascular dysregulation during hypotension occurs after fluid percussion brain injury (FPI) in the newborn pig owing to impaired K channel function. This study was designed to (1) determine the role of gender and K channel activation in adrenomedullin (ADM) cerebrovasodilation, (2) characterize the role of gender in the loss of hypotensive cerebrovasodilation after FPI, and (3) determine the role of gender in the ability of exogenous ADM to modulate hypotensive dysregulation after FPI. Lateral FPI (2 atm) was induced in newborn male and female newborn pigs (1 to 5 days old) equipped with a closed cranial window, n=6 for each protocol. Adrenomedullin-induced pial artery dilation was significantly greater in female than male piglets and blocked by the K(ATP) channel antagonist glibenclamide, but not by the K(ca) channel antagonist iberiotoxin. Cerebrospinal fluid ADM was increased from 3.8+/-0.7 to 14.6+/-3.0 fmol/mL after FPI in female but was unchanged in male piglets. Hypotensive pial artery dilation was blunted to a significantly greater degree in male versus female piglets after FPI. Topical pretreatment with a subthreshold vascular concentration of ADM (10(-10) mol/L) before FPI reduced the loss of hypotensive pial artery dilation in both genders, but protection was significantly greater in male versus female piglets. These data show that hypotensive pial artery dilation is impaired after FPI in a gender-dependent manner. By unmasking a gender-dependent endogenous protectant, these data suggest novel gender-dependent approaches for clinical intervention in the treatment of perinatal traumatic brain injury.     

Cyclooxygenase-2 inhibitor NS398 preserves neuronal function after hypoxia/ischemia in piglets.

Anoxic stress attenuates NMDA-induced pial arteriolar dilation via a mechanism involving actions of cyclooxygenase (COX)-derived reactive oxygen species (ROS). We examined whether the selective COX-2 inhibitor NS398 would protect neuronal function after global hypoxia/ischemia (H/I) in piglets. Pial arteriolar responses to NMDA (10-100 micromol/l) were determined using intravital microscopy in anesthetized piglets before and 1 h after H/I. Study groups received vehicle, 0.3, 1, or 5 mg/kg NS398, or 0.3 mg/kg indomethacin (n = 7, 6, 6, 5 and 8, respectively) i.v. 20 min prior to H/I. H/I reduced NMDA- induced dilation to 44 +/- 6% (100 micromol/l NMDA, mean +/- s.e.m.) of the pre-ischemic response in vehicle animals (p < 0.05). However, NS398 dose-dependently protected arteriolar dilation to NMDA (77 +/- 8, 81 +/- 16, and 102 +/- 10% preservation at 0.3, 1 and 5 mg/kg, respectively). Indomethacin caused similar preservation. However, indomethacin but not NS398 reduced serum thromboxane B(2) levels to undetectable values. In conclusion, COX-2 appears to be a major source of ROS in the piglet cerebral cortex after H/I.     

Plasminogen activators contribute to age-dependent impairment of NMDA cerebrovasodilation after brain injury

Previous studies have observed that fluid percussion brain injury (FPI) impaired NMDA induced pial artery dilation in an age-dependent manner. This study was designed to investigate the contribution of plasminogen activators to impaired NMDA dilation after FPI in newborn and juvenile pigs equipped with a closed cranial window. In the newborn pig, NMDA (10(-8), 10(-6) M) induced pial artery dilation was reversed to vasoconstriction following FPI, but pretreatment with the plasminogen activator inhibitor PAI-1 derived hexapeptide (EEIIMD) (10(-7) M) prevented post injury vasoconstriction (9 +/- 1 and 16 +/- 1, vs. -6 +/- 2 and-11 +/- 3, vs. 5 +/- 1 and 9 +/- 1% for responses to NMDA 10(-8), 10(-6) M prior to FPI, after FPI, and after FPI in EEIIMD pretreated animals, respectively). In contrast, in the juvenile pig, NMDA dilation was only attenuated following FPI and EEIIMD pretreatment partially prevented such inhibition (9 +/- 1 and 16 +/- 1 vs. 2 +/- 1 and 4 +/- 1 vs. 5 +/- 1 and 7 +/- 1% for responses to NMDA prior to FPI, after FPI, and after FPI in EEIIMD pretreated animals, respectively). Additionally, EEIIMD blunted age-dependent pial artery vasoconstriction following FPI. EEIIMD blocked dilation to the plasminogen activator agonists uPA and tPA while responses to SNP and papaverine were unchanged. Pretreatment with suPAR, which blocked dilation to uPA, elicited effects on pial artery diameter and NMDA vascular activity post FPI similar to that observed with EEIIMD. These data show that EEIIMD and suPAR partially prevented FPI induced alterations in NMDA dilation and reductions in pial artery diameter. EEIIMD and suPAR are efficacious and selective inhibitors of plasminogen activator induced dilation. These data suggest that plasminogen activators contribute to age-dependent impairment of NMDA induced dilation following FPI.     

Endothelin-1 contributes to normocapnic hyperoxic pial artery vasoconstriction

The present study was designed to determine if hyperoxia elicits pial artery vasoconstriction and to characterize the contribution of endothelin-1 (ET-1) to that vascular response in newborn pigs equipped with a closed cranial window. Hyperoxic conditions were established by ventilating the piglets with 100% O(2) during normocapnia and concomitantly topically applying artificial CSF that had been bubbled with 100% O(2). Hyperoxia elevated CSF ET-1 from 23+/-1 to 45+/-4 pg/ml. Hyperoxia also elicited pial artery vasoconstriction that was attenuated by BQ123 (10(-6) M), an ET-1 antagonist (-15+/-1 vs. -5+/-1%). These data indicate that ET-1 contributes to hyperoxic pial artery vasoconstriction.