Contribution of Kca channel activation to hypoxic cerebrovasodilation does not involve NO

Although nitric oxide (NO) and calcium sensitive K⁺ channel (K_ca) activation contribute to hypoxic pial artery dilation in the piglet, responses to the NO releasers SNP and SNAP are unchanged by the K_ca channel antagonist iberiotoxin. These data suggest that NO does not elicit dilation via K_ca channel activation. The present study was designed to determine if dilation by K_ca channel activation is mediated by NO in newborn pigs equipped with a closed cranial window. NS1619 (10⁻⁸, 10⁻⁶ M), a K_ca agonist, produced dilation that was unchanged by the NO synthase inhibitor, -NNA (10⁻⁶ or 10⁻³ M) (11±1 and 20±1 vs. 11±1 and 18±1% before and after -NNA 10⁻³ M). NS1619 dilation also was not associated with increased CSF cGMP and was unchanged by Rp 8-Bromo cGMPs, a cGMP antagonist (9±1 and 17±1 vs. 9±1 and 16±2% before and after Rp 8-Bromo cGMPs 10⁻⁵ M). Iberiotoxin (10⁻⁷ M) attenuated hypoxic dilation but hypoxia associated CSF cGMP release was unchanged (418±11 and 897±31 vs. 419±10 and 896±25 fmol/ml for control and moderate hypoxia before and after iberiotoxin). Coadministration of -NNA with iberiotoxin further decremented hypoxic pial dilation and blocked the hypoxia-associated rise in CSF cGMP. These data show that pial artery dilation by K_ca channel activation is not mediated by NO/cGMP. Further, these data suggest that NO and the K_ca channel act at different sites in their contributions to hypoxic pial artery dilation.     

Role of nitric oxide, adenosine,N-methyl-d-aspartate receptors, and neuronal activation in hypoxia-induced pial arteriolar dilation in rats

In this study, we tested the hypothesis that nitric oxide (NO) and adenosine (ADO) are the principal mediators of severe hypoxia-induced vasodilation. In addition, we examined whether activation ofN-methyl-d-aspartate (NMDA) receptors and/or perivascular nerves plays a role. A closed cranial window and intravital microscopy system was used to monitor diameter changes in pial arterioles ( 40 μm) in anesthetized rats. The relative contributions of ADO, NMDA, NO, and neuronal activation to hypoxic cerebrovasodilation were assessed using the blockers 8-sulfophenyltheophylline (8-SPT), MK-801, nitro-l-arginine methylester (LNAME), and tetrodotoxin (TTX). Two experimental series were studied. In the first, we tested the effects of NOS inhibition, via topical L-NAME (1 mM), on moderate (PaO₂ ≈ 46 mmHg)then severe (PaO₂ ≈ 34 mmHg) hypoxia-induced dilation. To confirm that L-NAME was affecting specifically NO-dependent responses, we also examined, in each experiment, the vasodilatory responses to topical applications of NOS-dependent (adenosine diphosphate (ADP); acetylcholine (ACh)(and -independent (sodium nitroprusside (SNP)) agents, in the presence of L-NAME or, in controls, the presence of D-NAME or no added analogue. In the second series, topical suffusions of ADP, ADO, and NMDA were sequentially applied, followed by 5 min exposure to severe hypoxia (PaO, ≈ 32 mmHg). Following return to normoxia, a suffusion of either 8-SPT (10 μM), MK-801 (10 μM), TTX (1 μM), or 8-SPT + MK-801 was initiated (or, in controls, application of a drug-free suffusate was maintained), and the above sequence repeated. In control, TTX, and 8-SPT + MK-801 experiments, baseline conditions were then restored and hypercapnia (PaCO₂ = 70–85 mmHg) was imposed. In the series 1 control groups, moderate and severe hypoxia elicited ≈ 20% and 35–40% increases in diameter, respectively. L-NAME attenuated ADP- and ACh-induced dilations, did not alter the arteriolar responses to SNP or moderate hypoxia, but prevented further dilation upon imposition of severe hypoxia. This suggested that 45–50% of the severe hypoxia response was NO-dependent. In series 2, 8-SPT blocked the adenosine response and reduced severe hypoxia-induced dilation by 46%. MK-801 predictably blocked NMDA-induced relaxation and reduced the hypoxic response by 42%. When combined, 8-SPT and MK-801 affected hypoxic vasodilation additively. After TTX, the ADP and ADO responses were normal, but NMDA and hypoxia responses were completely blocked. Hypercapnia-induced dilation was unaffected by TTX or 8-SPT + MK-801. The results imply that severe hypoxia-induced release of NO and ADO, and the accompanying pial arteriolar dilation, are wholly dependent on the capacity to generate action potentials in perivascular nerves. The similarity of the L-NAME and MK-801 effects on hypoxic cerebrovasodilation suggests that the NO-dependency, to a large degree, derives from NMDA receptor activation.     

Role of nitric oxide, adenosine,N-methyl-d-aspartate receptors, and neuronal activation in hypoxia-induced pial arteriolar dilation in rats

In this study, we tested the hypothesis that nitric oxide (NO) and adenosine (ADO) are the principal mediators of severe hypoxia-induced vasodilation. In addition, we examined whether activation ofN-methyl-d-aspartate (NMDA) receptors and/or perivascular nerves plays a role. A closed cranial window and intravital microscopy system was used to monitor diameter changes in pial arterioles (∼ 40 μm) in anesthetized rats. The relative contributions of ADO, NMDA, NO, and neuronal activation to hypoxic cerebrovasodilation were assessed using the blockers 8-sulfophenyltheophylline (8-SPT), MK-801, nitro-l-arginine methylester (LNAME), and tetrodotoxin (TTX). Two experimental series were studied. In the first, we tested the effects of NOS inhibition, via topical L-NAME (1 mM), on moderate (PaO₂ ≈ 46 mmHg)then severe (PaO₂ ≈ 34 mmHg) hypoxia-induced dilation. To confirm that L-NAME was affecting specifically NO-dependent responses, we also examined, in each experiment, the vasodilatory responses to topical applications of NOS-dependent (adenosine diphosphate (ADP); acetylcholine (ACh)(and -independent (sodium nitroprusside (SNP)) agents, in the presence of L-NAME or, in controls, the presence of D-NAME or no added analogue. In the second series, topical suffusions of ADP, ADO, and NMDA were sequentially applied, followed by 5 min exposure to severe hypoxia (PaO, ≈ 32 mmHg). Following return to normoxia, a suffusion of either 8-SPT (10 μM), MK-801 (10 μM), TTX (1 μM), or 8-SPT + MK-801 was initiated (or, in controls, application of a drug-free suffusate was maintained), and the above sequence repeated. In control, TTX, and 8-SPT + MK-801 experiments, baseline conditions were then restored and hypercapnia (PaCO₂ = 70–85 mmHg) was imposed. In the series 1 control groups, moderate and severe hypoxia elicited ≈ 20% and 35–40% increases in diameter, respectively. L-NAME attenuated ADP- and ACh-induced dilations, did not alter the arteriolar responses to SNP or moderate hypoxia, but prevented further dilation upon imposition of severe hypoxia. This suggested that 45–50% of the severe hypoxia response was NO-dependent. In series 2, 8-SPT blocked the adenosine response and reduced severe hypoxia-induced dilation by 46%. MK-801 predictably blocked NMDA-induced relaxation and reduced the hypoxic response by 42%. When combined, 8-SPT and MK-801 affected hypoxic vasodilation additively. After TTX, the ADP and ADO responses were normal, but NMDA and hypoxia responses were completely blocked. Hypercapnia-induced dilation was unaffected by TTX or 8-SPT + MK-801. The results imply that severe hypoxia-induced release of NO and ADO, and the accompanying pial arteriolar dilation, are wholly dependent on the capacity to generate action potentials in perivascular nerves. The similarity of the L-NAME and MK-801 effects on hypoxic cerebrovasodilation suggests that the NO-dependency, to a large degree, derives from NMDA receptor 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.     

Actinomycin D blocks interleukin-1α-induced pial arteriolar dilation and increased prostanoid production in newborn pigs

Effects of protein synthesis and cyclooxygenase inhibitors on interleukin-1α (IL-1α)- and histamine-induced pial arteriolar dilation and cerebrospinal fluid (CSF) prostanoid increases were examined in anesthetized piglets using closed cranial windows. Topical IL-la (10.8 μg) increased pial arteriolar diameter from 15 to 30 min after its infusion, and enhanced CSF prostanoids. Topical protein synthesis inhibitor, actinomycin D, at a concentration of 10⁻⁸ M attenuated and 10⁻⁸ M completely blocked both IL-1α-induced vasodilation and CSF prostanoid increase. Inhibition of prostaglandin H synthases with indomethacin blocked both vasodilation and CSF prostanoid increase by IL-1α. Topical histamine (10⁻⁶ M) also increased pial arteriolar diameter and CSF prostanoids but without the delay seen between IL-1α infusion and responses. These histamine effects were not modified by coinfusion of actinomycin D but blocked by indomethacin. These results suggest that, although IL-1α and histamine do share the same mechanism insofar as activation of prostaglandin synthesis is concerned, an additional step appears to be involved for IL-1α, likely involving de novo protein synthesis.     

Different mechanisms of -arginine induced dilation of brain arterioles in normotensive and hypertensive rats

We evaluated the response of pial arterioles to -arginine in anesthetized normotensive rats and spontaneously hypertensive rats equipped with a closed cranial window. Topical application of 10⁻⁶ − 10⁻⁴ mol/l -arginine, which is known to be the endogenous substrate for the synthesis of nitric oxide, induced dose-dependent arteriolar vasodilation. The response was more pronounced in hypertensive than in normotensive rats (at the concentration of 10⁻⁴ mol/l -arginine: 18.3 ± 3.3% vs. 6.7 ± 1.7%, respectively, means ± S.E.). The stereoisomer -arginine had no effect in hypertensive rats. Topical application of the nitric oxide synthase inhibitor N-nitro- -arginine converted -arginine-induced dilation to constriction in normotensive and hypertensive rats. The cyclooxygenase inhibitor indomethacin (5 μg/ml cerebrospinal fluid) also blocked the dilation in both strains. Photochemical endothelial injury blocked -arginine-induced dilation in normotensive rats, but only partly antagonized the response in hypertensive animals. Intravenous or topical pretreatment with the free radical scavenger superoxide dismutase significantly reduced the dilating response to 10⁻⁴ mol/l -arginine in hypertensive rats. Superoxide dismutase did not significantly change the response to -arginine in normotensive animals. It is concluded that nitric oxid formation in the endothelium and liberation of cyclooxygenase products cause -arginine-induced dilation in normotensive rats. While nitric oxide and cyclooxygenase products are also involved in -arginine-induced dilation in spontaneously hypertensive rats, superoxide radicals contribute to the enhanced response in this strain. This mechanism appears to be specific for the hypertensive animals and is only partly dependent on an intact endothelium.     

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.     

Vasopressin impairs KATP and Kca channel function after brain injury

This study was designed to characterize the role of vasopressin in impaired pial artery dilation to activators of the ATP sensitive K (K_ATP) and calcium sensitive K (K_ca) channel following fluid percussion brain injury (FPI) in newborn pigs equipped with a closed cranial window. Topical vasopressin was coadministered with the K_ATP and K_ca channel agonists cromakalim and NS1619 in a concentration approximating that observed in CSF following FPI. Vasopressin so administered attenuated pial artery dilation to these K⁺ channel activators under conditions of equivalent baseline diameter during non injury conditions (13±1 and 23±1 vs. 4±1 and 10±2% for cromakalim 10⁻⁸, 10⁻⁶ M before and after vasopressin, respectively). Attenuated responses were fully restored when these agonists were coadministered with vasopressin and the vasopressin antagonist [l-(β-mercapto-β,β-cyclopentamethylene propionic acid) 2-(o-methyl)-Tyr-AVP] (MEAVP). Cromakalim and NS1619 induced pial artery dilation was attenuated following FPI and MEAVP preadministration partially prevented such impairment (13±1 and 23±1, sham control; 2±1 and 5±1, FPI; and 9±1 and 15±2%, FPI-MEAVP pretreated for responses to cromakalim 10⁻⁸, 10⁻⁶ M, respectively). These data show that vasopressin blunts K_ATP and K_ca channel mediated cerebrovasodilation. These data suggest that vasopressin contributes to impaired K_ATP and K_ca channel function after brain injury.     

Hypotension dilates pial arteries by KATP and Kca channel activation

Hypotension induced pial artery dilation is prostaglandin-dependent in the newborn pig. Prostaglandins, in turn, elicit vasodilation through cGMP and cAMP dependent mechanisms and K⁺ channel activation contributes to cyclic nucleotide induced vasodilation. The present study was designed to characterize the role of ATP sensitive (K_ATP) and calcium sensitive (K_ca) channel activation in hypotension induced pial artery dilation in newborn pigs equipped with a closed cranial window. Glibenclamide and iberiotoxin, K_ATP and K_ca channel antagonists, attenuated hypotension induced dilation (36±1 vs. 14±2% before and after iberiotoxin). Combined administration of these K⁺ channel antagonists eliminated the vascular response. Hypotension induced dilation was associated with elevated cerebrospinal fluid (CSF) cAMP but not cGMP concentration (1023±29 vs. 1566±39 fmol/ml for cAMP). L-NNA, a nitric oxide (NO) synthase inhibitor, and Rp 8-Br cGMPs, a protein kinase G inhibitor, had no effect but Rp 8-Br cAMPs, a protein kinase A inhibitor, attenuated hypotensive dilation (35±1 vs. 16±2% before and after Rp 8-Br cAMPs). Dilation by the cAMP analogue 8-Bromo cAMP (10⁻⁸, 10⁻⁶ M) was attenuated by glibenclamide and iberiotoxin (8±1 and 17±1 vs. 4±1 and 9±1% before and after glibenclamide). These data show that both K_ATP and K_ca channel activation contribute to hypotension induced dilation. These data suggest that dilation during hypotension results from the sequential release of prostaglandins and cAMP, which, in turn, activates both the K_ATP and K_ca channel.     

Role of activation of calcium-sensitive K channels and cAMP in opioid-induced pial artery dilation

The present study was designed to investigate the role of activation of Kca⁺² channesl and cAMP in opioid-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Methionine enkephalin, an endogenous μ agonist, elicited dilation that was modestly attenuated by the Kca⁺² channel antagonist, iberiotoxin (10⁻⁷ M) (7 ± 1, 11 ± 1 and 16 ± 1 vs 4 ± 1, 7 ± 1, and 11 ± 1% for methionine enkephalin 10⁻¹⁰, 10⁻⁸, 10⁻⁶ M in the absence and presence of iberiotoxin, respectively). Dilator responses to leucine enkephalin and dynorphin, endogenous δ and κ agonists, as well as the synthetic analogues DAMGO, DPDPE, deltorphin and U50488H all were similarly attenuated by iberiotoxin. Dilation in response to methionine enkephalin was accompanied by increased CSF cAMP concentration (1170 ± 21, 1358 ± 22, 1473 ± 26, and 1575 ± 24 fmol/ml for control, 10⁻¹⁰, 10⁻⁸, 10⁻⁶ M methionine enkephalin, respectively). Methionine enkephalin-induced dilation was attenuated by Rp 8-bromo cAMPS (10⁻⁵ M), a cAMP antagonist (7 ± 1, 11 ± 1 and 17 ± 1 vs. 2 ± 1, 4 ± 1, and 7 ± 1% for methionine enkephalin 10⁻¹⁰, 10⁻⁸, and 10⁻⁶ M in the absence and presence of Rp 8-bromo cAMPs, respectively). Dilation by the other endogenous and synthetic opioid analogues was also accompanied by elevated CSF cAMP and attenuated by Rp 8-bromo cAMPs. Additionally, dilation produced by the cAMP analogue, 8-bromo cAMP, was blunted by iberiotoxin. These data show that both cAMP and activation of Kca⁺² channels contribute to opioid-induced pial artery dilation. Further, these data suggest that opioids elicit dilation, at least in part, via the sequential release of cAMP and subsequent activation of Kca⁺² channels by this second messenger.     

Nitric oxide contributes to opioid release from glia during hypoxia

Activation of neuronal nitric oxide (NO) synthase contributes to increased CSF concentrations of the opioids methionine enkephalin and leucine enkephalin during hypoxia in the newborn pig. NO and these opioids, in turn, contribute to hypoxic pial artery dilation. However, the cellular site of origin for opioids detected in CSF cannot be determined using this in vivo model. The present study, therefore, was designed to determine if NO contributes to opioid release from piglet glia grown in primary culture. Glial cell cultures produced more methionine enkephalin than leucine enkephalin under basal conditions. Administration of SNP and 8-Br cGMP to glial cells increased release of both opioids (471±58 vs. 1181±148 pg/mg protein methionine enkephalin before and after SNP 10⁻⁶ M). SNP also increased release of cGMP. Exposure of piglet glial cells to lower than normal O₂ increased the release of both opioids (503±61 vs. 1488±186 pg/mg protein methionine enkephalin before and after hypoxia, (P_O2≈15 mmHg). Hypoxia also increased the release of cGMP from glia while the NO synthase inhibitor N-nitro- -arginine blocked that release. These data show that NO/cGMP and hypoxia release opioids from glia. Additionally, hypoxia releases NO/cGMP from glia. These data therefore suggest that NO contributes to opioid release from glia during hypoxia.     

NMDA induces a biphasic change in intracellular pH in rat hippocampal slices

As alterations in intracellular pH (pH_i) tend to exert a profound effect on the properties of cells, this study was undertaken to examine NMDA-induced changes in pH_i in rat hippocampal slices using the BCECF fluorescent technique. The ‘resting' pH_i in the CA1 pyramidal cell layers was 6.93±0.07 (mean±S.D., n=72 slices) in 25 mM HCO₃⁻/5% CO₂-buffered solution at 37°C. Exposure of hippocampal slices to NMDA in the range of 10–1000 μM produced a biphasic change in pH_i: an initial transient alkaline shift was followed by a long-lasting acid shift. Dizocilpine (10 μM) but not CNQX (40 μM) blocked the NMDA-induced changes in pH_i. In 0 Ca medium (0 mM Ca²⁺ supplemented 1 mM EGTA, referred to as 0 Ca), pH_i acid shift caused by NMDA (20 μM) declined by about 11%, whereas the initial alkaline shift almost completely disappeared. In an independent experiment, the NMDA-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i) was reduced by more than 80% in 0 Ca medium. Glucose substitution using equimolar pyruvate (as an energy-yielding substrate) suppressed this NMDA-induced pH_i acid shift by two-thirds, while the NMDA-induced pH_i alkaline shift was enhanced. Fluoride (10 mM), a glycolytic inhibitor, abolished NMDA-induced pH_i acid shift. Furthermore, the lactate content of hippocampal slices was markedly increased following exposure to NMDA. In conclusion, activation of NMDA receptors in rat hippocampal slices evokes a biphasic change in pH_i. The initial alkaline shift is suggested to be associated with calcium influx, and the following acid shift may be caused by an increase in lactate production through the acceleration of glycolysis, as well as the increased [Ca²⁺]_i. The pH_i acid shift produced by the increased lactate may contribute to proton modulation of the NMDA receptor and NMDA-induced cell injury or death.     

Role of impaired cAMP and calcium-sensitive K channel function in altered cerebral hemodynamics following brain injury

Previous studies have shown that pial arteries constricted and responses to dilator opioids were blunted after fluid percussion injury (FPI) in newborn pigs. Membrane potential of vascular muscle is a major determinant of vascular tone and activity of K⁺ channels is a major regulator of membrane potential. Recent data show that opioids elicit dilation via the sequential production of cAMP and subsequent activation of calcium-sensitive K⁺ (

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) channels by this second messenger. The present study was designed to investigate the effect of FPI on cAMP and

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channel function. Chloralose-anesthetized piglets equipped with a closed cranial window were connected to a percussion device consisting of a saline-filled cylindrical reservoir and a metal pendulum. Brain injury of moderate severity (1.9–2.1 atm) was produced by allowing the pendulum to strike a piston on the cylinder. FPI blunted dilation to the cAMP analogs 8-Bromo cAMP and Sp 8-Bromo cAMPs (10⁻⁸, 10⁻⁶ M), (9±1 and 16±1 vs. 2±1 and 3±1% dilations to 8-Bromo cAMP before and after FPI, respectively, n=8). Similarly, FPI attenuated dilation to pituitary adenylate cyclase activating peptide (PACAP), an endogenous activator of adenylate cyclase, and NS 1619, a

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channel agonist (9±1 and 16±1 vs. 3±1 and 5±1% for NS 1619 10⁻⁸, 10⁻⁶ M before and after FPI, respectively, n=8). Moreover, FPI attenuated PACAP, methionine enkephalin, leucine enkephalin, and dynorphin induced elevations in CSF cAMP concentration (940±2, 1457±50, and 2191±53 vs. 810±17, 1033±36, and 1218±49 fmol/ml for control, PACAP 10⁻⁸, 10⁻⁶ M before and after FPI, respectively, n=8). These data show that cAMP and

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channel function is impaired after FPI. Further these data suggest that impaired cAMP and

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channel function contribute to altered cerebral hemodynamics following FPI.     

Interactions of dextromethorphan with theN-methyl-d-aspartate receptor-channel complex: single channel recordings

The actions of dextromethorphan (DXM) on the 50 pS conductance state of theN-methyl-d-aspartate (NMDA) receptor-operated channel were studied using outside-out patches obtained from cultured rat hippocampal pyramidal neurons. DXM (5–50 μM) had no effect on the amplitudes of unitary currents but caused concentration-dependent reductions in channel mean open times and the frequency of channel openings. Channel open probability was reduced in a concentration-dependent manner by DXM and was one-half of the control value at a DXM concentration of 6 μM, with the patch potential held at −60 mV. An IC₅₀ value of 4 μM was obtained for the reduction by DXM of NMDA-evoked rises in [Ca²⁺]_i in cultured rat hippocampal pyramidal neurons loaded with Fura-2. The results were consistent with drug block of the open NMDA channel with an onward (blocking) rate constant of 7.7 × 10⁶ M⁻¹ · s⁻¹ (at −60 mV). The estimated unblocking rate constant was about 10 s⁻¹, a value considerably higher compared to the off-rate constant found for dizocilpine block of the NMDA channel.     

Receptor types mediating the excitatory actions of exogenousl-aspartate andl-glutamate in rat olfactory cortex

Changes in potential between the pial and cut surfaces of rat olfactory cortex slices evoked by N-methyl-d-aspartate (NMDA), quisqualate, kainate,l-glutamate andl-aspartate and also by γ-aminobutyric acid (GABA) have been monitored using extracellular electrodes. All agonists produced a pial-negative potential response when superfused onto the pial surface, GABA,l-aspartate andl-glutamate being less potent than the others. Repeated applications of NMDA, but not of the other agonists, led to a progressive reduction in response to approximately 30% of the initial depolarization. The responses to NMDA (100 μM) were selectively abolished by(±)2-amino-5-phosphonopentanoic acid (APP; 100 μM) while depolarizations evoked byl-glutamate andl-aspartate (both at 10 mM) were only antagonized by21 ± 2 (n = 12) and36 ± 3 (n = 12) percent respectively (means ± S.E.M.). γ-d-Glutamylglycine (γ-DGG; 1 mM) and(±)cis-2,3-piperidine dicarboxylate (cis-PDA; 2 and 5 mM), in addition to antagonizing responses to NMDA, also partially blocked quisqualate- and kainate-evoked depolarizations. When a mixture of APP (100 μM), γ-DGG (1 mM) and cis-PDA (5 mM) was applied to preparations, although NMDA receptors were completely blocked and responses to both quisqualate and kainate antagonized by approximately 80%,l-glutamate andl-aspartate evoked depolarizations were only reduced by51 ± 7 (n = 4) and 49 ± 4 (n = 4) percent respectively (means ± S.E.M.). The results are discussed in terms of the contributions made by NMDA, quisqualate and kainate receptors to the composite responses evoked byl-aspartate andl-glutamate.     

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg²⁺, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 μM ACPD and in 7 of 12 cells with 0.5 μM ACPD. The EPSP amplitudes with 5 and 0.5 μM ACPD were 142±10% (mean±S.E.M., n=19) and 130±13% (n=12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 μM ACPD were not significantly different in cells perfused with the low Ca²⁺-high Mg²⁺ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence ofd-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg²⁺. Five μM ACPD potentiated monosynaptic EPSPs in 4 of 6 cells with 1 mM Mg²⁺ in the solution and in 2 of 17 cells with 4 mM Mg²⁺, and the EPSP amplitude was 123±9% (n=6) and 98±3% (n=17) of those before application of ACPD, respectively. These results suggest that activation of metabotropic glutamate receptors potentiates LC-EPSPs via mechanisms sensitive to Mg²⁺ and may work as a positive feedback mechanism at the excitatory amino acid-mediated synapses between the descending fibers and lumbar spinal motoneurons.     

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.     

N-methyl-d-aspartate (NMDA) stimulates release of α-MSH from the rat hypothalamus through release of nitric oxide

Superfusion of rat hypothalamic slices with 10⁻⁴ MN-methyl-d-aspartic acid (NMDA) resulted in increased release of α-melanocyte-stimulating hormone (α-MSH). Peptide release was blocked by 10⁻⁶ MN^G-nitro-l-arginine methyl ester (l-NAME) a specific competitive inhibitor of nitric oxide synthase but not by the inactive enantiomerd-NAME at 10⁻⁶ M. The inhibition byl-NAME was reversed by the addition of 10⁻⁵ Ml-arginine, an excess of enzyme substrate. Release of nitric oxide products into tissue superfusates was stimulated by a 50 mM concentration of potassium ions and by 10⁻⁴ M NMDA. Potassium-stimulated release was blocked byl-NAME. Basal, potassium-stimulated and NMDA-stimulated release of nitric oxide products were significantly inhibited by the NMDA-receptor antagonistd-(−)-2-amino-5-phosphopentanoic acid (AP5) at 10⁻⁴ M and by the NMDA-channel blocker ketamine at 10⁻⁴ M. We conclude that nitric oxide mediates the stimulatory action of glutamic acid ont he release of α-MSH from the rat hypothalamus.     

Excitatory action of N-methyl- -aspartate on the rat locus coeruleus is mediated by nitric oxide: an in vivo voltammetric study

Biochemical, electrophysiological and behavioural studies have provided evidence that activation of N-methyl- -aspartate (NMDA) receptors contributes to the hyperactivity of noradrenergic neurons of the locus coeruleus (LC) in precipitated opioid withdrawal. Recently, it was demonstrated that central administration of nitric oxide (NO) synthase inhibitors suppresses this hyperactivity suggesting that NO mediates the NMDA receptor activation of LC in opioid withdrawal. Using a combination of microdialysis and in vivo voltammetry, this study examined whether local application of NMDA to the LC in opioid naive animals mimics the NO-dependent LC response seen in opioid withdrawal. In the urethane anaesthetized rat, perfusion of the LC (2 μl min⁻¹) with a solution of NMDA (5 mmol) via a microdialysis probe for 9 min resulted in a rapid and robust increase (290.1±32.2% above baseline) in the catechol oxidation current (CA·OC) recorded from the LC using differential normal pulse voltammetry (DNPV). The NMDA microdialysis also produced a large increase in the blood pressure (150.4±6.9% above baseline). An injection of the non-competitive NMDA receptor antagonist (+)MK-801 (0.5 mg kg⁻¹ i.v.), given 45 min after the start of NMDA application, rapidly returned both the CA·OC signal and the blood pressure response to baseline levels. Pretreatment of animals with intraventricular nitric oxide synthase (NOS) inhibitor, N^ω-nitro- -arginine methyl ester ( -NAME) (100 μg) significantly inhibited NOS activity in the LC, PAG-PVG and cerebellum. This dose of -NAME, administered prior to application of NMDA by microdialysis abolished the NMDA-induced rise in the CAOC recorded in the LC and the increase in systolic blood pressure. The results show that in voltammetry experiments, NMDA produces hyperactivity of LC and hypertension, responses that are dependent upon the synthesis of NO. Thus, in opioid naive rats, regional NMDA application via microdialysis mimics characteristics of the LC response that occur during the antagonist-precipitated opioid withdrawal.     

Neuronal plasticity in hippocampal mossy fiber–CA3 synapses of mice lacking the inositol-1,4,5-trisphosphate type 1 receptor

In the present study, we used inositol-1,4,5-trisphosphate (IP₃) type 1 receptor (IP₃R1) knockout mice to examine the role of this receptor in the induction of LTP, LTD, and DP at mossy fiber–CA3 synapses. No difference in synaptically induced field-EPSPs was seen between the wild-type (IP₃R1^+/+) and IP₃R1 knockout mice (IP₃R1^−/−), showing that basic synaptic transmission does not involve IP₃R1 activation. Tetanus induced LTP in both wild-type and IP₃R1^−/− mice, but the magnitude of LTP was significantly greater in IP₃R1^−/− mice (149.8±3.5%, mean±S.E.M., n=15) than in wild-type mice (132.4±1.5%, n=17), suggesting that the IP₃R1 has a suppressive effect on LTP induction. To determine whether this effect involved N-methyl- -aspartate receptor (NMDAR)-dependent LTP, the effect of tetanus was tested in the present of the NMDAR antagonist, -AP5 (50 μM); under these conditions, the LTP in both IP₃R1^−/− and IP₃R1^+/+ mice was not significantly reduced. In addition, group I mGluR activation was shown to be necessary for LTP induction, as the LTP was almost blocked by the group I mGluR antagonist, RS-4CPG (500 μM) in both IP₃R1^−/− (117.6±1.7%, n=8) and IP₃R1^+/+ (116.9±1.8%, n=5) mice. The IP₃R1 also plays an essential role in LTD induction, as low-frequency stimulation (LFS) failed to induce LTD in the mutant mice (104.5±2.1%, n=10). DP was induced in both IP₃R1^−/− and wild-type mice.