Cloning of cyclooxygenase-1b (putative COX-3) in mouse.

OBJECTIVES: To clone and sequence cyclooxygenase-1b (COX-1b, also known as COX-3) mRNA and to generate an antibody against the mouse COX-1b protein and to demonstrate its existence in vivo in mouse tissues. ANIMALS: 10 C57BL/6 mice, 4 COX-1 knockout mice and 4 COX-1 wild type mice were used. METHODS: COX-1b mRNA sequence was determined by RT-PCR amplification using specific primers followed by DNA sequencing. COX-1b protein expression was determined by Western blotting. RESULTS: The mouse COX-1b mRNA is a splice variant of the COX-1 mRNA generated by the retention of intron-1. COX-1b mRNA encodes a 127 amino acid protein with no similarity with known COX sequences. We generated an anti-mouse COX-1b antibody and demonstrated the existence of COX-1b protein in vivo with the highest expression in kidney, heart, and neuronal tissues. We also detected COX-1b mRNA and protein expression in COX-1 knockout mice. CONCLUSIONS: In mouse, COX-1b encodes a protein with a completely different amino acid sequence than COX-1 or COX-2; therefore it is improbable that COX-1b in this species plays a role in prostaglandin-mediated fever and pain. In addition, the COX-1(-/-) mouse is not a COX-1b(-/-) mouse, therefore it cannot be used to elucidate the function of the COX-1b protein.

Cyclooxygenase-2 mediates the development of cortical spreading depression-induced tolerance to transient focal cerebral ischemia in rats.

We examined the role of cyclooxygenase-2 in the development of ischemic tolerance induced by cortical spreading depression against transient, focal brain ischemia. Cortical spreading depression was continuously induced for 2 h with topical KCl (13+/-1 depolarizations/2 h) in male Wistar rats. At 1, 2, 3, 4, and 5 days following recovery, the middle cerebral artery was transiently occluded for 120 min. Four days later, the animals were killed and infarct volume was determined. Additionally, cyclooxygenase-2 levels in the cerebral cortex and 15 deoxy-Delta(12, 14) PGJ2 levels in cerebrospinal fluid were determined at these times with Western blotting and immunoassay, respectively. Infarct volume was reduced compared with non-cortical spreading depression control animals (274.3+/-15.3 mm3) when cortical spreading depression was performed 3 and 4 days before middle cerebral artery occlusion (163.9+/-14.2 mm3, 154.9+/-14.2 mm3) but not at 1, 2 and 5 days (280.4+/-17.3 mm3, 276.3+/-16.9 mm3 and 268.5+/-17.3 mm3). Cyclooxygenase-2 levels increased most dramatically starting at 2 days, peaked at 3 days, and started to return toward baseline at 4 days after cortical spreading depression. 15 Deoxy-Delta(12, 14) PGJ2 levels increased from 134.7+/-83 pg/ml at baseline to 718+/-98 pg/ml at 3 days. Administration of N-[2-cyclohexyloxy-4-nitrophenyl] methanesulphonamide (10 mg/kg, i.v.), a selective cyclooxygenase-2 inhibitor, at 1 h prior to middle cerebral artery occlusion in cortical spreading depression preconditioned animals did not affect infarct volume (162.6+/-62.1 mm3). However, administration of N-[2-cyclohexyloxy-4-nitrophenyl] methanesulphonamide given three times prior to middle cerebral artery occlusion prevented the reduced infarct volume induced by cortical spreading depression preconditioning (272.9+/-63.2 mm3). Administration of L-nitro-arginine methyl ester (4 mg/kg, i.v.) prior to cortical spreading depression blocked increases in cyclooxygenase-2 normally seen at 3 and 4 days. We conclude that NO-mediated cyclooxygenase-2 upregulation by cortical spreading depression protects the brain against ischemic damage.

Insulin resistance and associated dysfunction of resistance vessels and arterial hypertension.

Insulin resistance (IR) has profound, negative effects on the function of arteries and arterioles throughout the body and leads to arterial hypertension and vascular occlusive diseases such as heart attacks and strokes. IR affects arteries and arterioles at both the endothelium and smooth muscle levels. One major, underlying mechanism of vascular dysfunction appears to involve the augmented generation, availability and subsequent actions of reactive oxygen species (ROS). Thus, application of superoxide dismutase (SOD), a specific scavenger of superoxide anion, is able to immediately restore normal dilator responsiveness in IR arteries. In some but not all circulations, however, other factors such as increased production of and actions by constrictor agents such as endothelin also appear to restrict normal dilator responses. The basis of ROS-mediated vascular dysfunction in IR is not completely understood, but inflammatory processes throughout the arterial wall appear to be involved. Treatments involving behavioral approaches, such as changes in diet, weight loss, and regular exercise, and pharmacological approaches, involving the use of insulin-sensitizing agents or statins, appear to offer benefits against the detrimental vascular effects of IR. Nonetheless, the most effective approach appears to involve prevention of IR via adoption of a healthy lifestyle by young people.

Chronic adrenomedullin treatment improves blood-brain barrier function but has no effects on expression of tight junction proteins.

We previously found that the production of adrenomedullin (AM) is one magnitude higher in cerebral endothelial cells (CECs) than in the peripheral endothelium and the AM concentration in the cerebral circulation is significantly higher than in other tested parts of the circulation. We also showed that CECs express AM receptors, and AM as an autocrine hormone is important to regulate the intracellular cAMP level in CECs. Further we reported that acute AM treatment has cAMP-like effects on specific BBB functions: AM decreased endothelial fluid phase endocytosis, activated the P-glycoprotein, increased transendothelial electrical resistance (TEER) and reduced endothelial permeability for sodium fluorescein, which suggests a tightening of intercellular junctions. In the present study, we found chronic AM exposure also increased TEER. In contrast, we could not detect significant effect of AM on the expression of tight junction proteins (claudin-1, occludin and zonula occludens-1). While not affecting expression of tight junction proteins, chronic AM treatment may influence the localization of these proteins which has been reported to correlate with functional changes of the BBB without a change in protein expression.

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.

Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia.

Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K(ATP) channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia.

Epoxyeicosatrienoic acid-induced relaxation is impaired in insulin resistance.

We assessed the effect of epoxyeicosatrienoic acids (EETs) in intact mesenteric arteries and Ca(2+)-activated K(+) (BK(Ca)) channels of isolated vascular smooth muscle cells from control and insulin-resistant (IR) rats. The response to 11,12-EET and 14,15-EET was assessed in small mesenteric arteries from control and IR rats in vitro. Mechanistic studies were performed in endothelium intact or denuded arteries and in the presence of pharmacological inhibitors. Moreover, EET-induced activation of the BK(Ca) channel was assessed in myocytes in both the cell-attached and the inside-out (I/O) patch-clamp configurations. In control arteries, both EET isomers induced relaxation. Relaxation was impaired by endothelium denudation, N(omega)-nitro-L-arginine, or iberiotoxin (IBTX), whereas it was abolished by IBTX + apamin or charybdotoxin + apamin. In contrast, the EETs did not relax IR arteries. In control myocytes, the EETs increased BK(Ca) activity in both configurations. Conversely, in the cell-attached mode, EETs had no effect on BK(Ca) channel activity in IR myocytes, whereas in the I/O configuration, BK(Ca) channel activity was enhanced. EETs induce relaxation in small mesenteric arteries from control rats through K(Ca) channels. In contrast, arteries from IR rats do not relax to the EETs. Patch-clamp studies suggest impaired relaxation is due to altered regulatory mechanisms of the BK(Ca) channel.

Ischemia increases prostaglandin H synthase-2 levels in retina and visual cortex in piglets.

BACKGROUND: Ischemia increases levels of prostaglandin H synthase-2 (PGHS-2) in neonatal brain and cerebral vasculature, but effects on the developing visual system are unknown. We examined the effects of ischemia on PGHS-2 mRNA and protein levels in the retina and visual cortex in anesthetized piglets. METHODS: Ten minutes of complete retinal and brain ischemia was induced by increasing intracranial pressure. After 2-12 h of reperfusion, samples of retina and visual cortex were collected for determinations of levels of PGHS-2 mRNA (RNase protection assay) or protein (immunohistochemistry and western blotting). Tissues also were obtained from control animals. RESULTS: Levels of PGHS-2 mRNA were undetectable in control animals but showed a dramatic increase at 2-4 h in the cortex and retina in animals exposed to ischemia. Detectable but limited PGHS-2 immunoreactivity (IR) was present in the retina and visual cortex from control animals. In piglets not subjected to ischemia, PGHS-2 IR was localized mainly to the outer limiting membrane and to the Muller cells. Ischemia induced a marked increase in PGHS-2 IR in the neural retina, with the greatest increase in the photoreceptor layer. PGHS-2 levels in whole retina also increased at 8 h after ischemia. In the intact visual cortex PGHS-2 IR was evident in layers II and V. Ischemia increased the intensity of IR in layers II/III as well as layer V. CONCLUSIONS: Detectable amounts of PGHS-2 protein are present in the piglet retina and visual cortex under normal conditions, but levels are markedly increased 8-12 h after ischemic stress. Enhanced PGHS-2 levels after ischemic stress may contribute to delayed pathological changes of the visual system in the neonate.

Kainic acid rapidly induces cyclooxygenase (COX)-2 in piglet cerebral cortex.

Ischemia/reperfusion (I/R) results in a robust induction of cyclooxygenase (COX)-2 in the newborn brain via unknown mechanisms, but glutamate release and activation of KA receptors may be involved. We examined effects of local KA (3-300 micromol/l for 10 min) treatment on cortical COX-2 expression in anesthetized piglets using a closed cranial window. Treated and corresponding control tissue samples were collected 0.5-10 h after treatment. COX-2 mRNA and protein levels were assessed using RNase protection assay and immunohistochemistry, respectively. KA elicited reproducible dose-dependent increases in cortical COX-2 mRNA unaffected by indomethacin or N(G)-nitro-L-arginine methyl ester pretreatment. COX-2 mRNA levels were elevated at 30 min, peaked at 2 h, but remained enhanced for up to 10 h after KA. Neuronal COX-2 immunoreactivity was also enhanced compared with the control side in all cortical layers 8h after KA. In summary, activation of KA receptors may be involved in the neuronal induction of COX-2 after I/R in the newborn.

Glibenclamide enhances cortical spreading depression-associated hyperemia in the rat.

We investigated the contribution of ATP-sensitive potassium channels (K(ATP)) and calcium-activated potassium channels (Kca2+) to cortical spreading depression (CSD)-associated hyperemia using the rat closed cranial window model. The peak CBF response was enhanced by 12 +/- 5, 13 +/- 4, and 28 +/- 8% (p<0.01) of the control with 10(-6), 10(-5) and 10(-4) mol/l glibenclamide (glyb), a K(ATP) antagonist, respectively. We also calculated the area under the CBF curve to fully represent the extent of hyperemia during CSD. The area increased by 30 +/- 8 (p<0.05), 72 +/- 31 (p<0.05) and 88 +/- 20% (p<0.05) of the control with 10(-6), 10(-5) and 10(-4) mol/l glyb, respectively. However, charybdotoxin (CTX), a Kca2+ antagonist showed no effect. The effect of glyb was inhibited by pretreatment with 5 mg/kg indomethacin. We conclude that activation of K(ATP), perhaps associated with neurons, plays an inhibitory role in the CSD-associated hyperemia via an indomethacin-sensitive mechanism.

Spinal cyclooxygenase-2 is involved in development of allodynia after nerve injury in rats.

Increased spinal cyclooxygenase activity is associated with nociception induced by tissue inflammation. In the present study, we examined the changes of cyclooxygenase-1 and cyclooxygenase-2 protein expression in several regions of the CNS associated with pain perception, and the role of spinal cyclooxygenase activity in the development of allodynia following nerve injury. Allodynia was induced by ligation of the left L5 and L6 spinal nerves in rats. Using western blot analysis, we found that the cyclooxygenase-2 protein levels in the dorsal spinal cord and thalamus (but not in the ventral spinal cord, cingulate cortex and locus coeruleus) increased significantly one day after nerve ligation, compared with those in the sham animals. The cyclooxygenase-2 protein levels in the above tissues were similar in nerve-injured and sham animals three and 14 days after surgery. In contrast, cyclooxygenase-1 protein was not detectable in any of the neural tissues examined one, three, and 14 days after nerve injury. In the behavioral experiments, we observed that intrathecal injection of 100microg of indomethacin immediately or one day after nerve ligation attenuated the development of tactile allodynia. However, intrathecal injection of indomethacin had no effect on established allodynia two weeks after nerve injury.Collectively, our results suggest that cyclooxygenase-2 is preferentially up-regulated in the dorsal spinal cord and thalamus in response to nerve injury in rats. Spinal cyclooxygenase-2 probably plays an important role in the early development, but not in the maintenance, of tactile allodynia caused by the nerve injury in this rat model of neuropathic pain.

Characteristics of induced spreading depression after transient focal ischemia in the rat.

We examined characteristics of spreading depression (SD) induced on the rat cortex 1 day after transient focal ischemia. Male Wistar rats (n=21) were subjected to transient intraluminal thread occlusion of the right middle cerebral artery for 75 min. Twenty-four hours after the reperfusion, cerebral blood flow (CBF) was determined using laser Doppler flowmeter during multiple SDs elicited on both non-stroke (left) and stroke (right) cortex by the topical application of 2 M KCl. We also examined CBF responses before and after the intravenous administration of the nonspecific NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) in normal and stroke cortex. Animals were divided into two groups; Group 1 (n=12), animals with subcortical infarction and Group 2 (n=9), animals with subcortical plus cortical infarction. There were no differences between non-stroke and stroke sides in the duration or amplitude of the DC potential shifts in either group. The transient CBF hyperemia during SD was not different between non-stroke (372+/-23% of baseline, mean+/-S.E.) and stroke sides (383+/-30%) in Group 1. However, in Group 2, CBF was significantly restricted on the stroke side (192+/-15% vs. non-stroke side, 374+/-33%). In four normal animals without ischemia, there were no differences in CBF response between both sides. L-NAME had no effect on the transient CBF hyperemia during SD in any of the groups. These data suggest that the CBF responses during SD in the peri-infarction area is restricted 1 day after the transient focal ischemia, while CBF responses are intact in normal cortex overlapping a subcortical infarct. Further, our results indicate that nitric oxide does not promote CBF responses during SD in normal cortex or in tissue surrounding infarction.

Hyperbaric oxygen decreases infarct size and behavioral deficit after transient focal cerebral ischemia in rats.

Cerebral hypoxia is a major component of immediate and secondary cell damage caused by ischemia. Hyperbaric oxygen (HBO) is a potent means to increase the amount of oxygen dissolved in blood plasma. The effectiveness of HBO in clinical and experimental cerebral ischemia, however, is controversial. We sought to determine whether treatment with HBO initiated early after focal cerebral ischemia-onset protects the brain when experimental conditions such as brain temperature are controlled. Male Wistar rats (n=57) underwent reversible filament occlusion of the right middle cerebral artery (MCA) for 75 min. Animals were awakened after filament introduction and assessed for presence of forelimb paresis. Rats then underwent a 60-min course of either 100% O(2) at 1.0 atmosphere absolute (ata; control group), HBO 1.5 ata, or HBO 2.5 ata in a customized HBO chamber allowing physiological monitoring and pericranial temperature control. The filament was then removed. Seven days after ischemia, rat behavior was scored from 3-18 (18=normal) and brains were removed for histological analysis of infarct volume. Rats treated with HBO 2.5 ata had better mean+/-standard deviation (S.D.) behavioral scores (14+/-2; p<0.05) than control (10+/-3) or HBO 1.5-ata-treated animals (11+/-3). Similarly, total infarct volumes (mean+/-S.D.) were smaller in animals receiving HBO at 2.5 ata (76+/-65 mm(3); p<0.05) compared to control (129+/-83 mm(3)) and HBO 1.5-ata (119+/-68 mm(3))-treated groups. Cortical infarction occurred less frequently in HBO 2. 5-ata-treated than in control animals (44% vs. 71%; p<0.05). We conclude that HBO can improve outcome after temporary focal ischemia when treatment is started early after ischemia-onset but HBO dose appears important. Potential mechanisms include enhanced oxygen supply to marginally perfused cells.

Mitochondrial potassium channel opener diazoxide preserves neuronal-vascular function after cerebral ischemia in newborn pigs.

BACKGROUND AND PURPOSE: N-Methyl-D-aspartate (NMDA) elicits neuronally mediated cerebral arteriolar vasodilation that is reduced by ischemia/reperfusion (I/R). This sequence has been preserved by pretreatment with the ATP-sensitive potassium (K(ATP)) channel opener aprikalim, although the mechanism was unclear. In the heart, mitochondrial K(ATP) channels (mitoK(ATP)) are involved in the ischemic preconditioning-like effect of K(+) channel openers. We determined whether the selective mitoK(ATP) channel opener diazoxide preserves the vascular dilation to NMDA after I/R. METHODS: Pial arteriolar diameters were determined with the use of closed cranial window/intravital microscopy in anesthetized piglets. Vascular responses to NMDA were assessed before and 1 hour after 10 minutes of global cerebral ischemia induced by raising intracranial pressure. Subgroups received 1 of the following pretreatments before I/R: vehicle; 1 to 10 micromol/L diazoxide; and coapplication of 100 micromol/L 5-hydroxydecanoic acid (5-HD), a K(ATP) antagonist with diazoxide. RESULTS: NMDA-induced dose-dependent pial arteriolar dilation was not affected by diazoxide treatment only but was severely attenuated by I/R. In contrast, diazoxide dose-dependently preserved the NMDA vascular response after I/R; at 10 micromol/L, diazoxide arteriolar responses were unaltered by I/R. The effect of diazoxide was antagonized by coapplication of 5-HD with diazoxide. Percent preservation of 100 micromol/L NMDA-induced vasodilation after I/R was 53+/-19% (mean+/-SEM, n=8) in vehicle-treated controls versus 55+/-10%, 85+/-5%, and 99+/-15% in animals pretreated with 1, 5, and 10 micromol/L diazoxide (n=8, n=8, and n=12, respectively) and 60+/-15% in the group treated with 5-HD+diazoxide (n=5). CONCLUSIONS: The mitoK(ATP) channel opener diazoxide in vivo preserves neuronal function after I/R, shown by pial arteriolar responses to NMDA, in a dose-dependent manner. Thus, activation of mitoK(ATP) channels may play a role in mediating the protective effect of other K(+) channel openers.

Cycloheximide rapidly inhibits cortical COX activity and COX-dependent pial arteriolar dilation in piglets.

We have previously shown that cycloheximide (CHX) preserved neuronal function after global cerebral ischemia in piglets, in a manner similar to indomethacin. To elucidate the mechanism of this protection, we tested the hypothesis that CHX would inhibit cyclooxygenase (COX) activity in the piglet cerebral cortex and vasculature. Pial arteriolar responses to hypercapnia, arterial hypotension, and sodium nitroprusside (SNP) were determined before and 20 min after treatment with CHX (0.3-1 mg/kg iv) using a closed cranial window and intravital microscopy. We also determined baseline and arachidonic acid (AA)-stimulated cortical PGF(2alpha) and 6-keto-PGF(1alpha) production before and 20-60 min after CHX (1 mg/kg iv) treatment, using ELISA kits. CHX did not affect baseline diameters (approximately 100 microm) but significantly decreased arteriolar dilation to COX-dependent stimuli, such as hypercapnia and hypotension, but not to COX-independent SNP. In the 1 mg/kg CHX-treated group, increases in vascular diameters were reduced from 22 +/- 2 to 10 +/- 2%, from 49 +/- 5 to 31 +/- 3% (means +/- SE, 5 and 10% CO2, respectively, n = 8), from 12 +/- 3 to 3 +/- 1%, and from 26 +/- 5 to 6 +/- 2% ( approximately 25 and 40% decreases in blood pressure, respectively, n = 6). CHX also inhibited conversion of exogenous AA to both PGF(2alpha) and 6-keto-PGF(1alpha); for example, 20 min after CHX treatment 10 microg/ml AA-stimulated PGF(2alpha) concentrations in the artificial cerebrospinal fluid decreased from 14.28 +/- 3.04 to 5.90 +/- 1.26 ng/ml (n = 9). Thus CHX rapidly decreases COX activity in the piglet cerebral cortex. This result may explain in part the preservation of neuronal function of CHX in cerebral ischemia.

Ischemia-reperfusion rapidly increases COX-2 expression in piglet cerebral arteries.

In the newborn, cyclooxygenase (COX)-derived products play an important role in the cerebrovascular dysfunction after ischemia-reperfusion (I/R). We examined effects of I/R on expression of COX-1 and COX-2 isoforms in large cerebral arteries of anesthetized piglets. The circle of Willis, the basilar, and the middle cerebral arteries were collected from piglets at 0.5-12 h after global ischemia (2.5-10 min, n = 50), hypoxia (n = 3), or hypercapnia (n = 2) and from time-control (n = 19) or untreated animals (n = 7). Tissues were analyzed for COX-1 and COX-2 mRNA and protein using RNase protection assay and immunoblot analysis, respectively. Ischemia increased COX-2 mRNA by 30 min, and maximal levels were reached at 2 h. Hypoxia or hypercapnia had minimal effects on COX-2 mRNA. COX-2 protein levels were also consistently elevated by 8 h after I/R. Increases in COX-2 mRNA or protein were not influenced by pretreatment with either indomethacin (5 mg/kg iv, n = 5) or nitro-L-arginine methyl ester (15 mg/kg iv, n = 7). COX-1 mRNA levels were low in time controls, and ischemic stress had no significant effect on COX-1 expression. Thus ischemic stress leads to relatively rapid, selective induction of COX-2 in cerebral arteries.

Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries.

We determined the expression and functional correlate of in vitro transfection with a recombinant adenoviral vector encoding the gene for bovine endothelial nitric oxide synthase (AdCMVeNOS) or Escherichia coli beta-galactosidase (AdCMVLacZ) in pulmonary endothelial cells (EC), vascular smooth muscle cells (VSMC), and pulmonary arteries (PA) from newborn piglets. AdCMVeNOS and AdCMVeLacZ vectors, grown in 293-cell monolayers, were purified by double-cesium gradient ultracentrifugation. Cell cultures and PA were incubated with increasing vector titers for 30 or 60 min, followed by incubation in fresh medium for 18 h at 37 degrees C. LacZ expression was assessed by histochemical staining; eNOS expression was evaluated by Western blot analysis. Functional eNOS expression was determined by measurement of cGMP and quantification of the relaxation response to bradykinin (BK). In PA, LacZ transgene expression was preferentially localized to the adventitia and endothelium. Increased eNOS protein expression was observed in EC and VSMC transfected with AdCMVeNOS. Functional studies revealed increased cGMP abundance in cultured cells and enhanced relaxation to BK in AdCMVeNOS-transfected PA. These studies demonstrate that gene transfer with AdCMVeNOS results in functional expression and altered vasoactive responses in the neonatal pulmonary vasculature. Gene transfer with replication-deficient adenovirus vectors is a useful tool for the study of targeted genes in vascular biology.

Cerebrovascular reactivity remains intact after cortical depolarization in newborn piglets.

Cerebrovascular reactivity is severely affected by ischemia, and changes in vascular responses have been reported after cortical spreading depression and head trauma as well. Cortical depolarization (CD) occurs during ischemia, cortical spreading depression, and head trauma, but its effects on cerebrovascular reactivity are unclear. We tested the hypothesis that CD induced by KCl diminishes the vascular responsiveness to various vasodilatory stimuli in piglets. Responses of pial arterioles were determined by changes in vascular diameter by use of a closed cranial window and intravital microscopy. Baseline arteriolar diameters were 105 +/- 3 microm (mean +/- SEM, n = 27). CD was elicited by topical administration of 1 mol/L KCl for 3 min. Vascular responses were measured before and 1 h after CD. KCl elicited CD and constricted arterioles by 54 +/- 4% (n = 27). N-methyl-D-aspartate induced dose-dependent vasodilation that was unaffected by CD; the percent changes were 9 +/- 1 versus 8 +/- 1 (before and after CD) at 10(-5) mol/L, 19 +/- 2 versus 18 +/- 3 at 5 x 10(-5) mol/L, and 29 +/- 2 versus 26 +/- 3 at 10(-4) mol/L (n = 9). Hypercapnic vasodilation was not diminished by CD; the percent changes were 15 +/- 2 versus 16 +/- 4 at 5%, and 27 +/- 5 versus 27 +/- 6 at 10% inspired CO2 (n = 8). Aprikalim and forskolin caused dilation that was also resistant to prior CD; the percent change values were 21 +/- 4 versus 18 +/- 3 and 16 +/- 2 versus 16 +/- 4 at 10(-6) mol/L, 36 +/- 5 versus 34 +/- 5 and 34 +/- 7 versus 37 +/- 7 at 10(-5) mol/L (n = 8), respectively. Finally, calcitonin gene-related peptide-induced vasodilation was unaffected by CD; percent changes were 15 +/- 3 versus 16 +/- 2 at 10(-7) mol/L and 26 +/- 4 versus 22 +/- 3 at 10(-6) mol/L (n = 8). The intact vascular responses after CD suggest that this component is not responsible for decreased cerebrovascular reactivity after ischemia, head trauma, or cortical spreading depression.

Cerebrovascular responses to therapeutic dose of indomethacin in newborn pigs.

The aims of this study were 1) to compare the effects of low versus high doses of indomethacin on cerebral blood flow (CBF) responses to hypercapnia and 2) to investigate the effects of low-dose indomethacin on the cerebral vasculature during resting conditions and during vasodilator stimuli. In the first experiment, 27 piglets were randomized into three groups to receive 5 mg/kg indomethacin, 0.2 mg/kg indomethacin, or normal saline. Ninety minutes later, CBF was measured by radioactive microspheres at baseline, during hypercapnia [PaCO2 > or = 70 mm Hg (> or =9.3 kPa)] and normocapnia. Total CBF was comparable among the three groups at baseline. CBF increased during hypercapnia in all groups, but the hyperemic response was significantly attenuated in the high-dose indomethacin group compared with the saline group but not in the group treated with 0.2 mg/kg. CBF returned toward baseline during normocapnia in all piglets. In the second experiment, a closed cranial window was implanted over the parietal cortex of nine piglets. Cerebrovascular responses to hypercapnia and topical application of isoproterenol (10(-7) and 10(-6) M) and histamine (10(-6) and 10(-5) M) were investigated before and after administration of 0.2 mg/kg indomethacin. Within 10 min of indomethacin administration, pial arteriolar diameters decreased from 72 +/- 8 to 58 +/- 6 microm (p < 0.05), and 6-keto-PGF1alpha concentration decreased from 1440 +/- 250 to 570 +/- 30 pg/mL (p < 0.05). Two hours (138 +/- 21 min) later, pial arteriolar diameters had returned toward baseline values (65 +/- 5 microm), whereas 6-keto-PGF1alpha values remained considerably lower than preindomethacin values (530 +/- 30 pg/mL). Cerebrovascular responses to dilator stimuli were preserved after 0.2 mg/kg indomethacin. We conclude that 0.2 mg/kg indomethacin does not markedly affect the cerebral hyperemic responses to hypercapnia in contrast with a very prominent inhibition by 5 mg/kg indomethacin. Also, although indomethacin at a low dose constricts pial arterioles transiently and attenuates cerebral prostanoid production, it does not inhibit the pial arteriolar responsiveness to prostanoid-associated dilator stimuli. This observation may be due to the permissive role that prostacyclin plays in cerebral vasodilatory responses to some vasogenic stimuli such as hypercapnia and histamine.

Inhibitors of protein synthesis preserve the N-methyl-D-aspartate-induced cerebral arteriolar dilation after ischemia in piglets.

BACKGROUND AND PURPOSE--Cerebral arteriolar dilation to N-methyl-D-aspartate (NMDA) is a neuronally mediated process that is sensitive to cerebral ischemia. We tested the hypothesis that pretreatment with transcription or translation inhibitors preserves the vascular response to NMDA after global cerebral ischemia. METHODS--Pial arteriolar diameters were measured in anesthetized piglets by use of a closed cranial window and intravital microscopy. Arteriolar responses to NMDA (10(-5) and 10(-4) mol/L) were measured before and 1, 2, and 4 hours after 10 minutes of ischemia. Ischemia was induced by increasing intracranial pressure. Subgroups were pretreated with vehicle, topical actinomycin D (Act-D) 10(-5) or 10(-6) mol/L, or intravenous cycloheximide (CHX) 1.0 or 0.3 mg/kg 15 minutes before ischemia. The effects of Act-D and CHX on vascular responses to NMDA without preceding ischemia were also examined. RESULTS--In the vehicle group, arteriolar responses to NMDA were clearly attenuated 1 hour after ischemia but returned to baseline at 2 to 4 hours. Preischemic compared with 1 hour postischemic arteriolar dilation to NMDA was 10+/-2% versus 1+/-0% at 10(-5) mol/L and 40+/-4% versus 20+/-4% at 10(-4) mol/L NMDA (mean+/-SEM; both P<0.05, n=7). In contrast, pretreatment with Act-D resulted in preservation of the arteriolar responses to NMDA 1 hour after ischemia. For 10(-6) mol/L (n=5) of Act-D, dilations were 6+/-2% versus 6+/-2% at 10(-5) mol/L and 51+/-9% versus 39+/-10% at 10(-4) mol/L of NMDA. For 10(-5) mol/L (n=5) of Act-D, arterioles dilated by 7+/-2% versus 7+/-2% at 10(-5) mol/L and 38+/-4% versus 35+/-4% at 10(-4) mol/L NMDA. Similarly, CHX preserved NMDA-induced vasodilation. For 0.3 mg/kg of CHX (n=5), dilations were 8+/-2% versus 8+/-1% at 10(-5) mol/L and 39+/-4% versus 28+/-6% at 10(-4) mol/L NMDA. For 1.0 mg/kg of CHX (n=5), arterioles dilated by 10+/-2% versus 6+/-2% at 10(-5) mol/L and 37+/-7% versus 35+/-6% at 10(-4) mol/L NMDA. In experiments without ischemia, NMDA-induced vasodilation before and 85 minutes after administration of Act-D or CHX was not significantly different. CONCLUSIONS--Vascular responses of cerebral arterioles to NMDA after ischemia are preserved by pretreatment with either Act-D or CHX. Without preceding ischemia, Act-D and CHX do not potentiate neuronal-vascular responses to NMDA. Our results suggest that continued or augmented protein synthesis is involved in the transient attenuation of NMDA-induced dilation during the early reperfusion phase and that inhibitors of protein synthesis may protect neurons against ischemic stress.