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(Circulation Research. 2000;87:1149.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Prolonged Hypercapnia-Evoked Cerebral Hyperemia via K⁺ Channel– and Prostaglandin E₂–Dependent Endothelial Nitric Oxide Synthase Induction

Taline Najarian¹, Anne Marilise Marrache¹, Isabelle Dumont, Pierre Hardy, Martin H. Beauchamp, Xin Hou, Krishna Peri, Fernand Gobeil, Jr, Daya R. Varma, Sylvain Chemtob

From the Department of Pharmacology and Therapeutics (T.N., A.M.M., D.R.V., S.C.), McGill University, Montreal; Departments of Pediatrics, Ophthalmology, and Pharmacology (T.N., A.M.M., I.D., P.H., X.H., F.G., S.C.), Research Center, Hôpital Ste-Justine, Montreal; and Theratechnologies (K.P.), St Laurent, Quebec, Canada.

Correspondence to Sylvain Chemtob, MD, PhD, FRCPC, Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Hôpital Ste-Justine, 3175 Côte Sainte-Catherine, Montreal, Quebec, Canada, H3T 1C5. E-mail chemtobs{at}ere.umontreal.ca

	Abstract

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Abstract—Mechanisms for secondary sustained increase in cerebral blood flow (CBF) during prolonged hypercapnia are unknown. We show that induction of endothelial NO synthase (eNOS) by an increase in prostaglandins (PGs) contributes to the secondary CBF increase during hypercapnic acidosis. Ventilation of pigs with 6% CO₂ (PaCO₂65 mm Hg; pH 7.2) caused a 2.5-fold increase in CBF at 30 minutes, which declined to basal values at 3 hours and gradually rose again at 6 and 8 hours; the latter increase was associated with PG elevation, nitrite formation, eNOS mRNA expression, and in situ NO synthase (NOS) reactivity (NADPH-diaphorase staining). Subjecting free-floating brain sections to acidotic conditions increased eNOS expression, the time course of which was similar to that of CBF increase. Treatment of pigs with the cyclooxygenase inhibitor diclofenac or the NOS inhibitor N-nitro-L-arginine blunted the initial rise and prevented the secondary CBF increase during hypercapnic acidosis; neuronal NOS blockers 1-(2-trifluoromethylphenyl) imidazole and 3-bromo-7-nitroindazole were ineffective. Diclofenac abolished the hypercapnia-induced rise in cerebrovascular nitrite production, eNOS mRNA expression, and NADPH-diaphorase reactivity. Acidosis (pH 7.15, PCO₂40 mm Hg; 6 hours) produced similar increases in prostaglandin E₂ (PGE₂) and eNOS mRNA levels in isolated brain microvessels and in NADPH-diaphorase reactivity of brain microvasculature; these changes were prevented by diclofenac, by the receptor-operated Ca²⁺ channel blocker SK&F96365, and by the K_ATP channel blocker glybenclamide. Acidosis increased Ca²⁺ transients in brain endothelial cells, which were blocked by glybenclamide and SK&F96365 but not by diclofenac. Increased PG-related eNOS mRNA and NO-dependent vasorelaxation to substance P was detected as well in rat brain exposed to 6 hours of hypercapnia. PGE₂ was the only major prostanoid that modulated brain eNOS expression during acidosis. Thus, in prolonged hypercapnic acidosis, the secondary CBF rise is closely associated with induction of eNOS expression; this seems to be mediated by PGE₂ generated by a K_ATP and Ca²⁺ channel–dependent process.

Key Words: hypercapnia • acidosis • endothelial nitric oxide synthase • prostaglandin E₂ • potassium channels

	Introduction

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Chronic obstructive pulmonary disease in adults and bronchopulmonary dysplasia in premature infants usually result in hypercapnia. Elevation in CO₂ tension causes a rapid increase in cerebral blood flow (CBF), which wanes in 3 to 5 hours.^{1 2 3} Few studies have examined effects of prolonged hypercapnia. These revealed that it is associated with a secondary CBF rise,^{2 3} which persists on resumption of normocapnia. Humans with chronic lung disease and hypercapnia also have increased CBF after correcting for age, decrease in cerebral venous outflow, and increased blood viscosity.^{4 5 6 7} Findings imply that separate mechanisms may operate in acute and prolonged hypercapnic-induced CBF increases.

Prostaglandins (PGs), mainly PGI₂ and prostaglandin E₂ (PGE₂), and NO exert a variable contribution on acute hypercapnia-induced increase in CBF.^{8 9 10 11 12} On the other hand, the mechanisms of the secondary sustained CBF increase are not known. Hypercapnic acidosis increases PGs^{11 13} and NO.^{11 14} However, NO synthase (NOS) as well as phospholipase A₂ and cyclooxygenase (COX) activities are optimal at basic pH_i^{15 16} ; along these lines, pH_o seems to be a major determinant of hypercapnia-induced cerebral vasomotor tone,^{17 18 19} and bicarbonate can restore CBF to basal levels.¹⁸ Acidosis can also stimulate K⁺ channel opening^{20 21 22 23} in endothelial cells, leading to increased activities of phospholipase A₂²⁴ and constitutive NOS.^{20 25}

Complex acute interactions between PGs, NO, and K⁺ channels have been described^{26 27 28} ; long-term interactions have also been reported, such as PG induction of endothelial NOS (eNOS) transcription.^{29 30} We hypothesized that the second CBF rise during prolonged hypercapnia may be triggered by early increases in PGs, which would induce eNOS expression and in turn increase NO release. Our findings disclose for the first time that the second cerebral hyperemia during prolonged hypercapnia is largely contributed by augmented NO release associated with increased eNOS expression, mediated by PGE₂ involving K⁺ and Ca²⁺ channels.

	Materials and Methods

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Chemicals
The following agents were purchased: diclofenac, glybenclamide, -chloralose, and pancuronium (Sigma); 16,16-dimethyl-PGE₂, PGD₂, carbaprostacyclin, U46619, and BW245C (Cayman); 1-(2-trifluoromethylphenyl) imidazole (TRIM) and 3-bromo-7-nitroindazole (Br-7-NI) (Tocris); charybdotoxin and cromakalim (Calbiochem); SK&F96365 (Biomol); PGE₂ RIA kits (Advanced Magnetics); fluorescent microspheres (Interactive Medical Technologies); and other chemicals (Fisher).

Animals
Yorkshire piglets (4 to 6 days old) and Sprague-Dawley rats (2 to 3 months old) were used according to a protocol of the Ste-Justine Hospital Animal Care Committee.

CBF Measurements
CBF was measured in pigs under chloralose sedation by the microsphere technique as previously described.^{31 32} After catheterization^{31 32} and stabilization (1.5 hours), fluorescent microspheres were injected in the left ventricle. After baseline CBF measurements, the gas mixture was changed to 6% CO₂, 73% N₂, and 21% O₂ to obtain stable PaCO₂ values (65 mm Hg) frequently encountered in the clinical setting; CBF measurements were repeated 0.5, 3, 6, and 8 hours after initiating hypercapnia.

Animals were randomly assigned to pretreatment with effective doses of diclofenac (5 mg/kg), L-nitro-arginine (L-NA, 3 mg/kg), neuronal NOS (nNOS) inhibitors TRIM (1 mg/kg followed by 50 µg/kgxmin^-1) and Br-7-NI (1 mg/kg followed by 50 µg/kgxmin^-1), or saline.^{32 33 34} At the end of the experiments, animals were killed with pentobarbital (120 mg/kg), brains were removed, and cortex and periventricular regions were weighed. Fluorescence in tissues and reference blood samples were analyzed by Interactive Medical Technologies, and regional CBF was calculated.^{31 32} Brain PGE₂ levels, nitrite production, eNOS mRNA expression, and NADPH-diaphorase reactivity were determined in some animals.

Tissue Preparation and Treatments
Brains (from pig and rat) were quickly placed in ice-cold artificial cerebral spinal fluid (aCSF) of the following composition (in mmol/L): 3.0 KCl, 1.5 MgCl₂, 1.5 CaCl₂, 132 NaCl, 6.6 urea, 1.2 KH₂PO₄, 24.6 NaHCO₃, and 10 glucose; 0.5% FBS; and 0.05% BSA. Free-floating coronal brain sections (2 to 3 mm) were incubated at 38°C in aCSF for 6 hours under normocapnic, hypercapnic acidotic, hypercapnic nonacidotic, and normocapnic acidosis, which simulated in vivo conditions; these conditions were achieved by bubbling CO₂ and adjusting pH with HCl or NaHCO₃. Brain slices were treated with diclofenac (100 µmol/L) alone or diclofenac plus 1 µmol/L 16,16-dimethyl-PGE₂, fenprostalene, PGD₂, BW245C, carbaprostacyclin, or U46619.

Cerebral Microvessel Preparation and Treatments
Cerebral microvessels (>70 µm) were prepared^{29 35} and incubated in aCSF for 6 hours under normocapnic or normocapnia acidotic conditions. Tissues were treated with one of the following (in µmol/L): 100 diclofenac, 10 glybenclamide, 0.1 charybdotoxin, or 10 SK&F96365. The incubation medium was assayed for PGE₂.^{26 36}

Vasomotor Response of Brain Microvessels
The brain parenchyma microvascular relaxant response to the NO-dependent substance P^{37 38} was studied by a video-imaging technique as described.²⁹

eNOS mRNA Detection by Ribonuclease Protection Assay and Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR)
eNOS and destrin (control) ribonuclease protection assays were conducted as described.^{29 30 32} To confirm changes in eNOS expression, RT-PCR was also used.^{29 30} Total RNA extracted from rat and pig tissues was transcribed, and eNOS and 18S (internal standard) cDNA fragments were amplified with Taq DNA polymerase (QuantumRNA Alternate 18S kit, Ambion). Primers for rat eNOS were 5'-GGAAACGCCAGAGGTACCGG-3' and 5'-AGCCTGGCGC-ACGGTACCTG-3', and those for pig were 5'-GCTTTTCCC- TGCAGGAGCGAC-3' and 5'-GCCAGTCTCTGCAGACTCTGG-3'.^{28 29} Amplified fragments were labeled with [-³²P]ATP, separated by PAGE, and quantified using a PhosphorImager (Molecular Dynamics).

Nitrite Production
NO production was estimated by measuring nitrite³⁹ as reported.^{29 30 32} Cerebrovascular nitrite production in vivo was calculated as the difference in sagittal sinus and arterial blood concentrationsxtotal CBF, expressed in ng/min per 100 g tissue, as reported.³²

NADPH-Diaphorase Histochemistry
NADPH-diaphorase reactivity was performed according to a previously described method^{29 40} on the brain cortex of pigs subjected to high CO₂ as well as on brain slices exposed for 6 hours to normocapnic nonacidotic and acidotic conditions and treated or not with (in µmol/L) 10 SK&F96365, 100 diclofenac, or 10 glybenclamide. The intensity of staining in blood vessels was analyzed digitally with ImagePro +4.1 software (Media Cybernetics). After normalizing for background tone, equal numbers of pixels taken from cortical microvessels were compared for significant differences in different treatment groups.^{29 30}

Calcium Transients in Cultured Endothelial Cells
Porcine brain microvascular endothelial cells were prepared as previously described.³⁶ [Ca²⁺]_i was measured by a fura-2–acetoxymethyl ester technique³⁶ in cells pretreated for 15 minutes with (in µmol/L) 100 diclofenac, 10 glybenclamide, 10 SK&F96365, and 10 SK&F96365 plus 1 µmol/L K⁺ channel opener cromakalim. The [Ca²⁺]_i was calculated as reported.^{36 41}

Statistics
CBF data were analyzed by 2-way ANOVA factoring for time and treatment group. Other data were analyzed by 1-way ANOVA. Comparison among means was performed by the Dunnett test. Statistical significance was set at P<0.05. Data are presented as mean±SEM.

	Results

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CBF, PGE₂ Levels, and Nitrite Production in Response to Hypercapnia
PaCO₂ remained high during the 8-hour ventilation of animals with 6% CO₂ (Table); pH initially decreased and then tended to increase slightly (P<0.05). Mean arterial blood pressure and PaO₂were unchanged. There was no difference in these parameters between treatment groups other than an expected rise in blood pressure by L-NA. Hypercapnic acidosis produced a marked increase in cortical and periventricular CBF at 30 minutes (Figures 1A and 1B) in control animals. CBF decreased at 3 hours and then increased gradually at 6 and 8 hours during hypercapnia (Figures 1A and 1B); CBF remained unchanged at all times when pH was normalized with bicarbonate.

View this table: [in this window] [in a new window]   Table 1. Arterial Blood Pressure and Gas Values in Pigs Before and After Hypercapnia

View larger version (49K): [in this window] [in a new window]   Figure 1. Figure 1. Time course of cortical (A) and periventricular (B) CBF response of pigs to 8 hours of hypercapnia by ventilation with 6% CO2 (indicated by bar). One group of animals was injected with diclofenac (5 mg/kg IV) 5.5 hours after exposure to hypercapnia, and another group was given sodium bicarbonate to normalize pH. All other animals were pretreated with diclofenac (5 mg/kg IV), L-NA (3 mg/kg), TRIM (1 mg/kg followed by 50 µg/kgxmin-1), Br-7-NI (1 mg/kg followed by 50 µg/kgxmin-1), or saline. At time 0, CBF was basal (normocapnia). Values are mean±SEM of 4 or 5 animals in each treatment group. *P<0.05 compared with basal values; P<0.05 compared with corresponding values in animals treated with diclofenac or L-NA. C and D, PGE2 levels (C) and in vivo cerebrovascular nitrite production (D) during hypercapnia and normocapnia. Values are mean±SEM of 3 or 4 animals. *P<0.05 compared with values without asterisks; P<0.05 compared with 0.5-hour value in saline-treated animals.

Brain PG levels increased acutely but tended to decrease for the remaining 8 hours of hypercapnia, although values remained higher than basal levels (Figure 1C). Pretreatment with diclofenac and L-NA blunted the early rise in CBF and totally prevented the second CBF increase noted at 6 and 8 hours of hypercapnia (Figures 1A and 1B); injection of diclofenac 5.5 hours after exposure to hypercapnia did not affect CBF. The nNOS inhibitors TRIM and Br-7-NI³² decreased nitrite production during normocapnia but not hypercapnia but did not affect the hypercapnia-induced CBF rise (Figures 1A, 1B, and 1D). PGE₂ levels were diminished by diclofenac (Figure 1C) but not by L-NA. Hypercapnia also caused an increase in cerebrovascular nitrite production, which was blocked by diclofenac (Figure 1D).

Brain NADPH-Diaphorase Reactivity and eNOS mRNA in Response to Prolonged Hypercapnia
Hypercapnic acidosis of 8 hours’ duration increased NADPH-diaphorase reactivity, which was mainly localized in microvasculature (Figure 2A); eNOS mRNA also increased (Figures 2B and 2C). These changes were inhibited by diclofenac pretreatment (Figures 2A through 2C). Diclofenac starting at 5.5 hours of hypercapnia did not modify the increase in eNOS mRNA; diclofenac exerted a negligible effect on NADPH-diaphorase reactivity and eNOS mRNA under normocapnia.

View larger version (77K): [in this window] [in a new window]   Figure 2. Figure 2. In vivo modulation of NADPH-diaphorase staining and eNOS mRNA expression by hypercapnia and diclofenac. Newborn pigs were ventilated and treated as in Figure 1. A, Brain slices were fixed for NADPH-diaphorase staining of blood vessels (arrows). Individual pixel tonality of densitometry was analyzed by correcting for background tone; higher arbitrary tonality units correspond to reduced densitometry (histogram). B, eNOS mRNA blots after RNase protection assay. Unprotected and protected fragments for RNase protection assays are, respectively, 414 and 356 nt for eNOS, and 237 and 165 nt for destrin. C, eNOS mRNA densitometry relative to destrin (control). Values in histograms are mean±SEM; n=3 for each condition. *P<0.05 compared with values without asterisks.

Effects of High CO₂ on eNOS mRNA Expression in Incubated Brain Slices and on NO-Dependent Relaxant Response of Brain Vasculature
Effects of high CO₂ on eNOS mRNA expression were studied on pig as well as rat brain sections; to assess whether these changes were reflected in function, vasomotor effects of the NO-dependent substance P^{37 38} were also tested. Both pig and rat brain sections exposed to high CO₂ for 6 hours exhibited increased eNOS mRNA and vasorelaxation to substance P, which were prevented by diclofenac (Figure 3); relaxation in response to NO-independent isoproterenol was unaltered by high CO₂. Acute (30 minutes) exposure to high CO₂ had no effect on the response to substance P.

View larger version (36K): [in this window] [in a new window]   Figure 3. Figure 3. Effects of hypercapnic acidosis on eNOS mRNA expression (A) and on cerebral vasorelaxant response to substance P (B) in brain sections. Brain sections from pig and rat were incubated in aCSF for 6 hours with 5% CO2 (PCO2=38±1 mm Hg, pH 7.39±0.02) or 9% to 10% CO2 (PCO2=64±4 mm Hg, pH 7.13±0.04) in the absence or presence of (in 1 µmol/L) 100 diclofenac, 10 glybenclamide, or 10 SK&F96365. Some tissue preparations (high and normal CO2) were treated with L-NA (1 mmol/L) 20 minutes before determining vasorelaxant response; other preparations (normal CO2) were treated with cromakalim (1 µmol/L). Tissues used for measurement of eNOS mRNA (detected by RT-PCR to ascertain RNase protection assay) were the same tested for vasomotor response. H-CO2 and N-CO2 refer to high and normal CO2 tensions, respectively. Values are mean±SEM of 3 or 4 experiments each. *P<0.05 compared with values without asterisks.

Concentration- and Time-Dependent Effect of CO₂ on eNOS mRNA Expression in Incubated Brain Slices
Exposure of brain to increasing CO₂ tension for 6 hours caused a concentration-dependent increase in eNOS mRNA (Figure 4A). Acidosis in the presence of normal CO₂ also induced a comparable time-dependent increase in eNOS expression (Figure 4B). Normalization of pH prevented changes in eNOS mRNA (Figure 4A).

View larger version (48K): [in this window] [in a new window]   Figure 4. Figure 4. A, Concentration- and time-dependent effects of hypercapnic acidosis on eNOS mRNA expression in brain sections. Brain sections were incubated in aCSF for 6 hours with 3% CO2 (PCO2=23±1 mm Hg, pH 7.51±0.02), 9% to 10% CO2 (PCO2=66±3 mm Hg, pH 7.15±0.03), 9% to 10% CO2 with normalized pH (PCO2=63±3 mm Hg, pH 7.4±0.03), and 5% CO2 with acidosis (PCO2=39±1 mm Hg, pH 7.15±0.03), or were not incubated (basal). B, Time-dependent changes in eNOS mRNA expression after exposure to normal CO2 acidotic conditions (see panel A). Values are mean±SEM of 3 or 4 experiments; *P<0.05 compared with values without asterisks. C and D, PGE2 levels (C) and eNOS expression (D) in brain microvessels exposed to acidosis. Brain microvessels (>70 µm) were incubated for 6 hours in normal pH (pH 7.38±0.03, PCO2=37±2 mm Hg) in absence or presence of glybenclamide (10 µmol/L) or cromakalim (1 µmol/L), or in normocapnic acidotic conditions (pH 7.1±0.05, PCO2=38±1 mm Hg) with or without (in 1 µmol/L) 100 diclofenac, 10 SK&F96365, 10 glybenclamide, or 0.1 charybdotoxin. PGE2 in incubation medium was measured at the end of the experiment; RNA (10 µg) was subjected to RNase protection assay for eNOS as in Figure 2; destrin was used as control. Values are mean±SEM of 5 experiments. *P<0.05 compared with all other values.

PG Levels and eNOS Expression in Brain Microvessels
Acidification of the incubation medium containing brain microvessels caused an increase in PGE₂ in the 6-hour period (Figure 4C). Diclofenac, as well as putative receptor-operated Ca²⁺ channel blocker SK&F96365, and K_ATP (glybenclamide) but not K_Ca blocker (charybdotoxin) prevented increases in PGE₂ levels, eNOS mRNA (Figures 4C and 4D), and NO-dependent substance P–evoked vasorelaxation (Figure 3B). SK&F96365 and glybenclamide did not affect eNOS expression and vasorelaxation under normal pH, whereas cromakalim increased PGE₂, eNOS mRNA, and substance P–elicited vasorelaxation (Figures 3B, 4C, and 4D). Modulation of acidosis-induced changes in eNOS mRNA expression in isolated brain microvessels by COX and channel blockers were in conformity with NADPH-diaphorase reactivity in brain slices (Figure 5).

View larger version (112K): [in this window] [in a new window]   Figure 5. Figure 5. Ex vivo modulation of acidosis-induced NADPH-diaphorase staining of brain slices by PGs and K+ and calcium channels. Brain slices were incubated in normal pH or normocapnic acidotic conditions (pH 7.12±0.05, PCO2=38±1 mm Hg) with or without (in 1 µmol/L) 100 diclofenac, 10 SK&F96365, or 10 glybenclamide. Higher arbitrary tonality units correspond to reduced densitometry (histogram). Values are mean±SEM of 3 experiments for each treatment group. *P<0.05 compared with all other values.

Effect of Acidosis on Calcium Signaling in Endothelial Cells
Calcium transients were measured directly on brain microvascular endothelial cells; smooth muscle and glial cells do not generate PGs to acidosis.⁴² Lowering pH of media to 7.1 by HCl or NaH₂PO₄ caused a rapid and marked increase in calcium transients (Figures 6A and 6B) prevented by bicarbonate and dependent on extracellular calcium entry (blocked by EGTA). SK&F96365 and glybenclamide, but not diclofenac, prevented acidosis-evoked calcium transients (Figures 6A and 6C). In addition, the K_ATP-channel opener cromakalim stimulated Ca²⁺ transients under normal pH; these effects were also blocked by SK&F96365 (Figures 6B and 6C).

View larger version (19K): [in this window] [in a new window]   Figure 6. Figure 6. Effects of acidosis on calcium transients in endothelial cells. Calcium transients were measured by the fura-2–acetoxymethyl ester technique. Medium was acidified by addition of HCl or NaH2PO4 (pH 7.1±0.05); effects of HCl are shown on typical tracings (A and B). Acidified media were also pretreated with (in µmol/L) 100 diclofenac, 10 SK&F96365, or 10 glybenclamide. In addition, cells were treated with KATP opener cromakalim (1 µmol/L) in the absence or presence of SK&F96365 (10 µmol/L). Arrows indicate time of administration of acidifying agents (H+) in panel A or K+ channel openers in panel B. C, Histogram presenting peak [Ca2+]i; values are mean±SEM of 3 or 4 experiments. *P<0.01 compared with all other values without asterisks.

Effects of PG Analogs on Acidosis-Induced Changes in eNOS mRNA
The COX product involved in acidosis-induced eNOS mRNA modulation was investigated; because reactive oxygen species that can be produced by COX do not play a role in hypercapnia-induced vasomotor response,⁴³ we focused on major prostanoids.^{29 30} The inhibitory effect of diclofenac on acidosis-induced increase in eNOS mRNA in brain slices was prevented by concurrent treatment with 16,16-dimethyl-PGE₂ (stable PGE₂ analog) but not with PGD₂, fenprostalene, U46619, carbaprostacyclin or BW245C (these last 4 are stable analogs of PGF₂, thromboxane A₂, PGI₂, and PGD₂) (Figure 7).

View larger version (48K): [in this window] [in a new window]   Figure 7. Figure 7. Effects of PG analogs on eNOS mRNA expression on porcine brain slices during HCl-induced acidosis (pH 7.15±0.04). Tissues were treated with diclofenac with or without 16,16-dimethyl–PGE2, fenprostalene, PGD2, BW245C, carbaprostacyclin, or U46619 (1 µmol/L each). Values are mean±SEM of 3 experiments. *P<0.05 compared with all other values without asterisks.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanisms of acute hypercapnic acidosis–induced changes in cerebral vasomotor tone have been extensively investigated,^{8 9 10 11} and our findings are generally consistent with a dissimilar contribution for PGs and NO in nonrodent species in which NO plays a variable role^{11 13 20 44 45 46} ; although absence of a role for nNOS in the early hypercapnia-induced CBF rise in Yorkshire pigs differs from that in rats and mice,^{8 10} the dominant role for NO seen in rodents is not uniformly observed in other species.^{11 20 44 45 46 47} In contrast to mechanisms of acute hypercapnia-induced hyperemia, factors responsible for changes in CBF during prolonged hypercapnia were so far unknown. The present study suggests the involvement of K_ATP channels in stimulation of PGE₂ formation during prolonged hypercapnic acidosis, which in turn induces eNOS expression resulting in increased NO-mediated second hyperemia; this would explain a biphasic and more extended action of prolonged hypercapnic acidosis on CBF regulation.³ The data from this study suggest that PGs trigger the secondary CBF rise through associated induction of eNOS expression.

The effect of acute hypercapnia on CBF is known to be pH dependent^{17 18} ; this also seems to be the case for prolonged hypercapnia, given that normalization of pH abrogated all changes in CBF. However, despite a (slight) increase in pH over time during hypercapnia (Table), CBF continued to increase (Figure 1A and 1B); this implies that under these conditions the effects of acidosis on CBF are delayed consistently with induction of a slower process such as gene transcription of eNOS. Evidence that the second hypercapnia-induced hyperemia is largely NO dependent and appears to result from increased eNOS expression is suggested by the following observations: (1) cerebrovascular nitrite production rose; (2) the nonselective NOS inhibitor L-NA prevented the second increase in CBF (Figure 1A and 1B); (3) in contrast, Br-7-NI and TRIM, preferential inhibitors of nNOS (as well as of inducible NOS),⁴⁸ did not affect the second rise in CBF; (4) maximum eNOS-dependent vasorelaxation to substance P^{37 38} was augmented by prolonged hypercapnia (Figure 3B); (5) the late (at 6 hours) increase in CBF paralleled the time-course profile of eNOS mRNA expression (Figure 4); (6) increased eNOS expression was manifested functionally by concurrent augmented NADPH-diaphorase activity and maximum eNOS-dependent vasorelaxation to substance P,^{37 38} consistent with the relation between expression of eNOS and hemodynamics^{49 50} ; and (7) more importantly, pretreatment but not late treatment with the COX inhibitor diclofenac abrogated eNOS expression and associated increased CBF.

A major feature of this study is the role of PGs in regulating eNOS expression during prolonged hypercapnic acidosis. This is supported by generation of PGE₂ by the acidosis and effects of the COX inhibitor diclofenac in vivo in animals ventilated with 6% CO₂ (Figure 2), ex vivo on brain sections (Figures 3A, 4A, and 4B), and in vitro on isolated microvessels (Figures 4C and 4D). PGE₂ was found to be the principal PG modulating brain eNOS expression during acidosis. Furthermore, involvement of K⁺ channels during acidosis was itself dependent on PGs (Figures 4 through 6); K_ATP channel blockers prevented acidosis-induced calcium transients (required for PG formation), PGE₂ generation, and as a result eNOS expression. One could presume endothelium to be a major source of PGs during acidosis, because in contrast to endothelial cells,^{19 42} smooth muscle and astroglial cells do not generate PGs in response to acidosis.⁴²

Few factors are known to modulate eNOS expression. For example, estrogen⁵¹ and shear stress⁵² augment and TNF-⁵³ and possibly hypoxia⁵⁴ decrease eNOS mRNA expression. More recently, PGE₂ and PGD₂ (but not PGI₂ or PGF₂) were found to contribute to developmental regulation of eNOS mRNA.^{29 30} Our findings are consistent with the latter; moreover, they reveal for the first time a dominant role for a specific PG, noteworthy PGE₂ in present case, in regulating eNOS expression during pathophysiological adaption.

Data suggest the apparent activation of K⁺ channels by acidosis, which seems to set off the cascade of intracellular calcium entry, increased PG formation, and eNOS expression, and the latter in turn results in augmented CBF. In cerebrovascular cells, acidosis-evoked PGE₂ generation and calcium transients were abolished by K_ATP (but not K_Ca) channel blocker and were reproduced by K_ATP openers (Figures 4 through 6), implying hyperpolarization of cells in this process. Interestingly, hyperpolarization of endothelial cells can lead to calcium influx^{55 56 57} and PG formation²⁶ ; a posthyperpolarization depolarization may be implicated in this trigger.⁵⁸ K_ATP channels are found on endothelial cells,⁵⁹ and acidosis can stimulate them, but not K_ir or K_Ca channels^{21 22 23} ; in some species, this may contribute to the early hypercapnia-induced hemodynamic changes.^{20 22}

The effects of prolonged hypercapnia on CBF cannot simply be explained by activation of PG and NO synthase enzymes. This inference is supported by the failure of COX and NOS inhibitors to further reduce CBF at 3 hours of hypercapnia and by the inefficacy of late (at 5.5 hours) versus early diclofenac administration on CBF (Figures 1A and 1B). Altogether, the data once more point to triggering of de novo expression on a pathway involved in control of cerebral circulation, namely eNOS. The mechanisms for CBF decline after its first peak are not clear but may be contributed to by decreased formation of PGs (and possibly NO) (Figures 1C and 1D) before augmented eNOS expression.

In conclusion, the present study discloses a major and previously unexplored mechanism for a second cerebral hyperemia during sustained hypercapnia, which is mediated by NO through a process dependent on interaction between K_ATP channels and PGs and involving the induction of eNOS expression. We postulate that in chronic lung disease, the mechanisms identified may provide protection to the brain by enhancing cerebral hemodynamics when the latter is being compromised by impaired venous outflow due to augmented lung volume and by polycythemic hyperviscosity.^{4 6}

	Acknowledgments

 
We are grateful to the Medical Research Council of Canada, the Heart & Stroke Foundation of Quebec, and the March of Dimes Foundation for financial support. We thank Hendrika Fernandez for technical assistance.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received May 26, 2000; revision received October 13, 2000; accepted October 13, 2000.

	References

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