May 2002
Volume 43, Issue 5
Free
Retina  |   May 2002
PGE2-Mediated eNOS Induction in Prolonged Hypercapnia
Author Affiliations
  • Daniella Checchin
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
    Department of Pharmacology and Therapeutics, McGill University, Montreal, Québec, Canada.
  • Xin Hou
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
  • Pierre Hardy
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
  • Daniel Abran
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
  • Taline Najarian
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
    Department of Pharmacology and Therapeutics, McGill University, Montreal, Québec, Canada.
  • Martin H. Beauchamp
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
  • Sylvie G. Bernier
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
  • Fernand Gobeil, Jr
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
  • Christiane Quiniou
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
  • Daya R. Varma
    Department of Pharmacology and Therapeutics, McGill University, Montreal, Québec, Canada.
  • Sylvain Chemtob
    From the Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center, Hôpital Ste. Justine, Montreal, Québec; and the
    Department of Pharmacology and Therapeutics, McGill University, Montreal, Québec, Canada.
Investigative Ophthalmology & Visual Science May 2002, Vol.43, 1558-1566. doi:
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      Daniella Checchin, Xin Hou, Pierre Hardy, Daniel Abran, Taline Najarian, Martin H. Beauchamp, Sylvie G. Bernier, Fernand Gobeil, Christiane Quiniou, Daya R. Varma, Sylvain Chemtob; PGE2-Mediated eNOS Induction in Prolonged Hypercapnia. Invest. Ophthalmol. Vis. Sci. 2002;43(5):1558-1566.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Because prostaglandins (PGs) are implicated in acute hypercapnia-induced hyperemia, this study was conducted to test the hypothesis that prolonged hypercapnia may cause a sustained increase in retinal blood flow (RBF) through a PG-dependent induction of endothelial nitric oxide synthase (eNOS).

methods. Time-dependent RBF (microsphere technique), PGE2, nitrite (NO2 ), and NOS protein (reduced nicotinamide adenine dinucleotide phosphate [NADPH]-diaphorase staining) production were measured in hypercapnia (6% CO2)-treated piglets. From the same species, PGE2, eNOS mRNA, NOS protein, and vasomotor responses were measured in eyecup preparations, as were Ca2+ transients in neuroretinovascular endothelial cells.

results. Hypercapnia caused biphasic (at 0.5 hours and 6–8 hours) increases in RBF that were abolished with normalization of the pH. The early phase (0.5 hour) was associated with an increase in PGE2 levels and the latter phase (6–8 hours) with an increase in NO2 and NOS protein. Inhibition of cyclooxygenase by diclofenac prevented the early and late increase in RBF. NOS inhibitor l-nitro-arginine prevented only the latter. Hypercapnic acidosis increased retinal PGE2 levels and eNOS-dependent vasorelaxation ex vivo. The ex vivo time course of eNOS mRNA expression corresponded with the late-phase increase in RBF and was blocked by the transcription inhibitor actinomycin D and the receptor-operated Ca2+ channel blocker SK&F96365. In neuroretinovascular cells, acidosis increased Ca2+ transients, which were inhibited by SK&F96365, but not diclofenac.

conclusions. This study discloses a previously unexplored mechanism for late retinal hyperemia during sustained hypercapnia that appears secondary to the induced expression of eNOS mediated by PGE2.

Hyperoxia and ensuing oxidant stress are major factors involved in the pathogenesis of retinopathy of prematurity (ROP). Other factors regularly encountered in neonates with bronchopulmonary dysplasia, such as hypercapnia and associated acidosis, have also been shown to play a role in the development of ROP. 1 2 3 4 Hypercapnia causes retinal vasodilatation leading to increased retinal blood flow (RBF) and, in turn, oxygenation. 5 6 7 8 9 10 11 12 However, the ocular hemodynamic effects of hypercapnia have been reported only in acute conditions that are insufficient to account for the development of ROP. Evidence in brain tissue suggests that acute hypercapnia-induced hyperemia is transient. 13 14 Because sustained hypercapnic acidosis has been alleged to increase retinal oxygenation, 3 15 a delayed second phase of hyperemia is thought to explain the induced retinovascular injury. 3 15 Prolonged exposure to hypercapnia has recently been shown in the brain to be associated with a second increase in cerebral blood flow, and both the initial and second cerebral hyperemias are significantly accounted for by increased NO generation. 16 Although the retina is part of the central nervous system, its vasculature may not respond similarly to physiological adaptations because of differential expression of receptors and enzymes involved in these processes. 17 18 19 Thus, the response of the eye to prolonged hypercapnia and the relative role of NO in this process remains unknown. 
Acute hypercapnia-induced ocular hyperemia appears to be largely mediated by relaxant prostaglandins (PGs), 8 20 whereas the involvement of NO is minimal. 20 21 22 Complex interactions have been described between PGs and NO. 23 24 Among these interactions, PGE2 has been found to induce endothelial nitric oxide synthase (eNOS) in brain microvessels, 16 25 whereas a dominant role for PGD2 has been documented in the choroidal vasculature of the eye. 26 We therefore hypothesized that sustained hypercapnia may cause PG-dependent early and late phases of increased RBF. Our findings disclose for the first time that sustained hypercapnia induces a late retinal hyperemia that is in large part due to augmented NO release secondary to increased eNOS expression mediated by PGE2
Methods
Animals
Yorkshire piglets (≤6 days old) were used according to a protocol approved by the Animal Care Committee of Hôpital Ste. Justine, in conformity with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
RBF Measurements
RBF was measured by the microsphere technique, as previously described by us. 27 28 29 Briefly, catheterization of the left ventricle through the right subclavian artery for injection of fluorescent microspheres; the left subclavian artery for the 70-second withdrawal of reference blood samples, beginning 10 seconds before the injection of each type of fluorescent microsphere; and the femoral artery for blood pressure recording were performed in animals under halothane (2.5%) anesthesia. In addition, a 27-gauge butterfly needle, attached to a catheter, was introduced into the anterior chamber of the eye, through the cornea, to measure intraocular pressure. 
Upon cessation of halothane, sedation was continued with α-chloralose (50 mg/kg bolus followed by an infusion of 10 mg/kg · h). Animals were allowed to stabilize for 1.5 hours before experiments began. After baseline RBF measurements under 21% O2 and 79% N2, the gas mixture was changed to 6% CO2, 73% N2, and 21% O2 to obtain stable Paco 2 (≈65 mm Hg), which is commonly encountered in the clinical setting. RBF measurements were repeated 0.5, 3, 6, and 8 hours after hypercapnia was initiated. 
Animals were randomly assigned to pretreatment (30 minutes before blood flow measurements) with one of the following: the PG synthase inhibitor diclofenac (5 mg/kg), the NOS inhibitor l-nitro-arginine (l-NA; 3 mg/kg), the inducible and neuronal NOS (iNOS and nNOS, respectively) inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM; 1 mg/kg followed by 50 μg/kg · min), 30 bicarbonate (8.4% solution) to normalize the pH, or saline. 16 31 32 After the experiment, the animals were killed with intravenous pentobarbital (120 mg/kg) and the eyes dissected to remove the retina. Tissues were digested, and flow cytometric analysis (Interactive Medical Technologies, Inc., Los Angeles, CA) was used to count the fluorescence intensities in the retinal and reference blood samples. Blood flow was calculated as the product of the microsphere count per minute per gram tissue and reference blood withdrawal rate, divided by the microsphere count in the reference blood. 27 28 29 Retinal PGE2 levels, nitrite (NO2 ) production, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity were also measured in some animals. 
Tissue Preparations
Isolated eyecups were placed in buffer of the following composition (in millimolar): 132 NaCl, 3.0 KCl, 1.5 CaCl2, 1.5 MgCl2, 24.6 NaHCO3, 1.2 KH2PO4, 20 glucose, 6.6 urea, and 0.5% fetal bovine serum. Tissues were incubated at 37°C for 6 hours under normocapnic, hypercapnic acidosis, hypercapnic nonacidosis, and normocapnic acidosis, as in in vivo conditions. Conditions were set by bubbling CO2 (3%–10%) and adjusting pH with HCl and NaHCO3. Preparations were treated with diclofenac (100 μM), with or without (1 μM) 16,16-dimethyl PGE2, the selective PGD2 agonist BW245C, PGF, or carbaprostacyclin or with the nonselective Ca2+ channel blocker SK&F96365 (10 μM) or the transcription inhibitor actinomycin D (2 μM). Tissues were used to measure eNOS mRNA and PGE2 levels. 
Vasomotor Response of Retinal Vessels
Eye cups were prepared, to study vasomotor responses of the relatively undisturbed retinal vasculature, as previously described, 28 29 33 and were incubated for 4 hours in normocapnic or hypercapnic conditions, with or without diclofenac (100 μM), as described earlier. Thereafter, they were placed in physiological buffer (as described in Tissue Preparations) with normal CO2, to determine vasorelaxant responses to eNOS-dependent substance P, 34 using video imaging techniques. 28 29 Effects of substance P were also tested in some eyecups treated with l-NA (1 mM) 20 minutes before determining the vasomotor response to substance P. 
NADPH-Diaphorase Histochemistry
In situ NOS expression was assessed using NADPH-diaphorase staining, as described. 35 The intensity of blood vessel staining was analyzed digitally by computer (ImagePro Plus, ver. 4.1; Media Cybernetics, Silver Spring, MD). After normalizing for background tone, densitometry of tonality was determined in an equal number of pixels from each of the treatment groups, as reported by us. 16  
eNOS mRNA Detection by RNase Protection Assay
eNOS and destrin (loading control) RNase protection assays were conducted as described. 25 26 The primer pair for porcine eNOS was 5′-GCTTTTCCCTGCAGGAGCGAC-3′ and 5′-GCCAGTCTCTGCAGACTCTGG-3′; the primer pair for porcine destrin was 5′-ATGATGCAAGCTTTGAAACC-3′ and 5′-GGAAGCTTTCGATCTGTGG-3′. 25 The amplified products (0.4 kb) were digested with appropriate restriction enzymes (italic sequences in the primers denote the restriction sites) and cloned into the pGEM4 vector. The 32P-labeled cRNA probes for eNOS and destrin were prepared with an in vitro transcription kit (Promega, Madison, WI). 
Briefly, 20 μg total retinal RNA was mixed with 105 cpm eNOS and destrin probes in 20 μL hybridization buffer (80% deionized formamide, 40 mM piperazine-N,N′-bis(2-ethanesulfonic acid) [pH 6.8], 1 mM EDTA, and 0.4 M NaCl), denatured for 5 minutes at 90°C, and incubated overnight at 50°C. The RNA hybrids were digested with RNase A (10 μg/mL) and RNase T1 (200 U/mL) in 200 μL digestion buffer (10 mM Tris-HCl [pH 7.5], 5 mM EDTA, and 0.3 M NaCl) for 30 minutes at 25°C, followed by precipitation of protected fragments. The protected RNA fragments were resolved on urea-6% polyacrylamide gels, and the bands were visualized by phosphorimaging (Molecular Dynamics, Sunnyvale, CA) and quantified by densitometry. 
PGE2 and Nitrite Measurements
PGE2 levels were determined with a radioimmunoassay (RIA) kit (Advanced Magnetics, Boston, MA) as previously described. 27 32 Formation of NO by the retinas of piglets ventilated and treated as detailed was estimated by measuring the stable metabolite NO2 . 36  
Calcium Transients in Cultured Endothelial Cells
Porcine neuroretinovascular endothelial cells were prepared as previously described. 32 37 [Ca2+]i was measured by the fura-2-acetoxymethyl ester technique 32 in cells pretreated for 15 minutes with diclofenac (100 μM), SK&F96365 (10 μM), or EGTA (5 mM). The [Ca2+]i was calculated according to a published method. 32 38  
Chemicals
The following chemicals were purchased: α-chloralose, actinomycin D, diclofenac, EGTA, l-NA, and substance P (Sigma-Aldrich, Oakville, Ontario, Canada); BW245C, carbaprostacyclin, 16,16-dimethyl-PGE2, PGF, and U46619 (Cayman, Ann Arbor, MI); TRIM (Tocris, Ballwin, MO); SK&F96365 (Biomol, Plymouth Meeting, PA); fura-2-acetoxymethyl (Calbiochem, La Jolla, CA); PGE2 RIA kits (Cedarlane, Hornby, Ontario, Canada); fluorescent microspheres (Interactive Medical Technologies); pGEM4 plasmid vector (Promega); [α-32P]CTP (3000 Ci/mmol; Amersham, Mississauga, Ontario, Canada); and RNase A (Pharmacia Biotech, Montreal, Québec Canada). All other high-purity chemicals were from Fisher Scientific (Montreal, Québec, Canada). 
Statistical Analysis
RBF was analyzed by 2-way ANOVA, factoring for time and treatment followed by Dunnett’s multiple comparison test. All other data were analyzed by one-way ANOVA with comparison among means being performed by Dunnett or Tukey multiple comparison test, as appropriate. Statistical significance was set at P < 0.05. Data are expressed as the mean ± SEM. 
Results
Time Course of Retinal Response to Hypercapnia
During the 8-hour ventilation of animals with 6% CO2, Paco 2 remained high, whereas pH decreased initially and then tended to increase over time (Table 1) . Mean arterial blood pressure (MABP) and Pao 2 remained unchanged (Table 1) , and intraocular pressure remained constant (14.5 ± 1.53 mm Hg). There was no difference in these parameters between treatment groups, with the exception of the expected increase in blood pressure with l-NA (Table 1) . Hypercapnia produced a marked increase in RBF at 0.5 hour in control animals (Fig. 1A ), which decreased significantly by 3 hours and gradually increased again between 6 and 8 hours (Fig. 1A) . Normalization of the pH with bicarbonate abolished the retinal hemodynamic changes to hypercapnia at all time points (Fig. 1A) . Both the early and late increases in RBF were blunted by diclofenac. l-NA prevented only the late-phase increase in RBF (Fig. 1A) ; whereas, the selective iNOS and nNOS inhibitor TRIM did not affect retinal hemodynamics. Injection of diclofenac at 5.5 hours of hypercapnia also did not alter the hemodynamic response (Fig. 1A)
The early increase in RBF coincided with an increase in retinal PGE2 levels under hypercapnic conditions, which diminished by 8 hours, but remained higher than basal values (Fig. 1B) . Under normocapnia, PGE2 levels and NO2 production remained stable up to 8 hours. NO2 production was also increased by hypercapnia (Fig. 1C) . Diclofenac reduced PGE2 levels and prevented the increase in NO2 production observed under hypercapnia (Figs. 1B 1C) ; l-NA reduced NO2 production but not PGE2 levels (Figs. 1B 1C) ; and TRIM reduced (slightly) NO2 production during normocapnia but negligibly during hypercapnia (Fig. 1C)
Retinal NADPH-Diaphorase Reactivity and eNOS mRNA
In vivo and ex vivo exposure to 6 hours of hypercapnia evoked increased NADPH-diaphorase reactivity that was largely localized to the vasculature, compared with the normocapnic control (Figs. 2 3A ). These changes in NOS expression were inhibited by diclofenac (Figs. 2 , top right, 3A , top right). eNOS mRNA also increased in the retinas of eye cups exposed to hypercapnia. This was blocked by diclofenac, but not by l-NA (Fig. 3B , bottom). Diclofenac did not affect NOS expression during normocapnia (data not shown). 
Effects of High CO2 on NO-Dependent Retinal Vasorelaxation
Experiments were conducted to assess whether changes in hypercapnia-induced eNOS expression were reflected functionally. The retinal vessels of eye cups exposed to high CO2 for 4 hours exhibited a marked increase in vasorelaxation to eNOS-dependent substance P when compared with the response of retinal vasculature maintained in normal CO2 tension (Fig. 4) . Cotreatment (4 hours) with diclofenac or actinomycin D prevented this augmented vasorelaxation induced by high CO2. Agents did not affect vasomotor response during normal CO2. Acute (15–30 minutes) exposure to high CO2 did not modify the response to substance P. 
Concentration- and Time-Dependent Effects of CO2 on eNOS mRNA Expression
Exposure of isolated eye cups to increasing CO2 tension for 6 hours produced a concentration-dependent increase in retinal eNOS mRNA (Fig. 5A) . Acidosis in the presence of normal CO2 induced a comparable time-dependent increase in eNOS expression (Figs. 5A 5B) , which was blocked by the transcription inhibitor actinomycin D. Normalization of the pH prevented changes in eNOS mRNA (Fig. 5A) . The acidosis-induced increase in eNOS expression was also associated with an increase in PGE2 levels, and both were prevented by the PG synthase inhibitor diclofenac, as well as the Ca2+ channel blocker SK&F96365 (Figs. 5C 5D) consistent with calcium requirements for formation of PG. 
Effect of Acidosis on Ca2+ Transients in Endothelial Cells
Acidosis-induced Ca2+ influx, an essential cofactor for phospholipase A2-dependent yield of PGs, was studied in endothelial cells, which are an important source of PGs under these conditions. 39 Acidification of the medium with HCl or NaH2PO4 to an approximate pH of 7.10 to 7.15 caused a rapid and marked increase in Ca2+ transients measured directly on neuroretinovascular endothelial cells (Figs. 6A 6B ). This was prevented by bicarbonate (data not shown), SK&F96365, and EGTA, but not by diclofenac (Figs. 6A 6B) , further suggesting that the Ca2+ transients precede the increase in PGs (Fig. 5C)
Effects of PG Analogues on Acidosis-Induced Changes in eNOS mRNA Levels
To establish which PG is involved in the acidosis-induced eNOS expression, eyecup preparations were exposed to acidified buffer (pH ∼7.15) and pretreated, or not, with diclofenac in the absence or presence of PGs. Inhibitors of selective PGE2, PGF, PGD2, and PGI2 synthases are not yet available. The inhibitory effect of diclofenac on the acidosis-induced retinal eNOS mRNA increase was prevented by concurrent treatment with 16,16-dimethyl-PGE2, but not with PGF or the stable analogues of PGD2 and PGI2, BW245C and carbaprostacyclin, respectively (Fig. 7)
Discussion
Although hypercapnia has been found to affect ocular blood flow, 5 6 7 8 9 10 11 12 the hemodynamic changes that occur during prolonged hypercapnia and the underlying mechanisms of these changes have been unknown thus far. Piglets were used in this study to measure hemodynamic parameters more accurately, because the larger size of their ocular tissues allows for the minimum number of microspheres (∼400) to be present without impairing tissue blood flow. 40 Although piglets, in contrast to rats, have a nearly mature retina at birth, oxygen-induced retinopathy has been produced in these animals. 41 42 Moreover, the early changes we observed in RBF in piglets and the role of PGs in this process are consistent with those previously reported in the same and other (monkeys) species. 8 20 During prolonged hypercapnia, the present study showed a second increase in RBF and expected O2 delivery that was NO-mediated and associated with increased eNOS expression induced by an earlier augmentation in PGE2 levels. 
The effects of high CO2 on vascular tone have largely been ascribed to acidosis. 43 44 45 The same appears to be true of prolonged exposure to hypercapnia, because normalization of the pH abolished all changes in RBF (Fig. 1A) . During the 8-hour exposure to high CO2, pH increased slightly, and MABP remained stable (Table 1) , yet RBF continued to increase (Fig. 1A) . This suggests that changes in RBF can be attributed to local vasomotor alterations. Once the hyperemic process is triggered, it cannot be prevented by a late normalization of the pH, and the effects of hypercapnic acidosis on RBF are delayed, perhaps because of a relatively slower process, such as gene transcription. 
A number of observations imply a major role for eNOS induction in the second hypercapnia-induced hyperemia. A direct effect of acidosis on NOS activity is unlikely, because acidosis would tend to reduce it. 46 However, retinal NO2 production increased late into the hypercapnic exposure (Fig. 1C) . Also, the nonselective NOS inhibitor l-NA prevented the late increases in RBF (Fig. 1A) , whereas TRIM, a selective inhibitor of iNOS and nNOS, 30 did not affect the retinal hemodynamics (Fig. 1A) . The second increase in RBF parallels the time course profile of eNOS mRNA expression (Fig. 5B) , which was further manifested functionally by augmented NADPH-diaphorase activity in retinal vasculature (Figs. 2 3) and eNOS-dependent retinal vasorelaxation to substance P (Fig. 4) . Along these lines, the mechanisms underlying the effects of prolonged hypercapnia on RBF cannot be explained by the mere activation of PG and NO synthases. The inefficacy of late (at 5.5 hours) diclofenac administration on RBF, compared with its efficacy when administered at the onset of hypercapnia (Fig. 1A) , supports this inference. Furthermore, the transcription inhibitor actinomycin D prevented both the delayed hypercapnia-induced increase in eNOS mRNA and vasorelaxation to substance P (Figs. 4 5) . Thus, collectively, the data are indicative of the triggering of de novo eNOS expression in retinal vasculature by hypercapnic acidosis. 
The role of PGs in regulating eNOS expression in the retina during prolonged hypercapnic acidosis is a salient feature of this study. Evidence supporting this major role for PGE2 includes an early increase in PGE2 levels during hypercapnia and prevention by diclofenac of the hypercapnia-induced eNOS expression (mRNA and in situ protein activity), as well as retinal hyperemia and increased vasorelaxation in response to substance P (Figs. 1 2 3 4 5) . In addition, PGE2 was found to be the major PG that modulates eNOS expression in retina (Fig. 7) , as reported in brain vasculature. 25 The mechanisms that lead to stimulation of PG formation are not clear from this study. Phospholipase A2 and cyclooxygenase are unlikely targets, because their activities are optimal at basic pH. 47 However, hypercapnia-induced Ca2+ influx as we observed (Fig. 5 6) , possibly mediated directly through stimulation of Ca2+ channels 48 49 or indirectly by K+ channels, 50 would provide a necessary cofactor for formation of PG. Although endothelium is a likely important source of PGs during acidosis, 39 participation by specialized neurons 48 49 and vascular musculature 51 cannot be fully excluded; however, smooth muscle, astroglia, and neurons including those in the retina, either do not form PGs or exhibit a calcium influx. 52 53 54  
In conclusion, the present study disclosed a previously unexplored mechanism for late retinal hyperemia during sustained hypercapnia that appears secondary to the induction of eNOS expression and activity mediated by PGE2, as recently described in brain tissue. 16 Therefore, PGs seem to be involved in both the acute and prolonged hypercapnia-induced increased retinal blood flow through direct and indirect vascular effects, respectively. The findings provide a mechanism for extended hypercapnia in predisposing to ROP 1 2 3 4 and are consistent with a role for PGs 55 and eNOS 56 in the development of this oculovascular disorder. 
 
Table 1.
 
Arterial Blood Pressure and Gas Levels in Piglets before and after Hypercapnia
Table 1.
 
Arterial Blood Pressure and Gas Levels in Piglets before and after Hypercapnia
Treatment Baseline 0.5 h 3 h 6 h 8 h
Control
 MABP (mm Hg) 58.3 ± 5.4 63.1 ± 3.8 60.2 ± 4.7 63.5 ± 5.3 65.2 ± 3.5
 Arterial pH 7.38 ± 0.04 7.19 ± 0.03* 7.23 ± 0.02* 7.26 ± 0.03* 7.29 ± 0.02* , ‡
 PaCO2 (mm Hg) 41.2 ± 1.5 68.7 ± 2.1* 65.1 ± 1.6* 66.8 ± 2.7* 64.6 ± 1.8*
PaO2 (mm Hg) 90.0 ± 8.6 89.3 ± 4.3 88.4 ± 4.1 86.1 ± 8.5 91.1 ± 8.5
Diclofenac
 MABP (mm Hg) 63.2 ± 5.4 62.7 ± 3.4 68.2 ± 6.3 69.1 ± 2.5 64.0 ± 2.6
 Arterial pH 7.40 ± 0.02 7.19 ± 0.02* 7.23 ± 0.03* 7.25 ± 0.06* 7.27 ± 0.03* , ‡
 PaCO2 (mm Hg) 41.8 ± 3.4 65.3 ± 3.1* 67.1 ± 3.4* 68.2 ± 2.5* 66.3 ± 2.6*
 PaO2 (mm Hg) 96.9 ± 7.9 91.8 ± 8.5 91.5 ± 4.5 86.1 ± 8.8 88.5 ± 8.0
l-NA
 MABP (mm Hg) 86.7 ± 3.1, † 85.5 ± 2.9, † 84.2 ± 3.9, † 84.9 ± 4.1, † 86.7 ± 2.7, †
 Arterial pH 7.39 ± 0.03 7.20 ± 0.01* 7.22 ± 0.01* 7.23 ± 0.02* 7.28 ± 0.03* , ‡
 PaCO2 (mm Hg) 42.4 ± 1.8 68.7 ± 3.3* 67.5 ± 3.3* 69.5 ± 2.6* 69.3 ± 2.8*
 PaO2 (mm Hg) 96.8 ± 9.5 97.8 ± 9.0 99.2 ± 8.4 86.7 ± 9.0 90.3 ± 8.4
TRIM
 MABP (mm Hg) 61.4 ± 2.3 65.3 ± 3.4 66.5 ± 2.2 61.4 ± 1.9 63.5 ± 3.5
 Arterial pH 7.41 ± 0.03 7.21 ± 0.02* 7.21 ± 0.03* 7.19 ± 0.02* 7.25 ± 0.02* , ‡
 PaCO2 (mm Hg) 44.8 ± 3.1 71.0 ± 3.1* 72.4 ± 2.4* 72.7 ± 2.6* 70.5 ± 3.3*
 PaO2 (mm Hg) 91.4 ± 7.6 93.3 ± 8.4 91.3 ± 5.1 93.5 ± 5.2 92.4 ± 4.5
Bicarbonate
 MABP (mm Hg) 63.4 ± 2.9 66.1 ± 2.1 61.9 ± 4.0 65.5 ± 3.3 62.3 ± 2.5
 Arterial pH 7.40 ± 0.03 7.41 ± 0.01 7.39 ± 0.03 7.38 ± 0.02 7.40 ± 0.02
 PaCO2 (mm Hg) 43.4 ± 2.0 69.9 ± 2.7* 71.9 ± 3.0* 70.5 ± 2.1* 72.1 ± 3.3*
 PaO2 (mm Hg) 88.3 ± 9.6 91.1 ± 6.4 89.3 ± 6.2 93.5 ± 5.6 91.3 ± 7.5
Figure 1.
 
(A) Time course of RBF in piglets during an 8-hour exposure to hypercapnia by ventilation with 6% CO2 (indicated by horizontal bar). One group of animals was treated with intravenous diclofenac (5 mg/kg) 5.5 hours after exposure to hypercapnia, and another group was given sodium bicarbonate (8.4%) to normalize pH. All other animals were pretreated with diclofenac (5 mg/kg), l-NA (3 mg/kg), TRIM (1 mg/kg followed by 50 μg/kg · min), or saline. At time 0, RBF was basal (normocapnia). Data are the mean ± SEM of four or five eyes of different animals. *P < 0.05 compared with basal levels; †P < 0.05 compared with corresponding levels in animals treated with diclofenac or l-NA. Retinal PGE2 levels (B) and NO2 production (C) are from animals exposed in vivo to normocapnic and hypercapnic conditions. Data are the mean ± SEM of three or four eyes from different piglets. *P < 0.05 compared with 8-hour values at Paco 2 of approximately 40 mm Hg.
Figure 1.
 
(A) Time course of RBF in piglets during an 8-hour exposure to hypercapnia by ventilation with 6% CO2 (indicated by horizontal bar). One group of animals was treated with intravenous diclofenac (5 mg/kg) 5.5 hours after exposure to hypercapnia, and another group was given sodium bicarbonate (8.4%) to normalize pH. All other animals were pretreated with diclofenac (5 mg/kg), l-NA (3 mg/kg), TRIM (1 mg/kg followed by 50 μg/kg · min), or saline. At time 0, RBF was basal (normocapnia). Data are the mean ± SEM of four or five eyes of different animals. *P < 0.05 compared with basal levels; †P < 0.05 compared with corresponding levels in animals treated with diclofenac or l-NA. Retinal PGE2 levels (B) and NO2 production (C) are from animals exposed in vivo to normocapnic and hypercapnic conditions. Data are the mean ± SEM of three or four eyes from different piglets. *P < 0.05 compared with 8-hour values at Paco 2 of approximately 40 mm Hg.
Figure 2.
 
In vivo modulation of NADPH-diaphorase staining of retinas by exposure to hypercapnia. Piglets were ventilated and treated as in Figure 1 , and retinal wholemounts were fixed for NADPH-diaphorase reactivity (top). Individual pixel tonality of densitometry of vasculature was analyzed by correcting for background tone (bottom). Data are the mean ± SEM of four retinal preparations from eye cups of different animals. *P < 0.05 compared with data without asterisks.
Figure 2.
 
In vivo modulation of NADPH-diaphorase staining of retinas by exposure to hypercapnia. Piglets were ventilated and treated as in Figure 1 , and retinal wholemounts were fixed for NADPH-diaphorase reactivity (top). Individual pixel tonality of densitometry of vasculature was analyzed by correcting for background tone (bottom). Data are the mean ± SEM of four retinal preparations from eye cups of different animals. *P < 0.05 compared with data without asterisks.
Figure 3.
 
Ex vivo modulation of NADPH-diaphorase staining and eNOS mRNA expression after exposure to hypercapnia. Isolated eyecups were exposed for 6 hours to 5% CO2 (Paco 2 ≈ 40 mm Hg, pH 7.4) or 9% to 10% CO2 (Paco 2 ≈ 65 mm Hg, pH 7.15–7.2) in absence or presence of diclofenac (100 μM) or l-NA (1 mM). (A) Retinal wholemounts were fixed for NADPH-diaphorase reactivity (top) and individual pixel tonality of densitometry of vasculature was analyzed by correcting for background tone (bottom). (B) eNOS mRNA blot from RNase protection assay (top) and semiquantitative analysis with densitometry (bottom) relative to destrin (control). Unprotected and protected fragments for eNOS are 414 and 356 nucleotides (nt), and for destrin, 237 and 165 nt. Data in both histograms are the mean ± SEM from three retinas of different animals. *P < 0.05 compared with data without asterisks.
Figure 3.
 
Ex vivo modulation of NADPH-diaphorase staining and eNOS mRNA expression after exposure to hypercapnia. Isolated eyecups were exposed for 6 hours to 5% CO2 (Paco 2 ≈ 40 mm Hg, pH 7.4) or 9% to 10% CO2 (Paco 2 ≈ 65 mm Hg, pH 7.15–7.2) in absence or presence of diclofenac (100 μM) or l-NA (1 mM). (A) Retinal wholemounts were fixed for NADPH-diaphorase reactivity (top) and individual pixel tonality of densitometry of vasculature was analyzed by correcting for background tone (bottom). (B) eNOS mRNA blot from RNase protection assay (top) and semiquantitative analysis with densitometry (bottom) relative to destrin (control). Unprotected and protected fragments for eNOS are 414 and 356 nucleotides (nt), and for destrin, 237 and 165 nt. Data in both histograms are the mean ± SEM from three retinas of different animals. *P < 0.05 compared with data without asterisks.
Figure 4.
 
Effects of exposure to high CO2 on the retinal vasorelaxant response to substance P. Eye cups were incubated for 4 hours in buffer bubbled with 5% or 9% to 10% CO2 in absence or presence of diclofenac (100 μM) or actinomycin D (2 μM), as described in Figure 3 . Some preparations were treated with l-NA (1 mM) 20 minutes before administration of substance P. Data are the mean ± SEM of three or four eyes of different animals. *P < 0.01 compared with other curves.
Figure 4.
 
Effects of exposure to high CO2 on the retinal vasorelaxant response to substance P. Eye cups were incubated for 4 hours in buffer bubbled with 5% or 9% to 10% CO2 in absence or presence of diclofenac (100 μM) or actinomycin D (2 μM), as described in Figure 3 . Some preparations were treated with l-NA (1 mM) 20 minutes before administration of substance P. Data are the mean ± SEM of three or four eyes of different animals. *P < 0.01 compared with other curves.
Figure 5.
 
Concentration- and time-dependent effects of hypercapnic acidosis on eNOS mRNA expression in piglet retinas. (A) Eye cups were incubated in physiological buffer for 6 hours with 3% CO2 (Paco 2 ≈ 25 mm Hg, pH ∼7.5), 10% CO2 (Paco 2 ≈ 65 mm Hg, pH ∼7.2), 10% CO2 with normalized pH (Paco 2 ≈ 65 mm Hg, pH ∼7.4) and 5% CO2 with acidosis (Paco 2 ≈ 40 mm Hg, pH ∼7.2) or not incubated (basal). Acidosis with normal CO2 was adjusted by addition of NaH2PO4 or HCl. (B) Time-dependent changes in eNOS mRNA after 2, 4, and 6 hours of exposure to normocapnic acidosis. PGE2 levels (C) and expression of eNOS (D) in retinas isolated from eyecups incubated for 6 hours in normocapnic acidosis conditions in the presence or absence of diclofenac (100 μM), SK&F96365 (10 μM), or actinomycin D (2 μM). mRNA was subjected to RNase protection assay for eNOS. Data are the mean ± SEM of five experiments on eyes from different piglets. *P < 0.05 compared with all other data.
Figure 5.
 
Concentration- and time-dependent effects of hypercapnic acidosis on eNOS mRNA expression in piglet retinas. (A) Eye cups were incubated in physiological buffer for 6 hours with 3% CO2 (Paco 2 ≈ 25 mm Hg, pH ∼7.5), 10% CO2 (Paco 2 ≈ 65 mm Hg, pH ∼7.2), 10% CO2 with normalized pH (Paco 2 ≈ 65 mm Hg, pH ∼7.4) and 5% CO2 with acidosis (Paco 2 ≈ 40 mm Hg, pH ∼7.2) or not incubated (basal). Acidosis with normal CO2 was adjusted by addition of NaH2PO4 or HCl. (B) Time-dependent changes in eNOS mRNA after 2, 4, and 6 hours of exposure to normocapnic acidosis. PGE2 levels (C) and expression of eNOS (D) in retinas isolated from eyecups incubated for 6 hours in normocapnic acidosis conditions in the presence or absence of diclofenac (100 μM), SK&F96365 (10 μM), or actinomycin D (2 μM). mRNA was subjected to RNase protection assay for eNOS. Data are the mean ± SEM of five experiments on eyes from different piglets. *P < 0.05 compared with all other data.
Figure 6.
 
Effects of acidosis on Ca2+ transients in neuroretinovascular endothelial cells. Ca2+ transients were measured by the fura-2-acetoxymethyl ester technique after acidification of the medium by the addition of HCl or NaH2PO4 (pH ∼7.10–7.15). (A) Typical tracing. (B) Histogram presenting peak [Ca2+]i. Media were pretreated with vehicle, diclofenac (100 μM), SK&F96365 (10 μM), or EGTA (5 mM). Arrow: time of administration of acidifying agents (H+). Data are the mean ± SEM of three or four experiments. *P < 0.01 compared with all other data without asterisks.
Figure 6.
 
Effects of acidosis on Ca2+ transients in neuroretinovascular endothelial cells. Ca2+ transients were measured by the fura-2-acetoxymethyl ester technique after acidification of the medium by the addition of HCl or NaH2PO4 (pH ∼7.10–7.15). (A) Typical tracing. (B) Histogram presenting peak [Ca2+]i. Media were pretreated with vehicle, diclofenac (100 μM), SK&F96365 (10 μM), or EGTA (5 mM). Arrow: time of administration of acidifying agents (H+). Data are the mean ± SEM of three or four experiments. *P < 0.01 compared with all other data without asterisks.
Figure 7.
 
Effects of PG analogues on retinal eNOS mRNA expression from eyecups exposed to HCl-induced acidosis. Tissues were exposed for 6 hours to acidosis, as described in Figure 5 and treated with diclofenac (100 μM), with or without 16,16-dimethyl-PGE2, BW245C, PGF, or carbaprostacyclin (1 μM each). Data are the mean ± SEM of three experiments conducted on eyes from different piglets. *P < 0.05 compared with all other data without asterisks.
Figure 7.
 
Effects of PG analogues on retinal eNOS mRNA expression from eyecups exposed to HCl-induced acidosis. Tissues were exposed for 6 hours to acidosis, as described in Figure 5 and treated with diclofenac (100 μM), with or without 16,16-dimethyl-PGE2, BW245C, PGF, or carbaprostacyclin (1 μM each). Data are the mean ± SEM of three experiments conducted on eyes from different piglets. *P < 0.05 compared with all other data without asterisks.
The authors thank Hendrika Fernandez for skillful technical assistance and Ferme Ménard, Inc. (Ange-Gardien, De Rouville, Québec, Canada) for generously supplying the piglets. 
Shohat M, Reisner SH, Krikler R, Nissenkorn I, Yassur Y, Ben-Sira I. Retinopathy of prematurity: incidence and risk factors. Pediatrics. 1983;72:159–163. [PubMed]
Holmes JM, Leske DA, Zhang S. The effect of raised inspired carbon dioxide on normal retinal vascular development in the neonatal rat. Curr Eye Res. 1997;16:78–81. [CrossRef] [PubMed]
Holmes JM, Zhang S, Leske DA, Lanier WL. Carbon dioxide-induced retinopathy in the neonatal rat. Curr Eye Res. 1998;17:608–616. [CrossRef] [PubMed]
Ajayi OA, Raval D, Lucheese N, Pildes RS. Ophthalmological morbidity in very-low-birthweight infants with bronchopulmonary dysplasia. J Natl Med Assoc. 1997;89:679–683. [PubMed]
Sponsel WE, DePaul KL, Zetlan SR. Retinal hemodynamic effects of carbon dioxide, hyperoxia, and mild hypoxia. Invest Ophthalmol Vis Sci. 1992;33:1864–1869. [PubMed]
Alm A, Bill A. The oxygen supply to the retina. II: effects of high intraocular pressure and of increased arterial carbon dioxide tension on uveal and retinal blood flow in cats—a study with radioactively labeled microspheres including flow determinations in brain and some other tissues. Acta Physiol Scand. 1972;84:306–319. [CrossRef] [PubMed]
Milley JR, Rosenberg AA, Jones MD, Jr. Retinal and choroidal blood flows in hypoxic and hypercarbic newborn lambs. Pediatr Res. 1984;18:410–414. [CrossRef] [PubMed]
Stiris T, Suguihara C, Hehre D, et al. Effects of cyclooxygenase inhibition on retinal and choroidal blood flow during hypercarbia in newborn piglets. Pediatr Res. 1992;31:127–130. [CrossRef] [PubMed]
Yu DY, Cringle SJ, Alder V, Su EN. Intraretinal oxygen distribution in the rat with graded systemic hyperoxia and hypercapnia. Invest Ophthalmol Vis Sci. 1999;40:2082–2087. [PubMed]
Harris A, Arend O, Wolf S, Cantor LB, Martin BJ. CO2 dependence of retinal arterial and capillary blood velocity. Acta Ophthalmol Scand. 1995;73:421–424. [PubMed]
Tsacopoulos M, Baker R, Johnson M, Strauss J, David NJ. The effect of arterial Pco 2 on inner-retinal oxygen availability in monkeys. Invest Ophthalmol Vis Sci. 1973;12:449–455.
Berkowitz BA. Adult and newborn rat inner retinal oxygenation during carbogen and 100% oxygen breathing: comparison using magnetic resonance imaging delta Po 2 mapping. Invest Ophthalmol Vis Sci. 1996;37:2089–2098. [PubMed]
Warner DS, Turner DM, Kassell NF. Time-dependent effects of prolonged hypercapnia on cerebrovascular parameters in dogs: acid-base chemistry. Stroke. 1987;18:142–149. [CrossRef] [PubMed]
Brubakk AM, Oh W, Stonestreet BS. Prolonged hypercarbia in the awake newborn piglet: effect on brain blood flow and cardiac output. Pediatr Res. 1987;21:29–33. [CrossRef] [PubMed]
Holmes JM, Zhang S, Leske DA, Lanier WL. Metabolic acidosis-induced retinopathy in the neonatal rat. Invest Ophthalmol Vis Sci. 1999;40:804–809. [PubMed]
Najarian T, Marrache AM, Dumont I, et al. Prolonged hypercapnia-evoked cerebral hyperemia via K+ channel- and prostaglandin E2-dependent endothelial nitric oxide synthase induction. Circ Res. 2000;87:1149–1156. [CrossRef] [PubMed]
Hardy P, Dumont I, Bhattacharya M, et al. Oxidants, nitric oxide and prostanoids in the developing ocular vasculature: a basis for ischemic retinopathy. Cardiovasc Res. 2000;47:489–509. [CrossRef] [PubMed]
Hardy P, Nuyt AM, Dumont I, et al. Developmentally increased cerebrovascular NO in newborn pigs curtails cerebral blood flow autoregulation. Pediatr Res. 1999;46:375–382. [CrossRef] [PubMed]
Hardy P, Lamireau D, Hou X, et al. Major role for neuronal nitric oxide synthase in curtailing choroidal blood flow autoregulation in the newborn pig. J Appl Physiol. 2001;91:1655–1662. [PubMed]
Pournaras C, Tsacopoulos M. The metabolic regulation of the retinal blood flow and the role of prostaglandins. Klin Monatsbl Augenheilkd. 1978;172:445–448. [PubMed]
Schmetterer L, Findl O, Strenn K, et al. Role of NO in the O2 and CO2 responsiveness of cerebral and ocular circulation in humans. Am J Physiol. 1997;273:R2005–R2012. [PubMed]
Gidday JM, Zhu Y. Nitric oxide does not mediate autoregulation of retinal blood flow in the newborn pig. Am J Physiol. 1995;269:H1065–H1072. [PubMed]
Hardy P, Abran D, Hou X, et al. A major role for prostacyclin in nitric oxide-induced ocular vasorelaxation in the piglet. Circ Res. 1998;83:721–729. [CrossRef] [PubMed]
Goodwin DC, Landino LM, Marnett LJ. Effects of nitric oxide and nitric oxide-derived species on prostaglandin biosynthesis. FASEB J. 1999;13:1121–1136. [PubMed]
Dumont I, Hou X, Hardy P, et al. Developmental regulation of endothelial nitric oxide synthase in cerebral vessels of newborn pig by prostaglandin E2. J Pharmacol Exp Ther. 1999;291:627–633. [PubMed]
Dumont I, Hardy P, Peri KG, et al. Regulation of endothelial nitric oxide synthase by PGD2 in the developing choroid. Am J Physiol Heart Circ Physiol. 2000;278:H60–H66. [PubMed]
Parys-Van Ginderdeuren R, Malcolm D, Varma DR, Aranda JV, Chemtob S. Dissociation between prostaglandin levels and blood flow to the retina and choroid in the newborn pig following nonsteroidal anti-inflammatory drugs. Invest Ophthalmol Vis Sci. 1992;33:3378–3384. [PubMed]
Hardy P, Abran D., Li D-Y, Fernandez H, Varma DR, Chemtob S. Free radicals in retinal and choroidal blood flow autoregulation in the piglet interaction with prostaglandins. Invest Ophthalmol Vis Sci. 1994;35:580–591. [PubMed]
Hardy P, Nuyt AM, Abran D, St-Louis J, Varma DR, Chemtob S. Nitric oxide in retinal and choroidal blood flow autoregulation in newborn pigs: interactions with prostaglandins. Pediatr Res. 1996;39:487–493. [CrossRef] [PubMed]
Handy RL, Harb HL, Wallace P, Gaffen Z, Whitehead KJ, Moore PK. Inhibition of nitric oxide synthase by 1-(2-trifluoromethylphenyl) imidazole (TRIM) in vitro: antinociceptive and cardiovascular effects. Br J Pharmacol. 1996;119:423–431. [CrossRef] [PubMed]
Salter M, Duffy C, Hazelwood R. Determination of brain nitric oxide synthase inhibition in vivo: ex vivo assays of nitric oxide synthase can give incorrect results. Neuropharmacology. 1995;34:327–334. [CrossRef] [PubMed]
Lahaie I, Hardy P, Hou X, et al. A novel mechanism for vasoconstrictor action of 8-isoprostaglandin F on retinal vessels. Am J Physiol. 1998;274:R1406–R1416. [PubMed]
Abran D, Varma DR, Li D-Y, Chemtob S. Reduced responses of the newborn pig retinal vessels to prostaglandins but not to thromboxane. Can J Physiol Pharmacol. 1994;72:168–173. [CrossRef] [PubMed]
Kitamura Y, Okamura T, Kani K, Toda N. Nitric oxide-mediated retinal arteriolar and arterial dilatation induced by substance P. Invest Ophthalmol Vis Sci. 1993;34:2859–2865. [PubMed]
Sagar SM. NADPH diaphorase Histochemistry in the rabbit retina. Brain Res. 1986;373:153–158. [CrossRef] [PubMed]
Verdon CP, Burton BA, Prior RL. Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interference by NADP when Greiss reaction is used to assay nitrite. Anal Biochem. 1995;224:502–508. [CrossRef] [PubMed]
Beauchamp MH, Martinez-Bermudez AK, Gobeil F, Jr, et al. Role of thromboxane in retinal microvascular degeneration in oxygen-induced retinopathy. J Appl Physiol. 2001;90:2279–2288. [PubMed]
Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–3450. [PubMed]
Hsu P, Shibata M, Leffler CW. Prostanoid synthesis in response to high CO2 in newborn pig brain microvascular endothelial cells. Am J Physiol. 1993;264:H1485–H1492. [PubMed]
Heymann MA, Payne BD, Hoffman JIE, et al. Blood flow measurements with radionuclide-labeled particles. Prog Cardiovasc Dis. 1977;20:55–79. [CrossRef] [PubMed]
Sisson T. The effect of light and various concentrations of oxygen on the retina of the newborn pig. Retinopathy of Prematurity Conference Syllabus. 1981;581–599. Washington, DC.
Tasman W. Vitreoretinal changes in cicatricial retrolental fibroplasia. Trans Am Ophthalmol Soc. 1970;68:548–594. [PubMed]
Stiris T, Hall C, Bratlid D. Retinal, choroidal and total ocular blood flow response to hypercarbia during spontaneous breathing and mechanical ventilation. Biol Neonate. 1991;59:86–92. [CrossRef] [PubMed]
Aalkjaer C, Poston L. Effects of pH on vascular tension: which are the important mechanisms?. J Vasc Res. 1996;33:347–359. [CrossRef] [PubMed]
Tsacopoulos M, David NJ. The effect of arterial Pco 2 on relative retinal blood flow in monkeys. Invest Ophthalmol Vis Sci. 1973;12:335–347.
Anderson RE, Meyer FB. Is intracellular brain pH a dependent factor in NOS inhibition during focal cerebral ischemia?. Brain Res. 2000;856:220–226. [CrossRef] [PubMed]
Rordorf G, Uemura Y, Bonventre JV. Characterization of phospholipase A2 (PLA2) activity in gerbil brain: enhanced activities of cytosolic, mitochondrial, and microsomal forms after ischemia and reperfusion. J Neurosci. 1991;11:1829–1836. [PubMed]
Buckler KJ, Vaughan-Jones RD. Effects of hypercapnia on membrane potential and intracellular calcium in rat carotid body type I cells. J Physiol. 1994;478:157–171. [CrossRef] [PubMed]
Sato M. Response of cytosolic Ca2+ to hypercapnic acidosis in cultured glomus cells of the adult rabbit carotid body. Brain Res. 1994;662:251–254. [CrossRef] [PubMed]
Xu H, Cui N, Yang Z, Qu Z, Jiang C. Modulation of kir4.1 and kir5.1 by hypercapnia and intracellular acidosis. J Physiol. 2000;524:725–735. [CrossRef] [PubMed]
Cairns SP, Westerblad H, Allen DG. Changes in myoplasmic pH and calcium concentration during exposure to lactate in isolated rat ventricular myocytes. J Physiol. 1993;464:561–574. [CrossRef] [PubMed]
Iwasawa K, Nakajima T, Hazama H, et al. Effects of extracellular pH on receptor-mediated Ca2+ influx in A7r5 rat smooth muscle cells: involvement of two different types of channel. J Physiol. 1997;503:237–251. [CrossRef] [PubMed]
Tombaugh GC, Somjen GG. Effects of extracellular pH on voltage-gated Na+, K+ and Ca2+ currents in isolated rat CA1 neurons. J Physiol. 1996;493:719–732. [CrossRef] [PubMed]
Takahashi K, Dixon DB, Copenhagen DR. Modulation of a sustained calcium current by intracellular pH in horizontal cells of fish retina. J Gen Physiol. 1993;101:695–714. [CrossRef] [PubMed]
Nandgaonkar BN, Rotschild T, Yu K, Higgins RD. Indomethacin improves oxygen-induced retinopathy in the mouse. Pediatr Res. 1999;46:184–188. [CrossRef] [PubMed]
Brooks SE, Gu X, Samuel S, et al. Reduced severity of oxygen-induced retinopathy in eNOS-deficient mice. Invest Ophthalmol Vis Sci. 2001;42:222–228. [PubMed]
Figure 1.
 
(A) Time course of RBF in piglets during an 8-hour exposure to hypercapnia by ventilation with 6% CO2 (indicated by horizontal bar). One group of animals was treated with intravenous diclofenac (5 mg/kg) 5.5 hours after exposure to hypercapnia, and another group was given sodium bicarbonate (8.4%) to normalize pH. All other animals were pretreated with diclofenac (5 mg/kg), l-NA (3 mg/kg), TRIM (1 mg/kg followed by 50 μg/kg · min), or saline. At time 0, RBF was basal (normocapnia). Data are the mean ± SEM of four or five eyes of different animals. *P < 0.05 compared with basal levels; †P < 0.05 compared with corresponding levels in animals treated with diclofenac or l-NA. Retinal PGE2 levels (B) and NO2 production (C) are from animals exposed in vivo to normocapnic and hypercapnic conditions. Data are the mean ± SEM of three or four eyes from different piglets. *P < 0.05 compared with 8-hour values at Paco 2 of approximately 40 mm Hg.
Figure 1.
 
(A) Time course of RBF in piglets during an 8-hour exposure to hypercapnia by ventilation with 6% CO2 (indicated by horizontal bar). One group of animals was treated with intravenous diclofenac (5 mg/kg) 5.5 hours after exposure to hypercapnia, and another group was given sodium bicarbonate (8.4%) to normalize pH. All other animals were pretreated with diclofenac (5 mg/kg), l-NA (3 mg/kg), TRIM (1 mg/kg followed by 50 μg/kg · min), or saline. At time 0, RBF was basal (normocapnia). Data are the mean ± SEM of four or five eyes of different animals. *P < 0.05 compared with basal levels; †P < 0.05 compared with corresponding levels in animals treated with diclofenac or l-NA. Retinal PGE2 levels (B) and NO2 production (C) are from animals exposed in vivo to normocapnic and hypercapnic conditions. Data are the mean ± SEM of three or four eyes from different piglets. *P < 0.05 compared with 8-hour values at Paco 2 of approximately 40 mm Hg.
Figure 2.
 
In vivo modulation of NADPH-diaphorase staining of retinas by exposure to hypercapnia. Piglets were ventilated and treated as in Figure 1 , and retinal wholemounts were fixed for NADPH-diaphorase reactivity (top). Individual pixel tonality of densitometry of vasculature was analyzed by correcting for background tone (bottom). Data are the mean ± SEM of four retinal preparations from eye cups of different animals. *P < 0.05 compared with data without asterisks.
Figure 2.
 
In vivo modulation of NADPH-diaphorase staining of retinas by exposure to hypercapnia. Piglets were ventilated and treated as in Figure 1 , and retinal wholemounts were fixed for NADPH-diaphorase reactivity (top). Individual pixel tonality of densitometry of vasculature was analyzed by correcting for background tone (bottom). Data are the mean ± SEM of four retinal preparations from eye cups of different animals. *P < 0.05 compared with data without asterisks.
Figure 3.
 
Ex vivo modulation of NADPH-diaphorase staining and eNOS mRNA expression after exposure to hypercapnia. Isolated eyecups were exposed for 6 hours to 5% CO2 (Paco 2 ≈ 40 mm Hg, pH 7.4) or 9% to 10% CO2 (Paco 2 ≈ 65 mm Hg, pH 7.15–7.2) in absence or presence of diclofenac (100 μM) or l-NA (1 mM). (A) Retinal wholemounts were fixed for NADPH-diaphorase reactivity (top) and individual pixel tonality of densitometry of vasculature was analyzed by correcting for background tone (bottom). (B) eNOS mRNA blot from RNase protection assay (top) and semiquantitative analysis with densitometry (bottom) relative to destrin (control). Unprotected and protected fragments for eNOS are 414 and 356 nucleotides (nt), and for destrin, 237 and 165 nt. Data in both histograms are the mean ± SEM from three retinas of different animals. *P < 0.05 compared with data without asterisks.
Figure 3.
 
Ex vivo modulation of NADPH-diaphorase staining and eNOS mRNA expression after exposure to hypercapnia. Isolated eyecups were exposed for 6 hours to 5% CO2 (Paco 2 ≈ 40 mm Hg, pH 7.4) or 9% to 10% CO2 (Paco 2 ≈ 65 mm Hg, pH 7.15–7.2) in absence or presence of diclofenac (100 μM) or l-NA (1 mM). (A) Retinal wholemounts were fixed for NADPH-diaphorase reactivity (top) and individual pixel tonality of densitometry of vasculature was analyzed by correcting for background tone (bottom). (B) eNOS mRNA blot from RNase protection assay (top) and semiquantitative analysis with densitometry (bottom) relative to destrin (control). Unprotected and protected fragments for eNOS are 414 and 356 nucleotides (nt), and for destrin, 237 and 165 nt. Data in both histograms are the mean ± SEM from three retinas of different animals. *P < 0.05 compared with data without asterisks.
Figure 4.
 
Effects of exposure to high CO2 on the retinal vasorelaxant response to substance P. Eye cups were incubated for 4 hours in buffer bubbled with 5% or 9% to 10% CO2 in absence or presence of diclofenac (100 μM) or actinomycin D (2 μM), as described in Figure 3 . Some preparations were treated with l-NA (1 mM) 20 minutes before administration of substance P. Data are the mean ± SEM of three or four eyes of different animals. *P < 0.01 compared with other curves.
Figure 4.
 
Effects of exposure to high CO2 on the retinal vasorelaxant response to substance P. Eye cups were incubated for 4 hours in buffer bubbled with 5% or 9% to 10% CO2 in absence or presence of diclofenac (100 μM) or actinomycin D (2 μM), as described in Figure 3 . Some preparations were treated with l-NA (1 mM) 20 minutes before administration of substance P. Data are the mean ± SEM of three or four eyes of different animals. *P < 0.01 compared with other curves.
Figure 5.
 
Concentration- and time-dependent effects of hypercapnic acidosis on eNOS mRNA expression in piglet retinas. (A) Eye cups were incubated in physiological buffer for 6 hours with 3% CO2 (Paco 2 ≈ 25 mm Hg, pH ∼7.5), 10% CO2 (Paco 2 ≈ 65 mm Hg, pH ∼7.2), 10% CO2 with normalized pH (Paco 2 ≈ 65 mm Hg, pH ∼7.4) and 5% CO2 with acidosis (Paco 2 ≈ 40 mm Hg, pH ∼7.2) or not incubated (basal). Acidosis with normal CO2 was adjusted by addition of NaH2PO4 or HCl. (B) Time-dependent changes in eNOS mRNA after 2, 4, and 6 hours of exposure to normocapnic acidosis. PGE2 levels (C) and expression of eNOS (D) in retinas isolated from eyecups incubated for 6 hours in normocapnic acidosis conditions in the presence or absence of diclofenac (100 μM), SK&F96365 (10 μM), or actinomycin D (2 μM). mRNA was subjected to RNase protection assay for eNOS. Data are the mean ± SEM of five experiments on eyes from different piglets. *P < 0.05 compared with all other data.
Figure 5.
 
Concentration- and time-dependent effects of hypercapnic acidosis on eNOS mRNA expression in piglet retinas. (A) Eye cups were incubated in physiological buffer for 6 hours with 3% CO2 (Paco 2 ≈ 25 mm Hg, pH ∼7.5), 10% CO2 (Paco 2 ≈ 65 mm Hg, pH ∼7.2), 10% CO2 with normalized pH (Paco 2 ≈ 65 mm Hg, pH ∼7.4) and 5% CO2 with acidosis (Paco 2 ≈ 40 mm Hg, pH ∼7.2) or not incubated (basal). Acidosis with normal CO2 was adjusted by addition of NaH2PO4 or HCl. (B) Time-dependent changes in eNOS mRNA after 2, 4, and 6 hours of exposure to normocapnic acidosis. PGE2 levels (C) and expression of eNOS (D) in retinas isolated from eyecups incubated for 6 hours in normocapnic acidosis conditions in the presence or absence of diclofenac (100 μM), SK&F96365 (10 μM), or actinomycin D (2 μM). mRNA was subjected to RNase protection assay for eNOS. Data are the mean ± SEM of five experiments on eyes from different piglets. *P < 0.05 compared with all other data.
Figure 6.
 
Effects of acidosis on Ca2+ transients in neuroretinovascular endothelial cells. Ca2+ transients were measured by the fura-2-acetoxymethyl ester technique after acidification of the medium by the addition of HCl or NaH2PO4 (pH ∼7.10–7.15). (A) Typical tracing. (B) Histogram presenting peak [Ca2+]i. Media were pretreated with vehicle, diclofenac (100 μM), SK&F96365 (10 μM), or EGTA (5 mM). Arrow: time of administration of acidifying agents (H+). Data are the mean ± SEM of three or four experiments. *P < 0.01 compared with all other data without asterisks.
Figure 6.
 
Effects of acidosis on Ca2+ transients in neuroretinovascular endothelial cells. Ca2+ transients were measured by the fura-2-acetoxymethyl ester technique after acidification of the medium by the addition of HCl or NaH2PO4 (pH ∼7.10–7.15). (A) Typical tracing. (B) Histogram presenting peak [Ca2+]i. Media were pretreated with vehicle, diclofenac (100 μM), SK&F96365 (10 μM), or EGTA (5 mM). Arrow: time of administration of acidifying agents (H+). Data are the mean ± SEM of three or four experiments. *P < 0.01 compared with all other data without asterisks.
Figure 7.
 
Effects of PG analogues on retinal eNOS mRNA expression from eyecups exposed to HCl-induced acidosis. Tissues were exposed for 6 hours to acidosis, as described in Figure 5 and treated with diclofenac (100 μM), with or without 16,16-dimethyl-PGE2, BW245C, PGF, or carbaprostacyclin (1 μM each). Data are the mean ± SEM of three experiments conducted on eyes from different piglets. *P < 0.05 compared with all other data without asterisks.
Figure 7.
 
Effects of PG analogues on retinal eNOS mRNA expression from eyecups exposed to HCl-induced acidosis. Tissues were exposed for 6 hours to acidosis, as described in Figure 5 and treated with diclofenac (100 μM), with or without 16,16-dimethyl-PGE2, BW245C, PGF, or carbaprostacyclin (1 μM each). Data are the mean ± SEM of three experiments conducted on eyes from different piglets. *P < 0.05 compared with all other data without asterisks.
Table 1.
 
Arterial Blood Pressure and Gas Levels in Piglets before and after Hypercapnia
Table 1.
 
Arterial Blood Pressure and Gas Levels in Piglets before and after Hypercapnia
Treatment Baseline 0.5 h 3 h 6 h 8 h
Control
 MABP (mm Hg) 58.3 ± 5.4 63.1 ± 3.8 60.2 ± 4.7 63.5 ± 5.3 65.2 ± 3.5
 Arterial pH 7.38 ± 0.04 7.19 ± 0.03* 7.23 ± 0.02* 7.26 ± 0.03* 7.29 ± 0.02* , ‡
 PaCO2 (mm Hg) 41.2 ± 1.5 68.7 ± 2.1* 65.1 ± 1.6* 66.8 ± 2.7* 64.6 ± 1.8*
PaO2 (mm Hg) 90.0 ± 8.6 89.3 ± 4.3 88.4 ± 4.1 86.1 ± 8.5 91.1 ± 8.5
Diclofenac
 MABP (mm Hg) 63.2 ± 5.4 62.7 ± 3.4 68.2 ± 6.3 69.1 ± 2.5 64.0 ± 2.6
 Arterial pH 7.40 ± 0.02 7.19 ± 0.02* 7.23 ± 0.03* 7.25 ± 0.06* 7.27 ± 0.03* , ‡
 PaCO2 (mm Hg) 41.8 ± 3.4 65.3 ± 3.1* 67.1 ± 3.4* 68.2 ± 2.5* 66.3 ± 2.6*
 PaO2 (mm Hg) 96.9 ± 7.9 91.8 ± 8.5 91.5 ± 4.5 86.1 ± 8.8 88.5 ± 8.0
l-NA
 MABP (mm Hg) 86.7 ± 3.1, † 85.5 ± 2.9, † 84.2 ± 3.9, † 84.9 ± 4.1, † 86.7 ± 2.7, †
 Arterial pH 7.39 ± 0.03 7.20 ± 0.01* 7.22 ± 0.01* 7.23 ± 0.02* 7.28 ± 0.03* , ‡
 PaCO2 (mm Hg) 42.4 ± 1.8 68.7 ± 3.3* 67.5 ± 3.3* 69.5 ± 2.6* 69.3 ± 2.8*
 PaO2 (mm Hg) 96.8 ± 9.5 97.8 ± 9.0 99.2 ± 8.4 86.7 ± 9.0 90.3 ± 8.4
TRIM
 MABP (mm Hg) 61.4 ± 2.3 65.3 ± 3.4 66.5 ± 2.2 61.4 ± 1.9 63.5 ± 3.5
 Arterial pH 7.41 ± 0.03 7.21 ± 0.02* 7.21 ± 0.03* 7.19 ± 0.02* 7.25 ± 0.02* , ‡
 PaCO2 (mm Hg) 44.8 ± 3.1 71.0 ± 3.1* 72.4 ± 2.4* 72.7 ± 2.6* 70.5 ± 3.3*
 PaO2 (mm Hg) 91.4 ± 7.6 93.3 ± 8.4 91.3 ± 5.1 93.5 ± 5.2 92.4 ± 4.5
Bicarbonate
 MABP (mm Hg) 63.4 ± 2.9 66.1 ± 2.1 61.9 ± 4.0 65.5 ± 3.3 62.3 ± 2.5
 Arterial pH 7.40 ± 0.03 7.41 ± 0.01 7.39 ± 0.03 7.38 ± 0.02 7.40 ± 0.02
 PaCO2 (mm Hg) 43.4 ± 2.0 69.9 ± 2.7* 71.9 ± 3.0* 70.5 ± 2.1* 72.1 ± 3.3*
 PaO2 (mm Hg) 88.3 ± 9.6 91.1 ± 6.4 89.3 ± 6.2 93.5 ± 5.6 91.3 ± 7.5
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