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The Role of Nitric Oxide in the Cerebrovascular Response to Hypercapnia

Smith, Jeremy J. MB, FFARCSI; Lee, Joseph G. MB, FFARCSI; Hudetz, Antal G. BMD, PhD; Hillard, Cecilia J. PhD; Bosnjak, Zeljko J. PhD; Kampine, John P. MD, PhD

Neurosurgical Anesthesia

Laser Doppler flowmetry was used to further investigate the role of nitric oxide (NO) in CO2-induced cerebrocortical hyperemia in rats. A second objective was to elucidate the source(s) of the NO involved in the response to hypercapnia. We used the L-arginine analogue Nomega-nitro-L-arginine methyl ester (L-NAME) to inhibit NO synthase (NOS) and 7-nitroindazole (7-NI) to selectively inhibit brain or nonendothelial NOS. Rats were anesthetized with a single dose of intraperitoneal (IP) pentobarbital (65 mg/kg) for surgery; 60-90 min later they were ventilated with 1.0% halothane in 30% O2 for 1 h to achieve a steady state. The animals were assigned to one of five groups. A control group (n = 9) was infused with 1 mL of saline. The second group (n = 10) received 20 mg/kg of L-NAME intravenously (IV). A third group (n = 9) also received L-NAME; in addition, cerebrocortical laser Doppler flow (LDF) and mean arterial pressure (MAP) were restored to baseline using the NO donor sodium nitroprusside (SNP). In a fourth group (n = 9), MAP was increased to the level usually seen after L-NAME with an infusion of phenylephrine (0.5-5 micro g [center dot] kg-1 [center dot] min-1). A fifth group (n = 11) received 7-NI at 40 mg/kg IP. The hypercapnic response of LDF was tested in all groups by adding 5% CO2 to the inspired gas at 30-45 min posttreatment; all changes in LDF were significant. In the control group, hypercapnia induced a 70% +/- 24% increase in LDF. In the L-NAME-treated group, the response was decreased to 36% +/- 22% at a posttreatment LDF that was 25% +/- 13% lower than the pre-L-NAME level. In the group where baseline LDF and MAP were restored with SNP, the CO2 response was 56% +/- 15% (not significant versus control). In the group in which MAP was increased with phenylephrine, the response to hypercapnia was 48% +/- 22% at a posttreatment LDF unchanged from pretreatment. These data suggest that increased vascular tone or the absence of basal NO after NOS inhibition influenced the vasodilator response to hypercapnia. In the 7-NI-treated group the response to hypercapnia was 38% +/- 3%, significantly attenuated at a posttreatment flow only 14% +/- 7% lower than pre-7-NI. We conclude that 1) endothelial NO does not mediate the response to hypercapnia but may have a permissive role in the response and 2) that brain NO may have an important role in the response to hypercapnia.

(Anesth Analg 1997;84:363-9)

Departments of (Smith, Lee, Hudetz, Bosnjak, Kampine) Anesthesiology, (Hudetz, Bosnjak, Kampine) Physiology, and (Hillard) Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin.

Section Editor: Donald S. Prough.

Supported in part by Anesthesiology Training Grant GM 08377, National Science Foundation Grant BES-9411631, and American Heart Association Grant 95009340.

Accepted for publication September 18, 1996.

Address correspondence and reprint requests to Antal G. Hudetz, BMD, PhD, Department of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226.

Increased arterial CO2 increases cerebrocortical blood flow [1]. However, the mechanisms involved are unclear. Part of the vasodilation is due to a direct effect of the extracellular acidosis associated with hypercapnia on the vascular smooth muscle [2]. The mediators of hypercapnic cerebral vasodilation remain elusive. Recent studies suggest a role for nitric oxide (NO) in this response while a few others refute this claim [3-7]. NO is produced in the endothelium from L-arginine by the constitutive form of NO synthase (NOS) in the presence of oxygen, calcium, calmodulin, and reduced form of nicotinamide adenine dinucleotide phosphate [8]. Other sources of NO that may influence cerebral vascular smooth muscle include neurons and astrocytes [9,10]. CO2 or the associated acidosis may stimulate NOS to produce NO, which activates guanylate cyclase to increase production of cyclic guanylic acid [11] and relax vascular smooth muscle. Niwa et al. [12] demonstrated that NOS blockade strongly attenuates the response of the cerebral vasculature to increased hydrogen ion concentration. Therefore the hyperemic response to increased arterial CO2 may, at least in part, depend on the activation of NOS.

Previous studies that demonstrated a role for NO in hypercapnic vasodilation have used pharmacological inhibition of NOS. NOS is competitively inhibited by various L-arginine analogs such as Nomega-nitro L-arginine methyl ester (L-NAME) [13]. 7-Nitroindazole (7-NI) is a relatively specific inhibitor of brain NOS [14]. Infusion of L-NAME increases mean arterial pressure (MAP) and decreases cerebral blood flow resulting in an increase in cerebrovascular resistance [15,16]. The studies that demonstrated a role for NO in the hypercapnic response do not distinguish whether a decrease in NO production or altered baseline flow and MAP after NOS inhibition influenced the response to hypercapnia. The response of a vascular bed to a vasodilator depends on the tone of the vessel at the time of the stimulus. A vessel under high tone may relax more than a vessel under low tone or, conversely, may be resistant to relaxation, as occurs in vasospasm [17]. Restoration of initial vessel tone with a NO donor, such as sodium nitroprusside (SNP) [18], after NOS inhibition may provide a model for testing the involvement of NO in CO2-induced cerebrovasodilation.

Assessment of the cerebrovascular response to hypercapnia by continuous measurement of blood flow is advantageous because it reflects the net response of all vessels, especially resistance vessels. Laser Doppler flowmetry is now a widely used technique for continuous monitoring of cerebral tissue perfusion in both experimental animals and humans [19,20]. We have used the technique in a minimally invasive rat model to investigate the effects of halothane and isoflurane on cerebrocortical microcirculation and autoregulation [19] on CO2 reactivity [21] and to study factors that affect cortical flow oscillations [22]. We hypothesized that NO was involved in the response to hypercapnia. In this study we used laser Doppler flowmetry to evaluate whether the role of NO in CO2-induced increases in cerebral perfusion was as a mediator or as a permissive modulator. A permissive role would mean that the presence of basal NO was required for the response but that hypercapnia did not vasodilate through an increase in NO synthesis. We also attempted to elucidate the source or sources of the NO involved in the response to hypercapnia.

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All experimental procedures and protocols used in this investigation were reviewed and approved by our animal care committee.

Adult male Sprague-Dawley rats (250-400 g body weight) were anesthetized for surgery with intraperitoneal pentobarbital (65 mg/kg body weight). A tracheotomy was performed and the trachea was intubated. One or both femoral arteries and veins were cannulated to allow continuous blood pressure measurement and drug administration. The lungs were ventilated (Model 707; Harvard Apparatus, South Natick, MA) with 30% O2 in air. Body temperature was monitored with a rectal temperature probe and maintained at 37 +/- 0.2 degrees C with a water-circulated heating pad. Once ventilation commenced, 1.0% halothane was added to the inspired gases. Inspired and expired O (2), CO2, and volatile anesthetic concentrations were measured continuously (POET II[TM]; Criticare Systems, Inc., Milwaukee, WI). In recognition of the well described difficulties of measuring ETCO2 in small animals that are ventilated, in a previous study [19] we tested the relationship of PaCO2 and ETCO2 in a group of rats at different concentrations of inspired halothane and with and without phenylephrine infusion. We found in all cases that the difference between end-tidal and arterial CO2 was less than 3 mm Hg. Increased halothane concentration or phenylephrine infusion did not seem to influence this difference. ETCO2, MAP, laser Doppler flow (LDF), and concentration of anesthetic vapor were displayed and recorded on an eight-channel polygraph recorder (Astro-Med, Inc., West Warwick, RI).

In all experiments the head of the rat was secured in a stereotaxic apparatus (Model 900; David Kopf, Tujunga, CA). The scalp and connective tissue were removed in a 1-cm2 area. A burr hole 2 mm in diameter was drilled in the cranium, over the right parietal cortex, using a low speed (0-8000 rpm) air drill (Model Rhino XP; Midwest Dental, DesPlaines, IL) with the aid of a stereomicroscope. The drilling was performed approximately 3 mm posterior to the bregma and 3 mm to the right of the midline while the surface was cooled by continuous airflow through a suction tip. The burr hole was made as deep as possible without penetrating the skull, as originally described by Hudetz et al. [22]. At the end of the experimental protocol the rats were killed with an intravenous (IV) injection of pentobarbital (50 mg) and gallamine (20 mg) followed by a surgical pneumothorax.

Halothane (Anaquest Inc., Madison, WI) was vaporized into the animal's breathing system using vaporizer Model 100F (Ohio Medical Products, Madison, WI). Pentobarbital, gallamine, L-NAME, SNP, and phenylephrine were obtained from Sigma Chemical Co. (St. Louis, MO). 7-NI was obtained from ICN Biomedicals, Inc. (Aurora, OH). All IV drugs were reconstituted on the day of experiment with a balanced salt solution, Plasmalyte[R] (Baxter, Deerfield, IL). 7-NI was dissolved in peanut oil.

LDF was monitored throughout the study with a laser Doppler flowmeter (PF3; Perimed, Stockholm, Sweden) and a flow probe (PF316, "dental probe," from the same company) with a tip diameter of 1 mm. The probe was lowered into the bottom of the cranial burr hole using a micromanipulator. A drop of mineral oil was applied at the probe tip to provide optical coupling between the probe and the tissue. Care was taken to position the probe over a tissue area devoid of large blood vessels which were visible through the thinned bone. Inadvertent placement of the probe over large surface vessels was indicated by extraordinary high flow values in which case the probe was repositioned. Once established, the probe position was not altered for the duration of the experimental protocol.

After a preparation time of approximately 60-90 min, the rat was allowed to stabilize for 1 h at 1.0% halothane, while maintaining ETCO2 between 34 and 37 mm Hg. At the end of this stabilization period the animals were sequentially assigned to one of five groups. A control group (n = 9) received 1 mL of saline over 10 min. In a second group (n = 10), L-NAME 20 mg/kg dissolved in 1 mL of saline was infused IV over 10 min. A third group (n = 9) also received L-NAME 20 mg/kg and was infused with SNP (0.5-5 micro g [center dot] kg-1 [center dot] min-1) to restore LDF to baseline values. A fourth group (n = 9) received an IV infusion of phenylephrine (0.5-5 micro g [center dot] kg-1 [center dot] min-1) to increase MAP to the level seen after treatment with L-NAME. A fifth group (n = 11) was treated with 7-NI 40 mg/kg dissolved in 1 mL of peanut oil intraperitoneally. The response to CO2 was tested 30-45 min after treatment in each group by increasing the inspired CO2 to 5% which resulted in an ETCO2 between 66 and 70 mm Hg. The response occurred within minutes and was recorded when a 5-min steady state was achieved. SNP was continuously infused in Group 3 as was phenylephrine in Group 4. To confirm the inhibition of endothelial enzyme activity by L-NAME, we tested the LDF response to a 1-min infusion of IV adenosine diphosphate (ADP) (1.5 micro M [centered dot] kg-1 [center dot] min-1) a vasodilator requiring an intact endothelial NO-generating system [23]. Similarly, we used IV SNP (0.5-5 micro g [centered dot] kg-1 [center dot] min-1), a NO donor that directly stimulates guanylate cyclase [20], to demonstrate the continued ability of the vascular smooth muscle to relax in the group of the rats treated with L-NAME + SNP. We also reversed the effects of L-NAME with L-arginine (200 mg/kg) in a separate group (n = 3). L-Arginine would not be expected to reverse the noncompetitive inhibition produced by 7-NI. Time control experiments were performed on another group (n = 3) of animals.

Representative sections of the LDF recording in the steady state were averaged over a 5-min period. The LDF response was, in all protocols, expressed as a change in percent of the baseline. Baseline LDF was taken at the end of the stabilization hour, at steady state conditions at 1.0% halothane at normocapnia (ETCO2 = 34-37 mm Hg). Cerebrovascular resistance was estimated by dividing MAP by LDF and the venous pressure was assumed to be 0. Data were expressed as mean +/- SEM. Statistical difference was obtained using one-way analysis of variance with repeated measures for within-group comparisons, and using analysis of variance plus Student-Newman-Keuls test for between-group comparisons. All reported changes were significant at the P < 0.05 level unless otherwise stated.

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At the end of the equilibration period with 1.0% halothane, all rats had achieved hemodynamic stability, as indicated by steady state values of LDF and MAP. Average baseline MAP (Table 1) and LDF (Figure 1) were similar in each group before treatment. Sampling of arterial blood gases and hemoglobin in some animals (n = 22) at the end of the equilibration period gave PaO2 values of 141 +/- 9 mm Hg, PaCO2 of 36 +/- 1 mm Hg, pH of 7.42 +/-.05, and hemoglobin of 17.7 +/- 0.3 g/100 mL. The corresponding average value for ETCO2 was 37 +/- 1 mm Hg, indicating maintenance of a close relationship between PaCO2 and ETCO2. In a sample (n = 8) of the L-NAME-treated group, blood variables were 148 +/- 13 and 35 +/- 1 mm Hg for PaO2 and PaCO2, respectively, and 7.40 +/- 0.01 for pH before the infusion of L-NAME, and 141 +/- 9 and 34 +/- 1 mm Hg for PaO2 and PaCO2 and 7.44 +/- 0.01 for pH after the infusion of L-NAME demonstrating no significant difference in these variables due to treatment with the inhibitor.

Table 1

Table 1

Figure 1

Figure 1

(Figure 1) illustrates the LDF in perfusion units at baseline, posttreatment, and during hypercapnia in the various treatment groups. Infusion with 1 mL of saline had no effect on baseline LDF or MAP. Treatment with L-NAME increased MAP by 12 +/- 3 mm Hg (Table 1), decreased LDF by 25% +/- 13%, and increased cerebrovascular resistance (CVR) by 49% +/- 22%. In Group 3, SNP infusion (0.5-5 micro g [centered dot] kg-1 [center dot] min-1) restored flow and MAP to levels not different from the original baseline. IV phenylephrine (0.5-5 micro g [center dot] kg-1 [center dot] min-1), used to increase MAP to levels similar to those attained after treatment with L-NAME, had no significant effect on baseline LDF but increased CVR by 20% +/- 1%. Intraperitoneal 7-NI (40 mg/kg) had no effect on MAP, decreased baseline LDF by 14% +/- 7%, and increased CVR by 20% +/- 17%.

The changes in LDF in response to inhaling 5% CO2 occurred within seconds, were complete in minutes, and all were significant. Figure 2 shows the percent increase of baseline flow in response to hypercapnia in each group. The control response to hypercapnia was a 70% +/- 24% increase in LDF. L-NAME alone decreased the response to 36% +/- 22%. In the group treated with L-NAME + SNP, the response to hypercapnia was 56% +/- 15%, which was not significantly different from that in the control group. In the phenylephrine-treated group, hypercapnia induced a 48% +/- 22% increase in LDF. After 7-NI treatment, the LDF response to hypercapnia was 38% +/- 9%, which was significantly attenuated compared to the control response.

Figure 2

Figure 2

In five rats, prior to treatment with L-NAME, we determined the response to a 1-min IV infusion of 1.5 micro M [center dot] kg-1 [center dot] min (-1) of ADP. A 15% +/- 5% increase in LDF was observed. Thirty minutes after administration of L-NAME, ADP infusion elicited no change in LDF. L-Arginine at a dose of 200 mg/kg restored LDF and MAP to values not different from the baseline prior to treatment with L-NAME. The time control animals demonstrated no significant change in LDF over the duration of the experiments.

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The results of this study indicate that the CO2-induced increase in cerebrocortical LDF is attenuated by IV L-NAME (20 mg/kg), that infusion of SNP to return cerebral blood flow and MAP to baseline values restores the response to hypercapnia to a level not different from control, and that 7-NI, a specific blocker of brain NOS, attenuates the hypercapnic response. Therefore, our findings may indicate that endothelial NO is not directly involved in mediating the response, or that its role is permissive in nature. Although brain NO appears to participate in the cerebral hyperemic response to CO2, from our present data it is not possible to say whether this role is as a mediator or as a permissive agent.

Recent studies indicate a critical role for NO in modulating the increase in cerebral blood flow associated with hypercapnia [4,5]. However, the exact role and the source(s) of the NO involved remain elusive. Toda et al. [24] demonstrated, in an in vitro study using isolated vessel strips, that removing the endothelium in dog middle cerebral arteries did not affect the response to hypercapnia. This indicates that neither brain NOS nor endothelial NOS are involved in the hypercapnic response of this vessel preparation. Fabricius and Lauritzen [5] investigated the cerebral microcirculation using laser Doppler flowmetry in halothane-anesthetized rats and concluded that IV L-NAME attenuated the response to hypercapnia. They used tetrodotoxin, a sodium channel blocker to block the effect of perivascular nerves and found that the CO2 response was unaffected. They concluded that the NO involved in the response to hypercapnia was endothelial in origin. Our control response was similar to that found in their study and we had a similar degree of attenuation of the response to hypercapnia after IV L-NAME treatment. L-NAME inhibits all types of NOS activity, including both brain and endothelial NOS. The 20-mg/kg dose in our experiments effectively blocked the endothelium-dependent response to an ADP infusion. Iadecola et al. [25], using a larger dose of L-NAME (40 mg/kg), achieved a 64% inhibition of total brain NOS activity when measured using the conversion of radiolabeled arginine to citrulline. Higher rates of inhibition are difficult to attain and are usually only achieved with chronic administration of the inhibitor. This may lead to confounding problems due to upregulation of other vasodilator systems [26].

In our study, infusion of SNP restored the response to hypercapnia, suggesting a permissive rather than a mediator role for NO in the CO2 response. A permissive role would mean that the presence of basal NO was required for the response but that hypercapnia did not vasodilate via an increase in the rate of NO synthesis. Previously, we suggested a permissive role for NO in the hyperemic response to increasing inspired halothane concentration [27]. Bryan et al. [28] have concluded that NO has a permissive role in alpha2-mediated dilations in rat cerebral arteries. Farrell and Bishop [29] suggest a permissive role for NO in the active thermoregulatory vasodilation in the rabbit ear.

Some studies [6,7] failed to demonstrate an attenuating effect of NOS inhibition on the CO2 response. Different anesthetic techniques used in these studies may explain the conflicting results. The effects of inhalational and IV anesthetics on cerebral blood flow and metabolism have been well described. Halothane is a cerebral vasodilator and decreases cerebral metabolism. Barbiturates result in decreased metabolism and vasoconstriction. Inhibition of NOS may also have an effect on the minimum alveolar concentration of an inhalational drug [30]. We used intraperitoneal sodium pentobarbital to induce anesthesia for surgical preparation of the animals. Our experiments were performed at least two and a half hours after this, at which time the pentobarbital would be well distributed to all body tissues and fluid compartments. If there was any residual effect of barbiturate in our experiments, then it was common to all animals. Our control response was almost identical to that found in Fabricius and Lauritzen's study [5], in which the rats were anesthetized with halothane only. Furthermore, time control experiments demonstrated no significant change in cerebral blood flow over the duration of our experiments. A decreasing barbiturate effect would usually result in a slowly increasing LDF. This was not evident in our experiments. The contradictory results seen by others may be due to the hypercapnic hyperemic response being dependent on the degree of hypercapnia. Iadecola and Zhang [31] postulated a nonuniform hyperemic response to CO2 with the greatest inhibition of the response by L-NAME occurring between 40 and 80 mm Hg PaCO2. Adachi et al. [6] used 10% CO2 to test the response to hypercapnia and found no attenuation in their study. PaCO2 was not measured in Adachi et al.'s study but it is likely that the PaCO2 levels achieved were higher than in most other studies.

As in the studies by Tanaka et al. [15] using NG-monomethyl-L-arginine and Wei et al. [16] using L-NAME in rats, NOS inhibition induced a significant increase in MAP and a decrease in cortical LDF in our experiments, resulting in a large, 49% increase in CVR. When we attempt to understand the mechanism of CO2-induced hyperemia, the altered baseline in the animals treated only with L-NAME makes a comparison between the response to hypercapnia in this group and that in the control group difficult to interpret. Restoration of baseline flow and tone in the L-NAME + SNP group allows a more useful comparison to be made. In the L-NAME + SNP group the response to hypercapnia was not significantly different from control indicating that NO may have a permissive role in the cerebrovasodilator response to hypercapnia. Iadecola et al. [25] demonstrated that the post-L-NAME attenuation of the hypercapnic response could be reversed by an intracarotid infusion of the NO donor 3-morpholinosydnonimine. They concluded that the mechanism by which NO inhibitors attenuate the response may be related to a decrease in basal levels of NO rather than to an inhibition of hypercapnia-induced NOS activation. This finding is compatible with the permissive role which our study indicates for NO in the response to CO2.

The constitutive form of NOS is found in the brain in endothelial cells [8], in astrocytes [9], and in perivascular neurons [10]. The individual contribution of each of these sources of NO to the maintenance of resting cerebral blood flow and the response to vasodilators such as CO2 remains unclear. Kovach et al. [32], using 7-NI, have observed a 68% reduction in resting cerebral blood flow in cats. In our study, 7-NI decreased baseline LDF by 14%, indicating that brain NO has a role in the control of resting cerebral blood flow in rats.

Wang et al. [3] showed that NOS inhibition with nitro-L-arginine but not endothelial injury attenuated hypercapnia-induced pial arteriolar vasodilation and concluded that the dilatory response was partly NO-dependent but the source of NO was nonendothelial. Using 7-NI, we demonstrated a significant attenuation of the cerebrovasodilator response to CO2, suggesting a role for neuronal NOS in this response. The increase in baseline vascular resistance (20%) after 7-NI treatment was substantially less than that seen in the L-NAME only treatment group (49%) where a similar attenuation in the response to hypercapnia was observed. A nonspecific effect of 7-NI is unlikely, as Wang et al. [34] in their recent paper on hypercapnia found that after treatment with 80 mg/kg of 7-NI the vasodilator response to oxotremorine was unchanged, suggesting no significant inhibition of endothelial NOS. Yoshida et al. [35] using doses of 25 and 50 mg/kg of 7-NI demonstrated no effect on blood pressure or on the response to acetylcholine suggesting no effect on endothelial NOS activity.

The mechanism of increased NO production in hypercapnia is unclear at present. CO2 or the acidosis associated with hypercapnia may stimulate NOS enzyme activity [36] to produce NO, which activates guanylate cyclase to increase production of cyclic guanylic acid [14]. Niwa et al. [12], using a closed cranial window and infusing acidic artificial cerebrospinal fluid, found that NOS inhibition strongly attenuated the response of the cerebral vasculature to changes in hydrogen ion concentration. Wang et al. [37] used acetazolamide to induce extracellular acidosis and found little attenuation of the cerebral blood flow response to decreased pH in the presence of NOS blockade with nitro-L-arginine. Wang speculated that intracellular acidification produced by CO2 may trigger the production of NO. Moreover, acidosis increases neuronal NOS activity while alkalosis increases endothelial NOS activity [38]. The latter observation may explain the attenuation seen in our study with 7-NI, a specific inhibitor of brain NOS.

L-NAME increased CVR by increasing MAP and decreasing LDF, and the vasodilator response to hypercapnia in this group was attenuated. In another experimental group, we increased MAP levels with an adrenergic agonist to produce MAP levels similar to those seen after treatment with L-NAME. Phenylephrine has negligible effects on cerebral blood flow [39] and infusion of phenylephrine resulted in MAP similar to that seen in the L-NAME group but baseline LDF was not altered. In the phenylephrine group, the response to hypercapnia was significantly attenuated compared to the control response and similar to the attenuation seen in the L-NAME and 7-NI groups. The increase in resistance in the phenylephrine group was due to an increase in MAP only without any change in flow, indicating that the increase in vascular resistance associated with an increased MAP may have a role in the attenuation of the hypercapnic response.

Inhibition of NOS by 7-NI does not completely abolish the vasodilation elicited by hypercapnia as a residual increase in LDF is still present. It is possible that 7-NI did not fully block brain NOS. Recent studies have found that 80 mg/kg 7-NI induces a 57% inhibition of total NOS activity [34]. Alternatively, a response as ubiquitous in nature as hypercapnic hyperemia may have more than one mediator. Other endothelium-dependent and independent vasodilator pathways may work in parallel with NO in the hyperemic response to increased CO2. Thus, indomethacin, a potent cyclooxygenase inhibitor, significantly attenuates the response to hypercapnia in rats and other species [40], although other inhibitors of prostaglandin synthesis do not reproduce this effect [41]. However, the attenuation of the hypercapnic response after treatment with indomethacin may be due in part to an altered baseline. There is evidence that, in neonatal pigs [42], prostaglandins may have a permissive rather than mediator role in hypercapnia-induced cerebral vasodilation.

In summary, our results suggest that the cerebral hyperemic response to CO2 is attenuated by IV L-NAME and that this attenuation can be reversed using the NO donor SNP. This finding may indicate a permissive role for endothelial NO in the response. 7-NI, a specific inhibitor of brain NOS, significantly attenuates the response to CO2 without a major change in baseline flow, which suggests that nonendothelial NO may play an important role in hypercapnic vasodilation in the rat cerebral cortex; however, it is not possible to conclude at this time whether this role of brain NO is permissive or mediator.

The authors express their gratitude to Dr. Arisztid G. B. Kovach, University of Pennsylvania, for his expert advice on the use of 7-nitroindazole. The useful suggestions of Drs. Dale A. Pelligrino and Qiong Wang, University of Illinois, are greatly appreciated. The authors express their thanks to Anita Tredeau and Angela M. Barnes for secretarial assistance and to James D. Wood for his help in preparing the figures.

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