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(Stroke. 1996;27:729-736.)
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Articles

Impaired Pial Arteriolar Reactivity to Hypercapnia During Hyperammonemia Depends on Glutamine Synthesis

Takahiko Hirata, MD; Raymond C. Koehler, PhD; Tetsu Kawaguchi, MD; Saul W. Brusilow, MD Richard J. Traystman, PhD

From the Departments of Anesthesiology/Critical Care Medicine (T.H., R.C.K., T.K., R.J.T.) and Pediatrics (S.W.B.), The Johns Hopkins Medical Institutions, Baltimore, Md.

	Abstract

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Background and Purpose Acute hyperammonemia causes glutamine and water accumulation in astrocytes and loss of the cerebral blood flow response selectively to CO₂. We tested whether extraparenchymal pial arterioles not subjected directly to mechanical compression by swollen astrocyte processes also lose hypercapnic reactivity and whether any such loss can be attenuated by inhibiting glutamine synthesis during hyperammonemia.

Methods Pentobarbital-anesthetized rats were pretreated intravenously with either saline vehicle, methionine sulfoximine (0.83 mmol/kg), which inhibits glutamine synthetase and potentially -glutamylcysteine synthetase, or buthionine sulfoximine (4 mmol/kg), which inhibits -glutamylcysteine synthetase. Three hours after pretreatment, cohorts received an intravenous infusion of either sodium or ammonium acetate for 6 hours. Pial arteriolar diameter was measured through a closed cranial window, and blood flow was measured with radiolabeled microspheres during normocapnia and 10 minutes of hypercapnia.

Results With sodium acetate infusion, pial arteriolar diameter increased during hypercapnia in groups pretreated with vehicle (23±3% [mean±SE]; n=6), methionine sulfoximine (37±11%; n=5), and buthionine sulfoximine (32±3%; n=5). With ammonium acetate infusion, pial arteriolar diameter increased only in the group pretreated with methionine sulfoximine (31±4%; n=8) but not in those pretreated with vehicle (-2±4%; n=8) or buthionine sulfoximine (4±4%; n=6). Methionine sulfoximine, but not buthionine sulfoximine, also prevented loss of the cerebral blood flow response to hypercapnia, an increase in cortical tissue water content, and an increase in pressure under the cranial window during normocapnia in hyperammonemic rats. In contrast to hypercapnia, hypoxemia increased arteriolar diameter 30±7% (n=5) during ammonium acetate infusion.

Conclusions Loss of the blood flow response to hypercapnia during acute hyperammonemia is not due simply to swollen astrocyte processes passively impeding blood flow because extraparenchymal resistance arterioles also lose their reactivity selectively to hypercapnia. Lost reactivity depends on glutamine synthesis rather than on ammonium ions per se and may reflect indirect effects of astrocyte dysfunction associated with glutamine accumulation or possibly effects of glutamine on nitric oxide production.

Key Words: ammonia • carbon dioxide • cerebral arteries • cerebral blood flow • glutamine synthetase

	Introduction

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Experimental increases in plasma ammonia to levels attained clinically in patients with severe liver disease, urea cycle disorders, and inborn errors of metabolism cause increased brain glutamine concentration,¹ increased cortical water content,^{2 3} and swelling of astrocytes, including perivascular end-feet.^{4 5 6 7} Glutamine synthetase rapidly incorporates plasma-labeled ammonia into glutamine,⁸ and the enzyme is highly localized in astrocytes.⁹ In glial cell culture, inhibition of glutamine synthetase with MSO prevents the cell swelling seen with exposure to 5 mmol/L ammonium salts.¹⁰ In vivo, we have found that MSO treatment prevents the increase in cortical glutamine concentration, water content,³ and intracranial pressure,¹¹ as well as the swelling of astrocyte perivascular processes¹² that is seen when plasma ammonia concentration is increased to 500 to 600 µmol/L for 6 hours. Thus, astrocyte swelling sufficient to increase tissue water content and intracranial pressure during acute ammonia exposure is related to glutamine synthesis and accumulation.

Acute hyperammonemia also leads to vascular abnormalities. Depressed CBF responsivity to CO₂ has been reported in primates,¹³ dogs,¹⁴ cats,¹⁵ and rats.¹¹ We found that the depressed CBF response to hypercapnia could be prevented by glutamine synthetase inhibition with MSO.¹¹ One explanation for the loss of CO₂ responsivity is that glutamine-dependent astrocyte swelling causes compression of intraparenchymal vessels. Nonspecific cerebral edema formation caused by plasma osmolarity decreased to 255 mOsm/L also reduced the CBF response to hypercapnia.¹¹ Thus, loss of blood flow responsivity to hypercapnia during hyperammonemia could be secondary to the mechanical effects of compression on intraparenchymal blood vessels. Cerebral capillary distortion can be observed in perfusion-fixed tissue of hyperammonemic rats.¹² If the loss of blood flow responsivity to CO₂ is related to a local effect of swollen astrocytes, then extraparenchymal pial arterioles not subjected to the direct compressive forces of astrocyte processes would be expected to retain vascular reactivity to CO₂.

In the present study, we measured both pial arteriolar diameter and CBF responses to hypercapnia during 6 hours of hyperammonemia. Three hypotheses were tested. First, we tested whether hyperammonemia blunts pial arteriolar responsivity to hypercapnia to the same degree that the CBF response is blunted. Second, we tested whether inhibition of glutamine synthesis with MSO prevented any attenuation of the pial arteriolar diameter response to hypercapnia. In addition to inhibiting glutamine synthetase, MSO can also inhibit -glutamylcysteine synthetase,¹⁶ an enzyme involved in glutathione synthesis. Administration of MSO does not decrease glutathione in brain.¹⁷ However, to control for this potential effect of MSO, we also used BSO, an analogue of MSO that is a more potent inhibitor of -glutamylcysteine synthetase but that does not inhibit glutamine synthetase.¹⁸ We tested a third hypothesis that treatment with BSO does not prevent attenuation of pial arteriolar diameter or CBF responsivity to hypercapnia.

	Materials and Methods

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All procedures were approved by the institutional animal care and use committee. Male Wistar rats (350 to 450 g) were anesthetized intraperitoneally with sodium pentobarbital (65 mg/kg plus 20 mg/kg every 1.5 hours). Rectal temperature was maintained at 37°C with a warm water circulating blanket. The lungs were mechanically ventilated with 25% to 30% oxygen, and catheterization was performed for infusion of salt solutions and microsphere measurement of blood flow as previously described.^{11 19} Catheterization included femoral veins for salt solution and donor blood infusions, the left ventricle through the right subclavian artery for microsphere injection, the femoral artery for obtaining the microsphere reference sample, and the tail artery for monitoring arterial pressure continuously.

We constructed a cranial window over the parietal cortex by performing a 3x4-mm craniotomy inside a 7-mm-diameter plastic ring cemented to the skull with acrylic as previously described.²⁰ The dura was incised and retracted under a well of artificial cerebrospinal fluid. A glass coverslip was cemented over the fluid-filled ring to seal the cranial window. A catheter connected to a side port of the ring was used to monitor pressure inside the window. Pial vessels were observed through the window with an Olympus binocular microscope (model BHMJ) connected to a video camera. Images were stored on videotape and played back on a video monitor for later analysis. In each rat, the diameters of two to four arterioles (baseline diameter, 11 to 60 µm) were measured. The percent changes for individual arteriolar segments were averaged for each rat, and this average value was used in the statistical analysis.

CBF was measured with radiolabeled microspheres.^{11 19} Approximately 0.2 million spheres (15 µm in diameter) labeled with ¹⁵³Gd, ¹¹³Sn, or ⁴⁶Sc (Dupont-NEN Products) were injected into the left ventricle. The microsphere reference sample was withdrawn at a rate of 0.68 mL/min and was replaced with blood from a donor rat. After the anesthetized rat was killed with potassium chloride, cortical gray matter samples were quickly dissected, weighed, placed in a dry oven at 100°C for 48 hours, and reweighed. Percent water content of the tissue was calculated. The remaining brain was sectioned into remaining cerebrum, diencephalon, midbrain, medulla plus pons, and cerebellum. Tissue and reference blood samples were counted for radioactivity. The dried gray matter sample was also counted and included in the values for total cerebrum. Counts per minute were corrected for spectral overlap, and blood flow was calculated as 0.68 tissue counts/blood counts. Values were normalized by wet weight.

Plasma ammonia was measured by a cation exchange–visible spectrophotometric technique,²¹ and osmolarity was measured by freezing-point depression (Advanced Instruments, model 3DII). Arterial blood gases and pH were measured with an ABL3 electrode system, and hemoglobin concentration was measured with an OSM3 Hemoximeter (Radiometer).

Rats received an intravenous infusion of either sodium acetate or ammonium acetate at a rate of approximately 50 µmol/kg per min (0.1 mL/min). Because sodium acetate causes metabolic alkalosis, hydrochloric acid was added to the infusate 30 minutes after the start of the infusion to control arterial pH. For each of the two salt infusions, rats were pretreated with either the saline vehicle (3 mL/kg), MSO (150 mg/kg; 0.83 mmol/kg), or BSO (880 mg/kg; 4 mmol/kg). Pretreatments were given as a 1-hour continuous intravenous infusion starting approximately 3 hours before baseline measurements were made. This dose of MSO is the same as that previously used to inhibit glutamine accumulation in rats.^{3 22} This dose of BSO has been shown by others to decrease brain glutathione concentration within 24 hours.²³ The sample size of groups was n=6, n=5, and n=5 for the sodium acetate rats pretreated with vehicle, MSO, and BSO, respectively, and n=8, n=8, and n=6 in the ammonium acetate rats pretreated with vehicle, MSO, and BSO, respectively.

Pial arteriolar diameter was measured during a 10-minute period of hypercapnia before the start of salt infusion, at hourly intervals during salt infusion under normocapnic conditions, and during a second 10-minute period of hypercapnia at 6 hours of salt infusion. The salt infusion continued through the second hypercapnic period. Microsphere measurements of CBF were made at 6 hours of salt infusion during normocapnia and the second hypercapnic period. Plasma ammonium and osmolarity and arterial blood pH, PO₂, PCO₂, and hemoglobin concentration were measured at baseline and at 2 and 6 hours of salt infusion. Blood gases were also measured during each hypercapnic period.

To test whether pial arterioles were capable of dilation to another physiological stimulus during hyperammonemia, diameter was measured during 10 minutes of hypoxic hypoxia at 6 hours of ammonium acetate infusion in a separate cohort of 5 rats.

Statistical comparisons among groups were made with ANOVA and the Newman-Keuls multiple-range test. For measurements repeated over time, comparisons were made with baseline values by repeated-measures ANOVA and Dunnett's test. Comparisons of the percent diameter response with hypercapnia between the first and second hypercapnic challenge were made by paired t test. Comparison of the normocapnic and hypercapnic CBF values at 6 hours were made by paired t test. The level of statistical significance was set at P<.05 in all tests. Data are expressed as mean±SE.

	Results

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Continuous ammonium acetate infusion increased plasma ammonia concentration to approximately 600 µmol/L by 6 hours (Table 1). Groups pretreated with MSO started at higher concentrations before the salt infusion. In all groups, values were stable between 2 and 6 hours, indicative of a steady state. Infusion of sodium acetate plus hydrochloric acid to control metabolic alkalosis caused a small increase in plasma osmolarity, whereas infusion of ammonium acetate caused a small decrease in plasma osmolarity (Table 1).

View this table: [in this window] [in a new window]   Table 1. Plasma Ammonia Concentration and Osmolarity During 6-Hour Salt Infusion

There was little change in arterial pH or PCO₂ during the 6-hour salt infusion. The level of induced hypercapnia was similar among groups during the first hypercapnic challenge before salt infusion and during the second hypercapnic challenge at 6 hours of salt infusion (Table 2). The rats were well oxygenated throughout the experiment. Modest hemodilution occurred during the salt infusion, but there was no difference among groups.

View this table: [in this window] [in a new window]   Table 2. Arterial pH, Blood Gases, and Hemoglobin Concentration Before and During Hypercapnic Challenges at Baseline and at 6 Hours of Salt Infusion

Hypercapnia caused a moderate reduction in mean arterial pressure, but there was no significant difference among groups (Table 3). As expected, pressure within the closed cranial window increased during hypercapnia. Ammonium acetate infusion caused an increase in normocapnic intracranial pressure in groups pretreated with vehicle or BSO but not with MSO (Table 3). Likewise, cortical tissue water content measured at the end of the experiment was elevated in ammonium acetate–infused groups pretreated with either vehicle or BSO but not with MSO (Table 4).

View this table: [in this window] [in a new window]   Table 3. Mean Arterial Pressure and Pressure in Cranial Window Before and During Hypercapnic Challenges at Baseline and at 6 Hours of Salt Infusion

View this table: [in this window] [in a new window]   Table 4. Cortical Tissue Water Content

During the 6-hour period of normocapnic hyperammonemia, significant increases in pial arteriolar diameter occurred by 5, 3, and 1 hours in groups pretreated with vehicle, MSO, and BSO, respectively (paired t test). With sodium acetate infusion, increases occurred by 3 hours in the group pretreated with BSO (Fig 1). At 6 hours, the increase in diameter in the vehicle and BSO–ammonium acetate groups was greater than that in the MSO–sodium acetate group, but other group comparisons were not significant (Newman-Keuls test).

View larger version (26K): [in this window] [in a new window]   Figure 1. Pial arteriole diameter (mean±SE) as a percent of baseline during the 6-hour infusion period of NaAcetate or NH4Acetate under normocapnic conditions. There was no significant change over time in the NaAcetate groups pretreated with vehicle (VEH) or MSO. Significant increases occurred by 3, 5, 3, and 1 hours in the BSO+NaAcetate, VEH+NH4Acetate, MSO+NH4Acetate, and BSO+NH4Acetate groups, respectively.

During the first hypercapnic exposure before salt infusion but approximately 3 hours after pretreatment, pial arterioles dilated in all groups, and there was no difference in the response among groups (Fig 2). During the second hypercapnic exposure at 6 hours of sodium acetate infusion, the dilatory response was unchanged from baseline dilatory response in groups pretreated with vehicle, MSO, or BSO. However, with 6 hours of ammonium acetate infusion, the dilatory response was lost in groups pretreated with vehicle or BSO. In contrast, the response was not significantly different from the first hypercapnic response in the hyperammonemic group pretreated with MSO. Comparisons among groups of the 6-hour hypercapnic response indicated significant differences between the corresponding ammonium acetate versus sodium acetate groups pretreated with vehicle or BSO but not with MSO. The response in the MSO–ammonium acetate group was greater than that in the vehicle and BSO–ammonium acetate groups.

View larger version (27K): [in this window] [in a new window]   Figure 2. Percent change in pial arteriole diameter (mean±SE) during 10 minutes of hypercapnia before infusion of sodium acetate or ammonium acetate but after treatment with vehicle, MSO, or BSO (baseline response) and during 10 minutes of hypercapnia at 6 hours of salt infusion in the same rats. The percent change of the second hypercapnic challenge is normalized by the normocapnic values at 6 hours. *P<.05 from baseline response by paired t test; P<.05 from 6-hour response in sodium acetate group with corresponding pretreatment by Newman-Keuls test; and #P<.05 from 6-hour response in MSO+ammonium acetate group by Newman-Keuls test.

CBF and cerebrovascular resistance at 6 hours of salt infusion during normocapnia were similar among the six groups (Fig 3). During hypercapnia, CBF increased in the three sodium acetate groups and in the ammonium acetate group pretreated with MSO. However, CBF did not increase in the hyperammonemic groups pretreated with vehicle and BSO, and the response was significantly less than those in the corresponding sodium acetate groups and in the ammonium acetate groups pretreated with MSO. Cerebrovascular resistance decreased during hypercapnia in all groups, but the decrease was less in the hyperammonemic groups pretreated with vehicle and BSO.

View larger version (35K): [in this window] [in a new window]   Figure 3. CBF and cerebral vascular resistance (mean±SE) during normocapnia and 10 minutes of hypercapnia at 6 hours of sodium acetate or ammonium acetate in groups pretreated with vehicle, MSO, or BSO. *P<.05 from normocapnia by paired t test; P<.05 from hypercapnic value in sodium acetate group with corresponding pretreatment by Newman-Keuls test; and #P<.05 from hypercapnic values in MSO+ammonium acetate group by Newman-Keuls test.

In cerebellum, hypercapnia produced an increase in CBF in hyperammonemic rats pretreated with MSO but not with vehicle or BSO (Fig 4). In diencephalon and medulla plus pons, there was a small increase in flow during hypercapnia with vehicle pretreatment, but the increase was less than that in the sodium acetate controls. In midbrain, diencephalon, and medulla plus pons, hypercapnic blood flow in hyperammonemic rats with MSO pretreatment exceeded that in rats with vehicle pretreatment.

View larger version (44K): [in this window] [in a new window]   Figure 4. Blood flow (mean±SE) to cerebellum, diencephalon, midbrain, and combined medulla and pons during normocapnia and 10 minutes of hypercapnia at 6 hours of sodium acetate or ammonium acetate in groups pretreated with vehicle, MSO, or BSO. *P<.05 from normocapnia by paired t test; P<.05 from hypercapnic value in sodium acetate group with corresponding pretreatment by Newman-Keuls test; and #P<.05 from hypercapnic values in MSO+ammonium acetate group by Newman-Keuls test.

In a separate group of 5 rats, arterial PO₂ was decreased from 161±5 to 45±2 mm Hg at 6 hours of ammonium acetate infusion. Pial arteriolar diameter increased 30±7%.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
There are several new findings in this study. First, acute hyperammonemia in a clinically relevant range²⁴ caused extraparenchymal pial arterioles to lose their dilatory response to hypercapnia but not to hypoxia. Cerebral edema formation, intracranial hypertension, and the loss of the blood flow response to hypercapnia throughout the brain also occurred in the same animals in which arteriolar reactivity was lost. Second, inhibition of glutamine synthesis during hyperammonemia by MSO pretreatment prevented loss of pial arteriolar reactivity to hypercapnia as well as preventing cerebral edema, intracranial hypertension, and loss of the CBF response to hypercapnia. Third, BSO, an analogue of MSO that inhibits -glutamylcysteine synthetase but not glutamine synthetase, failed to prevent any of the physiological changes observed during hyperammonemia.

Loss of the CBF response to hypercapnia during acute hyperammonemia has been described in several species, including the rat.^{11 13 14 15} We previously found that acute hyperammonemia caused increased cortical tissue water content³ and that swelling was localized in astrocytes, particularly in perivascular processes.¹² In perfusion-fixed tissues, capillaries appeared more elliptical, suggesting some compression. Thus, it was possible that the compressive forces of swollen astrocyte processes mechanically limited an increase in blood flow. If this were the sole explanation, then one would expect that extraparenchymal arterioles not surrounded by astrocytes would retain vascular reactivity to hypercapnia. However, we found that pial arterioles lost their dilatory response in concert with the loss of the blood flow response to hypercapnia. Thus, the loss of the CBF response is caused by a loss of CO₂ reactivity in resistance arterioles and not simply an increase in resistance in downstream capillaries.

It is possible that compression of downstream capillaries caused an autoregulatory dilation of upstream arterioles and that loss of CO₂ reactivity was secondary to an exhaustion of the vasodilatory reserve of pial arterioles. Indeed, the 34±9% increase in pial arteriolar diameter at 6 hours of ammonium acetate infusion (Fig 1) with normal levels of CBF (Fig 4) may represent, at least in part, an autoregulatory response to intraparenchymal vascular compression. However, we found that pial arterioles still dilated in response to hypoxia during acute hyperammonemia. This result is consistent with the intact CBF response to hypoxia and to hypotension previously reported in this model.¹⁹ Thus, loss of vasodilation is specific for hypercapnia.

We have previously shown in this model of acute hyperammonemia that MSO inhibits cortical glutamine synthetase by 64%, prevents the 13-mmol/kg increase in cortical glutamine concentration, prevents the increase in tissue water content, prevents the increase in cisterna magna pressure, and prevents the loss of the CBF response to hypercapnia.^{3 11} The results with MSO in the present study confirm our previous findings on tissue water content, intracranial pressure, and CBF. The increases in tissue water content to 80.3% and in pressure under the cranial window to 13.6 mm Hg in vehicle-treated hyperammonemic rats are similar to the values obtained previously for water content (80.4%) and cisterna magna pressure (13 mm Hg). Thus, the model is reproducible and causes diffuse increases in intracranial pressure. Furthermore, the small decrease in plasma osmolarity with ammonium acetate infusion is inadequate to explain the increase in tissue water content.³

The major new finding with MSO in the present study is that MSO prevents loss of the pial arteriolar dilator response to hypercapnia. This result indicates that the loss of arteriolar reactivity is not attributable to ammonium ions per se but is dependent on glutamine synthesis and accumulation associated with hyperammonemia. It also indicates that upstream arterioles that are not in immediate contact with astrocytes, where glutamine synthetase is enriched,⁹ are functionally altered by a glutamine-dependent mechanism. There are several possible explanations for this result.

First, swollen astrocytes may reflect astrocyte dysfunction. Dysfunctional astrocytes may no longer control the extracellular constituents in a tight fashion. Treatment with MSO reduces the appearance of watery cytoplasm in astrocyte processes¹² and may preserve astrocyte function. For example, a preliminary study with ion-sensitive microelectrodes indicates increased extracellular potassium activity that is attenuated by MSO pretreatment.²⁵ Such alterations in potassium activity could modulate CO₂ reactivity.²⁶

Second, astrocytes may release nonionic substances that are vasoactive and can diffuse to the pial surface without significant metabolic degradation. However, regulation of vascular smooth muscle tone by astrocyte-derived substances has not been well studied.

Third, glutamine itself might alter vascular reactivity. Although glutamine is a neutral amino acid with no known vascular receptors, it might exert indirect effects. In cultured endothelial cells, glutamine but not ammonium chloride inhibits agonist-induced NO release by interacting with arginine in the signal transduction pathway linked to NO synthase,²⁷ or possibly by inhibiting citrulline uptake,²⁸ and recycling of citrulline to arginine.^{29 30 31} However, hypercapnic reactivity is not endothelium dependent,³² and it is unclear whether glutamine inhibits NO production in neurons sufficiently to reduce smooth muscle guanylate cyclase. Preliminary work by others³³ suggests that glutamine can inhibit vasorelaxation elicited by transmural nerve stimulation. Whether such mechanisms can account for the complete loss of hypercapnic reactivity seen during acute hyperammonemia is unclear because NO synthase inhibition usually does not completely abolish hypercapnic reactivity.³⁴

In addition to inhibiting glutamine synthetase irreversibly,³⁵ MSO can inhibit -glutamylcysteine synthetase reversibly.¹⁶ -Glutamylcysteine is the precursor of glutathione. Administration of MSO has been reported to decrease glutathione in kidney but not in whole brain.¹⁷ However, astrocytes have a relatively high glutathione turnover rate,³⁶ and it is possible that MSO could decrease glutathione in this compartment. To control for this possibility, we administered BSO, a reversible inhibitor of -glutamylcysteine synthetase¹⁸ that can decrease brain glutathione levels within 24 hours²³ and augment focal ischemia damage.³⁷ We found that BSO did not prevent the increase in tissue water content and intracranial hypertension or the loss of pial arteriolar and CBF responsivity to hypercapnia associated with hyperammonemia. Furthermore, we previously found that BSO did not prevent swelling of perivascular astrocyte processes in hyperammonemic rats.¹² Therefore, the positive physiological effects of MSO are not attributable to potential inhibition of -glutamylcysteine synthetase.

In general, there was concordance among groups in the pial arteriolar diameter changes with the calculated changes in cerebrovascular resistance during hypercapnia. This observation is consistent with the premise that alterations occurred in both intraparenchymal and extraparenchymal resistance vessels. However, cerebrovascular resistance decreased somewhat during hypercapnia without a significant increase in pial arteriolar diameter in hyperammonemic rats pretreated with vehicle or BSO. Mean arterial blood pressure also decreased during hypercapnia. Because autoregulation remains intact in this experimental model,¹⁹ the small decrease in resistance may reflect an autoregulatory response in large arteries and arterioles³⁸ proximal to the small-to-medium arterioles (11 to 60 µm) monitored in the present study.

In other brain regions, hyperammonemia attenuated the CBF response to hypercapnia and MSO improved the response. These results agree with our previous study.¹¹ In the present study, we also found that BSO did not significantly improve the regional blood flow response to hypercapnia, again supporting the role of glutamine synthesis. Therefore, the role of glutamine accumulation during hyperammonemia is not restricted to cortical pial arterioles.

In summary, both the pial arteriolar dilatory and global CBF responses to hypercapnia were completely abolished within 6 hours of the elevation of plasma ammonia concentration to approximately 600 µmol/L. Lost reactivity was not attributable to vasoparalysis because pial arterioles still dilated during hypoxia. These pathophysiologic alterations depended on glutamine synthesis and accumulation rather than on ammonium ions per se. Because cerebral edema localized in astrocytes and moderate intracranial hypertension during acute hyperammonemia also depend on glutamine accumulation, changes in vascular reactivity may result indirectly from changes in astrocyte function. It is also possible that glutamine may affect vascular reactivity by modulating NO production.

	Selected Abbreviations and Acronyms

 

BSO = buthionine sulfoximine CBF = cerebral blood flow MSO = methionine sulfoximine NaAcetate = sodium acetate NH4Acetate = ammonium acetate NO = nitric oxide

	Acknowledgments

 
This work was supported by a grant from the National Institutes of Health (NS-25275). The authors thank Ying Wu for her technical assistance and Lisa DeLoriers for her help in preparing the manuscript.

	Footnotes

 
Reprint requests to Raymond C. Koehler, PhD, Department of Anesthesiology/Critical Care Medicine, The Johns Hopkins Medical Institutions, 600 N Wolfe St, Blalock 1408, Baltimore, MD 21287-4961.

Received September 14, 1995; revision received December 18, 1995; accepted January 16, 1996.

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