INTRODUCTION

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SEVERAL STUDIES HAVE SHOWN that glutamine at physiological concentrations can inhibit release of endothelium-derived relaxing factor in cell cultures (1, 11, 23, 37) and in isolated aortic rings and strips (1, 27). The mechanism of glutamine is thought to involve inhibition of argininosuccinate synthetase, an enzyme that is part of a two-step recycling of citrulline to arginine for use by nitric oxide synthase (NOS) (12, 24, 25). In isolated cerebral arteries, glutamine inhibits nitric oxide (NO)-dependent relaxation evoked by transmural nerve stimulation (21) by inhibiting the conversion of citrulline to arginine and the uptake of citrulline in perivascular nerves (6). Thus in vitro evidence in endothelial cells and perivascular nerves indicates that glutamine acts by inhibiting recycling of citrulline to arginine, thereby limiting arginine availability for NO production. In support of this concept, adding excess arginine overcomes the inhibitory effect of glutamine in these studies.

The aforementioned studies were performed on in vitro systems. There has been less work performed on establishing a role for glutamine in modulating vascular reactivity in vivo. One pathophysiological condition in which glutamine increases is hyperammonemia, such as occurs with liver disease or urea cycle disorders (8). The increase in glutamine is prominent in brain where glutamine synthetase is enriched in glia. The cerebral blood flow response to hypercapnia is suppressed during acute hyperammonemia in a variety of species (2, 7, 9, 17, 30). Inhibition of glutamine synthetase during hyperammonemia preserved the cerebral blood flow (30) and pial arteriolar responses (13, 15) to CO₂. Thus the impaired response was related to glutamine synthesis rather than to the increased concentration of ammonium ions. However, inhibition of glutamine synthesis during hyperammonemia also ameliorates ammonia-induced astrocyte swelling (36), intracranial hypertension (30), elevated extracellular potassium concentration in cortex (26), and depressed glucose consumption (10). Therefore, the impaired vascular response to CO₂ could be attributed to several factors associated with glutamine accumulation rather than to direct effects of glutamine on cerebral vessels.

In the present study, we determined whether increasing glutamine concentration can impair cerebrovascular reactivity to hypercapnia in the absence of hyperammonemia. Because the in vitro evidence implies that glutamine acts by limiting recycling of citrulline to arginine rather than by acting on vascular ionic channels, we assumed that an effect on vascular reactivity would take several hours to develop rather than several minutes. We therefore tested reactivity of pial arterioles at 3 and 6 h of intravenous infusion of glutamine. We also determined whether coinfusion of arginine prevented any effect of glutamine. Lastly, we determined whether endogenous increases in glutamine associated with hyperammonemia can be reversed by arginine infusion. Hence, three hypotheses were tested: 1) intravenous infusion of glutamine decreases pial arteriolar dilation to hypercapnia, 2) coinfusion of arginine with glutamine preserves hypercapnic vasodilation, and 3) intravenous infusion of arginine during hyperammonemia preserves hypercapnic vasodilation.

	METHODS

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All procedures were approved by the institutional animal care and use committee. Male Wistar rats weighing ~400 g were anesthetized intraperitoneally with pentobarbital sodium (65 mg/kg). The lungs were mechanically ventilated through a tracheostomy with ~30% O₂. Catheters were inserted into a femoral vein, femoral artery, and tail artery. A cranial window was constructed on the right parietal bone as described (15). This procedure involved removing a 3 × 4-mm section of bone, securing a plastic ring around the hole with acrylic cement, retracting the dura under a well of artificial cerebrospinal fluid (CSF), and gluing a glass coverslip on top of the fluid-filled plastic ring. Fluid pressure in the window was monitored continuously. Rectal temperature was maintained with a heating pad.

Pial arterioles were observed through a microscope video recording system (15). Internal diameter was measured on several arterioles per rat. Most arterioles were in the range of 25-50 µm. The percent change in diameter was calculated for each arteriole. An average percent response was calculated from these arterioles for each rat. Statistics were performed on these average responses with n equal to the number of rats per group.

Baseline measurements of arterial pH, arterial partial pressure of CO₂ (Pa_CO2) and O₂ (Pa_O2), arterial blood pressure, CSF pressure in the window, and pial arterolar diameter were measured 30 min after the surgery was completed. In the first experiment, hypercapnia was produced for 10 min by increasing the fractional inspired CO₂ concentration to ~0.07-0.08. The fractional O₂ concentration was maintained at ~0.3-0.4 to prevent arterial O₂ desaturation as a result of the Bohr effect. Measurements were repeated at 10 min of hypercapnia. After normocapnic recovery, rats were divided into three groups: 1) a control group (n = 11) receiving intravenous lactated Ringer solution (6 ml/h); 2) a group receiving 3 mmol · kg¹ · h¹ L-glutamine in lactated Ringer solution (n = 10); and 3) a group receiving 3 mmol · kg¹ · h¹ L-glutamine plus 2 mmol · kg¹ · h¹ L-arginine in lactated Ringer solution (n = 8). Hypercapnic challenges were repeated at 3 and 6 h of intravenous infusion. Measurements were repeated during normocapnia and 10 min of hypercapnia at 3 and 6 h. Plasma samples (100 µl) were obtained at baseline and 3 and 6 h of infusion for enzymatic determination of glutamine and glutamate based on the fluorometric analysis of glutamate after deamidation of glutamine by glutaminase (3). Fluid in the window was not flushed during the 6-h infusion period. At the end of the experiment, a sample of CSF was collected from the cranial window for analysis of glutamine and glutamate.

In the second experiment, hyperammonemia was produced by infusing 60-70 µmol · kg¹ · min¹ ammonium acetate intravenously (6 ml/h). This infusion produces a threefold increase in cortical tissue glutamine concentration (14, 29). Control groups received a solution of 200 mmol/l sodium acetate plus 140 mmol/l hydrochloric acid to prevent metabolic alkalosis. Rats were divided into four groups: 1) sodium acetate (n = 8); 2) sodium acetate plus L-arginine (n = 5); 3) ammonium acetate (n = 9); and 4) ammonium acetate plus L-arginine (n = 8). The rate of intravenous infusion of L-arginine was 2 mmol · kg¹ · h¹. Hypercapnia was produced for 10 min before the start of the infusion period and again at 7 h of infusion. Measurements were made during normocapnia and at 10 min of hypercapnia. Plasma ammonia concentration was measured by a cation-exchange visible spectrophotometric technique (5). Because high concentrations of L-arginine interfered with the resin exchange at low ammonium concentrations, ammonia levels are not reported in the sodium acetate plus L-arginine group.

In each experiment, comparisons were made among groups by ANOVA and the Newman-Keuls multiple range test with P < 0.05 considered significant. Data are presented as means ± SE.

	RESULTS

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Intravenous glutamine infusion. With continuous intravenous infusion of L-glutamine, the concentrations of glutamine in the plasma and CSF were nearly three times the values in the control group receiving lactated Ringer solution (Fig. 1). With coinfusion of L-arginine and L-glutamine, the concentrations of glutamine in plasma and CSF were similar to that of L-glutamine infusion alone. Glutamine can be metabolized to glutamate by glutaminase. However, there were no differences in plasma or CSF glutamate concentration among groups during the infusion.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Concentration of glutamate (A) and glutamine (B) in plasma preinfusion and at 3 and 6 h of infusion, and in cerebrospinal fluid (CSF) under cranial window at end of experiment, in groups receiving continuous intravenous infusion of vehicle (Ringer lactate), L-glutamine, or L-arginine+L-glutamine. Values are means ± SE. *P < 0.05 from Ringer lactate group; +P < 0.05 from glutamine group.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Percent change in basal pial arteriolar diameter under normocapnic conditions during intravenous infusion of lactated Ringer solution (n = 11), L-glutamine (n = 10), or L-arginine+L-glutamine (n = 8). Values are means ± SE; n = no. of rats. *P < 0.05 from Ringer lactate group and arginine group.

View larger version (46K): [in this window] [in a new window]   Fig. 3.   Percent change in pial arteriolar diameter during 10-min hypercapnic exposures preinfusion and at 3- and 6-h intravenous infusion of vehicle (Ringer lactate; n = 11), L-glutamine (n = 10), or L-arginine+L-glutamine (n = 8). Values are means ± SE. *P < 0.05 from Ringer lactate group.

Hyperammonemia. Plasma ammonia concentration was 23 ± 3 µmol/l at 7 h of sodium acetate infusion. With ammonium acetate infusion, ammonia concentration increased to a similar extent with (621 ± 70 µmol/l) and without (664 ± 65 µmol/l) coinfusion of L-arginine. Arterial pH and Pa_CO2 were nearly equivalent among groups during normocapnia and hypercapnia, except for a slightly lower Pa_CO2 normocapnic value in the sodium acetate group before infusion (Table 3). There were no differences in mean arterial blood pressure among groups during normocapnia or hypercapnia. At 7 h, hypercapnia decreased arterial pressure in both groups receiving sodium acetate, but not in either of the ammonium acetate groups. There was no effect of coinfusion of arginine on the arterial pressure response to hypercapnia (Table 3).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Percent change in basal pial arteriolar diameter under normocapnic conditions during intravenous infusion of Na acetate (n = 8), Na acetate+L-arginine (Arg; n = 5), NH4 acetate (n = 9), or NH4 acetate+L-arginine (n = 8). Values are means ± SE. *P < 0.05 from Na acetate group at 7 h.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Percent change in pial arteriolar diameter during 10-min hypercapnic exposures preinfusion and at 7-h infusion of Na acetate (n = 8), Na acetate+L-arginine (n = 5), NH4 acetate (n = 9), or NH4 acetate+L-arginine (n = 8). Values are means ± SE. *P < 0.05 from Na acetate group at 7 h; +P < 0.05 from NH4 acetate group at 7 h.

	DISCUSSION

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There are three major findings of this study on pial arteriolar reactivity to hypercapnia. First, threefold increases in plasma glutamine concentration attenuated hypercapnic reactivity, but the effect required more than 3 h to develop. Second, infusion of arginine counteracted the attenuating effect of exogenous glutamine administration. Third, infusion of arginine partially counteracted the loss of hypercapnic reactivity during hyperammonemia known to produce threefold increases in tissue glutamine concentration. These results demonstrate that glutamine can exert a modulatory effect on cerebrovascular CO₂ reactivity in vivo and that arginine can counter the inhibitory effects of endogenously or exogenously produced increases in glutamine.

Previous investigations of the vascular effects of glutamine have focused primarily on the recycling of citrulline to arginine in endothelial cell cultures and on NO release. A decrease in the synthesis of arginine from citrulline produced by an increase in intracellular glutamine has been described in endothelial cells derived from bovine aorta (24), bovine coronary venules (23), pig pulmonary artery (25), and human mesentric microvessels (37). The mechanism is thought to depend on inhibition of argininosuccinate synthase by glutamine (12, 24, 25), although a decrease in cellular uptake of citrulline by glutamine may also be important (37). Release of endothelium-derived relaxing factor by agonist stimulation of endothelial cells (11) and aortic strips (27) is inhibited by glutamine. Because glutamine does not directly inhibit NOS catalytic capacity (1, 21, 23), the effect of glutamine is presumed to be mediated by a limitation of arginine availability. However, in contrast to glutamine inhibition of NO release evoked by agonist stimulation, glutamine does not inhibit NO release induced by calcium ionophore application (1). Moreover, direct evidence (28) for glutamine inhibition of isolated argininosuccinate synthetase indicates that inhibition is modest compared with other amino acids at a 10 mmol/l concentration. Thus the mechanism of action of glutamine on NO release is not completely understood and could involve alternative pathways.

In brain, NO is derived from neuronal NOS (nNOS) as well as from endothelial NOS (eNOS). Inhibition of both isoforms with N-nitro-L-arginine (35) as well as inhibition of nNOS with 7-nitroindazole (34) reduces cerebrovascular reactivity to CO₂ in the rat. Hypercapnic reactivity is intact after endothelial injury (35) and in eNOS-deficient mice (22), whereas N-nitro-L-arginine inhibits CO₂ reactivity after endothelial injury (35) and in eNOS-deficient mice (22). Thus nNOS activity appears to be more important for CO₂ reactivity than eNOS activity. The effect of glutamine on NO derived from neuronally based NOS has not been as well studied as that based in endothelium, where much of the enzyme is compartmentalized in plasma membrane-associated caveolae. In perivascular nerves surrounding cerebral arteries, glutamine inhibits citrulline uptake and arginine synthesis, leading to inhibition of smooth muscle relaxation evoked by transmural nerve stimulation both in the presence and absence of endothelium (6, 21). Thus glutamine can act to inhibit NO release from isolated perivascular nerves as well as from endothelial cells in vitro.

Our results with intravenous glutamine infusion are novel in demonstrating that glutamine can modulate vascular reactivity in vivo and that this modulation does not require logarithmic changes in glutamine concentrations. Increasing plasma and CSF concentrations by a factor of three reduced CO₂ vascular reactivity by nearly one-half. In vitro studies demonstrated inhibitory effects of glutamine on both endothelium-dependent and neuron-dependent vascular relaxation in the extracellular glutamine concentration range of 200-1,000 µmol/l (1, 11, 21, 27). The increases in plasma glutamine concentration (from ~500 to ~1,700 µmol/l) and in CSF concentration (from ~240 to ~640 µmol/l) are in a range comparable to that in in vitro studies. Thus changes in glutamine concentration in the pathophysiological range are capable of modulating CO₂ reactivity.

In the rat, inhibiting NOS with either N-nitro-L-arginine or 7-nitroindazole attenuates cerebrovascular CO₂ reactivity, whereas application of an NO donor or a cGMP analog after NOS inhibition restores CO₂ reactivity (16, 32). Thus a minimal amount of NO and cGMP appears to be required for full expression of CO₂ reactivity. We found that the decrease in cerebrovascular reactivity to CO₂ with a 6-h infusion of glutamine was prevented by coinfusion of arginine. This observation is consistent with the hypothesis that glutamine acts by limiting arginine availability for basal NO production in brain.

Another consideration is the effect of arginine on vasodilation by ATP-sensitive K⁺ (K_ATP) channel agonists. The presence of arginine has been reported to be required for the pial arteriolar dilation evoked by agonists of K_ATP channels (19). Antagonists of K_ATP channels inhibit CO₂-induced dilation of pial arterioles (18). Thus preservation of CO₂ reactivity by arginine in our experiments conceivably could be due to an effect of arginine on K_ATP channels. However, this hypothesis presumes that glutamine inhibits K_ATP channels in order for arginine to have a disinhibitory effect. We are not aware of studies showing an inhibitory effect of glutamine directly on K_ATP channels. Furthermore, we did not observe an inhibitory effect of glutamine on CO₂ reactivity at 3 h of infusion. A direct allosteric effect of glutamine on K_ATP channels should have been present by this time. An increase in tissue glutamine concentration and a consequent decrease in arginine concentration below the Michaelis-Menten constant (K_m) for NOS via argininosuccinate synthetase inhibition and/or cellular uptake inhibition is likely to require a few hours. Thus the 6-h requirement for inhibiting CO₂ reactivity by glutamine is more consistent with a metabolic depletion of the arginine pool subserving NOS-containing neurons when arginine recycling is inhibited.

We also found that arginine infusion helped preserve CO₂ reactivity of pial arterioles during hyperammonemia. This effect of arginine was not simply due to a generalized augmentation of CO₂ reactivity because arginine infusion did not enhance hypercapnic vasodilation in a control group receiving sodium acetate infusion. We have previously measured a threefold increase in brain tissue glutamine concentration at comparable levels of increased plasma ammonia achieved in the present study (14, 29). The impaired vascular reactivity to CO₂ is related to glutamine accumulation rather than ammonium ions, because inhibiting glutamine synthesis preserves vascular reactivity at elevated ammonia levels (15). Ammonium ions do not affect NO release from endothelial cells (11, 23) or perivascular nerves (21). Thus the greater hypercapnic reactivity of pial arterioles in the group receiving arginine plus ammonium acetate compared with the group receiving ammonium acetate alone is more likely related to an interaction of arginine and glutamine than an interaction of arginine and ammonium ions.

Although arginine infusion increased CO₂ reactivity during hyperammonemia, vascular reactivity was not completely preserved at a level equivalent to that of the sodium acetate control group. Without arginine infusion, hypercapnic reactivity was lost during hyperammonemia in this study and in our previous studies (15, 30), whereas intravenous glutamine infusion alone reduced reactivity by only one-half. These results suggest that there may be other effects of glutamine that are not counteracted by arginine when the increase in glutamine is endogenously derived. During hyperammonemia, most of the increase in glutamine is thought to be generated by glial-based glutamine synthetase (8). Hyperammonemia causes swelling of astrocyte processes and accumulation of extracellular K⁺ activity, both of which are ameliorated by glutamine synthetase inhibition (26, 36). In the present study, arginine infusion did not reduce cortical tissue water content during hyperammonemia. Thus ammonia-induced glutamine accumulation may cause dysfunction of astrocytes that leads to impairment of vascular reactivity other than through a decrease in arginine availability for NOS. For example, when extracellular K⁺ is increased to ~10 mmol/l, a level attained during hyperammonemia (26), pial arteriolar dilation to acidosis is attenuated (4, 20). Hence, there may be two components of the glutamine-dependent loss of CO₂ reactivity during hyperammonemia: one that can be counteracted by arginine and that may depend on glutamine depletion of arginine availability or some other interaction of glutamine and arginine directly on the blood vessels, and a second component that is related to glutamine-induced astrocyte dysfunction and impaired K⁺ homeostasis.

Basal diameter under normocapnic conditions increased during both ammonium acetate and glutamine infusions. The difference in the increase in basal diameter between the glutamine (11%) and Ringer (5%) groups is unlikely to account for the decreased CO₂ reactivity, because the increase in basal diameter is too small to exhaust the vasodilatory reserve. For example, Tuor and Farrar (31) reported that the percent increase in diameter with hypercapnia was unabated when baseline diameter increased ~40% during arterial hypotension. In the case of hyperammonemia, cerebral vessels still dilate normally to hypoxia (14, 15). In hyperammonemic rats pretreated with a glutamine synthetase inhibitor, basal diameter increased by 14% but the percent increase in diameter with hypercapnia was normal (15). Moreover, arginine infusion during hyperammonemia in the present study did not attenuate the increase in basal diameter but did increase CO₂ reactivity. Therefore, the increases in baseline diameter observed over the 6- to 7-h infusion period are unlikely to be responsible for the observed decreases in CO₂ reactivity.

However, the increase in basal diameter is at variance with the hypothesis that glutamine decreases basal NO production, which would normally be expected to decrease basal diameter. In the case of hyperammonemia, the increase in extracellular K⁺ (26) and autoregulatory responses to downstream vascular compression arising from astrocytic swelling (36) may contribute to pial arteriolar dilation and override any constrictor influence of decreased NO production. In the case of intravenous glutamine infusion, any decrease in basal NO production arising from reduced arginine availability would be expected to occur gradually over the last few hours of the 6-h infusion period. A gradual decrease in NO could permit time for upregulation of other vasodilatory pathways that prevent vasoconstriction. Alternatively, arginine depletion by glutamine may not be sufficient to decrease basal NO production, but only to attenuate any increases evoked by hypercapnia (33). Our observation that elevated glutamine did not completely inhibit hypercapnic vasodilation, but reduced the response by only 45%, is consistent with this possibility.

In summary, the present study indicates that moderate increases in glutamine concentration are capable of inhibiting cerebrovascular CO₂ reactivity in vivo. Elevated glutamine may explain part of the depressed vascular CO₂ reactivity seen under conditions associated with elevated ammonia. Both the gradual time course of this inhibitory effect of glutamine and the reversal by arginine infusion are consistent with a metabolic mechanism of action of glutamine on a recycling of citrulline to arginine and a consequent reduction of arginine availability for NO release.