Adachi, Takehiko MD; Shinomura, Tetsutaro MD; Nakao, Shin-ichi MD; Kurata, Jiro MD; Murakawa, Masahiro MD; Shichino, Tsutomu MD; Seo, Norimasa MD; Mori, Kenjiro MD, FRCA
Nitric oxide (NO) is an inorganic free radical gas  and has divergent biologic roles, including vascular smooth muscle relaxation, humoral transmission in the central and peripheral nervous system, and mediation of host-defense mechanisms. It is synthesized from L-arginine (L-Arg) with formation of L-citrulline by a family of enzymes, namely nitric oxide synthases (NOSs). The actions of NO are exerted mainly through activation of soluble guanylate cyclase and consequent increase of cyclic guanosine monophosphate (cGMP) levels in target cells . Interactions of anesthetics with the NO-cGMP system are of considerable interest to anesthesiologists .
Halothane inhibits NOS activity  and decreases cGMP levels  in the brain. These findings suggest that the anesthetic action of halothane might be exerted, at least partly, through suppression of the L-Arg-NO-cGMP pathway. A major role of the L-Arg-NO pathway in nociception has also been postulated. Intraperitoneal, oral, and intracerebroventricular administration of a NOS inhibitor (Nomega-nitro-L-arginine methyl ester [L-NAME]) is antinociceptive in the mouse . Spinal NO mediates thermal hyperalgesia produced by loose ligation of the sciatic nerve with chromic gut sutures . Inhibition of spinal NO synthesis blocks N-methyl-D-aspartate-induced thermal hyperalgesia  and produces antinociception in the formalin test [8,9], although it does not block the responses to noxious thermal stimulation [7,8,10], noxious mechanical stimulation [10,11], or mechanical hyperalgesia  in rats. If the anesthetic action of halothane is exerted through suppression of the L-Arg-NO pathway, and/or if inhibition of NO synthesis has antinociceptive effects, a NO synthase inhibitor might reduce the minimum alveolar anesthetic concentration (MAC) of halothane. From this viewpoint, we studied the effects of acutely administered intravenous, intracerebroventricular, and intrathecal L-NAME on halothane MAC in rats. MAC reduction did not occur .
Recently it has been reported that brain NOS activity could not be inhibited by more than approximate equals 50% by acute intravenous administration of L-NAME . Our previous failure to reduce halothane MAC by L-NAME might be attributed to insufficient inhibition of NOS activity by acute administration of L-NAME. It has been reported that chronic administration of Nomega-nitro-L-arginine, a NOS inhibitor, results in marked inhibition of brain NOS in rats . Therefore, the first aim of the present study was to investigate the effect of chronic administration of L-NAME on brain NOS activity. Brain NOS activity was assessed by cerebellar NOS catalytic activity (L-citrulline formation) and cGMP levels. After confirming the marked reduction of brain NOS activity by chronic administration of L-NAME, we investigated the effect of that treatment on halothane MAC in rats.
Thirty-six male Sprague-Dawley rats were used for the study, which was approved by our institutional committee on animal research. Rats were divided randomly into either a saline (n = 18, body weight at the beginning of experiment 334 +/- 18 g, mean +/- SE) or L-NAME (n = 18, body weight 335 +/- 21 g, shown to be not significantly different from the saline control by Student's t-test) group. Animals were injected intraperitoneally twice a day with saline, 1 mL/kg, or L-NAME, 50 mg/kg in a volume of 1 mL/kg, for 4 consecutive days. On the 5th day, between 12 and 24 h after the last injection of L-NAME, determination of cerebellar NOS activity (n = 6 in each group), cerebellar cGMP levels (n = 6 in each group), or halothane MAC (n = 6 in each group) was performed.
Measurement of Cerebellar NOS Activity
Rats were stunned and killed by decapitation. The cerebella were removed and homogenized in 50 mM Tris-HCl (pH 7.4) buffer containing 1 mM EDTA and 1 mM EGTA using a Teflon glass homogenizer. NOS activity was measured by monitoring the Ca (2+) dependent conversion of [(3) H]arginine to [(3) H]citrulline [15-17]. Twenty-five microliters of tissue homogenate and 25 micro Liter of 100 micro Meter [(3) H]arginine was added to 75 micro Liter of 20 mM Hepes buffer (pH 7.4), supplemented with either 25 micro Liter of 6 mM nicotinamide adenine dinucleotide phosphate (reduced form), 6 mM CaCl2, or 25 micro Liter of 6 mM EGTA. After incubation for 15 min at 37 degrees C, the assay was terminated by the addition of 2 mL of ice-cold 20 mM Hepes (pH 5.5), containing 2 mM EDTA, and was applied to 1-mL Dowex AG Registered Trademark 50W-X8 (Na+ form) columns (Bio-Rad Laboratories, Hercules, CA), and eluted with 2 mL of water. Radioactivity of the total of 4 mL was quantified by liquid-scintillation counting. The activity of the Ca2+-dependent NOS was determined from the difference of radioactivity between samples containing Ca2+ and samples without Ca2+. The protein concentration was measured using the Bradford method .
Measurement of Cerebellar cGMP Levels
Rats were killed with microwave irradiation at 9.0 kW for 1 s (NJE 2607; New Japan Radio Co Ltd., Tokyo, Japan) . The cerebella were removed, weighed, and rapidly frozen. The frozen tissue was homogenized in cold 6% trichloroacetic acid to give a 10% wt/vol homogenate and then centrifuged at 2000g for 15 min at 4 degrees C. The supernatant fluids were washed four times with 5 vol of water saturated with diethyl ether and then lyophilized. The dried extract was stored at -20 degrees C until assayed. The radioimmunoassay of cGMP was performed using a cGMP assay kit.
Determination of Halothane MAC
The method to determine halothane MAC has been described in detail previously  and is briefly summarized here. The rats were anesthetized with halothane in oxygen and artificially ventilated. The right femoral vein and artery were cannulated for fluid administration and continuous monitoring of arterial blood pressure and heart rate. A rectal thermometer was inserted, and the temperature was maintained at approximately 38 degrees C with a warm water mattress and a heating lamp. A fine polyethylene catheter, 0.8 mm outside diameter, was inserted through the endotracheal tube until obstruction occurred and then was withdrawn approximately 1 cm. This catheter was connected to an anesthetic gas monitor (Type 1304; Bruel & Kjaer, Naerum, Denmark) for continuous monitoring of the concentrations of halothane and CO2.
For MAC determination, the end-tidal concentration of halothane was adjusted initially to 1.0%, which was then increased or decreased, depending on the response of rats to tail pinch, in steps of 0.1%, allowing at least 15 min for equilibration. The rats' tails were clamped with a hemostat to the second ratchet lock. Noxious stimulation to the animals was applied until gross positive movement of the head, extremities, or body occurred, or for 1 min. The tail was always stimulated proximal to a previous test site. MAC was determined as the point midway between the end-tidal concentration of halothane at which the animals did and did not move. At the end of the experiment, analysis of arterial blood gas, hemoglobin, and electrolytes (Na, K, Ca) was always performed.
All data were described as mean +/- SEM. Statistical analysis was performed using Student's t-test. P < 0.05 was considered significant.
[(3) H]Arginine (1.5 TBq/mmol) was obtained from DuPont/NEN (Boston, MA). cGMP assay kits were obtained from Yamasa (Tokyo, Japan). All other chemicals were obtained from Sigma (St. Louis, MO).
During 4 days of chronic treatment, animals treated with L-NAME did not show any apparently different behavior compared to the animals treated with saline.
Cerebellar NOS activity was 67 +/- 19 pmol centered dot mg-1 protein centered dot min-1 in the L-NAME group, whereas it was 487 +/- 160 pmol centered dot mg-1 protein centered dot min-1 in the control group. This difference was statistically significant (P < 0.05; Figure 1A).
The cerebellar cGMP levels were significantly lower in the L-NAME group, 8 +/- 4 fmol/mg tissue, than in the saline control, 299 +/- 92 fmol/mg tissue (P < 0.05; Figure 1B).
(Table 1) shows the physiologic variables of animals under halothane anesthesia. Mean arterial pressure and heart rate were measured during a stabilized state at 1.0% end-tidal concentration of halothane before the first tail pinch. As shown in Table 1, only mean arterial pressure was significantly higher in the L-NAME group than in the saline group. Figure 1C summarizes the MAC of halothane in both groups. There was no significant difference in the halothane MAC between the saline and L-NAME group: 0.93% +/- 0.05% in the control animals and 0.92% +/- 0.02% in the L-NAME group.
The present study reveals that the MAC of halothane was not modified by chronic treatment with L-NAME, despite the fact that cerebellar NOS activity and the cGMP levels were profoundly decreased, suggesting that neither NOS activity nor cGMP levels in the brain modifies the anesthetic potency (MAC) of halothane. It seems less likely that the action of halothane in reducing brain NOS activity  or cGMP levels  contributes to the anesthetic action of halothane as measured by MAC. The findings on MAC values are consistent with our previous study which showed that an acute administration of L-NAME did not change the MAC of halothane in rats . This is in agreement with studies which showed the absence of contribution of the spinal L-Arg-NO pathway in response to noxious mechanical stimulation [10,11].
In the central nervous system (CNS), NOS activity, NOS protein densities, NOS-messenger RNA , and cGMP levels  are all highest in the cerebellum, and changes in NOS activity and cGMP levels induced by pharmacologic manipulations of NOS may be best represented by those in the cerebellum. To eliminate the effects of different experimental durations on the measurement of brain NOS activity and cGMP levels, we measured cerebellar NOS activity and cGMP levels in an awake state in animals different from those used for MAC determination.
The rats treated with L-NAME showed systemic hypertension but not any apparent abnormal behaviors. This confirmed early studies which reported that chronic oral administration of L-NAME induced a dose-dependent increase of arterial blood pressure , that rats chronically treated with L-NAME (75 mg/kg intraperitoneally daily) did not show any sensory or motor dysfunction , and that the targeted disruption of the neuronal NOS gene did not result in any gross behavioral abnormalities .
The depression of NOS activity by chronic treatment with L-NAME in this study is 86% and comparable to an earlier study using another NOS inhibitor, Nomega-nitro-L-arginine . Recently, endothelial NOS was reported to have an important role in the CNS especially in synaptic plasticity [25,26]. Residual NOS activity (presumably endothelial NOS) of approximately 4% in the hippocampus of the neuronal NOS knockout mouse  may have a significant role in hippocampal long-term potentiation . As L-NAME inhibits both neuronal and endothelial NOS, it seems unlikely that the residual NOS activity in our study has a significant role in the CNS.
To our knowledge, this is the first study to demonstrate that chronic in vivo treatment with L-NAME reduces basal cerebellar cGMP levels significantly. A similar reduction of basal aortic cGMP levels occurs after chronic blockade of NOS with L-NAME . As cGMP is also regulated by atrial natriuretic peptide and particulate guanylyl cyclase, this result suggests that in vivo basal cerebellar cGMP levels are mainly dependent on NO and soluble guanylyl cyclase activity and to a minor extent on atrial natriuretic peptide and particulate guanylyl cyclase activity.
As discussed in our previous study , surgery-related noxious stimulation is protracted and has extensive spatial and temporal summating effects. It causes not only mechanical noxious stimulation but also production of chemical mediators secondary to local tissue ischemia/hypoxia and acute inflammation, all of which sensitize the nociceptive neural mechanism in the spinal cord dorsal horn. Such a sensitization is known as "CNS plasticity" and is possibly mediated by the L-Arg-NO-cGMP system. The present study does not necessarily rule out the significance of the L-Arg-NO-cGMP system in pain perception in the clinical situation of surgery.
In conclusion, the present study demonstrates that chronic treatment with L-NAME profoundly reduces cerebellar NOS activity and cGMP levels without altering the MAC of halothane in rats. These results suggest that the anesthetic potency (MAC) of halothane is not related to brain NOS activity or cGMP levels.
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