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ANESTHETIC PHARMACOLOGY

Nitric Oxide Is Not a Mediator of Inflammation-Induced Resistance to Atracurium

Heidrun Fink, MD^*, Ralph Bogdanski, MD^*, Peter Luppa, MD^#, J. A. Jeevendra Martyn, MD, and Manfred Blobner, MD^*

*Klinik für Anaesthesiologie der Technischen Universität München and #Institut für Klinische Chemie und Pathobiochemie der Technischen Universität München, Klinikum rechts der Isar, Munich, Germany; and Department of Anesthesiology and Critical Care, Harvard Medical School and Anesthesia Services, Massachusetts General Hospital, and Shriners Hospital for Children, Boston, MA

Address correspondence to Heidrun Fink, MD, Klinik für Anaesthesiologie der Technischen Universität München*, Klinikum rechts der Isar, Ismaninger Str. 22, 81675 Munich, Germany. Address electronic mail to h.fink{at}lrz.tum.de.

	Abstract

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Resistance to atracurium as a result of increased drug binding to ₁-acid glycoprotein is associated with increased inducible nitric oxide synthase activity and increased nitric oxide levels in plasma. We investigated if the inhibition of inducible nitric oxide synthase and suppression of nitric oxide can reverse the resistance to atracurium. As a model of ₁-acid glycoprotein and nitric oxide increase, 84 male Sprague-Dawley rats received an IV injection of either 60 mg/kg Corynebacterium parvum (CP) or saline (control). The 2 groups (CP/Control) were further divided into subgroups, receiving the selective inducible nitric oxide synthase inhibitor, N-Iminolysine, via drinking water at different concentrations. On day 4 post-CP injection, the pharmacodynamics of atracurium were determined. Plasma concentrations of nitric oxide, atracurium, and ₁-acid glycoprotein were measured and acetylcholine receptor numbers were quantified. In the CP groups, N-Iminolysine suppressed nitric oxide levels in a dose-dependent manner. Resistance to atracurium persisted. ₁-acid glycoprotein serum levels remained increased in all CP groups with no differences in acetylcholine receptor expression. Our results suggest that the mechanism leading to increased expression of ₁-acid glycoprotein and consecutive increased protein binding of atracurium is not mediated by inducible nitric oxide synthase induction and nitric oxide expression.

	Introduction

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Nitric oxide (NO) is a key signal transducing molecule of many aberrant responses observed during systemic inflammation, including hemodynamic instability (1), decreased organ function (2), decreased xenobiosis (3), and reduced drug action (4,5). Previously we had shown that action of muscle relaxants is also impaired at several levels by NO-related effects (4,5). The plasma clearance of vecuronium, for example, depends on hepatic cytochrome p450 activity, which is inhibited by NO but can be restored if NO synthesis is suppressed by the nonspecific NO synthase inhibitor N^G-monomethyl-L-arginine (5). Normalizing the hepatic clearance of vecuronium, however, revealed an additional effect of sepsis or NO, evidenced as a resistance towards its neuromuscular blocking effect. Studies with atracurium showed that the observed resistance to its action as muscle relaxant was attributable to an inflammation-associated increase in ₁-acid glycoprotein (₁-AGP), to which atracurium binds in serum (4).

The present study, therefore, addresses the question of whether suppression of NO can reverse the acute phase response to inflammation at a blood chemical level, i.e., the increase of ₁-AGP levels and at a functional level, i.e., by neutralizing the resistance to atracurium. Therefore, with the specific inducible nitric oxide synthase (iNOS) inhibitor N-Iminolysine (NIL), dose-response studies were performed on both targets.

	Methods

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After governmental approval of the study (Regierung von Oberbayern, AZ 211–2531–70/97), 84 male Sprague-Dawley rats (Charles River GmbH, Kisslegg, Germany) weighing 250–300 g were allowed to accommodate to the standard conditions of our animal facility with free access to chow and water for 14 days. Rats were divided into 2 groups: rats of the Corynebacterium parvum (CP) group (n = 42) received an IV injection of 60 mg/kg of a whole cell preparation of heat-killed CP (Roche, Penzberg, Germany) in 0.5 mL saline; rats of the control group (n = 42) received the solvent. The injection of heat-killed CP induces a granulomatous liver inflammation and systemic inflammatory response syndrome evidenced as increased NO and ₁-AGP levels peaking at the fourth day postinjection (4). To examine the role of NO in the altered biochemical and pharmaceutical changes, the two groups of rats (CP/control) were further divided into six subgroups and received six different doses of the selective iNOS inhibitor NIL via drinking water (2.2, 0.22, 0.022, 0.0022, 0.00022, and 0 mol/L of NIL) in drip-free drinking bottles. NIL treatment was begun 1 day before the injection and was continued until the fourth day after bacterial injection. Body weight and water intake were recorded daily. On the fourth day post-CP injection, effects of atracurium on neuromuscular function were evaluated. After these measurements the animals were killed by exsanguination. The blood was centrifuged (3500 rpm, 4°C, 10 min) and the serum was stored for later analysis of nitrite/nitrate (NO₂/NO₃) as well as ₁-AGP levels. The gastrocnemius muscle was excised to determine the expression of acetylcholine receptors.

Anesthesia was induced by inhalation of sevoflurane in a glass cylinder. After loss of consciousness, the rats were endotracheally intubated and mechanically ventilated with oxygen in nitrous oxide (ratio 1:2). Anesthesia was maintained with 4%–6% sevoflurane. After cannulation of the left external jugular vein, anesthesia was switched to a continuous infusion of propofol (20–40 mg · kg^–1 · h^–1) and fentanyl (4 µg · kg^–1 · h^–1). The left carotid artery was cannulated to measure mean arterial blood pressure and perform blood gas analyses. After the start of IV anesthesia, all animals were allowed to stabilize over a period of 60 min to eliminate effects of sevoflurane on neuromuscular transmission. At all times the administration of anesthesia was adjusted according to cardiovascular signs of adequate anesthesia.

Throughout the experiment, arterial carbon dioxide partial pressure (Paco₂) was maintained between 36 and 44 mm Hg. Whenever necessary, base excess was corrected with 1 mM sodium bicarbonate to values between –2 ± 2 mM. Arterial oxygen partial pressure (Pao₂), heart rate, and mean arterial blood pressure were continuously monitored to ensure stable oxygenation and hemodynamic conditions throughout the experiment. Rectal temperature was controlled between 36.8°C and 37.2°C with a warming blanket and heating lamp. Rats were excluded from the experiment if they were hemodynamically unstable (mean arterial blood pressure <80 mm Hg) at the beginning of the experiments or if their blood gas status throughout the experiment was not within the defined predetermined ranges (Pao₂ >100 mm Hg; pH, 7.36–7.44; Paco₂: 36–44 mm Hg; base excess –2 ± 2 mM).

Neuromuscular function was monitored by evoked mechanomyography (Myograph, Biometer, Copenhagen, Denmark). The sciatic nerve of the left leg was exposed at its exit from the lumbosacral plexus and stimulated using the train-of-four pattern (2 Hz for 2 s every 12 s). The knee was pinned and firmly fixed. A force transducer was connected to the Achilles’ tendon and the contraction of the gastrocnemius muscle was measured. Supramaximal stimulus and control twitch height (T0) were established. The mechanomyographic response was stabilized over a period of at least 10 min before determination of the individual dose-response relation of atracurium (ED₅₀) in each rat using the cumulative method described previously (4). Bolus doses of atracurium were given IV in increments between 0.2 and 0.8 mg/kg until the first twitch of the train-of-four (T1) was below 5% of baseline value. Each incremental dose was given only when the previous dose had produced maximal effect, as indicated by 3 equal (±2%) consecutive T1 twitches. After the last dose of atracurium, twitch response was allowed to recover to baseline values. The recovery interval was calculated as time at (T1/T0 = 75%) – time at (T1/T0 = 25%). After complete recovery of T1, a continuous infusion of atracurium was started, and the infusion rate adjusted to achieve a constant T1/T0 of 50%. After 10 min of stable T1/T0 = 50% at a fixed infusion rate, steady-state conditions were assumed. The required infusion rate was documented ("infusion rate at 50% neuromuscular block") and 1 mL of heparinized blood was withdrawn to determine total plasma concentrations of atracurium. The blood was immediately transferred to Eppendorff tubes containing 20 µL 1M H₂SO₄ (to avoid degradation of atracurium) and centrifuged (3500 rpm, 10 min, 4°C). The supernatant was collected and 0.2 mL portions were aliquoted into Eppendorff tubes containing 0.8 mL 15 mM H₂SO₄. The samples were immediately frozen at –70°C. After blood sampling, both gastrocnemius muscles were dissected from the surrounding structures and rapidly frozen in isopentane pre-cooled in liquid nitrogen and stored at –70°C. After this, animals were killed by exsanguination. The collected blood was centrifuged (3500 rpm, 10 min, 4°C) and the gained serum immediately stored at –70°C for determination of ₁-AGP and NO₂/NO₃-plasma concentrations.

Activity of iNOS and production of NO was assessed by measuring its stable metabolites, NO₂ and NO₃ (NO₂/NO₃), in plasma. Samples were deproteinized with 0.5 M NaOH and 10% ZnSO₄. NO₃ was then converted to NO₂ using HPLC on a cadmium column. NO₂ concentrations were determined spectrophotometrically at 540 nm using a method based on the Griess reaction (6). Methemoglobin (MetHb) levels as a result of NO binding to hemoglobin were measured oximetrically as part of the blood gas analyses (Rapidlab 860, Bayer Diagnostics, Munich, Germany).

₁-AGP was measured using a competitive chemiluminescence immunoassay (7). Signaling was induced by applying horseradish peroxidase-conjugated streptavidin and quantifying the enzyme activity using an enhanced chemiluminometric method (Amerlite System, Ortho Clinical Diagnostics, Neckargemünd, Germany). Polyclonal antibodies against rat ₁-AGP were raised in rabbits using pure commercially available rat ₁-AGP (Sigma, Deisenhofen, Germany).

Atracurium plasma concentrations were determined by HPLC as previously described (4). In brief, the method has a between-day imprecision of 3.7% (coefficient of variation) at a mean value of 9.6 µg/mL atracurium. The recovery of spiked plasma samples varied between 96% and 98.7%. The functional sensitivity of the method, being a measure of the lower limit of quantitation, was found to be 0.8 µg/mL. Linearity of the HPLC determination could be demonstrated up to 120 µg/mL. The plasma clearance of atracurium during steady-state conditions was calculated as clearance = infusion rate/plasma level.

The muscle was homogenized, the protein extracted and the amount of membrane acetylcholine receptors quantified by the ¹²⁵I- -bungarotoxin binding assay (8). The protein concentration of the muscle extract was assayed using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA), and the content of acetylcholine receptors was calculated and expressed in fmol/mg protein.

Data are described as mean values and 95% confidence intervals. Variables were compared between groups using factorial analyses of variance with the 2 independent factors infection (CP versus control) and NIL dose (0, 0.00022, 0.0022, 0.022, 0.22 mol/L). An effect of NIL treatment on a respective variable was considered if NIL or NIL x infection proved to be significant (P < 0.05) and was followed post hoc by factorial analyses of variance in the control groups and, independently, in the CP groups to evaluate dose dependency. If infection proved to be significant, groups with the same NIL dose were compared by unpaired Student’s t-tests post hoc. The dose-response of atracurium was evaluated by linear regressions of the degree of the blockade in logit scale and the respective cumulative dose of atracurium on log scale in each animal. Slopes and intercepts of the so calculated individual regressions served as determinates for the dose-response and were subjected to statistical analysis. The doses of atracurium for a 50% neuromuscular block (ED₅₀) were interpolated using the means of slope and intercept of each group and their 95% confidence borders.

	Results

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There was a 50% mortality rate in rats receiving 2.2 mM NIL regardless of CP or control group, making it impossible to include data from these animals in the statistical analysis. Rats of the 2.2 mM NIL groups that were still alive on day four were not included in the neuromuscular function tests because of hemodynamic instability and were killed by exsanguination. Therefore, only some blood chemistry could be performed.

There were no differences in water intake between the different NIL groups of the CP or the control rats at any day. However, from the third day postinjection on, there was a significant difference in water intake between CP (34 [32–36], 33 [32–34], 32 [30–34], 25 [24–26], 26 [24–27] mL/day from 1 day before NIL administration till day 4) and control rats (36 [34–37], 35 [34–36], 34 [32–36], 34 [32–36]*, 34 [32–35]* mL/day from 1 day before NIL administration till day 4, *P < 0.05 compared with CP rats). Injection of CP resulted in an increase in NO₂/NO₃ and MetHb levels in plasma. Treatment with NIL reduced NO₂/NO₃ and MetHb plasma concentrations in the experimental group in a dose-dependent fashion. NO₂/NO₃ levels in CP rats receiving 2.2 mM NIL had returned to values of control rats. NIL had no effect on NO₂/NO₃ and MetHb plasma concentrations in control rats. After CP injection ₁-AGP levels were increased. Administration of NIL did not have any effect on the increased ₁-AGP levels of the CP group. There was no difference in expression of nicotinic acetylcholine receptors in the gastrocnemic muscle between groups (Table 1).

View this table: [in this window] [in a new window]   Table 1. Nitrite/Nitrate, MetHemoglobin, 1-acid glycoprotein Levels, and Acetylcholine Receptor Concentration in Corynebacterium Parvum and Control Animals During Treatment With Various Concentrations of N-Iminolysine

The slopes of the dose-response curves did not differ between groups. The ED₅₀ of all CP groups were significantly larger compared with control and placebo rats (Table 2). Likewise the atracurium plasma concentrations to maintain a 50% neuromuscular block were also increased in the CP groups. Consistent with these findings, the recovery index was shortened in all groups with a larger requirement for atracurium (Table 2). Treatment with 0.22 mM NIL shifted the atracurium dose-response curve leftwards in both the CP and the control group (Table 2).

	Discussion

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In this study, the inflammation-induced increase in ₁-AGP levels could not be reversed with the iNOS inhibitor NIL despite dose-dependent attenuation of the release of NO. However, in nonlethal doses NIL was not able to suppress increased NO levels in animals with systemic inflammation to the levels of control animals. The increase in ₁-AGP resulted in resistance to the neuromuscular blocking actions of atracurium because of increased binding of atracurium to ₁-AGP.

Resistance to nondepolarizing neuromuscular blocking drugs can be seen in patients with systemic inflammation or sepsis. In a previous study in rats, we demonstrated that this resistance was not a result of altered target-organ, i.e., acetylcholine receptor, sensitivity but was attributed to an increase in drug binding to ₁-AGP (4). The present study confirms these results. Atracurium resistance was evidenced as increased effective doses, increased plasma concentration requirement to achieve a constant neuromuscular block, and decreased recovery times from complete neuromuscular block. The increased ₁-AGP levels persisted in all CP groups receiving NIL, despite attenuation of inflammation, evidenced as decreasing NO levels. Accordingly, the resistance to atracurium persisted in the all CP groups, demonstrated by a rightward shift of the dose-response curves compared with control animals. Interestingly, however, both CP and control groups receiving 0.22 mM NIL had a shift of their dose-response curve to the left again. Practically, the rightward shift (i.e., resistance to atracurium) of the dose-response curve induced by CP could be counteracted by NIL. Because, however, ₁-AGP levels and atracurium plasma concentrations remained increased, a direct inhibitory effect of NIL on neuromuscular transmission is very likely. NIL probably displays the same direct open or closed acetylcholine receptor channel block as the other NOS inhibitors L-NG-monomethyl arginine and L-nitroargine methylester (9).

The injection of CP induces a granulomatous liver inflammation with excessive production of NO. This endogenous NO reduces hepatic cytochrome p450 activity that can be restored by inhibiting iNOS activity (10). Atracurium, however, is a quaternary ammonium compound degraded primarily via Hoffmann elimination and is therefore independent of hepatic metabolism (11). Uniform distribution and degradation of atracurium were attempted by maintaining hemodynamic variables, acid-base balance, and temperature constant, resulting in comparable plasma clearance among groups. We are therefore confident that the observed differences in the effective doses and plasma concentrations of atracurium are unrelated to altered metabolism of atracurium.

NIL is a specific iNOS inhibitor. However, in animals receiving the larger doses of NIL, an effect on endothelial NO synthase (eNOS) and neural NO synthase (nNOS) activity cannot be excluded. A decrease in muscle perfusion on the microcirculatory level through an eNOS-inhibition mediated vasoconstriction might have affected our results. Furthermore, NO decreases submaximal force by modulating excitation-contraction coupling and therefore promotes relaxation of the skeletal muscle through the cyclic guanosine monophosphate (cGMP) pathway (12). Moreover, NO takes part in regulating acetylcholinesterase expression in the synaptic cleft and ion channel properties by activating cGMP (13). Finally, NO donors inhibit the evoked release of acetylcholine reducing the end-plate potential (14). Overall, it is impossible to speculate to what extent the various eNOS or nNOS effects on synaptic processes might have been suppressed. The NO levels remained increased NIL groups up to 0.22 M. All these effects of increased NO levels in our model, however, would have made the muscle more susceptible rather than resistant to muscle relaxants, i.e., leading to a leftward shift of the dose-response curve.

Conflicting results have been published regarding possible links between iNOS induction and production of ₁-AGP. Cultured and activated liver cells increasingly produce ₁-AGP during conditions of iNOS pathway (15). In contrast, iNOS expression in hepatocytes in vitro after lipopolysaccharide stimulation is not part of the acute-phase response and is differentially regulated from ₁-AGP (16). In our in vivo experiments the specific iNOS inhibitor NIL was able to dose-dependently suppress NO production without affecting increased ₁-acid glycoprotein levels. Although we did not measure iNOS activity, the approximately normalized NO metabolites NO₂/NO₃ prove the efficacy of the drug administration. NO₂/NO₃ levels in the 2.2 M CP group were restored to normal again. Because of the frequent lethality, however, the rats were excluded from analysis. Reasons for the deaths in these rats were not examined, although it can be speculated that they were related to hypertensive vasoconstriction, especially in the pulmonary circuit. Nevertheless, our results therefore support the notion of independent regulation of iNOS activity and ₁-acid glycoprotein expression.

NO has a high affinity to hemoglobin, resulting in the generation of MetHb. The effective suppression of increased NO levels was also reflected in a normalization of pathologically increased MetHb levels. In the CP groups receiving placebo or small-dose NIL treatment, however, MetHb levels of more than 5% posed the possibility of oxidative stress. Normal Pao₂ and pH ranges and the absence of any increased lactate levels, however, make the presence of hypoxia and an effect on our results unlikely.

In summary, we conclude that targeting increased NO levels to reverse altered pharmacodynamics of drugs binding to ₁-AGP, e.g., atracurium, does not seem effective. The only practically feasible way to deal with increased plasma protein binding is to administer more drug, in our case, more atracurium.

	Footnotes

 
Supported, in part, by a personal stipend from the Deutscher Akademischer Austauschdienst (DAAD), Bonn, Germany to Heidrun Fink, a grant to Manfred Blobner from GlaxoWellcome, Germany, and by grants # GM 31569–22, GM 55082–08, and GM 611411–06 from the National Institutes of Health and from Shriners Hospital for Children to J.A. Jeevendra Martyn.

Accepted for publication May 3, 2005.

	References

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fink, H. Articles by Blobner, M. Search for Related Content PubMed PubMed Citation Articles by Fink, H. Articles by Blobner, M. Related Collections Mechanisms Pharmacology