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CARDIOVASCULAR ANESTHESIA

Nitric Oxide Synthesis Inhibition Modifies the Cardiotoxicity of Tetracaine and Lidocaine

James E. Heavner, DVM, PhD^*,, Bing Shi, MD, PhD, and Mikko Pitkänen, MD, PhD^*,

Departments of *Anesthesiology and Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas; and Department of Anesthesiology, Töölö Hospital, Helsinki, Finland

Address correspondence and reprint requests to James E. Heavner, DVM, PhD, Department of Anesthesiology, Texas Tech University Health Sciences Center, 3601 4th St., Rm. 1C-258, Lubbock, TX 79430. Address e-mail to anejeh{at}ttuhsc.edu

	Abstract

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Suppression of nitric oxide (NO) production alters the toxicity of cocaine and bupivacaine. We undertook this study to determine whether the systemic toxicity of two other local anesthetics that differ in antiarrhythmic activity, plasma clearance, and biotransformation are similarly affected by nitric oxide synthase (NOS) inhibition. Sprague-Dawley rats anesthetized with 70% N₂O and 0.5% halothane mixed with O₂ were pretreated with saline (0.2 mL · kg^-1 · min^-1 IV) or N-nitro-L-arginine methyl ester (L-NAME; a competitive inhibitor of NOS) (2 mg · kg^-1 · min^-1 IV) for 30 min. The animals were then given tetracaine (3 mg · kg^-1 · min^-1 IV) or lidocaine (8 mg · kg^-1 · min^-1 IV) until cardiac arrest (asystole). Doses of lidocaine or tetracaine that produced arrhythmias, seizures, isoelectric encephalogram, and asystole were determined. Hemodynamic recordings were performed throughout the experiments, and plasma was collected to measure the concentration of lidocaine or tetracaine. L-NAME decreased tetracaine and lidocaine doses that produced arrhythmias (2° atrioventricular conduction block) (tetracaine 14 ± 2 mg/kg; lidocaine 102 ± 9 mg/kg) versus saline treatment (tetracaine 28 ± 2 mg/kg; lidocaine 136 ± 9 mg/kg; P < 0.05). The tetracaine and lidocaine doses required to produce asystole were also smaller in animals with L-NAME pretreatment than those in saline-pretreated animals. L-NAME reduced the arrhythmia dose of tetracaine more than the arrhythmia dose of lidocaine (28 of 14 = 2.0 fold and 136 of 102 = 1.3-fold). The plasma concentration of lidocaine, but not tetracaine, was significantly higher at each sample time in L-NAME–pretreated animals than in saline-pretreated animals. Inhibition of NOS by L-NAME enhances the cardiotoxicity of lidocaine and tetracaine, with a greater effect on tetracaine than on lidocaine. Altered drug clearance by L-NAME was insufficient to explain these findings because L-NAME pretreatment increased the plasma levels of only lidocaine, not tetracaine.

Implications: Inhibition of nitric oxide production in rats markedly enhances the cardiovascular toxicity of lidocaine and tetracaine. Altered drug clearance by N-nitro-L-arginine methyl ester was insufficient to explain these findings because N-nitro-L-arginine methyl ester pretreatment increased the plasma levels of only lidocaine, not tetracaine.

	Introduction

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Systemic effects of local anesthetics include cardiovascular depression, cardiac arrhythmias, and grand mal seizures (1). The cellular and subcellular mechanisms involved in these side effects are poorly understood. It is likely that nitric oxide (NO), an important neurotransmitter and intracellular messenger present in all organs of the body, is directly or indirectly involved in systemic toxic effects of local anesthetics. Like local anesthetics, NO produces vasodilation, myocardial depression, and central nervous system (CNS) excitation. NO and local anesthetics both affect calcium shifts in the cardiovascular and CNS, which indicates that local anesthetics and NO have some common actions (2,3). Therefore, investigating the effects of altered NO production on systemic responses to local anesthetics could provide new insight into cellular and subcellular processes that participate in systemic responses to local anesthetics.

We have demonstrated that suppression of the production of NO by the nonselective nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME) enhances the cardiovascular and CNS toxicity of cocaine and bupivacaine (4,5). In the present study, we investigated the effects of inhibition of NO synthesis by L-NAME on the toxicity of lidocaine and tetracaine. One consequence of NOS inhibition by L-NAME is constriction of renal, mesenteric, and hind-quarter blood vessels, which presumably alters the distribution of blood flow (6). Such an alteration might affect the plasma clearance of a drug such as lidocaine, whose plasma clearance is dependent on hepatic extraction and biotransformation, but not the clearance of a drug such as tetracaine, which is biotransformed by enzymes in the blood (7). Our hypothesis was that the effects of L-NAME on the toxicity of lidocaine and tetracaine would differ because of the clinically useful antiarrhythmic action of lidocaine and because of differences in biotransformation of the two local anesthetics.

	Methods

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The study was approved by the Texas Tech University Health Sciences Center Animal Care and Use Committee. Thirty-one male Sprague-Dawley rats approximately 8–10 wk of age were assigned to one of four groups. After preparation, the lightly anesthetized animals were given a constant IV infusion of the NOS inhibitor L-NAME (n = 15) 2 mg · kg^-1 · min^-1 (1% solution) or saline (n = 16, same rate as L-NAME solution) for 30 min. Lidocaine 8 mg · kg^-1 · min^-1 (2.0% solution) or tetracaine 3 mg · kg^-1 · min^-1 (1.0% solution) was then infused IV until electrical function of the heart ceased.

Surgical preparation was performed while the animals spontaneously breathed halothane/O₂ via a mask. The trachea was cannulated, and mechanical ventilation was instituted using a rodent ventilator. A catheter transducer was placed in the left ventricle via the right carotid artery to monitor left ventricular pressure. A cannula was placed through the left femoral vein into the vena cava for local anesthetic and saline or L-NAME infusion, and another cannula was placed into the right femoral vein for the administration of the neuromuscular blocking drug. The right femoral artery was cannulated for arterial pressure measurements and for sampling blood for blood gas and plasma tetracaine or lidocaine analysis. Electrocardiogram (ECG) (leads I, II, and V_I) and frontooccipital electroencephalogram (EEG) were recorded with needle electrodes.

After the surgical preparation, rats were given doxacurium 0.1–0.2 mg/kg to induce muscle paralysis, the halothane concentration was decreased to 0.5%, and N₂O (70%) mixed with O₂ was given. N₂O/halothane was used to provide postoperative analgesia and subdued consciousness. The doxacurium administration was repeated as needed to prevent spontaneous ventilation. ETCO₂ was measured using a microcapnometer, and ventilation of the lungs was adjusted so that PaCO₂ was between 30 and 35 mm Hg. Rectal temperature was maintained at 37.8–38.2°C using a warming blanket and radiant heat if needed. ECG, EEG, left ventricular pressure (LVP), arterial blood pressure (BP), and maximal rate of LVP change (LVP dP/dt_max) were recorded. Left ventricular end-diastolic pressure (LVEDP) was obtained from a high amplification of the LVP.

After surgical preparation, halothane/N₂O was administered, and the saline or L-NAME infusion was begun and continued for 30 min. Lidocaine (n = 8 in each group) or tetracaine (n = 8 in the saline group and n = 7 in the L-NAME groups) was then infused. We determined whether the following events were observed, and, if so, the dose of local anesthetic required to induce each event: arrhythmia (ARR), grand mal seizures (SZ), isoelectric EEG (isoEEG), and cardiac death (asystole; ASYS).

Arterial blood gases were analyzed before the beginning of local anesthetic infusion to confirm that ETCO₂ reflected PaCO₂. Blood samples (0.5 mL) for local anesthetic plasma concentration measurement were taken during the stabilization period and periodically during local anesthetic infusion. Local anesthetic concentrations were measured by using high-pressure liquid chromatography using ultraviolet detection (sensitivity <1 µg/mL; interday coefficient of variation [50 µg/mL] <0.645%) (8).

Student’s t-test for unpaired data (two-tailed) was used to determine whether there were significant differences between the saline and L-NAME groups. P < 0.05 was considered statistically significant.

	Results

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Body weights and baseline blood gases were similar among the groups (Table 1). Baseline left ventricular systolic pressure (LVSP) and LVEDP were significantly higher in the L-NAME groups than in the saline groups. Increased LVSP in L-NAME–treated groups was not accompanied by a corresponding decrease in heart rate (HR). Other hemodynamic variables were not significantly different between the L-NAME- and saline-treated groups.

View larger version (25K): [in this window] [in a new window]   Figure 1. Cardiovascular responses to lidocaine 8 mg · kg-1 · min-1 or tetracaine 3 mg · kg-1 · min-1 in the 5 min preceding asystole (ASYS; e.g., A-1 = 1 min before ASYS) in both the L-NAME and saline groups. Baseline values were recorded just before the start of the lidocaine or tetracaine infusion. Specific responses recorded include heart rate, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and the maximal rate of left ventricular pressure increase (+dP/dtmax), and the maximal rate of left ventricular pressure decrease (-dP/dtmax). *P < 0.05; two-tailed t-test for unpaired data.

The average doses of lidocaine or tetracaine that produced ARR and ASYS were significantly smaller in L-NAME- versus saline-treated rats (Fig. 2). The reduction of ARR doses by L-NAME was greater for tetracaine (28 vs 14 mg/kg = 2.0-fold) than for lidocaine (136 vs 102 mg/kg = 1.3-fold) (Table 2). The doses of lidocaine or tetracaine required to produce SZ were not altered by L-NAME treatment. The dose of lidocaine, but not tetracaine, that produced isoEEG was smaller, on average, in L-NAME- versus saline-treated animals (P < 0.05).

View larger version (39K): [in this window] [in a new window]   Figure 2. Doses of lidocaine 8 mg · kg-1 · min-1 IV or tetracaine 3 mg · kg-1 · min-1 IV that produced seizures, arrhythmias, isoelectric electroencephalograph (isoEEG), and asystole in the saline and N-nitro-L-arginine methyl ester (L-NAME) treatment groups. Values are means ± SE. *P < 0.05; two-tailed t -test for unpaired data.

As shown in Figure 3, the plasma concentrations of lidocaine were significantly higher at each sample time in the L-NAME–pretreated animals compared with the saline-pretreated animals. By contrast, the tetracaine plasma concentrations did not differ between the L-NAME and saline groups.

View larger version (40K): [in this window] [in a new window]   Figure 3. Plasma concentration of lidocaine 8 mg · kg-1 · min-1 or tetracaine 3 mg · kg-1 · min-1 in saline- and N-nitro-L-arginine methyl ester (L-NAME)-treated rats. Values are means ± SE.

	Discussion

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The reduction by L-NAME of the tetracaine and lidocaine doses required to produce ARRs and ASYS is consistent with our previous finding that L-NAME reduces the doses of cocaine and bupivacaine required to produce these effects (4,5). The finding that tetracaine pharmacokinetics were not altered by L-NAME, however, seems to be unique for tetracaine.

Our data point to pharmacokinetic and pharmacodynamic factors being involved in the modification of systemic toxic responses to local anesthetics by L-NAME. Pharmacokinetic factors influencing outcomes of this study may be divided into two categories: those resulting from L-NAME alone and those resulting from an interaction between L-NAME and tetracaine or lidocaine. When first injected into animals pretreated with L-NAME, the distribution of lidocaine or tetracaine through the body (i.e., first pass) is influenced (compared with the control) by the cardiovascular action of L-NAME. Via their cardiovascular actions, lidocaine or tetracaine then participate with L-NAME in influencing their own disposition kinetics.

Substantial experimental evidence shows that endogenous NO has a tonic vasodilatory effect on resistance vessels, thereby modulating the distribution of cardiac output (6,9). In addition to direct vascular action, NO modulates neuroregulation of the cardiovascular system and apparently is involved in the regulatory mechanisms intrinsic to the heart that control HR and contractile force (3,9,10). L-NAME removes the influences of NO on cardiovascular function, thereby producing a significant and sustained increase in arterial BP due primarily to increased peripheral vascular resistance and depression of cardiac output (6). Vascular beds of the heart and the brain are affected (11,12).

Among the anticipated alterations in the pharmacokinetics of tetracaine and lidocaine resulting from the cardiovascular action of L-NAME are reduced volume of distribution and alteration of tissue distribution. Reduced volume of distribution and altered tissue blood flow can have complimentary or antagonistic consequences. For example, delivery of drug to an organ may not change if the blood concentration increases resulting from a reduced volume of distribution are offset by a decrease in organ blood low. However, delivery of drug to an organ (e.g., the liver) may increase markedly if the concentration of drug in the blood is increased and organ blood flow also increases. Increased liver blood flow would increase the rate of clearance of a drug such as lidocaine, which has high hepatic extraction (13). Because lidocaine is biotransformed to less toxic compounds by the liver, clearance and biotransformation of lidocaine by the liver can markedly influence the systemic toxicity of lidocaine (1). However, because tetracaine is biotransformed by esterases in the blood, its systemic toxicity is less influenced by alteration in liver blood flow than is the toxicity of lidocaine (1). Our data show that the disposition kinetics of lidocaine, but not tetracaine, are influenced by L-NAME. Apparently, the concentration of tetracaine in the blood is controlled primarily by the rate of hydrolysis of tetracaine by blood esterases.

The dominant hemodynamic effect of tetracaine and lidocaine is decreased BP as a result of depression of the cardiac output and vasodilation produced by these drugs (1). Thus, in L-NAME–treated animals, local anesthetic is injected into a reduced volume of distribution, then conditions change to an increased volume of distribution (vasodilation) and reduced hepatic blood flow (secondary to reduced cardiac output). These hemodynamic shifts apparently influence the disposition kinetics (as reflected in arterial blood concentration) of lidocaine, but not tetracaine, because of differences in biotransformation. We propose that the overall picture from a pharmacokinetic perspective is injection of drug into an animal with a reduced volume of distribution and altered distribution of blood flow, then progressively reduced cardiac output, increased volume of distribution, and redistribution of blood flow.

There are compelling reasons why, in addition to pharmacokinetic factors, pharmacodynamic interactions between lidocaine or tetracaine and L-NAME contributed to the outcomes of our study. Local anesthetics decrease calcium fluxes across cell membranes and directly influence the processes involved in intracellular translocation of calcium (1,2,13)—so, too, does NO (3). Movement of Ca²⁺ into cells stimulates the production of NO (3).

Through a chain of reactions, NO produces either neuronal depolarization, vasodilation, or myocardial depression, depending on the cell type (neuronal, muscular) in which NO production is stimulated (3,12). L-NAME stops NO production and, hence, the effects of NO. Although not proven, local anesthetics may block NO synthesis by inhibiting the production of NO by blocking Ca²⁺ flux across cell membranes. However, the transmembrane flux of Ca²⁺ also initiates Ca²⁺-stimulated release of Ca²⁺ from the sarcoplasmic reticulum, which initiates muscle contraction. Thus, we propose that NO (by modulating intracellular calcium concentration) and local anesthetics (by blocking calcium-stimulated calcium movement across the sarcolemna) produce vasodilation. Compounds that inhibit NO production in muscle (e.g., L-NAME) and local anesthetic should therefore have antagonistic effects on muscle contraction (e.g., muscle in blood vessel walls). Our data clearly support the conclusion that L-NAME–constricted blood vessels in our experiments were relaxed by toxic doses of lidocaine and tetracaine.

NO also modulates the flux of ions such as sodium and potassium across cell membranes, thereby altering the electrical properties of the membranes (3). NO influences both voltage-gated and ligand-gated ion channels, and local anesthetics block ion fluxes across cell walls. Thus, local anesthetics and NO affect the electrical and mechanical functions of cardiac and vascular muscle cells (1,3). Blocking NO production would remove influences of NO on the ion channel-blocking action of local anesthetics. Through its action on voltage- and ligand-gated ion channels and intrinsic cardiac neuronal activity, NO modulates HR and rhythm (3,14).

Our data suggest that NO influences the effects of local anesthetics on heart rhythm as the arrhythmogenic activity of tetracaine and lidocaine is enhanced by inhibition of NO synthesis. Differences in the kinetic interactions between sodium channels in the heart and lidocaine or tetracaine may explain the greater effect of L-NAME on arrhythmogenic activity. However, the relevance of this kinetic difference to atrioventricular conduction block, which was the first ARR that met the definition we used to define the ARR threshold dose of tetracaine or lidocaine, is unclear.

Ion fluxes in neurons, as well as in myocytes, are affected by NO and by local anesthetics. There is overwhelming evidence that NO is involved in the CNS excitatory action of glutamate mediation via N-methyl-D-aspartate receptors (12). Blockade of N-methyl-D-aspartate receptors, as well as inhibition of NO synthesis, has anticonvulsant effects (2,16).¹ Local anesthetics have both anti- and proconvulsant activities (1). The mechanism by which local anesthetics affect SZ thresholds is poorly understood. One theory is that local anesthetics produce convulsions by blocking inhibitory influences, thereby leaving excitatory activity unopposed, and raise SZ thresholds by blocking sodium flux-mediated membrane depolarization (1). NO synthesis inhibition did not significantly change the doses of lidocaine or tetracaine required to produce SZ in our experiments. NO synthesis inhibition and local anesthetics most likely exert complex multiple interacting influences on CNS excitation.

The results of this study clearly substantiate that the nonselective NOS inhibitor L-NAME can profoundly affect the systemic toxicity of local anesthetics. The mechanisms responsible for this effect are complex and involve multiple pharmacokinetic and pharmacodynamic actions. This observation has important and obvious clinical implications relative to the development of NOS inhibitors for therapeutic purposes (e.g., treatment of pain or drug refractory hypotension).

	Acknowledgments

 
The authors thank Dani Joyner for assistance in the production of this manuscript and Katherine McDaniel for technical assistance in conducting the investigation.

	Footnotes

 
¹ Mcfarlane C, Warner DS, Dexter F, et al. Glutamatergic antagonism: effects on lidocaine-induced seizures in the rat [abstract]. Anesth Analg 1994;78:S278.

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