Amitriptyline versus bupivacaine in rat sciatic nerve blockade

BACKGROUND: Amitriptyline, a tricyclic antidepressant, is frequently used orally for the management of chronic pain. To date there is no report of amitriptyline producing peripheral nerve blockade. The authors therefore investigated the local anesthetic properties of amitriptyline in rats and in vitro. METHODS: Sciatic nerve blockade was performed with 0.2 ml amitriptyline or bupivacaine at selected concentrations, and the motor, proprioceptive, and nociceptive blockade was evaluated. Cultured rat GH3 cells were externally perfused with amitriptyline or bupivacaine, and the drug affinity toward inactivated and resting Na+ channels was assessed under whole-cell voltage clamp conditions. In addition, use-dependent blockade of these drugs at 5 Hz was evaluated. RESULTS: Complete sciatic nerve blockade for nociception was obtained with amitriptyline for 217 +/- 19 min (5 mM, n = 8, mean +/- SEM) and for 454 +/- 38 min (10 mM, n = 7) versus bupivacaine for 90 +/- 13 min (15.4 mM, n = 6). The time to full recovery of nociception for amitriptyline was 353 +/- 12 min (5 mM) and 656 +/- 27 min (10 mM) versus 155 +/- 9 min for bupivacaine (15.4 mM). Amitriptyline was approximately 4.7-10.6 times more potent than bupivacaine in binding to the resting channels (50% inhibitory concentration [IC50] of 39.8 +/- 2.7 vs. 189.6 +/- 22.3 microM) at - 150 mV, and to the inactivated Na+ channels (IC50 of 0.9 +/- 0.1 vs. 9.6 +/- 0.9 microM) at -60 mV. High-frequency stimulation at 3 microM caused an additional approximately 14% blockade for bupivacaine, but approximately 50% for amitriptyline. CONCLUSION: Amitriptyline is a more potent blocker of neuronal Na+ channels than bupivacaine in vivo and in vitro. These findings suggest that amitriptyline could extend its clinical usefulness for peripheral nerve blockade.     

Local Anesthetic Properties of Prenylamine

Background : Local anesthetics that produce analgesia of long duration with minimal impairment of autonomic functions are highly desirable for pain management in the clinic. Prenylamine is a known calcium channel blocker, but its local anesthetic blocking effects on voltage-gated sodium channels have not been studied thus far.

Methods : The authors characterized the tonic and use-dependent prenylamine block of native Na+ channels in cultured rat neuronal GH3 cells during whole cell voltage clamp conditions and the local anesthetic effect of prenylamine by neurologic evaluation of sensory and motor functions of sciatic nerve during neural block in rats.

Results : Prenylamine elicits both use-dependent block of Na+ channels during repetitive pulses (3 [mu]m prenylamine produced 50% block at 5 Hz) and tonic block for both resting and inactivated Na+ channels. The 50% inhibitory concentration for prenylamine was 27.6 +/- 1.3 [mu]m for resting channels and 0.75 +/- 0.02 [mu]m for inactivated channels. Furthermore, in vivo data show that 10 mm prenylamine produced a complete sciatic nerve block of motor function, proprioceptive responses, and nociceptive responses that lasted approximately 27, 34, and 24 h, respectively. Rats injected with 15.4 mm bupivacaine, a known local anesthetic currently used for pain management, had a significantly shorter duration of blockade (< 2 h) compared with rats injected with prenylamine.     

N-Phenylethyl Amitriptyline in Rat Sciatic Nerve Blockade

Background: The antidepressant amitriptyline is commonly used orally for the treatment of chronic pain, particularly neuropathic pain, which is thought to be caused by high-frequency ectopic discharge. Among its many properties, amitriptyline is a potent Na+ channel blocker in vitro, has local anesthetic properties in vivo, and confers additional blockade at high stimulus-discharge rates (use-dependent blockade). As with other drug modifications, adding a phenylethyl group to obtain a permanently charged quaternary ammonium derivative may improve these advantageous properties.

Methods: The electrophysiologic properties of N-phenylethyl amitriptyline were assessed in cultured neuronal GH3 cells with the whole cell mode of the patch clamp technique, and the therapeutic range and toxicity were evaluated in the rat sciatic nerve model.

Results: In vitro, N-phenylethyl amitriptyline at 10 [mu]m elicits a greater block of Na+ channels than amitriptyline (resting block of approximately 90%vs. approximately 15%). This derivative also retains the attribute of amitriptyline in evoking high-degree use-dependent blockade during repetitive pulses. In vivo, duration to full recovery of nociception in the sciatic nerve model was 1,932 +/- 72 min for N-phenylethyl amitriptyline at 2.5 mm (n = 7) versus 72 +/- 3 min for lidocaine at 37 mm (n = 4; mean +/- SEM). However, there was evidence of neurotoxicity at 5 mm.     

N-phenylethyl amitriptyline in rat sciatic nerve blockade

BACKGROUND: The antidepressant amitriptyline is commonly used orally for the treatment of chronic pain, particularly neuropathic pain, which is thought to be caused by high-frequency ectopic discharge. Among its many properties, amitriptyline is a potent Na(+) channel blocker in vitro, has local anesthetic properties in vivo, and confers additional blockade at high stimulus-discharge rates (use-dependent blockade). As with other drug modifications, adding a phenylethyl group to obtain a permanently charged quaternary ammonium derivative may improve these advantageous properties. METHODS: The electrophysiologic properties of N-phenylethyl amitriptyline were assessed in cultured neuronal GH(3) cells with the whole cell mode of the patch clamp technique, and the therapeutic range and toxicity were evaluated in the rat sciatic nerve model. RESULTS: In vitro, N-phenylethyl amitriptyline at 10 microm elicits a greater block of Na(+) channels than amitriptyline (resting block of approximately 90% vs. approximately 15%). This derivative also retains the attribute of amitriptyline in evoking high-degree use-dependent blockade during repetitive pulses. In vivo, duration to full recovery of nociception in the sciatic nerve model was 1,932 +/- 72 min for N-phenylethyl amitriptyline at 2.5 mm (n = 7) versus 72 +/- 3 min for lidocaine at 37 mm (n = 4; mean +/- SEM). However, there was evidence of neurotoxicity at 5 mm. CONCLUSION: N-phenylethyl amitriptyline appears to have a narrow therapeutic range but is much more potent than lidocaine, providing a block duration several times longer than any clinically used local anesthetic. Further work in animal models of neuropathic pain will assess the potential use of this drug.     

Ephedrine Blocks Rat Sciatic Nerve In Vivo and Sodium Channels In Vitro

Background: The sympathomimetic drug ephedrine has been used intrathecally as the sole local anesthetic for labor and delivery. Because ephedrine may be a useful adjuvant to local anesthetics, the authors investigated the local anesthetic properties of ephedrine in a rat sciatic nerve block model and the underlying mechanism in cultured cells stably expressing Na+ channels.

Methods: After approval of the animal protocol, the sciatic nerves of anesthetized rats were exposed by lateral incision of the thighs, 0.2 ml ephedrine at 0.25, 1, 2.5, or 5% and/or bupivacaine at 0.125% was injected, and the wound was closed. Motor and sensory/nociceptive functions were evaluated by the force achieved by pushing against a balance and the reaction to pinch, respectively. The whole cell configuration of the patch clamp technique was used to record Na+ currents from human embryonal kidney cells stably transfected with Nav1.4 channels.

Results: The nociception blockade was significantly longer than the motor blockade at test doses of 2.5 and 5% of ephedrine, or when 1% ephedrine was combined with 0.125% bupivacaine (analysis of variance with repeated measures, P < 0.001, n = 8/group). In vitro, the 50% inhibitory concentrations of ephedrine at -150 and -60 mV were 1,043 +/- 70 and 473 +/- 13 [mu]m, respectively. High-frequency stimulation revealed a use-dependent block of 18%, similar to most local anesthetics.     

Assessment of differential blockade by amitriptyline and its N-methyl derivative in different species by different routes

BACKGROUND: Increasing the duration of local anesthesia and/or creating greater differential blockade (i.e., selective block of pain-transmitting nerve fibers) has been attempted by modifying currently available agents. Most drugs show a different profile depending on the model or species studied. This study was designed to investigate the differential nerve-blocking properties of amitriptyline and its quaternary ammonium derivative in rats and sheep. METHODS: The Na+ channel-blocking properties of N-methyl amitriptyline were determined with the patch clamp technique in cultured GH(3) cells. Various functions (motor, nociception, proprioception-ataxia) were compared in rats (spinal and sciatic nerve blockade) and sheep (spinal blockade) with amitriptyline, N-methyl amitriptyline, lidocaine, and bupivacaine (partially from historical data). RESULTS: In vitro testing revealed N-methyl amitriptyline to be a potent Na+ channel blocker similar to amitriptyline but with a much longer duration of action. All drug concentrations tested in both the sciatic nerve model and the spinal block model produced no significant differential blockade in rats. Three of six rats in the 20-mM N-methyl amitriptyline group showed residual blockade 4 days after sciatic nerve injection. However, in the sheep spinal model, amitriptyline and in particular N-methyl amitriptyline displayed significant differential blockade at most time points. Sheep data for lidocaine and bupivacaine seemed to be more comparable to the clinical experience in humans than did rat data. CONCLUSIONS: Amitriptyline and N-methyl amitriptyline are potent Na+ channel blockers and show greater differential blockade in sheep than in rats. This differential blockade in sheep is greater than that produced by lidocaine or bupivacaine.     

Local anesthetic properties of prenylamine.

BACKGROUND: Local anesthetics that produce analgesia of long duration with minimal impairment of autonomic functions are highly desirable for pain management in the clinic. Prenylamine is a known calcium channel blocker, but its local anesthetic blocking effects on voltage-gated sodium channels have not been studied thus far. METHODS: The authors characterized the tonic and use-dependent prenylamine block of native Na(+) channels in cultured rat neuronal GH3 cells during whole cell voltage clamp conditions and the local anesthetic effect of prenylamine by neurologic evaluation of sensory and motor functions of sciatic nerve during neural block in rats. RESULTS: Prenylamine elicits both use-dependent block of Na(+) channels during repetitive pulses (3 microm prenylamine produced 50% block at 5 Hz) and tonic block for both resting and inactivated Na(+) channels. The 50% inhibitory concentration for prenylamine was 27.6 +/- 1.3 microm for resting channels and 0.75 +/- 0.02 microm for inactivated channels. Furthermore, in vivo data show that 10 mm prenylamine produced a complete sciatic nerve block of motor function, proprioceptive responses, and nociceptive responses that lasted approximately 27, 34, and 24 h, respectively. Rats injected with 15.4 mm bupivacaine, a known local anesthetic currently used for pain management, had a significantly shorter duration of blockade (< 2 h) compared with rats injected with prenylamine. CONCLUSIONS: The data presented here demonstrate that prenylamine possesses local anesthetic properties in vitro and elicits prolonged local anesthesia in vivo.     

Amitriptyline for prolonged cutaneous analgesia in the rat.

BACKGROUND: Amitriptyline has been reported to be a more potent local anesthetic than bupivacaine. In keeping with the objective of identifying drugs for prolonged cutaneous analgesia, the authors compared the cutaneous analgesic effectiveness of amitriptyline and bupivacaine in rats. METHODS: Rats were subcutaneously injected on shaved dorsal skin. The skin wheal raised after injection of 0.6 ml of various concentrations of either amitriptyline or bupivacaine with and without epinephrine (1:200,000) was marked. Inhibition of the cutaneous trunci muscle reflex was evaluated quantitatively by the fraction of times a total of six pinpricks applied to the marked area failed to elicit a nocifensive motor response compared with control responses. No responses out of six pinpricks was defined as 100% maximum possible effect. RESULTS: Complete recovery from the cutaneous analgesia elicited by 0.05% and 0.5 amitriptyline versus 0.05 and 0.5% bupivacaine occurred in 9.9 +/- 0.2 and 19.3 +/- 0.4 h versus 2.2 +/- 0.1 and 16.1 +/- 0.2 h, respectively (mean +/- SE). Addition of epinephrine increased this duration to 14.1 +/- 0.1 and 21.4 +/- 0.2 h versus 3.2 +/- 0.1 and 17.0 +/- 0.3 h, respectively. Complete nociceptive blockade after coinjection of 0.25% amitriptyline, 0.25% bupivacaine, and epinephrine lasted 24 +/- 0.5 h, and complete recovery from this block took 33 +/- 0.5 h. Areas under the percent maximum possible effect versus time curve were 1,770 +/- 24 and 1,471 +/- 50% h for 0.5% amitriptyline and bupivacaine with epinephrine, respectively, whereas this value was 2,836 +/- 62% h for the coinjected 0.25% amitriptyline, 0.25% bupivacaine, and epinephrine admixture. CONCLUSION: Amitriptyline is a longer-acting local anesthetic compared with bupivacaine for cutaneous infiltration. Its analgesic effectiveness is significantly enhanced by epinephrine. Coinjection of amitriptyline and bupivacaine with epinephrine enhances the analgesic duration of both drugs.     

Structural Requirements of Human Ether-a-go-go-related Gene Channels for Block by Bupivacaine

Background: Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine.

Methods: Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels.

Results: Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 [mu]m (n = 33). Bupivacaine (300 [mu]m) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 [mu]m) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001).     

The relative toxicity of amitriptyline,bupivacaine, and levobupivacaine administered as rapid infusions in rats

Intravascular injection of local anesthetics carries the risk of cardiovascular (CV) and central nervous system (CNS) toxicity. Amitriptyline, a tricyclic antidepressant, has local anesthetic potency that is more than that of bupivacaine. In this study, we compared the CV and CNS toxicity of the local anesthetics bupivacaine and levobupivacaine with that of amitriptyline. Twenty-nine Sprague-Dawley rats had their right external jugular vein and carotid artery cannulated under general anesthesia. On Day 2, rats were sedated with midazolam (0.375 mg/kg intraperitoneally) and received rapid infusions of either 1) bupivacaine, levobupivacaine, or amitriptyline at 2 mg x kg(-1) x min(-1) (5 mg/mL concentration) or 2) normal saline (400 micro L x kg(-1) x min(-1)) through an external jugular vein cannula. Electrocardiogram and arterial blood pressure were measured until the dose to cause impending death was reached (heart rate 50 bpm/asystole or apnea for >30 s). The mean dose required to cause apnea and impending death was significantly larger for amitriptyline (74.0 +/- 21 mg/kg and 74.5 +/- 21 mg/kg, respectively) than for levobupivacaine (32.2 +/- 20 mg/kg and 33.9 +/- 22 mg/kg, respectively) or bupivacaine (21.5 +/- 7 mg/kg and 22.7 +/- 7 mg/kg, respectively) (P < 0.05). A significantly larger dose of amitriptyline, given by rapid infusion, is required to cause CV and CNS toxicity in rats, when compared with bupivacaine and levobupivacaine. IMPLICATIONS:Amitriptyline, a tricyclic antidepressant, has local anesthetic properties and is more potent than bupivacaine. Significantly larger doses of amitriptyline, given by rapid infusion, are required to cause cardiovascular and central nervous system toxicity in rats, when compared with bupivacaine and levobupivacaine.     

Effects of Bupivacaine and Ropivacaine on High-voltage-activated Calcium Currents of the Dorsal Horn Neurons in Newborn Rats

Background: Local anesthetics, such as bupivacaine, have been reported to block calcium currents in primary sensory neurons and to interfere with the release of neurotransmitters in central nervous system neurons. However, it is unknown whether local anesthetics affect the calcium current activity of central nervous system neurons.

Methods: Using a traditional whole cell voltage clamp technique, effects of bupivacaine and ropivacaine on high-voltage-activated calcium currents (HVA-Ica) were investigated in enzymatically dissociated dorsal horn neurons of neonatal rats. Calcium currents were evoked by testing pulses from a holding potential of -90 to 0 mV.

Results: Bupivacaine significantly reduced HVA-Ica in a dose-dependent manner. The peak HVA-Ica decreased by 24.5 +/- 2.5, 32.0 +/- 6.8, 59.4 +/- 6.2, 88.3 +/- 1.5, and 91.6 +/- 1.1% in response to 10, 30, 50, 100 and 200 [mu]m bupivacaine, respectively. Unlike bupivacaine, ropivacaine markedly increased HVA-Ica at lower concentrations (< 50 [mu]m) but decreased HVA-Ica at higher concentrations (>= 50 [mu]m). The percent increases in peak HVA-Ica induced by 10 and 30 [mu]m ropivacaine were 95 +/- 19.1 and 41.6 +/- 8.3%, respectively. The percent decreases in response to 50, 100, and 200 [mu]m ropivacaine were 21.1 +/- 2.1, 63.2 +/- 6.0 and 79.1 +/- 7.6%, respectively. Results indicate that the inhibitory potency of ropivacaine on HVA-Ica was significantly lower than that of bupivacaine at the same concentrations.     

Amitriptyline for Prolonged Cutaneous Analgesia in the Rat

Background: Amitriptyline has been reported to be a more potent local anesthetic than bupivacaine. In keeping with the objective of identifying drugs for prolonged cutaneous analgesia, the authors compared the cutaneous analgesic effectiveness of amitriptyline and bupivacaine in rats.

Methods: Rats were subcutaneously injected on shaved dorsal skin. The skin wheal raised after injection of 0.6 ml of various concentrations of either amitriptyline or bupivacaine with and without epinephrine (1:200,000) was marked. Inhibition of the cutaneous trunci muscle reflex was evaluated quantitatively by the fraction of times a total of six pinpricks applied to the marked area failed to elicit a nocifensive motor response compared with control responses. No responses out of six pinpricks was defined as 100% maximum possible effect.

Results: Complete recovery from the cutaneous analgesia elicited by 0.05% and 0.5 amitriptyline versus 0.05 and 0.5% bupivacaine occurred in 9.9 +/- 0.2 and 19.3 +/- 0.4 h versus 2.2 +/- 0.1 and 16.1 +/- 0.2 h, respectively (mean +/- SE). Addition of epinephrine increased this duration to 14.1 +/- 0.1 and 21.4 +/- 0.2 h versus 3.2 +/- 0.1 and 17.0 +/- 0.3 h, respectively. Complete nociceptive blockade after coinjection of 0.25% amitriptyline, 0.25% bupivacaine, and epinephrine lasted 24 +/- 0.5 h, and com-plete recovery from this block took 33 +/- 0.5 h. Areas under the percent maximum possible effect versus time curve were 1,770 +/- 24 and 1,471 +/- 50% h for 0.5% amitriptyline and bupivacaine with epinephrine, respectively, whereas this value was 2,836 +/- 62% h for the coinjected 0.25% amitriptyline, 0.25% bupivacaine, and epinephrine admixture.     

Stereoselectivity of Bupivacaine in Local Anesthetic-sensitive Ion Channels of Peripheral Nerve

Background: The local anesthetic bupivacaine exists in two stereoisomeric forms, R(+)- and S(-)-bupivacaine. Because of its lower cardiac and central nervous system toxicity, attempts were made recently to introduce S(-)-bupivacaine into clinical anesthesia. We investigated stereoselective actions of R(+)- and S(-)-bupivacaine toward two local anesthetic-sensitive ion channels in peripheral nerve, the Na+ and the flicker K+ channel.

Methods: In patch-clamp experiments on enzymatically demyelinated peripheral amphibian nerve fibers, Na+ and flicker K+ channels were investigated in outside-out patches. Half-maximum inhibiting concentrations (IC50) were determined. For the flicker K+ channel, simultaneous block by R(+)-bupivacaine and S(-)-bupivacaine was analyzed for competition and association (k1) and dissociation rate constants (k-1) were determined.

Results: Both channels were reversibly blocked by R(+)- and S(-)-bupivacaine. The IC50 values (+/-SEM) for tonic Na+ channel block were 29 +/- 3 [mu]M and 44 +/- 3 [mu]M, respectively. IC50 values for flicker K+ channel block were 0.15 +/- 0.02 [mu]M and 11 +/- 1 [mu]M, respectively, resulting in a high stereopotency ratio (+/-) of 73. Simultaneously applied enantiomers competed for a single binding site. Rate constants k1 and k-1 were 0.83 +/- 0.13 x 106 M-1 [middle dot] s-1 and 0.13 +/- 0.03 s-1, respectively, for R(+)-bupivacaine and 1.90 +/- 0.20 x 106 M-1 [middle dot] s-1 and 8.3 +/- 1.0 s-1, respectively, for S(-)-bupivacaine.     

Tissue injury from tricyclic antidepressants used as local anesthetics

Neurotoxicity has been reported with tricyclic antidepressants (TCAs) used as local anesthetics. We examined the hypothesis that TCAs cause tissue injury, particularly myotoxicity, as occurs with many local anesthetics. Animals were given sciatic nerve injections with 0-80 mM doxepin, amitriptyline, or bupivacaine (1.5 mL for histological studies, 0.3 mL for neurobehavioral studies). Four days after injection, the TCAs caused ischemic tissue injury. Subcutaneous tissue showed expansion and hardening, with hemorrhage and adhesion to overlying skin. Muscle was diffusely pale. Histopathology showed coagulative necrosis of muscle and surrounding soft tissues, with thrombus formation in vasculature near affected areas. These findings were much reduced with bupivacaine. TCA-injected and bupivacaine-injected animals also developed characteristic local anesthetic myotoxicity. Amitriptyline proved less potent than bupivacaine as a local anesthetic: the concentrations required to provide 100 min of nerve block were 20 mM and 3 mM, respectively. Some animals receiving large concentrations of amitriptyline developed spontaneous recrudescence of nerve blockade or had irreversible nerve blockade, both of which may reflect nerve injury. Neither finding occurred in animals injected with bupivacaine. TCAs do not appear to offer any advantages over conventional local anesthetics and do appear to risk substantially increased toxicity.     

Kvβ1.3 Reduces the Degree of Stereoselective Bupivacaine Block of Kv1.5 Channels

Background: Kv[beta]1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kv[beta]1.3 competes with them for a binding site at Kv1.5 channels.

Methods: Currents generated by the activation of Kv1.5 and Kv1.5 + Kv[beta]1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques.

Results: Block of Kv1.5, but not that produced on Kv1.5 + Kv[beta]1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 [mu]m (n = 15) and 39.8 +/- 8.2 [mu]m (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 [mu]m) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 [mu]m) in Kv1.5 + Kv[beta]1.3 channels. Therefore, the degree of stereoselectivity ([theta]) decreased from 9 to 4 in the presence of Kv[beta]1.3. The decrease in potency to block Kv1.5 + Kv[beta]1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kv[beta]1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kv[beta]1.3 binding) but not for L510A (part of Kv[beta]1.3 binding site).     

Phase Ia and Ib study of amitriptyline for ulnar nerve block in humans: side effects and efficacy

BACKGROUND: The antidepressant amitriptyline is used as an adjuvant in the treatment of chronic pain conditions. Among its many actions, this drug also blocks ion channels, such as Na channels. Preliminary animal studies suggested that amitripty-line would be a longer-lasting local anesthetic than bupivacaine, with potentially fewer side effects. Therefore, the authors investigated the adverse effects and effectiveness of this drug when given for ulnar nerve blockade in human volunteers. METHODS: After obtaining written institutional review board approval and informed consent, a typical phase Ia trial was conducted by administration to the ulnar nerve at the level of the wrist in an open-label, dose-escalating fashion. Amitripty-line hydrochloride, 4 ml, at concentrations of 5, 10, and 20 mM (n = 4-9/group) was used for each volunteer. If no major side effects and nerve block were encountered, comparison in a randomized, double-blinded trial of amitriptyline (20 mM) to placebo and bupivacaine (4 mM) (n = 4-9/group), was to follow. A blunt needle was used to grade the pain, and motor blockade was assessed by the Froment test. RESULTS: There was no significant statistical difference in terms of side effects (pain, swelling, erythema, and sedation) among any groups. The analgesic effects of 20 mM amitriptyline and 4 mm bupivacaine solution were significantly higher than those of the placebo solution. CONCLUSIONS: Because of the lack of evidence that amitripty-line provides better nerve blockade than current local anesthetics and the potential for neurotoxicity, its use for peripheral nerve blockade in humans seems limited.     

Halothane Inhibits Contraction and Action Potential Duration to a Greater Extent in Subendocardial than Subepicardial Myocytes from the Rat Left Ventricle

Background : Halothane inhibits the 4-aminopyridine-sensitive transient outward K+ current (Ito), which in many species, including humans, plays an important role in determining action potential duration. As Ito is greater in the ventricular subepicardium than subendocardium, halothane may have differential effects on action potential duration and, therefore, contraction in cells isolated from these two regions.

Methods : Myocytes were isolated from the subendocardium and subepicardium of the rat left ventricle. Myocytes from each region were electrically stimulated at 1 Hz to measure contractions and action potentials and exposed to 0.6 mm halothane (approximately 2 x minimum alveolar concentration50 for the rat) for 1 min. The time from the peak of the action potential to repolarization at 0 and -50 mV was measured to assess the effects of halothane on action potential duration.

Results : Halothane inhibited contraction to a significantly (P = 0.002) greater extent in subendocardial myocytes than in subepicardial myocytes: the amplitude of contraction during control conditions was 3.6 +/- 0.4 [mu]m and 3.2 +/- 0.7 [mu]m in subendocardial and subepicardial cells, respectively, and this was reduced to 1.1 +/- 0.2 [mu]m (29 +/- 2% of control, P < 0.0001, n = 10) and 1.4 +/- 0.3 [mu]m (46 +/- 3% of control, P = 0.007, n = 7), respectively, after a 1-min exposure to 0.6 mm halothane. Control action potential duration (at -50 mV) was 67 +/- 10 and 28 +/- 4 ms in subendocardial and subepicardial myocytes, respectively, and these values were reduced to 39 +/- 6 ms (58 +/- 3% of control, P < 0.001) and 20 +/- 3 ms (73 +/- 5% of control, P = 0.009) by halothane, respectively.     

Effect of bupivacaine and levobupivacaine on exocytotic norepinephrine release from rat atria

BACKGROUND: The cardiotoxic mechanism of local anesthetics may include interruption of cardiac sympathetic reflexes. The authors undertook this investigation to determine if clinically relevant concentrations of bupivacaine and levobupivacaine interfere with exocytotic norepinephrine release from cardiac sympathetic nerve endings. METHODS: Rat atria were prepared for measurements of twitch contractile force and [H]-norepinephrine release. After nerve endings were loaded with [H]-norepinephrine, the tissue was electrically stimulated in 5-min episodes during 10 10-min sampling periods. After each period, a sample of bath fluid was analyzed for radioactivity and [H]-norepinephrine release was expressed as a fraction of tissue counts. Atria were exposed to buffer alone during sampling periods 1 and 2 (S1 and S2). Control atria received saline (100 microl each, n = 6 atria) in S3-S10. Experimental groups (n = 6 per group) received either bupivacaine or levobupivacaine at concentrations (in microM) of 5 (S3-S4), 10 (S5-S6), 30 (S7-S8), and 100 (S9-S10). RESULTS: Bupivacaine and levobupivacaine decreased stimulation-evoked fractional [H]-norepinephrine release with inhibitory concentration 50% values of 5.1 +/- 0.5 and 6.1 +/- 1.3 microM. The inhibitory effect of both local anesthetics (approximately 70%) approached that of tetrodotoxin. Local anesthetics abolished the twitch contractions of atria with inhibitory concentration 50% values of 12.6 +/- 5.0 microM (bupivacaine) and 15.7 +/- 3.9 microM (levobupivacaine). In separate experiments, tetrodotoxin inhibited twitch contractile force by only 30%. CONCLUSIONS: The results indicate that clinically relevant cardiotoxic concentrations of bupivacaine and levobupivacaine markedly depress cardiac sympathetic neurotransmission. A possible mechanism of local anesthetics in reducing evoked norepinephrine release from sympathetic endings is blockade of tetrodotoxin-sensitive fast sodium channels.     

Effect of Bupivacaine and Levobupivacaine on Exocytotic Norepinephrine Release from Rat Atria

Background: The cardiotoxic mechanism of local anesthetics may include interruption of cardiac sympathetic reflexes. The authors undertook this investigation to determine if clinically relevant concentrations of bupivacaine and levobupivacaine interfere with exocytotic norepinephrine release from cardiac sympathetic nerve endings.

Methods: Rat atria were prepared for measurements of twitch contractile force and 3[H]-norepinephrine release. After nerve endings were loaded with 3[H]-norepinephrine, the tissue was electrically stimulated in 5-min episodes during 10 10-min sampling periods. After each period, a sample of bath fluid was analyzed for radioactivity and 3[H]-norepinephrine release was expressed as a fraction of tissue counts. Atria were exposed to buffer alone during sampling periods 1 and 2 (S1 and S2). Control atria received saline (100 [mu]l each, n = 6 atria) in S3-S10. Experimental groups (n = 6 per group) received either bupivacaine or levobupivacaine at concentrations (in [mu]M) of 5 (S3-S4), 10 (S5-S6), 30 (S7-S8), and 100 (S9-S10).

Results: Bupivacaine and levobupivacaine decreased stimulation-evoked fractional 3[H]-norepinephrine release with inhibitory concentration 50% values of 5.1 +/- 0.5 and 6.1 +/- 1.3 [mu]m. The inhibitory effect of both local anesthetics (~70%) approached that of tetrodotoxin. Local anesthetics abolished the twitch contractions of atria with inhibitory concentration 50% values of 12.6 +/- 5.0 [mu]m (bupivacaine) and 15.7 +/- 3.9 [mu]m (levobupivacaine). In separate experiments, tetrodotoxin inhibited twitch contractile force by only 30%.     

Ephedrine blocks rat sciatic nerve in vivo and sodium channels in vitro

BACKGROUND: The sympathomimetic drug ephedrine has been used intrathecally as the sole local anesthetic for labor and delivery. Because ephedrine may be a useful adjuvant to local anesthetics, the authors investigated the local anesthetic properties of ephedrine in a rat sciatic nerve block model and the underlying mechanism in cultured cells stably expressing Na channels. METHODS: After approval of the animal protocol, the sciatic nerves of anesthetized rats were exposed by lateral incision of the thighs, 0.2 ml ephedrine at 0.25, 1, 2.5, or 5% and/or bupivacaine at 0.125% was injected, and the wound was closed. Motor and sensory/nociceptive functions were evaluated by the force achieved by pushing against a balance and the reaction to pinch, respectively. The whole cell configuration of the patch clamp technique was used to record Na currents from human embryonal kidney cells stably transfected with Nav1.4 channels. RESULTS: The nociception blockade was significantly longer than the motor blockade at test doses of 2.5 and 5% of ephedrine, or when 1% ephedrine was combined with 0.125% bupivacaine (analysis of variance with repeated measures, P < 0.001, n = 8/group). In vitro, the 50% inhibitory concentrations of ephedrine at -150 and -60 mV were 1,043 +/- 70 and 473 +/- 13 mum, respectively. High-frequency stimulation revealed a use-dependent block of 18%, similar to most local anesthetics. CONCLUSIONS: Because ephedrine's properties are at least partly due to Na channel blockade, detailed histopathologic investigations are justified to determine the potential of ephedrine as an adjuvant to clinically used local anesthetics.