Biomedical subjects

G K Wang

Publications and source records attributed to G K Wang.

At least 19 recordsLinked to original sources

PubMed2005

State-dependent block of human cardiac hNav1.5 sodium channels by propafenone.

State-dependent blockade of human cardiac hNav1.5 sodium channels by propafenone was studied using whole-cell patch clamp techniques. Both a direct investigation using cells with inactivation-deficient sodium channels and an algorithmic approach used on cells with wild-type channels revealed a rapid binding of propafenone to the open state. This occurs approximately 4000 and 700 times faster than the binding to the resting and inactivated state, respectively. An established mathematical "gating" model was modified to represent the experimental data.

Anti-Arrhythmia Agents↗

PubMed2005

The signaling network of tumor invasion.

The ability of a cell to invade its surroundings is an important hallmark of malignant tumors and results from aberrant cell signaling mechanisms. The signal transduction that leads to tumor invasion can be broken down into major pathways. Even though the pathway systems are distinct in themselves, none of these pathways operate independently when it comes to transmitting signals that culminate in an invasive phenotype. That is, the malignant change in one receptor not only leads to malignant changes directly downstream but can also affect the molecules of many other pathways. Three major pathway systems involved in tumor invasion are discussed in this review: the integrin system, the insulin-like growth factor system, and the Rho family GTPases. Here we see that although the individual signaling systems can each contribute to invasion, each system is networked to others and should not be considered isolated. Each system is first reviewed as independent contributors to an invasive phenotype and then discussed in the context of interacting pathways that collectively result in tumor invasion.

Animals↗

PubMed2004

Interactions of local anesthetics with voltage-gated Na+ channels.

Voltage-gated Na+ channels are dynamic transmembrane proteins responsible for the rising phase of the action potential in excitable membranes. Local anesthetics (LAs) and structurally related antiarrhythmic and anticonvulsant compounds target specific sites in voltage-gated Na+ channels to block Na+ currents, thus reducing excitability in neuronal, cardiac, or central nervous tissue. A high-affinity LA block is produced by binding to open and inactivated states of Na+ channels rather than to resting states and suggests a binding site that converts from a low- to a high-affinity conformation during gating. Recent findings using site-directed mutagenesis suggest that multiple S6 segments together form an LA binding site within the Na+ channel. While the selectivity filter may form the more extracellular-located part of this binding site, the role of the fast inactivation gate in LA binding has not yet been resolved. The receptor of the neurotoxin batrachotoxin (BTX) is adjacent to or even overlaps with the LA binding site. The close proximity of the LA and BTX binding sites to residues critical for inactivation, together with gating transitions through S6 segments, might explain the strong impact of LAs and BTX on inactivation of voltage-gated Na+ channels and might help elucidate the mechanisms underlying voltage- and frequency-dependent LA block.

Anesthetics, Local↗

PubMed2003

Methanethiosulfonate-modification alters local anesthetic block in rNav1.4 cysteine-substituted mutants S1276C and L1280C.

We previously showed that lysine substitutions at two residues in segment 6 of domain 3 in voltage-gated Na(+) channel rNav1.4 (S1276K, L1280K) reduced steady-state inactivated local anesthetic block. Here we studied cysteine substitutions at the same residues (S1276C, L1280C). We used whole-cell recordings to determine local anesthetic block (100 microM bupivacaine) before and after cysteine modification with 1.5 mM 2-aminoethyl methanethiosulfonate (MTSEA). Compared with rNav1.4, steady-state resting bupivacaine block at -180 mV was increased in S1276C, while inactivated block at -50 mV was not different in the mutants. After application of MTSEA at -160 mV, rNav1.4 showed enhanced bupivacaine block and a negative shift in V(1/2) of the bupivacaine affinity curve, while L1280C and S1276C showed a decrease in inactivated bupivacaine block after MTSEA. Application of MTSEA at 0 mV produced similar results in rNav1.4 and L1280C, but an opposite effect in S1276C, i.e., enhancement of bupivacaine block, with a large negative shift in V(1/2) of the bupivacaine affinity curve similar to that found in rNav1.4. We conclude that 1) MTSEA modification of 1276C or 1280C decreases inactivated bupivacaine block similar to that found in L1280K and S1276K, 2) residue 1276C is only accessible to MTS-modification in the resting state, and 3) MTSEA may modify a native cysteine in rNav1.4 that produces an allosteric, indirect effect on bupivacaine affinity.

Anesthetics, Local↗

PubMed2002

Modifications of human cardiac sodium channel gating by UVA light.

Voltage-gated Na(+) channels are membrane proteins responsible for the generation of action potentials. In this report we demonstrate that UVA light elicits gating changes of human cardiac Na+ channels. First, UVA irradiation hampers the fast inactivation of cardiac Nav1.5 Na(+) channels expressed in HEK293t cells. A maintained current becomes conspicuous during depolarization and reaches its maximal quasi steady-state level within 5-7 min. Second, the activation time course is slowed by UVA light; modification of the activation gating by UVA irradiation continues for 20 min without reaching steady state. Third, along with the slowed activation time course, the peak current is reduced progressively. Most Na(+) currents are eliminated during 20 min of UVA irradiation. Fourth, UVA light increases the holding current nonlinearly; this phenomenon is slow at first but abruptly fast after 20 min. Other skeletal muscle Nav1.4 isoforms and native neuronal Na(+) channels in rat GH(3) cells are likewise sensitive to UVA irradiation. Interestingly, a reactive oxygen metabolite (hydrogen peroxide at 1.5%) and an oxidant (chloramine-T at 0.5 mM) affect Na(+) channel gating similarly, but not identically, to UVA. These results together suggest that UVA modification of Na(+) channel gating is likely mediated via multiple reactive oxygen metabolites. The potential link between oxidative stress and the impaired Na(+) channel gating may provide valuable clues for ischemia/reperfusion injury in heart and in CNS.

Animals↗

PubMed2001

Regulation of the cycling of timeless (tim) RNA.

Circadian rhythms in Drosophila depend upon expression of the timeless (tim) and period (per) genes, which encode interacting components of the endogenous clock. These two clock genes show a robust circadian oscillation in transcription rate as well as RNA and protein levels. Transcriptional activation of both genes requires the basic helix-loop-helix (bHLH) PAS transcription factors dCLOCK (dCLK) and CYCLE (CYC), which bind E-box elements. We investigated the role of E-box elements in regulating behavioral rhythmicity and tim gene expression. We show that mutation of the upstream E-box in the tim gene prevents the rescue by tim cDNA sequences of the arrhythmic tim(01) phenotype. RNA encoded by this mutated tim transgene fails to cycle and is expressed at low levels. While a tim transgene carrying a wild-type E-box restores behavioral rhythms, tim RNA levels are intermediate to those of the mutant E-box transgenic lines and wild type, and do not display high amplitude cycling. On the other hand, high-amplitude RNA cycling was consistently obtained with a tim transgene that contains genomic, rather than cDNA, sequences. To identify additional sequences that may be required for tim cycling, we investigated the role of an E-box in the first intron of the tim gene through cell culture experiments. In these experiments, the presence of this intron did not have any effect on the activation of tim transcription by dCLK/CYC. As the upstream E-box was implicated in activation by dCLK/CYC in cell culture, we assayed sequences containing this E-box for association with proteins in fly head extracts. These studies provide the first biochemical evidence for an in vivo complex containing dCLK and CYC that binds the tim upstream sequence and is detected at all times of day. Together, these data highlight molecular mechanisms that are critical for behavioral rhythms.

ARNTL Transcription Factors↗

PubMed2001

Single point mutations affect fatty acid block of human myocardial sodium channel alpha subunit Na+ channels.

Suppression of cardiac voltage-gated Na(+) currents is probably one of the important factors for the cardioprotective effects of the n-3 polyunsaturated fatty acids (PUFAs) against lethal arrhythmias. The alpha subunit of the human cardiac Na(+) channel (hH1(alpha)) and its mutants were expressed in human embryonic kidney (HEK293t) cells. The effects of single amino acid point mutations on fatty acid-induced inhibition of the hH1(alpha) Na(+) current (I(Na)) were assessed. Eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced I(Na) in HEK293t cells expressing the wild type, Y1767K, and F1760K of hH1(alpha) Na(+) channels. The inhibition was voltage and concentration-dependent with a significant hyperpolarizing shift of the steady state of I(Na). In contrast, the mutant N406K was significantly less sensitive to the inhibitory effect of EPA. The values of the shift at 1, 5, and 10 microM EPA were significantly smaller for N406K than for the wild type. Coexpression of the beta(1) subunit and N406K further decreased the inhibitory effects of EPA on I(Na) in HEK293t cells. In addition, EPA produced a smaller hyperpolarizing shift of the V(1/2) of the steady-state inactivation in HEK293t cells coexpressing the beta(1) subunit and N406K. These results demonstrate that substitution of asparagine with lysine at the site of 406 in the domain-1-segment-6 region (D1-S6) significantly decreased the inhibitory effect of PUFAs on I(Na), and coexpression with beta(1) decreased this effect even more. Therefore, asparagine at the 406 site in hH1(alpha) may be important for the inhibition by the PUFAs of cardiac voltage-gated Na(+) currents, which play a significant role in the antiarrhythmic actions of PUFAs.

Animals↗

PubMed2001

Point mutations in alpha-subunit of human cardiac Na+ channels alter Na+ current kinetics.

Dietary polyunsaturated fatty acids (PUFAs) prevent ischemia-induced fatal cardiac arrhythmias in animals and probably in humans. This action results from inhibition of ion currents for Na+, Ca2+, and possibly other ions. To extend understanding of this protection we are seeking a possible binding site for the PUFAs on the alpha-subunit of the human cardiac Na+ channel, hH1alpha, transiently expressed in HEK293t cells. Three mutated single amino acid substitutions with lysine were made in the alpha-subunit at Domain 4-Segment 6 (D4-S6) for F1760, Y1767 and at D1-S6 for N406. These are in the putative sites of binding of local anesthetics and batrachotoxin, respectively. The mutants F1760K, Y1767K, and N406K, separately and to different extents, affected the current density, the steady-state inactivation potential, accelerated inactivation, delayed recovery from inactivation, and affected voltage-dependent block, but did not affect activation of the hH1alpha. It is essential to learn that single point mutations in D1-S6 and D4-S6 alone significantly modify the kinetics of human cardiac hH1alpha Na+ currents. The effects of PUFAs on these mutant channels will be the subject of subsequent reports.

Amino Acids↗

PubMed2001

Residue-specific effects on slow inactivation at V787 in D2-S6 of Na(v)1.4 sodium channels.

Slow inactivation in voltage-gated sodium channels (NaChs) occurs in response to depolarizations of seconds to minutes and is thought to play an important role in regulating membrane excitability and action potential firing patterns. However, the molecular mechanisms of slow inactivation are not well understood. To test the hypothesis that transmembrane segment 6 of domain 2 (D2-S6) plays a role in NaCh slow inactivation, we substituted different amino acids at position V787 (valine) in D2-S6 of rat skeletal muscle NaCh mu(1) (Na(v)1.4). Whole-cell recordings from transiently expressed NaChs in HEK cells were used to study and compare slow inactivation phenotypes between mutants and wild type. V787K (lysine substitution) showed a marked enhancement of slow inactivation. V787K enters the slow-inactivated state approximately 100x faster than wild type (tau(1) approximately 30 ms vs. approximately 3 s), and occurs at much more hyperpolarized potentials than wild type (V(1/2) of s(infinity) curve approximately -130 mV vs. approximately -75 mV). V787C (cysteine substitution) showed a resistance to slow inactivation, i.e., opposite to that of V787K. Entry into the slow inactivation state in V787C was slower (tau(1) approximately 5 s), less complete, and less voltage-dependent (V(1/2) of s(infinity) curve approximately -50 mV) than in wild type. Application of the cysteine modification agent methanethiosulfonate ethylammonium (MTSEA) to V787C demonstrated that the 787 position undergoes a relative change in molecular conformation that is associated with the slow inactivation state. Our results suggest that the V787 position in Na(v)1.4 plays an important role in slow inactivation gating and that molecular rearrangement occurs at or near residue V787 in D2-S6 during NaCh slow inactivation.

Amino Acid Motifs↗

PubMed2001

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.

Amitriptyline↗

PubMed2001

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.

Anesthetics, Local↗

PubMed2001

Disparate role of Na(+) channel D2-S6 residues in batrachotoxin and local anesthetic action.

Batrachotoxin (BTX) stabilizes the voltage-gated Na(+) channels in their open conformation, whereas local anesthetics (LAs) block Na(+) conductance. Site-directed mutagenesis has identified clusters of common residues at D1-S6, D3-S6, and D4-S6 segments within the alpha-subunit Na(+) channel that are critical for binding of these two types of ligands. In this report, we address whether segment D2-S6 is similarly involved in both BTX and LA actions. Thirteen amino acid positions from G783 to L795 of the rat skeletal muscle Na(+) channel ((mu)1/Skm1) were individually substituted with a lysine residue. Four mutants (N784K, L785K, V787K, and L788K) expressed sufficient Na(+) currents for further studies. Activation and/or inactivation gating was altered in mutant channels; in particular, mu1-V787K displays enhanced slow inactivation and exhibited use-dependent inhibition of peak Na(+) currents during repetitive pulses. Two of these four mutants, (mu)1-N784K and (mu)1-L788K, were completely resistant to 5 microM BTX. This BTX-resistant phenotype could be caused by structural perturbations induced by a lysine point mutation in the D2-S6 segment. However, these two BTX-resistant mutants remained quite sensitive to bupivacaine block with affinity for inactivated Na(+) channels (K(I)) of approximately 10 microM or less, which suggests that (mu)1-N784 and (mu)1-L788 residues are not in close proximity to the LA binding site.

Amino Acid Substitution↗

PubMed2001

Redox properties of local anesthetics: A structural determinant of closed channel blockers in BTX-modified Na+ channels.

Single channel analyses and macroscopic current measurements have shown that benzocaine is a predominantly closed channel blocker in BTX-modified Na+ channels; cocaine is an open channel blocker; and tetracaine, a dual channel blocker (Wang & Wang, 1994; Wang et al., 1994). The reason for such a selective state-dependent block by local anesthetics in BTX-modified Na+ channels is not clear. We assessed the redox properties of tetracaine, benzocaine, cocaine, and various derivatives by their ability to donate electrons to radical intermediates of eosin dye excited by visible light. Electron-donor properties of the drugs were previously proposed to be involved in Na+ channel blockade (Marinov, 1991). Our results provide evidence that redox properties of tetracaine, benzocaine, and their homologs correlate with their ability to enhance Na+ channel inactivation in BTX-modified Na+ channels. This correlation may be explained in terms of the previously proposed redox model of ion channels.

Anesthetics, Local↗

PubMed2001

A phenylalanine residue at segment D3-S6 in Nav1.4 voltage-gated Na(+) channels is critical for pyrethroid action.

Mammalian voltage-gated Na(+) channels were less sensitive to pyrethroids than their insect counterparts by 2 to 3 orders of magnitude. Deltamethrin at 10 microM elicited weak gating changes in rat skeletal muscle alpha-subunit Na(+) channels (Nav1.4) after > 30 min of perfusion. About 10% of the peak current was maintained during the 8-ms, +50-mV pulse and, upon repolarization to -140 mV, the amplitude of the slow tail current corresponded to less than 3% of total Na(+) channels modified by deltamethrin. A background mutation, Nav1.4-I687M (within D2:S4-S5 cytoplasmic linker), enhanced the deltamethrin-induced maintained current by approximately 2.5-fold, whereas Nav1.4-I687T, a homologous superkdr mutation, reduced it by approximately 2-fold. Repetitive pulses at 2 Hz further augmented the effects of deltamethrin on Nav1.4-I687M mutant channels so that approximately 75% of peak currents were maintained. A second mutation, Nav1.4-I687M/F1278I at the middle of D3-S6, rendered the channel greatly resistant to deltamethrin. This double mutant channel remained sensitive to batrachotoxin (BTX), even though nearby residues S1276 and L1280 were critical for BTX action. We hypothesize that the deltamethrin receptor and the BTX receptor are situated at the middle but opposite surface of the D3-S6 alpha-helical structure. Another mutant, Nav1.4-I687M/N784K, exhibited a partial deltamethrin-resistant phenotype but was completely resistant to BTX. Consistent with the BTX-resistant phenotype of N784K and the known adjacent kdr mutation at position L785F, deltamethrin and BTX were probably situated next to each other upon binding at D2-S6. Evidently, distinct residues from multiple S6 segments were critical for deltamethrin and BTX actions.

Amino Acid Motifs↗

PubMed2000

Modification of wild-type and batrachotoxin-resistant muscle mu1 Na+ channels by veratridine.

Biochemical evidence indicates that veratridine (VTD) and batrachotoxin (BTX) share a common binding site in Na+ channels. Under whole-cell voltage-clamp conditions, we examined this single receptor hypothesis by studying the VTD phenotype in BTX-resistant muscle Na+ channels, microl-I433K, N434K, L437K, F1579K, and N1584K. Derived from point mutations at segments D1-S6 and D4-S6, these mutant Na+ channels are resistant to 5 microM BTX when expressed in human embryonic kidney cells. In contrast to the wild-type phenotype, VTD at 200 microM elicits little or no maintained current during a test pulse at +50 mV, and little or no "tail" current after the test pulse in all BTX-resistant mutant channels. Paradoxically, VTD retains its ability to inhibit the peak Na+ current in BTX-resistant mutant Na+ channels. To explain these mutant phenotypes, we propose a two-step binding reaction scheme. An initial VTD-binding interaction with the Na+ channel results in the inhibition of peak current amplitude, and a second binding reaction results in the trapping of VTD within the D1-S6 and D4-S6 domain interface. The failure of BTX-resistant mutant Na+ channels to trap VTD suggests that segments of D1-S6 and D4-S6 form a common receptor for VTD and BTX.

Batrachotoxins↗

PubMed2000

Residues in Na(+) channel D3-S6 segment modulate both batrachotoxin and local anesthetic affinities.

Batrachotoxin (BTX) alters the gating of voltage-gated Na(+) channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na(+) conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na(+) channel alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display approximately 10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues mu1-S1276 and mu1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na(+) channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.

Amino Acid Sequence↗

PubMed2000

A point mutation in domain 4-segment 6 of the skeletal muscle sodium channel produces an atypical inactivation state.

We compared wild-type rat skeletal muscle NaChs (micro1) and a mutant NaCh (Y1586K) that has a single amino acid substitution, lysine (K) for tyrosine (Y), at position 1586 in the S6 transmembrane segment of domain 4. In Y1586K, macroscopic current decay is faster, the V(1/2) of the activation curve is shifted in the depolarized direction, and the fast-inactivation curve is less steep compared with mu1. After an 8-ms depolarization pulse, Y1586K recovers from inactivation much more slowly than mu1. The recovery is double exponential, suggesting recovery from two inactivation states. Varying the depolarization protocols isolates entry into an additional, "atypical" inactivation state in Y1586K that is distinct from typical fast or slow inactivation. Substitution of positively charged arginine (R) at Y1586 produces an inactivation phenotype similar to that of Y1586K. Substitution by negatively charged aspartic acid (D) or uncharged alanine (A) at Y1586 produces an inactivation phenotype similar to mu1. Our results suggest that the positive charge of lysine (K) produces the atypical inactivation state in Y1586K. We propose that a conformational change during depolarization alters the relative position of the 1586K residue in the D4-S6 segment and that atypical inactivation in Y1586K occurs via an electrostatic interaction in or near the inner pore region.

Animals↗

PubMed2000

Spinal tonicaine: potency and differential blockade of sensory and motor functions.

BACKGROUND: Long-acting local anesthetics are beneficial for the management of postoperative pain and chronic pain. The authors recently reported that a single injection of N-beta-phenylethyl-lidocaine (tonicaine), a quaternary lidocaine derivative, effectively blocks rat sciatic nerve function four to nine times longer than lidocaine, with a predominance of sensory versusmotor blockade. The purposes of this study were to measure directly the potency of this charged drug by internal perfusion of cultured neuronal cells, and to evaluate the differential blockade of sensory versus motor function via spinal route in rats. METHODS: The tonic and additional use-dependent blockade of Na+ currents by internal tonicaine was assayed in cultured GH3 cells during whole cell voltage-clamp conditions. In addition, tonicaine was injected into the intrathecal space of rats at intervertebral space L4-L5, and the proprioceptive, motor, and sensory functions, and tissue integrity, subsequently were evaluated. RESULTS: Internal application of tonicaine in GH3 cells revealed that it was approximately 80 times more potent in blocking Na+ currents than was externally applied lidocaine. In vivotesting in a rat neuraxial anesthesia model showed that tonicaine at 0.5 mm produced blockade that lasted much longer than that produced by bupivacaine even at approximately a 55 times higher concentration (28.8 mm). Tonicaine spinal block also produced a longer duration of sensory than motor blockade (112.5 +/- 16.3 min vs. 45.8 +/- 7.1 min). Evidence of neurotoxicity was seen at a concentration of 1.0 mm. CONCLUSION: In vitro testing shows that tonicaine displays a higher affinity for the local anesthetic binding site than does lidocaine; in vivotesting indicates that tonicaine elicits sensory blockade of a duration significantly longer than that elicited by bupivacaine. Tonicaine, however, has a narrow therapeutic index, with substantial neurotoxicity at 1 mm in rats, and may have limited clinical value.

Analysis of Variance↗