Effects of tetracaine on sarcoplasmic calcium release in mammalian skeletal muscle fibres. (1/340)

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum communis muscle of rats. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions in the presence and absence of the local anaesthetic tetracaine. 2. Changes in intracellular calcium concentration ([Ca2+]i) were monitored using the calcium sensitive dyes antipyrylazo III and fura-2 and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. Tetracaine decreased the maximal attained [Ca2+]i and suppressed, in a dose-dependent manner, both the early peak and the steady level of Rrel in the voltage range examined. 3. The concentration dependence of the effects on the two kinetic components of Rrel were almost identical with a half-effective concentration (K50) of 70 and 71 microM and a Hill coefficient (nH) of 2.7 and 2.3 for the peak and the steady level, respectively. Furthermore, the drug did not alter the peak to steady level ratio up to a concentration (50 microM) that caused a 35 +/- 5 % reduction in calcium release. Higher concentrations did suppress the ratio but the degree of suppression was voltage independent. 4. Tetracaine (50 microM) neither influenced the total available intramembrane charge nor altered its membrane potential dependence. It shifted the transfer function, the normalized SR permeability versus normalized charge to the right, indicating that similar charge transfer caused a smaller increase in SR permeability. 5. To explore the site of action of tetracaine further the ryanodine receptor (RyR) calcium release channel of the SR was purified and reconstituted into planar lipid bilayers. The reconstituted channel had a conductance of 511 +/- 14 pS (n = 8) in symmetric 250 mM KCl that was not affected by tetracaine. Tetracaine decreased the open probability of the channel in a concentration-dependent manner with K50 = 68 microM and nH = 1.5. 6. These experiments show that tetracaine suppresses SR calcium release in enzymatic isolated mammalian skeletal muscle fibres. This effect is due, presumably, to the decreased open probability of the RyR in the presence of the drug. Since both the inactivating peak and the steady level of Rrel were equally affected by tetracaine, our observations suggest that there is a tight coupling between these kinetic components of SR calcium release in mammalian skeletal muscle.  (+info)

Interaction of bupivacaine and tetracaine with the sarcoplasmic reticulum Ca2+ release channel of skeletal and cardiac muscles. (2/340)

BACKGROUND: Although various local anesthetics can cause histologic damage to skeletal muscle when injected intramuscularly, bupivacaine appears to have an exceptionally high rate of myotoxicity. Research has suggested that an effect of bupivacaine on sarcoplasmic reticulum Ca2+ release is involved in its myotoxicity, but direct evidence is lacking. Furthermore, it is not known whether the toxicity depends on the unique chemical characteristics of bupivacaine and whether the toxicity is found only in skeletal muscle. METHODS: The authors studied the effects of bupivacaine and the similarly lipid-soluble local anesthetic, tetracaine, on the Ca2+ release channel-ryanodine receptor of sarcoplasmic reticulum in swine skeletal and cardiac muscle. [3H]Ryanodine binding was used to measure the activity of the Ca2+ release channel-ryanodine receptors in microsomes of both muscles. RESULTS: Bupivacaine enhanced (by two times at 5 mM) and inhibited (66% inhibition at 10 mM) [3H]ryanodine binding to skeletal muscle microsomes. In contrast, only inhibitory effects were observed with cardiac microsomes (about 3 mM for half-maximal inhibition). Tetracaine, which inhibits [3H]ryanodine binding to skeletal muscle microsomes, also inhibited [3H]ryanodine binding to cardiac muscle microsomes (half-maximal inhibition at 99 microM). CONCLUSIONS: Bupivacaine's ability to enhance Ca2+ release channel-ryanodine receptor activity of skeletal muscle sarcoplasmic reticulum most likely contributes to the myotoxicity of this local anesthetic. Thus, the pronounced myotoxicity of bupivacaine may be the result of this specific effect on Ca2+ release channel-ryanodine receptor superimposed on a nonspecific action on lipid bilayers to increase the Ca2+ permeability of sarcoplasmic reticulum membranes, an effect shared by all local anesthetics. The specific action of tetracaine to inhibit Ca2+ release channel-ryanodine receptor activity may in part counterbalance the nonspecific action, resulting in moderate myotoxicity.  (+info)

Topical anaesthesia of intact skin: liposome-encapsulated tetracaine vs EMLA. (3/340)

In this randomized, double-blind study, we have compared the ability of 5% liposome-encapsulated tetracaine (amethocaine) (LET) vs 5% eutectic mixture of local anaesthetics (EMLA) to produce local anaesthesia of intact skin in 40 healthy volunteers. Volunteers had both preparations applied to their forearms under an occlusive dressing for 1 h. Superficial anaesthesia was measured by a total of nine 1-mm pinpricks on each arm. Deeper anaesthesia was assessed by single insertion of a sterile 22-gauge needle to a depth of 3 mm and pain was reported on a visual analogue scale (VAS). If the volunteer perceived greater than four of the 1-mm pinpricks, the 3-mm insertion was not performed. Results showed that the number of pinpricks perceived was significantly less (P < 0.01) for LET (median 1.0; range 0-9) vs EMLA (1.5; 0-9). In volunteers who had deeper anaesthesia assessed, there was no significant difference (P = 0.065) in VAS scores for LET (mean 1.5 (SD 1.4); n = 34) vs EMLA (2.4 (2.1); n = 28). Overall anaesthetic effect, as ranked by all of the subjects, was significantly better for LET compared with EMLA (P = 0.024). We have demonstrated that when applied in equal volumes, 5% LET produced better superficial local anaesthesia than EMLA.  (+info)

Isolation and characterization of the yeast las21 mutants, which are sensitive to a local anestheticum, tetracaine. (4/340)

We isolated and characterized yeast mutants whose growth is sensitive to a local anestheticum tetracaine and, at the same time, temperature sensitive. These mutants were collectively called las mutants (local anestheticum sensitive). The las21 mutants were analyzed in this study. The wild type LAS21 gene was cloned by exploiting temperature sensitivity of the las21 mutants and we found that LAS21 encodes ORF YJL062w which has not been analyzed before. Las21p is putative membrane protein belonging to the major facilitator super family containing plural membrane spanning domains. Complete elimination of the LAS21 ORF did not kill the cells but made their growth temperature sensitive. Interestingly, the complete loss of the LAS21 gene canceled the sensitivity to tetracaine. The ability of the las21 mutants to grow at a higher temperature was recovered in the various media containing an osmotic stabilizer or salts. Furthermore, temperature sensitivity of the las21 mutants was partially suppressed by introduction of PKC1, encoding protein kinase C, on a high copy vector. We found some genetic interactions between LAS21 and Ras/cAMP cascade genes. These results suggest that LAS21 defines unknown pathway regulating the stress response of yeast.  (+info)

Photoaffinity labeling the torpedo nicotinic acetylcholine receptor with [(3)H]tetracaine, a nondesensitizing noncompetitive antagonist. (5/340)

Tetracaine (N,N-dimethylaminoethyl-4-butylaminobenzoate) and related N,N-dialkylaminoethyl substituted benzoic acid esters have been used to characterize the high-affinity binding site for aromatic amine noncompetitive antagonists in the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Tetracaine binds at equilibrium to a single site with a K(eq) value of 0.5 microM in the absence of agonist or presence of alpha-bungarotoxin and with a K(eq) value of 30 microM in the presence of agonist (i.e., for nAChR in the desensitized state). Preferential binding to nAChR in the absence of agonist is also seen for N,N-DEAE and N,N-diethylaminopropyl esters, both binding with 10-fold higher affinity in the absence of agonist than in the presence, and for the 4-ethoxybenzoic acid ester of N, N-diethylaminoethanol, but not for the 4-amino benzoate ester (procaine). Irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine resulted in covalent incorporation with similar efficiency into nAChR alpha, beta, gamma, and delta subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [(3)H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [(3)H]tetracaine-binding site. Within alpha subunit, >/=95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser(173) that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [(3)H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain.  (+info)

Identification of amino acids of the torpedo nicotinic acetylcholine receptor contributing to the binding site for the noncompetitive antagonist [(3)H]tetracaine. (6/340)

[(3)H]Tetracaine is a noncompetitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds with high affinity in the absence of cholinergic agonist (K(eq) = 0.5 microM) and weakly (K(eq) = 30 microM) in the presence of agonist (i.e., to nAChR in the desensitized state). In the absence of agonist, irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine results in specific photoincorporation of [(3)H]tetracaine into each nAChR subunit. In this report, we identify the amino acids of each nAChR subunit specifically photolabeled by [(3)H]tetracaine that contribute to the high-affinity binding site. Subunits isolated from nAChR-rich membranes photolabeled with [(3)H]tetracaine were subjected to enzymatic digestion, and peptides containing (3)H were purified by SDS-polyacrylamide gel electrophoresis followed by reversed phase HPLC. N-terminal sequence analysis of the isolated peptides demonstrated that [(3)H]tetracaine specifically labeled two sets of homologous hydrophobic residues (alphaLeu(251), betaLeu(257), gammaLeu(260), and deltaLeu(265); alphaVal(255) and deltaVal(269)) as well as alphaIle(247) and deltaAla(268) within the M2 hydrophobic segments of each subunit. The labeling of these residues establishes that the high-affinity [(3)H]tetracaine-binding site is located within the lumen of the closed ion channel and provides a definition of the surface of the M2 helices facing the channel lumen.  (+info)

Tetracaine can inhibit contractions initiated by a voltage-sensitive release mechanism in guinea-pig ventricular myocytes. (7/340)

1. Effects of tetracaine on membrane currents and cell shortening were measured with high resistance electrodes, single-electrode voltage clamp (switch clamp) and a video edge detector at 37 C in cardiac ventricular myocytes. 2. Sequential voltage steps from -65 mV to -40 and 0 mV were used to activate two mechanisms of excitation-contraction (EC) coupling separately. The step to -40 mV activated the voltage-sensitive release mechanism (VSRM); the step to 0 mV1 activated Ca2+-induced Ca2+ release (CICR) coupled to inward Ca2+ current (IL). 3. Exposure to 100-300 microM tetracaine inhibited VSRM contractions but not CICR contractions. Inhibition of VSRM contractions was independent of INa blockade. In contrast, 100 microM Cd2+ blocked IL and CICR contractions, but not VSRM contractions. Simultaneous application of both agents blocked both mechanisms of EC coupling. 4. Contraction-voltage relationships were sigmoidal when the VSRM was available. However, when the VSRM was inhibited with 100-300 microM tetracaine, contraction-voltage relationships became bell-shaped. The tetracaine-insensitive contractions were abolished by 0.1 microM ryanodine, indicating that they were dependent on release of SR Ca2+. 5. At a higher concentration (1 mM) tetracaine also inhibited IL and contractions triggered by IL; however, the time course of effects on IL and associated contractions were different than for VSRM contractions. 6. With continuous application of tetracaine, the VSRM remained inhibited although SR Ca2+ stores increased 4-fold as assessed with caffeine. CICR contractions were not inhibited and maximum amplitude of contraction was not reduced. 7. Rapid application of tetracaine just before and during test steps also inhibited VSRM contractions, but without significantly affecting sarcoplasmic reticulum (SR) Ca2+ stores or CICR contractions. Maximum amplitude of contraction was reduced. 8. Rapid application of tetracaine (100-300 microM) allows preferential inhibition of the VSRM and provides a pharmacological method to assess the contribution of the VSRM to EC coupling.  (+info)

Tetracaine gel vs EMLA cream for percutaneous anaesthesia in children. (8/340)

We have evaluated the anaesthetic effect of tetracaine gel 1 g, applied for 45 min, compared with EMLA cream 2 g, applied for 60 min, in a randomized, double-blind study in 60 children aged 3-15 yr. Venous cannulation was performed 15 min after removal of the EMLA cream (n = 20) and tetracaine gel (n = 20). Cannulation was performed up to 215 min after removal of the tetracaine gel in another 20 patients. Significantly lower pain scores were recorded by the children treated with tetracaine gel compared with EMLA cream (P < 0.02). Forty to 45% of children in the tetracaine groups reported no pain compared with only 10% in the EMLA group. Only minor adverse effects were observed. We conclude that tetracaine gel provided effective, rapid, long-lasting and safe local anaesthesia, and was significantly better than EMLA cream in reducing pain during venous cannulation in children using the recommended application periods for both formulations.  (+info)

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