The effect of caffeine and tetracaine on the time course of potassium contractures of single muscle fibres.
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1. The time course of potassium contractures can be significantly prolonged by low concentrations of caffeine. 2. This effect of caffeine is not due to impairment of the fibre relaxing system. 3. Under conditions were contractile repriming is delayed (low temperature) an extra amount of activator can be released by caffeine, in addition to that released by potassium. 4. The source of this extra amount of activator is intracellular since its release can be shown in a O calcium EGTA medium. 5. Local anaesthetics, tetracaine, and to a lesser extent procaine, affect the release of contractile activator, without impairing the contractile machinery itself. 6. The results of the present paper support the view that the time course of potassium contracture is controlled by a membrane mechanism which is activated upon depolarization and later inactivates with time. 7. The effect of caffeine and local anaesthetics can be explained by assuming that the former prolongs the inactivation time course while the latter shortens it. (+info)
Effects of tetracaine on displacement currents and contraction of frog skeletal muscle.
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The kinetics of mechanical activation of intact fibres were examined with a voltage-clamp technique. Tetracaine (2 mM) increases fifteen- to seventyfold the time required to produce a just visible contraction by cell membrane depolarization. 2. Displacement currents thought to be related to contractile activation remain in 2 mM tetracaine. Their characteristics are virtually identical to those found in the absence of the drug. Displacement currents also remain in fibres immobilized by treatment with 10 mM formaldehyde. 3. Despite its effect on contraction of intact fibres, tetracaine does not diminish contraction tension when Ca is applied directly to the contractile proteins of 'skinned' muscle fibres. The sensitivity of the myofilaments to Ca2+ also remains undiminished. 4. When acting on intact fibres the drug must therefore inhibit Ca2+-release from the sarcoplasmic reticulum. It is estimated that 2 mM tetracaine diminishes more than tenfold the capacity for Ca2+-release in response to cell membrane depolarization.5. If muscle displacement currents represent events linking depolarization to Ca2+-release, then tetracaine must be able to block the release without affecting the potential-sensing portion of the release regulating mechanism. 6. Further experiments on skinned fibres show that tetracaine blocks or greatly diminishes caffeine contractions, but that Cl-induced contractions of normal amplitude are still possible. (+info)
Differential effects of tetracaine on delayed potassium channels and displacement currents in frog skeletal muscle.
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1. Delayed K+-currents and displacement currents were studied with a voltage-clamp technique. 2. In normal fibres, the conductance of the delayed channel grows e-fold per 3 millivolts at sufficiently negative potentials and reaches a limiting value of 2-10 m-mho/cm2 (mean 5-8 m-mho/cm2) at positive potentials. Adding tetracaine (2 mM) reduces the limiting conductance, shifts the voltage-dependence of the delayed channel to +25 mV more positive potentials and slows the kinetics fourfold. 3. By contrast, the displacement currents are virtually unaltered by 2 mM tetracaine. Their voltage-dependence is shifted by less than 5 mV and their kinetics are unaffected. 4. Tetraethylammonium ions (TEA) are known to slow the kinetics of delayed K+-channels fivefold but fail, like tetracaine, to change the kinetics of the displacement currents. 5. Both tetracaine and TEA have thus large effects on the 'gating' of the delayed channel, yet little or none on the displacement currents. This suggests that the displacement currents in skeletal muscle are for the most part unrelated to the opening and closing of delayed channels. It is estimated that 'gating' the delayed channel in muscle may require no more than 1 or 2% of the observed charge displacement. (+info)
Presence of functional sarcoplasmic reticulum in the developing heart and its confinement to chamber myocardium.
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During development fast-contracting atrial and ventricular chambers develop from a peristaltic-contracting heart tube. This study addresses the question of whether chamber formation is paralleled by a matching expression of the sarcoplasmic reticulum (SR) Ca(2+) pump. We studied indo-1 Ca(2+) transients elicited by field stimulation of linear heart tube stages and of explants from atria and outflow tracts of the prototypical preseptational E13 rat heart. Ca(2+) transients of H/H 11+ chicken hearts, which constitute the prototypic linear heart tube stage, were sensitive to verapamil only, indicating a minor contribution of Ca(2+)-triggered SR Ca(2+) release. Outflow tract transients displayed sensitivity to the inhibitors similar to that of the linear heart tube stages. Atrial Ca(2+) transients disappeared upon addition of ryanodine, tetracaine, or verapamil, indicating the presence of Ca(2+)-triggered SR Ca(2+) release. Quantitative radioactive in situ hybridization on sections of E13 rat hearts showed approximately 10-fold higher SERCA2a mRNA levels in the atria compared to nonmyocardial tissue and approximately 5-fold higher expression in compact ventricular myocardium. The myocardium of atrioventricular canal, outflow tract, inner curvature, and ventricular trabecules displayed weak expression. Immunohistochemistry on sections of rat and human embryos showed a similar pattern. The significance of these findings is threefold. (i) A functional SR is present long before birth. (ii) SR development is concomitant with cardiac chamber development, explaining regional differences in cardiac function. (iii) The pattern of SERCA2a expression underscores a manner of chamber development by differentiation at the outer curvature, rather than by segmentation of the linear heart tube. (+info)
Binding properties of agonists and antagonists to distinct allosteric states of the nicotinic acetylcholine receptor are incompatible with a concerted model.
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Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR. (+info)
Does topical amethocaine gel reduce the pain of venepuncture in newborn infants? A randomised double blind controlled trial.
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BACKGROUND: Topical amethocaine provides effective pain relief during venepuncture in children, and has been shown to have a local anaesthetic action in the newborn. AIM: To investigate the effect of topical amethocaine on the pain of venepuncture in the newborn. DESIGN: Randomised double blind placebo controlled trial. SUBJECTS: Forty newborn infants, gestation 27-41 weeks (median 33), age 2-17 days (median 7), undergoing routine venepuncture. METHOD: A 1.5 g portion of 4% (w/w) amethocaine gel (Ametop; Smith and Nephew, Hull, UK) or placebo was applied to the skin under occlusion for one hour, then wiped away. Venepuncture was performed five minutes later. Facial reaction and cry were recorded on videotape. Pain was assessed using a validated adaptation of the neonatal facial coding system. Five features were scored at one second intervals for five seconds before and after venepuncture. No or minimal pain was defined as a cumulative score of below 10 (out of 25) in the five seconds after needle insertion. Each author scored the tapes independently. RESULTS: There was close agreement on scoring of the tapes. One infant was excluded because of restlessness before the venepuncture. Of 19 amethocaine treated infants, 16 (84%) showed little or no pain compared with six of 20 (30%) in the placebo group (p = 0.001). The median cumulative neonatal facial coding system score over five seconds after needle insertion was 3 compared with 16 in the placebo group (p = 0.001). Of the 19 amethocaine treated infants, 15 (79%) did not cry compared with five of 20 (25%) placebo treated infants (p = 0.001). No local reaction to amethocaine was seen. CONCLUSION: Topical amethocaine provides effective pain relief during venepuncture in the newborn. (+info)
Topical amethocaine gel in the newborn infant: how soon does it work and how long does it last?
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AIM: To explore the time of onset and duration of action of topical amethocaine gel in the newborn infant. DESIGN: A randomised double blind placebo controlled trial. SUBJECTS: Thirty six infants were studied after 30 minutes application and 36 after 60 minutes application. A total of 56 infants (gestation 27-42 weeks, weight 0. 79-4.1 kg) were studied in the first two weeks after delivery. METHOD: 1.5 g amethocaine or placebo was applied to the dorsum of either foot, occluded, and then left for 30 or 60 minutes. Local anaesthesia was assessed by observing the cutaneous withdrawal response to graded nylon filaments (von Frey hairs). If there was a difference between feet in filament thickness required to elicit a response, the infant was studied in an identical manner at hourly intervals until the difference had disappeared. RESULTS: Evidence of local anaesthetic action of amethocaine was seen in 23 of 36 (64%) infants after 30 minutes and 26 of 36 (72%) infants after 60 minutes application (no significant difference). Evidence of local anaesthetic action was independent of gestation and order of testing. Amethocaine responders showed a significantly deeper anaesthetic action than placebo responders. The median duration of action was 1.5 hours (range 0.5-3.5) after 30 minutes and three hours (range 1-5) after 60 minutes (p<0.001). CONCLUSION: Topical amethocaine gel has a local anaesthetic action after 30 minutes application, but application for 60 minutes results in longer duration of action. (+info)
Regulation of a voltage-sensitive release mechanism by Ca(2+)-calmodulin-dependent kinase in cardiac myocytes.
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A role for Ca(2+)-calmodulin-dependent kinase (CamK) in regulation of the voltage-sensitive release mechanism (VSRM) was investigated in guinea pig ventricular myocytes. Voltage clamp was used to separate the VSRM from Ca(2+)-induced Ca(2+) release (CICR). VSRM contractions and Ca(2+) transients were absent in cells dialyzed with standard pipette solution but present when 2-5 microM calmodulin was included. Effects of calmodulin were blocked by KN-62 (CamK inhibitor), but not H-89, a protein kinase A (PKA) inhibitor. Ca(2+) current and caffeine contractures were not affected by calmodulin. Transient-voltage relations were bell-shaped without calmodulin, but they were sigmoidal and typical of the VSRM with calmodulin. Contractions with calmodulin exhibited inactivation typical of the VSRM. These contractions were inhibited by rapid application of 200 microM of tetracaine, but not 100 microM of Cd(2+), whereas CICR was inhibited by Cd(2+) but not tetracaine. In undialyzed myocytes (high-resistance microelectrodes), KN-62 or H-89 each reduced amplitudes of VSRM contractions by approximately 50%, but together they decreased VSRM contractions by 93%. Thus VSRM is facilitated by CamK or PKA, and both pathways regulate the VSRM in undialyzed cells. (+info)