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Biochimica Et Biophysica Acta Nov 1996The dynamics of structural changes in pea chloroplasts in the presence of 25-50 microM dibucaine or tetracaine has been examined using electron microscopy. The...
The dynamics of structural changes in pea chloroplasts in the presence of 25-50 microM dibucaine or tetracaine has been examined using electron microscopy. The light-induced uptake of anesthetic cations by thylakoids is attended by the appearance of local fusions of stroma-exposed thylakoid membranes. The first membrane protrusions and interthylakoid contacts are observed after 4 s illumination and they become numerous by 10 s. As a result, a network of anastomoses is formed which is maintained during at least 10 min. These effects are reversible in the dark and can be reproduced several times. The formation of membrane fusions is inhibited by the addition of protonophore. It is supposed that the energy-dependent uptake of protonated anesthetics by thylakoids leads to an increase in positive surface charge and thus a lateral pressure on the inner side of the thylakoid membrane. The appearance of membrane protrusions (crinkles) having the positive curvature of their inner surface may be considered as a way of compensating for lateral pressure. Presumably, anastomoses result from the fusion of crinkles to adjacent thylakoids.
Topics: Anesthetics, Local; Chloroplasts; Dibucaine; Hydrogen-Ion Concentration; Intracellular Membranes; Light; Membrane Fusion; Microscopy, Electron; Pisum sativum; Tetracaine
PubMed: 8948472
DOI: 10.1016/s0005-2736(96)00143-5 -
The Journal of Biological Chemistry Oct 1996The effects of both neutral and anionic lipids on the structure of the nicotinic acetylcholine receptor (nAChR) have been probed using infrared difference spectroscopy....
The effects of both neutral and anionic lipids on the structure of the nicotinic acetylcholine receptor (nAChR) have been probed using infrared difference spectroscopy. The difference between infrared spectra of the nAChR recorded using the attenuated total reflectance technique in the presence and absence of the neurotransmitter analog, carbamylcholine, exhibits a complex pattern of positive and negative bands that provides a spectral map of the structural changes that occur in the nAChR upon ligand binding and subsequent desensitization. This spectral map is essentially identical in difference spectra recorded from native, native alkaline-extracted, and affinity-purified nAChR reconstituted into either soybean asolectin or egg phosphatidylcholine membranes containing both neutral and anionic lipids. This result suggests both a similar structure of the nAChR and a similar resting to desensitized conformational change in each membrane environment. In contrast, difference spectra recorded from the nAChR reconstituted into egg phosphatidylcholine membranes lacking neutral and/or anionic lipids all exhibit an essentially identical pattern of band intensity variations, which is similar to the pattern of variations observed in difference spectra recorded in the continuous presence of the desensitizing local anesthetic, dibucaine. The difference spectra suggest that the main effect of both neutral and anionic lipids in a reconstituted egg phosphatidylcholine membrane is to help stabilize the nAChR in a resting conformation. In the absence of neutral and/or anionic lipids, the nAChR is converted into an alternate conformation that appears to be analogous to the local anesthetic-induced desensitized state. Significantly, the proportion of receptors found in the resting versus the putative desensitized state appears to be dependent upon the final lipid composition of the reconstituted membrane. A lipid-dependent modulation of the equilibrium between a channel-active resting and channel-inactive desensitized state may account for the modulations of nAChR activity that are observed in different lipid membranes.
Topics: Anions; Cell Membrane; Hydrogen-Ion Concentration; Ion Transport; Lipids; Protein Structure, Secondary; Receptors, Nicotinic; Spectroscopy, Fourier Transform Infrared
PubMed: 8798723
DOI: 10.1074/jbc.271.40.24590 -
Biophysical Journal Sep 1996To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel...
Locations of local anesthetic dibucaine in model membranes and the interaction between dibucaine and a Na+ channel inactivation gate peptide as studied by 2H- and 1H-NMR spectroscopies.
To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel inactivation gate peptide have been studied by 2H- and 1H-NMR spectroscopies. The 2H-NMR spectra of dibucaine-d9 and dibucaine-d1, which are deuterated at the butoxy group and at the 3 position in its quinoline ring, respectively, have been observed in multilamellar dispersions of the lipid mixture composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. 2H-NMR spectra of deuterated palmitic acids incorporated, as a probe, into the lipid mixture containing cholesterol have also been observed. An order parameter, SCD, for each carbon segment was calculated from the observed quadrupole splittings. Combining these results, we concluded that first, the butoxy group of dibucaine is penetrating between the acyl chains of lipids in the model membranes, and second, the quinoline ring of dibucaine is located at the polar region of lipids but not at the hydrophobic acyl chain moiety. These results mean that dibucaine is situated in a favorable position that permits it to interact with a cluster of hydrophobic amino acids (Ile-Phe-Met) within the intracellular linker between domains III and IV of Na+ channel protein, which functions as an inactivation gate. To confirm whether the dibucaine molecule at the surface region of lipids can really interact with the hydrophobic amino acids, we synthesized a model peptide that includes the hydrophobic amino acids (Ac-GGQDIFMTEEQK-OH, MP-1), the amino acid sequence of which corresponds to the linker part of rat brain type IIA Na+ channel, and the one in which Phe has been substituted by Gln (MP-2), and measured 1H-NMR spectra in both phosphate buffer and phosphatidylserine liposomes. It was found that the quinoline ring of dibucaine can interact with the aromatic ring of Phe by stacking of the rings; moreover, the interaction can be reinforced by the presence of lipids. In conclusion, we wish to propose that local anesthesia originates from the pi-stacking interaction between aromatic rings of an anesthetic molecule located at the polar headgroup region of the so-called boundary lipids and of the Phe in the intracellular linker between domains III and IV of the Na+ channel protein, prolonging the inactivated state and consequently making it impossible to proceed to the resting state.
Topics: Amino Acid Sequence; Anesthetics, Local; Animals; Biophysical Phenomena; Biophysics; Cholesterol; Deuterium; Dibucaine; In Vitro Techniques; Liposomes; Magnetic Resonance Spectroscopy; Membrane Lipids; Membranes, Artificial; Models, Molecular; Molecular Structure; Oligopeptides; Palmitic Acids; Phenylalanine; Phosphatidylserines; Protons; Rats; Sodium Channel Blockers; Sodium Channels
PubMed: 8873993
DOI: 10.1016/S0006-3495(96)79327-X -
Biochimica Et Biophysica Acta Jun 1996Alkanols and tertiary amine derivative local anesthetics modify the activity of Ca(2+)-ATPase. In order to investigate the primary binding sites, associated to the...
Alkanols and tertiary amine derivative local anesthetics modify the activity of Ca(2+)-ATPase. In order to investigate the primary binding sites, associated to the functional changes, sarcoplasmic reticulum (SR) Ca(2+)-ATPase was labeled with maleimide derivative spin labels which bind covalently to SH groups of cysteine residues and allow to probe the regions of the protein close to those residues. The EPR measurements showed motional constraints induced by drug-treatment which indicate changes in the enzyme dynamics and structure. n-Alkanols are shown to affect some of the protein-bound labels by restricting their motion. There is, however, no correlation between the functional effects and the observed motional restriction, in the sense that concentrations of the different alcohols leading to the same functional effects do not induce the same degree of restriction. Dibucaine and tetracaine at functional relevant concentrations also restrict the movement of protein bound labels. But, in this case, correlation between spectral changes and functional effects is observed.
Topics: 1-Butanol; Anesthetics, Local; Animals; Butanols; Calcium-Transporting ATPases; Dibucaine; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Maleimides; Muscle, Skeletal; Rabbits; Sarcoplasmic Reticulum; Spin Labels; Tetracaine
PubMed: 8664313
DOI: 10.1016/0005-2736(95)00291-x -
Biophysical Journal May 1996A recently completed model of Ca concentration and movements in the cardiac cell diadic cleft space predicts that removal or neutralization of inner sarcolemmal (SL)...
A recently completed model of Ca concentration and movements in the cardiac cell diadic cleft space predicts that removal or neutralization of inner sarcolemmal (SL) leaflet anionic Ca-binding sites at the sarcolemmal border of this space will greatly diminish Na/Ca exchange-mediated Ca efflux. The present study tests this prediction using the local anesthetic dibucaine as a probe. It is shown, in isolated SL, that dibucaine competitively displaces Ca specifically from anionic phospholipid headgroups. Dibucaine also displaces Ca from the SL when applied to intact cells. It does not affect the content or release of Ca from sarcoplasmic reticulum (SR) in these cells. This eliminates a primary effect on SR Ca as a contributing factor to dibucaine's effect on Na/Ca exchange-mediated Ca efflux. Measurement of this efflux from whole cells shows a highly significant reduction of 58% (p < 0.001) by 0.5 mM dibucaine. The inhibiting effect of dibucaine on Na/Ca exchange-mediated Ca efflux can be significantly reversed by augmentation of Ca release from SR by caffeine at the time of activation of Na/Ca exchange. This supports the contention that the dibucaine-SL interaction is a competitive one vis-a-vis Ca. The results are supportive of the model in which inner SL leaflet Ca-binding sites account for the delay of Ca diffusion from the diadic cleft, thereby prolonging the time for which [Ca] remains elevated in the cleft. The prolonged increased [Ca] significantly enhances the ability of Na/Ca exchange to remove Ca from the cell during the excitation-contraction cycle.
Topics: Animals; Animals, Newborn; Binding Sites; Caffeine; Calcium; Calcium Radioisotopes; Carrier Proteins; Cell Fractionation; Cells, Cultured; Dibucaine; Heart; Heart Rate; Models, Cardiovascular; Myocardial Contraction; Myocardium; Procaine; Rats; Rats, Sprague-Dawley; Sarcolemma; Sodium; Sodium-Calcium Exchanger
PubMed: 9172750
DOI: 10.1016/S0006-3495(96)79792-8 -
Kidney International Mar 1996Lactate dehydrogenase (LDH) leaks from the perfused rat kidney under the artificial conditions of a Ca(2+)-paradox protocol, namely Ca(2+)-repletion following a 20...
Lactate dehydrogenase (LDH) leaks from the perfused rat kidney under the artificial conditions of a Ca(2+)-paradox protocol, namely Ca(2+)-repletion following a 20 minute period of Ca(2+)-depletion. LDH leakage was markedly suppressed by perfusion at 25 degrees C or with 0.1 mM dibucaine or 2 mM lidocaine. Lidocaine inhibited leakage only during Ca(2+)-depletion. Lowering the perfusion rate significantly reduced LDH escape. No LDH loss occurred if the osmotic pressure of the perfusion fluid was raised by 420 mOsm during either Ca(2+)-depletion or Ca(2+)-repletion. Amiloride (2 mM) significantly reduced LDH leakage to 43%. Reduction of the pH of the perfusion fluid to 6.8 significantly inhibited LDH loss, and at pH 6.4 this leakage was almost completely suppressed. LDH loss was equally suppressed at pH 6.4 only during Ca(2+)-depletion, whereas pH 6.4 was markedly less effective when perfused only during Ca(2+)-repletion. Ouabain (5 x 10(-6) M) had only a limited effect in exacerbating LDH leakage. Raising [K+]o significantly protected against LDH leakage, which fell to 36% at 16 mM [K+]. These features correspond with the Ca(2+)-paradox of the perfused rat heart an it is suggested that: (i) a Ca(2+)-paradox can be produced in the rat kidney; (ii) a similar mechanism governs the release of cytosolic proteins in these two preparations; and (iii) the damage mechanism of the plasmalemma is a transmembrane oxidoreductase-diaphorase molecular complex which generates H+ when activated by Ca(2+)-depletion.
Topics: Amiloride; Anesthetics, Local; Animals; Calcium; Cell Membrane; Cold Temperature; Diuretics; Enzyme Inhibitors; Extracellular Space; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney; L-Lactate Dehydrogenase; Osmotic Pressure; Ouabain; Perfusion; Potassium; Rats; Rats, Wistar
PubMed: 8648904
DOI: 10.1038/ki.1996.92 -
Proceedings of the National Academy of... Feb 1996The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding...
The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n = 9 conjugated double bonds (e.g., violaxanthin) had no effect on fluorescence. When quenching in the light-harvesting complex of photosystem II was induced by a lowering of pH to 5.5, carotenoids with n = 9 conjugated double bonds (including violaxanthin) caused a noticeable inhibition of fluorescence quenching relative to the control. Of the 10 carotenoids tested, quenching induced by the addition of the tertiary amine compound, dibucaine, to isolated light-harvesting complex of photosystem II could only be reversed by violaxanthin. These results are discussed in terms of the two theories developed to explain the role of zeaxanthin and violaxanthin in nonphotochemical quenching of chlorophyll fluorescence.
PubMed: 11607629
DOI: 10.1073/pnas.93.4.1492 -
Journal of Biochemistry Feb 1996Effects of basic glycoside antibiotic aculeximycin (ACM) on the oxidative phosphorylation of rat-liver mitochondria were examined. ACM was shown to be a potent uncoupler...
Effects of basic glycoside antibiotic aculeximycin (ACM) on the oxidative phosphorylation of rat-liver mitochondria were examined. ACM was shown to be a potent uncoupler of the oxidative phosphorylation. To cause the same extent of respiration release, higher concentration of ACM was required in phosphate (Pi)-free medium than in Pi medium. During the uncoupling caused by ACM in Pi medium, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, indicating that ACM remarkably enhances permeability of the inner mitochondrial membrane. The Pi uptake via Pi/H+ symporter was shown to play an important, but not essential, role in the uncoupling by ACM, indicating the increase in membrane permeability is mostly due to acceleration of Pi/H+ influx through Pi/H+ symporter activated by ACM. ACM is the first naturally occurring antibiotic, to our knowledge, which activates Pi/H+ symporter. However, since the inhibition of Pi/H+ symporter by N-ethylmaleimide did not completely abolish the uncoupling activity of ACM, and ACM induced the uncoupling even in Pi-free medium, an increase in the membrane permeability for other ions, such as Na+ and K+, due to a different action mechanism has also to be considered. On the other hand, positively charged amine local anesthetics, like dibucaine, prevented the uncoupling activity by ACM in both Pi and Pi-free medium. The uncoupling activity of N-diacetylated ACM lacking free amino groups was ca. 1/120th that of ACM, indicating that positively charged amino groups are important for the uncoupling activity. It is suggested that some specific interactions between positively charged amino groups of ACM and the binding site, which is probably negatively charged, are triggers that affect the permeability of the inner mitochondrial membrane. Amine local anesthetics may mask the negative charge of the binding site, thereby interfering with ACM binding.
Topics: Animals; Anti-Bacterial Agents; Dibucaine; Macrolides; Male; Mitochondria, Liver; Mitochondrial Swelling; Oxygen; Rats; Rats, Wistar; Uncoupling Agents
PubMed: 8882718
DOI: 10.1093/oxfordjournals.jbchem.a021235 -
Anesthesiology Oct 1995Mivacurium chloride is a bis-benzylisoquinolinium nondepolarizing neuromuscular blocking agent, hydrolyzed by butyrylcholinesterase (PCHE). The dose-response... (Clinical Trial)
Clinical Trial Randomized Controlled Trial
BACKGROUND
Mivacurium chloride is a bis-benzylisoquinolinium nondepolarizing neuromuscular blocking agent, hydrolyzed by butyrylcholinesterase (PCHE). The dose-response relationships for PCHE after mivacurium have not been studied. Therefore, this study was designed to establish dose-response relationships for PCHE as an antagonist of mivacurium-induced neuromuscular blockade.
METHODS
Forty-eight physical status 1 adults were given 0.15 mg/kg mivacurium during fentanyl-thiopental-nitrous oxide-isoflurane anesthesia. Train-of-four (TOF) stimulation was applied to the ulnar nerve every 12 s, and the force of contraction of the adductor pollicis muscle was recorded. When spontaneous recovery of first twitch height (T1) reached 10% of its initial control value, exogenous PCHE equivalent to activity present in 2.5, 5, 7.5, 15, or 25 ml/kg of human plasma was administered by random allocation to 40 patients. Neuromuscular function in another eight subjects was allowed to recover spontaneously. Two blood samples were taken for determination of plasma cholinesterase activity. The first sample was taken before induction of anesthesia, and the second sample was taken when the TOF ratio had recovered to 0.75. Dibucaine and fluoride numbers were determined from the first assay.
RESULTS
Administration of PCHE produced significant increases in PCHE activity in all patients. The larger the dose, the greater was the resultant plasma activity. Human PCHE produced a dose-dependent antagonism of mivacurium-induced neuromuscular blockade and the recovery times correlated inversely with PCHE activity (P < 0.01). The recovery of T1 was greater (P < 0.01) and time to attain a TOF ratio of 0.75 was shorter (P < 0.01) with any dose of PCHE than that observed in the spontaneous recovery group. After the administration of exogenous PCHE equivalent to activity present in 25 ml/kg of human plasma, recovery of TOF ratio to 0.75 or more was observed in all patients in less than 10 min and time to attain a TOF ratio of 0.75 was 55% shorter than the spontaneous recovery group (8.4 [7.1-9.7] vs. 18.7 [15.4-22] min; mean and 95% confidence intervals).
CONCLUSIONS
Administration of exogenous PCHE equivalent to activity present in 25 ml/kg of human plasma (in a 65-kg patient, this dose is equivalent to PCHE activity of 1,625 ml of adult human plasma) resulted in reliable antagonism of mivacurium-induced neuromuscular blockade. Nevertheless, because of the prohibitive cost of this compound, this reversal modality is unlikely to have a routine practical application at this time.
Topics: Adolescent; Adult; Cholinesterases; Dose-Response Relationship, Drug; Female; Humans; Isoquinolines; Male; Middle Aged; Mivacurium; Neuromuscular Junction; Neuromuscular Nondepolarizing Agents
PubMed: 7574048
DOI: 10.1097/00000542-199510000-00008 -
The Journal of Biological Chemistry Mar 1995Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta...
Two membrane-associated phosphoinositide-specific phospholipase Cs (mPI-PLC-1 and mPI-PLC-2) and a cytosolic enzyme (cPI-PLC) that were activated by brain G-protein beta gamma subunits have been isolated from human platelets. The truncation of mPI-PLC-1 that was mediated by mu-calpain induced much higher activation by beta gamma subunits (Banno, Y., Asano, T., and Nozawa, Y. (1994) FEBS Lett. 340, 185-188). On the basis of size and immunological cross-reactivity, mPI-PLC-1 (155 kDa) was PLC-beta 3, and mPI-PLC-2 (100 kDa) was its truncated form. The cPI-PLC (140 kDa) was recognized by the antibody selective for internal sequences of PLC-beta 3 but not by the antibody raised against its carboxyl terminus, indicating that it may be related to PLC-beta 3. Treatment of human platelets with A23187 and dibucaine, activators of calpain, caused cleavage of actin-binding protein and talin in a time-dependent manner. At the same time, decrease of PLC-beta 3 (155 and 140 kDa) and concomitant increase of the 100-kDa product of cleavage were observed on immunoblots with the antibody to internal sequences of PLC-beta 3. Furthermore, stimulation of platelets by natural agonists, thrombin and collagen, caused the cleavage of PLC-beta 3 (155 and 140 kDa) and an increase of 100 kDa PLC-beta 3 in a time- and dose-dependent manner. The cleavage of these PLC-beta 3 enzymes was completely blocked by calpain inhibitor, calpeptin, indicating that the PLC-beta 3 modification may be a consequence of platelet activation leading to activation of calpain. This is the first demonstration that PLC-beta 3 is indeed cleaved by calpain upon platelet activation by physiological agonists. The cleavage of PLC-beta 3 evoked by thrombin and collagen but not ADP was correlated with irreversible aggregation, suggesting that the PLC-beta 3 modification may play a role in secondary irreversible aggregation in agonist-stimulated human platelets.
Topics: Amino Acid Sequence; Blood Platelets; Blotting, Western; Calpain; Collagen; Enzyme Activation; GTP-Binding Proteins; Humans; Hydrolysis; Isoenzymes; Molecular Sequence Data; Phospholipase C beta; Thrombin; Type C Phospholipases
PubMed: 7876193
DOI: 10.1074/jbc.270.9.4318