P. R. Krishna

Structure-selective anesthetic action of steroids : anesthetic potency and effects on lipid and protein

Alphaxalone was a clinically used steroid anesthetic. Its analog δ16-alphaxalone is nonanesthetic. The only difference between the two is the presence of a double bond at the hydrophobic end of the δ16-alphaxalone molecule. This study determined the anesthetic potency of alphaxalone and δ16-alphaxalone in goldfish and compared it with their effects on dipalmitoylphos-phatidylcholine (DPPC) membranes and an α-helix polypeptide, poly(L-lysine). The goldfish EC50 values were: alphaxalone 5 μmol/L and δ16-alphaxalone 80 μmol/L. Because these steroids are insoluble to water, the bulk of the steroid in water is absorbed by the fish. Larger containers hold more steroids than smaller containers at the same steroid concentrations. Then, EC50 values vary according to the size of the container. By assuming that the total amount of steroids in the container is distributed into the fish, the EC50 values expressed by the concentration in the fish body become 1.9 mmol/L for alphaxalone, and 30.5 mmol/L for δ16-alphaxalone. A monoamino acid peptide, poly(L-lysine), can be formed into random-coil, α-helix, or β-sheet. Addition of 0.07 mmol/L alphaxalone to the α-helix poly(L-lysine) partially transformed it to a β-sheet structure. An equivalent change was observed with 3.0 mmol/L δ16-alphaxalone. These values translate into 3.5 mmol/L for alphaxalone and 0.15 mol/L for δ16-alphaxalone, when expressed by the concentration in the peptide. The change from α-helix to β-sheet is accompanied by dehydration of the surface of poly(L-lysine). The steroids decreased the phase-transition temperature of DPPC membrane. At 1:10 steroid:DPPC mole ratio, alphaxalone and δ16-alphaxalone decreased the main transition temperature 0.6 and 0.2°C, respectively, and the pretransition temperature 4.5 and 1.0°C, respectively. When expressed by the steroid concentration in DPPC, 1:10 mole ratio equals 0.14 mmol/L. Addition of alphaxalone to the DPPC membrane released water molecules from the membrane surface. δ16-Alphaxalone did not. Lipid membranes cannot be formed without water. A string of amino acids transforms into biologically meaningful protein structure only when it is dissolved in water. These structures are supported by water molecules hydrogen-bonded to the macromolecular surfaces. We contend that the debate on the anesthetic action site between proteins and lipid membranes is futile. Although the affinity of anesthetics to neuroreceptors may vary, the basic interaction force is the tendency to be excluded from water to bind hydrophobic surfaces of proteins and lipid membranes. The present study demonstrated that alphaxalone induces nonspecific conformational changes of lipid membranes and proteins by releasing the supporting water molecules.

Does Pressure Antagonize Anesthesia? Opposite Effects on Specific and Nonspecific Inhibitors of Firefly Luciferase

Ueda and Suzuki (1998. Biochim. Biophys. Acta. 1380:313-319; 1998. Biophys. J. 75:1052-1057) reported that myristic acid inhibited firefly luciferase in microM range in competition with luciferin, whereas anesthetics inhibited it in millimeter ranges noncompetitively with luciferin. Myristate increased, whereas anesthetics decreased, the thermal denaturation temperature. The present study showed that high pressure increased the steady-state light intensity of the halothane-doped firefly luciferase but decreased that of the myristate-doped firefly luciferase. The steady-state light intensity showed a maximum at 19.1 degrees C. At 19.1 degrees C, high pressure did not affect the light intensity in the absence of the inhibitors. In the presence of 0.5 mM halothane, however, 25 MPa pressure (maximum effect) increased the light intensity to 106.0% of the control without the inhibitor. In the presence of 2.5 microM myristate, 40 MPa pressure decreased the light intensity to 90.9% of the control. When the temperature was 25 degrees C in the absence of inhibitors, 40 MPa pressure increased the light intensity 119.2% of the ambient value. At 0.5 mM halothane, 40 MPa pressure further increased the light intensity to 106.1% above the control 40 MPa value. At 2.5 microM myristate, 40 MPa pressure decreased the light intensity to 90.1% of the control 40 MPa value. From the pressure dependence of the light intensity, the volume change DeltaV of the enzyme was estimated at 25 degrees C: 0.5 mM halothane increased DeltaV = +3.93 cm3 mol-1, whereas 2.5 microM myristate decreased DeltaV = -7.66 cm3 mol-1. Present results show that there are distinct differences between the specific and nonspecific ligands in their response to high pressure. Myristate, which competes with luciferin, decreased the protein volume and stabilized the conformation against thermal perturbation. Halothane, which does not compete with the substrate, increased the protein volume and destabilized the conformation.

Malignant hyperthermia and calcium-induced heat production

The abnormal increase in intracellular Ca++ in malignant hyperthermia (MH) is well documented, but the link between the increased Ca++ concentration and high temperature remains speculative. We investigated the possibility that the Ca++-induced change in the state of cell membranes may contribute to the temperature elevation. Calcium ion transforms phosopholipid membranes from the fluid to solid state. This is analogous to the freezing of water, and liberates latent heat. Differential titration calorimetry (DTC) measures heat production or absorption during ligand binding to macromolecules. When CaCl2 solution was added to anionic dimyristoylphosphatidic acid (DMPA) and dimyristoylphosphatidylglycerol (DMPG) vesicle membranes in incremental doses, DTC showed that the heat production suddenly increased when the Ca++ concentration exceeded about 120/μM. At this Ca++ concentration range, these lipid membranes underwent phase transition. The latent heat of transition was measured by differential scanning calorimetry (DSC). The values were 7.1 ± 0.7 (SD, n = 4) kcal · mol−1 of DMPA and 6.8 ± 0.7 (SD, n = 4) kcal · mol−1 of DMPG. The study shows that Ca++ produces heal when bound to lipid membranes. We are not proposing, however, that this is the sole source of heat. We contend that the lipid phase transition is one of the heat sources and it may trigger a hypermetabolic state by elevating the temperature of cell membranes. Because Ca++ is implicated as the second messenger in signal transduction, multiple systems may be involved. More studies are needed to clarify how Ca++ increases body temperature.RésuméDans l’hyperthermie maligne (HM) l’augmentation anormale du Ca++ intracellulaire constitue une réalité bien établie mais le lien entre cette augmentation et celle de la température haute demeure spéculatif. Nous avons examiné la possibilité que les changements initiés par le Ca++ sur l’état de la membrane cellulaire puissent contribuer à l’élévation de la température. L’ion calcium transforme les membranes de phospholipides de l’état liquide à l’état solide. Ce phénomène est semblable au gel de l’eau et il libère de la chaleur latente. Le titrage différentiel par calorimétrie (DTC) mesure la production de chaleur ou son absorption pendant la liaison du ligand aux macromolécules. Quand la solution de CaCl2 est ajoutée à l’acide dimyristoylphosphatidique (DMPA) et des vésicules de membranes de dimyristoylphosphatidylglycérol (DMPG) à doses croissantes, le DTC montre que la production de chaleur augmente soudainement au moment où la concentration dépasse 120 μM. A cette concentration de Ca++, les membranes lipidiques subissent une transition de phase. La chaleur latente de transition est mesurée par calorimétrie scintigraphique différentielle (DSC). Les valeurs sont de 7,1 ± 0,7 (SD, n = 4) kcal · mol−1 de DMPA et 6,8 ± 0,7 (SD, n = 4)kcal · mol−1 de DMPF. L’étude montre que le Ca++ produit de la chaleur lorsqu’il se lie aux membranes lipidiques. Toutefois, nous ne suggérons pas que ce soit la seule source de chaleur. Nous prétendons que la transition de phase lipidique est une des sources de chaleur et qu’elle peut déclencher un état hypermétabolique en élevant la température des membranes cellulaires. Parce que le Ca++ joue le rôle de second messager dans la transduction du signal, plusieurs systèmes peuvent être impliqués. Des études supplémentaires sont requises pour éclaicir le phénomène de l’élévation de la température corporelle par le Ca++.