Propofol inhibits volume-sensitive chloride channels in human coronary artery smooth muscle cells

Volume-sensitive chloride channels (VSCC) play an important role in regulation of cell volume and electrical activity. Activation of vascular smooth muscle VSCC causes smooth muscle depolarization and contraction. We investigated the effects of propofol on VSCC in cultured human coronary artery smooth muscle cells by using the chloride-sensitive dye 6-methoxy-N-ethylquinolinium (MEQ). To activate VSCC, cells were superfused for 2 min with hypotonic gluconate solutions and then potassium thiocyanate solution. The percentage reduction in MEQ fluorescence during 60 s in the presence of potassium thiocyanate was measured and used as an index of VSCC activity. 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a well characterized chloride channel blocker, and propofol were dissolved in hypotonic gluconate solution to test their effect on VSCC activity. The reduction in fluorescence was inversely related to osmolality, indicating that activation of VSCC is osmolality dependent. Hypotonic gluconate solution (210 mOsm/kg H(2)O) reduced fluorescence by 38.9% +/- 2.6% of the baseline value. The reduction in fluorescence was dose-dependently inhibited by NPPB. Propofol at 0.3, 1, 3, 10, 30, and 100 micro g/mL significantly inhibited the reduction in fluorescence to 23.6% +/- 4.8%, 19.7% +/- 7.4%, 18.2% +/- 3.5%, 17.6% +/- 5.0%, 15.8% +/- 3.1%, and 10.3% +/- 3.9% of the baseline value, respectively. Our results indicate that propofol inhibits VSCC in a dose-dependent manner in human coronary artery smooth muscle cells.     

Single sodium channels from human skeletal muscle in planar lipid bilayers: characterization and response to pentobarbital

Purpose To investigate the response to general anesthetics of different sodium-channel subtypes, we examined the effects of pentobarbital, a close thiopental analogue, on single sodium channels from human skeletal muscle and compared them to existing data from human brain and human ventricular muscle channels.Methods Sodium channels from a preparation of human skeletal muscle were incorporated into planar lipid bilayers, and the steady-state behavior of single sodium channels and their response to pentobarbital was examined in the presence of batrachotoxin, a sodium-channel activator. Single-channel currents were recorded before and after the addition of pentobarbital (0.34–1.34mM).Results In symmetrical 500mM NaCl, human skeletal muscle sodium channels had an averaged single-channel conductance of 21.0 ± 0.6pS, and the channel fractional open time was 0.96 ± 0.04. The activation midpoint potential was –96.2 ± 1.6mV. Extracellular tetrodotoxin blocked the channel with a half-maximal concentration (k_1/2) of 60nM at 0mV. Pentobarbital reduced the time-averaged conductance of single skeletal muscle sodium channels in a concentration-dependent manner (inhibitory concentration 50% [IC₅₀] = 0.66mM). The steady-state activation was shifted to more hyperpolarized potentials (–16.7mV at 0.67mM pentobarbital).Conclusion In the planar lipid bilayer system, skeletal muscle sodium channels have some electrophysiological properties that are significantly different compared with those of sodium channels from cardiac or from central nervous tissue. In contrast to the control data, these different human sodium channel subtypes showed the same qualitative and quantitative response to the general anesthetic pentobarbital. The implication of these effects for overall anesthesia will depend on the role the individual channels play within their neuronal networks, but suppression of both central nervous system and peripheral sodium channels may add to general anesthetic effects.     

High-affinity blockade of voltage-operated skeletal muscle sodium channels by 2,6-dimethyl-4-chlorophenol

BACKGROUND AND OBJECTIVE: The aromatic alcohol most closely resembling the aromatic tail of lidocaine is 2,6-dimethylphenol. This agent is as potent as lidocaine in blocking voltage-operated sodium channels. The aim of this study was to show the effect of halogenation in the para-position on the potency of this compound to block voltage-operated sodium channels. METHODS: Insertion of the halogen chloride into the para-position of the molecule 2,6-dimethylphenol yielded 2,6-dimethyl-4-chlorophenol. Block of sodium currents by this compound was studied using heterologously expressed voltage-operated rat neuronal (rat IIa) sodium channels. RESULTS: 2,6-dimethyl-4-chlorophenol reversibly suppressed depolarization-induced whole-cell sodium inward currents. The ECR50 for block of resting channels at a hyperpolarized holding potential (-150 mV) was 127 micromol, the Hill coefficient nH 1.7. Membrane depolarization inducing either fast or slow-inactivation strongly increased the blocking potency. This is an important feature of a local-anaesthetic-like action. The estimated half-maximum effect concentration for the fast-inactivated channel state ECI50 was 28 micromol, the Hill coefficient nH 3.8. When 20-30% of channels were slow-inactivated using long (2.5 s) prepulses, followed by a 10 ms repolarization period to allow recovery from fast inactivation, the IC50 at -100 mV holding potential was reduced to 53 micromol. CONCLUSION: These results, which show that 2,6-dimethyl-4-chlorophenol blocks voltage-operated sodium channels in a lidocaine-like manner while having a several fold higher potency than the non-halogenated parent compound, highlight a potentially meaningful principle of increasing the sodium channel blocking potency of phenol derivatives.     

Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol

BACKGROUND AND OBJECTIVE: Thymol is a naturally occurring phenol derivative used in anaesthetic practice as a stabilizer and preservative of halothane, usually at a concentration of 0.01%. Although analgesic effects have long been described for thymol and its structural homologue menthol, a molecular basis for these effects is still lacking. We studied the blocking effects of thymol and menthol on voltage-activated sodium currents in vitro as possible molecular target sites. METHODS: Whole cell sodium inward currents via heterologously (HEK293 cells) expressed rat neuronal (rat type IIA) and human skeletal muscle (hSkM1) sodium channels were recorded in the absence and presence of definite concentrations of either thymol or menthol. RESULTS: When depolarizing pulses to 0 mV were started from a holding potential of -70 mV, half-maximum blocking concentrations (IC50) for the skeletal muscle and the neuronal sodium channel were 104 and 149 mumol for thymol and 376 and 571 mumol for menthol. The blocking potency of both compounds increased at depolarized holding potentials with the fraction of inactivated channels. The estimated dissociation constant Kd for thymol and menthol from the inactivated state was 22 and 106 mumol for the neuronal and 23 and 97 mumol for the skeletal muscle sodium channel, respectively. CONCLUSIONS: The results suggest that antinociceptive and local anaesthetic effects of thymol and menthol might be mediated via blockade of voltage-operated sodium channels with the phenol derivative thymol being as potent as the local anaesthetic lidocaine.     

Photobiomodulation delays the onset of skeletal muscle fatigue in a dose-dependent manner

Photobiomodulation (PBM) therapy has been implicated as an effective ergogenic aid to delay the onset of muscle fatigue. The purpose of this study was to examine the dose–response ergogenic properties of PBM therapy and its ability to prolong time to task failure by enhancing muscle activity and delaying the onset of muscle fatigue using a static positioning task. Nine participants (24.3?±?4.9 years) received three doses of near-infrared (NIR) light therapy randomly on three separate sessions (sham, 240, and 480 J). For the positioning task, participants held a 30 % one-repetition maximum (1-RM) load using the index finger until volitional fatigue. Surface electromyography (sEMG) of the first dorsal interosseous muscle was recorded for the length of the positioning task. Outcomes included time to task failure (TTF), muscle fatigue, movement accuracy, motor output variability, and muscle activity (sEMG). The 240-J dose significantly extended TTF by 26 % (p?=?0.032) compared with the sham dose. TTF for the 240-J dose was strongly associated with a decrease in muscle fatigue (R ²?=?0.54, p?=?0.024). Our findings show that a 240-J dose of NIR light therapy is efficacious in delaying the onset and extent of muscle fatigue during submaximal isometric positioning tasks. Our findings suggest that NIR light therapy may be used as an ergogenic aid during functional tasks or post-injury rehabilitation.     

Lidocaine increases intracellular sodium concentration through voltage-dependent sodium channels in an identified lymnaea neuron

BACKGROUND: The local anesthetic lidocaine affects neuronal excitability in the central nervous system; however, the mechanisms of such action remain unclear. The intracellular sodium concentration ([Na]i) and sodium currents (INa) are related to membrane potential and excitability. Using an identifiable respiratory pacemaker neuron from Lymnaea stagnalis, the authors sought to determine whether lidocaine changes [Na]i and membrane potential and whether INa is related to these changes. METHODS: Intracellular recording and sodium imaging were used simultaneously to measure membrane potentials and [Na]i, respectively. Measurements for [Na]i were made in normal, high-Na, and Na-free salines, with membrane hyperpolarization, and with tetrodotoxin pretreatment trials. Furthermore, changes of INa were measured by whole cell patch clamp configuration. RESULTS: Lidocaine increased [Na]i in a dose-dependent manner concurrent with a depolarization of the membrane potential. In the presence of high-Na saline, [Na]i increased and the membrane potential was depolarized; the addition of lidocaine further increased [Na]i, and the membrane potential was further depolarized. In Na-free saline or in the presence of tetrodotoxin, lidocaine did not change [Na]i. Similarly, hyperpolarization of the membrane by current injections also prevented the lidocaine-induced increase of [Na]i. In the patch clamp configuration, membrane depolarization by lidocaine led to an inward sodium influx. A persistent reduction in membrane potential, resulting from lidocaine, brings the cell within the window current of INa where sodium channel activation occurs. CONCLUSION: Lidocaine increases intracellular sodium concentration and promotes excitation through voltage-dependent sodium channels by altering membrane potential in the respiratory pacemaker neuron.     

New index of pain triggered by spinal activation of voltage-dependent sodium channels

Voltage-dependent sodium channels (VDSCs) are crucial for pain generation. Here, to develop a new behavioral index of pain induced by spinal VDSC activation, we examined whether intrathecal veratridine injection produced nociceptive behavior. Intrathecal injection of the VDSC opener veratridine in mice dose-dependently induced nociceptive responses, with response times subsequently reduced by administration of morphine or pregabalin. Systemic administration of lidocaine and mexiletine, but not amitriptyline, also decreased this response time. Taken together, these results demonstrated that response time of nociceptive behavior induced by intrathecal veratridine injection is a quantitative index of pain triggered by spinal VDSC activation.     

Ephedrine blocks rat sciatic nerve in vivo and sodium channels in vitro

BACKGROUND: The sympathomimetic drug ephedrine has been used intrathecally as the sole local anesthetic for labor and delivery. Because ephedrine may be a useful adjuvant to local anesthetics, the authors investigated the local anesthetic properties of ephedrine in a rat sciatic nerve block model and the underlying mechanism in cultured cells stably expressing Na channels. METHODS: After approval of the animal protocol, the sciatic nerves of anesthetized rats were exposed by lateral incision of the thighs, 0.2 ml ephedrine at 0.25, 1, 2.5, or 5% and/or bupivacaine at 0.125% was injected, and the wound was closed. Motor and sensory/nociceptive functions were evaluated by the force achieved by pushing against a balance and the reaction to pinch, respectively. The whole cell configuration of the patch clamp technique was used to record Na currents from human embryonal kidney cells stably transfected with Nav1.4 channels. RESULTS: The nociception blockade was significantly longer than the motor blockade at test doses of 2.5 and 5% of ephedrine, or when 1% ephedrine was combined with 0.125% bupivacaine (analysis of variance with repeated measures, P < 0.001, n = 8/group). In vitro, the 50% inhibitory concentrations of ephedrine at -150 and -60 mV were 1,043 +/- 70 and 473 +/- 13 mum, respectively. High-frequency stimulation revealed a use-dependent block of 18%, similar to most local anesthetics. CONCLUSIONS: Because ephedrine's properties are at least partly due to Na channel blockade, detailed histopathologic investigations are justified to determine the potential of ephedrine as an adjuvant to clinically used local anesthetics.     

Propofol decreases the amplitude of motor-evoked potentials in a concetration-dependent manner

Canadian Journal of Anesthesia/Journal canadien d'anesthésie -     

Halothane effects on human malignant hyperthermia skeletal muscle single calcium-release channels in planar lipid bilayers.

Malignant hyperthermia (MH) may be life-threatening when genetically predisposed individuals are administered triggering anesthetic agents that are believed to produce intracellular calcium release. To test this theory, the effects of halothane on normal and MH human skeletal muscle calcium-release channels were studied. Single calcium-release channels were incorporated from isolated sarcoplasmic reticulum membrane vesicles into a planar lipid bilayer, and halothane effects on the conductance and gating properties were measured by electrophysiologic techniques. Among the subjects studied, seven were MH-susceptible, and 13 channels were recorded from this group. Five subjects were negative for MH, and 10 channels were recorded from this group. Among the 13 channels recorded from the MH group, 7 were affected by halothane, which increased the probability of the channel to change from the inactive, closed state to an open state. This effect of halothane to increase open-state probability was associated with an overall increase in channel conductance. Thus, halothane affected the activation/inactivation process of the halothane-sensitive calcium-release channel from MH muscle as well as the gating properties of the MH calcium-release channel, as evidenced by the increased conductance. In 6 of the 13 channels recorded from MH muscle, halothane (2.2-17.6 microM) was without effect on these properties of the channel. Halothane (2.2-17.6 microM or 0.0057-0.0456 vol%) also had no measurable effect on the 10 channels from the negatively diagnosed subjects. Results of this study support a defect in the ryanodine-sensitive calcium-release channel from MH human muscle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sublytic complement attack increases intracellular sodium in rat skeletal muscle

BACKGROUND: Although excessive complement activation and deranged sodium homeostasis in skeletal muscle are characteristic in sepsis, their relationship has not been examined. This study was designed to determine if sublytic complement activation can directly mediate changes in myocellular sodium content. MATERIALS AND METHODS: Fast-twitch extensor digitorum longus muscles were freshly isolated from infant rats. Unsensitized muscles were incubated at 30 degrees C for 60 min in the media containing 10% human or rat serum under conditions of no complement activation, activation by zymosan, inactivation by heat, C7 or C9 deficiency, selective inhibition of complement pathway, and inhibition of Na(+)-K(+) ATPase by ouabain. Intracellular sodium ([Na(+)](i)) and potassium ([K(+)](i)) contents of the muscles, myocellular ATP, and LDH release from the muscles were then determined. RESULTS: Normal human serum significantly increased [Na(+)](i) and the [Na(+)](i)/[K(+)](i) ratio in the muscles as well as zymosan-activated serum. Heat inactivation, C7 deficiency, and inhibition of the alternative pathway completely abolished the cationic changes. Average LDH release was identical in all groups and less than 6%. Complement activation did not impair ouabain-sensitive Na(+)-K(+) ATPase activity in the muscles or alter myocellular ATP. Thus, the observed alterations are not likely due to dysfunction of Na(+)-K(+) pump or depletion of myocellular energy. Instead, alterations in [Na(+)](i) were dependent upon the amount of C9 added to C9-deficient serum, which suggests that the alterations are likely dependent on transmembrane pores created by membrane attack complexes (MAC). CONCLUSIONS: Sublytic amounts of MAC formed as a result of complement activation can directly alter [Na(+)](i) in ex vivo skeletal muscle.     

Hexokinase isozyme distribution in human skeletal muscle

Two isoforms of hexokinase (type I and type II) are expressed in skeletal muscle; however, the intracellular distribution of these hexokinase isoforms in human skeletal muscle is unclear. The current study was undertaken to assess this issue because binding of hexokinase to subcellular structures is considered to be an important mechanism in the regulation of glucose phosphorylation. Vastus lateralis muscle was obtained from healthy lean individuals. Muscle homogenate was separated at 45,000g into particulate and cytosolic fractions. The activity and subcellular distribution of hexokinase isozymes in human skeletal muscle was determined using ion-exchange chromatography and a highly sensitive high-performance liquid chromatography-based hexokinase assay. This criterion method was used to validate a modified thermal inactivation method for distinguishing type I and type II isoforms. Mean hexokinase activity was 3.88 +/- 0.65 U/g wet wt or 0.64 +/- 0.11 U/mU creatine kinase (CrK) in the particulate fraction and 0.45 +/- 0.22 U/g wet wt or 0.07 +/- 0.03 U/mU CrK in the cytosolic fraction. Hexokinase I and II accounted for 70-75 and 25-30% of total hexokinase activity, respectively. Nearly all (95%) of hexokinase I activity (0.52 +/- 0.09 U/mU CrK) was found in the particulate fraction, consistent with the known high affinity of hexokinase I for mitochondria. Hexokinase II activity was also largely bound to the particulate fraction (72%), but 28% was found within the cytosolic fraction. Thus, within the particulate fraction, the relative contributions of hexokinase I and hexokinase II were 81 and 19%, whereas within the cytosolic fraction, the relative contributions for hexokinase I and hexokinase II were 37 and 63%.     

Ischemic tolerance of human skeletal muscle

Until now, the ischemic tolerance of muscle tissue has not been adequately understood. Even when muscle vitality is lost, the perfusion matrix of the muscle flaps is retained. Because of toxic decomposition, however, irreversibly damaged muscle cells almost certainly increase the rate of complications. The retention of the vitality of the transplanted muscle tissue is absolutely essential for the myokinetic substitute operations, currently in the development stage, involving the free transplantation of muscles. Investigations into vitality reserves were carried out on skeletal muscle specimens. Nuclear magnetic resonance spectroscopy was used to establish that, in ischemia, the ATP pool remained topped up to a large extent as long as phosphocreatine was available. As long as the ATP pool was retained, rearterialization led to the complete restoration of the essential preischemic metabolite concentrations. After the ATP had been exhausted, biochemical restitution through arterial reperfusion did not occur. The time by which the established vitality threshold was reached because of the loss of the ATP pool is called the critical ischemia time; it depends on muscle temperature. The critical ischemia time of human skeletal muscles was determined between 26 degrees and 38 degrees C. A normothermia of 34 degrees C yielded a critical ischemia time of 2.25 hours, which is shorter than that previously reported in the literature. An ischemic tolerance of 5 hours presupposes a muscle temperature of less than 26 degrees C.     

Blockade of voltage-operated neuronal and skeletal muscle sodium channels by S(+)- and R(-)-ketamine

Besides its general anesthetic effect, ketamine has local anesthetic-like actions. We studied the voltage- and use-dependent interaction of S(+)- and R(-)-ketamine with two different isoforms of voltage-operated sodium channels, with a special emphasis on the difference in affinity between resting and inactivated channel states. Rat brain IIa and human skeletal muscle sodium channels were heterologously expressed in human embryonic kidney 293 cells. S(+)- and R(-)-ketamine reversibly suppressed whole-cell sodium inward currents; the 50% inhibitory concentration values at -70 mV holding potential were 240 +/- 60 microM and 333 +/- 93 microM for the neuronal isoform and 59 +/- 10 microM and 181 +/- 49 microM for the skeletal muscle isoform. S(+)-ketamine was significantly more potent than R(-)-ketamine in the skeletal muscle isoform only. Ketamine had a higher affinity to inactivated than to resting channels. However, the estimated difference in affinity between inactivated and resting channels was only 8- to 10-fold, and the time course of drug equilibration between inactivated and resting channels was too fast to cause use-dependent block at 10 Hz up to a concentration of 300 microM. These results suggest that ketamine is less effective than lidocaine-like local anesthetics in stabilizing the inactivated channel state. IMPLICATIONS: Blockade of sodium channels by ketamine shows voltage dependency, an important feature of local anesthetic action. However, ketamine is less effective than lidocaine-like local anesthetics in stabilizing the inactivated state. Because it does not elicit phasic blockade at small concentrations, its ability to reduce the firing frequency of action potentials may be small.     

Fuel selection in human skeletal muscle in insulin resistance: a reexamination

For many years, the Randle glucose fatty acid cycle has been invoked to explain insulin resistance in skeletal muscle of patients with type 2 diabetes or obesity. Increased fat oxidation was hypothesized to reduce glucose metabolism. The results of a number of investigations have shown that artificially increasing fat oxidation by provision of excess lipid does decrease glucose oxidation in the whole body. However, results obtained with rodent or human systems that more directly examined muscle fuel selection have found that skeletal muscle in insulin resistance is accompanied by increased, rather than decreased, muscle glucose oxidation under basal conditions and decreased glucose oxidation under insulin-stimulated circumstances, producing a state of "metabolic inflexibility." Such a situation could contribute to the accumulation of triglyceride within the myocyte, as has been observed in insulin resistance. Recent knowledge of insulin receptor signaling indicates that the accumulation of lipid products in muscle can interfere with insulin signaling and produce insulin resistance. Therefore, although the Randle cycle is a valid physiological principle, it may not explain insulin resistance in skeletal muscle.     

Tourniquet-induced ischemia and reperfusion in human skeletal muscle

Microdialysis conceivably enables longitudinal and simultaneous investigation of several metabolites by repeated measurements in skeletal muscle. We used and evaluated microdialysis as an in vivo method to characterize the time-course and relative kinetics of pyruvate, glucose, lactate, glycerol, hypoxanthine, uric acid, and urea, in skeletal muscles, exposed to ischemia and reperfusion, in eight patients having arthroscopic-assisted anterior cruciate ligament reconstruction. A dialysis probe was implanted before surgery in the rectus femoris muscle. Dialysate samples were collected at 10-minute intervals at a flow rate of 1 microL/minute until 2 hours after tourniquet deflation. Ninety minutes of ischemia resulted in accumulation of lactate (234% +/- 38%), hypoxanthine (582% +/- 166%), and glycerol (146% +/- 46%), consumption of glucose (54% +/- 9%) and pyruvate (16% +/- 44%), and a slight decrease of urea (78% +/- 11%) compared with baseline (100%). Uric acid was unchanged (95% +/- 12%). Within 90 minutes after tourniquet deflation the concentrations were virtually normalized for all measured metabolites, suggesting that the duration of ischemia was well tolerated by the patients. The results indicate that the use of microdialysis for monitoring energy metabolic events during orthopaedic surgery that requires ischemia and reperfusion is feasible and safe.     

Succinylcholine metabolite succinic acid alters steady state activation in muscle sodium channels

BACKGROUND: Animal experiments revealed that succinylcholine produced masseter muscle rigidity and activated myotonic discharges despite neuromuscular blockade with a nondepolarizing blocker. These results suggest that either succinylcholine or its metabolites might interfere directly with voltage-operated ion channels of the sarcolemma. The aim of this study was to examine effects of one product of succinylcholine hydrolysis, succinic acid, on voltage-gated muscle sodium (Na+) channels. METHODS: Alpha subunits of human muscle sodium channels were heterologously expressed in HEK293 cells. Activation of Na+ currents was examined applying standard whole-cell voltage-clamp protocols in the absence (control and washout) and presence of succinic acid in different concentrations (0.05-10 mm). RESULTS: Succinic acid shifted the midpoints of steady state activation plots in the direction of more negative test potentials, indicating that channels open during smaller depolarizations in the presence of the drug. The maximum amount of the negative shift in 10 mm succinic acid was -6.3 +/- 1.7 mV; the EC50 for this effect was 0.39 mm. In addition, succinic acid (10 mm) significantly enhanced maximum currents after depolarizations with respect to a series of control experiments. CONCLUSION: Succinic acid facilitates voltage-dependent activation in muscle sodium channels in vitro. This might lead to muscle hyperexcitability in vivo.