Hirochika Komai

Interaction of Bupivacaine and Tetracaine with the Sarcoplasmic Reticulum Ca2+Release Channel of Skeletal and Cardiac Muscles

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 Bupivacaines 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.

Local Anesthetic Inhibition of Voltage-activated Potassium Currents in Rat Dorsal Root Ganglion Neurons

Background Local anesthetic actions on the K+ channels of dorsal root ganglion (DRG) and dorsal horn neurons may modulate sensory blockade during neuraxial anesthesia. In dorsal horn neurons, local anesthetics are known to inhibit transient but not sustained K+ currents. The authors characterized the effects of local anesthetics on K+ currents of isolated DRG neurons. Methods The effects of lidocaine, bupivacaine, and tetracaine on K+ currents in isolated rat DRG neurons were measured with use of a whole cell patch clamp method. The currents measured were fast-inactivating transient current (IAf), slow-inactivating transient current (IAs), and noninactivating sustained current (IKn). Results One group of cells (type 1) expressed IAf and IKn. The other group (type 2) expressed IAs and IKn. The diameter of type 2 cells was smaller than that of type 1 cells. Lidocaine and bupivacaine inhibited all three K+ currents. Tetracaine inhibited IAs and IKn but not IAf. For bupivacaine, the concentration for half-maximal inhibition (IC50) of IKn in type 2 cells was lower than that for IKn in type 1 cells (57 vs. 121 &mgr;m). Similar results were obtained for tetracaine (0.6 vs. 1.9 mm) and for lidocaine (2.2 vs. 5.1 mm). Conclusions Local anesthetics inhibited both transient and sustained K+ currents in DRG neurons. Because K+ current inhibition is known to potentiate local anesthetic–induced impulse inhibition, the lower IC50 for IKn of small type 2 cells may reflect preferential inhibition of impulses in nociceptive neurons. The overall modulatory actions of local anesthetics probably are determined by their differential effects on presynaptic (DRG) and postsynaptic (dorsal horn neurons) K+ currents.

Negative inotropic effects of isoflurane and halothane in rabbit papillary muscles

The possibility that the negative inotropic effect of isoflurane is primarily due to a competitive inhibition of the influx of extracellular Ca2+ with little effect on the availability of Ca2+ stored intracellularly in the sarcoplasmic reticulum was examined in rabbit papillary muscle. The negative inotropic effect of isoflurane (1.4%) on steady state contractions (primarily dependent on the influx of extracellular Ca2+) was significantly greater than that on potentiated-state contractions (primarily dependent on Ca2+ released from the sarcoplasmic reticulum). In previous work from this laboratory we found that halothane has an opposite effect in this regard. Increasing stimulation frequency in the presence of isoproterenol (0.1, 1 μM) completely reversed the negative inotropic effect of isoflurane (1.4%) but not that of halothane (0.6%). These results suggest that isoflurane inhibits Ca2+ influx with little effect on the availability of activator Ca2+ stored in and released from the sarcoplasmic reticulum, and that the effect of isoflurane but not that of halothane can be effectively counteracted by conditions that are known to increase Ca2+ influx in the absence of an anesthetic. These properties of isoflurane may in part account for the minimal myocardial depressant effect of the anesthetic on the intact heart in the presence of a functional autonomic system.

Effects of Bupivacaine and Lidocaine on Av Conduction in the Isolated Rat Heart: Modification by Hyperkalemia

The intrinsic cardiotoxicities of bupivacaine and lidocaine were examined in the isolated, perfused rat heart. The perfusates contained no protein and were equilibrated with a gas mixture of 95 per cent O2 and 5 per cent CO2. Autonomic activity, competitive binding, and postseizure hypoxia and acidosis were absent in this experimental model. The effects of the two local anesthetics were evaluated at normokalemia (5.9 mEq/l) and hyperkalemia (9.0 mEq/l). For normokalemia, the ratio of the potency of bupivacaine to that of lidocaine was 14 for slowing ventricular rate to 50 per cent of control, 6 for slowing atrial rate to 50 per cent of control, and 17 for doubling of the PR interval. The action of bupivacaine to slow ventricular rate was due to an inhibitory effect on both AV conduction and atrial rate. For lidocaine, ventricular slowing was mediated mainly by an inhibition of atrial rate with decreased AV conduction playing a minor role. Hyperkalemia of 9.0 mEq/l had little effect on heart rate or AV conduction in the absence of bupivacaine or lidocaine. It did, however, greatly potentiate the effect of both local anesthetics to slow ventricular rate. For bupivacaine, ventricular slowing to 50 per cent of control during hyperkalemia was accomplished almost entirely via an inhibition of AV conduction, while for lidocaine it occurred because of inhibition of both AV conduction and atrial rate. Regardless of the mechanism, hyperkalemia of this degree increased the ventricular slowing effect of both bupivacaine and lidocaine.

Functional properties of ryanodine receptors from rat dorsal root ganglia

The properties of ryanodine receptors (RyRs) from rat dorsal root ganglia (DRGs) have been studied. The density of RyRs (B max) determined by [3H]ryanodine binding was 63 fmol/mg protein with a dissociation constant (K d) of 1.5 nM. [3H]Ryanodine binding increased with caffeine, decreased with ruthenium red and tetracaine, and was insensitive to millimolar concentrations of Mg2+ or Ca2+. DRG RyRs reconstituted in planar lipid bilayers were Ca2+‐dependent and displayed the classical long‐lived subconductance state in response to ryanodine; however, unlike cardiac and skeletal RyRs, they lacked Ca2+‐dependent inactivation. Antibodies against RyR3, but not against RyR1 or RyR2, detected DRG RyRs. Thus, DRG RyRs are immunologically related to RyR3, but their lack of divalent cation inhibition is unique among RyR subtypes.

Negative inotropic effect of ketamine in rabbit ventricular muscle.

The effect of ketamine on myocardial contractile force was examined in rabbit papillay muscles in order to determine the underlying mechanism of action of the anesthetic. Ketamine HCl (20 and 40 mg/L) inhibited rested-state contractions that are dependent on the transsarcolemmal influx of Ca2+ for activation and reduced the upstroke velocity of the slow action potential, which reflects Ca2+ influx through the slow Ca2+ channel. On the other hand, ketamine had a relatively small effect on potentiated-state contractions and no effect on rapid cooling induced contractures, both of which are activated by the release of Ca2+ stored in the sarcoplasmic reticulum. These results suggest that ketamine inhibition of transsarcolemmal Ca2+ influx plays a major role in the negative inotropic action of the anesthetic.

Direct effect of halothane and isoflurane on the function of the sarcoplasmic reticulum in intact rabbit atria.

The negative inotropic effect of halothane and isoflurane on potentiated-state contractions of isolated rabbit atria in a normal Ca2+ (2.5 mM) medium was compared with the force depression in low Ca2+ media without an anesthetic. When this comparison was made in the presence of 1 microM ryanodine so that the force of contraction was dependent only upon transsarcolemmal Ca2+ influx with no Ca2+ contribution from the sarcoplasmic reticulum (SR), the force of contraction was depressed equally by 0.6% halothane in a normal Ca2+ medium and by a 1.5 mM Ca2+ medium without the anesthetic. Similarly, 1.0% halothane or 1.5% isoflurane and a 1.0 mM Ca2+ medium were equally depressant as were 2.4% isoflurane and a 0.5 mM Ca2+ medium. In the absence of ryanodine, where the atrial contractile activity is largely dependent on Ca2+ released from the SR, 0.6% halothane in the normal Ca2+ medium depressed contractile force by 32%, whereas the force was depressed by only 16% in the 1.5 mM Ca2+ medium without the anesthetic. Similar results were obtained when the effects of 1.0% halothane and of 1.0 mM Ca2+ were compared. In contrast, the force of contraction measured in the absence of ryanodine was not at all inhibited by 1.5% isoflurane and minimally (11%) inhibited by 2.4% isoflurane. Consequently, the force depression by isoflurane was less than that found in the low Ca2+ media.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of a major hydrophobic protein of the mitochondrial inner membrane.

Abstract A protein of molecular weight 29,000 has been isolated from the mitochondrial inner membrane. It is a major component of Rackers hydrophobic protein mixture and is also rather selectively released from the inner membrane by lysolecithin treatment. Data indicate that the 29,000 component may be as much as 10% of the total protein of the inner membrane.

Effect of lysolecithin treatment on the structure and functions of the mitochondrial inner membrane

Abstract Lysolecithin treatment of electron transport particles (ETP) generated membrane fragments capable of catalyzing ATP- 32 P i exchange, which was resistant to the uncoupling action of Valinomycin plus Nigericin or Valinomycin plus Monensin A in the presence of K + . Electron micrographs of ultrathin, positively stained sections of lysolecithin treated ETP were virtually devoid of circular patterns characteristic of closed vesicles. The results suggest that the closed vesicular structure of the mitochondrial inner membrane demanded by the chemiosmotic hypothesis of energy transduction (1) may not be essential for the ATP- 32 P i exchange reaction.

Differences in the myocardial depressant action of thiopental and halothane.

The negative inotropic effects of thiopental (10–30 mg/L) and halothane (0.5–1.5%) were compared in rabbit papillary muscles under various stimulation conditions to gain insight into the action of these anesthetics on the availability of Ca2+ for the activation of myocardial contractile activity. The negative inotropic effect of thiopental was more pronounced at short (0.5 sec) than at long (1 sec) beat-to-beat intervals under steady-state conditions, and thiopentals effect on potentiated state contractions was less than that on steady-state contractions. For all variables studied, the effect of halothane was opposite that of thiopental. These results suggest that thiopental reduces the influx of extracellular Ca2+ and the amount of Ca2+ in sarcolemmal sites and slows the transport of intracellular Ca2+ within the sar-coplasmic reticulum from sites of uptake to sites of release without markedly diminishing the amount of intracellular Ca2+ Halothane does not appreciably affect the transport but does diminish the amount of Ca2+ within the sarco-plasmic reticulum that is available for the activation of myocardial contractile activity.