Ichiro Uchida

Evidence for the involvement of GABAA receptor blockade in convulsions induced by cephalosporins

There is accumulating evidence that most beta-lactam antibiotics (i.e., cephalosporins and penicillins) have some degree of convulsive activity, both in laboratory animals as well as in clinical settings. The proposed mechanism is suppression of inhibitory postsynaptic responses, mainly mediated by gamma-amino butyric acid (GABA)(A)-receptors (GABA(A)-R). However, comprehensive studies on the convulsive activities of various beta-lactam antibiotics in vivo and in vitro have not been performed. We have therefore examined the convulsive activities of seven different cephalosporins using both in vivo and in vitro models: intracerebroventricular (ICV) administration in mouse; [(3)H]muscimol binding assay (BA) in mouse brain synaptosome; and inhibition of recombinant mouse alpha1beta2gamma2s GABA(A)-Rs in Xenopus oocyte (GR). The rank orders of convulsive activities in mouse (cefazolin>cefoselis>cefotiam>cefpirome>cefepime>ceftazidime>cefozopran) correlated with those of inhibitory potencies on [(3)H]muscimol binding and GABA-induced currents of GABA(A)-R in vitro, with correlation coefficients of ICV:GR, ICV:BA and BA:GR of 0.882, 0.821 and 0.832, respectively. In contrast, none of the antibiotics had affinities for N-methyl-D-aspartate (NMDA) receptors nor facilitatory actions on NMDA receptor-mediated current in oocytes. These results clearly demonstrate that the mechanism of cephalosporin-induced convulsions is mediated predominantly through the inhibition of GABA(A)-R function and not through NMDA receptor modulation.

Prolongation of canine epidural anesthesia by liposome encapsulation of lidocaine.

The purpose of our study was to produce a long-acting lidocaine by using a liposome that would entrap the drug. Egg yolk phosphatidylcholine and cholesterol were used as liposome materials. After epidural administration, the pharmacodynamics and pharmacokinetics of liposomal and free lidocaine were studied in 20 dogs. Two percent liposomal or free lidocaine (3.0 mL) was injected into the lumbar epidural space. Nerve blocking effects were estimated by measuring somatosensory evoked potentials. Recovery time from the epidural block in the liposomal lidocaine group (170 +/- 49.5 min) was approximately three times longer than that in the free lidocaine group (61 +/- 18.1 min). The areas under the drug concentration-time curves (AUC0-infinity) and time to maximal concentration (Tmax) in the liposomal lidocaine group were significantly larger than those in the free lidocaine group. These results suggest that the prolongation of epidural blockade by liposomal lidocaine is caused by a slow release of the drug from liposomes. The present study suggests that liposomal lidocaine can be used as a long-acting local anesthetic.

Local anaesthetics have different mechanisms and sites of action at the recombinant N‐methyl‐D‐aspartate (NMDA) receptors

Although the principal pharmacological targets of local anaesthetics (LAs) are voltage‐gated Na+ channels, other targets have also been suggested. Here we examined the effects of LAs on the N‐methyl‐D‐aspartate (NMDA) receptor, a receptor involved in the process of nociception. LAs (bupivacaine, lidocaine, procaine, and tetracaine) reversibly and concentration‐dependently inhibited recombinant ε1/ζ1 and ε2/ζ1 NMDA receptors expressed in Xenopus oocytes (IC50s for bupivacaine, lidocaine, procaine, and tetracaine were 1032.0, 1174.1, 642.1 and 653.8 μM at the ε1/ζ1 receptor; and 1090.8, 1821.3, 683.0 and 662.5 μM respectively (at the ε2/ζ1 receptor). Bupivacaine and procaine were non‐competitive antagonists; bupivacaine possesses non‐competitive and competitive actions when interacting with glycine, whereas procaine has only non‐competitive action. Mutation of asparagine residue at position 598 (Asp598) in the ζ1 subunit, a residue associated with the blockade site for Mg2+ and ketamine, to glutamine or arginine reduced the sensitivity to procaine but not to bupivacaine. Thus, procaine may interact with sites of action that are closely related to those of Mg2+ and ketamine blockade. These results suggest that LAs inhibit the NMDA receptor by various mechanisms.

The diverse actions of volatile and gaseous anesthetics on human-cloned 5-hydroxytryptamine3 receptors expressed in Xenopus oocytes

Background General anesthetics can modulate the 5-hydroxytryptamine type 3 (5-HT3) receptor, which may be involved in processes mediating nausea and vomiting, and peripheral nociception. The effects of the new volatile anesthetic sevoflurane and the gaseous anesthetics nitrous oxide (N2O) and xenon (Xe) on the 5-HT3 receptor have not been well-characterized. Methods Homomeric human-cloned 5-HT3A receptors were expressed in Xenopus oocytes. The effects of halothane, isoflurane, sevoflurane, N2O, and Xe on 5-HT-induced currents were studied using a two-electrode, voltage clamping technique. Results Halothane (1%) and isoflurane (1%) potentiated 1 &mgr;m 5-HT–induced currents to 182 ± 12 and 117 ± 2%, respectively. In contrast, sevoflurane (1%), N2O (70%), and Xe (70%) inhibited 5-HT–induced currents to 76 ± 1, 77 ± 4, and 34 ± 4%, respectively. The inhibitory effects were noncompetitive for sevoflurane and competitive for N2O and Xe. None of these inhibitory effects showed voltage dependency. Conclusion Inhalational general anesthetics produce diverse effects on the 5-HT3 receptor. Both halothane and isoflurane enhanced 5-HT3 receptor function in a concentration-dependent manner, which is consistent with previous studies. Sevoflurane inhibited the 5-HT3 receptor noncompetitively, whereas N2O and Xe inhibited the 5-HT3 receptor competitively, suggesting the inhibitory mechanism of sevoflurane might be different from those of N2O and Xe.

The agonistic action of pentobarbital on GABAA β-subunit homomeric receptors

Murine γ-aminobutyric acid type A (GABAA) receptor β1, β2, and β3 subunits were expressed in Xenopus oocytes and studied using the two electrode voltage clamp technique. Although all three β-subunits were unresponsive to GABA when expressed as homomers, the intravenous general anaesthetics pentobarbital, etomidate and propofol induced currents in β2 and β3 homomers. The pentobarbital-induced currents in β3 homomers showed a dose dependence with an ED50 of 89 ± 8.9 μM and a Hill coefficient of 0.94 ± 0.08. Zinc (50 μM) blocked (61.1 ± 5.6% of control) and 200 μM lanthanum potentiated (139 ± 8.6% of control) the pentobarbital-induced current. This current was also blocked by picrotoxin but was insensitive to the GABAA receptor antagonist bicuculline. These observations indicate that the full expression of the agonistic action of GABA requires the presence of an α-subunit, in contrast to the agonistic action of intravenous general anesthetics, where the presence of a β2 or β3-subunit is sufficient. The difference in the agonistic action of intravenous anaesthetics among these highly homologous β-subunits suggests that the β-subunit homomeric receptors may be useful to further define the molecular sites of action of intravenous general anaesthetics and other functional domains on GABAA receptors.

The β-lactam antibiotics, penicillin-G and cefoselis have different mechanisms and sites of action at GABAA receptors

The action of the β‐lactam antibiotics, penicillin‐G (PCG) and cefoselis (CFSL) on GABAA receptors (GABAA‐R) was investigated using the two‐electrode voltage clamp technique and Xenopus oocyte expressed murine GABAA‐R. Murine GABAA‐Rs were expressed in Xenopus oocytes by injecting cRNA that encoded for each subunit (α1, β2, and γ2) and the effects of PCG and CFSL on the α1β2γ2s subunit receptors were examined using two‐electrode voltage clamp. Using the α1β2γ2s GABAA‐R, PCG and CFSL inhibited GABA‐induced currents in a concentration‐dependent manner, with IC50s of 557.1±125.4 and 185.0±26.6 μM, respectively. The inhibitory action of PCG on GABA‐induced currents was non‐competitive whereas that of CFSL was competitive. Mutation of tyrosine to phenylalanine at position 256 in the β2 subunit (β2Y256F), which is reported to abolish the inhibitory effect of picrotoxin, drastically reduced the potency of PCG (IC50=28.4±1.42 mM) for the α1β2Y256Fγ2s receptor without changing the IC50 of CFSL (189±26.6 μM). These electrophysiological data indicate that PCG and CFSL inhibit GABAA‐R in a different manner, with PCG acting non‐competitively and CFSL competitively. The mutational study indicates that PCG might act on an identical or nearby site to that of picrotoxin in the channel pore of the GABAA‐R.

The α and γ subunit-dependent effects of local anesthetics on recombinant GABAA receptors

Although convulsions due to local anesthetic systemic toxicity are thought to be due to inhibition of GABA(A) receptor-linked currents in the central nervous system, the mechanism of action remains unclear. We therefore examined the effects of local anesthetics on gamma-aminobutyric acid (GABA)-induced currents using recombinant GABA(A) receptors with specific combinations of subunits. Murine GABA(A) receptors were expressed by injection of cRNAs encoding each subunit into Xenopus oocytes. The effects of local anesthetics (lidocaine, bupivacaine, procaine and tetracaine) on GABA-induced currents of receptors expressing different subunit combinations (alpha1beta2, alpha1beta2gamma2s, alpha4beta2gamma2s and beta2) were examined via the two electrode voltage clamp method. At alpha1beta2, alpha1beta2gamma2s and alpha4beta2gamma2s GABA(A) receptors, all local anesthetics inhibited GABA-induced currents in a dose-dependent manner. The presence of the gamma2s subunit resulted in a greater inhibition by all local anesthetics, but the presence of the alpha4 subunit resulted in less inhibition. At beta2 homomeric receptors, local anesthetics directly induced an outward current similar to that of picrotoxin. These data indicated that (1) the alpha and gamma subunits of GABA(A) receptors modulated the inhibitory effects of local anesthetics on GABA(A) function, and (2) local anesthetics can activate the beta2 subunit and may block the GABA(A) receptor channel pore.

Nitrous oxide and xenon inhibit the human (α7)5 nicotinic acetylcholine receptor expressed in Xenopus oocyte

The neuronal nicotinic acetylcholine (nACh) receptor is one of the ligand-gated ion channels that regulate the synaptic release of neurotransmitters in the central nervous system. Recently, neuronal nACh receptors have received attention as a potential target for general anesthetics because many general anesthetics inhibit their functions at clinical concentrations. Several general anesthetics are known to inhibit the homomeric (&agr;7)5 nACh receptor, a subtype of neuronal nACh receptors, but the effects of two gaseous anesthetics, nitrous oxide (N2O) and xenon (Xe), remain unknown. Using the two-electrode voltage-clamping technique, we investigated the effects of N2O and Xe at the human (&agr;7)5 nACh receptor expressed in Xenopus oocytes. At clinically relevant concentrations, N2O and Xe reversibly inhibited the ACh-induced currents of the (&agr;7)5 nACh receptor in a concen-tration-dependent manner. The inhibitory actions of both anesthetics at the (&agr;7)5 nACh receptor were noncompetitive and voltage-independent. Our results suggest that inhibition of the (&agr;7)5 nACh receptor by N2O and Xe may play a role in their anesthetic effects.

Gamma subunit dependent modulation by nitric oxide (no) in recombinant Gabaa receptor

The effect of nitric oxide (NO) on GABA induced Cl− current of recombinant GABAA receptors was studied. Either α1β2γ2s or α1β2 subunit mRNAs synthesized from cDNA of mouse brain were injected into Xenopus oocytes and functional GABAA receptors were expressed. GABA-induced Cl− current was measured with the two electrode voltage clamp technique. The NO donor NOC-18 reduced the GABA-induced Cl− current in the α1β2γ2s subunit receptor in a dose-dependent manner. In α1,β2 subunit receptor, NOC-18 had no effects on GABA-induced currents at low concentrations but showed potentiation at high concentration. These effects were antagonized by the NO extinguisher, carboxy-PTIO. The cGMP analogue 8-Br-cGMP failed to induce NO-like effects. NO directly acts at the GABAA receptor and the γ2s subunit is involved in its action.

Inhibitory effect of glucocorticoids on human-cloned 5-hydroxytryptamine3A receptor expressed in xenopus oocytes.

Background: Methylprednisolone, dexamethasone, and other glucocorticoids have been found effective against nausea and vomiting induced by chemotherapy and surgery. Although the specific 5-hydroxytriptamine3 (5-HT3) receptor antagonists such as ondansetron and ramosetron are used as antiemetics, reports show that the use of 5-HT3 receptor antagonists with some glucocorticoids brings additional effects. Glucocorticoids are reported to be antiemetic. The effect of glucocorticoids on 5-HT3 receptor, however, has not been well characterized. This study was designed to examine whether dexamethasone and methylprednisolone had direct effects on human-cloned 5-HT3A receptor expressed in Xenopus oocytes. Methods: Homomeric human-cloned 5-HT3A receptor was expressed in Xenopus oocytes. The authors used the two-electrode voltage-clamping technique to study the effect of methylprednisolone and dexamethasone on 5-HT-induced current. Results: Both dexamethasone and methylprednisolone concentration-dependently attenuated 5-HT-induced current. Dexamethasone inhibited 2 &mgr;m 5-HT-induced current, which was equivalent to EC30 concentration for 5-HT3A receptor, with an inhibitory concentration 50% of 5.29 ± 1.02 &mgr;m. Methylprednisolone inhibited 2 &mgr;m 5-HT-induced current with an inhibitory concentration 50% of 1.07 ± 0.15 mm. The mode of inhibition with either dexamethasone or methylprednisolone was noncompetitive and voltage-independent. When administered together with the 5-HT3 receptor antagonists, ramosetron or metoclopramide, both glucocorticoids showed an additive effect on 5-HT3 receptor. Conclusion: The glucocorticoids had a direct inhibitory effect on 5-HT3 receptors. The combined effect of glucocorticoids and the 5-HT3 receptor antagonists seems additive.