Protein Kinase A Is Responsible for the Presynaptic Inhibition of Glycinergic and Glutamatergic Transmissions by Xenon in Rat Spinal Cord and Hippocampal CA3 Neurons.

The effects of a general anesthetic xenon (Xe) on spontaneous, miniature, electrically evoked synaptic transmissions were examined using the "synapse bouton preparation," with which we can clearly evaluate pure synaptic responses and accurately quantify pre- and postsynaptic transmissions. Glycinergic and glutamatergic transmissions were investigated in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xe presynaptically inhibited spontaneous glycinergic transmission, the effect of which was resistant to tetrodotoxin, Cd2+, extracellular Ca2+, thapsigargin (a selective sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor), SQ22536 (an adenylate cyclase inhibitor), 8-Br-cAMP (membrane-permeable cAMP analog), ZD7288 (an hyperpolarization-activated cyclic nucleotide-gated channel blocker), chelerythrine (a PKC inhibitor), and KN-93 (a CaMKII inhibitor) while being sensitive to PKA inhibitors (H-89, KT5720, and Rp-cAMPS). Moreover, Xe inhibited evoked glycinergic transmission, which was canceled by KT5720. Like glycinergic transmission, spontaneous and evoked glutamatergic transmissions were also inhibited by Xe in a KT5720-sensitive manner. Our results suggest that Xe decreases glycinergic and glutamatergic spontaneous and evoked transmissions at the presynaptic level in a PKA-dependent manner. These presynaptic responses are independent of Ca2+ dynamics. We conclude that PKA can be the main molecular target of Xe in the inhibitory effects on both inhibitory and excitatory neurotransmitter release. SIGNIFICANCE STATEMENT: Spontaneous and evoked glycinergic and glutamatergic transmissions were investigated using the whole-cell patch clamp technique in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xenon (Xe) significantly inhibited glycinergic and glutamatergic transmission presynaptically. As a signaling mechanism, protein kinase A was responsible for the inhibitory effects of Xe on both glycine and glutamate release. These results may help understand how Xe modulates neurotransmitter release and exerts its excellent anesthetic properties.

Depression of Synaptic N-methyl-D-Aspartate Responses by Xenon and Nitrous Oxide.

In "synapse bouton preparation" of rat hippocampal CA3 neurons, we examined how Xe and N2O modulate N-methyl-D-aspartate (NMDA) receptor-mediated spontaneous and evoked excitatory post-synaptic currents (sEPSCNMDA and eEPSCNMDA). This preparation is a mechanically isolated single neuron attached with nerve endings (boutons) preserving normal physiologic function and promoting the exact evaluation of sEPSCNMDA and eEPSCNMDA responses without influence of extrasynaptic, glial, and other neuronal tonic currents. These sEPSCs and eEPSCs are elicited by spontaneous glutamate release from many homologous glutamatergic boutons and by focal paired-pulse electric stimulation of a single bouton, respectively. The s/eEPSCAMPA/KA and s/eEPSCNMDA were isolated pharmacologically by their specific antagonists. Thus, independent contributions of pre- and postsynaptic responses could also be quantified. All kinetic properties of s/eEPSCAMPA/KA and s/eEPSCNMDA were detected clearly. The s/eEPSCNMDA showed smaller amplitude and slower rise and 1/e decay time constant (τ Decay) than s/eEPSCAMPA/KA Xe (70%) and N2O (70%) significantly decreased the frequency and amplitude without altering the τ Decay of sEPSCNMDA They also decreased the amplitude but increased the Rf and PPR without altering the τ Decay of the eEPSCNMDA These data show clearly that "synapse bouton preparation" can be an accurate model for evaluating s/eEPSCNMDA Such inhibitory effects of gas anesthetics are primarily due to presynaptic mechanisms. Present results may explain partially the powerful analgesic effects of Xe and N2O. SIGNIFICANCE STATEMENT: We could record pharmacologically isolated NMDA receptor-mediated spontaneous and (action potential-evoked) excitatory postsynaptic currents (sEPSCNMDA and eEPSCNMDA) and clearly detect all kinetic parameters of sEPSCNMDA and eEPSCNMDA at synaptic levels by using "synapse bouton preparation" of rat hippocampal CA3 neurons. We found that Xe and N2O clearly suppressed both sEPSCNMDA and eEPSCNMDA. Different from previous studies, present results suggest that Xe and N2O predominantly inhibit the NMDA responses by presynaptic mechanisms.

Xenon modulates synaptic transmission to rat hippocampal CA3 neurons at both pre- and postsynaptic sites.

KEY POINTS: Xenon (Xe) non-competitively inhibited whole-cell excitatory glutamatergic current (IGlu ) and whole-cell currents gated by ionotropic glutamate receptors (IAMPA , IKA , INMDA ), but had no effect on inhibitory GABAergic whole-cell current (IGABA ). Xe decreased only the frequency of glutamatergic spontaneous and miniature excitatory postsynaptic currents and GABAergic spontaneous inhibitory postsynaptic currents without changing the amplitude or decay times of these synaptic responses. Xe decreased the amplitude of both the action potential-evoked excitatory and the action potential-evoked inhibitory postsynaptic currents (eEPSCs and eIPSCs, respectively) via a presynaptic inhibition in transmitter release. We conclude that the main site of action of Xe is presynaptic in both excitatory and inhibitory synapses, and that the Xe inhibition is much greater for eEPSCs than for eIPSCs. ABSTRACT: To clarify how xenon (Xe) modulates excitatory and inhibitory whole-cell and synaptic responses, we conducted an electrophysiological experiment using the 'synapse bouton preparation' dissociated mechanically from the rat hippocampal CA3 region. This technique can evaluate pure single- or multi-synapse responses and enabled us to accurately quantify how Xe influences pre- and postsynaptic aspects of synaptic transmission. Xe inhibited whole-cell glutamatergic current (IGlu ) and whole-cell currents gated by the three subtypes of glutamate receptor (IAMPA , IKA and INMDA ). Inhibition of these ionotropic currents occurred in a concentration-dependent, non-competitive and voltage-independent manner. Xe markedly depressed the slow steady current component of IAMPA almost without altering the fast phasic IAMPA component non-desensitized by cyclothiazide. It decreased current frequency without affecting the amplitude and current kinetics of glutamatergic spontaneous excitatory postsynaptic currents and miniature excitatory postsynaptic currents. It decreased the amplitude, increasing the failure rate (Rf) and paired-pulse rate (PPR) without altering the current kinetics of glutamatergic action potential-evoked excitatory postsynaptic currents. Thus, Xe has a clear presynaptic effect on excitatory synaptic transmission. Xe did not alter the GABA-induced whole-cell current (IGABA ). It decreased the frequency of GABAergic spontaneous inhibitory postsynaptic currents without changing the amplitude and current kinetics. It decreased the amplitude and increased the PPR and Rf of the GABAergic action potential-evoked inhibitory postsynaptic currents without altering the current kinetics. Thus, Xe acts exclusively at presynaptic sites at the GABAergic synapse. In conclusion, our data indicate that a presynaptic decrease of excitatory transmission is likely to be the major mechanism by which Xe induces anaesthesia, with little contribution of effects on GABAergic synapses.

Modifications of excitatory and inhibitory transmission in rat hippocampal pyramidal neurons by acute lithium treatment.

The acute effects of high-dose Li(+) treatment on glutamatergic and GABAergic transmissions were studied in the "synaptic bouton" preparation of isolated rat hippocampal pyramidal neurons by using focal electrical stimulation. Both action potential-dependent glutamatergic excitatory and GABAergic inhibitory postsynaptic currents (eEPSC and eIPSC, respectively) were dose-dependently inhibited in the external media containing 30-150 mM Li(+), but the sensitivity for Li(+) was greater tendency for eEPSCs than for eIPSCs. When the effects of Li(+) on glutamate or GABAA receptor-mediated whole-cell responses (IGlu and IGABA) elicited by an exogenous application of glutamate or GABA were examined in the postsynaptic soma membrane of CA3 neurons, Li(+) slightly inhibited both IGlu and IGABA at the 150 mM Li(+) concentration. Present results suggest that acute treatment with high concentrations of Li(+) acts preferentially on presynaptic terminals, and that the Li(+)-induced inhibition may be greater for excitatory than for inhibitory transmission.

Chemotherapy using hybrid liposomes along with induction of apoptosis.

We have produced hybrid liposomes (HL) which can be prepared by ultrasonicating a mixture of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(23)dodecyl ether in a buffer solution. The fifty-percent inhibitory concentration (IC50) of HL on the growth of human B lymphoma (RAJI) cells in vitro was determined. The IC50 of HL on the growth of RAJI cells was one half of that of DMPC liposomes. Induction of apoptosis by HL in RAJI cells was verified on the basis of flow cytometric analysis, agarose gel electrophoresis and fluorescence microscopy. Apoptotic DNA was observed in RAJI cells treated with HL. Fluorescence micrographs of RAJI cells after adding HL indicated the induction of apoptosis. The therapeutic effects of HL in vivo were examined using SCID mice inoculated with RAJI cells. Markedly prolonged survival of mice was obtained after treatment with HL. No adverse effects were observed in normal rats in toxicity tests carried out with HL. Clinical applications of HL for patients were examined after the approval of the Bioethics Committee. Remarkable reduction of a solid tumor and prolonged survival for one patient with advanced lymphoma were attained after treatment using HL. Chemotherapy with drug-free HL was established without any side effects for the first time.

Chemotherapy with hybrid liposomes for lymphoma without drugs in vivo.

Hybrid liposomes composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene (n) dodecyl ether (C(12)(EO)(n), n=21 and 25) were prepared with the method of sonication. Clear solution of hybrid liposomes having hydrodynamic diameter of 80-100 nm could be maintained over 3 weeks. Hybrid liposomes induced apoptosis for human lymphoma (MOLT-4 and RAJI) cells in vitro. No toxicity was observed in the rats after intravenously injecting hybrid liposomes in vivo. We clearly demonstrated that a mouse model of lymphoma was established and prolonged survival was obtained in mice models of lymphoma after the treatment with hybrid liposomes without drugs in vivo. The results in this study should support the prolonged survival for patients with lymphoma in clinical applications.

Induction of apoptosis by hybrid liposomes for human breast tumor cells along with activation of caspases.

Highly inhibitory effects of hybrid liposomes on the growth of human breast tumor cells in vitro were obtained. It is worthy to note that induction of apoptosis through activation of caspases by hybrid liposomes was clearly observed.

Two methylene groups in phospholipids distinguish between apoptosis and necrosis for tumor cells.

High inhibitory effects of hybrid liposomes (HL) themselves composed of DMPC/10 mol% C12(EO)23 on the growth of HL-60 cells were obtained without any drug. Induction of apoptosis was obtained by the HL of DMPC/10 mol% C12(EO)23. On the other hand, necrosis was observed for the HL of DLPC/10 mol% C12(EO)23. In the case of DPPC/10 mol% C12(EO)23, neither apoptosis nor necrosis was observed.

[Chemotherapy with hybrid liposomes without any drug in vivo].

Prolonged survival was seen in a carcinoma model in mice intraperitoneally inoculated with B-16 melanoma cells after the intraperitoneal treatment with hybrid liposomes composed of L-alpha-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylenedodecyl ether (C12(EO)n, n = 10 and 23 respectivery) which had a uniform and stable structure. No drug was administered. The therapeutic effects of the single-component liposomes composed of lipids with a variety of hydrophilic head groups and different hydrophobic alkyl chains were investigated. Markedly prolonged survival (248%) of mice was achieved after treatment with DMPC liposomes. However, DMPC liposomes have the disadvantage of an unstable structure, requiring daily sonication. On the other hand, no life-prolonging effects or toxicity occurred with the administration of the other single-component liposome employed in this study. Next, we successfully prepared stable, uniform liposomes composed of 90 mol% DMPC and 10 mol% C12(EO)n (n = 10 and 23, respectively), which have diameters of 70 nm and 100 nm, respectively. Interestingly, prolonged survival (173-186%) of mice was achieved after treatment with hybrid liposomes of 90 mol% DMPC/10 mol% C12(EO)n (n = 10 and 23). Finally, we conducted toxicity tests using normal rats to determine hybrid liposome stability. There were no abnormal findings in blood chemistry or relative organ weights at autopsy of normal rats after hybrid liposome administration. In addition, hybrid liposomes were metabolized in the liver after intravenous administration to normal mice. These results suggest that hybrid liposomes could be used as a new single chemotherapeutic agent in the treatment of carcinoma with no side effects.