A comparison of the effect on QT interval between thiamylal and propofol during anaesthetic induction*.

SUMMARY: The aim of this study was to determine the effect of thiamylal and propofol on heart rate-corrected QT (QTc) interval during anaesthetic induction. We studied 50 patients undergoing lumbar spine surgery. Patients were administered 3 microgxkg(-1) fentanyl and were randomly allocated to receive 5 mgxkg(-1) thiamylal or 1.5 mgxkg(-1) propofol as an induction agent. Tracheal intubation was performed after vecuronium administration. Heart rate, mean arterial pressure, bispectral index score, and 12-lead electrocardiogram were recorded at the following time points: just before (T1) and 2 min after (T2) fentanyl administration; 2 min after anaesthetic administration (T3); 2.5 min after vecuronium injection (T4); and 2 min after intubation (T5). Thiamylal prolonged (p < 0.0001), but propofol shortened (p < 0.0001), the QTc interval.

Ground-water radon anomaly before the kobe earthquake in Japan.

Radon concentration in ground water increased for several months before the 1995 southern Hyogo Prefecture (Kobe) earthquake on 17 January 1995. From late October 1994, the beginning of the observation, to the end of December 1994, radon concentration increased about fourfold. On 8 January, 9 days before the earthquake, the radon concentration reached a peak of more than 10 times that at the beginning of the observation, before starting to decrease. These radon changes are likely to be precursory phenomena of the disastrous earthquake.

The effect of sevoflurane-induced hypotension in combination with acute hypervolaemic haemodilution on middle cerebral artery flow velocity in surgical patients.

BACKGROUND AND OBJECTIVE: This study was carried out to clarify the effect of the combination of acute hypervolaemic haemodilution and hypotensive anaesthesia induced with sevoflurane on human middle cerebral artery flow velocity using transcranial Doppler ultrasonography. METHODS: Thirty patients who were maintained with N2O-O2-sevoflurane anaesthesia undergoing hip surgery were randomly divided into two groups (no controlled hypotension group, Group A, and controlled hypotension group, Group B). Haemodilution was produced by acute preoperative infusion of 1000 mL of hydroxyethylstarch without removing blood in both groups. Mean arterial pressure was maintained at approximately 95 mmHg in Group A and at 55 mmHg for 80 min by increasing the inspired concentration of sevoflurane in Group B. Middle cerebral artery flow velocity was measured before haemodilution, after haemodilution, 80 min after starting hypotension, and 60 min after recovery from hypotension. RESULTS: Middle cerebral artery flow velocity significantly increased in both groups after haemodilution; by 28%, in Group A, P < 0.05 vs. before haemodilution and by 30% vs. before haemodilution in Group B, P < 0.05). During controlled hypotension, it decreased towards the pre-haemodilution value (P < 0.05 vs. after haemodilution). CONCLUSIONS: Sevoflurane-induced hypotension to a mean arterial pressure of 55 mmHg would reduce middle cerebral artery flow that had been increased by acute hypervolaemic haemodilution, such as haematocrit value of 26%, whereas it could preserve the flow in pre-haemodilution condition during normocapnia.

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes.

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.

Protective role of glutathione synthesis in response to oxidized low density lipoprotein in human vascular endothelial cells.

Impairment of endothelial cells by oxidized low density lipoprotein (OxLDL) is believed to be the first step in atherogenesis. It is also believed that oxidative stress/antioxidant imbalance is involved in the cell damage by OxLDL. However, little is known about the interaction between OxLDL and antioxidants. In this study, we show that treatment of human vascular endothelial cells with OxLDL caused a gradual increase of glutathione (gamma-glutamylcysteinyl glycine, GSH) levels in 24 h. OxLDL increased the intracellular levels of reactive oxygen species (ROS) and stimulated the expression of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme for the GSH synthesis, the mitogen-activated protein kinase (MAPK) activity, and the AP-1-DNA binding activity. The luciferase activity of gamma-GCS promoter containing AP-1 site was activated by OxLDL. Collectively, OxLDL induces gamma-GCS expression mediated by AP-1 resulting in an increase of GSH levels. The MAPK activity stimulated by ROS may be involved in the activation of AP-1. The increase in GSH by OxLDL may afford cellular protection against OxLDL-induced oxidative stress.

Sequences on the N-terminus of ACh receptor subunits regulate their assembly.

Mutant alpha subunits of Torpedo acetylcholine receptors (AChR) were constructed and expressed in Xenopus oocytes together with other normal subunits to investigate regions in the subunit that are required for subunit assembly. I have found that chimeric alpha subunits, consisting of the N-terminal extracellular domain of the AChR alpha subunit, followed either by the hydrophobic transmembrane segments of GABAA receptor or glutamate receptor subunits, were still recognized as the AChR subunit and associated with co-expressed other normal AChR subunits, suggesting that this part of the N-terminal extracellular domain contains 'assembly signals'.

The amino acid residues 1-128 in the alpha subunit of the nicotinic acetylcholine receptor contain assembly signals.

Expression of nicotinic acetylcholine receptor (AChR) involves complex processes including assembly of different receptor subunits into hetero-oligomers. To identify the minimal N-terminal region involved in AChR subunit association, we used a dominant negative assay. Co-expression of fragments of the alpha subunit, containing the N-terminal extracellular domain and transmembrane domain 1 (TM 1), with the parental AChR subunits in Xenopus oocytes blocked functional expression of the receptor. In contrast, co-expression of N-terminal extracellular fragments without TM1 failed to inhibit functional expression of AChRs, but altered the functional properties of co-expressed parental AChRs. Furthermore, when these alpha subunit fragments were co-expressed with the beta, gamma, and delta subunits, they were co-immunoprecipitated with a mixture of beta, gamma, and delta subunit specific antibodies. These results suggest that 'assembly signals' are confined to a local structure in the N-terminal extracellular domain. Our findings also indicate that an assembly step may be a target for genetic intervention not only to block the expression of functional receptors, but also to alter the function of the receptor.

A cAMP-dependent Ca2+ signalling pathway at the endplate provided by the gamma to epsilon subunit switch in ACh receptors.

During development of neuromuscular junctions there is a switch in the expression of acetylcholine receptors (AChR) from an embryonic form (gamma-AChR) to an adult form (epsilon-AChR). Studies with gamma- and epsilon-AChRs expressed in Xenopus oocytes showed that the gamma to epsilon subunit switch accelerates rates of desensitization and increases Ca2+ permeability. Site-directed mutagenesis of the gamma and epsilon subunits suggests that these changes are regulated by cAMP-dependent phosphorylation on the epsilon subunit. These results suggest that the gamma to epsilon subunit switch could provide for a cAMP-dependent Ca2+ signalling pathway at the endplate.

Lysophosphatidic acid potentiates ACh receptor currents by G-protein-mediated activation of protein kinase C.

The effect of lysophosphatidic acid (lysoPA) on acetylcholine (ACh)-evoked currents was examined using normal and mutant Torpedo nicotinic ACh receptors expressed in Xenopus oocytes. LysoPA enhanced ACh-evoked currents in a washing time- and dose-dependent manner at concentrations of 0.1-3 microM, reaching a maximum of 210% 30 min after treatment, and instead, higher concentrations of lysoPA potentiated to a lesser extent or inhibited the currents. Dose-response curve to ACh was not affected by treatment with lysoPA. Current potentiation by lysoPA was fully inhibited by a broad G-protein inhibitor, guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS), but not by a Gi/o-protein inhibitor, pertussis toxin (PTX). Additionally, the selective protein kinase C (PKC) inhibitor, GF109203X, blocked the potentiation, although the effect of lysoPA was not affected by the selective cAMP-dependent protein kinase (PKA) inhibitor, H-89, or mitogen-activated protein kinase inhibitor, PD98059. LysoPA (3 microM) enhanced currents to 130% in Ca2+-free extracellular solution, and to 150% still in the mutant ACh receptors lacking PKC phosphorylation sites. The potentiation was also completely blocked by GF109203X. These results indicate that lysoPA potentiates ACh receptor currents by PTX-insensitive G-protein-mediated activation of Ca2+-dependent/-independent PKCs with subsequent phosphorylation of the receptors and by an unknown factor or process activated by PKC activation.

Desensitization of junctional and extrajunctional nicotinic ACh receptors expressed in Xenopus oocytes.

Desensitization of nicotinic acetylcholine receptors (AChRs) was studied using the Xenopus oocyte expression system. Mouse and cat extrajunctional AChRs expressed in oocytes desensitized more slowly than Torpedo AChRs. Substitution of the mouse gamma subunit into the Torpedo AChR reduced the rate of desensitization, making it similar to the mouse AChR. Likewise, substitution of the Torpedo gamma subunit into the mouse extrajunctional AChR increased the rate of desensitization, making it similar to the Torpedo AChR. Furthermore, mouse junctional AChRs in which the gamma subunit was replaced by the epsilon subunit desensitized more rapidly than either mouse or cat extrajunctional AChRs, and resembled Torpedo AChRs. Thus, in addition to other properties, junctional and extrajunctional AChRs differ with respect to desensitization, suggesting a possible role of desensitization in normal development of neuromuscular junction.

Assembly and N-glycosylation of all ACh receptor subunits are required for their efficient insertion into plasma membranes.

Various combinations of synthetic acetylcholine receptor (AChR) subunit mRNAs were injected into Xenopus oocytes, and assembly of incomplete AChRs and their insertion into the plasma membrane was studied. Assembly of incomplete AChRs is not greatly affected by the absence of one or two of the other subunits. In contrast, the membrane insertion of incomplete AChRs is profoundly reduced as compared with complete AChRs. The role of N-glycosylation on the assembly of AChR subunits, and on their insertion into plasma membranes, was also studied by using the Xenopus oocyte expression system and tunicamycin. Assembly of non-N-glycosylated AChR subunits occurs in tunicamycin-treated oocytes, but these subunits remain in intracellular compartments, suggesting that N-glycosylation of AChR subunits is not a prerequisite for receptor assembly, but is required for their efficient insertion into the plasma membrane.

Nicotinic receptors are regulated by protein kinase C activated via a nicotinic receptors-mediated signaling pathway.

The present study was conducted to examine the effect of protein kinase C (PKC) on nicotinic acetylcholine (ACh) receptors expressed in Xenopus oocytes by monitoring single-channel currents. In an outside-out patch-clamp configuration, ACh (1 microM) elicited single channel currents with a slope conductance of 31 pS (control) in normal Torpedo ACh receptors. Activation of PKC via an endogenous phosphatidylinositol signaling pathway elevated the slope conductance to 41 pS, which effect was blocked by the selective PKC inhibitor, staurosporine. Mutant ACh receptor channels, which mimic PKC phosphorylation of the receptors, exhibited a slope conductance of 41 pS. Notably, pretreatment with a higher concentration of ACh (100 microM) caused an increase in the slope conductance of the channels for 1 microM ACh (43 pS), which was the same level as obtained with either PKC activation or mutant ACh receptors, and this effect was also inhibited by staurosporine. In addition, the control slope conductance was reduced by PKC inhibitor peptide (24 pS), which corresponded to that obtained with another mutant ACh receptors lacking PKC phosphorylation sites (18 pS). Mouse muscle ACh receptors were also regulated by the same mechanism. The results of the present study suggest that ACh activates PKC via nicotinic ACh receptors, which alternatively, modulates the properties of the receptor channels.

Antibiotics cause changes in the desensitization of ACh receptors expressed in Xenopus oocytes.

ACh receptors (AChRs) synthesized in Xenopus oocytes which were cultured in medium containing gentamicin desensitized much more rapidly than those expressed in the absence of gentamicin. The effect caused by 24 h incubation in gentamicin could not be reversed by leaving oocytes in culture medium without gentamicin for 24 h. In addition, gentamicin exhibited a direct reversible blocking action on the function of AChRs. Our experiments indicate that antibiotics should be used cautiously in culturing oocytes when studying the function of induced neurotransmitter receptors and ion channels.

A conserved disulfide loop facilitates conformational maturation in the subunits of the acetylcholine receptor.

To examine the structural determinants for the assembly of ligand-gated receptors, we constructed mutant alpha, beta, gamma and delta subunits of the Torpedo acetylcholine receptor (AChR), lacking one of the conserved cysteine residues which forms a 13-amino acid disulfide loop in the amino terminal domain of each subunit. Mutant subunits were co-expressed with complementary wild-type subunits in Xenopus oocytes. Using subunit-specific antisera and monoclonal antibodies that recognize the two distinct alpha-bungarotoxin (alpha-BuTX) sites on the AChR, we were able to distinguish immature subunit associations from conformationally mature AChR complexes. Removal of the disulfide loop on the alpha subunit completely destroyed the formation of the two toxin-binding sites, while removal of the structure on the beta subunit had little effect. While mutant gamma and delta subunits were capable of forming associations (immature assembly) with other subunits, the formation of alpha-BTX sites between alpha and mutant gamma or mutant delta subunits was diminished. Interestingly, assembly of alpha beta gamma subunits remained efficient in the presence of mutant delta subunits, whereas assembly of alpha beta delta subunits was inefficient in the presence of mutant gamma subunits. Thus, these results indicate that the formation of the disulfide loop facilitates the conformational maturation of alpha gamma and alpha delta complexes, which may be conditional for correct subunit coupling in assembling receptors. Furthermore, it seems likely that the correct coupling between the alpha and gamma subunits is the most important step in subunit assembly.

Site-directed mutagenesis of the conserved N-glycosylation site on the nicotinic acetylcholine receptor subunits.

The role of the conserved N-glycosylation site on each subunit of the Torpedo acetylcholine receptor (AChR) in the biogenesis and function of the receptor was examined by expressing site-directed mutant subunits in Xenopus oocytes. Different mutant subunits caused different effects. The most striking effect was seen with the mutant gamma subunit which, when co-expressed with alpha, beta, and delta subunits, caused degradation of all the subunits. N-Glycosylation of the other subunits appears to contribute to stability of the subunits and/or efficient insertion of the receptor into the plasma membrane, but is not required for assembly. The AChRs containing the mutant alpha subunit formed functional ion channels in the plasma membrane and their activity could be blocked by alpha-bungarotoxin (alpha-BuTX). Thus, attachment of a carbohydrate moiety at the conserved N-glycosylation site is not an absolute requirement for the formation of ACh and alpha-BuTX binding sites.

Positive- and negative-regulation of Ca2+ entry through ACh receptor channels by phosphorylation.

During development of mammalian neuromuscular junctions, the expression of ACh receptors (AChRs) switches from an embryonic form to an adult form, which provides a higher Ca2+ permeable channel. The lack of this switch can prevent normal development of neuromuscular junctions. In non-mammalian species, however, the switch appears not to occur. The results of the present study show that Ca2+ entry through non-mammalian AChR channels was controlled by differential phosphorylation rather than a subunit switch. This finding provides support for the hypothesis that localized Ca2+ influx through AChR channels has a critical role in the formation and maintenance of neuromuscular junctions.

Modulation of ACh receptor currents by arachidonic acid.

The present study investigated the effects of arachidonic acid on Torpedo (alpha beta gamma delta) and neuronal nicotinic acetylcholine (ACh) receptors (chick alpha7; rat alpha7, alpha3 beta2, alpha3 beta4, alpha4 beta2, and alpha4 beta4). Arachidonic acid (10 microM) depressed currents through normal Torpedo ACh receptors during treatment and afterward, persistently (>/=30 min) potentiated the currents. The potentiation was blocked by the selective protein kinase C (PKC) inhibitor, GF109203X or PKC inhibitor peptide (PKCI). The depression was not inhibited by any protein kinase inhibitor examined here, but greater in Ca2+-free extracellular solution. Arachidonic acid also potentiated currents through mutant Torpedo ACh receptors lacking PKC phosphorylation sites at Ser333 on the alpha subunit and Ser377 on the delta subunit without depression, but otherwise, it depressed currents through mutant receptors replacing of each Ser by negatively charged amino acid residue, possibly that mimics PKC phosphorylation of the receptors. These results suggest that the depression was due to the direct blocking effect on Ca2+-modulatory sites, which was accelerated under conditions of the receptors phosphorylated by PKC, and that the potentiation was caused by PKC activation, independently of PKC phosphorylation of the receptors. Arachidonic acid reduced currents through chick alpha7 receptors by a mechanism independent of protein kinase activation. In contrast, arachidonic acid potentiated currents through rat alpha7, alpha3 beta2, alpha4 beta2, and alpha4 beta4 receptors, perhaps by the same mechanism as the potentiation observed in Torpedo ACh receptors, although it had no effect on rat alpha3 beta4 receptors. The results of the present study thus demonstrate that arachidonic acid exerts diverse actions on nicotinic ACh receptors by different mechanisms.

Long-lasting enhancement of ACh receptor currents by lysophospholipids.

Lysophosphatidylcholine (LysoPtdCho) and lysophosphatidylethanolamine (LysoPtdEtn), which are formed by phospholipase A2-catalyzed hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn), respectively, are proposed to be involved in protein kinase C (PKC) activation. Their physiological significance, however, remains unclear. We examined the effects of lysoPtdCho and lysoPtdEtn on acetylcholine (ACh) receptor currents using oocytes expressing Torpedo nicotinic ACh receptors. LysoPtdCho enhanced the currents in a washing time- and dose-dependent manner (10 nM-1 microM), reaching a maximum of 191% at 20 min after treatment. The currents were enhanced to a lesser extent at higher concentrations, and instead, inhibited to 81% at 10 microM. Likewise, lysoPtdEtn also potentiated the currents to 200% at 10 microM, although its dose-dependent curve shifted to right as compared with that of lysoPtdCho. The current potentiation was blocked by a PKC inhibitor, PKC inhibitor peptide (PKCI), or removal of extracellular Ca2+. In addition, lysoPtdCho and lysoPtdEtn enhanced the currents in mutant ACh receptors lacking PKC phosphorylation sites on the alpha and delta subunits. These results suggest that lysophospholipids such as lysoPtdCho and lysoPtdEtn potentiated ACh receptor currents by Ca2+-dependent PKC activation, but that this effect did not require PKC phosphorylation of the ACh receptor.

Differential interactions of gentamicin with mouse junctional and extrajunctional ACh receptors expressed in Xenopus oocytes.

The nicotinic acetylcholine receptors (AChRs) from Torpedo electric organ and mouse muscles when expressed in Xenopus oocytes desensitize with different time courses. Initially, the role of cAMP-dependent phosphorylation on the gamma subunits in the different desensitization rates was investigated by expressing normal and mutant AChRs in the oocytes cultured in the presence of gentamicin. Mutant Torpedo AChRs lacking the potential cAMP-dependent phosphorylation sites in the gamma subunit appear to desensitize like normal Torpedo AChRs. Similarly, mutant mouse extrajunctional AChRs containing a newly created phosphorylation site in the gamma subunit appeared to desensitize like normal mouse AChRs, which lack the potential cAMP-dependent phosphorylation site in the gamma subunit. These results suggest that different rates of desensitization between the Torpedo and muscle extrajunctional AChRs are not attributable to differential cAMP-dependent phosphorylation of these AChRs. Subsequently, to determine whether gentamicin used in culturing oocytes differentially interacts with muscle junctional and extrajunctional AChRs, we analyzed rates of current decay following different gentamicin treatments. Both chronic and acute treatment with gentamicin profoundly accelerated the decay of whole-cell currents mediated by both types of AChR. The effect of prolonged gentamicin treatment on junctional AChRs was long lasting when compared to treatment on extrajunctional AChRs. Although the two types of AChR still desensitize differently in the absence of gentamicin, these results suggest that the characteristic desensitization of junctional and extrajunctional AChRs observed previously is largely due to differential interactions of gentamicin with the two types of AChR.