Transcript expression of vesicular glutamate transporters in lumbar dorsal root ganglia and the spinal cord of mice - effects of peripheral axotomy or hindpaw inflammation.

Using specific riboprobes, we characterized the expression of vesicular glutamate transporter (VGLUT)1-VGLUT3 transcripts in lumbar 4-5 (L4-5) dorsal root ganglions (DRGs) and the thoracolumbar to lumbosacral spinal cord in male BALB/c mice after a 1- or 3-day hindpaw inflammation, or a 7-day sciatic nerve axotomy. Sham animals were also included. In sham and contralateral L4-5 DRGs of injured mice, VGLUT1-, VGLUT2- and VGLUT3 mRNAs were expressed in ∼45%, ∼69% or ∼17% of neuron profiles (NPs), respectively. VGLUT1 was expressed in large and medium-sized NPs, VGLUT2 in NPs of all sizes, and VGLUT3 in small and medium-sized NPs. In the spinal cord, VGLUT1 was restricted to a number of NPs at thoracolumbar and lumbar segments, in what appears to be the dorsal nucleus of Clarke, and in mid laminae III-IV. In contrast, VGLUT2 was present in numerous NPs at all analyzed spinal segments, except the lateral aspects of the ventral horns, especially at the lumbar enlargement, where it was virtually absent. VGLUT3 was detected in a discrete number of NPs in laminae III-IV of the dorsal horn. Axotomy resulted in a moderate decrease in the number of DRG NPs expressing VGLUT3, whereas VGLUT1 and VGLUT2 were unaffected. Likewise, the percentage of NPs expressing VGLUT transcripts remained unaltered after hindpaw inflammation, both in DRGs and the spinal cord. Altogether, these results confirm previous descriptions on VGLUTs expression in adult mice DRGs, with the exception of VGLUT1, whose protein expression was detected in a lower percentage of mouse DRG NPs. A detailed account on the location of neurons expressing VGLUTs transcripts in the adult mouse spinal cord is also presented. Finally, the lack of change in the number of neurons expressing VGLUT1 and VGLUT2 transcripts after axotomy, as compared to data on protein expression, suggests translational rather than transcriptional regulation of VGLUTs after injury.

The diverse roles of vesicular glutamate transporter 3.

The expression of vesicular glutamate transporters (VGLUTs) 1 and 2 accounts for the ability of most traditionally accepted excitatory neurons to release glutamate by exocytosis. However, several cell populations (serotonin and dopamine neurons) have been demonstrated to release glutamate in vitro and do not obviously express these transporters. Rather, these neurons express a novel, third isoform that in fact appears confined to neurons generally associated with a transmitter other than glutamate. They include serotonin and possibly dopamine neurons, cholinergic interneurons in the striatum, and GABAergic interneurons of the hippocampus and cortex. Although the physiological role of VGLUT3 remains largely conjectural, several observations in vivo suggest that the glutamate release mediated by VGLUT3 has an important role in synaptic transmission, plasticity, and development.

Thoracic epidural catheter placement via the caudal approach in infants by using electrocardiographic guidance.

UNLABELLED: We examined the success of inserting epidural catheters via the caudal route in infants by using electrocardiographic guidance. A case series of 20 patients with thoracic epidural analgesia was studied. After the induction of general anesthesia, an 18-gauge IV catheter was inserted into the caudal space to allow threading of a 20-gauge epidural catheter. The electrocardiogram (ECG) tracings via the epidural catheter, as well as the surface ECG at the target spine level, were recorded simultaneously with a modified two-channel five-lead ECG system. The epidural catheter was advanced from the caudal space until the tip reached the target level as demonstrated by a match in the configuration of the epidural ECG tracing to that of the surface ECG tracing at the target level. The catheter tip location was verified by postoperative radiographs. All catheter tips were located within two vertebrae of the target level, and satisfactory intraoperative epidural anesthesia was achieved in all subjects. IMPLICATIONS: Epidural electrocardiography may be used to guide the positioning of the thoracic epidural catheter tip via the caudal approach to the appropriate dermatome for optimum analgesia.

Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance.

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released L-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC(50)s for L-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC(50) for L-glutamate to activate the anion conductance, without affecting the EC(50) for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation.

Effects of threo-beta-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5).

D,L-threo-beta-Benzyloxyaspartate (D,L-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.

Thoracic epidural analgesia via the caudal approach in pediatric patients undergoing fundoplication using nerve stimulation guidance.

IMPLICATIONS: Epidural catheter placement using electrical stimulation guidance is an alternative approach for positioning the catheter into the thoracic region via the caudal space. This easily performed clinical assessment provides optimization of catheter tip positioning for achieving effective pain control.

Proton pump inhibitors: a study of GPs' prescribing.

BACKGROUND: There has been a rapid increase in proton pump inhibitor (PPI) prescribing in recent years, and controlling the cost and improving the quality of prescribing is an issue of concern to many GPS: OBJECTIVE: Our aim was to compare GPs' usage of different PPIs and explore how GPs' PPI prescribing changes following the introduction of a cheaper competitor. METHODS: PPI prescribing data (PACT) for 53 GPs, who were selected as regular users of a teaching hospital, were monitored from January 1995 to December 1997. The GPs were located in two adjoining health districts and had been interviewed about influences on their decisions to begin prescribing lansoprazole. The PPI prescribing data were collected for the teaching hospital and the general hospital in the adjoining district. RESULTS: Complete prescribing data were available for 50 GPS: Total PPI prescribing increased throughout the study due mainly to increasing use of the new PPIS: Use of the new PPIs increased from 6 to 24% over 3 years. The proportion of maintenance doses prescribed increased from 3 to 12%. There was a 23-fold difference in total PPI prescribing and an 87-fold difference in lansoprazole prescribing between the highest and lowest prescribers. The uptake of pantoprazole was slower than that of lansoprazole. A rapid increase in the use of lansoprazole by the GPs followed an increase in use in the teaching hospital. CONCLUSION: Hospital prescribing was an important influence on the choice of PPI used by GPS: The wide variation in PPI prescribing suggests that there is scope for improvement in the quality and cost of PPI prescribing.

A model for the topology of excitatory amino acid transporters determined by the extracellular accessibility of substituted cysteines.

Excitatory amino acid transporters (EAATs) function as both substrate transporters and ligand-gated anion channels. Characterization of the transporter's general topology is the first requisite step in defining the structural bases for these distinct activities. While the first six hydrophobic domains can be readily modeled as conventional transmembrane segments, the organization of the C-terminal hydrophobic domains, which have been implicated in both substrate and ion interactions, has been controversial. Here, we report the results of a comprehensive evaluation of the C-terminal topology of EAAT1 determined by the chemical modification of introduced cysteine residues. Our data support a model in which two membrane-spanning domains flank a central region that is highly accessible to the extracellular milieu and contains at least one reentrant loop domain.

Thoracic epidural analgesia via the caudal approach using nerve stimulation in an infant with CATCH22.

PURPOSE: To illustrate insertion of an epidural catheter via caudal route in a small infant under electrical stimulation guidance. CLINICAL FEATURES: A six month old boy, weighting 4.25 kg, with a diagnosis of CATCH22 (Cardiac abnormality/abnormal faces, T cell deficit due to thymic hypoplasia, cleft palate, hypocalcemia due to hypoparathyroidism resulting from 22q11 deletion) was scheduled for fundoplication and gastrostomy tube (G-tube) insertion. A combined light general anesthesia and continuous epidural anesthesia technique was selected. Following induction of general anesthesia and tracheal intubation with 1.5 mg midazolam, 10 microg fentanyl and 10 mg succinylcholine, a 16G intravenous catheter was inserted into the caudal space. A 19G epidural catheter (Arrow Flextip Plus) epidural catheter was then inserted up cranially. A low electrical current (1-10mA) was then applied through the catheter. The level of motor movement was advanced from the lower limb muscles to the upper abdominal muscles as the catheter was threaded cranially. After 19 cm of epidural catheter had been inserted, intercostal muscle movement (T9-10 level) was observed at 4.2mA. The tip of the catheter was later confirmed to be at the T9-10 interspace by radiographical imaging. The patient awakened without distress and the trachea was extubated the same evening. The infant was discharged to the ward next morning with good pain relief from a continuous epidural infusion of bupivacane 0.1% with 1 microg x ml(-1) at 1.6 ml(-1). CONCLUSION: Epidural stimulation may help placement of the epidural catheter at the appropriate dermatome for effective anesthesia and analgesia.

Excitatory amino acid transporters: a family in flux.

As the most predominant excitatory neurotransmitter, glutamate has the potential to influence the function of most neuronal circuits in the central nervous system. To limit receptor activation during signaling and prevent the overstimulation of glutamate receptors that can trigger excitotoxic mechanisms and cell death, extracellular concentrations of excitatory amino acids are tightly controlled by transport systems on both neurons and glial cells. L-Glutamate is a potent neurotoxin, and the inadequate clearance of excitatory amino acids may contribute to the neurodegeneration seen in a variety of conditions, including epilepsy, ischemia, and amyotrophic lateral sclerosis. To establish the contributions of carrier systems to the etiology of neurological disorders, and to consider their potential utility as therapeutic targets, a detailed understanding of transporter function and pharmacology is required. This review summarizes current knowledge of the structural and functional diversity of excitatory amino acid transporters and explores how they might serve as targets for drug design.

Comparison of esophageal, rectal, axillary, bladder, tympanic, and pulmonary artery temperatures in children.

Our objective was to determine the most reliable site for temperature measurement in children. In anesthetized children esophageal temperature readings were closest to those in the pulmonary artery (mean difference 0.1 degree C +/- 0.5 degree C compared with Genius tympanic thermometer (mean difference 0.6 degree C +/- 1.0 degree C), IVAC tympanic thermometer (mean difference 0.8 degree C +/- 1.0 degree C), rectal probe (mean difference 0.7 degree C +/- 1.7 degrees C), bladder probe (mean difference 0.9 degree C +/- 1.4 degrees C), and axillary probe (mean difference 1.3 degrees C +/- 1.3 degrees C).

Identification and characterization of a cDNA encoding a neuronal glutamate transporter from Drosophila melanogaster.

Sodium-dependent glutamate transporters influence neurotransmission in the central nervous system by removing synaptically released glutamate from the extracellular space and by maintaining extracellular glutamate concentrations below neurotoxic levels. In insects, glutamate also serves as the neurotransmitter at the neuromuscular junction, but the mechanism for neurotransmitter clearance at this synapse has not well-established. Here we report that cloning and characterization of a sodium-dependent glutamate transporter, dEAAT, from Drosophila melanogaster. The 479 amino acid dEAAT gene product is 40-50% homologous to mammalian members of this carrier family. A 3.3 kilobase (kb) transcript for dEAAT was detected in adult fly heads and to a lesser extent in bodies by Northern-blot analysis and was also localized to neurons in the central nervous system by in situ hybridization. The transport activity observed following express of dEAAT in Xenopus oocytes or COS-7 cells shows a high affinity for L-glutamate, L-aspartate and D-aspartate, an absolute dependence on external sodium ions, and considerable stereoselectivity for the transport of L-glutamate over D-glutamate. As has been observed for the human carriers, EAAT 4 and EAAT 5, a significant component of the current activated by L-glutamate application to dEAAT-expressing oocytes appears to arise from the activation of a chloride channel associated with the carrier.

Oesophageal, rectal, axillary, tympanic and pulmonary artery temperatures during cardiac surgery.

PURPOSE: The gradient between temperatures measured at different body sites is not constant; one factor which will change this gradient is rapid changes in body temperature. Measurement of this gradient was done in patients undergoing rapid changes in body temperature to establish the best site to measure temperature and to compare two brands of commercial tympanic thermometers. METHOD: A total of 228 sets of temperatures were measured from probes in the oesophagus, rectum, and axilla and from two brands of tympanic thermometer and compared with pulmonary artery (PA) temperature in 18 adults during cardiac surgery. RESULTS: Measurements from the oesophageal site was closest to PA readings (mean difference 0.0 +/- 0.5 degree C) compared with IVAC tympanic thermometer (mean difference -0.3 +/- 0.5 degree C), Genius tympanic thermometer (mean difference -0.4 +/- 0.5 degree C), axillary (mean difference 0.2 +/- 1.0 degrees C) and rectal (mean difference -0.4 +/- 1.0 degree C) readings. When data during cooling were analysed separately, all sites had similar gradients from PA except for rectal, which was larger. On rewarming, oesophageal readings were closest to PA readings; tympanic readings were closer to PA than were rectal or axillary readings. Readings from the two brands of tympanic thermometer were equivalent. CONCLUSION: Oesophageal temperature is more accurate and will reflect rapid changes in body temperature better than tympanic, axillary, or rectal temperature. When oesophageal temperature cannot be measured, tympanic temperature done by a trained operator should become the reading of choice.

A reentrant loop domain in the glutamate carrier EAAT1 participates in substrate binding and translocation.

To investigate the structural determinants underlying transport by the glutamate transporter EAAT1, we mutated each of 24 highly conserved residues (P392 to Q415) to cysteine. A majority of these substituted cysteines react with the sulfhydryl-modifying reagent MTSEA, suggesting that they reside in an aqueous environment. The impermeant reagents MTSES and MTSET react with residues at each end of the domain (A395C and A414C), supporting a model that places these residues near the extracellular surface. Substrates and inhibitors block the reaction between MTS derivatives and A395C, and the cosubstrate, sodium, slows reaction of MTSEA with Y405C and E406C. From these results, we propose that this domain forms a reentrant membrane loop at the cell surface and may comprise part of the translocation pore for substrates and cotransported ions.