The diagnosis of brain death.
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Brain Death↗
Biomedical subjects
A T Gray
Publications and source records attributed to A T Gray.
Excessive voltage output?
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Anesthesia, Conduction↗
Localization of the tandem pore domain K+ channel TASK-1 in the rat central nervous system.
Recently, a new family of potassium channels with two pore domains in tandem and four transmembrane segments has been identified. Seven functional mammalian channels have been reported at this time. These channels give rise to baseline potassium currents because they are not gated by voltage and exhibit spontaneous activity at all membrane potentials. Although the physiological role of these ion channels has yet to be determined, three mammalian members of this family (TREK-1, TASK-1, TASK-2) are activated by volatile anesthetics and may therefore contribute to the central nervous system (CNS) depression produced by volatile anesthetics. In this study we used northern blot analysis and immunohistochemical localization to determine the expression of TASK-1 subunits in the CNS. TASK-1 immunoreactivity was prominently found in astrocytes of the hippocampus, in the median eminence, in the choroid plexus, and the granular layer, Purkinje cell layer, and molecular layer of the cerebellum. In the spinal cord, strong TASK-I immunoreactivity was seen in ependymal cells lining the central canal and in white matter. These findings suggest a role for the TASK-1 channel in the production of cerebrospinal fluid and function of hypothalamic neurosecretory cells.
Amino Acid Sequence↗
Amino acid transport system A resembles system N in sequence but differs in mechanism.
Classical amino acid transport System A accounts for most of the Na(+)-dependent neutral amino acid uptake by mammalian cells. System A has also provided a paradigm for short- and long-term regulation by physiological stimuli. We now report the isolation of a cDNA encoding System A that shows close similarity to the recently identified System N transporter (SN1). The System A transporter (SA1) and SN1 share many functional characteristics, including a marked sensitivity to low pH, but, unlike SN1, SA1 does not mediate proton exchange. Transport mediated by SA1 is also electrogenic. Amino acid transport Systems A and N thus appear closely related in function as well as structure, but exhibit important differences in ionic coupling.
Amino Acid Sequence↗
Additive inhibition of nicotinic acetylcholine receptors by corticosteroids and the neuromuscular blocking drug vecuronium.
BACKGROUND: Neuromuscular disorders associated with muscular weakness and prolonged paralysis are common in critically ill patients. Acute myopathy has been described in patients receiving a combination therapy of corticosteroids and nondepolarizing neuromuscular blocking drugs for treatment of acute bronchospasm. The cause of this myopathy is not fully established and may involve drug interactions that perturb neuromuscular transmission. To investigate the interaction of corticosteroids with neuromuscular blocking drugs, the authors determined the effects of methylprednisolone and hydrocortisone alone and in combination with vecuronium on fetal (gamma-subunit containing) and adult (epsilon-subunit containing) subtypes of the muscle-type nicotinic acetylcholine receptor. METHODS: Functional channels were expressed in Xenopus laevis oocytes and activated with 1 microM acetylcholine. The resulting currents were recorded using a whole cell two-electrode voltage clamp technique. RESULTS: Both forms of the muscle-type acetylcholine receptor were potently inhibited by methylprednisolone and hydrocortisone, with concentrations producing 50% inhibition in the range of 400-600 microM and 1-2 mM, respectively. The corticosteroids produced noncompetitive antagonism of the muscle-type nicotinic acetylcholine receptor at clinical concentrations. Both receptor forms were also inhibited, even more potently, by vecuronium, with a concentration producing 50% inhibition in the range of 1-2 nM. Combined application of vecuronium and methylprednisolone showed additive effects on both receptor forms, which were best described by a two-site model, with each site independent. CONCLUSIONS: The enhanced neuromuscular blockade produced when corticosteroids are combined with vecuronium may augment pharmacologic denervation and contribute to the pathophysiology of prolonged weakness observed in some critically ill patients.
Adrenal Cortex Hormones↗
Volatile anesthetics activate the human tandem pore domain baseline K+ channel KCNK5.
BACKGROUND: Previous studies have identified a volatile anesthetic-induced increase in baseline potassium permeability and concomitant neuronal inhibition. The emerging family of tandem pore domain potassium channels seems to function as baseline potassium channels in vivo. Therefore, we studied the effects of clinically used volatile anesthetics on a recently described member of this family. METHODS: A cDNA clone containing the coding sequence of KCNK5 was isolated from a human brain library. Expression of KCNK5 in the central nervous system was determined by Northern blot analysis and reverse-transcription polymerase chain reaction. Functional expression of the channel was achieved by injection of cRNA into Xenopus laevis oocytes. RESULTS: Expression of KCNK5 was detected in cerebral cortex, medulla, and spinal cord. When heterologously expressed in Xenopus oocytes, KCNK5 currents exhibited delayed activation, outward rectification, proton sensitivity, and modulation by protein kinase C. Clinical concentrations of volatile general anesthetics potentiated KCNK5 currents by 8-30%. CONCLUSION: Human KCNK5 is a tandem pore domain potassium channel exhibiting delayed activation and sensitivity to volatile anesthetics and may therefore have a role in suppressing cellular excitability during general anesthesia.
Anesthetics, Inhalation↗
Kinetic and selectivity differences between rodent, rabbit, and human organic cation transporters (OCT1).
Organic cation transporters play an important role in the absorption, distribution, and elimination of clinical agents, toxic substances, and endogenous compounds. In kidney preparations, significant differences in functional characteristics of organic cation transport between various species have been reported. However, the underlying molecular mechanisms responsible for these interspecies differences are not known. The goal of this study was to determine the kinetics and substrate selectivities of organic cation transporter (OCT1) homologs from mouse, rat, rabbit, and human that may contribute to interspecies differences in the renal and hepatic handling of organic cations. With a series of n-tetraalkylammonium (nTAA) compounds, a correlation between increasing alkyl chain length and affinity for the four OCT1 homologs was observed. However, the apparent affinity constants (K(i)) differed among the species homologs. For the mouse homolog mOCT1, apparent K(i) values ranged from 7 microM for tetrabutylammonium to 2000 microM for tetramethylammonium. In contrast, the human homolog hOCT1 exhibited weaker interactions with the nTAA compounds. Trans-stimulation studies and current measurements in voltage-clamped oocytes demonstrated that larger nTAA compounds were transported at greater rates in oocytes expressing hOCT1, whereas smaller nTAAs were transported at greater rates in oocytes expressing mOCT1 or rOCT1. The rabbit homolog rbOCT1 exhibited intermediate properties in its interactions with nTAAs compared with its rodent and human counterparts. This report demonstrates that the human OCT1 homolog has functional properties distinct from those of the rodent and rabbit OCT1 homologs. The study underscores potential difficulties in extrapolating data from preclinical studies in animal models to humans.
1-Methyl-4-phenylpyridinium↗
Electrophysiological analysis of the substrate selectivity of a sodium-coupled nucleoside transporter (rCNT1) expressed in Xenopus laevis oocytes.
Nucleoside transporters that mediate cellular uptake of therapeutic nucleoside analogs are major determinants of the pharmacokinetic properties of these compounds. Understanding the substrate selectivity of these transporters is critical in the development of therapeutic nucleoside analogs with optimal pharmacokinetic properties, including high oral bioavailability and tissue-specific distribution. In general, substrate selectivity of nucleoside transporters has been evaluated indirectly by inhibition studies. The purpose of this study was to directly measure the transport of nucleoside analogs by the sodium-coupled pyrimidine-selective transporter rCNT1 using electrophysiology methods. We used a two-electrode voltage clamp assay to investigate the substrate selectivity of rCNT1; 19 structurally diverse nucleosides and nucleoside analogs were studied. Uridine-induced currents in voltage-clamped oocytes expressing rCNT1 were sodium-, voltage-, and concentration-dependent (K(0.5) = 21 microM), and were blocked by adenosine. Uridine-induced currents increased approximately 5-fold upon hyperpolarization of membrane potential from -10 to -150 mV. Uridine, thymidine, and cytidine induced currents in rCNT1-expressing oocytes, whereas guanosine, inosine, and adenosine did not. Uridine, deoxyuridine, and cytidine analogs with modifications at the 3-, 4-, or 5-position were found to be substrates of rCNT1, whereas uridine and cytidine analogs modified at the 6-position were not. In addition, it was found that the 5'-hydroxyl group of the sugar is not required for transport by rCNT1. These results enhance our understanding of the structural basis for substrate selectivity of nucleoside transporters and should prove useful in the development of therapeutic nucleoside analogs.
Animals↗
TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family.
Potassium channels are found in all mammalian cell types, and they perform many distinct functions in both excitable and non-excitable cells. These functions are subserved by several different families of potassium channels distinguishable by primary sequence features as well as by physiological characteristics. Of these families, the tandem pore domain potassium channels are a new and distinct class, primarily distinguished by the presence of two pore-forming domains within a single polypeptide chain. We have cloned a new member of this family, TWIK-2, from a human brain cDNA library. Primary sequence analysis of TWIK-2 shows that it is most closely related to TWIK-1, especially in the pore-forming domains. Northern blot analysis reveals the expression of TWIK-2 in all human tissues assayed except skeletal muscle. Human TWIK-2 expressed heterologously in Xenopus oocytes is a non-inactivating weak inward rectifier with channel properties similar to TWIK-1. Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in their response to quinidine, quinine, and barium. TWIK-2 is inhibited by intracellular, but not extracellular, acidification. This new clone reveals the existence of a subfamily in the tandem pore domain potassium channel family with weak inward rectification properties.
Amino Acid Sequence↗
Local anesthetic inhibition of baseline potassium channels with two pore domains in tandem.
BACKGROUND: Recently, a new structural family of potassium channels characterized by two pore domains in tandem within their primary amino acid sequence was identified. These tandem pore domain potassium channels are not gated by voltage and appear to be involved in the control of baseline membrane conductances. The goal of this study was to identify mechanisms of local anesthetic action on these channels. METHODS: Oocytes of Xenopus laevis were injected with cRNA from five cloned tandem pore domain baseline potassium channels (TASK, TREK-1, TOK1, ORK1, and TWIK-1), and the effects of several local anesthetics on the heterologously expressed channels were assayed using two-electrode voltage-clamp and current-clamp techniques. RESULTS: Bupivacaine (1 mM) inhibited all studied tandem pore potassium channels, with TASK inhibited most potently. The potency of inhibition was directly correlated with the octanol: buffer distribution coefficient of the local anesthetic, with the exception of tetracaine, to which TASK is relatively insensitive. The approximate 50% inhibitory concentrations of TASK were 709 microM mepivacaine, 222 microM lidocaine, 51 microM R(+)-ropivacaine, 53 microM S(-)-ropivacaine, 668 microM tetracaine, 41 microM bupivacaine, and 39 microM etidocaine. Local anesthetics (1 mM) significantly depolarized the resting membrane potential of TASK cRNA-injected oocytes compared with saline-injected control oocytes (tetracaine 22+/-6 mV rs. 7+/-1 mV, respectively, and bupivacaine 31+/-7 mV vs. 6+/-4 mV). CONCLUSIONS: Local anesthetics inhibit tandem pore domain baseline potassium channels, and they could depolarize the resting membrane potential of cells expressing these channels. Whether inhibition of these channels contributes to conduction blockade or to the adverse effects of local anesthetics remains to be determined.
Anesthetics, Local↗
Assignment of KCNK6 encoding the human weak inward rectifier potassium channel TWIK-2 to chromosome band 19q13.1 by radiation hybrid mapping.
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Chromosomes, Human, Pair 19↗
Baseline K+ channels as targets of general anesthetics: studies of the action of volatile anesthetics on TOK1.
A large body of evidence has accumulated in recent years pointing towards the GABA(A) receptor as a primary determinant of volatile anesthetic action (Franks and Lieb, 1994). Nevertheless, our understanding of the function of the central nervous system (CNS) remains sufficiently incomplete that other mechanisms of CNS depression remain to be examined. We have studied a new family of potassium (K+) channels which function as regulators of the baseline excitability of neuronal tissue. As such they must be considered potential targets for volatile anesthetic action and as a possible mechanism by which volatile anesthetics act to allow patients to undergo noxious surgical stimulation.
Anesthetics, General↗
Molecular cloning and functional expression of a rabbit renal organic cation transporter.
A cDNA encoding an organic cation transporter (rbOCT1) was isolated from rabbit kidney. The cDNA encodes a 554 amino acid protein that is highly homologous to other mammalian organic cation transporters. rbOCT1 mediated 3H-1-methyl-4-phenylpyridinium (3H-MPP+) transport in Xenopus laevis oocytes was saturable, sensitive to membrane potential, and inhibited by various organic cations. rbOCT1 mRNA transcripts are expressed in the kidney, liver, and intestine.
Amino Acid Sequence↗
An open rectifier potassium channel with two pore domains in tandem cloned from rat cerebellum.
Tandem pore domain K+ channels represent a new family of ion channels involved in the control of background membrane conductances. We report the structural and functional properties of a TWIK-related acid-sensitive K+ channel (rTASK), a new member of this family cloned from rat cerebellum. The salient features of the primary amino acid sequence include four putative transmembrane domains and, unlike other cloned tandem pore domain channels, a PDZ (postsynaptic density protein, disk-large, zo-1) binding sequence at the C terminal. rTASK has distant overall homology to a putative Caenorhabditis elegans K+ channel and to the mammalian clones TREK-1 and TWIK-1. rTASK expression is most abundant in rat heart, lung, and brain. When exogenously expressed in Xenopus oocytes, rTASK currents activate instantaneously, are noninactivating, and are not gated by voltage. Because rTASK currents satisfy the Goldman-Hodgkin-Katz current equation for an open channel, rTASK can be classified an open rectifier. Activation of protein kinase A produces inhibition of rTASK, whereas activation of protein kinase C has no effect. rTASK currents were inhibited by extracellular acidity. rTASK currents also were inhibited by Zn2+ (IC50 = 175 microM), the local anesthetic bupivacaine (IC50 = 68 microM), and the anti-convulsant phenytoin ( approximately 50% inhibition at 200 microM). By demonstrating open rectification and open probability independent of voltage, we have established that rTASK is a baseline potassium channel.
Acids↗
Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.
Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.
Animals↗
Oleamide potentiates benzodiazepine-sensitive gamma-aminobutyric acid receptor activity but does not alter minimum alveolar anesthetic concentration.
UNLABELLED: A naturally occurring brain lipid, cis-9,10-octadeceamide--oleamide (OA), is found in increased concentrations in the cerebrospinal fluid of sleep-deprived cats, which suggests that it may be an endogenous sleep-inducing substance. We studied the effects of this fatty-acid derivative on the function of cloned gamma-aminobutyric acid (GABA(A)) receptors expressed in Xenopus oocytes. Oocytes were injected with cRNA synthesized in vitro to express simple GABA(A) receptors (alpha1beta1, alpha3beta1, alpha5beta1, and alpha1beta2 subunit combinations) and receptors in which the GABA-induced chloride currents were potentiated in the presence of benzodiazepines (alpha1beta1gamma2s and alpha1beta2gamma2s subunit combinations). OA only produced significant potentiation of the peak Cl- current when applied with GABA to benzodiazepine-sensitive GABA(A) receptors. The peak currents of the simple GABA(A) receptors in the presence of OA were either unaffected or slightly inhibited by OA, but the overall mean currents were not significantly altered. Oleic acid was also capable of potentiating benzodiazepine-sensitive GABA(A) receptor function. The function of other ligand-gated ion channels, such as the N-methyl-D-aspartate receptor (NR1 + NR2A or 2C) and the 5-HT3 receptor expressed in Xenopus oocytes, were unaffected by OA. Sprague-Dawley rats receiving intraperitoneal injections of oleamide (10, 20, or 100 mg/kg) showed no change in the minimum alveolar anesthetic concentration (MAC) of desflurane required to abolish movement in response to noxious (tail clamp) stimulation (control MAC 6.48% +/- 1.28% atm; 100 mg/kg OA MAC 7.05% +/- 0.42% atm). These results reinforce the view that oleyl compounds may be natural modulators of inhibitory ion channel function, but that these effects contribute little to the central nervous system depression produced by volatile anesthetics as measured by MAC. IMPLICATIONS: The putative sleep-inducing substance, oleamide, potentiates benzodiazepine-sensitive gamma-aminobutyric acid receptor function but does not alter desflurane minimum alveolar anesthetic concentration in rats.
Anesthetics, Inhalation↗
TOK1 is a volatile anesthetic stimulated K+ channel.
BACKGROUND: Volatile anesthetic agents can activate the S channel, a baseline potassium (K+) channel, of the marine mollusk Aplysia. To investigate whether cloned ion channels with electrophysiologic properties similar to the S channel (potassium selectivity, outward rectification, and activation independent of voltage) also are modulated by volatile anesthetic agents, the authors expressed the cloned yeast ion channel TOK1 (tandem pore domain, outwardly rectifying K+ channel) in Xenopus oocytes and studied its sensitivity to volatile agents. METHODS: Standard two-electrode voltage and patch clamp recording methods were used to study TOK1 channels expressed in Xenopus oocytes. RESULTS: Studies with two-electrode voltage clamp at room temperature showed that halothane, isoflurane, and desflurane increased TOK1 outward currents by 48-65% in barium Frog Ringer's perfusate. The concentrations at which 50% potentiation occurred (EC50 values) were in the range of 768-814 microM (0.016-0.044 atm) and had a rank order of potency in atm in which halothane > isoflurane > desflurane. The potentiation of TOK1 by volatile anesthetic agents was rapid and reversible (onset and offset, 1-20 s). In contrast, the nonanesthetic 1,2-dichlorohexafluorocyclobutane did not potentiate TOK1 currents in concentrations up to five times the MAC value predicted by the Meyer-Overton hypothesis based on oil/gas partition coefficients. Single TOK1 channel currents were recorded from excised outside-out patches. The single channel open probability increased as much as twofold in the presence of isoflurane and rapidly returned to the baseline values on washout. Volatile anesthetic agents did not alter the TOK1 single channel current-voltage (I-V) relationship, however, suggesting that the site of action does not affect the permeation pathway of the channel. CONCLUSION: TOK1 is a potassium channel that is stimulated by volatile anesthetic agents. The concentrations over which potentiation occurred (EC50 values) were higher than those commonly used in clinical practice (approximately twice MAC).
Anesthetics, Inhalation↗
An active-site histidine of NR1/2C mediates voltage-independent inhibition by zinc.
Endogenous zinc is an important modulator of ion channels of the central nervous system. To understand mechanisms of zinc inhibition, cloned heteromeric N-methyl-D-aspartate receptors (primary subunit NR1 with secondary subunits NR2A, NR2C or NR2D) were expressed in Xenopus oocytes and studied under two-electrode voltage-clamp. Voltage-independent inhibition of NR1/2A heteromers by nanomolar concentrations of extracellular zinc was observed in barium-containing perfusion solutions. In contrast, voltage-independent zinc inhibition of NR1/2C heteromers occurred with lower affinity. Zinc inhibition data from NR1/2D heteromers was fit well with a voltage-independent one-site model and resembled that previously reported for NR1/2B. Reduction of zinc inhibition of NR1/2C heteromers was seen after labeling with the histidine-modifying reagent diethylpyrocarbonate. This finding suggests that the NR1/2C heteromeric ion channel contains an active-site histidine responsible for zinc inhibition.
Animals↗