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1.
J Neonatal Perinatal Med ; 10(4): 445-450, 2017.
Article in English | MEDLINE | ID: mdl-29286931

ABSTRACT

OBJECTIVES: Federal law mandates states to have policies and procedures to identify newborns exposed to maternal substance use during pregnancy. National guidelines for newborn drug testing are lacking; therefore, procedures are variable and determined by state law and local practices. In Iowa, maternal substance use during pregnancy is considered child abuse and must be reported.The objective of this study was to identify newborn drug testing policies and procedures among birthing hospitals in Iowa. METHODS: This was a cross sectional survey of all birthing hospitals in Iowa identified via the Statewide Perinatal Care Program. An electronic survey was sent to the representative at each affiliated hospital. RESULTS: Sixty-nine of 76 hospitals completed the survey for a 90.8% response rate. Newborn drug testing is ordered in 97.1% of responding hospitals with most testing 25% or less of newborns annually. The majority utilized a risk assessment tool (89.6%), although many (62.7%) also allowed for provider discretion. No hospital performed universal testing of all newborns. 86.6% of hospitals reported all positive newborn drug test results including illicit and/or prescription drugs to child protective services. 35.0% of hospitals notified mothers of the report and 45.5% offered substance abuse services and/or treatment to the mothers. CONCLUSIONS: Most Iowa birthing hospitals perform newborn drug testing and report all positive test results to child protective services. The majority use risk assessment tools. Maternal notification practices and referral for substance use disorder treatment are suboptimal and represent an area for future improvement.


Subject(s)
Hospitals/statistics & numerical data , Neonatal Screening/organization & administration , Organizational Policy , Substance Abuse Detection/statistics & numerical data , Substance-Related Disorders/diagnosis , Child Protective Services , Clinical Decision-Making , Communication , Cross-Sectional Studies , Female , Humans , Infant, Newborn , Iowa , Mothers , Neonatal Screening/legislation & jurisprudence , Risk Assessment , Substance Abuse Detection/legislation & jurisprudence , Substance-Related Disorders/therapy
3.
ISBT Sci Ser ; 11(3): 153-162, 2016 Dec.
Article in English | MEDLINE | ID: mdl-30679944

ABSTRACT

BACKGROUND AND OBJECTIVES: Hematopoietic progenitor cell (HPC) counts from Sysmex hematology analyzers have been shown to correlate with peripheral blood (PB) CD34+ cell counts by flow cytometry. Algorithms utilizing HPC counts to guide stem cell collections have been proposed but rarely tested. This study describes the development and validation of algorithms utilizing HPC and PB CD34+cell counts to predict adequate peripheral blood stem cell (PBSC) collections for chemomobilized and cytokine-mobilized individuals. MATERIALS AND METHODS: Utilizing a test set of 83 PB samples from chemomobilized or cytokine-mobilized PBSC collection patients, PB CD34+ counts were correlated with HPC counts and a receiver operating characteristic curve was constructed. Cut-offs of ≤0.5 HPC/µl and ≥7 HPC/µl were established to maximize sensitivity and specificity for using HPC to predict PB CD34+ ≥ 10 cells/µl. These cut-offs were subsequently validated using a separate prospective validation set of 88 HPC/CD34+ cell sample pairs. RESULTS: Using the algorithms, all patients in the prospective validation data set achieved adequate collections of ≥1 × 106 CD34+ cells/kg, and a 67% reduction in the number of CD34+ cell counts performed was achieved. This lead to a direct cost savings of at least $18,700 USD over a 21-month period (88% reduction in direct costs). CONCLUSION: Use of the algorithms provides significant time and cost savings for the laboratory while accurately predicting (i) timing of PBSC collections to obtain adequate CD34+ product yields for chemomobilized patients and (ii) when to administer plerixafor to cytokine-mobilized patients to improve the likelihood of achieving adequate collections.

4.
Clin Toxicol (Phila) ; 51(2): 83-91, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23387345

ABSTRACT

INTRODUCTION: The increasing abuse of amphetamine-like compounds presents a challenge for clinicians and clinical laboratories. Although these compounds may be identified by mass spectrometry-based assays, most clinical laboratories use amphetamine immunoassays that have unknown cross-reactivity with novel amphetamine-like drugs. To date, there has been a little systematic study of amphetamine immunoassay cross-reactivity with structurally diverse amphetamine-like drugs or of computational tools to predict cross-reactivity. METHODS: Cross-reactivities of 42 amphetamines and amphetamine-like drugs with three amphetamines screening immunoassays (AxSYM(®) Amphetamine/Methamphetamine II, CEDIA(®) amphetamine/Ecstasy, and EMIT(®) II Plus Amphetamines) were determined. Two- and three-dimensional molecular similarity and modeling approaches were evaluated for the ability to predict cross-reactivity using receiver-operator characteristic curve analysis. RESULTS: Overall, 34%-46% of the drugs tested positive on the immunoassay screens using a concentration of 20,000 ng/mL. The three immunoassays showed differential detection of the various classes of amphetamine-like drugs. Only the CEDIA assay detected piperazines well, while only the EMIT assay cross-reacted with the 2C class. All three immunoassays detected 4-substituted amphetamines. For the AxSYM and EMIT assays, two-dimensional molecular similarity methods that combined similarity to amphetamine/methamphetamine and 3,4-methylenedioxymethampetamine most accurately predicted cross-reactivity. For the CEDIA assay, three-dimensional pharmacophore methods performed best in predicting cross-reactivity. Using the best performing models, cross-reactivities of an additional 261 amphetamine-like compounds were predicted. CONCLUSIONS: Existing amphetamines immunoassays unevenly detect amphetamine-like drugs, particularly in the 2C, piperazine, and ß-keto classes. Computational similarity methods perform well in predicting cross-reactivity and can help prioritize testing of additional compounds in the future.


Subject(s)
Amphetamines/analysis , Central Nervous System Stimulants/analysis , Immunoassay/methods , Amphetamines/immunology , Antibody Specificity , Area Under Curve , Central Nervous System Stimulants/immunology , Cross Reactions , Crystallography, X-Ray , Illicit Drugs , Models, Chemical , Models, Molecular , Molecular Conformation , Predictive Value of Tests , ROC Curve , Substance Abuse Detection
5.
Neuropharmacology ; 41(8): 952-64, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11747900

ABSTRACT

gamma-Aminobutyric acid type A (GABA(A)) receptors are an important target for general anesthetics in the central nervous system. Site-directed mutagenesis techniques have identified amino acid residues that are important for the positive modulation of GABA(A) receptors by general anesthetics. In the present study, we investigate the role of an amino acid residue in transmembrane (TM) domain 3 of the GABA(A) receptor beta(2) subunit for modulation by the general anesthetic 2,6-diisopropylphenol (propofol). Mutation of methionine 286 to tryptophan (M286W) in the beta(2) subunit abolished potentiation of GABA responses by propofol but did not affect direct receptor activation by propofol in the absence of GABA. In contrast, substitution of methionine 286 by alanine, cysteine, glutamate, lysine, phenylalanine, serine, or tyrosine was permissive for potentiation of GABA responses and direct activation by propofol. Using propofol analogs of varying molecular size, we show that the beta(2)(M286W) mutation resulted in a decrease in the 'cut-off' volume for propofol analog molecules to enhance GABA responses at GABA(A) alpha(1)beta(2)gamma(2s) receptors. This suggests that mutation of M286 in the GABA(A) beta(2) subunit alters the dimensions of a 'binding pocket' for propofol and related alkylphenol general anesthetics.


Subject(s)
Anesthetics, General/metabolism , Anesthetics, Intravenous/metabolism , Methionine/genetics , Propofol/metabolism , Receptors, GABA-A/physiology , Amino Acid Sequence , Amino Acid Substitution/genetics , Anesthetics, General/chemistry , Anesthetics, Intravenous/chemistry , Animals , Binding Sites/drug effects , Binding Sites/genetics , Cell Line , Dose-Response Relationship, Drug , Etomidate/pharmacology , Humans , Methionine/pharmacology , Methohexital/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Propofol/chemistry , Protein Structure, Tertiary/genetics , Rats , Receptors, GABA-A/genetics , gamma-Aminobutyric Acid/pharmacology
6.
Anesthesiology ; 94(6): 1050-7, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11465597

ABSTRACT

BACKGROUND: Positive modulation of gamma-aminobutyric acid type A (GABAA) receptor function is recognized as an important component of the central nervous system depressant effects of many general anesthetics, including propofol. The role for GABAA receptors as an essential site in the anesthetic actions of propofol was recently challenged by a report that the propofol analog 4-iodopropofol (4-iodo-2,6-diisopropylphenol) potentiated and directly activated GABAA receptors, yet was devoid of sedative-anesthetic effects in rats after intraperitoneal injection. Given the important implications of these findings for theories of anesthesia, the authors compared the effects of 4-iodopropofol with those of propofol using established in vivo and in vitro assays of both GABAA receptor-dependent and -independent anesthetic actions. METHODS: The effects of propofol and 4-iodopropofol were analyzed on heterologously expressed recombinant human GABAA alpha1beta2gamma2 receptors, evoked population spike amplitudes in rat hippocampal slices, and glutamate release from rat cerebrocortical synaptosomes in vitro. Anesthetic potency was determined by loss of righting reflex in Xenopus laevis tadpoles, in mice after intraperitoneal injection, and in rats after intravenous injection. RESULTS: Like propofol, 4-iodopropofol enhanced GABA-induced currents in recombinant GABAA receptors, inhibited synaptic transmission in rat hippocampal slices, and inhibited sodium channel-mediated glutamate release from synaptosomes, but with reduced potency. After intraperitoneal injection, 4-iodopropofol did not produce anesthesia in mice, but it was not detected in serum or brain. However, 4-iodopropofol did produce anesthesia in tadpoles (EC50 = 2.5 +/- 0.5 microM) and in rats after intravenous injection (ED50 = 49 +/- 6.2 mg/kg). CONCLUSIONS: Propofol and 4-iodopropofol produced similar actions on several previously identified cellular and molecular targets of general anesthetic action, and both compounds induced anesthesia in tadpoles and rats. The failure of 4-iodopropofol to induce anesthesia in rodents after intraperitoneal injection is attributed to a pharmacokinetic difference from propofol rather than to major pharmacodynamic differences.


Subject(s)
Anesthetics, Intravenous/pharmacology , Propofol/pharmacology , Receptors, GABA-A/drug effects , Animals , Behavior, Animal/drug effects , Electrophysiology , Excitatory Postsynaptic Potentials/drug effects , Glutamic Acid/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Hypnotics and Sedatives/pharmacology , In Vitro Techniques , Male , Mice , Neurotransmitter Agents/metabolism , Propofol/analogs & derivatives , Rats , Rats, Sprague-Dawley , Recombinant Proteins , Synaptosomes/metabolism , Xenopus laevis
7.
J Pharmacol Exp Ther ; 297(1): 338-51, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11259561

ABSTRACT

A series of 27 analogs of the general anesthetic propofol (2,6-diisopropylphenol) were examined for general anesthetic activity in Xenopus laevis tadpoles and for the ability to produce enhancement of submaximal GABA responses and/or direct activation at recombinant GABA(A) receptors. Fourteen of the propofol analogs produced loss of righting reflex in the tadpoles, whereas 13 were inactive as anesthetics. The same pattern of activity was noted with the actions of the compounds at the GABA(A) alpha(1)beta(2)gamma(2s) receptor. The potencies of the analogs as general anesthetics in tadpoles correlated better with potentiation of GABA responses than direct activation at the GABA(A) alpha(1)beta(2)gamma(2s) receptor. The calculated octanol/water partition coefficients for the analogs did not explain the lack of activity exhibited by the 13 nonanesthetic analogs, although this physicochemical parameter did correlate modestly with in vivo anesthetic potency. The actions of one nonanesthetic analog, 2,6-di-tert-butylphenol, were examined in detail. 2,6-Di-tert-butylphenol was inactive at GABA(A) receptors, did not function as an anesthetic in the tadpoles, and did not antagonize any of the actions of propofol at GABA(A) receptors or in tadpoles. A key influence on the potency of propofol analogs appears to be the size and shape of the alkyl groups at positions 2 and 6 of the aromatic ring relative to the substituent at position 1. These data suggest steric constraints for the binding site for propofol on the GABA(A) receptor.


Subject(s)
Anesthetics, Intravenous/pharmacology , Propofol/pharmacology , Receptors, GABA-A/drug effects , Allosteric Regulation , Animals , Dose-Response Relationship, Drug , Propofol/analogs & derivatives , Propofol/chemistry , Reflex/drug effects , Solubility , Structure-Activity Relationship , Xenopus laevis , gamma-Aminobutyric Acid/pharmacology
8.
Mol Psychiatry ; 5(3): 283-92, 2000 May.
Article in English | MEDLINE | ID: mdl-10889531

ABSTRACT

The dopamine transporter (DAT) provides major regulation of the synaptic levels of dopamine and is a principal target of psychostimulant drugs. Associations between DAT gene polymorphisms and human disorders with possible links to dopaminergic neurotransmission, including attention-deficit/hyperactivity disorder (ADHD) and consequences of cocaine and alcohol administration, have been reported. We now report approximately 60000 bp of genomic sequence containing the entire DAT gene. This sequence was used to amplify each of the 15 DAT gene exons and several introns and analyze these amplification products by single-stranded sequence conformation (SSCP) and/or direct sequencing. These results define silent allelic single nucleotide sequence variants in DAT gene exons 2, 6, 9 and 15. Rare conservative mutations are identified in amino acids encoded by DAT exons 2 and 8. Analyses of the common nucleotide variants and the previously reported VNTR in the non-coding region of exon 15 define the pattern of linkage disequilibrium across the DAT locus. These comprehensive analyses, however, fail to identify any common protein coding DAT sequence variant in more than 150 unrelated individuals free of neuropsychiatric disease, 109 individuals meeting City of Hope criteria for Tourette's syndrome, 64 individuals with DSM-IV diagnoses of ethanol dependence, or 15 individuals with ADHD. These data are consistent with substantial evolutionary conservation of the DAT protein sequence. They suggest that gene variants that alter levels of DAT expression provide the best current candidate mechanism for reported associations between DAT gene markers, ADHD and other more tentatively associated neuropsychiatric disorders.


Subject(s)
Alcoholism/genetics , Attention Deficit Disorder with Hyperactivity/genetics , Carrier Proteins/genetics , Membrane Glycoproteins , Membrane Transport Proteins , Nerve Tissue Proteins , Substance-Related Disorders/genetics , Tourette Syndrome/genetics , Adolescent , Base Sequence , Child , Conserved Sequence , Dopamine Plasma Membrane Transport Proteins , Exons , Genetic Variation , Humans , Introns , Linkage Disequilibrium , Minisatellite Repeats , Polymorphism, Single-Stranded Conformational
9.
Anesthesiology ; 93(2): 510-9, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10910502

ABSTRACT

BACKGROUND: The duration of action for many pharmaceutical agents is dependent on their breakdown by endogenous hydrolytic enzymes. Dietary factors that interact with these enzyme systems may alter drug efficacy and time course. Cholinesterases such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) hydrolyze and inactivate several anesthetic drugs, including cocaine, heroin, esmolol, local ester anesthetics, and neuromuscular blocking drugs. Natural glycoalkaloid toxins produced by plants of the family Solanaceae, which includes potatoes and tomatoes, inhibit both AChE and BuChE. Here the authors assess the extent to which two solanaceous glycoalkaloids (SGAs), alpha-solanine and alpha-chaconine, can alter the effects of neuromuscular blocking drugs and cholinesterase inhibitors in vivo and in vitro. METHODS: Inhibition of purified human AChE and BuChE by SGAs, neuromuscular blocking drugs, and cholinesterase inhibitors was assessed by an in vitro colorimetric cholinesterase assay. In vivo experiments were carried out using anesthetized rabbits to test whether SGAs affect recovery from mivacurium-induced paralysis. RESULTS: SGAs inhibited human BuChE at concentrations similar to those found in serum of individuals who have eaten a standard serving of potatoes. Coapplication of SGAs (30-100 nm) with neuromuscular blocking drugs and cholinesterase inhibitors produced additive cholinesterase inhibition. SGA administration to anesthetized rabbits inhibited serum cholinesterase activity and mivacurium hydrolysis. In addition, SGA prolonged the time needed for recovery from mivacurium-induced paralysis (149 +/- 12% of control; n = 12). CONCLUSIONS: These findings support the hypothesis that inhibition of endogenous enzyme systems by dietary factors can influence anesthetic drug metabolism and duration of action. Diet may contribute to the wide variation in recovery time from neuromuscular blockade seen in normal, healthy individuals.


Subject(s)
Cholinesterase Inhibitors/pharmacology , Isoquinolines/metabolism , Neuromuscular Nondepolarizing Agents/metabolism , Neuromuscular Nondepolarizing Agents/pharmacology , Solanine/analogs & derivatives , Solanine/pharmacology , Animals , Drug Interactions , Female , Humans , Isoquinolines/antagonists & inhibitors , Isoquinolines/blood , Male , Mivacurium , Neuromuscular Nondepolarizing Agents/blood , Rabbits , Solanum tuberosum
10.
Neuropharmacology ; 39(7): 1168-83, 2000 Apr 27.
Article in English | MEDLINE | ID: mdl-10760360

ABSTRACT

A challenge for theories of general anesthesia is the existence of compounds predicted to be anesthetics but which, instead, do not produce anesthesia and often elicit other behavioral effects such as convulsions. This study focused on flurothyl (bis[2,2, 2-trifluoroethyl] ether), a potent volatile convulsant, and its anesthetic isomer, 'iso-flurothyl' (1,1,1,3,3, 3-hexafluoro-2-methoxypropane). The effects of flurothyl and iso-flurothyl were studied using the whole-cell patch-clamp technique on agonist-activated chloride currents in human GABA(A), glycine, and GABA(C) rho(1) receptors expressed in HEK 293 cells. GABA(A) and glycine receptors are promising molecular targets for the actions of inhaled ether general anesthetics. Flurothyl acted as a non-competitive antagonist at GABA(A) alpha(2)beta(1) and alpha(2)beta(1)gamma(2s) receptors, but had no effect at glycine alpha(1) receptors. Flurothyl had biphasic actions on GABA responses at GABA(C) rho(1) receptors. In contrast, iso-flurothyl enhanced ('potentiated') submaximal agonist responses at GABA(A) and glycine receptors, but had no effect on GABA responses at GABA(C) rho(1) receptors. Point mutations in GABA(A) and glycine receptor subunits, which have been previously shown to abolish potentiation of agonist responses by the ether anesthetics enflurane and isoflurane, also ablated potentiation of agonist responses by iso-flurothyl. These same mutations in the GABA(A) receptor had only modest effects on the inhibitory actions of flurothyl. GABA(A) receptors with mutations conferring insensitivity to antagonism by picrotoxin were still inhibited by flurothyl, suggesting that picrotoxin and flurothyl antagonize GABA responses by distinct sites or mechanisms of action. In summary, antagonism of GABA(A) receptors is likely to account for the convulsant effects of flurothyl, while the general anesthetic actions of iso-flurothyl, like those of other ether anesthetics, may be related to positive modulation of GABA(A) and/or glycine receptors.


Subject(s)
Convulsants/pharmacology , Flurothyl/pharmacology , Ion Channel Gating/drug effects , Amino Acid Sequence , Cell Line , Chromatography, Gas , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Electrophysiology , Humans , Ion Channel Gating/genetics , Isomerism , Kidney/drug effects , Kidney/metabolism , Ligands , Membranes/drug effects , Membranes/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed/drug effects , Receptors, GABA-A/drug effects , Receptors, GABA-A/genetics , Receptors, Glycine/drug effects , Receptors, Glycine/genetics , Transfection/genetics
11.
Anesthesiology ; 92(5): 1418-25, 2000 May.
Article in English | MEDLINE | ID: mdl-10781289

ABSTRACT

BACKGROUND: Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are potently inhibited by volatile anesthetics, but it is not known whether they are affected by intravenous anesthetics. Ketamine potentiates gamma-aminobutyric acid type A (GABAA) receptors at high concentrations, but it is unknown whether there is potentiation at clinically relevant concentrations. Information about the effects of intravenous anesthetics with different behavioral profiles on specific ligand-gated ion channels may lead to hypotheses as to which ion channel effect produces a specific anesthetic behavior. METHODS: A heteromeric nAChR composed of alpha4 and beta4 subunits was expressed heterologously in Xenopus laevis oocytes. Using the two-electrode voltage clamp technique, peak ACh-gated current was measured before and during application of ketamine, etomidate, or thiopental. The response to GABA of alpha1beta2gamma2s GABAA receptors expressed in human embryonic kidney cells and Xenopus oocytes was compared with and without coapplication of ketamine from 1 microm to 10 mm. RESULTS: Ketamine caused potent, concentration-dependent inhibition of the alpha4beta4 nAChR current with an IC50 of 0.24 microm. The inhibition by ketamine was use-dependent; the antagonist was more effective when the channel had been opened by agonist. Ketamine did not modulate the alpha1beta2gamma2s GABAA receptor response in the clinically relevant concentration range. Thiopental caused 27% inhibition of ACh response at its clinical EC50. Etomidate did not modulate the alpha4beta4 nAChR response in the clinically relevant concentration range, although there was inhibition at very high concentrations. CONCLUSIONS: The alpha4beta4 nAChR, which is predominantly found in the central nervous system (CNS), is differentially affected by clinically relevant concentrations of intravenous anesthetics. Ketamine, commonly known to be an inhibitor at the N-methyl-D-aspartate receptor, is also a potent inhibitor at a central nAChR. It has little effect on a common CNS GABAA receptor in a clinically relevant concentration range. Interaction between ketamine and specific subtypes of nAChRs in the CNS may result in anesthetic behaviors such as inattention to surgical stimulus and in analgesia. Thiopental causes minor inhibition at the alpha4beta4 nAChR. Modulation of the alpha4beta4 nAChR by etomidate is unlikely to be important in anesthesia practice based on the insensitivity of this receptor to clinically used concentrations.


Subject(s)
Anesthetics, Intravenous/pharmacology , Ion Channel Gating/drug effects , Receptors, GABA-A/drug effects , Receptors, Nicotinic/drug effects , Anesthetics, Dissociative/pharmacology , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Etomidate/pharmacology , Humans , Ketamine/pharmacology , Membrane Potentials/drug effects , Oocytes/drug effects , Patch-Clamp Techniques , Rats , Thiopental/pharmacology , Xenopus
12.
Br J Pharmacol ; 129(4): 731-43, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10683198

ABSTRACT

The actions of 13 general anaesthetics (diethyl ether, enflurane, isoflurane, methoxyflurane, sevoflurane, chloral hydrate, trifluoroethanol, tribromoethanol, tert-butanol, chloretone, brometone, trichloroethylene, and alpha-chloralose) were studied on agonist-activated Cl(-) currents at human GABA(A) alpha(2)beta(1), glycine alpha(1), and GABA(C) rho(1) receptors expressed in human embryonic kidney 293 cells. All 13 anaesthetics enhanced responses to submaximal (EC(20)) concentrations of agonist at GABA(A) and glycine receptors, except alpha-chloralose, which did not enhance responses at the glycine alpha(1) receptor. None of the anaesthetics studied potentiated GABA responses at the GABA(C) rho(1) receptor. Potentiation of submaximal agonist currents by the anaesthetics was studied at GABA(A) and glycine receptors harbouring mutations in putative transmembrane domains 2 and 3 within GABA(A) alpha(2), beta(1), or glycine alpha(1) receptor subunits: GABA(A) alpha(2)(S270I)beta(1), alpha(2)(A291W)beta(1), alpha(2)beta(1)(S265I), and alpha(2)beta(1)(M286W); glycine alpha(1)(S267I) and alpha(1)(A288W). For all anaesthetics studied except alpha-chloralose, at least one of the mutations above abolished drug potentiation of agonist responses at GABA(A) and glycine receptors. alpha-Chloralose produced efficacious direct activation of the GABA(A) alpha(2)beta(1) receptor (a 'GABA-mimetic' effect). The other 12 anaesthetics produced minimal or no direct activation of GABA(A) and glycine receptors. A non-anaesthetic isomer of alpha-chloralose, beta-chloralose, was inactive at GABA(A) and glycine receptors and did not antagonize the actions of alpha-chloralose at GABA(A) receptors. The implications of these findings for the molecular mechanisms of action of general anaesthetics at GABA(A) and glycine receptors are discussed.


Subject(s)
Alcohols/pharmacology , Alkanes/pharmacology , Anesthetics, General/pharmacology , Ethers/pharmacology , Receptors, GABA-A/physiology , Receptors, Glycine/physiology , Anesthetics, Intravenous/pharmacology , Animals , Chloralose/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , GABA-A Receptor Agonists , GABA-A Receptor Antagonists , Humans , Mutagenesis, Site-Directed , Receptors, GABA-A/genetics , Receptors, Glycine/agonists , Receptors, Glycine/antagonists & inhibitors , Receptors, Glycine/genetics , Xenopus laevis
13.
Cell Mol Life Sci ; 55(10): 1278-303, 1999 Aug 15.
Article in English | MEDLINE | ID: mdl-10487207

ABSTRACT

The molecular mechanisms of general anaesthetics have remained largely obscure since their introduction into clinical practice just over 150 years ago. This review describes the actions of general anaesthetics on mammalian neurotransmitter-gated ion channels. As a result of research during the last several decades, ligand-gated ion channels have emerged as promising molecular targets for the central nervous system effects of general anaesthetics. The last 10 years have witnessed an explosion of studies of anaesthetic modulation of recombinant ligand-gated ion channels, including recent studies which utilize chimeric and mutated receptors to identify regions of ligand-gated ion channels important for the actions of general anaesthetics. Exciting future directions include structural biology and gene-targeting approaches to further the understanding of general anaesthetic molecular mechanisms.


Subject(s)
Anesthetics, General/pharmacology , Ion Channel Gating/physiology , Ion Channels/drug effects , Receptors, Cell Surface/drug effects , Animals , Cell Membrane/physiology , Cell Membrane/ultrastructure , Humans , Ion Channels/chemistry , Ion Channels/physiology , Ligands , Mammals , Protein Conformation , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/physiology
14.
Neuropharmacology ; 38(2): 253-65, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10218866

ABSTRACT

The gamma subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) is essential for bestowing both normal single channel conductance and sensitivity to benzodiazepines on native GABA(A)-Rs. The long splice variant of the gamma2 subunit (gamma2L) has been postulated to be essential in mediating the modulatory actions of ethanol at the GABA(A)-R. In order to evaluate this hypothesis, gene targeting was used to delete the 24bp exon which distinguishes gamma2L from the short splice variant (gamma2S). Mice homozygous for this exon deletion (gamma2L-/-) are viable and indistinguishable from wild-type (gamma2L+/+) mice. No gamma2L mRNA was detected in these mice, nor could gamma2L-containing GABA(A)-R protein be detected by specific antibodies. Radioligand binding studies showed the total amount of gamma2 subunit protein to be not significantly changed, suggesting that gamma2S replaces gamma2L in the brains of the knockout animals. Electrophysiological recordings from dorsal root ganglion neurons revealed a normal complement of functional receptors. There was no difference in the potentiation of GABA currents by ethanol (20-200 mM) observed in neurons from gamma2L+/+ or gamma2L-/- mice. Several behavioral effects of ethanol, such as sleep time, anxiolysis, acute functional tolerance, chronic withdrawal hyperexcitability and hyperlocomotor activity were also unaffected by genotype. It is concluded that gamma2L is not required for ethanol's modulatory action at the GABA(A)-R or whole animal behavioral effects.


Subject(s)
Alternative Splicing , Brain/metabolism , Ethanol/pharmacology , Ganglia, Spinal/physiology , Genetic Variation , Neurons/physiology , Receptors, GABA-A/physiology , Animals , Anxiety , Cell Membrane/metabolism , Chimera , Crosses, Genetic , Exons , Female , Flunitrazepam/pharmacokinetics , Macromolecular Substances , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Motor Activity/drug effects , Neurons/drug effects , Radioligand Assay , Receptors, GABA-A/deficiency , Receptors, GABA-A/genetics , Sequence Deletion , Sleep/drug effects , Substance Withdrawal Syndrome , Transcription, Genetic
15.
Mol Pharmacol ; 53(3): 530-8, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9495821

ABSTRACT

Both volatile and intravenous general anesthetics allosterically enhance gamma-aminobutyric acid (GABA)-evoked chloride currents at the GABA type A (GABAA) receptor. Recent work has revealed that two specific amino acid residues within transmembrane domain (TM)2 and TM3 are necessary for positive modulation of GABAA and glycine receptors by the volatile anesthetic enflurane. We now report that mutation of these residues within either GABAA alpha2 (S270 or A291) or beta1 (S265 or M286) subunits resulted in receptors that retain normal or near-normal gating by GABA but are insensitive to clinically relevant concentrations of another inhaled anesthetic, isoflurane. To determine whether receptor modulation by intravenous general anesthetics also was affected by these point mutations, we examined the effects of propofol, etomidate, the barbiturate methohexital, and the steroid alphaxalone on wild-type and mutant GABAA receptors expressed in human embryonic kidney 293 cells. In most cases, these mutations had little or no effect on the actions of these intravenous anesthetics. However, a point mutation in the beta1 subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct activation of the receptor by high concentrations of propofol. These data indicate that the receptor structural requirements for positive modulation by volatile and intravenous general anesthetics may be quite distinct.


Subject(s)
Anesthetics, Inhalation/pharmacology , Anesthetics, Intravenous/pharmacology , Isoflurane/pharmacology , Propofol/pharmacology , Receptors, GABA-A/drug effects , Binding Sites , Cells, Cultured , Drug Synergism , Etomidate/pharmacology , Humans , Methohexital/pharmacology , Mutagenesis, Site-Directed , gamma-Aminobutyric Acid/pharmacology
16.
Neurosci Lett ; 240(2): 81-4, 1998 Jan 09.
Article in English | MEDLINE | ID: mdl-9486477

ABSTRACT

Mice whose gamma-aminobutyric acid type A (GABA(A)) beta3 subunit gene is inactivated ('beta3 knockout mice') have been previously shown to have epilepsy, hypersensitive behavior, cleft palate, and a high incidence of neonatal mortality. In this study, we analyze whole-cell responses to GABA in neurons from beta3+/+, beta3+/- and beta3-/- mice. We demonstrate markedly decreased responses to GABA in both hippocampal and dorsal root ganglion neurons isolated from beta3-/- mice without major differences in the GABA concentration-response curves. We also utilize the subunit selective pharmacology of Zn2+ and the anticonvulsant drug loreclezole to help infer the presence of beta2 and gamma subunits in the GABA(A) receptors remaining in neurons from beta3-/- mice.


Subject(s)
Mice, Knockout/physiology , Neurons/metabolism , Receptors, GABA-A/deficiency , Receptors, GABA-A/genetics , Animals , Cells, Cultured , Drug Synergism , Ganglia, Spinal/drug effects , Ganglia, Spinal/metabolism , Ganglia, Spinal/physiology , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/physiology , Mice , Neurons/cytology , Neurons/physiology , Patch-Clamp Techniques , Receptors, GABA-A/drug effects
17.
J Pharmacol Exp Ther ; 284(3): 934-42, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9495852

ABSTRACT

The actions of 2,2,2,-trichloroethanol were studied on agonist-activated Cl- currents in gamma-aminobutyric acid type A (GABAA), glycine and GABA rho 1 receptors by use of the whole-cell patch-clamp technique. Recombinant wild-type and mutant receptor subunits were transiently expressed in human embryonic kidney (HEK) 293 cells. Trichloroethanol enhanced currents elicited by submaximal (EC20) agonist concentrations at GABAA alpha 2 beta 1 receptors and glycine alpha 1 homomeric receptors in a reversible, concentration-dependent manner. Trichloroethanol, at concentrations of < or = 2 mM, did not significantly alter the magnitude of submaximal GABA currents at GABA rho 1 receptors, whereas higher concentrations inhibited submaximal GABA currents. Recent work has identified residues within putative transmembrane domains 2 and 3 as critical for positive modulation of GABAA and glycine receptors by n-alkanols and volatile ether anesthetics. Submaximal glycine currents at receptors containing either of two specific mutations within the glycine receptor alpha 1 subunit (S267I and A288W) were not enhanced by low concentrations of trichloroethanol and were inhibited by higher concentrations of trichloroethanol. In the GABAA alpha 2 beta 1 receptor, a specific mutation within transmembrane domain 3 of the beta 1 subunit (M286W) also abolished positive modulation by trichloroethanol. Mutations within the GABAA alpha 2 receptor subunit did not alter positive modulation by TCEt, whereas such mutations ablate positive modulation by n-alkanols and volatile anesthetics. In summary, trichloroethanol modulation of GABAA, glycine and GABA rho 1 receptors shares some, but not all, features in common with the requirements for modulation by n-alkanols and volatile anesthetics.


Subject(s)
Ethylene Chlorohydrin/analogs & derivatives , Receptors, GABA-A/drug effects , Receptors, GABA/drug effects , Receptors, Glycine/drug effects , Amino Acid Sequence , Cells, Cultured , Dose-Response Relationship, Drug , Ethylene Chlorohydrin/pharmacology , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Structure-Activity Relationship
18.
Nature ; 389(6649): 385-9, 1997 Sep 25.
Article in English | MEDLINE | ID: mdl-9311780

ABSTRACT

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.


Subject(s)
Anesthetics, Inhalation/pharmacology , Enflurane/pharmacology , Ethanol/pharmacology , Receptors, GABA-A/drug effects , Receptors, Glycine/drug effects , Alanine/physiology , Amino Acid Sequence , Anesthetics, Intravenous/pharmacology , Animals , Binding Sites , Cell Line , Electrophysiology , Glycine/pharmacology , Humans , Molecular Sequence Data , Mutagenesis , Propofol/pharmacology , Receptors, GABA-A/genetics , Receptors, Glycine/genetics , Recombinant Fusion Proteins/drug effects , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid , Serine/physiology , Tryptophan/physiology , Xenopus
19.
Neuropharmacology ; 36(7): 941-9, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9257938

ABSTRACT

We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.


Subject(s)
Anesthetics, Intravenous/pharmacology , Propofol/pharmacology , Receptors, GABA-A/drug effects , Animals , Cell Line , Dose-Response Relationship, Drug , Ethylene Chlorohydrin/analogs & derivatives , Ethylene Chlorohydrin/pharmacology , Methohexital/pharmacology , Mice , Muscimol/pharmacology , Patch-Clamp Techniques , Stereoisomerism
20.
Can J Anaesth ; 44(5 Pt 1): 525-34, 1997 May.
Article in English | MEDLINE | ID: mdl-9161749

ABSTRACT

PURPOSE: Acetylcholinesterase and butyrylcholinesterase are two closely related enzymes important in the metabolism of acetylcholine and anaesthetic drugs, including succinylcholine, mivacurium, and cocaine. The solanaceous glycoalkaloids (SGAs) are naturally occurring steroids in potatoes and related plants that inhibit both acetylcholinesterase and butyrylcholinesterase. There are many clinical examples of direct SGA toxicity due to cholinesterase inhibition. The aim of this study was to review the hypotheses that (1) SGAs may be the evolutionary driving force for atypical butyrylcholinesterase alleles and that (2) SGAs may adversely influence the actions of anaesthetic drugs that are metabolized by acetylcholinesterase and butyrylcholinesterase. SOURCE: The information was obtained by Medline search and consultation with experts in the study of SGAs and cholinesterases. PRINCIPAL FINDINGS: The SGAs inhibit both acetylcholinesterase and butyrylcholinesterase in numerous in vitro and in vivo experiments. Although accurate assays of SGA levels are difficult, published data indicate human serum SGA concentrations at least ten-fold lower than required to inhibit acetylcholinesterase and butyrylcholinesterase in vitro. However, we review evidence that suggests the dietary ingestion of SGAs can initiate a cholinergic syndrome in humans. This syndrome appears to occur at SGA levels lower than those which interfere with anaesthetic drug catabolism. The world distribution of solanaceous plants parallels the distribution of atypical alleles of butyrylcholinesterase and may explain the genetic diversity of the butyrylcholinesterase gene. CONCLUSION: Correlative evidence suggests that dietary SGAs may be the driving force for atypical butyrylcholinesterase alleles. In addition, SGAs may influence the metabolism of anaesthetic drugs and this hypothesis warrants experimental investigation.


Subject(s)
Cholinesterase Inhibitors/adverse effects , Solanaceous Alkaloids/adverse effects , Animals , Butyrylcholinesterase/genetics , Cholinesterase Inhibitors/pharmacology , Humans , Solanaceous Alkaloids/pharmacology
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