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Biomedical subjects

L L Firestone

Publications and source records attributed to L L Firestone.

At least 37 records · Page 2Linked to original sources
PubMed1997

Gene knockout of the alpha6 subunit of the gamma-aminobutyric acid type A receptor: lack of effect on responses to ethanol, pentobarbital, and general anesthetics.

The alpha6 subunit of the gamma-aminobutyric acid type A receptor (GABA(A)-R) has been implicated in mediating the intoxicating effects of ethanol and the motor ataxic effects of general anesthetics. To test this hypothesis, we used gene targeting in embryonic stem cells to create mice lacking a functional alpha6 gene. Homozygous mice are viable and fertile and have grossly normal cerebellar cytoarchitecture. Northern blot and reverse transcriptase-polymerase chain reaction analyses demonstrated that the targeting event disrupted production of functional alpha6 mRNA. Autoradiography of histological sections of adult brains demonstrated that diazepam-insensitive binding of [3H]Ro15-4513 to the cerebellar granule cell layer of wild-type mice was completely absent in homozygous mice. Cerebellar GABA(A)-R density was unchanged in the mutant mice; however, the apparent affinity for muscimol was markedly reduced. Sleep time response to injection of ethanol after pretreatment with vehicle or Ro15-4513 did not differ between genotypes. Sleep time response to injection of pentobarbital and loss of righting reflex and response to tail clamp stimulus in mice anesthetized with volatile anesthetics also did not differ between genotypes. Thus, the alpha6 subunit of the GABA(A)-R is not required for normal development, viability, and fertility and does not seem to be a critical or unique component of the neuronal pathway mediating the hypnotic effect of ethanol and its antagonism by Ro15-4513 in mice. Similarly, the alpha6 subunit does not seem to be involved in the behavioral responses to general anesthetics or pentobarbital.

Anesthetics, Inhalation↗
PubMed1997

Cerebellar gamma-aminobutyric acid type A receptors: pharmacological subtypes revealed by mutant mouse lines.

The vast molecular heterogeneity of brain gamma-aminobutyric acid type A (GABAA) receptors forms the basis for receptor subtyping. Using autoradiographic techniques, we established the characteristics of cerebellar granule cell GABAA receptors by comparing wild-type mice with those with a targeted disruption of the alpha6 subunit gene. Cerebellar granule cells of alpha6(-/-) animals have severe deficits in high affinity [3H]muscimol and [3H]SR 95531 binding to GABA sites, in agonist-insensitive [3H]Ro 15-4513 binding to benzodiazepine sites, and in furosemide-induced increases in tert-[35S]butylbicyclophosphorothionate binding to picrotoxin-sensitive convulsant sites. These observations agree with the known specific properties of these sites on recombinant alpha6beta2/3gamma2 receptors. In the presence of GABA concentrations that fail to activate alpha1 subunit-containing receptors, methyl-6,7-dimethoxy-4-ethyl-beta-carboline (30 microM), allopregnanolone (100 nM), and Zn2+ (10 microM) are less efficacious in altering tert-[35S]butylbicyclophosphorothionate binding in the granule cell layer of the alpha6(-/-) than alpha6(+/+) animals. These data concur with the deficiency of the cerebellar alpha6 and delta subunit-containing receptors in the alpha6(-/-) animals and could also account for the decreased affinity of [3H]muscimol binding to alpha6(-/-) cerebellar membranes. Predicted additional alterations in the cerebellar receptors of the mutant mice may explain a surplus of methyl-6,7-dimethoxy-4-ethyl-beta-carboline-insensitive receptors in the alpha6(-/-) granule cell layer and an increased diazepam-sensitivity in the molecular layer. These changes may be adaptive consequences of altered GABAA receptor subunit expression patterns in response to the loss of two subunits (alpha and delta) from granule cells.

Animals↗
PubMed1996

Human brain activity response to fentanyl imaged by positron emission tomography.

Positron emission tomography (PET) is a noninvasive imaging technique that can be used to observe drug actions on human brain in vivo. We used 15O-water PET scanning in six volunteers to examine the effects on regional cerebral activity as reflected by regional cerebral blood flow (rCBF) of a small intravenous bolus of fentanyl. rCBF was compared between scans obtained after fentanyl or a placebo using three separate statistical criteria including a pixel-by-pixel t statistic; significance was stringently defined at P values < 0.01. Anatomic locations of regional cerebral activity changes were verified by aligning rCBF PET scans with cranial magnetic resonance images using mathematical coregistration. Fentanyl administration was associated with significant increases in rCBF consistent with regional neuronal activation in both cingulate and orbitofrontal and medial prefrontal cortices, as well as caudate nuclei. These areas are responsive to nociceptive stimuli and are involved in avoidance learning, reward and addiction, visceromotor control, maintenance of attention, and pain-related affective behavior. Significant decreases were noted in both frontal and temporal areas and the cerebellum, a distribution far less extensive than that of opiate receptors in general. These data indicate that fentanyl's effects are highly localized and specifically affect cerebral regions associated with a range of pain-related behaviors.

Adult↗
PubMed1996

In vivo imaging of human limbic responses to nitrous oxide inhalation.

Human behavioral studies have shown that nitrous oxide, in subanesthetic concentrations, impairs psychomotor function, cognitive performance, and learning and memory processes. However, the cerebral mechanisms of such effects remain unknown. Positron emission tomography (PET) was used to map the brain areas associated with nitrous oxide effects. Regional cerebral blood flow (rCBF) was measured in eight volunteers, during room air (control) or 20% nitrous oxide (nitrous oxide) inhalation using 15(O)-water, to reflect regional neuronal activity. To control for the possibility that 20% nitrous oxide uncoupled cerebral blood flow and metabolism, in four of the subjects, regional cerebral metabolic rate (rCMR) was also measured using 18F-deoxyglucose during the two experimental conditions. Results of rCBF and rCMR scans were compared between conditions using the statistical parametric mapping method, and areas of nitrous oxide-related activation or deactivation were identified at a significance level of 0.005. Percent changes in rCBF scan pixels from these activated or deactivated areas were then compared with those of stereotactically corresponding rCMR scan pixels with t statistics (P < 0.05 was defined as a significant difference). It was found that cerebral blood flow and metabolism were not uncoupled by 20% nitrous oxide, since percent changes in rCBF and rCMR, detected during nitrous oxide inhalation, did not differ significantly from each other (P < 0.05). Nitrous oxide inhalation was associated with significant activation in the anterior cingulate cortex, a limbic area known to mediate psychomotor and cognitive processes. Deactivation was found in the posterior cingulate, hippocampus, parahippocampal gyrus, and visual association cortices in both hemispheres; the former two regions are known to mediate learning and memory. These areas identified by PET in vivo may provide the neuroanatomical basis for the behavioral responses associated with subanesthetic nitrous oxide inhalation.

Adult↗
PubMed1995

Isoflurane's enhancement of chloride flux through rat brain gamma-aminobutyric acid type A receptors is stereoselective.

BACKGROUND: Recent evidence is consistent with the view that volatile anesthetics interact directly with excitable membrane-bound channel proteins. If these agents interact directly with chiral centers in the neuronal cell membrane, then their effects should be stereoselective. Using rat brain membranes enriched in gamma-aminobutyric acid type A (GABAA) receptors, we investigated the hypothesis that the permeability response of this well-characterized central nervous system channel protein to stereoisomers of isoflurane is stereoselective. METHODS: Rat brain synaptic microvesicles were prepared by differential centrifugation. Agonist-stimulated 36Cl- flux through membrane-bound GABAA receptors was assayed in the presence of (+)- and (-)-isoflurane and compared with control conditions. RESULTS: Both isomers increased the potency and efficacy of GABA; however, (+)-isoflurane was significantly more potent and efficacious than the (-)-isomer. For example, the (+)-isomer (140 microM) reduced the median effective concentration of GABA from 12.7 +/- 1.0 to 5.4 +/- 0.5 microM, whereas the (-)-isomer reduced it to 9.6 +/- 1.0 microM (P < 0.001). The (+)-isomer also was 1.6 times as potent as the (-)-isomer in augmenting 5 microM GABA-gated flux (79 +/- 11 vs. 130 +/- 17 microM, respectively; P = 0.01). In addition, the (+)-isomer produced significantly greater maximal enhancement of flux (9.4 +/- 0.4 vs. 7.0 +/- 0.3 nmol.mg-1.3 s-1; P < 0.001). CONCLUSIONS: Isoflurane's effects on GABA-gated chloride flux were stereoselective. This result supports direct interaction with a stereoselective site, possibly the GABAA channel protein itself, rather than a nonspecific perturbation of the surrounding membrane lipid. In addition, these findings, from a functional assay using mammalian brain, agree with recent observations in invertebrate ion channels and mammalian neuronal cell cultures.

Animals↗
PubMed1995

Staurosporine, a protein kinase inhibitor, increases the intoxicating potencies of ethanol and other n-alkanols in Rana pipiens tadpoles.

Central nervous system protein kinases are the intracellular effectors for many of the signal transduction pathways essential to neurotransmission. Although the in vitro activity of at least one of these important enzymes, protein kinase C, is diminished by therapeutic concentrations of ethanol and other central depressants, the relationship of this effect to intoxication in vivo is not known. If intoxication by ethanol involves central protein kinase inhibition, then other inhibitors of these enzymes should enhance ethanol's intoxicating potency. To test this hypothesis, we compared the median effective concentrations of ethanol and two other n-alkanols for loss-of-righting reflex in Rana pipiens tadpoles pretreated with staurosporine and in untreated controls. Alkanol concentrations were confirmed by gas chromatography and staurosporine concentrations by ultraviolet absorbance spectrophotometry. Results obtained with 650 animals demonstrate that pretreatment with staurosporine concentrations in the nanomolar range significantly decrease the median effective concentration for ethanol (56% of control; p < 0.001), butanol (38% of control; p < 0.001), and octanol (59% of control; p < 0.001). This finding supports that central protein kinase inhibition may be involved in the acute intoxicating effects of ethanol and other n-alkanols.

1-Butanol↗
PubMed1994

Halothane sensitivity in replicate mouse lines selected for diazepam sensitivity or resistance.

We have previously shown that mice selected for sensitivity to diazepam are also more sensitive to halothane, and that halothane augments the gamma-aminobutyric acid (GABA)-mediated chloride flux response in brain tissue from diazepam-sensitive (DS) mice to a greater degree than in diazepam-resistant (DR) mice. These findings suggest that the GABAA receptor is an important site of halothane action. To confirm this correlation, halothane requirement was determined in two independently developed replicate lines of DS and DR mice. Association of the traits of diazepam and halothane sensitivity in replicate lines of DS mice diminishes the probability that the original finding was due to a false-positive correlation, and instead suggests that it results from the common action of genes controlling diazepam sensitivity. Halothane median effective concentration (EC50) was determined by using the end-point of loss of righting reflex in two replicate lines of mice selected for diazepam sensitivity (resistant mice = diazepam high performance-1 and -2 [DHP-1 and DHP-2], sensitive mice = diazepam low performance-1 and -2 [DLP-1 and DLP-2]). DLP-1 and DLP-2 mice were sensitive to halothane, whereas DHP-1 and DHP-2 mice were resistant to halothane. Halothane EC50 in the DLP-1 and DHP-1 mice was 0.86 +/- 0.01 (SE) and 1.10 +/- 0.04 atm%, respectively (P < 0.0001), and that in the DLP-2 and DHP-2 mice was 0.88 +/- 0.01 and 0.97 +/- 0.02 atm%, respectively (P < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Does general anesthetic-induced desensitization of the Torpedo acetylcholine receptor correlate with lipid disordering?

We have tested the hypothesis that general anesthetics stabilize the desensitized state of the nicotinic acetylcholine receptor by disordering its surrounding lipids. Acetylcholine receptor-rich postsynaptic membranes from the electroplaques of Torpedo were used in this study to obtain the highest possible receptor specific activity in native membranes. We examined 18 general anesthetics, including six inhalation agents, eight 1-alcohols, the enantiomers of 2-octanol, and two intravenous general anesthetics (pentobarbital and ethylcarbamate). The degree of desensitization after preincubation with the general anesthetics was determined by brief exposure to [3H]acetylcholine, making use of the facts that desensitized receptors have much higher affinity than do those in the resting state and that interconversion between the states is slow. All of the general anesthetics desensitized the receptor within minutes, exhibiting steep concentration-response curves with Hill coefficients generally within the range of 2-4. At the highest general anesthetic concentrations, almost all receptors were desensitized. The concentrations that desensitized half of the resting state receptors varied by > 3000-fold. The 2-octanol enantiomers were without stereoselectivity. Membrane order was examined in parallel by using spin-labeled fatty acids doped into the native membranes. The spin label 5-doxylpalmitate reported from the most ordered part of the bilayer near the aqueous interface, whereas 12-doxylstearate reported from the less ordered region nearer the center of the bilayer. The spin label deeper in the membranes was 3 times more sensitive to a given anesthetic than was the other probe. At both depths in the membrane general anesthetics decreased lipid order linearly with increasing concentration. The range of disordering potencies (change in order parameter induced by a unit concentration of general anesthetic in the aqueous phase) was 5333 for 5-doxylpalmitate and 7143 for 12-doxylstearate, but the range of disordering compared at equally desensitizing concentrations was reduced by 875- and 1430-fold, respectively. The average degrees of disordering at concentrations that desensitized half of the resting state receptors were 1.5% and 4.4%, respectively. It is unlikely that changes in membrane order parameter per se cause desensitization, because the associated changes in order parameter can be reproduced by changes in cholesterol content or temperature that do not cause desensitization. We conclude that, although there is a strong association between anesthetic-induced membrane disordering and desensitization, more detailed tests of a mechanistic nature will be necessary to elucidate the mechanisms underlying the Meyer-Overton-type behavior we have observed.

Anesthesia, General↗
PubMed1993

Halothane's effects on GABA-gated chloride flux in mice selectively bred for sensitivity or resistance to diazepam.

The DS (diazepam-sensitive) and DR (diazepam-resistant) lines of mice, selected on the basis of their ataxic response to diazepam, also diverge in the physiologic response of their brain gamma-aminobutyric acidA (GABAA) receptors to benzodiazepines, as indicated by augmentation of GABA-mediated chloride flux. Cross-sensitivity and -resistance to other sedatives known to interact with the GABAA-receptor have also been demonstrated in DS and DR mice. Based on the finding that these mice also show cross-sensitivity and -resistance to obtundation by halothane, we predicted that their GABAA-receptors would also exhibit a differential response to halothane as assayed by an in vitro 36Cl- influx assay using purified brain microvesicles. Consistent with this prediction, therapeutic concentrations of halothane enhanced 1 mumol/l GABA-gated flux with significantly greater potency in DS than in DR mice (halothane EC50 336 +/- 64 mumol/l (S.E.M.) vs. 605 +/- 110 mumol/l, respectively, P = 0.03), but there was no difference in maximal flux enhancement between the two lines (DS 4.7 +/- 0.4 nmol.mg-1 x 3 s-1, vs. DR 4.7 +/- 0.5 nmol.mg-1 x 3 s-1). Halothane (500 mumol/l) also shifted the entire GABA concentration-flux relationship significantly to the left, decreasing the EC50 for GABA in both the DS and DR lines. Importantly, the shift in the GABA concentration-flux response in the presence of halothane was more pronounced in the DS mice (GABA EC50 1.8 +/- 0.4 mumol/l vs. 14.7 +/- 0.9 mumol/l without halothane) than in the DR mice (GABA EC50 4.7 +/- 0.6 mumol/l vs. 14.7 +/- 0.9 mumol/l without halothane).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Staurosporine, a protein kinase C inhibitor, decreases the general anesthetic requirement in Rana pipiens tadpoles.

Protein kinase C, the intracellular effector for the inositol phosphate-mediated signal transduction pathway, plays a key role in neurotransmission in the central nervous system. Although the in vitro activity of protein kinase C is inhibited by therapeutic concentrations of volatile anesthetics, the relation of this effect to in vivo obtundation has not been established. If obtundation by volatile anesthetics involves protein kinase C inhibition, then an inhibitor of this enzyme should decrease the anesthetic requirement. To test this hypothesis, we compared the EC50S of halothane and diethylether for loss of the righting reflex in Rana pipiens tadpoles pretreated with staurosporine and in untreated controls. Anesthetic concentrations were confirmed by gas chromatography and staurosporine concentrations by ultraviolet absorbance spectrophotometry. Results obtained in more than 1000 animals indicated that pretreatment with staurosporine concentrations in the nanomolar range significantly decreased the EC50 for both halothane (68% of control; P < 0.035) and diethylether (41% of control; P < 0.001). This finding implies that protein kinase C inhibition may play a role in general anesthetic-induced obtundation.

Alkaloids↗
PubMed1993

Volatile anesthetic requirements differ in mice selectively bred for sensitivity or resistance to diazepam: implications for the site of anesthesia.

One approach to elucidating the general anesthetic target has used genetic selection procedures, wherein animals are bred for sensitivity or resistance to general anesthetics and correlations are sought with a specific neuronal structural or functional defect. For example, murine strains have been developed that are either sensitive or resistant to the obtunding effects of diazepam, as assessed by their ability to maintain balance on a rotating rod. The present study explored whether diazepam-sensitive (DS) and diazepam-resistant (DR) mice might also be similarly divergent in the obtunding response to general anesthetics, by testing the requirements for halothane and enflurane in these strains. Using a carousel enclosed in a chamber, the end-point of loss-of-righting reflex was defined. For both anesthetics, the DS groups had a lower median effective dose (ED50, %atm) than did the DR group, and the reductions paralleled diazepam susceptibility. For example, with halothane, the ED50 for the DS group was 0.72 +/- 0.022 (SE); the ED50 for the DR group was 0.87 +/- 0.030 (P < 0.0001). Similar results were obtained with enflurane. Such findings associate an inbred difference in response to diazepam with altered volatile anesthetic requirement, suggesting that these two phenotypes are mediated by a common underlying mechanism.

Anesthesia, Inhalation↗
PubMed1992

Ligand-dependent effects of ethanol and diethylether at brain benzodiazepine receptors.

The GABAA receptor chloride channel complex interacts with various categories of sedatives, including the benzodiazepines, and possibly ethanol and volatile general anesthetics. Thus, specific binding of tritiated derivatives of a benzodiazepine antagonist, flumazenil, and an agonist, flunitrazepam, to rat brain membrane fragments was monitored at equilibrium in the presence and absence of anesthetizing concentrations of ethanol and diethylether. Ethanol produced a concentration-dependent inhibition of [3H]flumazenil binding, which was not reversed by the GABAA receptor competitive antagonist bicuculline, but had no effect on [3H]flunitrazepam binding. Both ethanol and diethylether decreased the affinity of the benzodiazepine site for [3H]flumazenil. These data indicate that ethanol and diethylether have GABA-independent effects at the benzodiazepine sites of the GABAA receptor. These findings are inconsistent with a two-state functional model of the benzodiazepine site and, instead, support a model containing a specific, antagonist-favored conformation.

Animals↗
PubMed1989

Nonanesthetic alcohols dissolve in synaptic membranes without perturbing their lipids.

While many theories of general anesthesia postulate a lipid site of action, there has been no adequate explanation for the lack of anesthetic potency of the highly hydrophobic primary alkanols with more than 12 carbons (the cut-off). Some work suggests that these nonanesthetic alcohols do not dissolve in membranes. Other work contradicts this and suggests that an anesthetic site on a protein provides a better explanation. Here we show that both the anesthetic dodecanol and the nonanesthetic tetradecanol are taken up equally well into the tissues of animals and into isolated postsynaptic membranes. When a group of Rana pipiens tadpoles were treated with dodecanol, half were anesthetized by 4.7 microM (free aqueos concentration), and the corresponding concentration in the tissues was found to be 0.4 mmol per kg wet weight. Prolonged exposure (92 hr) to tetradecanol produced even higher tissue concentrations (0.7 mmol per kg wet weight), yet no anesthetic effects were observed. Furthermore, general anesthetics are thought to act on postsynaptic membranes but both alkanols partitioned into postsynaptic membranes from Torpedo electroplaques. The spin label, 12-doxyl stearate, was incorporated into these membranes. The lipid order parameter it reported was decreased by the anesthetic alcohols (octanol, decanol, and dodecanol), whereas the nonanesthetic alcohols either did not change it significantly (tetradecanol) or actually increased it (hexadecanol and octadecanol). Thus, although lipid solubility is unable to account for the pharmacology of the cut-off in potency of the long-chain alcohols, lipid perturbations provide an accurate description.

Alcohols↗
PubMed1989

Anaesthetic potencies of primary alkanols: implications for the molecular dimensions of the anaesthetic site.

1. We have redetermined the anaesthetic potencies (EC50S) for a series of primary alkanols, to resolve uncertainties about the molecular dimensions of the anaesthetic site resulting from the use of data from different laboratories. 2. For each alkanol, concentration-response relationships for loss of righting reflex (LRR) were plotted for over one hundred tadpoles, and the median effective concentrations determined. Aqueous concentrations present during potency assays were determined independently, and for alkanols with chain length greater than nonanol, correction was made for depletion from the aqueous phase. 3. The EC50S were found to decrease logarithmically with increasing number of carbon atoms in the hydrocarbon chain of the alkanol (CN), such that, on average, each additional methylene group was associated with an approximately four fold increase in potency. 4. The relationship between log EC50 and CN was best described by the quadratic equation, log EC50 = 0.022 (+/- 0.0038) CN2 + 0.76 (+/- 0.051) CN + 3.7 (+/- 0.14) (r2 = 0.9951). 5. A previously described correlation between the apparent changes in the free energy of binding of an additional methylene group both to luciferase and to the sites for LRR in tadpoles was not confirmed. 6. A cut-off in potency beyond dodecanol was established in experiments where tadpoles were maintained in supersaturated solutions of tridecanol for 20 h without demonstrable LRR. 7. These findings indicate that the soluble enzyme firefly luciferase does not adequately model the anaesthetic site. Specifically, there are discrepancies in the position of cut-off, and the apparent changes in the free energy of binding, per methylene group, of an alkanol to luciferase do not parallel that for tadpoles.

Alcohols↗
PubMed1987

Is agonist self-inhibition at the nicotinic acetylcholine receptor a nonspecific action?

Agonist concentration-response relationships at nicotinic postsynaptic receptors were established by measuring 86Rb+ efflux from acetylcholine receptor rich native Torpedo membrane vesicles under three different conditions: integrated net ion efflux (in 10 s) from untreated vesicles, integrated net efflux from vesicles in which most acetylcholine sites were irreversibly blocked with alpha-bungarotoxin, and initial rates of efflux (5-100 ms) from vesicles that were partially blocked with alpha-bungarotoxin. Exposure to acetylcholine, carbamylcholine, suberyldicholine, phenyltrimethylammonium, or (-)-nicotine over 10(8)-fold concentration ranges results in bell-shaped ion flux response curves due to stimulation of acetylcholine receptor channel opening at low concentrations and inhibition of channel function at 60-2000 times higher concentrations. Concentrations of agonists that inhibit their own maximum 86Rb+ efflux by 50% (KB values) are 110, 211, 3.0, 39, and 8.9 mM, respectively, for the agonists listed above. For acetylcholine and carbamylcholine, KB values determined from both 10-s and 15-ms efflux measurements are the same, indicating that the rate of agonist-induced desensitization increases to maximum at concentrations lower than those causing self-inhibition. For all partial and full agonists studied, Hill coefficients for self-inhibition are close to 1.0. Concentrations of agonists up to 8 times KB did not change the order parameter reported by a spin-labeled fatty acid incorporated in Torpedo membranes. We conclude that agonist self-inhibition cannot be attributed to a general nonspecific membrane perturbation. Instead, these results are consistent with a saturable site of action either at the lipid-protein interface or on the acetylcholine receptor protein itself.

Acetylcholine↗