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Biomedical subjects

Roderic G Eckenhoff

Publications and source records attributed to Roderic G Eckenhoff.

At least 19 recordsLinked to original sources
PubMed2006

High throughput modular chambers for rapid evaluation of anesthetic sensitivity.

BACKGROUND: Anesthetic sensitivity is determined by the interaction of multiple genes. Hence, a dissection of genetic contributors would be aided by precise and high throughput behavioral screens. Traditionally, anesthetic phenotyping has addressed only induction of anesthesia, evaluated with dose-response curves, while ignoring potentially important data on emergence from anesthesia. METHODS: We designed and built a controlled environment apparatus to permit rapid phenotyping of twenty-four mice simultaneously. We used the loss of righting reflex to indicate anesthetic-induced unconsciousness. After fitting the data to a sigmoidal dose-response curve with variable slope, we calculated the MAC(LORR) (EC50), the Hill coefficient, and the 95% confidence intervals bracketing these values. Upon termination of the anesthetic, Emergence timeRR was determined and expressed as the mean +/- standard error for each inhaled anesthetic. RESULTS: In agreement with several previously published reports we find that the MAC(LORR) of halothane, isoflurane, and sevoflurane in 8-12 week old C57BL/6J mice is 0.79% (95% confidence interval = 0.78-0.79%), 0.91% (95% confidence interval = 0.90-0.93%), and 1.96% (95% confidence interval = 1.94-1.97%), respectively. Hill coefficients for halothane, isoflurane, and sevoflurane are 24.7 (95% confidence interval = 19.8-29.7%), 19.2 (95% confidence interval = 14.0-24.3%), and 33.1 (95% confidence interval = 27.3-38.8%), respectively. After roughly 2.5 MAC(LORR) x hr exposures, mice take 16.00 +/- 1.07, 6.19 +/- 0.32, and 2.15 +/- 0.12 minutes to emerge from halothane, isoflurane, and sevoflurane, respectively. CONCLUSION: This system enabled assessment of inhaled anesthetic responsiveness with a higher precision than that previously reported. It is broadly adaptable for delivering an inhaled therapeutic (or toxin) to a population while monitoring its vital signs, motor reflexes, and providing precise control over environmental conditions. This system is also amenable to full automation. Data presented in this manuscript prove the utility of the controlled environment chambers and should allow for subsequent phenotyping of mice with targeted mutations that are expected to alter sensitivity to induction or emergence from anesthesia.

Journal Article↗
PubMed2006

Partitioning of anesthetics into a lipid bilayer and their interaction with membrane-bound peptide bundles.

Molecular dynamics simulations have been performed to investigate the partitioning of the volatile anesthetic halothane from an aqueous phase into a coexisting hydrated bilayer, composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids, with embedded alpha-helical peptide bundles based on the membrane-bound portions of the alpha- and delta-subunits, respectively, of nicotinic acetylcholine receptor. In the molecular dynamics simulations halothane molecules spontaneously partitioned into the DOPC bilayer and then preferentially occupied regions close to lipid headgroups. A single halothane molecule was observed to bind to tyrosine (Tyr-277) residue in the alpha-subunit, an experimentally identified specific binding site. The binding of halothane attenuated the local loop dynamics of alpha-subunit and significantly influenced global concerted motions suggesting anesthetic action in modulating protein function. Steered molecular dynamics calculations on a single halothane molecule partitioned into a DOPC lipid bilayer were performed to probe the free energy profile of halothane across the lipid-water interface and rationalize the observed spontaneous partitioning. Partitioned halothane molecules affect the hydrocarbon chains of the DOPC lipid, by lowering of the hydrocarbon tilt angles. The anesthetic molecules also caused a decrease in the number of peptide-lipid contacts. The observed local and global effects of anesthetic binding on protein motions demonstrated in this study may underlie the mechanism of action of anesthetics at a molecular level.

Anesthetics, Inhalation↗
PubMed2006

Photoactive analogues of the haloether anesthetics provide high-resolution features from low-affinity interactions.

The difficulty in obtaining binding target and site information for low-affinity drugs, like the inhaled anesthetics, has limited identification of their molecular effectors. Because such information can be provided by photoactive analogues, we designed, synthesized, and characterized a novel diazirnyl haloether that closely mimics isoflurane, the most widely used clinical general anesthetic. This compound, H-diaziflurane, is a nontoxic, potent anesthetic that potentiates GABA-gated ion channels in primary cultures of hippocampal neurons. Calorimetric and structural characterizations show that H-diaziflurane binds a model anesthetic host protein with similar energetics as isoflurane and forms photoadducts with residues lining the isoflurane binding site. H-diaziflurane will be immediately useful for identifying targets and sites important for the molecular pharmacology of the inhaled haloether anesthetics.

Anesthetics↗
PubMed2006

A guest molecule-host cavity fitting algorithm to mine PDB for small molecule targets.

Inhaled anesthetic molecule occupancy of a protein internal cavity depends in part on the volumes of the guest molecule and the host site. Current algorithms to determine volume and surface area of cavities in proteins whose structures have been determined and cataloged make no allowance for shape or small degrees of shape adjustment to accommodate a guest. We developed an algorithm to determine spheroid dimensions matching cavity volume and surface area and applied it to screen the cavities of 6,658 nonredundant structures stored in the Protein Data Bank (PDB) for potential targets of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane). Our algorithm determined sizes of prolate and oblate spheroids matching dimensions of each cavity found. If those spheroids could accommodate halothane (radius 2.91 A) as a guest, we determined the packing coefficient. 394,766 total cavities were identified. Of 58,681 cavities satisfying the fit criteria for halothane, 11,902 cavities had packing coefficients in the range of 0.46-0.64. This represents 20.3% of cavities large enough to hold halothane, 3.0% of all cavities processed, and found in 2,432 protein structures. Our algorithm incorporates shape dependence to screen guest-host relationships for potential small molecule occupancy of protein cavities. Proteins with large numbers of such cavities are more likely to be functionally altered by halothane.

Algorithms↗
PubMed2006

Interactions of volatile anesthetics with neurodegenerative-disease-associated proteins.

The prevalence of the neurodegenerative disorders is increasing as life expectancy lengthens, and there exists concern that environmental influences may contribute to this increase. These disorders are varied in their clinical presentation, but appear to have a common biophysical initiation. At this level, it is both plausible and now proven that anesthetics can enhance aggregation of some disease-causing proteins. Although data in support of an interaction in animal models are still lacking, data from clinical studies indicate an association, which provides further cause for concern. Many opportunities exist for rapid progress at all levels on defining whether anesthetics do indeed contribute to the pathogenesis of these progressive, debilitating disorders.

Aged↗
PubMed2006

Rat brain DNA transcript profile of halothane and isoflurane exposure.

Inhaled anesthetics produce many effects and bind to a large number of brain proteins, but it is not yet clear if this is accompanied by widespread changes in gene expression of the biological targets. Such changes in expression might implicate functionally important targets from the large pool of binding targets. Both rats and isolated primary cortical neurons were exposed to anesthetics and DNA oligonucleotide microarrays were used to detect and quantify transcriptional changes in neural tissue. Using analysis of variance with multiple testing correction, multiple exposures of rats to 0.8 MAC (minimum alveolar concentration) halothane only produced significant changes in a few metabolic genes. No significant in-vivo gene transcriptional response to 0.8 MAC isoflurane was detected. The use of primary cortical neurons allowed exposure to 3 MAC anesthetics without evidence of toxicity. Isoflurane altered several genes involved with neurotransmitter transport, signaling and cellular structure, whereas halothane produced few detectable changes in these cultured cells. Selected genes were confirmed by quantitative reverse transcription-polymerase chain reaction. Although indicating only a small degree of transcriptional regulation, these data implicate several plausible targets, including synaptic vesicle handling, that might contribute to drug action. In addition, the data show different gene expression profiles for the two inhaled anesthetics, suggesting unique pharmacological targets and mechanisms in each case.

Anesthetics, Inhalation↗
PubMed2005

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography.

A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.

Chromatography, High Pressure Liquid↗
PubMed2005

Anesthetic potency of two novel synthetic polyhydric alkanols longer than the n-alkanol cutoff: evidence for a bilayer-mediated mechanism of anesthesia?

The polyhydroxyalkanes 1,6,11,16-hexadecanetetraol (1) and 2,7,12,17-octadecanetetraol (2) were synthesized utilizing the thiophene ring as a scaffold to affix the hydroxyalkyl chains by lithiation of the acidic alpha-hydrogens and subsequent desulfurization. Both compounds exhibited significant anesthetic potency, individually and in additivity studies with hexanol, using immobility in tadpoles as the phenotypic endpoint. These results, which contradict a protein-binding mechanism in which cutoff results from steric hindrance, are consistent with recent predictions of a membrane-mediated mechanism involving the lateral pressure profile.

Anesthetics, General↗
PubMed2005

Truncated human serum albumin retains general anaesthetic binding activity.

Multiple binding sites for anaesthetics in HSA (human serum albumin) make solution studies difficult to interpret. In the present study, we expressed the wild-type HSA domain 3 (wtHSAd3), a peptide with two known anaesthetic binding sites in a yeast expression system. We also expressed a site-directed mutant of domain 3 (Y411Wd3). The stability and secondary structure of the constructed fragments were determined by HX (hydrogen-tritium exchange) and CD spectroscopy. The binding of two general anaesthetics, 2-bromo-2-chloro-1,1,1-trifluoroethane and propofol, to wtHSAd3 and Y411Wd3 was determined using isothermal titration calorimetry, HX and intrinsic tryptophan fluorescence quenching. Although the expressed fragments are less stable than intact wtHSA as indicated by both CD and HX, they retain the secondary structure and anaesthetic-binding characteristics of an intact HSA molecule, but with fewer binding sites. Y411Wd3 had decreased affinity for propofol but not for 2-bromo-2-chloro-1,1,1-trifluoroethane, consistent with steric hindrance. Retention of structural features and anaesthetic binding properties with fewer binding sites in this truncated protein provide feasibility for using scaled-down models of otherwise intractable systems to gain an understanding of anaesthetic binding requirements and binding-stability relationships.

Anesthetics, General↗
PubMed2005

Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently.

Depletion of calcium from the neuronal endoplasmic reticulum (ER) induces apoptosis. Isoflurane depletes calcium from sarcoplasmic reticulum (SR) of muscle, an analogue of ER in neurons, while sevoflurane maintains or increases SR calcium. We hypothesized that isoflurane, but not sevoflurane, induces apoptosis by depleting the ER calcium. Rat PC12 pheochromocytoma cells and primary cortical neurons were treated with equipotent doses of isoflurane and sevoflurane. Isoflurane, but not sevoflurane, at equipotent doses induced cell damage determined by both LDH release and MTT reduction assays, dose and time dependently, in both types of cells. Isoflurane at 2.4% for 24 h induced cytotoxicity in both cell types, which was characterized by nuclear condensation and fragmentation and activation of caspases 3 and 9. Isoflurane cytotoxicity was suppressed by dantrolene, a ryanodine receptor antagonist that inhibits abnormal calcium release from the ER. Isoflurane decreased the Bcl-2/Bax ratio by as much as 36% (P < 0.05). However, sevoflurane did not cause neuronal damage by apoptosis nor did it decrease the Bcl-2/Bax ratio. These results suggest that isoflurane and sevoflurane differentially affect the Bcl-2/Bax ratio and cell survival. At equipotent concentrations, isoflurane, but not sevoflurane, induces cytotoxicity in both PC12 cells and primary cortical neurons and decreases the Bcl-2/Bax ratio.

Acridine Orange↗
PubMed2005

Structural basis for high-affinity volatile anesthetic binding in a natural 4-helix bundle protein.

Physiologic sites for inhaled anesthetics are presumed to be cavities within transmembrane 4-alpha-helix bundles of neurotransmitter receptors, but confirmation of binding and structural detail of such sites remains elusive. To provide such detail, we screened soluble proteins containing this structural motif, and found only one that exhibited evidence of strong anesthetic binding. Ferritin is a 24-mer of 4-alpha-helix bundles; both halothane and isoflurane bind with K(A) values of approximately 10(5) M(-1), higher than any previously reported inhaled anesthetic-protein interaction. The crystal structures of the halothane/apoferritin and isoflurane/apoferritin complexes were determined at 1.75 A resolution, revealing a common anesthetic binding pocket within an interhelical dimerization interface. The high affinity is explained by several weak polar contacts and an optimal host/guest packing relationship. Neither the acidic protons nor ether oxygen of the anesthetics contribute to the binding interaction. Compared with unliganded apoferritin, the anesthetic produced no detectable alteration of structure or B factors. The remarkably high affinity of the anesthetic/apoferritin complex implies greater selectivity of protein sites than previously thought, and suggests that direct protein actions may underlie effects at lower than surgical levels of anesthetic, including loss of awareness.

Anesthetics, Inhalation↗
PubMed2005

Weak polar interactions confer albumin binding site selectivity for haloether anesthetics.

BACKGROUND: Enflurane and isoflurane are structural isomers with different anesthetic potencies and side effects. It is not clear whether these differences are produced by differing occupancy of common protein binding sites or by occupancy of different sites, but the very similar molecular properties make the latter possibility unlikely. In this study, the authors examined binding site selectivity of these anesthetics in human serum albumin (HSA). METHODS: Binding of isoflurane and enflurane with HSA was determined with isothermal titration calorimetry. Competition with known ligands (propofol) allowed localization of binding sites within the HSA molecule. Molecular properties of isoflurane and enflurane were calculated. RESULTS: Isoflurane binds HSA with higher affinity but smaller total enthalpy than enflurane. Enthalpogram analysis suggested that isoflurane bound a single site, whereas enflurane bound two. Competition experiments indicated that enflurane and isoflurane share one binding site, which also binds propofol. The additional enflurane site binds propofol but not isoflurane. Increased salt concentration decreased the affinity for isoflurane but not for enflurane. The dipole moment of isoflurane is higher than that of enflurane, and the isoflurane binding site is more polar. CONCLUSION: These data indicate two binding sites of different character for the haloether anesthetics on HSA. One site is more polar and prefers isoflurane, presumably because of its larger dipole. The second site prefers the less polar enflurane. Therefore, weak polar interactions confer considerable selectivity, and differences in drug action may arise from occupancy of different protein sites.

Albumins↗
PubMed2005

Binding of the volatile general anesthetics halothane and isoflurane to a mammalian beta-barrel protein.

A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic-protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large beta-barrel internal cavity (515 +/- 30 angstroms(3)) lined predominantly by aromatic and aliphatic residues. Halothane binding to the beta-barrel cavity was determined using fluorescence quenching of Trp16, and a competitive binding assay with 1-aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using isothermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the beta-barrel of porcine odorant binding protein with dissociation constants of 0.46 +/- 0.10 mM and 0.43 +/- 0.12 mM determined using fluorescence quenching and competitive binding with 1-aminoanthracene, respectively. Isothermal titration calorimetry revealed that halothane and isoflurane bound with K(d) values of 80 +/- 10 microM and 100 +/- 10 microM, respectively. Halothane and isoflurane binding resulted in an overall stabilization of the folded conformation of the protein by -0.9 +/- 0.1 kcal.mol(-1). In addition to indicating specific binding to the native protein conformation, such stabilization may represent a fundamental mechanism whereby anesthetics reversibly alter protein function. Because porcine odorant binding protein has been successfully analyzed by X-ray diffraction to 2.25 angstroms resolution [1], this represents an attractive system for atomic-level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules.

Anesthetics, Inhalation↗
PubMed2004

Volatile anesthetic modulation of oligomerization equilibria in a hexameric model peptide.

To determine if occupancy of interfacial pockets in oligomeric proteins by volatile anesthetic molecules can allosterically regulate oligomerization equilibria, variants of a three-helix bundle peptide able to form higher oligomers were studied with analytical ultracentrifugation, hydrogen exchange and modeling. Halothane shifted the oligomerization equilibria towards the oligomer only in a mutation predicted to create sufficient volume in the hexameric pocket. Other mutations at this residue, predicted to create a too small or too polar pocket, were unaffected by halothane. Inhaled anesthetic modulation of oligomerization interactions is a novel and potentially generalizable biophysical basis for some anesthetic actions.

Allosteric Regulation↗
PubMed2004

Comparative binding character of two general anaesthetics for sites on human serum albumin.

Propofol and halothane are clinically used general anaesthetics, which are transported primarily by HSA (human serum albumin) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of these drugs. We characterized anaesthetic-HSA interactions in solution using elution chromatography, ITC (isothermal titration calorimetry), hydrogen-exchange experiments and geometric analyses of high-resolution structures. Binding affinity of propofol to HSA was determined to have a K(d) of 65 microM and a stoichiometry of approx. 2, whereas the binding of halothane to HSA showed a K(d) of 1.6 mM and a stoichiometry of approx. 7. Anaesthetic-HSA interactions are exothermic, with propofol having a larger negative enthalpy change relative to halothane. Hydrogen-exchange studies in isolated recombinant domains of HSA showed that propofol-binding sites are primarily found in domain III, whereas halothane sites are more widely distributed. Both location and stoichiometry from these solution studies agree with data derived from X-ray crystal-structure studies, and further analyses of the architecture of sites from these structures suggested that greater hydrophobic contacts, van der Waals interactions and hydrogen-bond formation account for the stronger binding of propofol as compared with the less potent anaesthetic, halothane.

Anesthetics, General↗
PubMed2004

Inhalational anesthetic-binding proteins in rat neuronal membranes.

Molecular targets of inhaled anesthetics must be represented in the group that specifically bind these drugs, but the paucity of direct binding data has limited the number of candidates for further evaluation. To find candidate targets, we used a combination of photolabeling, two-dimensional gel electrophoresis, and mass spectrometry to identify halothane-binding targets in rat neuronal membranes. Of the 265 spots detected on the two-dimensional gels, 90 were labeled by [(14)C]halothane, and 34 were identified. Mitochondrial proteins, especially respiratory complex and voltage-dependent anion channels, dominated the labeled group, and there were several examples of subunit- and state-dependent binding. A significant correlation was found between internal protein cavities and binding in a group of proteins with high resolution structures. Therefore, in addition to identifying novel neuronal targets, these data suggest a general molecular feature, the buried cavity, as a dominant attribute of volatile anesthetic-binding sites found in a limited number of neuronal membrane proteins.

Anesthetics↗
PubMed2004

Inhaled anesthetic enhancement of amyloid-beta oligomerization and cytotoxicity.

BACKGROUND: The majority of surgical patients receive inhaled anesthetics, principally small haloalkanes and haloethers. Long-term cognitive problems occur in the elderly subsequent to anesthesia and surgery, and previous surgery might also be a risk factor for neurodegenerative disorders like Alzheimer and Parkinson disease. The authors hypothesize that inhaled anesthetics contribute to these effects through a durable enhancement of peptide oligomerization. METHODS: Light scattering, filtration assays, electron microscopy, fluorescence spectroscopy and size-exclusion chromatography was used to characterize the concentration-dependent effects of halothane, isoflurane, propofol, and ethanol on amyloid beta peptide oligomerization. Pheochromocytoma cells were used to characterize cytotoxicity of amyloid oligomers with and without the above anesthetics. RESULTS: Halothane and isoflurane enhanced amyloid beta oligomerization rates and pheochromocytoma cytotoxicity in vitro through a preference for binding small oligomeric species. Ethanol and propofol inhibited oligomerization at low concentration but enhanced modestly at very high concentration. Neither ethanol nor propofol enhanced amyloid beta toxicity in pheochromocytoma cells. CONCLUSIONS: Inhaled anesthetics enhance oligomerization and cytotoxicity of Alzheimer disease-associated peptides. In addition to the possibility of a general mechanism for anesthetic neurotoxicity, these results call for further evaluation of the interaction between neurodegenerative disorders, dementia, and inhalational anesthesia.

Adrenal Gland Neoplasms↗