Competitive Interactions Between Propofol and Diazepam: Studies in GABAA Receptors and Zebrafish.

Although propofol is among the most commonly administered general anesthetics, its mechanism of action is not fully understood. It has been hypothesized that propofol acts via a similar mechanism as (R)-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate) by binding within the GABAA receptor transmembrane receptor domain at the two ß +/α - subunit interfaces with resultant positive allosteric modulation. To test this hypothesis, we leveraged the ability of diazepam to bind to those sites and act as a competitive antagonist. We used oocyte-expressed α 1 ß 3 γ 2L GABAA receptors to define the actions of diazepam (± flumazenil) on currents activated or potentiated by propofol and a zebrafish activity assay to define the impact of diazepam and flumazenil on propofol-induced anesthesia. We found that diazepam increased the amplitudes of GABAA receptor-mediated currents at nanomolar concentrations but reduced them at micromolar concentrations. The current amplitude changes produced by nanomolar diazepam concentrations were inhibited by flumazenil whereas those produced by micromolar diazepam concentrations were not. Studies of agonist potentiation showed that the micromolar inhibitory action of diazepam was surmountable by high concentrations of propofol and produced a rightward shift in the propofol concentration-response curve characterized by a Schild slope not statistically significantly different from 1, consistent with competition between diazepam and propofol. Although micromolar concentrations of diazepam (plus flumazenil) similarly reduced GABAA receptor currents modulated by propofol and etomidate, it only reduced the anesthetic actions of etomidate. We conclude that while both propofol and etomidate can modulate GABAA receptors by binding to the ß +/α - subunit interfacial sites, propofol-induced anesthesia likely involves additional target sites. SIGNIFICANCE STATEMENT: Although the drug combination of diazepam and flumazenil reverses the GABAA receptor positive modulatory actions of both propofol and (R)-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate (etomidate), it only reverses the in vivo anesthetic actions of etomidate. These results strongly suggest that distinct mechanisms of action account for the anesthetic actions of these two commonly administered anesthetic agents.

Photomotor Responses in Zebrafish and Electrophysiology Reveal Varying Interactions of Anesthetics Targeting Distinct Sites on γ-Aminobutyric Acid Type A Receptors.

BACKGROUND: Etomidate, barbiturates, alfaxalone, and propofol are anesthetics that allosterically modulate γ-aminobutyric acid type A (GABAA) receptors via distinct sets of molecular binding sites. Two-state concerted coagonist models account for anesthetic effects and predict supra-additive interactions between drug pairs acting at distinct sites. Some behavioral and molecular studies support these predictions, while other findings suggest potentially complex anesthetic interactions. We therefore evaluated interactions among four anesthetics in both animals and GABAA receptors. METHODS: The authors used video assessment of photomotor responses in zebrafish larvae and isobolography to evaluate hypnotic drug pair interactions. Voltage clamp electrophysiology and allosteric shift analysis evaluated coagonist interactions in α1ß3γ2L receptors activated by γ-aminobutyric acid (GABA) versus anesthetics [log(d, AN):log(d, GABA) ratio]. Anesthetic interactions at concentrations relevant to zebrafish were assessed in receptors activated with low GABA. RESULTS: In zebrafish larvae, etomidate interacted additively with both propofol and the barbiturate R-5-allyl-1-methyl m-trifluoromethyl mephobarbital (R-mTFD-MPAB; mean ± SD α = 1.0 ± 0.07 and 0.96 ± 0.11 respectively, where 1.0 indicates additivity), while the four other drug pairs displayed synergy (mean α range 0.76 to 0.89). Electrophysiologic allosteric shifts revealed that both propofol and R-mTFD-MPAB modulated etomidate-activated receptors much less than GABA-activated receptors [log(d, AN):log(d, GABA) ratios = 0.09 ± 0.021 and 0.38 ± 0.024, respectively], while alfaxalone comparably modulated receptors activated by GABA or etomidate [log(d) ratio = 0.87 ± 0.056]. With low GABA activation, etomidate combined with alfaxalone was supra-additive (n = 6; P = 0.023 by paired t test), but etomidate plus R-mTFD-MPAB or propofol was not. CONCLUSIONS: In both zebrafish and GABAA receptors, anesthetic drug pairs interacted variably, ranging from additivity to synergy. Pairs including etomidate displayed corresponding interactions in animals and receptors. Some of these results challenge simple two-state coagonist models and support alternatives where different anesthetics may stabilize distinct receptor conformations, altering the effects of other drugs.

Competitive Antagonism of Etomidate Action by Diazepam: In Vitro GABAA Receptor and In Vivo Zebrafish Studies.

BACKGROUND: Recent cryo-electron microscopic imaging studies have shown that in addition to binding to the classical extracellular benzodiazepine binding site of the α1ß3γ2L γ-aminobutyric acid type A (GABAA) receptor, diazepam also binds to etomidate binding sites located in the transmembrane receptor domain. Because such binding is characterized by low modulatory efficacy, the authors hypothesized that diazepam would act in vitro and in vivo as a competitive etomidate antagonist. METHODS: The concentration-dependent actions of diazepam on 20 µM etomidate-activated and 6 µM GABA-activated currents were defined (in the absence and presence of flumazenil) in oocyte-expressed α1ß3γ2L GABAA receptors using voltage clamp electrophysiology. The ability of diazepam to inhibit receptor labeling of purified α1ß3γ2L GABAA receptors by [H]azietomidate was assessed in photoaffinity labeling protection studies. The impact of diazepam (in the absence and presence of flumazenil) on the anesthetic potencies of etomidate and ketamine was compared in a zebrafish model. RESULTS: At nanomolar concentrations, diazepam comparably potentiated etomidate-activated and GABA-activated GABAA receptor peak current amplitudes in a flumazenil-reversible manner. The half-maximal potentiating concentrations were 39 nM (95% CI, 27 to 55 nM) and 26 nM (95% CI, 16 to 41 nM), respectively. However, at micromolar concentrations, diazepam reduced etomidate-activated, but not GABA-activated, GABAA receptor peak current amplitudes in a concentration-dependent manner with a half-maximal inhibitory concentration of 9.6 µM (95% CI, 7.6 to 12 µM). Diazepam (12.5 to 50 µM) also right-shifted the etomidate-concentration response curve for direct activation without reducing the maximal response and inhibited receptor photoaffinity labeling by [H]azietomidate. When administered with flumazenil, 50 µM diazepam shifted the etomidate (but not the ketamine) concentration-response curve for anesthesia rightward, increasing the etomidate EC50 by 18-fold. CONCLUSIONS: At micromolar concentrations and in the presence of flumazenil to inhibit allosteric modulation via the classical benzodiazepine binding site of the GABAA receptor, diazepam acts as an in vitro and in vivo competitive etomidate antagonist.

Nutrient Enrichment Alters Salt Marsh Fungal Communities and Promotes Putative Fungal Denitrifiers.

Enrichment of ecosystems with excess nutrients is occurring at an alarming rate and has fundamentally altered ecosystems worldwide. Salt marshes, which lie at the land-sea interface, are highly effective at removing anthropogenic nutrients through the action of macrophytes and through microbial processes in coastal sediments. The response of salt marsh bacteria to excess nitrogen has been documented; however, the role of fungi and their response to excess nitrogen in salt marsh sediments is not fully understood. Here, we document the response of salt marsh fungal communities to long-term excess nitrate in four distinct marsh habitats within a northern temperate marsh complex. We show that salt marsh fungal communities varied as a function of salt marsh habitat, with both fungal abundance and diversity increasing with carbon quantity. Nutrient enrichment altered fungal communities in all habitats through an increase in fungal abundance and the proliferation of putative fungal denitrifiers. Nutrient enrichment also altered marsh carbon quality in low marsh surface sediments where fungal response to nutrient enrichment was most dramatic, suggesting nutrient enrichment can alter organic matter quality in coastal sediments. Our results indicate that fungi, in addition to bacteria, likely play an important role in anaerobic decomposition of salt marsh sediment organic matter.

Measurement of extreme hyperfine fields in two-coordinate high-spin Fe2+ complexes by Mössbauer spectroscopy: essentially free-ion magnetism in the solid state.

Mössbauer studies of three two-coordinate linear high-spin Fe(2+) compounds, namely, Fe{N(SiMe3)(Dipp)}2 (1) (Dipp = C6H3-2,6-(i)Pr2), Fe(OAr')2 (2) [Ar' = C6H3-2,6-(C6H3-2,6-(i)Pr2)2], and Fe{C(SiMe3)3}2 (3), are presented. The complexes were characterized by zero- and applied-field Mössbauer spectroscopy (1-3), as well as zero- and applied-field heat-capacity measurements (3). As 1-3 are rigorously linear, the distortion(s) that might normally be expected in view of the Jahn-Teller theorem need not necessarily apply. We find that the resulting very large unquenched orbital angular momentum leads to what we believe to be the largest observed internal magnetic field to date in a high-spin iron(II) compound, specifically +162 T in 1. The latter field is strongly polarized along the directions of the external field for both longitudinal and transverse field applications. For the longitudinal case, the applied field increases the overall hyperfine splitting consistent with a dominant orbital contribution to the effective internal field. By contrast, 2 has an internal field that is not as strongly polarized along a longitudinally applied field and is smaller in magnitude at ca. 116 T. Complex 3 behaves similarly to complex 1. They are sufficiently self-dilute (e.g., Fe···Fe distances of ca. 9-10 Å) to exhibit varying degrees of slow paramagnetic relaxation in zero field for the neat solid form. In the absence of EPR signals for 1-3, we show that heat-capacity measurements for one of the complexes (3) establish a geff value near 12, in agreement with the principal component of the ligand electric field gradient being coincident with the z axis.

Selective actions of benzodiazepines at the transmembrane anaesthetic binding sites of the GABAA receptor: In vitro and in vivo studies.

BACKGROUND AND PURPOSE: In addition to binding to the classical high-affinity extracellular benzodiazepine binding site of the GABAA receptor, some benzodiazepines occupy transmembrane inter-subunit anaesthetic sites that bind etomidate (ß+ /α- sites) or the barbiturate derivative R-mTFD-MPAB (α+ /ß- and γ+ /ß- sites). We aimed to define the functional effects of these interactions on GABAA receptor activity and animal behaviour. EXPERIMENTAL APPROACH: With flumazenil blocking classical high-affinity extracellular benzodiazepine site effects, modulation of GABA-activated currents by diazepam, midazolam and flurazepam was measured electrophysiologically in wildtype and M2-15' mutant α1 ß3 γ2L GABAA receptors. Zebrafish locomotive activity was also assessed in the presence of each benzodiazepine plus flumazenil. KEY RESULTS: In the presence of flumazenil, micromolar concentrations of diazepam and midazolam both potentiated and inhibited wildtype GABAA receptor currents. ß3 N265M (M2-15' in the ß+ /α- sites) and α1 S270I (M2-15' in the α+ /ß- site) mutations reduced or abolished potentiation by these drugs. In contrast, the γ2 S280W mutation (M2-15' in the γ+ /ß- site) abolished inhibition. Flurazepam plus flumazenil only inhibited wildtype receptor currents, an effect unaltered by M2-15' mutations. In the presence of flumazenil, zebrafish locomotion was enhanced by diazepam at concentrations up to 30 µM and suppressed at 100 µM, suppressed by midazolam and enhanced by flurazepam. CONCLUSIONS AND IMPLICATIONS: Benzodiazepine binding to transmembrane anaesthetic binding sites of the GABAA receptor can produce positive or negative modulation manifesting as decreases or increases in locomotion, respectively. Selectivity for these sites may contribute to the distinct GABAA receptor and behavioural actions of different benzodiazepines, particularly at high (i.e. anaesthetic) concentrations.

Synthesis and electronic structure of cationic, neutral, and anionic bis(imino)pyridine iron alkyl complexes: evaluation of redox activity in single-component ethylene polymerization catalysts.

A family of cationic, neutral, and anionic bis(imino)pyridine iron alkyl complexes has been prepared, and their electronic and molecular structures have been established by a combination of X-ray diffraction, Mössbauer spectroscopy, magnetochemistry, and open-shell density functional theory. For the cationic complexes, [((iPr)PDI)Fe-R][BPh(4)] ((iPr)PDI = 2,6-(2,6-(i)Pr(2)-C(6)H(3)N═CMe)(2)C(5)H(3)N; R = CH(2)SiMe(3), CH(2)CMe(3), or CH(3)), which are known single-component ethylene polymerization catalysts, the data establish high spin ferrous compounds (S(Fe) = 2) with neutral, redox-innocent bis(imino)pyridine chelates. One-electron reduction to the corresponding neutral alkyls, ((iPr)PDI)Fe(CH(2)SiMe(3)) or ((iPr)PDI)Fe(CH(2)CMe(3)), is chelate-based, resulting in a bis(imino)pyridine radical anion (S(PDI) = 1/2) antiferromagnetically coupled to a high spin ferrous ion (S(Fe) = 2). The neutral neopentyl derivative was reduced by an additional electron and furnished the corresponding anion, [Li(Et(2)O)(3)][((iPr)PDI)Fe(CH(2)CMe(3))N(2)], with concomitant coordination of dinitrogen. The experimental and computational data establish that this S = 0 compound is best described as a low spin ferrous compound (S(Fe) = 0) with a closed-shell singlet bis(imino)pyridine dianion (S(PDI) = 0), demonstrating that the reduction is ligand-based. The change in field strength of the bis(imino)pyridine coupled with the placement of the alkyl ligand into the apical position of the molecule induced a spin state change at the iron center from high to low spin. The relevance of the compounds and their electronic structures to olefin polymerization catalysis is also presented.

Mechanistic investigation of the staudinger ligation.

The Staudinger ligation of azides and phosphines has found widespread use in the field of chemical biology, but the mechanism of the transformation has not been characterized in detail. In this work, we undertook a mechanistic study of the Staudinger ligation with a focus on factors that affect reaction kinetics and on the identification of intermediates. The Staudinger ligation with alkyl azides was second-order overall and proceeded more rapidly in polar, protic solvents. Hammett analyses demonstrated that electron-donating substituents on the phosphine accelerate the overall reaction. The electronic and steric properties of the ester had no significant impact on the overall rate but did affect product ratios. Finally, the structure of an intermediate that accumulates under anhydrous conditions was identified. These findings establish a platform for optimizing the Staudinger ligation for expanded use in biological applications.

C-H bond activation of hydrocarbons by an imidozirconocene complex.

Monomeric imidozirconocene complexes of the type Cp2(L)Zr=NCMe3 (Cp = cyclopentadienyl, L = Lewis base) have been shown to activate the carbon-hydrogen bonds of benzene, but not the C-H bonds of saturated hydrocarbons. To our knowledge, this singularly important class of C-H activation reactions has heretofore not been observed in imidometallocene systems. The M=NR bond formed on heating the racemic ethylenebis(tetrahydro)indenyl methyl tert-butyl amide complex, however, cleanly and quantitatively activates a wide range of n-alkane, alkene, and arene C-H bonds. Mechanistic experiments support the proposal of intramolecular elimination of methane followed by a concerted addition of the hydrocarbon C-H bond. Products formed by activation of sp2 C-H bonds are generally more thermodynamically stable than those formed by activation of sp3 C-H bonds, and those resulting from reaction at primary C-H bonds are preferred over secondary sp3 C-H activation products. There is also evidence that thermodynamic selectivity among C-H bonds is sterically rather than electronically controlled.