Imidazoleacetic Acid Research Articles

Discovery of α-Substituted Imidazole-4-acetic Acid Analogues as a Novel Class of ρ1 γ-Aminobutyric Acid Type A Receptor Antagonists with Effect on Retinal Vascular Tone.

The ρ-containing γ-aminobutyric acid type A receptors (GABAA Rs) play an important role in controlling visual signaling. Therefore, ligands that selectively target these GABAA Rs are of interest. In this study, we demonstrate that the partial GABAA R agonist imidazole-4-acetic acid (IAA) is able to penetrate the blood-brain barrier in vivo; we prepared a series of α- and N-alkylated, as well as bicyclic analogues of IAA to explore the structure-activity relationship of this scaffold focusing on the acetic acid side chain of IAA. The compounds were prepared via IAA from l-histidine by an efficient minimal-step synthesis, and their pharmacological properties were characterized at native rat GABAA Rs in a [3 H]muscimol binding assay and at recombinant human α1 β2 γ2S and ρ1 GABAA Rs using the FLIPR™ membrane potential assay. The (+)-α-methyl- and α-cyclopropyl-substituted IAA analogues ((+)-6 a and 6 c, respectively) were identified as fairly potent antagonists of the ρ1 GABAA R that also displayed significant selectivity for this receptor over the α1 β2 γ2S GABAA R. Both 6 a and 6 c were shown to inhibit GABA-induced relaxation of retinal arterioles from porcine eyes.

Potentiometric, Thermodynamics and Coordination Properties for Binary and Mixed Ligand Complexes of Copper(II) with Imidazole-4-acetic Acid and Tryptophan or Phenylalanine Aromatic Amino Acids

Binary and ternary complex formation equilibria of copper(II) with imidazole-4-acetic acid (IMA) and some aromatic amino acids such as tryptophan and phenylalanine have been studied from 15 to 45 °C by potentiometric titration. The pH-titrations of the reaction mixtures have shown 1:1:1 (Cu:IMA:amino acid) ternary complex formation. The stability of mixed-ligand complexes was quantitatively compared with the stability of the binary complexes as Δlog10 K, Δlog10 β and log10 X parameters. The speciation of different species in solution has been evaluated as a function of pH. The effect of temperature on protonation of the ligands and formation of mixed ligand complexes was investigated. Thermodynamic parameters were calculated and are discussed. The effect of solvent of the protonation of IMA and Cu–IMA complex formation was also investigated and discussed. Additionally, the stoichiometric protonation constants (log10 β) of imidazole-4-acetic acid and its binary Cu(II)–IMA complexes were determined potentiometrically over a wide range of solvent composition.

Comparison of templates for homology model of ρ1 GABAC receptors: More insights to the orthosteric binding site’s structure and functionality

Five sets of ρ1 GABAC homology models were generated based on X-ray crystal structures of the acetylcholine binding protein (AChBP), the ion channel from Caenorhabditis elegans (GLIC), the ion channel from Erwinia chrysanthemi (ELIC), the homomeric GABAA β3 ion channel, and the homomeric α-subunit of glutamate-gated homopentameric chloride channel (GluCl). The GluCl based model was found to the represent the structure of ρ1 GABAC receptors. The GABA pose docked in the selected best model was confirmed by QM-polarized ligand docking and induced fit docking protocol, and used to study molecular interactions in the ρ1 GABA binding site. The potential interactions of identified residues are discussed. This study identified several residues with potential ligand interactions located on loops F and G with their side chain oriented toward the binding site such as Ser215 and Gln83. The partial agonists muscimol and imidazole-4-acetic acid (I4AA) were docked into the binding site of the most reliable ‘GABA bound’ homology model. The potency and efficacy of these partial agonists in activating recombinant ρ1 receptors were correlated with their docking results. The model predicts that muscimol resembles GABA in the docking pose with similar interactions. However, I4AA has a very different docking pose to GABA and was predicted by the model to form π–π stacking with aromatic residues in the orthosteric binding site. A set of TPMPA bound ρ1 homology models based on the GluClα ‘apo state’ template was built in order to study a competitive antagonist in the ρ1 orthosteric binding site. The results demonstrated the ability of our model to explain most experimental findings and predict potential roles of residues within the orthosteric binding site.

A molecular characterization of the agonist binding site of a nematode cys-loop GABA receptor.

Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and are potential targets for novel anthelmintics (nematicides). However, compared with insect and mammalian receptors, little is known regarding the pharmacological characteristics of nematode Cys-loop GABA receptors. Here we have investigated the agonist binding site of the Cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus. We used two-electrode voltage-clamp electrophysiology to measure channel activation by classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes, along with site-directed mutagenesis and in silico homology modelling. The sulphonated molecules P4S and taurine had no effect on Hco-UNC-49. Other classical Cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > trans-4-aminocrotonic acid > isoguvacine > imidazole-4-acetic acid (IMA) > (R)-(-)-4-amino-3-hydroxybutyric acid [R(-)-GABOB] > (S)-(+)-4-amino-3-hydroxybutyric acid [S(+)-GABOB] > guanidinoacetic acid > isonipecotic acid > 5-aminovaleric acid (DAVA) (partial agonist) > β-alanine (partial agonist). In silico ligand docking revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response to DAVA and decreased twofold the EC50 for R(-)- and S(+)-GABOB. The pharmacological profile of Hco-UNC-49 differed from that of vertebrate Cys-loop GABA receptors and insect resistance to dieldrin receptors, suggesting differences in the agonist binding pocket. These findings could be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes.

Imidazole catalyzes chlorination by unreactive primary chloramines

Hypochlorous acid and simple chloramines (RNHCl) are stable biologically derived chlorinating agents. In general, the chlorination potential of HOCl is much greater than that of RNHCl, allowing it to oxidize or chlorinate a much wider variety of reaction partners. However, in this study we demonstrate by kinetic analysis that the reactivity of RNHCl can be dramatically promoted by imidazole and histidyl model compounds via intermediary formation of the corresponding imidazole chloramines. Two biologically relevant reactions were investigated—loss of imidazole-catalyzed chlorinating capacity and phenolic ring chlorination using fluorescein and the tyrosine analog, 4-hydroxyphenylacetic acid (HPA). HOCl reacted stoichiometrically with imidazole, N-acetylhistidine (NAH), or imidazoleacetic acid to generate the corresponding imidazole chloramines which subsequently decomposed. Chloramine (NH2Cl) also underwent a markedly accelerated loss in chlorinating capacity when NAH was present, although in this case N-α-acetylhistidine chloramine (NAHCl) did not accumulate, indicating that the catalytic intermediate must be highly reactive. Mixing HOCl with 1-methylimidazole (MeIm) led to very rapid loss in chlorinating capacity via formation of a highly reactive chlorinium ion (MeImCl+) intermediate; this behavior suggests that the reactive forms of the analogous imidazole chloramines are their conjugate acids, e.g., the imidazolechlorinium ion (HImCl+). HOCl-generated imidazole chloramine (ImCl) reacted rapidly with fluorescein in a specific acid-catalyzed second-order reaction to give 3′-monochloro and 3′,5′-dichloro products. Equilibrium constants for the transchlorination reactions HOCl + HIm = H2O + ImCl and NH2Cl + HIm = NH3 + ImCl were estimated from the dependence of the rate constants on [HIm]/[HOCl] and literature data. Acid catalysis again suggests that the actual chlorinating agent is HImCl+; consistent with this interpretation, MeIm markedly catalyzed fluorescein chlorination by HOCl. Time-dependent imidazole-catalyzed HPA chlorination by NH2Cl was also demonstrated by product analyses. Quantitative assessment of the data suggests that physiological levels of histidyl groups will react with primary chloramines to generate a flux of imidazole chloramine sufficient to catalyze biological chlorination via HImCl+, particularly in environments that generate high concentrations of HOCl such as the neutrophil phagosome.

Borne Identity: Leading Endogenous Suspects at Imidazoline Binding Sites

Over the past few years, a vast amount of research has shed light on the pharmacology of imidazoline binding sites (I-BS). To date, at least three classes of imidazoline binding sites have been characterised in accordance to their localisation, drug selectivity, proposed signalling pathways and functional roles. The existence of these sites raises the question as to whether an endogenous modulator exists. The identification of an endogenous extract denoted as clonidine displacing substance prompted the search for the active ingredient capable of mimicking the action of selective ligands at these sites. A number of candidates have been isolated and their functional activities have been assessed at these sites. Such endogenous ligands include agmatine, imidazoleacetic acid ribotide and the β-carboline harmane. As of yet, no consensus has been made to confirm the identity of the endogenous ligand at I-BS. The current review collates and reports what is known about these substances and their functional significance at I-BS.

Separation of native allophycocyanin and R-phycocyanin from marine red macroalga Polysiphonia urceolata by the polyacrylamide gel electrophoresis performed in novel buffer systems.

Three buffer systems of Imidazole−Acetic acid, HEPES−Imidazole/Bis-tris and Bis-tris−HEPES−MES were designed based on the principle of discontinuous polyacrylamide gel electrophoresis (PAGE) for the native PAGE which could be performed in pH 7.0 and 6.5 in order to analyze and prepare the minor components of allophycocyanin (AP) and R-phycocyanin (R-PC) from marine red macroalga Polysiphonia urceolata. These AP and R-PC phycobiliproteins are easily denatured in alkaline environments. The obtained results demonstrated that the PAGE modes performed in the buffer systems of HEPES−Imidazole/Bis-tris and Bis-tris−HEPES−MES gave the satisfactory resolution and separation of AP and R-PC proteins. The absorption and fluorescence spectra of the AP and R-PC proteins which were prepared by the established PAGE modes proved that they maintained natural spectroscopic characteristics. The established PAGE modes may also provide useful references and selections for some other proteins that are sensitive to alkaline environments or are not effectively separated by the classical PAGE modes performed normally in alkaline buffer systems.

Potentiometric and Thermodynamic Studies of Binary and Ternary Transition Metal(II) Complexes of Imidazole-4-acetic Acid and Some Bio-relevant Ligands

Proton–ligand association constants of imidazole-4-acetic acid (IMA) were determined potentiometrically in aqueous solution at different temperatures in the range 15–35 °C. The stepwise stability constants of IMA with some selected bivalent transition metal ions were also determined in 0.1 mol·dm−3 NaNO3. The stability of the complexes follows the trend Cu2+ > Ni2+ > Co2+ > Mn2+, which is in agreement with the Irving–Williams order of the metal ions. The thermodynamic parameters for Cu(II)–IMA complex formation were derived and discussed. The ternary complexes Cu(IMA)L (IMA = imidazole-4-acetic acid, HL = amino acid, amides or DNA constituents) have been investigated. Ternary complexes of amino acids or amides are formed by a simultaneous mechanism. Amino acids form the complex Cu(IMA)L, whereas amides form two complex species Cu(IMA)L and Cu(IMA)(LH−1). The DNA constituents form both 1:1 and 1:2 complexes. The stabilities of ternary complexes are quantitatively compared with their corresponding binary complexes. The concentration distribution of the complexes in solution was evaluated as a function of pH.

Optimization of the degradation of imazalil by photocatalysis: Comparison between commercial and lab-made photocatalysts

The elimination and mineralization of the fungicide imazalil and its by-products has been studied by means of heterogeneous photocatalysis. The activity of different photocatalysts, including the commercial TiO2 benchmark photocatalyst (Evonik P25) and several TiO2-based lab-made catalysts, is compared. The best results were achieved with the new lab-made photocatalyst synthesized by using sulphuric acid in the sol–gel process and calcined at a high temperature (EST-1023t). The apparent initial rate constant was up to 2 times faster for EST-1023t when compared to that obtained with Evonik P25. The effect of different operating parameters (pH, photocatalyst load and initial imazalil concentration) on the photocatalytic process were studied for these two photocatalysts and a degradation pathway was proposed considering data obtained from ion chromatography, LC–MS and FTIR studies. The formation of imidazole acetic acids was confirmed, as well as complete mineralization and release of stoichiometric nitrogen (as ammonium and nitrate) and chloride ions.

Imidazole Initiates Exsheathing of L3Teladorsagia circumcincta

The infective larvae of Teladorsagia circumcincta have a protective sheath that is lost soon after they reach the rumen of the sheep (the definitive host). Incubation in vitro with 50 mM imidazole caused more than 75% of L3 T. circumcincta to begin exsheathing within 2 hr. The initiation of exsheathing was less likely at pH 6.2 than at pH 7.8. The apparent pKa of this process was 7.08, similar to that for the conversion of imidazolium(+) to imidazole. Both the extent and the initial rate of exsheathing initiation increased with imidazole concentration (the apparent K(1/2) was about 50 mM). The initial rate of exsheathing initiation was stimulated by lactose and maltose, but not by some other carbohydrates, and by propylamine and imidazole acetic acid, but not by histidine.

Differentiating Enantioselective Actions of GABOB: A Possible Role for Threonine 244 in the Binding Site of GABAC ρ1 Receptors

Designing potent and subtype-selective ligands with therapeutic value requires knowledge about how endogenous ligands interact with their binding site. 4-Amino-3-hydroxybutanoic acid (GABOB) is an endogenous ligand found in the central nervous system in mammals. It is a metabolic product of GABA, the major inhibitory neurotransmitter. Homology modeling of the GABA(C) ρ(1) receptor revealed a potential H-bond interaction between the hydroxyl group of GABOB and threonine 244 (T244) located on loop C of the ligand binding site of the ρ(1) subunit. Using site-directed mutagenesis, we examined the effect of mutating T244 on the efficacy and pharmacology of GABOB and various ligands. It was found that mutating T244 to amino acids that lacked a hydroxyl group in their side chains produced GABA insensitive receptors. Only by mutating ρ(1)T244 to serine (ρ(1)T244S) produced a GABA responsive receptor, albeit 39-fold less sensitive to GABA than ρ(1)wild-type. We also observed changes in the activities of the GABA(C) receptor partial agonists, muscimol and imidazole-4-acetic acid (I4AA). At the concentrations we tested, the partial agonists antagonized GABA-induced currents at ρ(1)T244S mutant receptors (Muscimol: ρ(1)wild-type, EC(50) = 1.4 μM; ρ(1)T244S, IC(50) = 32.8 μM. I4AA: ρ(1)wild-type, EC(50) = 8.6 μM; ρ(1)T244S, IC(50) = 21.4 μM). This indicates that T244 is predominantly involved in channel gating. R-(-)-GABOB and S-(+)-GABOB are full agonists at ρ(1)wild-type receptors. In contrast, R-(-)-GABOB was a weak partial agonist at ρ(1)T244S (1 mM activates 26% of the current produced by GABA EC(50) versus ρ(1)wild-type, EC(50) = 19 μM; I(max) 100%), and S-(+)-GABOB was a competitive antagonist at ρ(1)T244S receptors (ρ(1)wild-type, EC(50) = 45 μM versus ρ(1)T244S, IC(50) = 417.4 μM, K(B) = 204 μM). This highlights that the interaction of GABOB with T244 is enantioselective. In contrast, the potencies of a range of antagonists tested, 3-aminopropyl(methyl)phosphinic acid (3-APMPA), 3-aminopropylphosphonic acid (3-APA), S- and R-(3-amino-2-hydroxypropyl)methylphosphinic acid (S-(-)-CGP44532 and R-(+)-CGP44533), were not altered. This suggests that T244 is not critical for antagonist binding. Receptor gating is dynamic, and this study highlights the role of loop C in agonist-evoked receptor activation, coupling agonist binding to channel gating.

Optimization of co‐agonism at GLP‐1 and glucagon receptors to safely maximize weight reduction in DIO‐rodents

The ratio of GLP-1/glucagon receptor (GLP1R/GCGR) co-agonism that achieves maximal weight loss without evidence of hyperglycemia was determined in diet-induced obese (DIO) mice chronically treated with GLP1R/GCGR co-agonist peptides differing in their relative receptor agonism. Using glucagon-based peptides, a spectrum of receptor selectivity was achieved by a combination of selective incorporation of GLP-1 sequences, C-terminal modification, backbone lactam stapling to stabilize helical structure, and unnatural amino acid substitutions at the N-terminal dipeptide. In addition to α-amino-isobutyric acid (Aib) substitution at position two, we show that α,α'-dimethyl imidazole acetic acid (Dmia) can serve as a potent replacement for the highly conserved histidine at position one. Selective site-specific pegylation was used to further minimize enzymatic degradation and provide uniform, extended in vivo duration of action. Maximal weight loss devoid of any sign of hyperglycemia was achieved with a co-agonist comparably balanced for in vitro potency at murine GLP1R and GCGR. This peptide exhibited superior weight loss and glucose lowering compared to a structurally matched pure GLP1R agonist, and to co-agonists of relatively reduced GCGR tone. Any further enhancement of the relative GCGR agonist potency yielded increased weight loss but at the expense of elevated blood glucose. We conclude that GCGR agonism concomitant with GLP1R agonism constitutes a promising approach to treatment of the metabolic syndrome. However, the relative ratio of GLP1R/GCGR co-agonism needs to be carefully chosen for each species to maximize weight loss efficacy and minimize hyperglycemia.

Enhancing Polysaccharide-Mediated Delivery of Nucleic Acids Through Functionalization with Secondary and Tertiary Amines

Chitosan is a polysaccharide that has generated significant interest as a non-viral gene delivery vehicle due to its cationic and biocompatible characteristics. However, transfection efficiency of chitosan is significantly lower compared to other cationic gene delivery agents, e.g. polyethyleneimine (PEI), dendrimers or cationic lipids. This is primarily attributed to its minimal solubility and low buffering capacity at physiological pH leading to poor endosomal escape of the gene carrier and inefficient cytoplasmic decoupling of the complexed nucleic acid. Here we have developed an imidazole acetic acid (IAA)-modified chitosan to introduce secondary and tertiary amines to the polymer in order to improve its endosomal buffering and solubility. The modified polymer was characterized by ninhydrin and (1)H NMR assays for degree of modification, while buffering and solubility were analyzed by acid titration. Nanocomplex formation, studied at various polymer-nucleic acid ratios, showed an increase in particle zeta potential for chitosan-IAA, as well as an increase in the effective diameter. Up to 100-fold increase in transfection efficiency of pDNA was seen for chitosan-IAA as compared to native chitosan, nearly matching that of PEI. In addition, transfection of siRNA by the modified polymers showed efficient gene knockdown equivalent to commercially available siPORT Amines. Collectively, these results demonstrate the potential of the imidazole-grafted chitosan as a biocompatible and effective delivery vehicle for both pDNA and siRNA.

A Catalytic Mechanism Revealed by the Crystal Structures of the Imidazolonepropionase from Bacillus subtilis

Imidazolonepropionase (EC 3.5.2.7) catalyzes the third step in the universal histidine degradation pathway, hydrolyzing the carbon-nitrogen bonds in 4-imidazolone-5-propionic acid to yield N-formimino-l-glutamic acid. Here we report the crystal structures of the Bacillus subtilis imidazolonepropionase and its complex at 2.0-A resolution with substrate analog imidazole-4-acetic acid sodium (I4AA). The structure of the native enzyme contains two domains, a TIM (triose-phosphate isomerase) barrel domain with two insertions and a small beta-sandwich domain. The TIM barrel domain is quite similar to the members of the alpha/beta barrel metallo-dependent hydrolase superfamily, especially to Escherichia coli cytosine deaminase. A metal ion was found in the central cavity of the TIM barrel and was tightly coordinated to residues His-80, His-82, His-249, Asp-324, and a water molecule. X-ray fluorescence scan analysis confirmed that the bound metal ion was a zinc ion. An acetate ion, 6 A away from the zinc ion, was also found in the potential active site. In the complex structure with I4AA, a substrate analog, I4AA replaced the acetate ion and contacted with Arg-89, Try-102, Tyr-152, His-185, and Glu-252, further defining and confirming the active site. The detailed structural studies allowed us to propose a zinc-activated nucleophilic attack mechanism for the hydrolysis reaction catalyzed by the enzyme.

Hair analysis of histamine and several metabolites in C3H/HeNCrj mice by ultra performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS): Influence of hair cycle and age

Background According to a previous study, the concentration of HA in the hair of SD rats was similar in each rat and the variation in HA concentration was not so great. However, the concentration in human hair was fairly different in each person. As possible reasons for the higher variation in human hair, the differences in hair cycles and age in each person may be considerable. Based on this idea, the studies using C3H/HeNCrj mice who can synchronize their hair cycle were performed for resolution of the influence of hair cycle and age. Methods The effects of hair cycle and age on the concentration of histamine (HA) and several metabolites, i.e., 1-methylhistamine (MHA), imidazole-4-acetic acid (IAA), and 1-methyl-4-imidazole-acetic acid (MIAA), in C3H/HeNCrj mice hair were investigated by ultra-performance liquid chromatography (UPLC) with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). HA and the metabolites were labeled with 4-( N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) and 4-( N, N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ). The resulting derivatives were separated by UPLC and determined with ESI-TOF-MS. Results A good linearity was achieved from the calibration curves, obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., histamine-α,α,β,β- d 4 (HA- d 4) or 4-imidazolecarboxylic acid (ICA), against the injected amounts of each compound. The detection limits of HA, MHA, IAA, and MIAA on mass chromatograms were 0.21, 1.0, 0.17, and 0.11 pmol, respectively. The concentrations of HA and the metabolites in the hair shafts and hair root of C3H/HeNCrj mice were determined by this method. The concentration of HA in the hair shaft was relatively higher in the telogen phase. In contrast, the HA content in the anagen phase was increased only in the hair root of old mice. Conclusion HA appears to possess some effect on hair growth, although the exact reason was not obvious. The HA metabolites, i.e., MHA, MIAA and IAA, were also determined the same as HA; however, the difference in the metabolite concentrations between the hair cycle and age was not clear in both hair shaft and hair root. Such studies of the effect of hair cycle and age on these concentrations are the first report. This analytical technique may be applicable to the determination of various biological compounds in hair.

OFF response of bullfrog cones is shaped by terminal ionotropic GABA receptors

We recently reported an ionotropic GABA receptor expressed at the bullfrog retinal cone terminal that is potentiated by the GABA A receptor antagonist bicuculline (BIC) and suppressed by the GABA C receptor antagonist imidazole-4-acetic acid (I4AA) [13]. In this study, by using the patch clamp technique in current clamp mode, we show that activation of this GABA receptor causes voltage changes of cones, which are closely dependent on the membrane potential level in relation to the chloride equilibrium potential of the cells. Furthermore, the OFF overshoot of cone light responses is enhanced or diminished when this receptor is potentiated by BIC or suppressed by I4AA, suggesting the involvement of this GABA receptor in shaping OFF light responses of bullfrog cones.

Rapid determination of histamine and its metabolites in mice hair by ultra-performance liquid chromatography with time-of-flight mass spectrometry

The rapid determination of histamine (HA) and several metabolites, i.e., 1-methylhistamine (MHA), imidazole-4-acetic acid (IAA), and 1-methyl-4-imidazole-acetic acid (MIAA), in mice hairs was performed by ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC-TOF-MS). HA and MHA, having a primary amino group ( NH 2) in their structures, were first labeled with 4-( N, N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 °C for 45 min in the mixture of 0.1 M borax (pH 9.3) and acetonitrile (CH 3CN). On the other hand, 4-( N, N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) was used for the labeling of a carboxylic acid group ( COOH) in IAA and MIAA in the presence of 2,2′-dipyridyl disulfide (DPDS) and triphenylphosphine (TPP). The reaction with DBD-PZ was completed at 50 °C after 2 h. The resulting derivatives of HA and the metabolites were perfectly separated using an ACQUITY UPLC™ BEH C 18 column (1.7 μm, 100 × 2.1 mm, i.d.) with the mixture of 20 mM HCOONH 4 and CH 3CN (8:2). The structures of HA and the metabolites were identified from the protonated-molecular ions [M + H] + and the de-protonated-molecular ions [M − H] − of authentic compounds, obtained from TOF-MS measurement. A good linearity was achieved from the calibration curves, obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., histamine-α,α,β,β- d 4 (HA- d 4) or 4-imidazolecarboxylic acid (ICA), against the injected amounts of each compound (1.0–25 pmol, r 2 = 0.998). The detection limits of HA and the metabolites were less than 1 pmol. The proposed method was applied to the determination in the hair shafts of C3H mice. The average concentrations of HA, MHA, IAA and MIAA in 1 mg of the hair shafts were 16.3 pmol ( n = 7), 21.6 pmol ( n = 3), 6.6 pmol ( n = 3) and 7.1 pmol ( n = 3), respectively. Because the proposed method provides good mass accuracy and the trace detection of HA and several metabolites in hair, this analytical technique seems to be applicable for the determination of various biological compounds in hair.

High pH accelerates GABA deactivation on perch-ρ1B receptors

The ionotropic GABA C receptor, formed by GABA ρ subunits, is known to be modulated by a variety of endogenous compounds, as well as by changes in pH. In this study, we explore the proton sensitivity of the GABA ρ subunits cloned from the perch retina, and report a novel action of high pH on the homomeric receptor formed by one of the GABA ρ subunits, the perch-ρ 1B subunit. Raising extracellular pH to 9.5 significantly accelerated GABA deactivation responses elicited from oocytes expressing the perch-ρ 1B subunit, and reduced its sensitivity to GABA. The change in the kinetics of the GABA-offset response occurred without altering the maximum response amplitude, and the reduced GABA sensitivity was independent of membrane potential. Although acidification of the extracellular solution also accelerated GABA deactivation for all other GABA ρ receptors examined in this study, the effects of high pH were unique to the homomeric receptor formed by the perch-ρ 1B subunit. In addition, we found that, unlike the effects on the response to the naturally occurring full agonist GABA, the responses elicited by partial agonists (imidazole-4-acetic acid (I4AA) and β-alanine) in the presence of the high pH solution showed a significant reduction in the maximum response amplitude. When considered in terms of a model describing the activation of GABA C receptors, in which pH can potentially affect either the binding affinity or the rate of channel closure, the results were consistent with the view that external alkalization reduces the gating efficiency of the receptor. To identify the proton sensitive domain(s) of the perch-ρ 1B receptor, chimeras were constructed by domain swapping with other perch-ρ subunits. Analysis of the pH sensitivities of the various chimeric receptors revealed that the alkaline-sensitive residues are located in the N-terminal region of the perch-ρ 1B subunit.

An Ionotropic GABA Receptor with Novel Pharmacology at Bullfrog Cone Photoreceptor Terminals

Characteristics of ionotropic γ-aminobutyric acid (GABA) receptors at bullfrog cone terminals were studied by patch clamp techniques in isolated cell and retinal slice preparations. GABA-induced inward currents from isolated cones reversed in polarity at a potential, very close to the chloride equilibrium potential, and they were completely suppressed by picrotoxin. Unexpectedly, the GABA current was dose-dependently potentiated by the well-known GABA_A receptor antagonist bicuculline (BIC), but was suppressed by gabazine, another GABA_A antagonist, and imidazole-4-acetic acid (I4AA), a GABA_C receptor antagonist. Similarly, currents induced by both GABA_A agonist muscimol and GABA_C agonist cis-4-aminocrotonic acid (CACA) were also potentiated by BIC. Furthermore, currents induced from cones by GABA and kainate-caused depolarization of horizontal cells in retinal slice preparations were both potentiated by BIC. All these results suggest that the ionotropic GABA receptor at the bullfrog cone terminal exhibits novel pharmacology, distinct from both traditional GABA_A and GABA_C receptors.

Imidazoleacetic acid-ribotide: an endogenous ligand that stimulates imidazol(in)e receptors.

We identified the previously unknown structures of ribosylated imidazoleacetic acids in rat, bovine, and human tissues to be imidazole-4-acetic acid-ribotide (IAA-RP) and its metabolite, imidazole-4-acetic acid-riboside. We also found that IAA-RP has physicochemical properties similar to those of an unidentified substance(s) extracted from mammalian tissues that interacts with imidazol(in)e receptors (I-Rs). ["Imidazoline," by consensus (International Union of Pharmacology), includes imidazole, imidazoline, and related compounds. We demonstrate that the imidazole IAA-RP acts at I-Rs, and because few (if any) imidazolines exist in vivo, we have adopted the term "imidazol(in)e-Rs."] The latter regulate multiple functions in the CNS and periphery. We now show that IAA-RP (i) is present in brain and tissue extracts that exhibit I-R activity; (ii) is present in neurons of brainstem areas, including the rostroventrolateral medulla, a region where drugs active at I-Rs are known to modulate blood pressure; (iii) is present within synaptosome-enriched fractions of brain where its release is Ca(2+)-dependent, consistent with transmitter function; (iv) produces I-R-linked effects in vitro (e.g., arachidonic acid and insulin release) that are blocked by relevant antagonists; and (v) produces hypertension when microinjected into the rostroventrolateral medulla. Our data also suggest that IAA-RP may interact with a novel imidazol(in)e-like receptor at this site. We propose that IAA-RP is a neuroregulator acting via I-Rs.