I1 Sites Research Articles

Temporal Variations of PCBs and Their Estimated Air-Soil Exchange Fluxes Measured in Seven Sites in Bursa-Turkey

ABSTRACT Polychlorinated biphenyls (PCBs) can travel in ambient air for long distances and exchange between different environments. Soil and ambient air samples were simultaneously gathered during the winter and summer months in 7 regions in Bursa, Turkey, to study spatio-temporal fluctuations and soil-air exchange of PCBs. Σ25 PCB concentrations for the winter and summer seasons were 0.007–0.04 ng/m3 and 0.03–0.24 ng/m3 in air, also 4.09–10.00 ng/g DM and 1.65–10.01 ng/g DM in soil, respectively. PCB concentrations showed spatial variations in air and soil samples. The lowest total PCB levels were measured both in air and soil samples in the R (rural) site. The highest Σ25 PCB concentrations were measured in the SR3 site in soil samples, while the I1 site was the most polluted in air samples. Ambient air concentrations of PCBs in the summer months were greater than those in winter. In SR1, SR2, SR3, and I1 sites, soil PCB levels in the summer were higher than in the winter. The opposite was observed in the other sites (R, U, I2). Net flux values and fugacity fraction (ff) values for all regions indicated that PCBs represented a transfer from soil to air. The air-soil net gas phase flux values changed between 0.04 - 1.01 ng/m2-day and 0.02–0.83 ng/m2-day in the summer and winter, respectively. The PCB homologous distributions of mean flux values differed depending on the regions and seasons. PCBs with 4- and 5-CBs were dominant in both seasons.

Increased monoamine oxidase activity and imidazoline binding sites in insulin-resistant adipocytes from obese Zucker rats.

BACKGROUNDDespite overt insulin resistance, adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIMTo investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide, since it is produced by monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in adipocytes.METHODS 3H-2-deoxyglucose uptake (2-DG) was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine. 14C-tyramine oxidation and binding of imidazolinic radioligands [3H-Idazoxan, 3H-(2-benzofuranyl)-2-imidazoline] were studied in adipocytes, the liver, and muscle. The influence of in vivo administration of tyramine + vanadium on glucose handling was assessed in lean and obese rats.RESULTS2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats, when compared to their lean littermates. Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited, while MAO was increased and SSAO decreased. These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat, when compared to the lean. In vitro, tyramine precluded the binding to I2 sites, while in vivo, its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese rats. CONCLUSIONThe adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number. However, probably as a consequence of SSAO down-regulation, the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes. The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.

First-principles study of the effect of boron on grain boundary in NiAl

The grain boundary (GB) is critical to the mechanical properties of NiAl. The addition of B in NiAl can lead to the increase of yield strength due to its segregation in GB. To elucidate the B-induced enhancement of GB, we investigate the segregation state and its strengthening mechanism of the additive B in the typical ∑5(310)[001] GB of NiAl at atomic scale using the first-principles method. Our calculations shown that the additive B atom prefers to segregate at the interstitial I1 site of GB, in which case it is surrounded by four first-nearest-neighboring (1NN) and two second-nearest-neighboring (2NN) Ni atoms. It is found the segregated B prefers to bond with its neighboring Ni instead of Al atoms. Our calculations further shown not only the intergranular cohesion is improved by the covalent bonding between the segregated B and its four 1NN Ni atoms, but also the intra-planar cohesion of GB is enhanced by the covalent bonding between the segregated B and its two 2NN Ni atoms. The covalent bonding between B and Ni atoms mainly stem from the contribution of B-p and Ni-d states, partly from the weak interactions of B-s and Ni-s states. Our first-principles tensile test shown that the yield stress and ductility of NiAl are indeed improved due to the segregation of B in GB.

Molecular aspects of monoamine oxidase B

Monoamine oxidases (MAO) influence the monoamine levels in brain by virtue of their role in neurotransmitter breakdown. MAO B is the predominant form in glial cells and in platelets. MAO B structure, function and kinetics are described as a background for the effect of alterations in its activity on behavior. The need to inhibit MAO B to combat decreased brain amines continues to drive the search for new drugs. Reversible and irreversible inhibitors are now designed using data-mining, computational screening, docking and molecular dynamics. Multi-target ligands designed to combat the elevated activity of MAO B in Alzheimer's and Parkinson's Diseases incorporate MAO inhibition (usually irreversible) as well as iron chelation, antioxidant or neuroprotective properties. The main focus of drug design is the catalytic activity of MAO, but the imidazoline I2 site in the entrance cavity of MAO B is also a pharmacological target. Endogenous regulation of MAO B expression is discussed briefly in light of new studies measuring mRNA, protein, or activity in healthy and degenerative samples, including the effect of DNA methylation on the expression. Overall, this review focuses on examples of recent research on the molecular aspects of the expression, activity, and inhibition of MAO B.

Highly crystalline, small sized, monodisperse α-NiS nanocrystal ink as an efficient counter electrode for dye-sensitized solar cells

α-NiS nanocrystals exhibit excellent catalytic activity for I3− reduction due to the strong interaction between I1 and α-NiS active sites.

Theoretical and experimental study of CO adsorption on Ca-FER zeolite

The interaction of CO with Ca-FER was investigated by a combination of IR spectroscopy and microcalorimetry and by periodic DFT calculations employing the ω/r correlation for calculations of CO stretching frequencies in order to gain more insight on Ca2+ cation coordination in the FER zeolite and site-specificity of CO stretching frequency in IR spectra of CO/Ca-FER system. Experimental data were interpreted on the basis of a good agreement between experimental and theoretical results. Detailed analysis of both theoretical and experimental results reveals that no individual band can be assigned to specific type of CO–Ca complex or to a one type of Ca2+ sites. On the contrary, several different type of (poly)carbonyl complexes contribute to each vibrational band discerned in the experimental IR spectra. The band at 2197cm−1 gradually shifting to 2194cm−1 with increasing coverage represents monocarbonyls at all Ca2+ sites and most stable dicarbonyls formed on Ca2+ cations at the intersection of channels. The band at 2188cm−1 is assigned to the dicarbonyl complexes on P6, M5, and M7 sites, whereas the band at 2184cm−1 belongs to the vibration of the less stable dicarbonyl complexes on P8 sites and to the tricarbonyl complexes on I2 sites. Comparison of results for Ca-FER with those reported previously for Mg-FER zeolite (R. Bulanek, I. Voleska, E. Ivanova, K. Hadjiivanov, P. Nachtigall, J. Phys. Chem. C 113 (2009) 11066) clearly showed that the differences in specific “effect from the bottom” are much smaller in the case of Ca2+ cations than in the case of Mg2+ cations due to the fact that large Ca2+ cations interacts with more framework oxygen atoms and they are farther from the zeolite framework than Mg2+ cations.

The Binding Parameters of Integration Host Factor to the Lambda Phage Cosi1 Site and Holliday Junction DNA

Integration Host Factor (IHF) is a heterodimeric DNA-binding protein produced by E.coli. IHF interacts with the minor groove and phosphodiester backbone of the DNA to create sharp bends in the DNA and is often referred to as an architectural protein. Although the protein binds sequence-specifically, it also recognizes DNA conformation. Prokaryotic cellular processes involving IHF include viral integration, replication and activation of gene expression. We have recently shown that IHF exhibits high binding affinity for the Holliday or four-way junction DNA (4WJ), an intermediate in homologous recombination where strands from different chromosomes cross over. Förster Resonance Energy Transfer (FRET), where the 5'-ends of DNA strands are labeled with fluorophores, is used to determine the conformational changes of the 4WJ when interacting with DNA-binding proteins. These measurements have shown that the 4WJ opens upon binding with IHF, which is consistent with data promoting the protein's involvement in recombinase action. Subsequent FRET experiments performed confirm this observation, and provide a value for the efficiency of binding and the degree of distortion induced. Interactions between IHF and the cos I1 site of DNA from the lambda bacteriophage will also be explored. It is known that IHF induces bending at cos sites. The affinity of binding to Δ cos DNA is compared to that between IHF and its consensus sequence (H1) in E. coli DNA. Gel mobility shift assay (GMSA) and solutions experiments will measure the binding and affinity of IHF with the cos I1 site. FRET experiments with labeled cosI1 DNA will reveal the distortion induced upon IHF binding and these results will be compared to those with IHF binding to the H1 site.

Adsorption of carbon monoxide on Ag(I)-ZSM-5 zeolite: An ab initio density functional theory study

Adsorption of carbon monoxide on different Ag+-exchange sites of Ag–ZSM-5 zeolite has been investigated using density functional theory. The coordination and local geometry of the Ag+ ion in Ag–ZSM-5 as well as adsorption structures and energies of CO adsorbed on these sites are explored extensively. The structure of Ag+-exchange sites, location of the Al atom on the T site, and number of the Al atoms contained in the sites are considered in the theoretical calculations. The calculated results show that the AgO coordination number of two is strongly preferred before and after CO adsorption. The AgO bond lengths are in a broad range of 2.2–2.9Å, and the AgC bond lengths for CO adsorbed on Ag–ZSM-5 zeolite are calculated to be 2.0–2.2Å. Both AgO and AgC bond lengths for CO–Ag–ZSM-5 complex are longer than those for CO–Cu–ZSM-5 complex. The calculated adsorption energy of CO adsorbed on the I2 sites is between 28.5 and 29.6kcal/mol, and that on the Z5, Z6, M5 and M6 sites containing one Al atom on the T position is between 11.3 and 18.9kcal/mol whereas the calculated adsorption energy of CO adsorbed on the M7 site containing one Al atom is 19.9kcal/mol. The introduction of the two Al atoms to the Ag+-exchange site results in a reduction of CO adsorption energy. In general, the adsorption energy of CO on Ag–ZSM-5 is lower than that on Cu–ZSM-5. The predicted coordination of the Ag+ ion, bond lengths of AgO and AgC as well as adsorption energy are in accord with available experimental results.

Analgesic efficacy of CR4056, a novel imidazoline-2 receptor ligand, in rat models of inflammatory and neuropathic pain

Two decades of investigations have failed to unequivocally clarify the functions and the molecular nature of imidazoline-2 receptors (I2R). However, there is robust pharmacological evidence for the functional modulation of monoamino oxidase (MAO) and other important enzyme activities by I2 site ligands. Some compounds of this class proved to be active experimental tools in preventing both experimental pain and opioid tolerance and dependence. Unfortunately, even though these compounds bind with high potency to central I2 sites, they fail to represent a valid clinical opportunity due to their pharmacokinetic, selectivity or side-effects profile. This paper presents the preclinical profile of a novel I2 ligand (2-phenyl-6-(1H-imidazol-1yl) quinazoline; [CR4056]) that selectively inhibits the activity of human recombinant MAO-A in a concentration-dependent manner. A sub-chronic four day oral treatment of CR4056 increased norepinephrine (NE) tissue levels both in the rat cerebral cortex (63.1% ±4.2%; P < 0.05) and lumbar spinal cord (51.3% ± 6.7%; P < 0.05). In the complete Freund’s adjuvant (CFA) rat model of inflammatory pain, CR4056 was found to be orally active (ED50 = 5.8 mg/kg, by mouth [p.o.]). In the acute capsaicin model, CR4056 completely blocked mechanical hyperalgesia in the injured hind paw (ED50 = 4.1 mg/kg, p.o.; ED100 = 17.9 mg/kg, p.o.). This effect was dose-dependently antagonized by the non-selective imidazoline I2/α2 antagonist idazoxan. In rat models of neuropathic pain, oral administration of CR4056 significantly attenuated mechanical hyperalgesia and allodynia. In summary, the present study suggests a novel pharmacological opportunity for inflammatory and/or neuropathic pain treatment based on selective interaction with central imidazoline-2 receptors.

Formation of an ATP-DnaA-specific Initiation Complex Requires DnaA Arginine 285, a Conserved Motif in the AAA+ Protein Family

Escherichia coli DnaA protein, a member of the AAA+ superfamily, initiates replication from the chromosomal origin oriC in an ATP-dependent manner. Nucleoprotein complex formed on oriC with the ATP-DnaA multimer but not the ADP-DnaA multimer is competent to unwind the oriC duplex. The oriC region contains ATP-DnaA-specific binding sites termed I2 and I3, which stimulate ATP-DnaA-dependent oriC unwinding. In this study, we show that the DnaA R285A mutant is inactive for oriC replication in vivo and in vitro and that the mutation is associated with specific defects in oriC unwinding. In contrast, activities of DnaA R285A are sustained in binding to the typical DnaA boxes and to ATP and ADP, formation of multimeric complexes on oriC, and loading of the DnaB helicase onto single-stranded DNA. Footprint analysis of the DnaA-oriC complex reveals that the ATP form of DnaA R285A does not interact with ATP-DnaA-specific binding sites such as the I sites. A subgroup of DnaA molecules in the oriC complex must contain the Arg-285 residue for initiation. Sequence and structural analyses suggest that the DnaA Arg-285 residue is an arginine finger, an AAA+ family-specific motif that recognizes ATP bound to an adjacent subunit in a multimeric complex. In the context of these and previous results, the DnaA Arg-285 residue is proposed to play a unique role in the ATP-dependent conformational activation of an initial complex by recognizing ATP bound to DnaA and by modulating the structure of the DnaA multimer to allow interaction with ATP-DnaA-specific binding sites in the complex.

Imidazoline Receptors and their Ligands as Potentiators of Nutrient-Induced Insulin Secretion

It is now well established that many tissues express binding sites that are defined pharmacologically as “imidazoline” receptors. These are still undergoing full molecular characterisation but candidate clones have been identified for both the I 1 and I2 subtypes. The physiological roles of these receptors remain to be the subject of continuing investigation although there is strong evidence that I1 sites play a role in the central regulation of blood pressure while I2 sites may control feeding and other aspects of behaviour. In molecular terms, the third imidazoline receptor subtype (I3) is the least well characterised although progress in understanding its functional pharmacology is continuing. This receptor plays a role in the regulation of insulin secretion and mediates glucose-dependent increases in secretion in response to synthetic ligands such as efaroxan and antazoline. These agents were originally developed as selective α2-adrenoceptor antagonists but it is now clear that they also act as I3 agonists. Molecular characterisation of the functional activity of I3 agonists has been complicated by the fact that most of the ligands also interact with other cellular proteins, leading to potential difficulties in the interpretation of experimental data. In particular, it was established more than a decade ago that many I3-agonists induce the closure of ATP-sensitive potassium (KATP) channels and it is now clear that an imidazoline binding site is located within the pore-forming subunit (Kir6.2) of the KATP channel. However, increasing evidence implies that closure of this channel is not primarily responsible for the ability of imidazoline drugs to enhance insulin secretion. In support of this, imidazoline compounds have recently been described that do not close KATP channels but which potentiate insulin secretion very effectively. These probably interact with intracellular targets within pancreatic β- cells and the results suggest that the I3 receptor may be distinct from the imidazoline binding site associated with Kir6.2. Rather, the I3-site appears to control a distal step in the exocytotic pathway. The present review examines recent progress in identifying the I 3 receptor and describes the pharmacology of I3-agonists that regulate insulin secretion. The possibility that these agonists might then serve as prototypes for the development of novel ligands having therapeutic potential is also considered. Keywords: type 2 diabetes, imidazoline receptors, efaroxan, clonidine-displacing substance, islets of langerhans, insulin secretion, glucagon secretion

Binding of β-carbolines at imidazoline I2 receptors: a structure–affinity investigation

A series of ring-substituted (i.e., methoxy and bromo) 3,4-dihydro- and 1,2,3,4-tetrahydro-β-carbolines was examined at I2 imidazoline receptors, as was the effect of ring-opening, ring-expansion, and translocation of the piperidinyl nitrogen atom. Several analogues were identified that bind with Ki <20 nM at I2 sites and with reduced affinity at α2-adrenergic receptors, and 1,2,3,4-tetrahydro-γ-carbolines were identified as a novel class of I2 imidazoline receptor ligand.

Imidazoline binding sites in the endocrine pancreas: can they fulfil their potential as targets for the development of new insulin secretagogues?

A variety of compounds containing an imidazoline ring have the ability to stimulate insulin secretion. Many of these also improve glycaemia in experimental models of type 2 diabetes and in man, suggesting that this class may be useful in the development of new orally active anti-diabetic drugs. However, the mechanisms by which imidazolines promote insulin secretion have not been clarified. The response does not appear to be due to the binding of ligands to either of the two major types of "imidazoline receptor" defined by pharmacological criteria (I1 and I2 sites) but may result from interaction with a novel imidazoline binding site. One such site has been identified in association with the ATP-sensitive potassium (K(ATP)) channel in the beta-cell and has been designated "I3". Electrophysiological and biochemical evidence suggest that the I3 site may be intrinsic to the ion-conducting pore component, Kir6.2, of the K(ATP) channel, but the effects of imidazoline ligands on insulin secretion can be dissociated from the regulation of Kir6.2. Indeed, there is increasing evidence that some imidazolines can control exocytosis directly, both in beta-cells and in pancreatic alpha-cells. Thus, it is proposed that a further imidazoline binding site is primarily responsible for control of hormone secretion. Evidence is reviewed which suggests that this site occupies a central position within an amplification pathway that also mediates the effects of cAMP in the beta-cell. Characterisation of this site should provide the stimulus for the design of new insulin secretagogues that are devoid of K(ATP) channel-blocking properties.

Selective imidazoline I2 ligands do not show antidepressant-like activity in the forced swim test in mice.

Clonidine is an adrenergic agonist with high affinity for alpha2 adrenoceptors that also has affinity for imidazoline receptors. Clonidine has previously been shown to reduce immobility in the forced swim test (FST) in mice. In the present study, this effect was blocked by idazoxan (0.06 mg/kg s.c.) and by yohimbine (1.0 mg/kg s.c.) suggesting that clonidine's effects in this test are mediated via its action at alpha2 sites. Imidazoline I2 site ligands have been shown to inhibit monoamine oxidase and thus may also have antidepressant activity. Three compounds with selective affinity for I2 receptors (BU224, BU239, BDF 8082) were also tested in the FST. These compounds showed no activity either alone or in combination with a subthreshold dose of imipramine in the FST. These results suggest that I2 receptor ligands do not show antidepressant-like activity in the FST in mice. Furthermore the activity of the mixed alpha2/I1 agonist clonidine is most likely to be due to its action at alpha2 sites.

Autoradiographic Comparison of [3H]-Clonidine Binding to Non-Adrenergic Sites and α2-Adrenergic Receptors in Human Brain

Clonidine is a partial agonist at brain α2-adrenoceptors (α2AR), but also has high affinity (KD = 51 nM) in homogenate binding assays for non-adrenergic imidazoline-binding sites (I-sites; imidazoline receptors). Herein, an autoradiographic comparison of [3H]-clonidine binding to I-sites and α2AR in sections of human brain is reported. For I-sites, the adrenergic component of 50 nM [3H]-clonidine binding was masked with either 60 μM norepinephrine (NE; α2AR agonist) or 12.5 μM methoxy-idazoxan (MIDX; selective α2AR antagonist), whereas the remaining non-adrenergic sites were studied by displacement with 20 μM cirazoline. Levels of [3H]-clonidine binding to α2AR and I-sites, determined in adjacent tissue sections, were positively correlated across 27 brain regions (p = 0.0003; r2 = 0.385). The principal olivary nucleus and the rostral portion of the ventrolateral medulla had highest ratios of I-sites: α2AR (>4:1). Quantitative transepts drawn across hippocampal images revealed α2AR enrichments in the CA-1 and inner molecular layers of the dentate gyrus—areas not enriched in I-sites. Competition curves were generated for I-sites in caudate sections using 10 ligands known to distinguish between I1 and I2 subtypes. The rank-order of affinities were cirazoline > harmane > BDF6143 > idazoxan = tizanidine (affinities of agmatine, efaroxan, moxonidine, NE, and oxymetazoline were too low to be reliable). Only the endogenous I-site ligand, harmane, had a monophasic displacement curve at the non-adrenergic sites (Ki = 521 ± 12 nM). In conclusion: 1) the distribution of non-adrenergic [3H]-clonidine binding sites in human brain sections was correlated with, but distinct from α2AR; and 2) the affinities of these sites was distinct from α1AR, α2AR, I1 or I2 sites as previously defined in membrane binding assays. The properties of this non-adrenergic [3H]-clonidine binding site are consistent with I-sites previously labeled by [3H]-cirazoline in rat brain.

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.

The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

Pharmacologic characterization of imidazoline receptor proteins identified by immunologic techniques and other methods.

Biochemical and pharmacologic evidence supports the heterogeneous nature of imidazoline receptors (IRs). However, only monoamine oxidase (MAO) (55- and 61-kD) isozymes have been identified as imidazoline binding site-containing proteins. Idazoxan-binding proteins of approximately 70- and approximately 45-kD of unknown amino acid sequences have been isolated from chromaffin cells and rat brain, respectively. Other proteins of approximately 27-30 to > 80 kD have been visualized by immunologic and photoaffinity labeling techniques in different tissues and species. The specific antiserum that recognizes the approximately 70-, approximately 45-, and approximately 29-kD IR proteins, but not MAO, was used to quantitate these proteins in the rat brain cortex. Treatments (7 days) with the I2-selective imidazoline drugs idazoxan (10 mg/kg), cirazoline (1 mg/kg), and LSL 60101 ([2-(2-benzofuranyl) imidazole; 10 mg/kg]) induced differential changes in these proteins: levels of the approximately 29-kD IR were increased by idazoxan and LSL 60101 (23%), levels of the approximately 45-kD protein only by cirazoline (44%), and those of the approximately 66-kD protein only by idazoxan (50%). These treatments also increased the densities of [3H]-idazoxan (I2) binding sites (32-42%). Chronic treatment with efaroxan, RX821002, and yohimbine (10 mg/kg), which possess very low affinity for I2-IRs, did not alter either their immunoreactivities or the density of I2 sites. Chronic treatment with MAO inhibitors clorgyline and phenelzine (10 mg/kg) and acute treatment with EEDQ (1.6 mg/kg, 6 h) induced decreases in the levels of these IR proteins (17-47%) and I2 sites (31-57%). Significant correlations were found when the mean percentage changes in immunoreactivity of IR proteins were related to the mean percentage changes in the density of I2 sites after treatment with the foregoing drug (r = 0.92, r = 0.69, and r = 0.75 for the approximately 29-, approximately 45-, and approximately 66-kD proteins, respectively). These results indicate that in the rat cerebral cortex, the I2 sites labeled by [3H]idazoxan are heterogeneous and that the related immunoreactive IR proteins contribute differently to the modulation of I2 sites after drug treatment.

'Seeing through a glass darkly': casting light on imidazoline 'I' sites.

Although imidazoline sites have been the subject of research for several years, there is still controversy about their structure, diversity and physiology. The I1 site is thought to exist principally as a binding site and is widely purported to play a role in controlling systemic blood pressure, although this is still unclear. The majority of I2 sites are widely accepted as being allosteric sites on monoamine oxidase; however, even with selective ligands, their exact function remains to be determined. A putative I3 site modulates insulin secretion and could represent the first functional site to be pharmacologically defined with selective agonists and antagonists. The structure and relevance of the proposed endogenous ligand 'clonidine-displacing substance' remains elusive. A potential candidate for this substance is agmatine; however, although it is capable of displacing bound clonidine from imidazoline sites, it lacks the functionality ascribed to the clonidine-displacing substance. In this review, Richard M. Eglen and colleagues assess our knowledge of imidazoline sites in the light of recent data.

Novel [3H]Clonidine Binding Sites in the Intestine of the Eel Acclimated to Sea Water

Novel clonidine binding sites were characterized in the intestinal membrane isolated from seawater eels. The specific clonidine binding sites consisted of at least two classes, high affinity (Kd = 1.4 ± 0.3 nM, n = 5) and low affinity (Kd = 175 ± 34 nM, n = 5) sites. The specific binding of 2 nM [3H]clonidinewas most enhanced at 20°C and at pH 7.5, and reversed by unlabelled clonidine. Such binding was hardly inhibited by adrenaline, yohimbine or rauwolscine, indicating that most binding sites are distinct from α2-adrenoceptor. The specific clonidine binding was inhibited by various imidazoline/guanidinium drugs, indicating existence of imidazoline/guanidinium receptive sites (IGRS) or imidazoline receptors in the eel intestine. Competition experiments revealed that rank order to displace 2 nM [3H]clonidine from their binding sites was as follows: guanabenz > cirazoline = naphazoline = UK14304 = ST587 ≥ clonidine ≥ idazoxan = RX821002 = tolazoline > ST93 = oxymetazoline = amiloride = ST91 > yohimbine = efaroxan = rauwolscine ≥ adrenaline = ST567 = histamine = agmatine. The rank order was different from those in I1 or I2 sites of IG RS reported in various mammalian tissues, suggesting existence of new IGRS, non I1 and non I2 sites, in the eel intestine. In addition, structure-affinity relationships are discussed from the results of competition experiments. Although physiological role of IGRS is not clear yet even in mammalian cells/tissues, eel intestine may be a good model to elucidate how the IGRS act in the cell and to decide what is the endogenous ligand for the IGRS, since eel intestine contains great amount of IGRS and it responds to guanabenz, an exogenous clonidine derivative.

Stimulation of insulin release from the MIN6 cell line by a new imidazoline compound, S-21663: evidence for the existence of a novel imidazoline site in beta cells.

1. The MIN6 cell line derived from in vivo immortalized insulin-secreting pancreatic beta cells was used to study the insulin-releasing capacity and the cellular mode of action of S-21663, a newly synthesized imadizoline compound known for its antidiabetic effect in vivo and its ability to release insulin from perfused pancreas. 2. S-21663, at concentrations ranging from 10(-5) M to 10(-3) M was able to release insulin from MIN6 cells; its activity peaked at 10(-4) M, a drop in the stimulant factor being noted between 10(-4) and 10(-3) M. Its efficacy, which did not differ whatever the glucose concentration (stimulant or not), was higher than that of the other secretagogues tested, glucose, sulphonylureas or the peptide tGLP-1. 3. In contrast to tGLP-1, S-21663 did not change the cyclic AMP content, whereas it increased Ca2+ influx via verapamil- and nifedipine-sensitive voltage-dependent calcium channels, the insulin release being a direct consequence of this Ca2+ entry. The S-21663-induced Ca2+ influx appears to be essentially the consequence of closure of K+ channels which differ from the ATP-dependent K+ (K-ATP) channels as determined by measurement of 86Rb efflux and use of a K-ATP channel opener. 4. Comparison of the effects of S-21663 to that of efaroxan, another imidazoline compound shown to act on insulin release in a glucose-dependent way via binding sites distinct from the imidazoline I1 and I2 sites, suggested that S-21663 acts through a novel site which displays a remarkably stable expression along the cell culture. 5. It is concluded that S-21663 is a very efficient, glucose-independent insulin secretagogue acting through a novel imidazoline site, linked to K+ channels, distinct from the I1, I2 and 'efaroxan' binding sites. In vitro and in vivo features of S-21663 indicate that this compound, or new drugs derived from it, might be the basis for a new pharmacological approach to the mangement of type II (non insulin-dependent) diabetes.