Lack of G protein-coupled sigma receptors in rat brain membranes: receptor-mediated high-affinity GTPase activity and [35S]GTPgammaS binding studies.

Although sigma (sigma) receptors have been identified as an independent receptor family distinct from opioid and phencyclidine receptors, the physiological roles of these receptors are largely unknown. It is controversial whether there exist metabotropic sigma receptors that are coupled with heterotrimeric G proteins. In the present study, the stimulatory effects of sigma ligands on high-affinity GTPase activity and [35S]GTPgammaS binding were determined in the membranes prepared from rat cerebral cortex, hippocampus, and striatum. In either G protein activation assay, none of the sigma ligands examined had stimulatory effect in any brain regions, except for unambiguous concentration-dependent increase in [35S]GTPgammaS binding by (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine [(+)-3-PPP] in striatal membranes. However, the competition study clearly showed this response was mediated through dopamine D2-like receptors, but not sigma receptors. It is concluded that sigma receptors are not coupled to heterotrimeric G proteins, at least those of Gi/o type.

Development of orally active nonpeptidic inhibitors of human neutrophil elastase.

5-Amino-2-phenylpyrimidin-6-ones, some of their desamino derivatives, and miscellaneous derivatives were synthesized and biologically evaluated on both in vitro activity and oral activity in an acute hemorrhagic assay. These compounds contained an alpha-keto-1,3,4-oxadiazole moiety to bind covalently to the Ser-195 hydroxy group of human neutrophil elastase (HNE). Among those tested, compounds 11a-c,e,i-l(F), 11d,e,k(H), 21d,e,k(F), and 21d,e(H) showed a good oral profile. RS-Mixture 3(H) was selected for clinical evaluation based on its oral potency, duration of action, enzyme selectivity, safety profile, and ease of synthesis. Structure-activity relationships (SARs) are discussed.

Cyclic AMP-mediated signaling components are upregulated in the prefrontal cortex of depressed suicide victims.

The components of cyclic AMP signaling cascade (catalytic (Calpha) subunit of cyclic AMP-dependent protein kinase (PKA) and cyclic AMP response element binding protein (CREB)) were quantitated by Western blotting in the prefrontal cortex of depressed suicide victims (n=23) and their matched controls (n=14). There was a significant increase in the levels of CREB, both in total (tCREB; 121+/-8% (mean+/-S.E.M.), P<0.02) and phosphorylated (pCREB; 128+/-9%, P<0.01) forms, but not in PKA Calpha levels (109+/-9%, ns), in brains of depressed suicides compared to those in control subjects. The increases in CREB were specifically observed in antidepressant drug-free subjects (tCREB: 137+/-11%, P<0.01; pCREB: 136+/-12%, P<0.02; n=9), but not in the antidepressant-treated subjects (tCREB: 108+/-18%, ns; pCREB: 111+/-17%, ns; n=8). There were significant correlations between the levels of PKA and those of tCREB and pCREB in the prefrontal cortex of depressed suicides. These results indicate that the components of cyclic AMP signaling are upregulated in a coordinated manner in brains of depressed suicides and that this alteration is not related to antidepressant treatment.

The crystal structure of the complex of non-peptidic inhibitor of human neutrophil elastase ONO-6818 and porcine pancreatic elastase.

The crystal structure of a new inhibitor of human neutrophil elastase (HNE), N-[2-[5-(tert-butyl)-1,3,4-oxadiazol-2-yl]-(IRS)-1-(methylethyl)-2-oxoethyl]-2-(5-amino-6-oxo-2-phenyl-6H-pyrimidin-1-ly)acetamide (ONO-6818, 1) complexed to porcine pancreatic elastase (PPE) has been determined at 1.86 A resolution. Analytical results provided evidence of a 1:1 complex in which the electrophilic ketone of 1 covalently bound to O gamma of Ser195 at the active site of PPE. The role of the unique electron-withdrawing ketone of 1 has been elucidated.

Conformational study of a highly specific CXCR4 inhibitor, T140, disclosing the close proximity of its intrinsic pharmacophores associated with strong anti-HIV activity.

We report the solution structure of T140, a truncated polyphemusin peptide analogue that efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 through its specific binding to a chemokine receptor, CXCR4. Nuclear magnetic resonance analysis and molecular dynamic calculations revealed that T140 has a rigidly structured conformation constituted by an antiparallel beta-sheet and a type II' beta-turn. A protuberance is formed on one side of the beta-sheet by the side-chain functional groups of the three amino acid residues (L-3-(2-naphthyl)alanine, Tyr5 and Arg14), each of which is indispensable for strong anti-HIV activity. These findings provide a rationale to dissect the structural basis for the ability of this compound to block the interaction between CXCR4 and envelope glycoproteins from T-tropic strains of HIV-1.

Identification of galpha subtype(s) involved in gamma-aminobutyric acid(B) receptor-mediated high-affinity guanosine triphosphatase activity in rat cerebral cortical membranes.

The ability of a series of specific Galpha carboxyl-terminal antisera, (i.e. anti-Gsalpha, anti-Gi1/2alpha, anti-Gi3alpha/Goalpha, anti-Goalpha/Gi3alpha, and anti-Gq/11alpha) to disrupt (+/-)-baclofen-stimulated high-affinity guanosine triphosphatase (GTPase) activity was explored in rat cerebral cortical membranes to identify the Galpha subunit(s) involved in gamma-aminobutyric acid(B) (GABA(B)) receptor-mediated signal transduction. Pretreatment of the membranes with the AS/7 (anti-Gi1/2alpha) antiserum inhibited GABA(B) receptor-mediated response without affecting the basal activity. The RM/1 (anti-Gsalpha) and QL (anti-Gq/11alpha) antisera failed to inhibit GABA(B) receptor-coupled responses. The results of the EC/2 (anti-Gi3alpha/Goalpha) and GO/1 (anti-Goalpha/Gi3alpha) antisera were difficult to interpret since the basal activities were influenced by these antisera. These results, in conjunction with the data in our previous reconstitution study, indicate that Gi2alpha is a main transducer of GABA(B) receptor-mediated signaling in rat cerebral cortex.

Functional coupling of GABA(B) receptors with G proteins that are sensitive to N-ethylmaleimide treatment, suramin, and benzalkonium chloride in rat cerebral cortical membranes.

Although it is known that GABA(B) receptors are negatively coupled to adenylyl cyclase, the detailed selectivity of functional interaction between GABA(B) receptors and Gi subfamily members is still ambiguous. (+/-)-Baclofen-stimulated high-affinity GTPase activity, which was competitively antagonized by 2-hydroxy-saclofen, was attenuated by pretreatment of the membranes with N-ethylmaleimide (NEM) in a concentration- and incubation period-dependent manner. The NEM-pretreated (50 microM, 15 min at 4 degrees C) membranes restored the (+/-)-baclofen-sensitive high-affinity GTPase activity when reconstituted with pertussis toxin-sensitive bovine brain G proteins. Among recombinant rat Galpha subunits, G(i alpha(-2)) appeared most effective as compared with other subunits (G(i alpha(-2)) > G(i alpha(-3) > G(i alpha(-1) = G(o alpha). The GABA(B) receptor-mediated high-affinity GTPase activity was also completely eliminated by 100 microM suramin and by 100 microM benzalkonium chloride. These results indicate that GABA(B) receptors in rat cerebral cortex couple to NEM-sensitive G proteins, in particular Gi2, which are sensitive to suramin and benzalkonium chloride.

Open pergolide treatment of tricyclic and heterocyclic antidepressant-resistant depression.

BACKGROUND: Recently, a dopamine hypothesis of depression was put forward, and several studies have demonstrated that direct and indirect dopamine agonists have antidepressant effects. METHODS: Using Clinical Global Impressions, we evaluated the efficacy of 4-week treatment of pergolide as an antidepressant adjuvant involving 20 unipolar depressed patients who were refractory to standard treatment with antidepressants. RESULTS: One patients (5%) were very much improved, seven (35%) much improved, four (20%) minimally improved, six (30%) no change or worse, and two (10%) not assessed. There was no significant difference in any clinical factors between the pergolide responder and non-responder group. LIMITATIONS: This study was a non-blind open trial, and pergolide was added to tricyclic and heterocyclic antidepressants. CONCLUSION: Pergolide may be useful as an antidepressant adjuvant, suggesting a potential role for dopamine-2 stimulation in the antidepressant response.

Pharmacological characterization of metabotropic glutamate receptor-mediated high-affinity GTPase activity in rat cerebral cortical membranes.

Activation of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) functionally coupled to metabotropic glutamate receptors (mGluRs) was assessed by agonist-induced high-affinity GTPase (EC3.6.1.-) activity in rat cerebral cortical membranes. L-Glutamate (1 mM) stimulated high-affinity GTPase activity to the same extent throughout the incubation period up to 20 min, in a Mg(2+)-dependent manner. The addition of 1 mM L-glutamate augmented V(max) of the enzyme activity (1670 to 3850 pmol mg(-1) protein 15 min(-1)) with slight increase in K(M) value (0.26 to 0.63 microM). The high-affinity GTPase activity was stimulated by the following compounds with a rank order of potency of (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl) glycine (DCG-IV) > (2S,1'S, 2'S)-2-(carboxycyclopyropyl)glycine (L-CCG-I) > L-glutamate > or = 2R, 4R-4-aminopyrrolidine-2,4-dicarboxylate [(2R,4R)-APDC] > 1S, 3R-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] > (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] > (S)-3-carboxy-4-hydroxyphenylglycine [(S)-3C4HPG] > ibotenate, but not by L-(+)-2-amino-4-phosphonobutyrate (L-AP4), (RS)-3, 5-dihydroxyphenylglycine [(RS)-3,5-DHPG], quisqualate, or L-serine-O-phosphate (L-SOP), indicative of involvement of group II mGluRs, in particular mGluR2. (2S)-alpha-Ethylglutamate (EGLU), a presumably selective antagonist against group II mGluRs, inhibited DCG-IV-stimulated high-affinity GTPase activity in a competitive manner with an apparent K(B) of 220 microM. L-Glutamate-stimulated activity was eliminated by pretreatment of the membranes with sulfhydryl alkylating agent N-ethylmaleimide (NEM) at 30-50 microM, indicating that G-proteins of the G(i) family are involved. These results indicate that mGluR agonist-induced high-affinity GTPase activity in rat cerebral cortical membranes may be used to detect the functional interaction between group II mGluRs, in particular mGluR2, and NEM-sensitive G(i) proteins.

Stimulation of high-affinity GTPase activity through group II metabotropic glutamate receptors in rat hippocampal and striatal membranes.

The stimulation of high-affinity GTPase activity through metabotropic glutamate receptors (mGluRs) was pharmacologically characterized with the use of a series of agonists for mGluRs in rat hippocampal and striatal membranes. The pharmacological profile of the response was almost identical to each other between both brain regions. Thus, the high-affinity GTPase activities were stimulated by several mGluR-related compounds with the following rank order of potency: (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) = (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I) > L-glutamate = 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate [(2R,4R)-APDC] > (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] = 1S,3R-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] > (S)-3-carboxy-4-hydroxyphenylglycine [(S)-3C4HPG] = ibotenate. The negative logarithmically transformed EC50 (pEC50) values of these compounds in both brain regions were significantly correlated with those reported previously in the cerebral cortical membranes (N. Nishi et al., Br. J. Pharmacol., 130, 1664-1670, 2000). On the contrary, other reagents including a selective group I mGluRs agonist, (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG], and selective group III mGluRs agonists such as L(+)-2-amino-4-phosphonobutylate (L-AP4) and L-serine-O-phosphate (L-SOP) had little or no effects even at the highest concentration examined. Quisqualate was also a very weak agonist in both regions. These results indicate that mGluR-mediated high-affinity GTPase activity derives from the Gi proteins associated with adenylyl cyclase inhibition through group II mGluRs, in particular the mGluR2 subtype, in rat hippocampal and striatal membranes.

Activation of G proteins by neuropeptide Y and gamma-aminobutyric acid(B) receptor agonists in rat cerebral cortical membranes through distinct modes of action.

The neuropeptide Y (NPY)-elicited increase in high-affinity GTPase activity in the rat cerebral cortical membranes was assayed and compared with the gamma-aminobutyric acid (GABA)(B) receptor-mediated response, representative of the conventional receptor-dependent mode of G protein activation. GABA and a selective GABA(B) receptor agonist, (+/-)-baclofen, stimulated the high-affinity GTPase activity in a concentration-dependent and saturable manner, with a strict Mg(2+) dependence. On the other hand, NPY (10 microM)-stimulated high-affinity GTPase activity was detectable even in the absence of Mg(2+). The concentration-response curve for NPY-induced increase in high-affinity GTPase activity in the presence of 2 mM MgCl(2) revealed a biphasic pattern, and NPY (100 nM)-stimulated activity was dependent on MgCl(2). In the presence of 2 mM MgCl(2), the increase in high-affinity GTPase activity by 100 nM NPY was almost fully inhibited by a selective NPY Y-1 receptor antagonist, (R)-N(2)-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]argininami de (BIBP3226), whereas the effect of 10 microM NPY was only partially antagonized by this compound. The increase in the activity by 10 microM NPY in the absence of MgCl(2) was not at all inhibited by BIBP3226. The high-affinity GTPase activity was augmented by [Leu(31),Pro(34)]NPY (porcine) but not by desamido-NPY, NPY(13-36) (porcine), or rat pancreatic polypeptide at submicromolar concentrations. These results indicate that NPY activates G proteins through two distinct modes of action: the conventional receptor-mediated pathway through NPY Y-1 receptor subtype dominant in the presence of the lower concentrations of NPY and receptor-independent, direct G protein activation driven by the higher concentrations of NPY.

The crystal structure of pyroglutamyl peptidase I from Bacillus amyloliquefaciens reveals a new structure for a cysteine protease.

BACKGROUND: The N-terminal pyroglutamyl (pGlu) residue of peptide hormones, such as thyrotropin-releasing hormone (TRH) and luteinizing hormone releasing hormone (LH-RH), confers resistance to proteolysis by conventional aminopeptidases. Specialized pyroglutamyl peptidases (PGPs) are able to cleave an N-terminal pyroglutamyl residue and thus control hormonal signals. Until now, no direct or homology-based three-dimensional structure was available for any PGP. RESULTS: The crystal structure of pyroglutamyl peptidase I (PGP-I) from Bacillus amyloliquefaciens has been determined to 1.6 A resolution. The crystallographic asymmetric unit of PGP-I is a tetramer of four identical monomers related by noncrystallographic 222 symmetry. The protein folds into an alpha/beta globular domain with a hydrophobic core consisting of a twisted beta sheet surrounded by five alpha helices. The structure allows the function of most of the conserved residues in the PGP-I family to be identified. The catalytic triad comprises Cys144, His168 and Glu81. CONCLUSIONS: The catalytic site does not have a conventional oxyanion hole, although Cys144, the sidechain of Arg91 and the dipole of an alpha helix could all stabilize a negative charge. The catalytic site has an S1 pocket lined with conserved hydrophobic residues to accommodate the pyroglutamyl residue. Aside from the S1 pocket, there is no clearly defined mainchain substrate-binding region, consistent with the lack of substrate specificity. Although the overall structure of PGP-I resembles some other alpha/beta twisted open-sheet structures, such as purine nucleoside phosphorylase and cutinase, there are important differences in the location and organization of the active-site residues. Thus, PGP-I belongs to a new family of cysteine proteases.

Three dimensional electron spin resonance (ESR) imaging of internal organs in living mice.

In vivo electron spin resonance imaging (ESR imaging) was applied to living mice after peroral administration of a nitroxide radical spin probe. A 3D ESR imaging procedure was applied in vivo in order to obtain the exact distribution of the spin probe in a living animal. The imaging pictures demonstrated that the administered spin probe was firstly located in the stomach, then delivered to the liver, kidney and heart of the animal.

Receptor-mediated and receptor-independent activation of G-proteins in rat brain membranes.

High-affinity GTPase activity intrinsic to G-proteins, which serves as an index of G-protein activation elicited through agonist-stimulated receptors as well as by receptor-independent direct G-protein activators like mastoparan, was measured in rat brain membranes. Receptor-mediated high-affinity GTPase activity was detectable preferentially for the Gi subfamily associated with adenylyl cyclase inhibition mediated by group II metabotropic glutamate, pirenzepine-insensitive muscarinic acetylcholine, GABA(B), adenosine A1, dopamine D2-like (striatum), and serotonin 5-HT1A (hippocampus) receptors. The pharmacological characteristics of such receptor-mediated high-affinity GTPase activities were presented. Mastoparan, a tetradecapeptide from wasp venom which has been shown to directly activate Gi and Go, inhibited low-affinity GTP hydrolyzing activity, probably due to its activating effect on nucleoside diphosphokinase (NDPK). When NDPK activity was inhibited completely by UDP, mastoparan stimulated high-affinity GTPase activity in a concentration-dependent manner. There are many compounds other than mastoparan with apparently diverse structural properties which have been shown to directly activate G-proteins. The relevance and possible participation of receptor-independent mode of G-protein activation for some neuropeptides were discussed.

Measurement of receptor-mediated functional activation of G proteins in postmortem human brain membranes.

Guanine nucleotide-binding regulatory proteins (G proteins) play a pivotal role in receptor-mediated transmembrane signal transduction, and have been implicated in modes of action of psychotropic drugs as well as in pathogenesis of psychiatric disorders. In the present investigation, functional activation of G proteins coupled with several receptors, in particular with GABAB receptors, was assessed by agonist-induced stimulation of high-affinity GTPase, an enzyme that is intrinsic to alpha subunit of G protein, in postmortem human frontal cortical membranes. High-affinity GTPase activity was stimulated by GABA as well as (+/-)-baclofen, a selective GABAB receptor agonist, with EC50 values of 60-150 and 10-40 microM, respectively, in a Mg(2+)-dependent manner. The (+/-)-baclofen-stimulated response was antagonized by the selective GABAB receptor antagonist, 2-hydroxy-saclofen, in a competitive manner with a KB value of 59 microM. Although the maximal percent increase above basal value (% Emax) for GABAB receptor-mediated high-affinity GTPase activity was varied from subject to subject, % Emax values for both agonists were highly correlated with each other, and replicable and stable in a given subject, indicating that this measure is trustworthy as an index of functional coupling between receptors and G proteins in future studies at the aim of elucidating possible alteration of receptor/G protein interaction in psychiatric disorders. The % Emax values for GABAB receptor-mediated responses were correlated inversely with brain storage duration, which should be critically considered in postmortem studies. The increases in high-affinity GTPase activity stimulated by several agonists other than GABAB receptor agonists seemed too low to quantify for making a comparison in future studies.

Lack of interfering effects of lithium on receptor/G protein coupling in human platelet and rat brain membranes.

To verify the theory that lithium exerts its multiple effects by altering the receptor-mediated G protein's activation, in vitro effects of lithium on agonist-induced guanosine triphosphate (GTP) hydrolysis were examined. The basal GTP hydrolyzing activity in human platelet membranes was decreased nonselectively by either LiCl or NaCl at millimolar concentrations, whereas (-)-epinephrine-stimulated increase in the activity (an index of alpha (2A)-adrenoceptor coupled Gi2 function) was unaltered. Furthermore, the stimulation of high-affinity GTPase activity induced by dopamine, carbachol, and R-N(6)-phenylisopropyladenosine in rat brain membranes (indices of the functional coupling between dopamine D2-like, pirenzepine-insensitive muscarinic, and adenosine A1 receptors and their respective Gi proteins) was substantially unaltered regardless of whether 0.5 mmol/L adenosine 5'-(beta, gamma-imido)triphosphate (i.e., 1.75 mmol/L lithium) was included in the assay mixture or not. These results indicate that lithium does not affect in vitro the receptor-mediated activational process of G proteins, at least not of Gi associated with adenylate cyclase inhibition.

Effects of the wasp venom peptide, mastoparan, on GTP hydrolysis in rat brain membranes.

1. The effects of mastoparan, a wasp venom toxin, on GTP hydrolyzing activity were examined in rat brain membranes. 2. Mastoparan inhibited the low-affinity GTPase activity, defined as the amount of 32Pi released from 0.3 microm [gamma-32P]-GTP in the presence of 100 microM unlabelled GTP, in a concentration-dependent manner. This inhibitory effect of mastoparan on low-affinity GTPase activity was diminished by increasing concentrations of UDP and was completely attenuated at 20 mM, indicating that activation of nucleoside diphosphokinase (NDPK) is inolved in the phenomenon. 3. In the presence of 20 mM UDP, mastoparan stimulated the high-affinity GTPase activity by increasing the Vmax value without affecting the apparent K(M) for GTP. Mastoparan-stimulated high-affinity GTPase activity was apparent at concentrations higher than 1 microM, in a concentration-dependent manner, but without saturation even at 100 microM. 4. Mastoparan-induced high-affinity GTPase activity showed a characteristic sensitivity to MgCl2, quite different from that seen in L-glutamate-stimulated activity, a representative of receptor-mediated G-protein activation. 5. There appeared to be a simple additive interaction between mastoparan- and L-glutamate-stimulated high-affinity GTPase activities, indicting that distinct pools of G-proteins are involved in receptor-independent and receptor-mediated G-protein activation. 6. These results suggest that G-proteins in brain membranes are functionally altered by mastoparan through multiple mechanisms of action and that the mastoparan-induced, direct G-protein activating process lacks a synergistic or antagonistic interaction with an agonist-induced, receptor-mediated activation of G-proteins.

Synthesis of structural analogues of leukotriene B4 and their receptor binding activity.

Structural analogues of leukotriene B4 (LTB4) were designed based on the plausible conformation of LTB4 (1). Joining C-7-C-9 of the conformer A or B into an aromatic ring system led to the discovery of benzene analogues 2, 4 and 6a. Joining C-4-C-9 of the conformer C or D into an aromatic ring system led to the discovery of analogues 3, 5 and 7. The compounds examined in this study were evaluated as to their inhibition of [3H] LTB4 binding to human neutrophils, and by a secondary intact human neutrophil functional assay for agonist/antagonist activity. The first analogues prepared, compounds 2-7, demonstrated moderate potency in the LTB4 receptor binding assay. The modification of these compounds by the introduction of another substituent into the aromatic ring produced a marked increase in receptor binding (28c, IC50 = 0.020 microM; 38c, IC50 = 0.020 microM; 52a, IC50 = 0.020 microM; 52b, IC50 = 0.018 microM). Most of these structural analogues of LTB4 demonstrated agonist activity. Of the analogues prepared in this study, only compound 57 demonstrated weak LTB4 receptor antagonist activity, at 10 microM.

Trisubstituted benzene leukotriene B4 receptor antagonists: synthesis and structure-activity relationships.

A series of trisubstituted benzenes which demonstrate leukotriene B4 (LTB4, 1) receptor affinity was prepared. Previous trisubstituted benzenes from our laboratory showed high affinity to the LTB4 receptor but demonstrated agonist activity in functional assays. Compound 3a, the initial lead compound of this new series, showed only modest affinity (IC50 = 0.20 microM). However, 3a was a receptor antagonist with no demonstrable agonist activity up to 30 microM. Further modification of the lipid tail and aryl head groups region led to the discovery of 3b (ONO-4057). This compound, free of agonist activity, possesses high affinity to the LTB4 receptor (Ki = 3.7 +/- 0.9 nM).