Pharmacological profile of histaprodifens at four recombinant histamine H1 receptor species isoforms

There are differences in the pharmacological properties of phenylhistamines and histaprodifens between guinea pig histamine H(1) receptor (gpH(1)R) and human histamine H(1) receptor (hH(1)R). The aim of this study was to analyze species differences in more detail, focusing on histaprodifen derivatives and including the bovine histamine H(1) receptor (bH(1)R) and rat histamine H(1) receptor (rH(1)R). H(1)R species isoforms were coexpressed with the regulator of G protein signaling RGS4 in Sf9 insect cells. We performed [(3)H]mepyramine binding assays and steady-state GTPase assays. For a novel class of histaprodifens, the chiral histaprodifens, unique species differences between hH(1)R, bH(1)R, rH(1)R, and gpH(1)R were observed. The chiral histaprodifens 8R and 8S were both partial agonists at gpH(1)R, but only 8R was a partial agonist at the other H(1)R species isoforms. An additional phenyl group in chiral histaprodifens 10R and 10S, respectively, resulted in a switch from agonism at gpH(1)Rto antagonism at hH(1)R, bH(1)R, and rH(1)R. In general, histaprodifens showed the order of potency hH(1)R < bH(1)R < rH(1)R < gpH(1)R. An active-state model of gpH(1)R was generated with molecular dynamics simulations. Dimeric histaprodifen was docked into the binding pocket of gpH(1)R. Hydrogen bonds and electrostatic interactions were detected between dimeric histaprodifen and Asp-116, Ser-120, Lys-187, Glu-190, and Tyr-432. We conclude the following: 1) chiral histaprodifens interact differentially with H(1)R species isoforms; 2) gpH(1)R and rH(1)R, on one hand, and hH(1)R and bH(1)R, on the other hand, resemble each other structurally and pharmacologically; and 3) histaprodifens interact with H(1)R at multiple sites.     

Ligand-specific contribution of the N terminus and E2-loop to pharmacological properties of the histamine H1-receptor

There are species differences between human histamine H(1) receptor (hH(1)R) and guinea pig (gp) histamine H(1) receptor (gpH(1)R) for phenylhistamines and histaprodifens. Several studies showed participation of the second extracellular loop (E2-loop) in ligand binding for some G protein-coupled receptors (GPCRs). Because there are large species differences in the amino acid sequence between hH(1)R and gpH(1)R for the N terminus and E2-loop, we generated chimeric hH(1)Rs with gp E2-loop (h(gpE2)H(1)R) and gp N terminus and gp E2-loop (h(gpNgpE2)H(1)R). hH(1)R, gpH(1)R, and chimeras were expressed in Sf9 insect cells. [(3)H]Mepyramine binding assays and steady-state GTPase assays were performed. In the series hH(1)R > h(gpE2)H(1)R > h(gpNgpE2)H(1)R, we observed a significant decrease in potency of histamine 1 in the GTPase assay. For phenoprodifen 5 and the chiral phenoprodifens 6R and 6S, a significant decrease in affinity and potency was found in the series hH(1)R > h(gpE2)H(1)R > h(gpNgpE2)H(1)R. In addition, we constructed new active-state H(1)R models based on the crystal structure of the human beta(2)-adrenergic receptor (hbeta(2)AR). Compared with the H(1)R active-state models based on the crystal structure of bovine rhodopsin, the E2-loop differs in its contact to the ligand bound in the binding pocket. In the bovine rhodopsin-based model, the backbone carbonyl of Lys187 (gpH(1)R) interacts with large histaprodifens in the binding pocket, but in the hbeta(2)AR-based model, Lys187 (gpH(1)R) is located distantly from the binding pocket. In conclusion, the differences in N terminus and E2-loop between hH(1)R and gpH(1)R exert an influence on affinity and/or potency for histamine and phenoprodifens 5, 6R, and 6S.     

Mutations of Cys-17 and Ala-271 in the human histamine H2 receptor determine the species selectivity of guanidine-type agonists and increase constitutive activity

In a steady-state GTPase activity assay, N-[3-(1H-imidazol-4-yl)propyl)]guanidines and N(G)-acylated derivatives are more potent and efficacious at fusion proteins of guinea pig (gpH(2)R-G(salphaS)) than human (hH(2)R-G(salphaS)) histamine H(2) receptor, coupled to the short splice variant of G(salpha), G(salphaS). Whereas Ala-271 (hH(2)R) and Asp-271 (gpH(2)R) in transmembrane domain 7 were identified to determine the potency differences of guanidine-type agonists, the molecular basis for the efficacy differences remains to be elucidated. A homology model of the gpH(2)R suggested that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation of the gpH(2)R. In the present study, we generated a mutant hH(2)R-G(salphaS) with Cys-17--> Tyr-17/Ala-271--> Asp-271 exchanges (hH(2)R-->gpH(2)R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH(2)R-G(salphaS) and gpH(2)R-G(salphaS). Potencies and efficacies of guanidines and N(G)-acylguanidines were increased at this mutant receptor compared with hH(2)R-G(salphaS), but they were still lower than at gpH(2)R-G(salphaS), suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH(2)R-G(salphaS) mutant with a Cys-17--> Tyr-17 exchange showed inefficient coupling to G(salphaS) as revealed by reduced agonist-stimulated GTPase and basal adenylyl cyclase activities. Collectively, our present pharmacological study confirms the existence of an H-bond between Tyr-17 and Asp-271 favoring the stabilization of an active receptor conformation. Distinct potencies and efficacies of agonists and inverse agonists further support the concept of ligand-specific conformations in wild-type and mutant H(2)R-G(salphaS) fusion proteins.     

Probing ligand-specific histamine H1- and H2-receptor conformations with NG-acylated Imidazolylpropylguanidines

Impromidine (IMP) and arpromidine (ARP)-derived guanidines are more potent and efficacious guinea pig (gp) histamine H(2)-receptor (gpH(2)R) than human (h) H(2)R agonists and histamine H(1)-receptor (H(1)R) antagonists with preference for hH(1)R relative to gpH(1)R. We examined N(G)-acylated imidazolylpropylguanidines (AIPGs), which are less basic than guanidines, at hH(2)R, gpH(2)R, rat H(2)R (rH(2)R), hH(1)R, and gpH(1)R expressed in Sf9 cells as probes for ligand-specific receptor conformations. AIPGs were similarly potent H(2)R agonists as the corresponding guanidines IMP and ARP, respectively. Exchange of pyridyl in ARP against phenyl increased AIPG potency 10-fold, yielding the most potent agonists at the hH(2)R-G(salpha) fusion protein and gpH(2)R-G(salpha) identified so far. Some AIPGs were similarly potent and efficacious at hH(2)R-G(salpha) and gpH(2)R-G(salpha). AIPGs stabilized the ternary complex in hH(2)R-G(salpha) and gpH(2)R-G(salpha) differently than the corresponding guanidines. Guanidines, AIPGs, and small H(2)R agonists exhibited distinct agonist properties at hH(2)R, gpH(2)R, and rH(2)R measuring adenylyl cyclase activity. In contrast to ARP and IMP, AIPGs were partial H(1)R agonists exhibiting higher efficacies at hH(1)R than at gpH(1)R. This is remarkable because, so far, all bulky H(1)R agonists exhibited higher efficacies at gpH(1)R than at hH(1)R. Collectively, our data suggest that AIPGs stabilize different active conformations in hH(2)R, gpH(2)R, and rH(2)R than guanidines and that, in contrast to guanidines, AIPGs are capable of stabilizing a partially active state of hH(1)R.     

Differential efficacies of somatostatin receptor agonists for G-protein activation and desensitization of somatostatin receptor subtype 4-mediated responses

Both the histamine H1-receptor (H1R) and H2-receptor (H2R) exhibit pronounced species selectivity in their pharmacological properties; i.e., bulky agonists possess higher potencies and efficacies at guinea pig (gp) than at the corresponding human (h) receptor isoforms. In this study, we examined the effects of NG-acylated imidazolylpropylguanidines substituted with a single phenyl or cyclohexyl substituent on H1R and H2R species isoforms expressed in Sf9 insect cells. N1-(3-Cyclohexylbutanoyl)-N2-[3-(1H-imidazol-4-yl)propyl]guanidine (UR-AK57) turned out to be the most potent hH2R agonist identified so far (EC50 of 23 nM in the GTPase assay at the hH2R-Gsalpha fusion protein expressed in Sf9 insect cells). UR-AK57 was almost a full-hH2R agonist and only slightly less potent and efficacious than at gpH2R-Gsalpha. Several NG-acylated imidazolylpropylguanidines showed similar potency at hH2R and gpH2R. Most unexpectedly, UR-AK57 exhibited moderately strong partial hH1R agonism with a potency similar to that of histamine, whereas at gpH1R, UR-AK57 was only a very weak partial agonist. Structure/activity relationship studies revealed that both the alkanoyl chain connecting the aromatic or alicyclic substituent with the guanidine moiety and the nature of the carbocycle (cyclohexyl versus phenyl ring) critically determine the pharmacological properties of this class of compounds. Collectively, our data show that gpH1R and gpH R do not necessarily exhibit preference for bulky agonists (2) compared with hH1R and hH2R, respectively, and that UR-AK57 is a promising starting point for the development of both potent and efficacious hH1R and hH2R agonists.     

Constitutive activity and ligand selectivity of human, guinea pig, rat, and canine histamine H2 receptors

Previous studies revealed pharmacological differences between human and guinea pig histamine H(2) receptors (H(2)Rs) with respect to the interaction with guanidine-type agonists. Because H(2)R species variants are structurally very similar, comparative studies are suited to relate different properties of H(2)R species isoforms to few molecular determinants. Therefore, we systematically compared H(2)Rs of human (h), guinea pig (gp), rat (r), and canine (c). Fusion proteins of hH(2)R, gpH(2)R, rH(2)R, and cH(2)R, respectively, and the short splice variant of G(salpha), G(salphaS), were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH(2)R-G(salphaS) but neither gpH(2)R-G(salphaS) nor rH(2)R-G(salphaS) showed the hallmarks of increased constitutive activity compared with hH(2)R-G(salphaS), i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH(2)R-G(salphaS), increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H(2)Rs without or together with mammalian G(salphaS) or H(2)R-G(salpha) fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH(2)R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH(2)R-G(salphaS), gpH(2)R-G(salphaS), and rH(2)R-G(salphaS) in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH(2)R-G(salphaS). In conclusion, the cH(2)R exhibits increased constitutive activity compared with hH(2)R, gpH(2)R, and rH(2)R, and there is evidence for ligand-specific conformations in H(2)R species isoforms.     

Evaluation of histamine H1-, H2-, and H3-receptor ligands at the human histamine H4 receptor: identification of 4-methylhistamine as the first potent and selective H4 receptor agonist

The histamine H(4) receptor (H(4)R) is involved in the chemotaxis of leukocytes and mast cells to sites of inflammation and is suggested to be a potential drug target for asthma and allergy. So far, selective H(4)R agonists have not been identified. In the present study, we therefore evaluated the human H(4)R (hH(4)R) for its interaction with various known histaminergic ligands. Almost all of the tested H(1)R and H(2)R antagonists, including several important therapeutics, displaced less than 30% of specific [(3)H]histamine binding to the hH(4)R at concentrations up to 10 microM. Most of the tested H(2)R agonists and imidazole-based H(3)R ligands show micromolar-to-nanomolar range hH(4)R affinity, and these ligands exert different intrinsic hH(4)R activities, ranging from full agonists to inverse agonists. Interestingly, we identified 4-methylhistamine as a high-affinity H(4)R ligand (K(i) = 50 nM) that has a >100-fold selectivity for the hH(4)R over the other histamine receptor subtypes. Moreover, 4-methylhistamine potently activated the hH(4)R (pEC(50) = 7.4 +/- 0.1; alpha = 1), and this response was competitively antagonized by the selective H(4)R antagonist JNJ 7777120 [1-[(5-chloro-1H-indol-2-yl)-carbonyl]-4-methylpiperazine] (pA(2) = 7.8). The identification of 4-methylhistamine as a potent H(4)R agonist is of major importance for future studies to unravel the physiological roles of the H(4)R.     

An 80-amino acid deletion in the third intracellular loop of a naturally occurring human histamine H3 isoform confers pharmacological differences and constitutive activity

In this article, we pharmacologically characterized two naturally occurring human histamine H3 receptor (hH3R) isoforms, hH3R(445) and hH3R(365). These abundantly expressed splice variants differ by a deletion of 80 amino acids in the intracellular loop 3. In this report, we show that the hH3R(365) is differentially expressed compared with the hH3R(445) and has a higher affinity and potency for H3R agonists and conversely a lower potency and affinity for H3R inverse agonists. Furthermore, we show a higher constitutive signaling of the hH3R(365) compared with the hH3R(445) in both guanosine-5'-O-(3-[35S]thio) triphosphate binding and cAMP assays, likely explaining the observed differences in hH3R pharmacology of the two isoforms. Because H3R ligands are beneficial in animal models of obesity, epilepsy, and cognitive diseases such as Alzheimer's disease and attention deficit hyperactivity disorder and currently entered clinical trails, these differences in H3R pharmacology of these two isoforms are of great importance for a detailed understanding of the action of H3R ligands.     

Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host

OBJECTIVES: Influenza A H3N2 viruses isolated recently have characteristic receptor binding properties that may decrease susceptibility to neuraminidase inhibitor drugs. A panel of clinical isolates and recombinant viruses generated by reverse genetics were characterized and tested for susceptibility to zanamivir. METHODS: Plaque reduction assays and neuraminidase enzyme inhibition assays were used to assess susceptibility to zanamivir. Receptor binding properties of the viruses were characterized by differential agglutination of red blood cells (RBCs) from different species. Sequence analysis of the haemagglutinin (HA) and neuraminidase (NA) genes was carried out. RESULTS: Characterization of a panel of H3N2 clinical isolates from 1968 to 2000 showed a gradual decrease in agglutination of chicken and guinea pig RBCs over time, although all isolates could agglutinate turkey RBCs equally. Sequence analysis of the HA and NA genes identified mutations in conserved residues of the HA1 receptor binding site, in particular Leu-226 --> Ile-226/Val-226, and modification of potential glycosylation site motifs. This may be indicative of changes in virus binding to sialic acid (SA) receptors in recent years. Although recent isolates had reduced susceptibility to zanamivir in MDCK cell based plaque reduction assays, no difference was found in an NA enzyme-inhibition assay. Assays with recombinant isogenic viruses showed that the recent HA, but not the NA, conferred reduced susceptibility to zanamivir. CONCLUSION: This study demonstrates that recent clinical isolates of influenza A H3N2 virus no longer agglutinate chicken RBCs, but despite significant receptor binding changes as a result of changes in HA, there was little variation in sensitivity of the NA to zanamivir.     

Reconstruction of the immunogenic peptide RNase(43-56) by identification and transfer of the critical residues into an unrelated peptide backbone

The involvement of each of the amino acid residues of the I-Ak-restricted T cell determinant RNase(43-56) was examined in detail using a series of peptides containing single amino acid substitutions. Four positions were identified as being essential for the formation of the determinant, Phe-46, Val-47, His-48, and Leu-51. When these four residues were substituted into the backbone of the unrelated peptide HA(130-144), a nonstimulatory peptide was obtained. The inclusion of an additional residue, Val-54, resulted in a chimeric peptide, RN/HA2, which was nearly as active as the native molecule. The peptide RN/HA2 was able to prime in vivo for RNase reactivity, confirming that these five residues contained all of the specificity of the RNase(43-56) determinant. The role of three of these critical residues was examined using both a functional competition assay and an in vivo priming assay. It was ascertained that the Phe-46 was directly involved in contacting the TCR, while the His-48 and Leu-51 were either involved in binding to the I-Ak molecule or in determining the conformation of the peptide. Thus, by critically evaluating the contribution of each of the amino acid residues in a T cell determinant, we were able to generate a chimeric peptide only containing 5 of 15 residues from the RNase(43-56) sequence that was functionally identical to the native RNase(43-56) molecule both in vitro and in vivo.     

Topoisomerase II and IV quinolone resistance-determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility

The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 microg/ml. GyrA (Leu-55 to Gln-155, Escherichia coli numbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa, with the degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa (70.9 to 88.6%) and E. coli (73.0 to 88.6%) were observed. Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs. The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected. The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 microg/ml) showed a ParC amino acid change (Ser-80-->Arg). In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE. The frequencies of alterations at positions 437, 465, 477, and 485 were higher (P < 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs. An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (0.5 microg/ml) or reserpine (10 microg/ml), was suspected in seven strains. These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S. maltophilia.     

Similar apparent constitutive activity of human histamine H(2)-receptor fused to long and short splice variants of G(salpha).

Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta(2)-adrenoceptor (beta(2)AR) fused to the long splice variant of G(salpha) (G(salphaL)) exhibits a higher apparent constitutive activity than the beta(2)-adrenoceptor fused to the short splice variant of G(salpha) (G(salphaS)). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the beta(2)AR fused to G(salphaL) relative to the beta(2)AR fused to G(salphaS), indicating that the agonist-free beta(2)AR and the beta(2)AR occupied by partial agonists promote GDP dissociation from G(salphaL) more efficiently than from G(salphaS). In fact, the GDP affinity of G(salphaS) fused to the beta(2)AR is higher than the GDP affinity of G(salphaL) fused to the beta(2)AR. We asked the question whether the histamine H(2)-receptor (H(2)R) exhibits similar coupling to G(salpha) splice variants as the beta(2)AR. To address this question, we studied H(2)R-G(salpha) fusion proteins expressed in Sf9 cells. In contrast to beta(2)AR-G(salpha) fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H(2)R fused to G(salphaL) and G(salphaS) as assessed by guanosine-5'-O-(3-thio)triphosphate binding and/or steady-state GTPase activity. However, the time course analysis of guanosine-5'-O-(3-thio)triphosphate binding indicated that G(salphaS) fused to the H(2)R possesses a higher GDP-affinity than G(salphaL) fused to the H(2)R. Our data show that the H(2)R fused to G(salphaL) and G(salphaS) possesses similar constitutive activity and is insensitive to differences in GDP affinity of G(salpha) splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors.     

Analysis of differential substrate selectivities of CYP2B6 and CYP2E1 by site-directed mutagenesis and molecular modeling

Human CYP2B6 and CYP2E1 were used to investigate the extent to which differential substrate selectivities between cytochrome P450 subfamilies reflect differences in active-site residues as opposed to distinct arrangement of the backbone of the enzymes. Reciprocal CYP2B6 and CYP2E1 mutants at active-site positions 103, 209, 294, 363, 367, and 477 (numbering according to CYP2B6) were characterized using the CYP2B6-selective substrate 7-ethoxy-4-trifluoromethylcoumarin, the CYP2E1-selective substrate p-nitrophenol, and the common substrates 7-ethoxycoumarin, 7-butoxycoumarin, and arachidonic acid. This report is the first to study the active site of CYP2E1 by systematic site-directed mutagenesis. One of the most intriguing findings was that substitution of CYP2E1 Phe-477 with valine from CYP2B6 resulted in significant 7-ethoxy-4-trifluoromethylcoumarin deethylation. Use of three-dimensional models of CYP2B6 and CYP2E1 based on the crystal structure of CYP2C5 suggested that deethylation of 7-ethoxy-4-trifluoromethylcoumarin by CYP2E1 is impeded by van der Waals overlaps with the side chain of Phe-477. Interestingly, none of the CYP2B6 mutants acquired enhanced ability to hydroxylate p-nitrophenol. Substitution of residue 363 in CYP2E1 and CYP2B6 resulted in significant alterations of the metabolite profile for the side chain hydroxylation of 7-butoxycoumarin. Probing of CYP2E1 mutants with arachidonic acid indicated that residues Leu-209 and Phe-477 are critical for substrate orientation in the active site. Overall, the study revealed that differences in the side chains of active-site residues are partially responsible for differential substrate selectivities across cytochrome P450 subfamilies. However, the relative importance of active-site residues appears to be dependent on the structural similarity of the compound to other substrates of the enzyme.     

Inverse agonism at heptahelical receptors: concept, experimental approach and therapeutic potential

Inverse agonists (negative antagonists) are ligands that stabilize the inactive conformation (R) of receptors according to the two-state receptor model. The active conformation (R*) of heptahelical receptors, i.e. G protein-coupled receptors, has high affinity for G proteins. According to ternary complex models of receptor activation, the R*G complex is in equilibrium with R + G, with spontaneous activity in the absence of agonist. Inverse agonists, having a higher affinity for R, shift R*G towards R + G, decreasing the spontaneous activity of receptors. Agonists have the opposite effect, with a higher affinity for R*. Neutral antagonists have the same affinity for R and R* and compete for both agonists and inverse agonists. Inverse agonists have been recently proposed for a variety of heptahelical receptors. Methods to detect inverse agonists among antagonists are based on the determination of ligand affinity at R and R* with binding experiments, and on the modulation of G protein activity (GTP binding and hydrolysis) or of effector activity. Receptor inverse agonists, but also G protein antagonists and GTPase inhibitors, decrease spontaneous G protein activity corresponding to R*G. Receptor agonists, G protein agonists and GTPase inhibitors increase effector basal activity, but receptor inverse agonists decrease it. The therapeutic potential of inverse agonists is proposed in human diseases ascribed to constitutively active mutant receptors and may be extended to diseases related to wild-type receptor over-expression leading to the increase of R*. Some of the therapeutic effects of presently used receptor antagonists may be related to their inverse agonist properties. Inverse agonists lead to receptor upregulation, offering new approaches to tolerance and dependence to drugs.     

Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF)

OBJECTIVES: YhiV (MdtF) is an resistance nodulation division (RND) type efflux pump in Escherichia coli with significant homology to AcrB but usually expressed at a low level in clinical isolates. When overexpressed the pump confers decreased susceptibility to a variety of substances including erythromycin and ethidium bromide (EtBr). We characterized two mutants of E. coli E12 (DeltaacrB DeltaacrF) overexpressing yhiV that showed surprising differences in their spectrum of multidrug resistance (MDR). METHODS: The two mutants obtained after repeated exposure of E. coli E12 to levofloxacin were tested for antimicrobial susceptibility to a variety of agents and for intracellular accumulation of selected pump substrates. Gene expression was studied by quantitative RT-PCR, and yhiV was sequenced. Gene inactivation and replacement were done by phage lambda-based homologous recombination. RESULTS: Mutant DKO20/1 overexpressed yhiV, showed a wild-type yhiV sequence and had >2-fold increased MICs of fluoroquinolones, novobiocin, macrolides/ketolides, EtBr, oxacillin and Phe-Arg-beta-naphthylamide (PAbetaN, a putative efflux pump inhibitor) compared with the E12 parent. A second mutant, strain DKO1/17 that had the Val-610-->Phe point mutation in YhiV differed from DKO20/1 by faster growth, >2-fold increased MICs of linezolid and tetracycline, but >2-fold decreased MICs of PAbetaN, azithromycin and telithromycin. Inactivation of yhiV in DKO1/17 and reintroduction of the wild-type and mutant yhiV sequence confirmed that the differing MICs of most of the drugs were associated with the observed single point mutation. Intracellular drug accumulation studies with linezolid and PAbetaN were consistent with the MIC results. CONCLUSIONS: The region around amino acid Val-610 in YhiV appears to be involved in determining recognition and efficiency of export of a number of MDR efflux pump substrates. This single point mutation in the periplasmic loop of the pump can increase resistance to a given drug such as a fluoroquinolone while decreasing resistance to another one.     

Angiotensin II effects upon the glomerular microcirculation and ultrafiltration coefficient of the rat.

The effects of both synthetic and biologically produced angiotensin II (AII) upon the process of glolerular filtration were examined in the plasma-expanded (2.5% body wt) Munich-Wistar rat, by micropuncture evaluation of pressures, nephron plasma flow (rpf) and filtration rate (sngfr). Plasma expansion was chosen as a control condition because (a) response to AII was uniform and predictable, (b) endogenous generation of AII was presumably suppressed, and (c) the high control values for rpf permitted accurate determination of values for the glomerular permeability coefficient (LpA) before and during AII infusion. With subpressor quantities of synthetic Asn-1, Val-5 AII (less than 5 ng/100 g body wt/min), sngfr fell from 47.7 in the control group to 39.8 nl/min/g kidney (P less than 0.005). The rpf fell to 60% of control values (P less than 0.001). Measurement of glomerular capillary (PG) and Bowman's space (Pt) hydrostatic pressures in surface glomeruli with a servo-nulling device permitted evaluation of the hydrostatic pressure gradient (deltaP = PG - Pi). DeltaP increased from 38.1 +/- 1.2 in control to 45.9 +/- 1.3 mm Hg after Asn-1, Val-5 AII and essentially neutralized the effect of decreased rpf in sngfr. The sngfr then fell as a result of a decreased in LpA from 0.063 +/- 0.008 in control to 0.028 +/- 0.004 nl/s/g kidney/mm Hg after Asn-1, Val-5 AII (P less than 0.02). Lower doses of Asp-1, Ile-5 AII (less than 3 ng/100 g body wt/min) had no effect on sngfr, rpf, deltaP, and afferent and efferent vascular resistance, but significantly elevated systemic blood pressure, suggesting peripheral effects on smooth muscle at this low dose. LpA was 0.044 +/- 0.007 nl/s/g kidney/mm Hg after low-dose Asp-1, Ile-5 AII, and 0.063 +/- 0.008 in the control group (0.02 greater than P greater than 0.1). Higher, equally pressor doses of native AII (5 ng/100 g body wt/min) produced effects almost identical to similar quantites of synthetic Asn-1, Val-5 AII upon rpf, deltaP, sngfr, and renal vascular resistance. LpA again fell to 0.026 +/- 0.004 nl/s/g kidney/mn Hg, a value almost identical to that after the synthetic AII. Paired studies with Asp-1, Ile-5 AII also demonstrated a consistent reduction in LpA.     

Block of human heart hH1 sodium channels by amitriptyline

Amitriptyline is a tricyclic antidepressant used to treat major depression and various neuropathic pain syndromes. This drug also causes cardiac toxicity in patients with overdose. We characterized the tonic and use-dependent amitriptyline block of human cardiac (hH1) Na(+) channels expressed in human embryonic kidney cells under voltage-clamp conditions. Our results show that, near the therapeutic plasma concentration of 1 microM, amitriptyline is an effective use-dependent blocker of hH1 Na(+) channels during repetitive pulses (approximately 55% block at 5 Hz). The tonic block for resting and for inactivated hH1 channels by amitriptyline (0.1-100 microM) yielded IC(50) values (50% inhibitory concentration) of 24.8 +/- 2.0 (n = 9) and 0.58 +/- 0.03 microM (n = 7), respectively. Substitution of phenylalanine with lysine at the hH1-F1760 position, a putative binding site for local anesthetics, eliminates the use-dependent block by amitriptyline at 1 microM. The time constants of recovery from the inactivated-state amitriptyline block in hH1 wild-type and hH1-F1760K mutant channels are 8.0 +/- 0. 5 (n = 6) and 0.45 +/- 0.07 s (n = 6), respectively. A substitution at either hH1-F1760K or hH1-Y1767K significantly increases the IC(50) values for resting and inactivated states of amitriptyline, but the increase is much more pronounced with the hH1-F1760K mutation. Because these two residues were proposed to form a part of the local anesthetic binding site, we conclude that amitriptyline and local anesthetics interact with a common binding site. Furthermore, at therapeutic concentrations, the ability of amitriptyline to act as a potent use-dependent blocker of Na(+) channels may, in part, explain its analgesic actions.     

Ligands regulate cell surface level of the human kappa opioid receptor by activation-induced down-regulation and pharmacological chaperone-mediated enhancement: differential effects of nonpeptide and peptide agonists

Two peptide agonists, eight nonpeptide agonists, and five nonpeptide antagonists were evaluated for their capacity to regulate FLAG (DYKDDDDK)-tagged human kappa opioid receptors (hKORs) stably expressed in Chinese hamster ovary cells after incubation for 4 h with a ligand at a concentration approximately 1000-fold of its EC(50) (agonist) or K(i) (antagonist) value. Dynorphins A and B decreased the fully glycosylated mature form (55-kDa) of FLAG-hKOR by 70%, whereas nonpeptide full agonists [2-(3,4-dichlorophenyl)-N-methyl-N-[(2R)-2-pyrrolidin-1-ylcyclohexyl-]acetamide (U50,488H), 17-cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[N-methyl-trans-3-(3-furyl) acrylamido] morphinan hydrochloride (TRK-820), ethylketocyclazocine, bremazocine, asimadoline, and (RS)-[3-[1-[[(3,4-dichlorophenyl)acetyl]-methylamino]-2-(1-pyrrolidinyl)ethyl]phenoxy] acetic acid hydrochloride (ICI 204,448) caused 10-30% decreases. In contrast, pentazocine (partial agonist) and etorphine (full agonist) up-regulated by approximately 15 and 25%, respectively. The antagonists naloxone and norbinaltorphimine also significantly increased the 55-kDa receptor, whereas selective mu, delta, and D(1) receptor antagonists had no effect. Naloxone up-regulated the receptor concentration- and time-dependently and enhanced the receptor maturation extent, without affecting its turnover. Treatment with brefeldin A (BFA), which disrupts Golgi, resulted in generation of a 51-kDa form that resided intracellularly. Naloxone up-regulated the new species, indicating that its action site is in the endoplasmic reticulum as a pharmacological chaperone. After treatment with BFA, all nonpeptide agonists up-regulated the 51-kDa form, whereas dynorphins A and B did not, indicating that nonpeptide agonists act as pharmacological chaperones, but peptide agonists do not. BFA treatment enhanced down-regulation of the cell surface receptor induced by nonpeptide agonists, but not that by peptide agonists, and unmasked etorphine- and pentazocine-mediated receptor down-regulation. These results demonstrate that ligands have dual effects on receptor levels: enhancement by chaperone-like effects and agonist-promoted down-regulation, and the net effect reflects the algebraic sum of the two.     

Regional characterization of agonist and depolarization-induced phosphoinositide hydrolysis in rat brain

The ability of various receptor agonists and elevated extracellular potassium to initiate inositol phospholipid hydrolysis in various regions of rat brain was examined by using a direct assay which involves prelabeling slices with [3H]inositol and assaying [3H]inositol phosphates ([3H]IPs) in the presence of lithium. Both carbachol and noradrenaline evoked an increase in [3H]IP accumulation in all cerebral regions, although there were marked topographical differences in maximal responsiveness. These marked differences do not seem to be due to regional differences in coupling as similar apparent affinities of full agonists and the relative intrinsic activities of partial agonists were obtained. Both carbachol and noradrenaline responses were antagonized equipotently in all the brain regions tested by the muscarinic and alpha-1 antagonists atropine and prazosin, respectively. However, the putatively selective muscarinic antagonist pirenzepine did show an (approximately 10-fold) apparent selectivity against the carbachol responses elicited in the forebrain regions from those in the pons-medulla. Evaluation of extracellular potassium to 18 mM resulted in an increased production of [3H]IPs in all brain regions except the cerebellum. Incubation of slices with the cholinesterase inhibitor physostigmine (50 microM) and the dihydropyridine Ca++ channel activator BAY-K-8644 (1 microM) greatly enhanced the responses produced by elevated K+ in the forebrain regions but had markedly weaker effects in the hindbrain regions. The elevated K+ response alone and the enhanced response in the presence of BAY-K-8644 were both antagonized significantly by the dihydropyridine antagonist (+)-PN-205-033 in all brain regions, by 70 to 80 and 70 to 95%, respectively.     

Susceptibility of coxsackievirus B3 laboratory strains and clinical isolates to the capsid function inhibitor pleconaril: antiviral studies with virus chimeras demonstrate the crucial role of amino acid 1092 in treatment

OBJECTIVES: At present, most promising compounds to treat enterovirus-induced diseases are broad-spectrum capsid function inhibitors which bind into a hydrophobic pocket in viral capsid protein 1 (VP1). Coxsackievirus B3 (CVB3) Nancy was the only prototypic enterovirus strain shown to be pleconaril-resistant. This study was designed to better understand the polymorphism of the hydrophobic pocket in CVB3 laboratory strains and clinical isolates and its implications for treatment with the capsid function inhibitor pleconaril. METHODS: Pleconaril susceptibility was determined in cytopathic effect-inhibitory, plaque reduction or virus yield assays. Sequence analysis of the genome region coding for VP1 and/or subsequent alignment of amino acids lining the hydrophobic pocket of five CVB3 laboratory strains and 20 clinical isolates were carried out. Virus chimeras and computational analysis were used to prove the role of amino acid 1092. RESULTS AND CONCLUSIONS: Despite high conservation of pocket amino acids, polymorphism was detected at positions 1092, 1094 and 1180. Neither Pro-1094-->Thr nor Val-1180-->Ile altered efficacy of pleconaril treatment. But the amino acid at position 1092 was strongly associated with susceptibility of CVB3 to the capsid inhibitor. Whereas leucine was involved in resistance, isoleucine and valine were detected in pleconaril-susceptible CVB3. Results from antiviral assays with hybrid viruses demonstrate the crucial role of amino acid 1092 in pleconaril susceptibility. A resistant cDNA-generated CVB3 became pleconaril-susceptible after accepting parts from the genome region encoding Ile-1092 into its capsid. Computational analysis suggests that conformational changes in the hydrophobic pocket occur when leucine is substituted for isoleucine or valine and that this change leads to susceptibility to pleconaril.