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Biochemical Pharmacology (v.71, #12)
Some implications of receptor theory for in vivo assessment of agonists, antagonists and inverse agonists by S. Stevens Negus (pp. 1663-1670).
Drug effects can be classified into three major phenotypes: agonist, antagonist and inverse agonist. Agonist and inverse agonist effects are associated with receptor activation and inactivation, respectively, whereas antagonism implies that a drug produces no effect when administered alone but blocks the effects of agonists and inverse agonists. Attention has only recently begun to focus on the theoretical and clinical implications of inverse agonists, and studies of inverse agonism have also stimulated revisions in receptor theory. This commentary addresses two specific issues related to the application of receptor theory to studies of inverse agonists in vivo. First, principles of receptor theory suggest that increasing drug doses produce a graded pharmacological stimulus that is transduced by receptor-containing tissue into a biological response. However, assays vary in their ability to detect those responses, and any given assay provides only a narrow window on the full range of underlying drug effects. Consequently, in vivo assessment of inverse agonists will benefit from development of assays sensitive to graded inverse agonist effects. Second, detection of inverse agonist effects requires some preexisting level of receptor activity (or tone). This tone can result from at least two sources: (a) endogenous ligands for the receptor, or (b) constitutive receptor activity. Strategies for discriminating these two sources of tone will also contribute to the in vivo assessment of inverse agonist effects. Studies with intermediate efficacy ligands may be especially helpful in this regard, because their effects are differentially influenced by endogenous agonist tone versus constitutive receptor tone.
Keywords: Inverse agonist; Agonist; Antagonist; Receptor theory; In vivo; Constitutive activity
Structure–activity relationship for nucleoside analogs as inhibitors or substrates of adenosine kinase from Mycobacterium tuberculosis by Mary C. Long; William B. Parker (pp. 1671-1682).
Adenosine kinase (Ado kinase, EC 2.7.1.20) is a purine salvage enzyme that phosphorylates adenosine (Ado) to AMP. Ado kinase from Mycobacterium tuberculosis also catalyzes an essential step in the conversion of 2-methyl-Ado to a compound with selective antimycobacterial activity. In order to aid in the design of more potent and selective Ado analogs, eighty nucleoside analogs with modifications to the adenine (Ade) moiety of Ado were evaluated as both substrates and inhibitors of Ado kinase from M. tuberculosis, and a subset was further tested with human Ado kinase for the sake of comparison. The best substrates were 2-aza-Ado, 8-aza-9-deaza-Ado, and 2-fluoro-Ado and the most potent inhibitors were N1-benzyl-Ado ( Ki=0.19μM), 2-fluoro-Ado ( Ki=0.5μM), 6-cyclopentyloxy-purine riboside ( Ki=0.15μM), and 7-iodo-7-deaza-Ado ( Ki=0.21μM). These studies revealed the presence of a hydrophobic pocket near the N6- and N1-positions that can accommodate substitutions at least as large as a benzyl group. The ability to fit into this pocket increased the likelihood that a compound would be an inhibitor and not a substrate. The 2-position was able to accommodate exocyclic substitutions as large as a methoxy group, although substrate activity was low. Similarly, the 7-position could bind an exocyclic group as large as a carboxamido moiety. However, all of the compounds tested with modifications at the 7-position were much better inhibitors than substrates. MIC studies performed with selected compounds have yielded several Ado analogs with promising antitubercular activity. Future studies will utilize this information for the design of new analogs that may be selective antitubercular agents.
Keywords: Abbreviations; Ade; adenine; Ado; adenosine; AD; Ado deaminase; Ado kinase; adenosine kinase; BSA; bovine serum albumin; DTT; dithiothreitol; formycin A; 8-aza-9-deaza-Ado; methyl-Ado; 2-methyl-adenosine; MIC; minimum inhibitory concentration; SAR; structure–activity relationshipAdenosine kinase; Mycobacterium tuberculosis; nucleoside analogs; Structure–activity relationship; Purine salvage; Methyl-adenosine
Radiolytic and cellular reduction of a novel hypoxia-activated cobalt(III) prodrug of a chloromethylbenzindoline DNA minor groove alkylator by G-One Ahn; K. Jane Botting; Adam V. Patterson; David C. Ware; Moana Tercel; William R. Wilson (pp. 1683-1694).
Metabolic reduction can be used to activate prodrugs in hypoxic regions of tumours, but reduction by ionising radiation is also theoretically attractive. Previously, we showed that a cobalt(III) complex containing 8-hydroxyquinoline (8-HQ) and cyclen ligands releases 8-HQ efficiently on irradiation in hypoxic solutions [Ahn G-O, Ware DC, Denny WA, Wilson WR. Optimization of the auxiliary ligand shell of cobalt(III)(8-hydroxyquinoline) complexes as model hypoxia-selective radiation-activated prodrugs. Radiat Res 2004;162:315–25]. Here we investigate an analogous Co(III) complex containing the potent DNA minor groove alkylator azachloromethylbenzindoline (azaCBI,1) to determine whether it releases1 on radiolytic and/or enzymatic reduction under hypoxia. Monitoring by HPLC, the azaCBI ligand in the Co(III)(cyclen)(azaCBI) complex (2) slowly hydrolysed in aqueous solution, in contrast to the free ligand1 which readily converted to its reactive cyclopropyl form. Irradiation of2 (30–50μM) in hypoxic solutions released1 with yields of 0.57μmol/J in formate buffer and 0.13μmol/J in human plasma. Using bioassay methods, cytotoxic activation by irradiation of2 at 1μM in hypoxic plasma was readily detectable at clinically relevant doses (≥1Gy), with a estimated yield of1 of 0.075μmol/J. Release of1 from2 was also observed in hypoxic HT29 cultures without radiation, with subsequent conversion of1 to its O-glucuronide. Surprisingly, overexpression of human cytochrome P450 reductase in A549 cells did not increase the rate of metabolic reduction of2, suggesting that other reductases and/or non-enzymatic reductants are responsible. Thus the cobalt(III) complex2 is a promising prodrug capable of being activated to release a very potent cytotoxin when reduced by either ionising radiation or cells under hypoxic conditions.
Keywords: Tumour hypoxia; Prodrug; Ionising radiation; Co(III) complex; CBI; DNA minor groove alkylator
Vincristine transcriptional regulation of efflux drug transporters in carcinoma cell lines by Rong Huang; Daryl J. Murry; Dhanashri Kolwankar; Stephen D. Hall; David R. Foster (pp. 1695-1704).
The increased expression of drug transporters following cancer chemotherapy contributes to resistance. This may reflect transcriptional up-regulation and/or clonal selection. We quantified the expression of mRNA for ABCB1 (mdr1), ABCC1 (mrp1), ABCC2 (mrp2) and ABCC3 (mrp3) to evaluate the potential contribution of induction. ABCB1, ABCC1– 3 mRNAs were quantified by real time RT-PCR and normalized to GAPDH. We used intestinal cells that express high pregnane X receptor (LS174T), low pregnane X receptor (Caco-2) and lung cells (A549) that express glucocorticoid receptor and low pregnane X receptor. Rifampin (10μM) caused significant induction of ABCB1 (595±263%, p<0.05) in LS174T cells but induction was absent in Caco-2 or A549 cells. ABCC1 was not induced in any cell at 24, 48 and 72h following rifampin treatment. In contrast, vincristine (10nM and 100nM), a ligand for ABCB1 and ABCC1– 3 and a potential PXR/CAR ligand, induced ABCC2 and ABCC3 expression in LS174T cells at 48h (372±87% and 303±42%, respectively, p<0.05). A similar induction of ABCC2 and ABCC3 genes was also seen with 10nM VCR in A549 cells following 48h treatment. In summary, there may be a significant contribution of transcriptional activation to multi-drug resistance. However, this is cell selective and is not necessarily dependent on PXR mediated effects.
Keywords: Abbreviations; ABCB1; multidrug resistance protein 1 (mdr1) or P-glycoprotein (P-gp); ABCC1; multidrug resistance associated protein 1 (mrp1); ABCC2; multidrug resistance associated protein 2 (mrp2) or canalicular multispecfic organic anion transporter (cMOAT); ABCC3; multidrug resistance associated protein 3 (mrp3); CAR; constitutive androstane receptor; CYP3A4; Cytochrome P450 3A4; GR; glucocorticoid receptor; PXR, hPXR, SXR; human pregnane X receptor; RIF; rifampin; RT-PCR; reverse transcriptase polymerase chain reaction; VCR; vincristineVincristine; ABCB1; ABCC1; ABCC2; ABCC3; Transcriptional regulation
γ-Aminobutyric acid (GABA)-C receptor stimulation increases prolactin (PRL) secretion in cultured rat anterior pituitary cells by Yasuhisa Nakayama; Naoki Hattori; Hitomi Otani; Chiyoko Inagaki (pp. 1705-1710).
γ-Aminobutyric acid (GABA) reportedly inhibits secretion of anterior pituitary hormones by directly acting on GABA-A and GABA-B receptors on anterior pituitary cells, but the roles of GABA-C receptors are little known. In this study, involvement of GABA-C receptors in the secretion of prolactin (PRL) was examined using cultured rat anterior pituitary cells. GABA-C receptor agonist, cis-4-aminocrotonic acid (CACA, 0.1–1mM) increased PRL secretion dose-dependently, while GABA-A receptor agonist, 100μM muscimol, but not GABA-B receptor agonist, 100μM baclofen, decreased the secretion. GABA-C receptor antagonist, 15μM (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA), and GABA-A receptor antagonist, 100μM bicuculline, not only reversed such an agonist-induced increase or decrease in PRL secretion, but also suppressed or enhanced spontaneous PRL secretion, raising a possibility of GABA-C or GABA-A receptor stimulation by intrinsic pituitary-derived GABA. GABA-C receptor subunits (Ï?1, Ï?2, Ï?3) and GABA synthesizing enzymes (GAD 65 and GAD 67) were shown to be expressed as assayed by RT-PCR, and GABA-C receptor stimulation by CACA obviously increased intracellular Ca2+ concentration in the anterior pituitary cells. Thus, PRL secretion from anterior pituitary cells appears to be enhanced via direct GABA-C receptor stimulation by GABA originating from the anterior pituitary cells besides well-known hypothalamic GABA.
Keywords: Abbreviations; GABA; γ-aminobutyric acid; CACA; cis; -aminocrotonic acid; PRL; prolactin; TPMPA; 1,2,5,6-tetrahydropyridin-4-yl methylphosphinic acid; GAD; glutamic acid decarboxylaseGABA; GABA-C receptor; Prolactin; Pituitary
Cross-talk between angiotensin II and glucagon receptor signaling mediates phosphorylation of mitogen-activated protein kinases ERK 1/2 in rat glomerular mesangial cells by Xiao C. Li; Oscar A. Carretero; Jia L. Zhuo (pp. 1711-1719).
We have recently shown that the pancreatic hormone glucagon-induced phosphorylation of mitogen-activated protein (MAP) kinase ERK 1/2 as well as growth and proliferation of rat glomerular mesangial cells (MCs) via activation of cAMP-dependent protein kinase A (PKA)-and phospholipase C (PLC)/Ca2+-mediated signaling pathways. Since circulating glucagon and tissue angiotensin II (Ang II) levels are inappropriately elevated in type 2 diabetes, we tested the hypothesis that glucagon induces phosphorylation of ERK 1/2 in MCs by interacting with Ang II receptor signaling. Stimulation of MCs by glucagon (10nM) induced a marked increase in intracellular [Ca2+] i that was abolished by [Des-His1, Glu9]-glucagon (1μM), a selective glucagon receptor antagonist. Both glucagon and Ang II-induced ERK 1/2 phosphorylation (glucagon: 214±14%; Ang II: 174±16%; p<0.001 versus control), and these responses were inhibited by the AT1 receptor blocker losartan (glucagon+losartan: 77±14%; Ang II+losartan: 84±18%; p<0.01 versus glucagon or Ang II) and the AT2 receptor blocker PD 123319 (glucagon+PD: 78±7%; Ang II+PD: 87±7%; p<0.01 versus glucagon or Ang II). Inhibition of cAMP-dependent PKA with H89 (1μM) or PLC with U73122 (1μM) also markedly attenuated the phosphorylation of ERK 1/2 induced by glucagon (glucagon+U73122: 109±15%; glucagon+H89: 113±16%; p<0.01 versus glucagon) or Ang II (Ang II+U73122: 111±13%; Ang II+H89: 86±10%; p<0.01 versus Ang II). Wortmannin (1μM), a selective PI 3-kinase inhibitor, also blocked glucagon- or Ang II-induced ERK 1/2 phosphorylation. These results suggest that AT1 receptor-activated cAMP-dependent PKA, PLC and PI 3-kinase signaling is involved in glucagon-induced MAP kinase ERK 1/2 phosphorylation in MCs. The inhibitory effect of PD 123319 on glucagon-induced ERK 1/2 phosphorylation further suggests that AT2 receptors also play a similar role in this response.
Keywords: Angiotensin II; Diabetes mellitus; Glucagon; Mesangial cells; Signaling cross-talk
The Cdc42 inhibitor secramine B prevents cAMP-induced K+ conductance in intestinal epithelial cells by Henry E. Pelish; William Ciesla; Nori Tanaka; Krishna Reddy; Matthew D. Shair; Tomas Kirchhausen; Wayne I. Lencer (pp. 1720-1726).
Cyclic AMP- (cAMP) and calcium-dependent agonists stimulate chloride secretion through the coordinated activation of distinct apical and basolateral membrane channels and ion transporters in mucosal epithelial cells. Defects in the regulation of Cl− transport across mucosal surfaces occur with cystic fibrosis and V. cholerae infection and can be life threatening. Here we report that secramine B, a small molecule that inhibits activation of the Rho GTPase Cdc42, reduced cAMP-stimulated chloride secretion in the human intestinal cell line T84. Secramine B interfered with a cAMP-gated and Ba2+-sensitive K+ channel, presumably KCNQ1/KCNE3. This channel is required to maintain the membrane potential that sustains chloride secretion. In contrast, secramine B did not affect the Ca2+-mediated chloride secretion pathway, which requires a separate K+ channel activity from that of cAMP. Pirl1, another small molecule structurally unrelated to secramine B that also inhibits Cdc42 activation in vitro, similarly inhibited cAMP-dependent but not Ca2+-dependent chloride secretion. These results suggest that Rho GTPases may be involved in the regulation of the chloride secretory response and identify secramine B an inhibitor of cAMP-dependent K+ conductance in intestinal epithelial cells.
Keywords: Abbreviations; CaCC; calcium activated chloride channel; cAMP; 3′,5′-cyclic AMP; CFTR; cystic fibrosis transmembrane regulator; ER; endoplasmic reticulum; Isc; short circuit current; Isc-b; l; K; basolateral membrane potassium Isc; Isc-a; p; Cl; apical membrane chloride Isc; I; –; V; current–voltage; NKCC; sodium potassium two chloride cotransporter; TER; transepithelial resistanceSecramine; Chloride secretion; Cdc42; cAMP; Potassium channel regulation; KCNQ1/KCNE3
Effects of capsaicin on P-gp function and expression in Caco-2 cells by Yi Han; Theresa May Chin Tan; Lee-Yong Lim (pp. 1727-1734).
Capsaicin is the pungent component of hot chilli, a popular spice in many populations. The aim of the present study was to evaluate the chronicity and reversibility of the modulating effect of capsaicin on both the P-gp expression and activity in the Caco-2 cell monolayers. Capsaicin at concentrations ranging from 10 to 100μM, which were found to be non-cytotoxic towards the Caco-2 cells, were observed to inhibit P-gp mediated efflux transport of [3H]-digoxin in the cells. The acute inhibitory effect was dependent on the capsaicin concentration and duration of exposure, with abolishment of polarity of [3H]-digoxin transport attained at 50μM of capsaicin. In contrast, longer term (48 and 72h) co-incubation of the Caco-2 cells with capsaicin (50 and 100μM) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by higher degree of [3H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. The induction of P-gp protein and mRNA levels was also influenced by capsaicin concentration and duration of exposure, with higher expression levels, in particular of the mRNA, seen at higher spice concentrations over prolonged period of incubation. Our data suggest that caution should be exercised when capsaicin is to be consumed with drugs that are P-gp substrates. In particular, the oral bioavailability of these drugs may be influenced by the P-gp status of populations that rely heavily on hot chilli in their diets.
Keywords: Abbreviations; MDR; multidrug resistance; P-gp; P-glycoprotein; PXR; Pregnane X Receptor; PBS; phosphate-buffered saline; DMSO; dimethyl sulfoxideCapsaicin; P-gp; MDR1; Digoxin transport; Caco-2 cells
Activation of phosphoinositide 3-kinase and Src family kinase is required for respiratory burst in rat neutrophils stimulated with artocarpol A by Yu-Hsiang Kuan; Ruey-Hseng Lin; Hui-Yi Lin; Li-Jiau Huang; Chi-Ren Tsai; Lo-Ti Tsao; Chun-Nan Lin; Ling-Chu Chang; Jih-Pyang Wang (pp. 1735-1746).
Artocarpol A (ART), a natural product isolated from Artocarpus rigida, stimulated superoxide anion (O2−) generation, which was inhibited by 2-(4-morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase (PI3K) inhibitor, in rat neutrophils. ART stimulated phosphorylation of protein kinase B (PKB/Akt) on both T308 and S473 residues, and LY 294002 inhibited these effects. Rat neutrophils expressed both class IA PI3K subunits (p85, p110α, p110β, and p110δ) and a class IB PI3K subunit (p110γ) as assessed by a combination of Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) approaches. Stimulation of neutrophils with ART evoked phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5) P3) formation, which reached a maximal level at 2min and was attenuated by LY 294002, as evidenced by immunofluorescence microscopy and by flow cytometry. Detectable membrane-association of class IA PI3Ks, class IB PI3K and Ras was seen as early as 1.5, 0.5 and 1.5min, respectively, after stimulation with ART. The kinetics of ART-induced Ras activation paralleled the kinetics of class IA PI3Ks recruitment to membrane caused by ART, and the p85 and p110γ immunoprecipitates contain Ras. ART stimulated Src family kinase activation, which was detectable within 1.5min of incubation with ART. Both Src kinase activity and PtdIns(3,4,5) P3 formation in ART-stimulated neutrophils were inhibited by 4-amino-1- tert-butyl-3-(1′-naphthyl)pyrazolo[3,4-d]pyrimidine (PP1 analog). PP1 analog also attenuated the ART-stimulated O2− generation in rat neutrophils. These results indicate that the stimulation of respiratory burst by ART in neutrophils implicates PI3K signaling.
Keywords: Abbreviations; ART; artocarpol A; dhCB; dihydrocytochalasin B; fMLP; formyl-Met-Leu-Phe; HBSS; Hanks’ balanced salt solution; MAPK; mitogen-activated protein kinase; MAPKAPK-2; MAPK-activated protein kinase-2; O; 2; −; superoxide anion; PDK1; phosphoinositide-dependent kinase 1; PI3K; phosphoinositide 3-kinase; PKB/Akt; protein kinase B; PKC; protein kinase C; PLC; phospholipase C; PtdIns(3,4,5); P; 3; phosphatidylinositol-3,4,5-trisphosphate; RT-PCR; reverse transcription-polymerase chain reactionArtocarpol A; Superoxide anion; Phosphoinositide 3-kinase; Akt; Ras; Src family kinase; Neutrophils
Superoxide radical production by allopurinol and xanthine oxidase by Chiara Galbusera; Peter Orth; David Fedida; Thomas Spector (pp. 1747-1752).
Oxypurinol, an inhibitor of xanthine oxidase (XO), is being studied to block XO-catalyzed superoxide radical formation and thereby treat and protect failing heart tissue. Allopurinol, a prodrug that is converted to oxypurinol by xanthine oxidase, is also being studied for similar purposes. Because allopurinol, itself, may be generating superoxide radicals, we currently studied the reaction of allopurinol with xanthine oxidase and confirmed that allopurinol does produce superoxide radicals during its conversion to oxypurinol.At pH 6.8 and 25°C in the presence of 0.02U/ml of XO, 10 and 20μM allopurinol both produced 10μM oxypurinol and 2.8μM superoxide radical (determined by cytochrome C reduction). The 10μM allopurinol was completely converted to oxypurinol, while the 20μM allopurinol required a second addition of xanthine oxidase to complete the conversion. Fourteen percent of the reducing equivalents donated from allopurinol or xanthine reacted with oxygen to form superoxide radicals. Superoxide dismutase prevented the reduction of cytochrome C by these substrates. At higher xanthine oxidase concentrations, or at lower temperatures, more of the 20μM allopurinol was converted to oxypurinol during the initial reaction. At lower xanthine oxidase concentrations, or higher temperatures, less conversion occurred. At pH 7.8, the amount of superoxide radicals produced from allopurinol and xanthine was nearly doubled. These results indicate that allopurinol is a conventional substrate that generates superoxide radicals during its oxidation by xanthine oxidase. Oxypurinol did not produce superoxide radicals.
Keywords: Abbreviations; ROS; reactive oxygen species; SOD; superoxide dismutase; XO; xanthine oxidase; XOR; xanthine oxidoreductase; XDH; xanthine dehydrogenase; FAD; flavine–adenine dinucleotideAllopurinol; Oxypurinol; Xanthine oxidase; Superoxide radical; Heart failure
Metabolism of oxidants by blood from different mouse strains by Daniela Giustarini; Isabella Dalle-Donne; Eleonora Cavarra; Silvia Fineschi; Giuseppe Lungarella; Aldo Milzani; Ranieri Rossi (pp. 1753-1764).
Haemoglobins bearing reactive sulfhydryl groups have been shown to be able to interplay with glutathione in some detoxification processes. Blood from different mouse strains commonly used as experimental animal models, i.e., C57, DBA and ICR, was treated with oxidants with the aim of evaluating: (i) the involvement of protein SH groups in oxido-reductive reactions that are commonly carried out by glutathione and (ii) the impact of this phenomenon on blood-mediated metabolism of thiol reactants. All the main forms of glutathione (reduced, disulfide, and mixed disulfide with haemoglobin) were measured after oxidant treatment. Significant differences were found among the studied strains: DBA mice formed preferably mixed disulfides instead of glutathione disulfide, whereas the opposite behaviour was shown by C57 mice. Unexpectedly, the ICR strain resulted to be composed of three different subgroups (ICRa, ICRb, and ICRc), with the ICRa behaving similarly to the DBA strain, ICRc to the C57 strain, and ICRc showing an intermediate behaviour. These results are due to the different number of haemoglobin SH groups in the studied mouse strains. In particular, additional fast-reacting SH groups were found in haemoglobin from DBA, ICRa, and ICRb mice, but not in the C57 and ICRc strain. These differences were also reflected in the susceptibility of haemoglobin to dimerize and in its ability to react with S-nitrosocysteine. Because of the widely different reactivity of haemoglobin cysteinyl residues, the mouse strains examined are an interesting but complicated model in which to study the pharmacological and toxicological action of some drugs.
Keywords: Blood; Reduced glutathione; Glutathione disulfide; Glutathionylation; Haemoglobin sulfhydryl groups; Mouse strains; Oxidative stress