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Biochemical Pharmacology (v.84, #8)

Editorial Board (pp. iii).

Extraneuronal activities and regulatory mechanisms of the atypical cyclin-dependent kinase Cdk5 by Abul Arif (pp. 985-993).

Cyclin-dependent kinase, Cdk5, is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases with no evident role in cell cycle progression. Cdk5 is present in post-mitotic and terminally differentiated neuronal/glial cells and is also known to arrest cell cycle. Also atypical is the activation of Cdk5 by binding of a non-cyclin activator protein, namely, the Cdk5 regulatory proteins Cdk5R1 (p35), truncated Cdk5R1 (p25), or Cdk5R2 (p39). Despite its ubiquitous presence in all cells and tissues, Cdk5 is often referred to as a neuron-specific kinase largely due to the abundant presence of the activator proteins in neuronal cells. Recently, this concept of a canonical neuronal function of Cdk5 has been extended, if not challenged, by the observation of p35 and p39 expression, as well as Cdk5 activity, in multiple non-neuronal cells. Extraneuronal Cdk5 regulates critical biological processes including transcript-selective translation control for regulation of macrophage gene expression, glucose-inducible insulin secretion, hematopoietic cell differentiation, vascular angiogenesis, cell migration, senescence, and wound-healing, among others. Recent advances in the extraneuronal functions of Cdk5 are reviewed and discussed here in the context of their physiological activities and pathophysiological implications with some speculative comments on the endogenous control mechanisms that might “turn on” Cdk5 activity. The potential importance of targeted inhibition of Cdk5 as therapeutic agents against glucotoxicity, diabetes, cardiovascular diseases, and cancer is also discussed.

Keywords: Abbreviations; APE1; apurinic-apyrimidinic endonuclease 1; Cdk; cyclin-dependent kinase; Cdk5R1 or p35; Cdk5 regulatory protein R1; Cdk5R2 or p39; Cdk5 regulatory protein R2; EPRS; glutamyl-prolyl tRNA synthetase; E-Syt1; extended synaptotagmin-1; FAK; focal adhesion kinase; GAIT; Interferon-γ-Activated inhibitor of translation; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HDAC; histone deacetylase; HMGB1; high mobility group box 1; L-VDCC; L-type voltage-dependent Ca; ²⁺; channel; NCoR; nuclear receptor co-repressor; NRG1; neuregulin; NSAP1; NS1-associated protein; PPARγ; peroxisome proliferator-activated receptor; PLD2; phospholipase D2; STAT3; signal transducer and activator of transcription 3; TC10α; Rho family GTP-binding protein; TonEBP/OREBP; tonicity-responsive enhancer-binding protein/osmotic response element-binding protein; UTR; untranslated regionCdk5; p35; p39; Extraneuronal; EPRS; Translation control; Inflammation

Cognitive enhancement by pharmacological and behavioral interventions: the murine Down syndrome model by Hanns Möhler (pp. 994-999).

The cognitive deficits in Down syndrome (DS) are attributed to an excessive hippocampal inhibition, which obstructs neuronal plasticity and normal learning and memory, a view which is largely based on studies of Ts65Dn mice, the best characterized mouse model of DS. The cognitive behavioral deficits of Ts65Dn mice can be rescued by reducing GABAergic inhibition, most selectively by partial inverse agonists acting on α₅ GABA-A receptors, of which one compound has recently entered clinical trials in DS. Most remarkably, the improved cognitive performance of Ts65Dn can persist for weeks and months after cessation of drug treatment, as demonstrated for the non-specific GABA antagonist pentylenetetrazole. The Alzheimer drugs, memantine and donepezil largely fail to show any benefit. Finally, repeated non-invasive sensory stimulation such as over-training or enriching the environment, are able to enhance the learning performance which underlines the reversibility of an obstructed neuronal plasticity in Ts65Dn mice.

Keywords: Alpha5 GABA-A receptor; Inverse agonist; RO4938581; Alpha5 IA; Ts65Dn mice

Human UDP-glucuronosyltransferases: Feedback loops between substrates and ligands of their transcription factors by Karl Walter Bock (pp. 1000-1006).

Expression profiles of human adult and fetal hepatic and intestinal UDP-glucuronosyltransferases (UGTs), information about their endo- and xenobiotic substrates, and their transcriptional regulation suggests regulatory circuits between some UGT substrates and ligands of their transcription factors. For examples: (i) bilirubin is solely conjugated by UGT1A1 and activates its transcription factors Ah receptor, PXR and CAR. (ii) Hepatotoxic lithocholic acid (LCA) is oxidized to hyodeoxycholic acid, the latter conjugated by UGT2B4 and UGT2B7. LCA is also an agonist of FXR and PPARα, which are controlling these UGTs. (iii) Similar feedback loops possibly exist between some eicosanoids, PPARα and UGTs. (iv) Regulatory circuits may also have evolved between dietary polyphenols, which are efficient substrates of UGTs and activators of the Ah receptor. Although many newly developed drugs are conjugated by promiscuous UGTs, the discussed regulatory circuits may provide hints to evolutionary important UGT substrates.

Keywords: Abbreviations; AhR; Ah receptor; CAR; constitutive androstane receptor; CYP; cytochrome P450; FXR; farnesoid receptor; GST; glutathione S-transferase; HETE; hydroxyeicosatetraenoic acid; LATF; ligand-activated transcription factor; LTB4; leucotriene B4; PPAR; peroxisome proliferator-activated receptor; SULT; sulfotransferase; UGT; UDP-glucuronosyltransferase; XRE; xenobiotic response elementUDP-glucuronosyltransferases; Ligand-activated transcription factors; Feedback loops; Bilirubin; Bile acids; Eicosanoids

Copper influx transporter 1 is required for FGF, PDGF and EGF-induced MAPK signaling by Cheng-Yu Tsai; J. Cameron Finley; Sameh S. Ali; Hemal H. Patel; Stephen B. Howell (pp. 1007-1013).

Copper transporter 1 (CTR1) is the major copper (Cu) influx transporter in mammalian cells. We report here that CTR1 is required for the activation of signaling to the MAPK pathway by the ligands of three major receptor tyrosine kinases (RTK) including FGF, PDGF and EGF. Induction of Erk1/2 phosphorylation was compared in isogenic wild type CTR1^+/+ and CTR1^−/− cells. Whereas all three ligands increased pErk1/2 in the CTR1^+/+ cells, they failed to do this in CTR1^−/− cells. While FGF did not enhance the phosphorylation of AKT in the CTR1^+/+ cells, both PDGF and EGF increased pAKT in the CTR1^+/+ but not CTR1^−/− cells. The deficit in Erk1/2 phosphorylation in the CTR1^−/− cells was rescued by adding Cu to the medium, and it was induced in CTR1^+/+ cells by treatment with a Cu chelator. Intracellular Cu availability was reduced in the CTR1^−/− cells as reflected by increased expression of the Cu chaperone CCS. The failure of RTK-induced signaling to both Erk1/2 and AKT suggested the presence of a Cu-dependent step upstream of Ras. The Cu-dependent enzyme SOD1 is responsible for generating the hydrogen peroxide in response to RTK activation that serves to inhibit phosphatases that normally limit RTK signaling. SOD1 activity was reduced by a factor of 17-fold in the CTR1^−/− cells, and addition of hydrogen peroxide restored signaling. We conclude that Cu acquired from CTR1 is required for signaling in pathways regulated by RTKs that play major roles in development and cancer.

Keywords: Abbreviations; SOD1; superoxide dismutase 1; Cu; copper; H; ₂; O; ₂; hydrogen peroxide; RTK; receptor tyrosine kinase; CCS; copper chaperone; EPR; electron paramagnetic resonance; MAPK; mitogen activated protein kinase; PTPase; protein tyrosine phosphatase; BCS; bathocuproine disulphonateCTR1; SOD1; Copper; Receptor tyrosine kinase

A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells by Juliana Vieira Dias; Zahia Benslimane-Ahmim; Marion Egot; Anna Lokajczyk; Françoise Grelac; Isabelle Galy-Fauroux; Luiz Juliano; Bernard Le-Bonniec; Cristina Maeda Takiya; Anne-Marie Fischer; Olivier Blanc-Brude; Verônica Morandi; Catherine Boisson-Vidal (pp. 1014-1023).

Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20μg/mL of TSP-HepI peptide for 18h enhanced cell migration ( p<0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC.

Keywords: Abbreviations; EPC; endothelial progenitor cells; ECFC; endothelial colony-forming cells; FGF-2; basic fibroblast growth factor; FACS; fluorescence-activated cell sorting; FCS; fetal calf serum; HUVEC; human umbilical vein endothelial cell; PBS; phosphate buffered saline; VEGF; vascular endothelial growth factorthrombospondin-1; endothelial colony-forming cells; glycosaminoglycans; angiogenesis

Store-independent pathways for cytosolic STIM1 clustering in the regulation of store-operated Ca²⁺ influx by Bo Zeng; Gui-Lan Chen; Shang-Zhong Xu (pp. 1024-1035).

STIM1 is a Ca²⁺ sensing molecule. Once the Ca²⁺ stores are depleted, STIM1 moves towards the plasma membrane (PM) (translocation), forms puncta (clustering), and triggers store-operated Ca²⁺ entry (SOCE). Although this process has been regarded as a main mechanism for store-operated Ca²⁺ channel activation, the STIM1 clustering is still unclear. Here we discovered a new phenomenon of STIM1 clustering, which is not triggered by endoplasmic reticulum (ER) Ca²⁺ depletion.STIM1 subplasmalemmal translocation and clustering can be induced by ER Ca²⁺ store depletion with thapsigargin (TG), G-protein-coupled receptor activator trypsin and ryanodine receptor (RyR) agonists caffeine and 4-chloro-3-ethylphenol (4-CEP) in the HEK293 cells stably transfected with STIM1–EYFP. The STIM1 clustering induced by TG was more sustained than that induced by trypsin and RyR agonists. Interestingly, 4-CEP-induced STIM1 clustering also happened in the cytosol without ER Ca²⁺ store depletion. Application of some pharmacological regulators including flufenamic acid, 2-APB, and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) at concentrations without affecting ER Ca²⁺ store also evoked cytosolic STIM1 clustering. However, the direct store-operated ORAI channel blockers (SKF-96365, Gd³⁺ and diethylstilbestrol) or the signaling pathway inhibitors (genistein, wortmannin, Y-27632, forskolin and GF109203X) did not change the STIM1 movement. Disruption of cytoskeleton by colchicine and cytochalasin D also showed no effect on STIM1 movement.We concluded that STIM1 clustering and translocation are two dynamic processes that can be pharmacologically dissociated. The ER Ca²⁺ store-independent mechanism for STIM1 clustering is a new alternative mechanism for regulating store-operated channel activity, which could act as a new pharmacological target.

Keywords: STIM1; 2-Aminoethoxydiphenyl borate; Store-operated Ca; ²⁺; channels; Mitochondrial Ca; ²⁺; release; ORAI

Quercetin and its metabolites improve vessel function by inducing eNOS activity via phosphorylation of AMPK by Yu Shen; Kevin D. Croft; Jonathan M. Hodgson; Reece Kyle; I-Ling E. Lee; Yutang Wang; Roland Stocker; Natalie C. Ward (pp. 1036-1044).

Quercetin is a major flavonoid in a wide range of fruits and vegetables. Consumption of quercetin may contribute to a reduction in risk of cardiovascular disease (CVD). Following ingestion, flavonoids are metabolized rapidly by methylation or glucuronidation, which can alter their biological activity. Certain dietary flavonoids have been shown to upregulate the expression of adenosine monophosphate-activated protein kinase (AMPK). AMPK is a conserved key enzyme in cellular energy homeostasis that affects fatty acid oxidation. The aim of the present study was to investigate the effects of supraphysiological concentrations of quercetin and its methyl and glucuronide metabolites (3′- O-methyl-quercetin and quercetin-3- O-glucuronide) on activation of AMPK and eNOS in human aortic endothelial cells (HAECs) and endothelial function in isolated aortic rings from C57BL mice. We found that 5 and 10μM quercetin and its metabolites, and pretreatment of arteries with quercetin and its metabolites can protect vessels against hypochlorous acid-induced endothelial dysfunction in isolated arteries ( P<0.05). Inhibition of AMPK blocked these protective effects. We also found that 5 and 10μM quercetin and its metabolites can induce activation of AMPK and eNOS in human aortic endothelial cells, and lead to an increase in the concentrations of S-nitrosothiols and nitrite in cell culture media ( P<0.05). These results provide further support for the cardioprotective effects of certain dietary flavonoids. They suggest that beneficial effects of quercetin on endothelial cell functions are in part mediated via AMPK pathway.

Keywords: Abbreviations; CVD; cardiovascular disease; HAECs; human aortic endothelial cells; AMPK; adenosine monophosphate-activated protein kinase; NO; nitric oxide; eNOS; endothelial nitric oxide synthase; ACC; acetyl-CoA carboxylaseQuercetin; Flavonoid; Endothelial function; AMPK; eNOS

Sildenafil stimulates the expression of gaseous monoxide-generating enzymes in vascular smooth muscle cells via distinct signaling pathways by Xiao-ming Liu; Kelly J. Peyton; Xinhui Wang; William Durante (pp. 1045-1054).

Sildenafil is a cGMP-specific phosphodiesterase type 5 inhibitor that augments cGMP accumulation following the activation of soluble guanylate cyclase (sGC). In this study, we investigated whether sildenafil promotes the production of the sGC-stimulatory gases, carbon monoxide and nitric oxide, by stimulating the expression of the inducible isoforms of heme oxygenase (HO-1) and nitric oxide synthase (iNOS) in vascular smooth muscle cells (SMCs). Sildenafil increased HO-1 expression and potentiated cytokine-mediated expression of iNOS and NO synthesis by SMCs. The induction of HO-1 was unaffected by the sGC inhibitor 1H-(1,2,4)oxadiazolo[4,3-α]quinozalin-1-one (ODQ) or the protein kinase G inhibitor (8R,9S,11S)-(-)-2-methyl-9-methoxyl-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta9(cde)trinen-1-one (KT 5823). However, the sildenafil-mediated increase in HO-1 promoter activity was abolished by mutating the antioxidant responsive elements in the promoter or by overexpressing a dominant-negative mutant of NF-E2-related factor-2 (Nrf2). Furthermore, the induction of HO-1 by sildenafil was accompanied by an increase in reactive oxygen species (ROS) and blocked by N-acetyl-l-cysteine and rotenone. In contrast, the enhancement of cytokine-stimulated NO synthesis by sildenafil was prevented by ODQ and the protein kinase A inhibitor (9S,10S,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo(1,2,3-fg:3′,2′,1′-kl)pyrrolo(3,4-i)(1,6)benzodiazocine-10-carboxylic acid hexyl ester (KT 5720) and duplicated by lipophilic analogs of cGMP. In conclusion, these studies demonstrate that sildenafil stimulates the expression of HO-1 and iNOS via the ROS-Nrf2 and sGC-cGMP pathway, respectively. The ability of sildenafil to block the catabolism of cGMP while stimulating the synthesis of sGC-stimulatory gaseous monoxides through the induction of HO-1 and iNOS provides a potent mechanism by which cGMP-dependent vascular actions of this drug are amplified.

Keywords: Heme oxygenase; Inducible nitric oxide synthase; Vascular smooth muscle cells

Effects of commonly used protein kinase inhibitors on vascular contraction and L-type Ca²⁺ current by Simona Saponara; Fabio Fusi; Giampietro Sgaragli; Maurizio Cavalli; Brian Hopkins; Sergio Bova (pp. 1055-1061).

Regulation of smooth muscle contraction is driven by a number of protein kinases: the evidence for this often originates from studies that investigate the effects of extracellularly added specific protein kinase inhibitors. Six compounds, thought to be selective inhibitors of various kinases, were analysed for their effects on vascular L-type Ca²⁺ channels because this potential subsidiary activity could strongly influence our understanding of the pathways involved in smooth muscle contraction. Whole-cell L-type Ba²⁺ currents [ I_Ba(L)] were recorded in single myocytes, and contractile responses were measured from endothelium-denuded rings taken from the rat tail artery. Although ML-7, ML-9, and wortmannin (MLCK inhibitors), HA-1077 and Y-27632 (Rho-associated kinase inhibitors), and GF-109203X (PKC inhibitor) relaxed rings pre-contracted with high KCl in a concentration-dependent manner, their effect on I_Ba(L) intensity was surprisingly variable. Wortmannin showed negligible effects while HA-1077 and Y-27632 were ineffective. I_Ba(L) was partly inhibited by GF-109203X and blocked by ML-7 and ML-9 in a concentration-dependent manner, with the blockade by ML-7 being voltage-dependent. Whilst ML-7, ML-9, and GF-109203X sped up the inactivation kinetics of I_Ba(L), GF-109203X did not modify ML-7- or ML-9-induced effects, with both intensity and kinetics of the current remaining unchanged. In contrast, application of Bay K 8644 on myocytes pre-treated with ML-7 or ML-9 raised I_Ba(L) beyond control values. In conclusion, ML-7 and ML-9 inhibit L-type Ca²⁺ channels via a mechanism independent of MLCK, PKC or Rho kinase activities, and as such caution should be used in employing these agents to elucidate the role of kinases in smooth muscle contraction.

Keywords: Abbreviations; Bay K 8644; (S)-(−)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate; GF-109203X; 3-(N-[dimethylamino]propyl-3-indolyl)-4-(3-indolyl)maleimide; HA-1077; hexahydro-1-(5-isoquinolinylsulfonyl)-1H-1,4-diazepine; I; _Ba(L); L-type Ba; ²⁺; current; ML-7; 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine; ML-9; 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine; MLCK; myosin light chain kinase; PKC; protein kinase C; V; _h; holding potential; Y-27632; (R)-(+)-; trans; -4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamideL-type Ca; ²⁺; channel; ML-7; ML-9; Myosin light chain kinase; Rat tail artery; Whole-cell patch-clamp

Nitric oxide-dependent bradycardia in mutant analbuminemic rats by Ali R. Mani; Silvia Ippolito; Miguel N. Centelles; Kevin P. Moore (pp. 1062-1069).

Nagase analbuminemic rats (NAR) are natural mutant Sprague-Dawley rats which do not express albumin due to a single splice mutation in the albumin gene. We accidentally discovered that NAR have a significant bradycardia compared with wild type Sprague-Dawley rats, and the present study was carried out to investigate the mechanism of bradycardia in analbuminemic rats. In vitro studies showed that the basal spontaneous beating rate of isolated atria is similar in NAR compared with wild type animals. However, the chronotropic responsiveness of isolated atria to cholinergic stimulation was markedly increased in NAR, an effect which was prevented by incubation with a nitric oxide synthase (NOS) or guanylyl cyclase inhibitor. NAR had a significant increase in plasma nitrite/nitrate concentrations. Administration of a NOS inhibitor for 5 days normalized heart rate in NAR. The level of NOS isoforms, caveolin-1 and caveolin-3 expression in the atria was assessed by real time PCR. There was no significant difference in the expression of NOS isoforms or caveolin-3 in NAR compared with wild type controls. However, NAR exhibited a significant decrease in caveolin-1 expression in the atria. Since caveolin-1 is known to inhibit endothelial NOS activity in cardiomyocytes, we suggest that decreased caveolin-1 levels may have a role in increased nitric oxide production in NAR. Our data suggest that a NOS/cGMP-dependent mechanism might be involved in increased responsiveness to vagal stimulation and bradycardia in analbuminemic condition.

Keywords: Bradycardia; Caveolin; Nagase analbuminemic rats; Nitric oxide

Enhanced expression of organic anion transporting polypeptides (OATPs) in androgen receptor-positive prostate cancer cells: Possible role of OATP1A2 in adaptive cell growth under androgen-depleted conditions by Hiroshi Arakawa; Takeo Nakanishi; Chihiro Yanagihara; Tomohiro Nishimoto; Tomohiko Wakayama; Atsushi Mizokami; Mikio Namiki; Keiichi Kawai; Ikumi Tamai (pp. 1070-1077).

The biological mechanisms underlying castration resistance of prostate cancer are not fully understood. In the present study, we examined the role of organic anion transporting polypeptides (OATPs) as importers of dehydroepiandrosterone sulfate (DHEAS) into cells to support growth under androgen-depleted conditions. Cell growth and mRNA expression of OATP genes were studied in human prostate cancer LNCaP and 22Rv1 cells under androgen-depleted conditions. The stimulatory effect of DHEAS on cell growth was investigated in LNCaP cells in which OATP1A2 had been silenced. Growth of both cell lines was stimulated by DHEAS and the effect was attenuated by STX64, an inhibitor of steroid sulfatase which can covert DHEAS to DHEA. OATP1A2 mRNA expression was increased most prominently among various genes tested in LNCaP cells grown in androgen-depleted medium. Similar results were obtained with 22Rv1 cells. Furthermore, the characteristics of [³H]DHEAS uptake by LNCaP cells were consistent with those of OATP-mediated transport. Knockdown of OATP1A2 in LNCaP cells resulted in loss of the DHEAS sensitivity of cell growth. Our results suggest that enhanced OATP1A2 expression is associated with adaptive cell growth of prostate cancer cells under androgen-depleted conditions. Thus, OATP1A2 may be a pharmacological target for prostate cancer treatment.

Keywords: Abbreviations; AR; androgen receptor; STX64; 6-oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl sulfamate; CRPC; castration-resistant prostate cancer; STS; steroid sulfatase; OATP; organic anion transporting polypeptide; DHEA; dehydroepiandrosterone; DHEAS; dehydroepiandrosterone sulfate; DHT; dihydrotestosterone; ADT; androgen deprivation therapy; SRB; sulforhodamine B; PSA; prostate specific antigen; HPRT1; hypoxanthine guaninephosphoribosyl-transferase; HSD; hydroxysteroid dehydrogenase; AP-1; activator protein-1; NTCP; Na; ⁺; -dependent taurocholate cotransporting polypeptide; E3S; estrone 3-sulfate; BSP; bromosulfophthalein; TCA; taurocholate; PRO; probenecid; SAL; salicylate; PAH; p; -aminohippurate; TEA; tetra-ethylammonium; MPP; ⁺; 1-methyl-4-phenylpyridiniumProstate cancer; OATP1A2; DHEAS; Androgen receptor; Steroid sulfatase

Kynurenic acid inhibits glutamatergic transmission to CA1 pyramidal neurons via α7 nAChR-dependent and -independent mechanisms by Jyotirmoy Banerjee; Manickavasagom Alkondon; Edson X. Albuquerque (pp. 1078-1087).

Glutamatergic hypofunction and elevated levels of kynurenic acid (KYNA) in the brain are common features of patients with schizophrenia. In vivo studies indicate that in the hippocampus KYNA decreases glutamate levels, presumably via inhibition of α7 nicotinic receptors (nAChRs). Here we tested the hypothesis that basal synaptic glutamate activity in the hippocampus is regulated by tonically active α7 nAChRs and is sensitive to inhibition by KYNA. To this end, spontaneous excitatory postsynaptic currents (EPSCs), sensitive to AMPA receptor antagonist CNQX (10μM), were recorded from CA1 pyramidal neurons at −70mV in rat hippocampal slices. The α7 nAChR antagonists α-bungarotoxin (α-BGT, 100nM) and methyllycaconitine (MLA, 1–50nM), and the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV, 50μM) reduced the frequency of EPSCs. MLA and α-BGT had no effect on miniature EPSCs (mEPSCs). The effect of MLA decreased in the presence of APV (50μM), with 1nM MLA becoming completely ineffective. KYNA (1–20μM) suppressed the frequency of EPSCs, without affecting mEPSCs. The effect of KYNA decreased in the presence of MLA (1nM) or α-BGT (100nM), with 1μM KYNA being devoid of any effect. In the presence of both MLA (10nM) and APV (50μM) higher KYNA concentrations (5–20μM) still reduced the frequency of EPSCs. These results suggest that basal synaptic glutamate activity in CA1 pyramidal neurons is maintained in part by tonically active α7 nAChRs and NMDA receptors and is inhibited by micromolar concentrations of KYNA, acting via α7 nAChR-dependent and -independent mechanisms.

Keywords: Abbreviations; α-BGT; α-Bungarotoxin; ACh; acetylcholine; ACSF; artificial cerebrospinal fluid; AMPA; α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid; APV; 2-amino-5-phosphonovaleric acid; CNQX; 6-Cyano-7-nitroquinoxaline-2,3-dione; EPSC; excitatory postsynaptic current; KYNA; kynurenic acid; MLA; methyllycaconitine; nAChR; nicotinic acetylcholine receptor; NMDA; N-methyl-; d; -aspartic acid; TTX; Tetrodotoxin; τ; _d; decay-time constantGlutamate; Nicotinic receptor; NMDA receptor; Kynurenic acid; Hippocampus

Potential for food–drug interactions by dietary phenolic acids on human organic anion transporters 1 (SLC22A6), 3 (SLC22A8), and 4 (SLC22A11) by Li Wang; Douglas H. Sweet (pp. 1088-1095).

Phenolic acids exert beneficial health effects such as anti-oxidant, anti-carcinogenic, and anti-inflammatory activities and show systemic exposure after consumption of common fruits, vegetables, and beverages. However, knowledge regarding which components convey therapeutic benefits and the mechanism(s) by which they cross cell membranes is extremely limited. Therefore, we determined the inhibitory effects of nine food-derived phenolic acids, p-coumaric acid, ferulic acid, gallic acid, gentisic acid, 4-hydroxybenzoic acid, protocatechuic acid, sinapinic acid, syringic acid, and vanillic acid, on human organic anion transporter 1 (hOAT1), hOAT3, and hOAT4. In the present study, inhibition of OAT-mediated transport of prototypical substrates (1μM) by phenolic acids (100μM) was examined in stably expressing cell lines. All compounds significantly inhibited hOAT3 transport, while just ferulic, gallic, protocatechuic, sinapinic, and vanillic acid significantly blocked hOAT1 activity. Only sinapinic acid inhibited hOAT4 (∼35%). For compounds exhibiting inhibition>∼60%, known clinical plasma concentration levels and plasma protein binding in humans were examined to select compounds to evaluate further with dose–response curves (IC₅₀ values) and drug–drug interaction (DDI) index determinations. IC₅₀ values ranged from 1.24 to 18.08μM for hOAT1 and from 7.35 to 87.36μM for hOAT3. Maximum DDI indices for gallic and gentisic acid (≫0.1) indicated a very strong potential for DDIs on hOAT1 and/or hOAT3. This study indicates that gallic acid from foods or supplements, or gentisic acid from salicylate-based drug metabolism, may significantly alter the pharmacokinetics (efficacy and toxicity) of concomitant therapeutics that are hOAT1 and/or hOAT3 substrates.

Keywords: Abbreviations; ACN; anthocyanin; BCRP; breast cancer resistance protein; C; _max; maximum plasma concentration; CHO; Chinese hamster ovary; DDI; drug–drug interaction; ES; estrone sulfate; f; _u; fraction unbound in plasma; FDA; Food and Drug Administration; HEK; human embryonic kidney 293; h; human; IC; ₅₀; median maximal inhibitory concentration; K; _i; inhibitory constant; K; _m; Michaelis constant; MDR1; multidrug resistance transporter 1; OAT; organic anion transporter; OATP1B1; organic anion transporting polypeptide 1B1; OCT2; organic cation transporter 2; PAH; p; -aminohippuric acid; SLC; solute carrierDrug–drug interactions; Drug–food interaction; Pharmacokinetics; Renal transport; Solute carriers

Interaction of the antiviral drug telaprevir with renal and hepatic drug transporters by Annett Kunze; Jörg Huwyler; Gian Camenisch; Heike Gutmann (pp. 1096-1102).

Telaprevir inhibits renal and hepatic drug transporters with corresponding IC₅₀ values in the range of 2.15–22.98μM.Telaprevir is a new, direct-acting antiviral drug that has been approved for the treatment of chronic hepatitis C viral infection. First data on drug–drug interactions with co-medications such as cyclosporine, tacrolimus and atorvastatin have been reported recently. Drug transporting proteins have been shown to play an important role in clinically observed drug–drug interactions. The aim of this study was therefore to systematically investigate the potential of telaprevir to inhibit drug transporting proteins. The effect of telaprevir on substrate uptake mediated by drug transporters located in human kidney and liver was investigated on a functional level in HEK293 cell lines that over-express single transporter. Telaprevir was shown to exhibit significant inhibition of the human renal drug transporters OCT2 and MATE1 with IC₅₀ values of 6.4μM and 23.0μM, respectively, whereas no inhibitory effect on OAT1 and OAT3 mediated transport by telaprevir was demonstrated. Liver drug transporters were inhibited with an IC₅₀ of 2.2μM for OATP1B1, 6.8μM for OATP1B3 and 20.7μM for OCT1. Our data show that telaprevir exhibited significant potential to inhibit human drug transporters. In view of the inhibitory potential of telaprevir, clinical co-administration of telaprevir together with drugs that are substrates of renal or hepatic transporters should be carefully monitored.

Keywords: OCT; MATE1; Telaprevir; OATP; Drug–drug interaction; Drug transporter

Corrigendum to “OATP1B1/1B3 activity in plated primary human hepatocytes over time in culture” [Biochem. Pharmacol. 82 (2011) 1219–1226] by Maria Ulvestad; Petter Björquist; Espen Molden; Anders Åsberg; Tommy B. Andersson (pp. 1103-1103).

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Biochemical Pharmacology (v.84, #8)