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Biochemical Pharmacology (v.72, #8)

Editorial Advisory Board (pp. iii).

Coincident signaling in mesolimbic structures underlying alcohol reinforcement by Tao A. Zhang; Regina E. Maldve; Richard A. Morrisett (pp. 919-927).

The medium spiny neurons (MSNs) of the nucleus accumbens function in a critical regard to examine and integrate information in the processing of rewarding behaviors. These neurons are aberrantly affected by drugs of abuse, including alcohol. However, ethanol is unlike any other common drug of abuse, due to its pleiotropic actions on intracellular and intercellular signaling processes. Intracellular biochemical pathways appear to critically contribute to long-term changes in the level of synaptic activation of these neurons, which have been implicated in ethanol dependence. Additionally, these neurons also display a fascinating pattern of up/down activity, which appears to be, at least in part, regulated by convergent activation of dopaminergic and glutamatergic (NMDA) inputs. Thus, dopaminergic and NMDA receptor-mediated synaptic transmission onto these neurons may constitute a critical site of ethanol action in mesolimbic structures. For instance, dopaminergic inputs alter the ability of ethanol to regulate NMDA receptor-mediated synaptic transmission onto accumbal MSNs. Prior activation of D1-signaling cascade through the cAMP-regulated phosphoprotein-32kD (DARPP-32) and protein phosphatase-1 (PP-1) pathway significantly attenuates ethanol inhibition of NMDA receptor function. Therefore, the interaction of D1-signaling and NMDA receptor signaling may alter NMDA receptor-dependent long-term synaptic plasticity, contributing to the development of ethanol-induced neuroadaptation of the reward pathway.

Keywords: Nucleus accumbens; Ventral tegmental area; Medium spiny neuron; Synaptic plasticity; Addiction; DARPP-32

TPA-induced up-regulation of activator protein-1 can be inhibited or enhanced by analogs of the natural product curcumin by Waylon M. Weber; Lucy A. Hunsaker; Amanda M. Gonzales; Justin J. Heynekamp; Robert A. Orlando; Lorraine M. Deck; David L. Vander Jagt (pp. 928-940).

The activator protein-1 (AP-1) family of transcription factors, including the most common member c-Jun-c-Fos, participates in regulation of expression of numerous genes involved in proliferation, apoptosis, and tumorigenesis in response to a wide array of stimuli including pro-inflammatory cytokines, growth factors, stress, and tumor promoters. A number of plant polyphenols including curcumin, a yellow compound in the spice turmeric, have been shown to inhibit the activation of AP-1. Curcumin is a polyphenolic dienone that is potentially reactive as a Michael acceptor and also is a strong anti-oxidant. Multiple activities reported for curcumin, including inhibition of the stress-induced activation of AP-1, have been suggested to involve the anti-oxidant properties of curcumin. In the present study, a library of analogs of curcumin was screened for activity against the TPA-induced activation of AP-1 using the Panomics AP-1 Reporter 293 stable cell line which is designed for screening potential inhibitors. Numerous analogs were identified that were more active than curcumin, including analogs that were not anti-oxidants and analogs that were not Michael acceptors. Clearly, anti-oxidant activity or reactivity as a Michael acceptor is not an essential feature of active compounds. In addition, a number of analogs were identified that enhanced the TPA-induced activation of AP-1. The results from screening were confirmed using BV-2 microglial cells where curcumin and analogs were shown to inhibit LPS-induced COX-2 expression; analogs identified as more potent than curcumin in the screening assay were also more potent than curcumin in preventing COX-2 expression.

Keywords: Abbreviations; AP-1; activator protein-1; TPA; 12-; O; -tetradecanoylphorbol-13-acetate; TRE; TPA-responsive elementAP-1; Curcumin; Inhibitors; Activators; Anti-oxidants

Receptor tyrosine kinase (RTK) inhibition is effective in chemosensitising EGFR-expressing drug resistant human ovarian cancer cell lines when used in combination with cytotoxic agents by Helen M. Coley; Christine F. Shotton; Abi Ajose-Adeogun; Helmout Modjtahedi; Hilary Thomas (pp. 941-948).

This study has focused on the use of RTK inhibitors in the treatment of ovarian cancer. We have used the human ovarian cancer cell line PEO1 alongside two in-house derived drug resistant variants: PEO1CarboR (8-fold acquired resistance to carboplatin and cisplatin) and the Pgp expressing PEO1TaxR (15-fold acquired resistance to paclitaxel). These variant cell lines were shown to have a higher expression of EGFR 1.6- and 2.0-fold increase, respectively, compared with the parental cell line.We have shown that the RTK inhibitor GW282974A (an analogue of GW2016; lapatinib) is effective in chemosensitisation of drug resistant EGFR over-expressing cells giving rise to a synergistic effect when used in combination with either cisplatin or paclitaxel in chemosensitivity assays. These effects were also seen at the level of apoptosis using the Annexin V assay and expression levels of the IAP Survivin. A reduction in the downstream signalling effector phosphorylated ERK was seen in both resistant cell lines when GW282974A was used in combination with either cisplatin or paclitaxel. This reduction was not so apparent in cells treated with the single agent GW282974A or cytotoxic agent. Interestingly, we did not show evidence for an enhanced sensitivity to the RTK inhibitor in our EGFR expressing resistant lines versus parental PEO1 cells. However, the paclitaxel resistant cell line appeared more sensitive to the chemosensitising effects of GW282974A, in line with its increased EGFR expression. Our data suggest that RTK inhibition is effective in circumvention of tumour cell drug resistance that occurs in conjunction with EGFR overexpression.

Keywords: Receptor tyrosine kinase inhibition; Ovarian cancer; EGFR expressing; Drug resistance

Uridine-5′-triphosphate (UTP) reduces infarct size and improves rat heart function after myocardial infarct by Smadar Yitzhaki; Asher Shainberg; Yelena Cheporko; Bernardo A. Vidne; Alex Sagie; Kenneth A. Jacobson; Edith Hochhauser (pp. 949-955).

We have previously found that uridine 5′-triphosphate (UTP) significantly reduced cardiomyocyte death induced by hypoxia via activating P2Y₂ receptors. To explore the effect of UTP following myocardial infarction (MI) in vivo we studied four groups: sham with or without LAD ligation, injected with UTP (0.44μg/kg i.v.) 30min before MI, and UTP injection (4.4μg/kg i.v.) 24h prior to MI. Left ventricular end diastolic area (LVEDA), end systolic area (LVESA) fractional shortening (FS), and changes in posterior wall (PW) thickness were performed by echocardiography before and 24h after MI. In addition, we measured different biochemical markers of damage and infarct size using Evans blue and TTC staining. The increase in LVEDA and LVESA of the treated animals was significantly smaller when compared to the MI rats ( p<0.01). Concomitantly, FS was higher in groups pretreated with UTP 30min or 24h (56±14.3 and 36.7±8.2%, p<0.01, respectively). Ratio of infarct size to area at risk was smaller in the UTP pretreated hearts than MI rats (22.9±6.6, 23.1±9.1%, versus 45.4±7.6%, respectively, p<0.001). Troponin T and ATP measurements, demonstrated reduced myocardial damage. Using Rhod-2-AM loaded cardiomyocytes, we found that UTP reduced mitochondrial calcium levels following hypoxia. In conclusion, early or late UTP preconditioning is effective, demonstrating reduced infarct size and superior myocardial function. The resulting cardioprotection following UTP treatment post ischemia demonstrates a reduction in mitochondrial calcium overload, which can explain the beneficial effect of UTP.

Keywords: Heart protection; Ischemia; P2Y receptors

Cellular pharmacokinetics and pharmacodynamics of dipyridamole in vascular smooth muscle cells by Weiwei Zhu; Takahisa Masaki; Alfred K. Cheung; Steven E. Kern (pp. 956-964).

Hemodialysis arteriovenous grafts are often plagued by stenosis at the vein-graft anastomosis, which is due to the proliferation of venous smooth muscle cells (SMCs). Perivascular delivery of dipyridamole, a potent antiproliferative agent, has been proposed for the prevention of graft stenosis. In order to develop an optimal delivery system for dipyridamole, we examined its pharmacokinetics and pharmacodynamics in human and porcine venous and arterial SMCs in vitro. SMCs were incubated with dipyridamole for various durations, and visualized for the uptake and release by fluorescence microscopy, which were further quantified by fluorospectrometry. The antiproliferative effect of dipyridamole was examined by cell counting or the methylthiazoletetrazolium (MTT) dye-reduction assay. Cytotoxicity was examined by the lactate dehydrogenase (LDH)-release assay. The kinetics of dipyridamole transport through the cell membrane was compatible with a passive diffusion mechanism. Dipyridamole inhibited SMC proliferation in a dose-dependent manner and was more effective in venous than arterial cells in both species. The inhibition was completely reversible at 15μg/ml upon drug removal from the medium. At 25μg/ml, however, the effect was partially irreversible, which might be attributed to the cytotoxicity of dipyridamole. These data support the need for sustained delivery of dipyridamole to achieve the long-term inhibition of SMC proliferation in the prevention of stenosis since SMCs are continuously stimulated at the anastomosis of hemodialysis arteriovenous grafts.

Keywords: Abbreviations; cAMP; cyclic adenosine monophosphate; cGMP; cyclic guanosine monophosphate; EC; ₅₀; 50% effective concentration; LC; ₅₀; 50% lethal concentration; LDH; lactate dehydrogenase; MTT; methylthiazoletetrazolium; PDE; phosphodiesterase; PTFE; polytetrafluoroethylene; SMC; smooth muscle cellHemodialysis arteriovenous graft stenosis; Smooth muscle cell; Dipyridamole; Antiproliferative effect; Cytotoxicity; Sustained delivery

The antiepileptic drug topiramate preserves metabolism-secretion coupling in insulin secreting cells chronically exposed to the fatty acid oleate by Francesca Frigerio; Gaelle Chaffard; Monique Berwaer; Pierre Maechler (pp. 965-973).

Topiramate (Topamax), primarily prescribed against epilepsy, was reported to reduce body weight and to ameliorate glycemic control in obese patients with diabetes. In rodent models of obesity and diabetes, topiramate treatment counteracts hyperglycemia and increases insulin levels upon glucose tolerance test. These observations suggest that topiramate might exert direct action on insulin secreting cells, in particular regarding obesity associated β-cell dysfunction. In this study, INS-1E β-cells were exposed for 3 days to the fatty acid oleate (0.4mM) and concomitantly treated with therapeutic concentrations of topiramate before measurements of insulin secretion and metabolic parameters. In healthy cells, topiramate had no acute or chronic effects on insulin release. Exposure of INS-1E cells to oleate for 3 days increased insulin release at basal 2.5mM glucose and blunted the response to stimulatory glucose concentration (15mM). Such lipotoxic effects were associated with impaired mitochondrial function, as evidenced by partial loss of resting mitochondrial membrane potential and reduced hyperpolarization in response to glucose. Oil-red-O staining and triglyceride measurements revealed lipid accumulation in oleate treated cells. Topiramate treatment counteracted oleate-induced lipid load and partially protected against mitochondrial membrane dysfunction. In particular, topiramate restored glucose stimulated insulin secretion, essentially by maintaining low insulin release at basal glucose. Topiramate increased expression of the nutrient sensor PPARα and of the mitochondrial fatty acid carrier CPT-1, correlating with enhancement of β-oxidation rate. The data demonstrate that a drug originally used as mood stabilizer exerts a direct action on β-cells, protecting against lipid-induced dysfunction.

Keywords: Abbreviations; CPT-1; carnitine palmitoyl transferase; FAT; fatty acid translocase; FCCP; carbonyl cyanide; p; -trifluoromethoxyphenylhydrazone; GSIS; glucose stimulated insulin secretion; PPARα; peroxisome proliferator-activated receptor α; TPM; topiramateEndocrine pancreas; Insulin secretion; Islet beta-cell; Obesity; Diabetes; Topiramate

Effect of salivary proteins on the transport of tannin and quercetin across intestinal epithelial cells in culture by Kuihua Cai; Anders Bennick (pp. 974-980).

Polyphenols including tannins and flavonoids are common in plant foods. While tannins may be deleterious to animals, flavonoids can have beneficial effects on the cardio-vascular system. Since salivary proteins can form complexes with polyphenols and thereby interfere with their intestinal absorption, the effect of salivary proteins on transport of tannins and flavonoids across intestinal epithelial cells (Caco-2 cells) was investigated. In presence of the salivary protein Histatin5 (Hst5) the rate of transport (d C/d t) of pentagalloyl glucose (5GG), a tannic acid, across Caco-2 cells in culture was reduced maximally 3.6-fold to 5×10⁻⁷μM/s in apical to basolateral direction. Replacing the basolateral salt solution with serum caused a 13-fold increase in d C/d t of 5GG in the absence of Hst5, but addition of Hst5 decreased d C/d t l4-fold. Transport of 5GG in basolateral to apical direction was decreased in half in the presence of Hst5. Decreases in d C/d t were closely paralleled by formation of insoluble 5GG–Hst5 complexes. In contrast, Hst5 and 1B4, a salivary proline-rich protein, had little if any effect on the transepithelial transport of the flavonoid quercetin in apical to basolateral as well as basolateral to apical direction. Taken together with previous studies [Cai K, Hagerman AE, Minto RE, Bennick A. Decreased polyphenol transport across cultured intestinal cells by a salivary proline-rich protein. Biochem Pharmacol 2006;71:1570–80] it appears that although Hst5 and 1B4 are synthesized in salivary glands they have an important biological function in the intestines as scavenger molecules preventing uptake of tannin but without notably affecting absorption of flavonoids.

Keywords: Abbreviations; PRP; proline-rich protein; Hst; histatin; DMEM; Dulbecco's modified Eagle's medium; 5GG; pentagalloyl glucose; DMSO; dimethyl sulfoxide; TEER; transepithelial electrical resistance; HBSS; Hank's balanced salt solution; HEPES; (; N; -2)hydroxyethylpiperazine-; N; ′-2-ethanesulfonic acid; d; C; /d; t; rate of transport; P; _app; apparent permeability coefficient; SGLT1; sodium-dependent glucose transporter; MRP2; multidrug resistance-associated protein 2Proline-rich protein; Histatin; Pentagalloyl glucose; Quercetin; Caco-2 cells; Intestinal absorption

Circumvention and reactivation of the p53 oncogene checkpoint in mouse colon tumors by Wataru Aizu; Glenn S. Belinsky; Christopher Flynn; Emily J. Noonan; Colleen C. Boes; Cassandra A. Godman; Bindi Doshi; Prashant R. Nambiar; Daniel W. Rosenberg; Charles Giardina (pp. 981-991).

The p53 tumor suppressor protein is sequence-normal in azoxymethane (AOM)-induced mouse colon tumors, making them a good model for human colon cancers that retain a wild type p53 gene. Cellular localization and co-immunoprecipitation experiments using a cell line derived from an AOM-induced colon tumor (AJ02-NM₀ cells) pointed to constitutively expressed Mdm2 as being an important negative regulator of p53 in these cells. Although the Mdm2 inhibitory protein p19/ARF was expressed in AJ02-NM₀ cells, its level of expression was not sufficient for p53 activation. We tested the response of AJ02-NM₀ cells to the recently developed Mdm2 inhibitor, Nutlin-3. Nutlin-3 was found to activate p53 DNA binding in AJ02-NM₀ cells, to a level comparable to doxorubicin and 5-fluorouracil (5-FU). In addition, Nutlin-3 increased expression of the p53 target genes Bax and PERP to a greater extent than doxorubicin or 5-FU, and triggered a G2/M phase arrest in these cells, compared to a G1 arrest triggered by doxorubicin and 5-FU. The differences in the cellular response may be related to differences in the kinetics of p53 activation and/or its post-translational modification status. In an ex vivo experiment, Nutlin-3 was found to activate p53 target gene expression and apoptosis in AOM-induced tumor tissue, but not in normal adjacent mucosa. Our data indicate that Mdm2 inhibitors may be an effective means of selectively targeting colon cancers that retain a sequence-normal p53 gene while sparing normal tissue and that the AOM model is an appropriate model for the preclinical development of these drugs.

Keywords: Colon cancer; p19/ARF; p53; Mdm2; Nutlin-3; Azoxymethane; Mouse model

Hypoxia-induced iNOS expression in microglia is regulated by the PI3-kinase/Akt/mTOR signaling pathway and activation of hypoxia inducible factor-1α by Dah-Yuu Lu; Houng-Chi Liou; Chih-Hsin Tang; Wen-Mei Fu (pp. 992-1000).

Exposure to hypoxia induced microglia activation and animal studies have shown that neuronal cell death is correlated with microglial activation following cerebral ischemia. Thus, it is likely that toxic inflammatory mediators produced by activated microglia under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. The hypoxia-inducible factor-1 (HIF-1) is primarily involved in the sensing and adapting of cells to changes in the O₂ level, which is regulated by many physiological functions.However, the role of HIF-1 in microglia activation under hypoxia has not yet been defined. In the current work, we investigate the signaling pathways of HIF-1α involved in the regulation of hypoxia-induced overexpression of inducible NO synthase (iNOS) in microglia. Exposure of primary rat microglial cultures as well as established microglial cell line BV-2 to hypoxia induced the expression of iNOS, indicating that hypoxia could lead to the inflammatory activation of microglia. iNOS induction was accompanied with NO production. Moreover, the molecular analysis of these events indicated that iNOS expression was regulated by the phosphatidylinositol 3-kinase (PI3-kinase)/AKT/ mammalian target of rapamycin (mTOR) signaling pathway and activation of hypoxia inducible factor-1α (HIF-1α). Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but also promote neuronal injury indirectly via microglia activation. In this study, we demonstrated that hypoxia induced iNOS expression by regulation of HIF-1α in microglia.

Keywords: Abbreviations; CNS; central nervous system; HIF-1α; hypoxia inducible factor-1alpha; IL-1; interleukin-1; iNOS; inducible nitric oxide synthase; LY294002; 2-(4-morpholinyl)-8-henyl-1(4H)-benzopyran-4-one; mTOR; mammalian target of rapamycin; NO; nitric oxide; PD980509; 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; PI3-kinase; phosphatidylinositol 3-kinase; ROS; reactive oxygen species; SB203580; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole; TNF-α; tumor necrosis factor-alphaHypoxia; Microglia; HIF-1α; iNOS; Akt; mTOR

Suppression of endotoxin-induced proinflammatory responses by citrus pectin through blocking LPS signaling pathways by Chien-Ho Chen; Ming-Thau Sheu; Tzeng-Fu Chen; Ying-Ching Wang; Wen-Chi Hou; Der-Zen Liu; Tsao-Chuen Chung; Yu-Chih Liang (pp. 1001-1009).

Pectin is composed of complex polysaccharides rich in galactoside residues, and it is most abundant in citrus fruits. Pectin and modified pectin have been found to exhibit anti-mutagenic activity and inhibit cancer metastasis and proliferation, with no evidence of toxicity or other serious side effects. In this study, we investigated the inhibitory effect of pectin at different degrees of esterification (DEs) on the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-activated macrophages. Western blot and RT-PCR analyses demonstrated that 30% esterified pectin (DE30), DE60 pectin, and DE90 pectin significantly inhibited the protein and mRNA expressions of iNOS and COX-2 in LPS-activated macrophages, and DE90 pectin was the most-potent inhibitor. To clarify the mechanisms involved, DE90 pectin was found to inhibit the phosphorylation of MAPKs and IKK kinase activity. In addition, DE90 pectin inhibited the activation of NF-κB and AP-1 by electrophoretic mobility shift assay and transient transfection experiments. Finally, we found that DE90 pectin could bind with LPS, and might result in decreased binding of LPS to its receptor. These results suggest that modulation of iNOS and COX-2 expressions by dietary pectin may be important in cancer chemoprevention and anti-inflammation.

Keywords: Citrus pectin; Inducible nitric oxide synthase (iNOS); Cyclooxygenase-2 (COX-2); Lipopolysaccharide (LPS); Mitogen-activated protein kinases (MAPKs); Nuclear factor-κB (NF-κB)

Inhibition of pro-inflammatory markers in primary bone marrow-derived mouse macrophages by naturally occurring flavonoids: Analysis of the structure–activity relationship by Mònica Comalada; Isabel Ballester; Elvira Bailón; Saleta Sierra; Jordi Xaus; Julio Gálvez; Fermín Sánchez de Medina; Antonio Zarzuelo (pp. 1010-1021).

Flavonoids possess several biological/pharmacological activities including anticancer, antimicrobial, antiviral, anti-inflammatory, immunomodulatory and antioxidant. The aim of this study was to evaluate the effect of flavonoids on macrophage physiology. For this purpose we selected some flavonoids belonging to the most common and abundant groups (flavonols—quercetin and kaempferol; flavones—diosmetin, apigenin, chrysin and luteolin; isoflavones—genistein and daidzein and flavanones—hesperetin). We decided to use primary bone marrow-derived macrophages (BMDM) as cellular model, since they represent a homogenous, non-transformed population of macrophages that can be stimulated in vitro to proliferate by macrophage colony-stimulating factor (M-CSF) or activated by LPS. In this regard, we demonstrated that most of the flavonoids assayed reduce macrophage M-CSF-induced proliferation without affecting cellular viability. Moreover, some flavonoids also inhibit TNFα production as well as iNOS expression and NO production in LPS-activated macrophages, an effect that has been associated with the inhibition of the NF-κB pathway. We also found that luteolin and quercetin are able to stimulate the expression of the anti-inflammatory cytokine IL-10 at low concentrations (<50μM). Analysis of the structure–activity relationship showed that four hydroxylations at positions 5, 7, 3′ and 4′, together with the double bond at C₂–C₃ and the position of the B ring at 2, seem to be necessary for the highest anti-inflammatory effect.

Keywords: Flavonoids; Inflammation; Macrophages; Structure–activity analysis; iNOS; TNF

Role of 5-lipoxygenase pathway in the regulation of RAW 264.7 macrophage proliferation by Diana Nieves; Juan José Moreno (pp. 1022-1030).

Arachidonic acid (AA) metabolites control cell proliferation, among other physiologic functions. RAW 264.7 macrophages can metabolise AA through the cyclooxygenase and lipoxygenase (LOX) pathways. We aimed to study the role of AA-metabolites derived from 5-LOX in the control of RAW 264.7 macrophage growth. Our results show that zileuton, a specific 5-LOX inhibitor, and nordihydroguaiaretic acid (NDGA), a non-specific LOX inhibitor, inhibit cell proliferation and [³H]-thymidine incorporation in a concentration-dependent fashion. Growth inhibition induced by NDGA can be explained by an apoptotic process, while zileuton does not seem to induce apoptosis. Moreover, these treatments delay the cell cycle, as analysed by flow cytometry. On the other hand, the leukotriene (LT) B₄ receptor antagonist U-75302, the LTD₄ receptor antagonists LY-171883 and MK-571, and the cysteinyl-LT receptor antagonist REV-5901 also inhibit cell proliferation and [³H]-thymidine incorporation in a concentration-dependent manner, and delay the RAW 264.7 cell cycle. However, these antagonists did not induce annexin V staining, caspase activation or DNA fragmentation. Furthermore, we demonstrated that exogenous addition of LTB₄ or LTD₄ revert the cell growth inhibition induced by zileuton or the leukotriene receptor antagonists mentioned above. Finally, we observed that LTB₄ and LTD₄, in the absence of growth factors, have pro-proliferative effects on macrophages, and we obtained preliminary evidences that this effect could be through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. In conclusion, our results show that the interaction between LTB₄ and LTD₄ with its respective receptor is involved in the control of RAW 264.7 macrophage growth.

Keywords: Abbreviations; AA; arachidonic acid; LOX; lipoxygenase; NDGA; nordihydroguaiaretic acid; LT; leukotriene; MAPK; mitogen-activated protein kinase; PI3K; phosphatydilinositol 3-kinase; PG; prostaglandin; COX; cyclooxygenase; HETE; hydroxyeicosatetraenoic acid; CysLT; cysteinyl leukotriene; CysLTR; cysteinyl leukotriene receptor; BLT; LTB; ₄; receptor; EGF; epidermal growth factor; DMEM; Dulbecco's modified Eagle's medium; FBS; foetal bovine serum; FACS; fluorescent-activated cell sortingLeukotriene; Leukotriene receptor; Cell growth; Cell cycle; Apoptosis

Regulation of death and survival in astrocytes by ADP activating P2Y₁ and P2Y₁₂ receptors by Liaman K. Mamedova; Zhan-Guo Gao; Kenneth A. Jacobson (pp. 1031-1041).

ADP is the endogenous agonist for both P2Y₁ and P2Y₁₂ receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5′-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y₁ receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y₁₂ receptor behaved similarly. We demonstrated here that, unlike with the G_q-coupled P2Y₁ receptor, activation of the G_i-coupled P2Y₁₂ receptor does not induce apoptosis. Furthermore, activation of the P2Y₁₂ receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor α (TNFα)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y₁₂ receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y₁ receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y₁₂ receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y₁₂ receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y₁₂ receptor activation. Apyrase did not affect the ability of TNFα to induce apoptosis in hP2Y₁₂-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y₁ and P2Y₁₂ receptors and their opposing effects on cell death pathways.

Keywords: Abbreviations; BrdUTP; 5-bromo-2′-deoxyuridine 5′-triphosphate; caspase-3; cytosolic aspartate-specific protease-3; DMEM; Dulbecco's modified Eagle's medium; Erk1/2; extracellular signal-regulated protein kinases 1 and 2; FACS; fluorescence-activated cell sorting; FBS; fetal bovine serum; HRP; horseradish peroxidase; IP; ₃; inositol trisphosphate; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; 2MeSADP; 2-methylthioadenosine 5′-diphosphate; MRS2179; N; ⁶; -methyl-2′-deoxyadenosine-3′,5′-bisphosphate; PI; propidium iodide; PI3-K; phosphatidylinositol 3-kinase; PKC; protein kinase C; PTX; pertussis toxin; TNF; tumor necrosis factor; TUNEL; Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling; U73122; 1-[6-((17b-3-Methoxyestra-1,3,5(10)-trien-17-yl)-amino)hexyl]-1Hpyrrole-2,5-dioneApoptosis; Nucleotides; G protein-coupled receptors; Tumor necrosis factor; Phospholipase C; Protein kinase C

Site-dependent contributions of P-glycoprotein and CYP3A to cyclosporin A absorption, and effect of dexamethasone in small intestine of mice by Mingji Jin; Tsutomu Shimada; Koichi Yokogawa; Masaaki Nomura; Junko Ishizaki; Yingshi Piao; Yukio Kato; Akira Tsuji; Ken-Ichi Miyamoto (pp. 1042-1050).

We examined whether the oral bioavailability of cyclosporin A is controlled primarily by P-glycoprotein (P-gp) or CYP3A in the small intestine. In situ loop method was used to evaluate the uptake of cyclosporin A (40nmol) at the upper and lower intestine of wild-type and mdr1a/1b knockout mice treated or not treated with dexamethasone (75mg/kg/day, 7 days, i.p.). Expression of CYP3A mRNA in the control group was higher in the upper than the lower intestine, while that of the multidrug resistance-1a ( mdr1a) mRNA was in the opposite order. Dexamethasone administration potently induced CYP3A and mdr1a mRNAs in the lower and upper intestine, respectively. At 45min after cyclosporin A administration into an upper intestinal loop of the control group of wild-type mice, the ratio of residual cyclosporin A to dose did not differ significantly from that of mdr1a/1b knockout mice, whereas in dexamethasone-treated wild-type mice, the residual ratio was increased significantly. The ratio of the cyclosporin A metabolite M17 to cyclosporin A in portal venous blood at an upper intestinal loop of mdr1a/1b knockout mice was much higher than that a lower intestinal loop. The M17/cyclosporin A ratio of portal venous blood at a lower intestinal loop in mdr1a/1b knockout mice was increased significantly by dexamethasone treatment. These results suggest that, under physiological conditions, the oral bioavailability of cyclosporin A is mainly controlled by CYP3A in the upper intestine, rather than liver, but when P-gp is induced by steroid, the intestinal absorption of cyclosporin A may be inhibited.

Keywords: Cyclosporin A; Bioavailability; P-glycoprotein; Dexamethasone; CYP3A; In situ; loop; mdr1a/1b; knockout mice

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Biochemical Pharmacology (v.72, #8)

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Biochemical Pharmacology (v.72, #8)