JPET current issue

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Pharmacological Profile of JNJ-27141491 [(S)-3-[3,4-Difluorophenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl Acid Methyl Ester], as a Noncompetitive and Orally Active Antagonist of the Human Chemokine Receptor CCR2

2008-09-19

The interaction between CC chemokine receptor 2 (CCR2) with monocyte chemoattractant proteins, such as MCP-1, regulates the activation and recruitment of inflammatory leukocytes. In this study, we characterized (S)-3-[3,4-difluoro-phenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl acid methyl ester (JNJ-27141491) as a noncompetitive and orally active functional antagonist of human (h)CCR2. JNJ-27141491 strongly suppressed hCCR2-mediated in vitro functions, such as MCP-1-induced guanosine 5'-O-(3-[³⁵S]thio)triphosphate binding; MCP-1, -3, and -4-induced Ca²⁺ mobilization; and leukocyte chemotaxis toward MCP-1 (IC₅₀ = 7–97 nM), whereas it had little or no effect on the function of other chemokine receptors tested. The inhibition of CCR2 function was both insurmountable and reversible, consistent with a noncompetitive mode of action. JNJ-27141491 blocked the binding of ¹²⁵I-MCP-1 to human monocytes (IC₅₀ = 0.4 µM), but it failed to affect MCP-1 binding to mouse, rat, and dog cells (IC₅₀ > 10 µM). Therefore, transgenic mice, in which the mouse (m)CCR2 gene was replaced by the human counterpart, were generated for in vivo testing. In these mice, oral administration of JNJ-27141491 dose-dependently [5–40 mg/kg q.d. (once daily) or b.i.d.] inhibited monocyte and neutrophil recruitment to the alveolar space 48 h after intratracheal mMCP-1/lipopolysaccharide instillation. Furthermore, treatment with JNJ-27141491 (20 mg/kg q.d.) significantly delayed the onset and temporarily reduced neurological signs in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Taken together, these results identify JNJ-27141491 as a noncompetitive, functional antagonist of hCCR2, capable of exerting oral anti-inflammatory activity in transgenic hCCR2-expressing mice.

[CELLULAR AND MOLECULAR] Expression of CYP4A1 in U251 Human Glioma Cell Induces Hyperproliferative Phenotype in Vitro and Rapidly Growing Tumors in Vivo

2008-09-19

Exogenous 20-hydroxyeicosatetraenoic acid (20-HETE) increases the growth of human glioma cells in vitro. However, glioma cells in culture show negligible 20-HETE synthesis. We examined whether inducing the expression of a 20-HETE synthase in a human glioma U251 cell line would increase proliferation. U251 cells transfected with CYP4A1 cDNA (termed U251 O) increased the formation of 20-HETE from less than 1 to over 60 pmol/min/mg proteins and increased their proliferation rate by 2-fold (p < 0.01). Compared with control U251, U251 O cells were rounded, smaller, showed a disorganized cytoskeleton, exhibited reduced vinculin staining, and were easily detached from the growing surface. They showed a marked increase in dihydroethidium staining, suggesting increased oxidative stress. The expression of phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1/2, and vascular endothelial growth factor was markedly elevated in U251 O. The hyperproliferative and signaling effects seen in U251 O cells are abolished by selective CYP4A inhibition of 20-HETE formation with HET0016 [N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine], by small interfering RNA against the enzyme, and by the putative 20-HETE antagonist, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid. In vivo, implantation of U251O cells in the brain of nude rats resulted in a ~10-fold larger tumor volume (10 days postimplantation) compared with animals receiving mock-transfected U251 cells. These data show that elevations in 20-HETE synthesis in U251 cells lead to an increased growth both in vitro and in vivo. This suggests that 20-HETE may have proto-oncogenic properties in U251 human gliomas. Further studies are needed to determine whether 20-HETE plays a role promoting growth of some human gliomas.

[NEUROPHARMACOLOGY] MDMA (3,4-Methylenedioxymethamphetamine)-Mediated Distortion of Somatosensory Signal Transmission and Neurotransmitter Efflux in the Ventral Posterior Medial Thalamus

Starr, M. A., Page, M. E., Waterhouse, B. D. — 2008-09-19

MDMA (3,4-methylenedioxymethamphetamine, Ecstasy) is reported to enhance tactile sensory perception, an effect that is believed to contribute to its popularity as a recreational drug. To date, no literature exists that addresses the neurophysiological mechanisms underlying the effects of MDMA on somatosensation. However, MDMA interactions with the serotonin transporter protein (SERT) are well known. The rat trigeminal somatosensory system has been studied extensively and receives serotonergic afferents from the dorsal raphe nucleus. Given that these fibers express SERT, they should be vulnerable to MDMA-induced effects. We found that short-term low-dose MDMA administration (3 mg/kg i.p.) led to a significant increase in 5-hydroxytryptamine (5-HT) efflux in the ventral posterior medial (VPM) thalamus, the main relay along the lemniscal portion of the rodent trigeminal somatosensory pathway. We further evaluated the potential for MDMA to modulate whisker-evoked discharge (WED) of individual neurons in this region. After surgically implanting stainless steel 8-wire multichannel electrode bundles, we recorded spike train activity from single cells of halothane-anesthetized rats while mechanically activating the whisker pathway. We found that short-term low-dose MDMA (3 mg/kg i.p.) increased the spontaneous firing rate but reduced the magnitude and duration of WED in individual VPM thalamic neurons. It is noteworthy that the time course of drug action on neuronal firing patterns was generally consistent with increased 5-HT efflux as shown from our microdialysis studies. Based on these results, we propose the working hypothesis that MDMA may "distort" rather than enhance tactile experiences in humans, in part, by disrupting normal spike firing patterns through somatosensory thalamic relay circuits.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Targeting of VX2 Rabbit Liver Tumor by Selective Delivery of 3-Bromopyruvate: A Biodistribution and Survival Study

2008-09-19

The aim of this study was to determine the biodistribution and tumor targeting ability of ¹⁴C-labeled 3-bromopyruvate ([¹⁴C]3-BrPA) after i.a. and i.v. delivery in the VX2 rabbit model. In addition, we evaluated the effects of [¹⁴C]3-BrPA on tumor and healthy tissue glucose metabolism by determining ¹⁸F-deoxyglucose (FDG) uptake. Last, we determined the survival benefit of i.a. administered 3-BrPA. In total, 60 rabbits with VX2 liver tumor received either 1.75 mM [¹⁴C]3-BrPA i.a., 1.75 mM [¹⁴C]3-BrPA i.v., 20 mM [¹⁴C]3-BrPA i.v., or 25 ml of phosphate-buffered saline (PBS). All rabbits (with the exception of the 20 mM i.v. group) received FDG 1 h before sacrifice. Next, we compared survival of animals treated with i.a. administered 1.75 mM [¹⁴C]3-BrPA in 25 ml of PBS (n = 22) with controls (n = 10). After i.a. infusion, tumor uptake of [¹⁴C]3-BrPA was 1.8 ± 0.2% percentage of injected dose per gram of tissue (%ID/g), whereas other tissues showed minimal uptake. After i.v. infusion (1.75 mM), tumor uptake of [¹⁴C]3-BrPA was 0.03 ± 0.01% ID/g. After i.a. administration of [¹⁴C]3-BrPA, tumor uptake of FDG was 26 times lower than in controls. After i.v. administration of [¹⁴C]3-BrPA, there was no significant difference in tumor FDG uptake. Survival analysis showed that rabbits treated with 1.75 mM 3-BrPA survived longer (55 days) than controls (18.6 days). Intra-arterially delivered 3-BrPA has a favorable biodistribution profile, combining a high tumor uptake resulting in blockage of FDG uptake with no effects on healthy tissue. The local control of the liver tumor by 3-BrPA resulted in a significant survival benefit.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Nonlinear Pharmacokinetics of ({+/-})3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") and Its Major Metabolites in Squirrel Monkeys at Plasma Concentrations of MDMA That Develop After Typical Psychoactive Doses

Mueller, M., Peters, F. T., Maurer, H. H., McCann, U. D., Ricaurte, G. A. — 2008-09-19

At certain doses, the psychoactive drug (±)3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy") destroys brain serotonin axon terminals. By causing increases in plasma MDMA concentrations that exceed those predicted by the increase in dose, nonlinear pharmacokinetics has the potential to narrow the range between safe and neurotoxic doses of MDMA. The present study sought to determine whether the pharmacokinetics of MDMA in nonhuman primates are nonlinear and, if they are, to identify plasma concentrations of MDMA at which nonlinear accumulation of MDMA occurs. Four different oral doses of MDMA were tested in the same six squirrel monkeys in random order. At each dose, pharmacokinetic parameters for MDMA and its metabolites 3,4-dihydroxymethamphetamine (HHMA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 3,4-methylenedioxyamphetamine were determined. Doses were selected to be equivalent to 0.4, 0.8, 1.6, and 2.8 mg/kg doses in humans. The maximal concentration (C_max) and area under the curve (AUC) of MDMA increased nonlinearly with dose, whereas the C_max and AUC of the metabolites HHMA and HMMA remained relatively constant. Nonlinear MDMA pharmacokinetics occurred at plasma MDMA concentrations of 100 to 300 ng/ml and above. The half-life (T_1/2) of MDMA and its metabolites also increased with dose. These results firmly establish nonlinear pharmacokinetics for MDMA in squirrel monkeys and indicate that nonlinear MDMA accumulation occurs at plasma MDMA concentrations that develop in humans taking typical doses. By raising MDMA concentrations and prolonging its action, nonlinear pharmacokinetics and T_1/2 prolongation, respectively, may influence the likelihood and severity of MDMA toxicities (including brain serotonin neurotoxicity).

[CARDIOVASCULAR] Anti-Inflammatory and Anti-Apoptotic Effects of Fumonisin B1, an Inhibitor of Ceramide Synthase, in a Rodent Model of Splanchnic Ischemia and Reperfusion Injury

2008-09-19

Ceramide is a sphingolipid with potent proinflammatory and proapoptotic properties. This study sought to determine whether pharmacological inhibition of ceramide biosynthesis in the intestine attenuates pathophysiological sequelae of shock induced by splanchnic artery occlusion and reperfusion. Ischemia and reperfusion injury was induced in anesthetized rats by clamping both the superior mesenteric artery and the celiac artery for 45 min followed by reperfusion. Within 6 min after reperfusion, animals developed significant systemic hypotension with 100% of the animals dying during the 4-h period of reperfusion. In parallel experiments, animals were necropsied after 60 min of reperfusion, and the ileum was harvested for histological examination and assessment of biochemical changes. Administration of fumonisin B1 (FB1), a competitive and reversible inhibitor of ceramide synthase (3 mg/kg, 15 min before reperfusion), significantly reduced i) the increased ceramide expression as detected by immunohistochemistry; ii) peroxynitrite-mediated protein nitration; iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils following a decrease in intercellular adhesion molecule-1 expression; iv) production of the proinflammatory cytokine tumor necrosis factor-; and v) apoptosis in the ileum. Overall, tissue-protective effects were clearly observed upon histological examination of the ileum. These beneficial events were ultimately linked to decreases in both the development of hypotension and overall mortality. These results implicate ceramide as a key signaling molecule in splanchnic arterial ischemia and reperfusion-induced shock. The broader implications of our results provide a pharmacological rationale for the development of inhibitors of ceramide biosynthesis as novel therapeutics for ischemia and reperfusion-induced shock of several etiologies.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Fispemifene [Z-2-{2-[4-(4-Chloro-1,2-diphenylbut-1-enyl)-phenoxy]ethoxy}-ethanol], a Novel Selective Estrogen Receptor Modulator, Attenuates Glandular Inflammation in an Animal Model of Chronic Nonbacterial Prostatitis

Yatkin, E., Bernoulli, J., Lammintausta, R., Santti, R. — 2008-09-19

The anti-inflammatory and antiestrogenic action of fispemifene [Z-2-{2-[4-(4-chloro-1,2-diphenylbut-1-enyl)phenoxy]ethoxy}-ethanol], a novel selective estrogen receptor modulator (SERM), was tested on the Noble rat model of chronic nonbacterial prostatic inflammation with cellular composition and inflammation patterns similar to those described in human prostatitis. Inflammation was assessed by counting perivascular and stromal infiltrates and the number of inflamed acini. Furthermore, the aggressiveness of inflammation was assessed on the basis of the relation of lymphocytes to the acinar epithelium. The immunohistochemical expression of progesterone receptor (PR) and Fos-related antigen 2 (Fra2), prolactin concentration in serum, and the weights of the seminal vesicles and pituitary glands were used as endpoints of estrogen action. Fispemifene significantly attenuated the glandular form of inflammation induced in the dorsolateral prostatic lobes (DLP) in the hormonal milieu of the decreased androgen/estrogen ratio. The anti-inflammatory action was seen in the decreased number of acini containing intraluminal neutrophils. As signs of antiestrogenic action, fispemifene blocked estrogen-induced expression of PR and Fra2 in the acinar epithelium of the DLP, and it decreased prolactin concentration in serum and the relative weights of the seminal vesicles and pituitary glands. Because fispemifene exhibited both antiestrogenic and anti-inflammatory action in the prostate, this experimental study suggests that SERMs could be considered as a new therapeutic option in the treatment and prevention of prostatic inflammation.

[CELLULAR AND MOLECULAR] The Pore Region of N-Methyl-D-aspartate Receptors Differentially Influences Stimulation and Block by Spermine

2008-09-19

The transmembrane and pore-forming regions of N-methyl-d-aspartate receptors containing the NR1 and NR2B subunits were studied by measuring the effects of various NR1 and NR2B mutants on stimulation and block by spermine. Block by spermine was predominantly affected by mutations in the M3 segment of NR1 and especially in the M1 and M3 segments of NR2B. These regions are in the outer vestibule of the channel pore and may contribute to a spermine binding site. Mutations in different regions—predominantly the M3 segment and M2 loop of NR1 and the M3 segment of NR2B—influenced spermine stimulation, a surprising finding because spermine stimulation is thought to involve a spermine binding site in the distal, extracellular regulatory domain. However, some of these mutations also influence sensitivity to ifenprodil and protons, and changes in spermine sensitivity may be secondary to changes in proton sensitivity. The results are consistent with the proposal that the relative positions of the M1 and M3 transmembrane segments and M2 loops are staggered or asymmetric in NR1 and NR2 subunits, and with the idea that stimulation and block by spermine involve separate binding sites and distinct mechanisms, although some residues in the receptor subunits can affect both stimulation and block.

[ENDOCRINE AND DIABETES] The Translational Pharmacology of a Novel, Potent, and Selective Nonsteroidal Progesterone Receptor Antagonist, 2-[4-(4-Cyano-phenoxy)-3,5-dicyclopropyl-1H-pyrazol-1-yl]-N-methylacetamide (PF-02367982)

de Giorgio-Miller, A., Bungay, P., Tutt, M., Owen, J., Goodwin, D., Pullen, N. — 2008-09-19

The progesterone receptor (PR) is an important regulator of endometrial function. Blockade of PR function has been recognized as the potential basis for preventing gynecological conditions such as endometriosis and uterine fibroids. In this study, we examine the in vitro and in vivo properties of a nonsteroidal PR antagonist, 2-[4-(4-cyano-phenoxy)-3,5-dicyclopropyl-1H-pyrazol-1-yl]-N-methylacetamide (PF-02367982) in comparison with the nonselective steroidal antagonist RU-486 (mifepristone). PF-02367982 was found to be a potent PR antagonist with far greater selectivity over the glucocorticoid receptor than RU-486. Both PF-02367982 and RU-486 blocked progesterone-induced arborization of the rabbit endometrium in a dose-dependent manner at unbound drug exposures that were commensurate with their potencies as PR antagonists in vitro. Translation of this pharmacology to a clinically relevant system was required to bridge the pharmacological gap between nonmenstruating rabbits and humans. Thus, the pharmacokinetic (PK) and pharmacodynamic (PD) data from the rabbit were combined to predict pharmacological effects on the naturally cycling cynomolgus macaque endometrium. PF-02367982 blocked the effect of progesterone on the cynomolgus macaque endometrium to the same degree as RU-486 and at exposures predicted by the rabbit PK-PD model. With such an efficacious and superior selectivity profile to the nonselective RU-486, PF-02367982 may have significant therapeutic value in the treatment of gynecological conditions such as endometriosis.

[CELLULAR AND MOLECULAR] Phenylalanine 169 in the Second Extracellular Loop of the Human Histamine H4 Receptor Is Responsible for the Difference in Agonist Binding between Human and Mouse H4 Receptors

2008-09-19

Using the natural variation in histamine H₄ receptor protein sequence, we tried to identify amino acids involved in the binding of H₄ receptor agonists. To this end, we constructed a variety of chimeric human-mouse H₄ receptor proteins to localize the domain responsible for the observed pharmacological differences between human and mouse H₄ receptors in the binding of H₄ receptor agonists, such as histamine, clozapine, and VUF 8430 [S-(2-guanidylethyl)-isothiourea]. After identification of a domain between the top of transmembrane domain 4 and the top of transmembrane domain 5 as being responsible for the differences in agonist affinity between human and mouse H₄Rs, detailed site-directed mutagenesis studies were performed. These studies identified Phe¹⁶⁹ in the second extracellular loop as the single amino acid responsible for the differences in agonist affinity between the human and mouse H₄Rs. Phe¹⁶⁹ is part of a Phe-Phe motif, which is also present in the recently crystallized β₂-adrenergic receptor. These results point to an important role of the second extracellular loop in the agonist binding to the H₄ receptor and provide a molecular explanation for the species difference between human and mouse H₄ receptors.

[CARDIOVASCULAR] Dissociation between Superoxide Accumulation and Nitroglycerin-Induced Tolerance

Tsou, P.-S., Addanki, V., Fung, H.-L. — 2008-09-19

We hypothesize that superoxide (O₂^·) accumulation is not a crucial causative factor in inducing nitroglycerin (NTG) tolerance. In LLC-PK1 cells, pre-exposure to NTG resulted in increased O₂^· accumulation and reduced cGMP response to NTG versus vehicle control. O₂^· stimulated by NTG was reduced by oxypurinol (100 µM), a xanthine oxidase inhibitor. Exposure to angiotensin II (Ang II) increased O₂^· but did not reduce cGMP response. The O₂^· scavenger tiron reduced Ang II-induced O₂^· production but did not increase NTG-stimulated cGMP production. Using p47^phox–/– and gp91^phox–/– mice versus their respective wild-type controls (WT), we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg s.c., t.i.d. for 3 days), as indicated by their ex vivo pEC₅₀ and cGMP accumulation upon NTG challenge. In vitro aorta O₂^· production was enhanced by NTG incubation in both p47^phox null and WT mice. Pre-exposure of isolated mice aorta to 100 µM NTG for 1 h resulted in vascular tolerance toward NTG and increased O₂^· accumulation. Oxypurinol (1 mM) reduced O₂^· but did not attenuate vascular tolerance. These results suggest that O₂^· does not initiate either in vitro and in vivo NTG tolerance, and that the p47^phox and gp91^phox subunits of NADPH oxidase are not critically required. Increased O₂^· accumulation may be an effect, rather than an initiating cause, of NTG tolerance.

[CARDIOVASCULAR] Sodium Hydrogen Exchange 1 (NHE-1) Regulates Connexin 43 Expression in Cardiomyocytes via Reverse Mode Sodium Calcium Exchange and c-Jun NH2-Terminal Kinase-Dependent Pathways

Stanbouly, S., Kirshenbaum, L. A., Jones, D. L., Karmazyn, M. — 2008-09-19

Connexin 43, the major connexin isoform in gap junctions of cardiac ventricular myocytes, undergoes changes in distribution and expression in cardiac diseases. The Na⁺-H⁺ exchanger (NHE-1), a key mediator of hypertrophy and heart failure, has been shown to be localized in the cardiomyocyte gap junctional regions; however, whether NHE-1 regulates gap junction proteins in the hypertrophied cardiomyocyte is not known. To address this question, neonatal rat ventricular myocytes were treated with phenylephrine (PE) for 24 h to induce hypertrophy. Increased Cx43 expression observed with PE treatment (132.4 ± 6.3% compared to control; P < 0.05) was further significantly augmented by the specific NHE-1 inhibitor EMD87580 [N-[2-methyl-4,5-bis(methylsulfonyl)-benzoyl]-guanidine hydrochloride] (173.2 ± 8.7% increase compared to control; P < 0.05 versus PE), an effect that was mimicked by another NHE-1 inhibitor cariporide [4-isopropyl-3-(methylsulfonyl)benzoyl-guanidine methanesulfonate]. PE-induced hypertrophy was associated with mitogen-activated protein kinase c-Jun NH₂-terminal kinase (JNK) 1/2 activation, whereas inhibition of JNK1/2 with either SP600125 [anthra(1,9-cd)pyrazol-6(2H)-one 1,9-pyrazoloanthrone] or small interfering RNA significantly increased PE-induced up-regulation of Cx43 protein levels. Inhibition of reverse mode Na⁺-Ca²⁺ exchange (NCX) with KB-R7943 [2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate] partially reversed JNK1/2 activation (195.2 ± 21.4 versus 143.7 ± 14.4% with KB-R7943; P < 0.05) and augmented up-regulation of Cx43 protein (121.1 ± 8.3 versus 215.9 ± 25.6% with KB-R7943; P < 0.05) in the presence of PE. Our results demonstrate that NHE-1 negatively regulates Cx43 protein expression in PE-induced cardiomyocyte hypertrophy via a JNK1/2-dependent pathway, which is probably activated by reverse mode NCX activity.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Levcromakalim and MgGDP Activate Small Conductance ATP-Sensitive K+ Channels of K+ Channel Pore 6.1/Sulfonylurea Receptor 2A in Pig Detrusor Smooth Muscle Cells: Uncoupling of cAMP Signal Pathways

Kajioka, S., Nakayama, S., Asano, H., Seki, N., Naito, S., Brading, A. F. — 2008-09-19

Pharmacological studies have suggested the existence of ATP-sensitive K⁺ (K_ATP) channel as a therapeutic target in urinary bladders; however, electrical properties have not yet been shown. Patch-clamp techniques were applied to investigate the properties of K_ATP channels in pig detrusor cells. In whole-cell configuration, levcromakalim, a K_ATP channel opener, induced a long-lasting outward current in a concentration-dependent manner. The current-voltage curve of the levcromakalim-induced membrane current intersected at approximately -80 mV. This current was abolished by glibenclamide. Intracellular application of 0.1 mM GDP significantly enhanced the levcromakalim-induced membrane current, whereas cAMP did not. Furthermore, neurotransmitters related to cAMP signaling, such as calcitonin gene-related peptide, vasointestinal peptide, adenosine, and somatostatin, had little effect on the membrane current. In cell-attached configuration, levcromakalim activated K⁺ channels with a unitary conductance of ~12 pS. When the patch configuration was changed to inside-out mode, the K⁺ channel activity ran down. Subsequent application of 1 mM GDP reactivated the channels. The openings of the ~12 pS K⁺ channels in the presence of 1 mM GDP was suppressed by ATP and glibenclamide. In reverse transcription-polymerase chain reaction, K⁺ channel pore 6.1 and sulfonylurea receptor (SUR)2A were predominant in pig detrusor cells. The 12 pS K⁺ channel activated by levcromakalim in pig detrusor smooth muscle cells is a K_ATP channel. The predominant expression of SUR2A can account for the lack of effect of neurotransmitters related to cAMP.

[NEUROPHARMACOLOGY] Paraquat Exposure Reduces Nicotinic Receptor-Evoked Dopamine Release in Monkey Striatum

2008-09-19

Paraquat, an herbicide widely used in the agricultural industry, has been associated with lung, liver, and kidney toxicity in humans. In addition, it is linked to an increased risk of Parkinson's disease. For this reason, we had previously investigated the effects of paraquat in mice and showed that it influenced striatal nicotinic receptor (nAChR) expression but not nAChR-mediated dopaminergic function. Because nonhuman primates are evolutionarily closer to humans and may better model the effects of pesticide exposure in man, we examined the effects of paraquat on striatal nAChR function and expression in monkeys. Monkeys were administered saline or paraquat once weekly for 6 weeks, after which nAChR levels and receptor-evoked [³H]dopamine ([³H]DA) release were measured in the striatum. The functional studies showed that paraquat exposure attenuated dopamine (DA) release evoked by 3/6β2^* (nAChR that is composed of the 3 or 6 subunits, and β2; the asterisk indicates the possible presence of additional subunits) nAChRs, a subtype present only on striatal dopaminergic terminals, with no decline in release mediated by 4β2^* (nAChR containing 4 and β2 subunits, but not 3 or 6) nAChRs, present on both DA terminals and striatal neurons. Paraquat treatment decreased 4β2^* but not 3/6β2^* nAChR expression. The differential effects of paraquat on nAChR expression and receptor-evoked [³H]DA release emphasize the importance of evaluating changes in functional measures. The finding that paraquat treatment has a negative impact on striatal nAChR-mediated dopaminergic activity in monkeys but not mice indicates the need for determining the effects of pesticides in higher species.

[CARDIOVASCULAR] Simvastatin Inhibits Catecholamine Secretion and Synthesis Induced by Acetylcholine via Blocking Na+ and Ca2+ Influx in Bovine Adrenal Medullary Cells

Matsuda, T., Toyohira, Y., Ueno, S., Tsutsui, M., Yanagihara, N. — 2008-09-19

Simvastatin, an inhibitor of HMG-CoA reductase, is a potent inhibitor of cholesterol biosynthesis and has beneficial effects in the primary and secondary prevention of cardiovascular diseases. In this study, we report the effects of simvastatin on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells used as a model of sympathetic neurons. Simvastatin inhibited catecholamine secretion induced by acetylcholine, an agonist of the nicotinic acetylcholine receptor; by veratridine, an activator of voltage-dependent Na⁺ channels; and by high K⁺, an activator of voltage-dependent Ca²⁺ channels (IC₅₀ = 3.8, 7.8, and 6.1 µM, respectively). Simvastatin also suppressed acetylcholine-induced ²²Na⁺ influx (IC₅₀ = 4.3 µM) and ⁴⁵Ca²⁺ influx (IC₅₀ = 6.1 µM), veratridine-induced ²²Na⁺ influx (IC₅₀ = 6.6 µM) and ⁴⁵Ca²⁺ influx (IC₅₀ = 12 µM), and high K⁺-induced ⁴⁵Ca²⁺ influx (IC₅₀ = 11 µM). The reduction of catecholamine secretion caused by simvastatin was not overcome by increasing the concentration of acetylcholine or by treatment with mevalonate, the first metabolite of HMG-CoA. The inhibitory effect of simvastatin on histamine-induced secretion of catecholamines was observed in the presence of extracellular Ca²⁺, but not in a Ca²⁺-free medium, suggesting that simvastatin does not interfere with histamine receptors nonselectively. Simvastatin also suppressed acetylcholine-induced [¹⁴C]catecholamine synthesis from [¹⁴C]tyrosine as well as tyrosine hydroxylase activity. These findings suggest that simvastatin inhibits catecholamine secretion and synthesis induced by acetylcholine through suppression of Na⁺ and Ca²⁺ influx in the adrenal medulla and probably in the sympathetic neurons.

[BEHAVIORAL PHARMACOLOGY] The Extensive Nitration of Neurofilament Light Chain in the Hippocampus Is Associated with the Cognitive Impairment Induced by Amyloid {beta} in Mice

2008-09-19

Tyrosine nitration of proteins at an extensive level is widely associated with the cognitive pathology induced by amyloid β peptide (Aβ). However, the precise identity and explicit consequences of protein nitration have scarcely been addressed. In this study, we examined the detectable nitration of proteins in the hippocampus of mice with cognitive impairment (day 5) induced by the i.c.v. injection of Aβ_25–35 (day 0). The intensity of the nitration of proteins was inversely associated with the level of recognition memory in mice. The detectable tyrosine nitrations were revealed in proteins with a single size of approximately 70 kDa. The specific nitrated proteins at this size were identified using the liquid chromatography/mass spectrometry/mass spectrometry analysis and immunodetection methods. Intense nitration of the neurofilament light chain (NFL) was observed. The increased nitration of NFL was associated with its serine hyperphosphorylation and weak interaction with the nuclear distribution element-like, a protein essential for the stable assembly of neurofilaments. No changes in cell numbers in the hippocampus were found (day 5) in mice that received Aβ_25–35 injections. These findings suggested that extensive nitration of NFL is associated with the Aβ-induced impairment of recognition memory in mice.

[CARDIOVASCULAR] Gap Junction Inhibitors Reduce Endothelium-Dependent Contractions in the Aorta of Spontaneously Hypertensive Rats

Tang, E. H. C., Vanhoutte, P. M. — 2008-09-19

Experiments were designed to determine the effect of gap junction inhibitors on endothelium-dependent contractions. Isolated aortic rings of spontaneously hypertensive rats (SHR) were suspended in vitro for isometric force recording. The nonselective gap junction inhibitor, carbenoxolone, reduced endothelium-dependent contractions to acetylcholine and the calcium ionophore A23187 [5-methylamino-2-(2S,3R,5R,8S,9S)-3,5,9-trimethyl-2-(1-oxo-(1H-pyrrol-2-yl)propan-2-yl)-1,7-dioxaspiro-(5,5)undecan-8-yl)methyl)benzooxazole-4-carboxylic acid]. There was no or modest effect of the gap peptides ⁴⁰Gap27, ^37,43Gap27, or ⁴³Gap26 when applied alone on endothelium-dependent contractions. However, the combined treatment with the three gap peptides significantly decreased endothelium-dependent contractions. The combined inhibition of the three connexins was not as effective as carbenoxolone, suggesting the involvement of other connexins in the process of endothelium-dependent contraction. The present study shows the involvement of gap junctions in endothelium-dependent contractions of the SHR aorta, presumably that of the combination of connexins 37, 40, and 43 rather than a single subtype of these proteins. Contractions of the vascular smooth muscle caused by 9,11-dideoxy-11, 9-epoxymethanoprostaglandin F₂ (U46619) and prostacyclin, but not to those of endoperoxides and phenylephrine, were reduced only minimally by carbenoxolone. Thus, if gap junction signaling is involved in the contraction of the vascular smooth muscle to thromboxane-prostanoid receptor agonists, their contribution is small. This suggests that the reduction of endothelium-dependent contractions by carbenoxolone and the gap peptides cannot be attributed to the homocellular gap junctions between vascular smooth muscle, but is more likely to involve the homocellular gap junctions between endothelial cells and/or myoendothelial gap junctions.

[NEUROPHARMACOLOGY] The Effects of Herkinorin, the First {micro}-Selective Ligand from a Salvinorin A-Derived Scaffold, in a Neuroendocrine Biomarker Assay in Nonhuman Primates

2008-09-19

Herkinorin is the first µ-opioid receptor-selective ligand from the salvinorin A diterpenoid scaffold. Herkinorin has relative µ > > binding selectivity, and it can act as an agonist at both µ- and -receptors, in vitro. These studies were the first in vivo evaluation of the effects of herkinorin in nonhuman primates, using prolactin release, a neuroendocrine biomarker assay that is responsive to both µ- and -agonists, as well as to compounds with limited ability to cross the blood-brain barrier. In cumulative dosing studies (0.01–0.32 mg/kg i.v.), herkinorin produced only small effects in gonadally intact males (n = 4), but a more robust effect in females (n = 4). Time course studies with herkinorin (0.32 mg/kg) confirmed this greater effectiveness in females and revealed a fast onset after i.v. administration (e.g., by 5–15 min). Antagonism experiments with different doses of nalmefene (0.01 and 0.1 mg/kg) caused dose-dependent and complete prevention of the effect of herkinorin in females. This is consistent with a principal µ-agonist effect of herkinorin, with likely partial contribution by -agonist effects. The peripherally selective antagonist quaternary naltrexone (1 mg/kg s.c.) caused approximately 70% reduction in the peak effect of herkinorin (0.32 mg/kg) in females, indicating that this effect of herkinorin is prominently mediated outside the blood-brain barrier.

[NEUROPHARMACOLOGY] Methylphenidate-Induced Increases in Vesicular Dopamine Sequestration and Dopamine Release in the Striatum: The Role of Muscarinic and Dopamine D2 Receptors

2008-09-19

Methylphenidate (MPD) administration alters the subcellular distribution of vesicular monoamine transporter-2 (VMAT-2)-containing vesicles in rat striatum. This report reveals previously undescribed pharmacological features of MPD by elucidating its receptor-mediated effects on VMAT-2-containing vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles) and on striatal dopamine (DA) release. MPD administration increased DA transport into, and decreased the VMAT-2 immunoreactivity of, the membrane-associated vesicle subcellular fraction. These effects were mimicked by the D2 receptor agonist quinpirole and blocked by the D2 receptor antagonist eticlopride. Both MPD and quinpirole increased vesicular DA content. However, MPD increased, whereas quinpirole decreased, K⁺-stimulated DA release from striatal suspensions. Like MPD, the muscarinic receptor agonist, oxotremorine, increased K⁺-stimulated DA release. Both eticlopride and the muscarinic receptor antagonist scopolamine blocked MPD-induced increases in K⁺-stimulated DA release, whereas the N-methyl-d-aspartate receptor antagonist (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) was without effect. This suggests that D2 receptors mediate both the MPD-induced redistribution of vesicles away from synaptosomal membranes and the MPD-induced up-regulation of vesicles remaining at the membrane. This results in a redistribution of DA within the striatum from the cytoplasm into vesicles, leading to increased DA release. However, D2 receptor activation alone is not sufficient to mediate the MPD-induced increases in striatal DA release because muscarinic receptor activation is also required. These novel findings provide insight into the mechanism of action of MPD, regulation of DA sequestration/release, and treatment of disorders affecting DA disposition, including attention-deficit hyperactivity disorder, substance abuse, and Parkinson's disease.

[CELLULAR AND MOLECULAR] 1-[1-Hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone, a Potent and Highly Selective Small Molecule Blocker of the Large-Conductance Voltage-Gated and Calcium-Dependent K+ Channel

Zeng, H., Gordon, E., Lin, Z., Lozinskaya, I. M., Willette, R. N., Xu, X. — 2008-09-19

The large-conductance voltage-gated and calcium-dependent K⁺ (BK) channels are widely distributed and play important physiological roles. Commonly used BK channel inhibitors are peptide toxins that are isolated from scorpion venoms. A high-affinity, nonpeptide, synthesized BK channel blocker with selectivity against other ion channels has not been reported. We prepared several compounds from a published patent application (Doherty et al., 2004) and identified 1-[1-hexyl-6-(methyloxy)-1H-indazol-3-yl]-2-methyl-1-propanone (HMIMP) as a potent and selective BK channel blocker. The patch-clamp technique was used for characterizing the activity of HMIMP on recombinant human BK channels ( subunit, +β1 and +β4 subunits). HMIMP blocked all of these channels with an IC₅₀ of ~2 nM. The inhibitory effect of HMIMP was not voltage-dependent, nor did it require opening of BK channels. HMIMP also potently blocked BK channels in freshly isolated detrusor smooth muscle cells and vagal neurons. HMIMP (10 nM) reduced the open probability significantly without affecting single BK-channel current in inside-out patches. HMIMP did not change the time constant of open states but increased the time constants of the closed states. More importantly, HMIMP was highly selective for the BK channel. HMIMP had no effect on human Na_V1.5 (1 µM), Ca_V3.2, L-type Ca²⁺, human ether-a-go-go-related gene potassium channel, KCNQ1+minK, transient outward K⁺ or voltage-dependent K⁺ channels (100 nM). HMIMP did not change the action potentials of ventricular myocytes, confirming its lack of effect on cardiac ion channels. In summary, HMIMP is a highly potent and selective BK channel blocker, which can serve as an important tool in the pharmacological study of the BK channel.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] Calcium Sensitization in Human Esophageal Muscle: Role for RhoA Kinase in Maintenance of Lower Esophageal Sphincter Tone

Sims, S. M., Chrones, T., Preiksaitis, H. G. — 2008-09-19

A rise in intracellular-free calcium ([Ca²⁺]_i) concentration is important for initiating contraction of smooth muscles, and Ca²⁺ sensitization involving RhoA kinase can sustain tension. We previously found that [Ca²⁺]_i was comparable in cells from the esophageal body (EB) and lower esophageal sphincter (LES) muscles, despite the fact that the LES maintains resting tone. We hypothesized that Ca²⁺ sensitization contributes to contraction in human esophageal muscle. Tension and [Ca²⁺]_i were measured simultaneously in intact human EB and LES muscles using the ratiometric Ca²⁺-sensitive dye fura-2. Spontaneous oscillations in EB muscle tension were associated with transient elevations of [Ca²⁺]_i. Carbachol caused a large increase in tension, compared with spontaneous oscillations, although the rise of [Ca²⁺]_i was similar, suggesting Ca²⁺ sensitization. The RhoA-kinase blockers (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632) and 1-(5-isoquinolinesulfonyl)-homopiperazine hydrochloride (HA-1077) reduced carbacholand nerve-evoked contraction of the EB, accompanied by smaller reduction in the rise of [Ca²⁺]_i. Protein kinase C inhibitors reduced force to a lesser extent. RhoA-kinase blockers caused concentration-dependent reduction of tension in spontaneously contracted LES muscles. Moreover, RhoA-kinase blockers reduced intrinsic nerve-evoked and carbachol-evoked contraction. However, there was no effect on nerve- or nitric oxide-mediated relaxation of LES. Ca²⁺ sensitization mediated by the RhoA-kinase pathway has an important role in contraction of human EB muscles and LES tonic contraction, a feature not previously recognized.

[NEUROPHARMACOLOGY] Alterations in Fos-Related Antigen 2 and {sigma}1 Receptor Gene and Protein Expression Are Associated with the Development of Cocaine-Induced Behavioral Sensitization: Time Course and Regional Distribution Studies

Liu, Y., Matsumoto, R. R. — 2008-09-19

Repeated exposure to cocaine results in neuroadaptations that can alter the way the brain responds to subsequent stimuli. Earlier studies demonstrated that acute administration of cocaine up-regulates the immediate-early gene fos-related antigen 2 (fra-2) followed by a later up-regulation of ₁ receptor gene and protein levels in brain regions involved in addiction and reward. To test whether such alterations could have long-term consequences on behavior, the present study was undertaken. Using a cocaine-induced behavioral sensitization model coupled with gene and protein expression studies in mice, the results show that cocaine induces the expression of fra-2, which leads to a progressive increase in ₁ receptor gene and protein expression over a period of days. This progressive increase in ₁ expression corresponds to the steady increase in the locomotor response to repeated cocaine administration in mice. The cocaine-induced changes in fra-2 and ₁ receptor gene and protein expression occur in brain regions that subserve drug abuse, such as the cortex, striatum, and hippocampus, but not the cerebellum. Moreover, the prototypic ₁ receptor antagonist 1-[2-(3,4-dichloropheny)ethyl]-4-methylpiperazine (BD1063) significantly attenuates both the molecular adaptations and behavioral sensitization induced by cocaine. These data suggest that repeated exposure to cocaine elicits alterations in fra-2 and ₁ receptor-mediated mechanisms, which ultimately manifest as altered behavioral responses to cocaine.

[NEUROPHARMACOLOGY] Purinergic Type 2 Receptors at GABAergic Synapses on Ventral Tegmental Area Dopamine Neurons Are Targets for Ethanol Action

Xiao, C., Zhou, C., Li, K., Davies, D. L., Ye, J. H. — 2008-09-19

The current study investigated whether ethanol alters ATP activation of purinergic type 2 receptors (P2Rs) in the ventral tegmental area (VTA). The VTA is a key region of the brain that has been implicated in the development of alcohol addiction. We investigated the effects of ATP and ethanol on spontaneous inhibitory postsynaptic currents (sIPSCs) and the spontaneous firings in the VTA dopaminergic neurons, obtained using an enzyme-free procedure. These neurons preserved some functional GABA-releasing terminals after isolation. We found that ATP (1–200 µM) either increased or decreased the frequency of sIPSCs and the activity of VTA dopaminergic neurons. The effects of ATP on sIPSC frequency inversely correlated with its effects on dopaminergic neuron activity. The ATP-induced changes in sIPSC frequency were blocked by tetrodotoxin (a sodium channel blocker) and by suramin (a nonselective P2R antagonist). Furthermore, ,β-methylene ATP, a selective P2X₁ and P2X₃ receptor agonist, increased sIPSC frequency, whereas adenosine 5'-[β-thio]diphosphate, a preferential agonist of P2Y receptors, decreased sIPSC frequency. In experiments testing the effects of ethanol (10 and 40 mM) on sIPSCs, we found that ethanol significantly attenuated ATP-induced increase and enhanced ATP-induced decrease in sIPSC frequency. Taken together, the results demonstrate that multiple subtypes of P2Rs exist on GABA-releasing terminals that make synapses on VTA dopaminergic neurons. It seems that ATP increases sIPSC frequency involving P2X₁ and/or P2X₃ receptors, and ATP decreases sIPSC frequency involving P2YRs. These findings are also consistent with the notion that P2Rs at GABA-releasing terminals on VTA dopaminergic neurons are important targets for ethanol action.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] 7-Epiclusianone, a Tetraprenylated Benzophenone, Relaxes Airway Smooth Muscle through Activation of the Nitric Oxide-cGMP Pathway

2008-09-19

This study was undertaken to investigate the putative mechanism(s) underlying the antispasmodic effect of 7-epiclusianone, a naturally occurring compound isolated from the plant Garcinia brasiliensis. Guinea pig tracheal rings were mounted in tissue baths filled with Krebs' solution, and the contractile response to distinct stimuli was measured in the presence or absence of 7-epiclusianone. We also tested the effect of 7-epiclusianone on methacholine-evoked airways obstruction in BALB/c mice using barometric plethysmography. 7-Epiclusianone (10 µM) inhibited epithelium-intact tracheal ring contraction induced by allergen, histamine, 5-hydroxytryptamine, or carbachol challenge. The relaxation effect was abrogated by epithelium removal, the presence of nitric-oxide synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) (100 µM), or soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM). 7-Epiclusianone (1–100 µM) induced a dose-dependent increase in the intracellular cGMP levels of cultured tracheal rings. The relaxation effect of 7-epiclusianone was also inhibited by K⁺ channel blockers tetraethylammonium (10 µM), glibenclamide (1 µM), or apamin (1 µM), but not by 9-(tetrahydro-2'-furyl)adenine (SQ22,536) (100 µM), an adenylate cyclase inhibitor. In epithelium-intact tracheal rings, 7-epiclusianone also inhibited Ca²⁺-induced contractions in K⁺ (60 mM)-depolarized preparations, but it seemed ineffective in assays in which epithelium-denuded tracheal ring preparations were used. Oral administration of 7-epiclusinone (25–100 mg/kg) dose-dependently inhibited airway obstruction triggered by aerosolized methacholine (6–25 mg/ml), in a mechanism sensitive to l-NAME (20 mg/kg). In conclusion, the relaxation effect of 7-epiclusinone seems to be mediated by epithelium-, nitric oxide-, and cGMP-dependent mechanisms. Furthermore, oral administration of 7-epiclusianone reduces episodes of bronchial obstruction, warranting further research on this compound regarding a putative application in asthma therapy.

[INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA] Thiorphan-Induced Survival and Proliferation of Rat Thymocytes by Activation of Akt/Survivin Pathway and Inhibition of Caspase-3 Activity

Amantini, C., Mosca, M., Lucciarini, R., Perfumi, M. C., Santoni, G. — 2008-09-19

The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK₁R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5⁺ thymocytes, namely on the CD4⁺CD8⁺ (double positive; DP) and CD4⁺ subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK₁R mRNA expression as well as SP and NK₁R protein levels in an NK₁R-dependent manner. Thiorphan increased CD10⁺CD4⁺ and CD10⁺DP thymocyte numbers, and an NK₁R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor chain expression, and concanavalin A-induced proliferation of CD5⁺ thymocytes, and it inhibited spontaneous and NK₁R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.

[CELLULAR AND MOLECULAR] Vasoactive Intestinal Peptide Increases Cystic Fibrosis Transmembrane Conductance Regulator Levels in the Apical Membrane of Calu-3 Cells through a Protein Kinase C-Dependent Mechanism

Chappe, F., Loewen, M. E., Hanrahan, J. W., Chappe, V. — 2008-09-19

Noncholinergic neurons contribute to innate airway defenses by releasing vasoactive intestinal peptides (VIP), which stimulates the submucosal glands to produce a bicarbonate-rich fluid containing mucins and antimicrobial factors. VIP elevates cAMP and activates cystic fibrosis transmembrane conductance regulator (CFTR) channels; however, its effects on surface expression have not been investigated. We studied CFTR levels in the apical membrane of polarized Calu-3 cell monolayers, a widely used model for submucosal gland serous cells. Biotinylation during VIP exposure revealed a significant increase in apical CFTR within 10 min, which reached a maximal 3.3-fold increase after 30 min. Total CFTR content of cell lysates was not altered during this time period; therefore, the increase in surface CFTR reflects redistribution from intracellular pools. Internalization assays revealed that apical accumulation was due, at least in part, to a reduction in the rate of CFTR endocytosis. VIP-induced accumulation of apical CFTR was mimicked by phorbol ester but not by forskolin, and it was blocked by the protein kinase (PK)C inhibitors bisindolylmaleimide X (BisX) or chelerythrine chloride but not by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89). Increases in surface expression were paralleled by enhanced iodide effluxes during cAMP stimulation. BisX inhibition of VIP responses was abrogated when monolayers were pretreated with tannic acid to inhibit endosome recycling. Thus, PKC increases the surface expression of CFTR channels in addition to potentiating their responsiveness to PKA phosphorylation. Integrated regulation through multiple signaling pathways may be a common feature of VIP and other physiological secretagogues.

[BEHAVIORAL PHARMACOLOGY] Continuous and Intermittent Nicotine Treatment Reduces L-3,4-Dihydroxyphenylalanine (L-DOPA)-Induced Dyskinesias in a Rat Model of Parkinson's Disease

Bordia, T., Campos, C., Huang, L., Quik, M. — 2008-09-19

The development of abnormal involuntary movements (AIMs) or dyskinesias is a serious complication of l-DOPA [l-3,4-dihydroxyphenylalanine] therapy for Parkinson's disease. Our previous work had shown that intermittent nicotine dosing reduced l-DOPA-induced dyskinetic-like movements in nonhuman primates. A readily available nicotine formulation is the nicotine patch, which provides a constant source of nicotine. However, constant nicotine administration more readily desensitizes nicotinic receptors, to possibly yield alternate behavioral outcomes. Therefore, we investigated whether constant nicotine administration reduced l-DOPA-induced AIMs in a rat parkinsonian model, with results compared with those with intermittent nicotine dosing. Rats with a unilateral 6-hydroxydopamine (6-OHDA) lesion were exposed to either intermittent (drinking water) or constant (minipump) nicotine for ≥2 weeks at doses that yielded plasma levels of the nicotine metabolite cotinine similar to those in smokers. The rats were next treated with l-DOPA/benserazide (8 or 12 mg/kg/15 mg/kg) for ≥3 weeks to allow for the development of AIMs, with nicotine treatment continued. Both modes of nicotine administration resulted in ≥50% decline in l-DOPA-induced AIMs. Nicotine treatment also significantly reduced AIMs in l-DOPA-primed rats using either dosing regimen, whereas nicotine removal led to an increase in AIMs. There was no effect of nicotine on various measures of motor performance in 6-OHDA-lesioned rats. In summary, nicotine provided either via the drinking water or minipump reduced l-DOPA-induced AIMs in a rat model of Parkinson's disease. These results suggest that either intermittent or constant nicotine treatment may be useful in the treatment of l-DOPA-induced dyskinesias in patients with Parkinson's disease.

[METABOLISM, TRANSPORT, AND PHARMACOGENOMICS] Apparent High CYP3A5 Expression Is Required for Significant Metabolism of Vincristine by Human Cryopreserved Hepatocytes

Dennison, J. B., Mohutsky, M. A., Barbuch, R. J., Wrighton, S. A., Hall, S. D. — 2008-09-19

Vincristine is metabolized to one primary metabolite, M1, by cDNA-expressed CYP3A4 and CYP3A5 and by CYP3A enzymes in human liver microsomes. For both systems, CYP3A5 is predicted to mediate approximately 80% of the CYP3A metabolism for individuals with high CYP3A5 expression (at least one CYP3A5^*1 allele). In the current study, the role of CYP3A5 was quantified in the metabolism of vincristine with human cryopreserved hepatocytes. The hepatocytes were genotyped for common CYP3A5 allelic variants (CYP3A5^*3, CYP3A5^*6, and CYP3A5^*7) to predict CYP3A5 expression. For each hepatocyte preparation, the rates of vincristine depletion and metabolite formation were quantified. Whereas human hepatocytes with predicted low CYP3A5 expression did not detectably metabolize vincristine, human hepatocytes with predicted high CYP3A5 expression metabolized vincristine to one primary metabolite, M1. In paired experiments using cryopreserved hepatocytes from the same donor, vincristine was incubated with intact cells and cell lysates supplemented with NADPH. The rates of M1 formation were 4 to 69-fold higher for the cell lysates compared with the intact cells. For one representative donor, the intact cells had a 3-fold higher K_m value and a 3-fold lower V_max value for M1 formation compared with the cell lysates. Thus, the rate of M1 formation in the hepatocytes may be influenced by the rate of vincristine translocation across the plasma membrane. We conclude that genetically determined CYP3A5 expression in human cryopreserved hepatocytes plays a major role in vincristine metabolism.

[GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL] E Series of Prostaglandin Receptor 2-Mediated Activation of Extracellular Signal-Regulated Kinase/Activator Protein-1 Signaling Is Required for the Mitogenic Action of Prostaglandin E2 in Esophageal Squamous-Cell Carcinoma

2008-09-19

The use of nonsteroidal anti-inflammatory drugs is associated with a lower risk for esophageal squamous cell carcinoma, in which overexpression of cyclooxygenase-2 (COX-2) is frequently reported. Prostaglandin E₂ (PGE₂), a COX-2-derived eicosanoid, is implicated in the promotion of cancer growth. However, the precise role of PGE₂ in the disease development of esophageal squamous cell carcinoma remains elusive. In this study, we investigated the effect of PGE₂ on the proliferation of cultured esophageal squamous cell carcinoma cells (HKESC-1). Results showed that HKESC-1 cells expressed all four series of prostaglandin (EP) receptors, namely, EP1 to EP4 receptors. In this regard, PGE₂ and the EP2 receptor agonist (±)-15-deoxy-16S-hydroxy-17-cyclobutyl PGE₁ methyl ester (butaprost) markedly increased HKESC-1 cell proliferation. Moreover, the mitogenic effect of PGE₂ was significantly attenuated by RNA interference-mediated knockdown of the EP2 receptor, indicating that this receptor mediated the mitogenic effect of PGE₂. In this connection, PGE₂ and butaprost induced phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), whose down-regulation by RNA interference significantly attenuated PGE₂-induced cell proliferation. In addition, PGE₂ and butaprost increased c-Fos expression and activator protein 1 (AP-1) transcriptional activity, which were abolished by the mitogen-activated protein kinase/Erk kinase inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)-butadiene ethanolate (U0126). AP-1-binding inhibitor curcumin also partially reversed the mitogenic effect of PGE₂. Taken together, these data demonstrate for the first time that the EP2 receptor mediates the mitogenic effect of PGE₂ in esophageal squamous cell carcinoma via activation of the Erk/AP-1 pathway. This study supports the growth-promoting action of PGE₂ in esophageal squamous cell carcinoma and the potential application of EP2 receptor antagonists in the treatment of this disease.

[ENDOCRINE AND DIABETES] Remogliflozin Etabonate, in a Novel Category of Selective Low-Affinity Sodium Glucose Cotransporter (SGLT2) Inhibitors, Exhibits Antidiabetic Efficacy in Rodent Models

2008-09-19

The low-affinity sodium glucose cotransporter (SGLT2) plays an important role in renal glucose reabsorption and is a remarkable transporter as a molecular target for the treatment of diabetes. We have discovered remogliflozin etabonate, which is a novel category of selective SGLT2 inhibitors. Remogliflozin etabonate is a prodrug based on benzylpyrazole glucoside and is metabolized to its active form, remogliflozin, in the body. We identified remogliflozin to be a potent and highly selective SGLT2 inhibitor by examining COS-7 cells transiently expressing either high-affinity sodium glucose cotransporter (SGLT1) or SGLT2. Orally administered remogliflozin etabonate increased urinary glucose excretion in a dose-dependent manner in both mice and rats. By increasing urinary glucose excretion, remogliflozin etabonate inhibited the increase in plasma glucose after glucose loading without stimulating insulin secretion in normal rats. Remogliflozin etabonate also showed antihyperglycemic effects in both streptozotocin-induced diabetic rats in oral glucose tolerance and in db/db mice in the fed condition. Chronic treatment with remogliflozin etabonate reduced the levels of fasting plasma glucose and glycated hemoglobin, and it ameliorated glucosuria in db/db mice. In high-fat diet-fed Goto-Kakizaki rats, remogliflozin etabonate improved hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and insulin resistance. This study demonstrates that treatment with remogliflozin etabonate exhibits antidiabetic efficacy in several rodent models and suggests that remogliflozin etabonate may be a new and useful drug for the treatment of diabetes.

[BEHAVIORAL PHARMACOLOGY] K+ Channel TASK-1 Knockout Mice Show Enhanced Sensitivities to Ataxic and Hypnotic Effects of GABAA Receptor Ligands

2008-09-19

TASK two-pore-domain leak K⁺ channels occur throughout the brain. However, TASK-1 and TASK-3 knockout (KO) mice have few neurological impairments and only mildly reduced sensitivities to inhalational anesthetics, contrasting with the anticipated functions and importance of these channels. TASK-1/-3 channel expression can compensate for the absence of GABA_A receptors in GABA_A 6 KO mice. To investigate the converse, we analyzed the behavior of TASK-1 and -3 KO mice after administering drugs with preferential efficacies at GABA_A receptor subtypes: benzodiazepines (diazepam and flurazepam, active at 1β2, 2β2, 3β2, and 5β2 subtypes), zolpidem (1β2 subtype), propofol (β2–3-containing receptors), gaboxadol (4β and 6β subtypes), pregnanolone, and pentobarbital (many subtypes). TASK-1 KO mice showed increased motor impairment in rotarod and beam-walking tests after diazepam and flurazepam administration but not after zolpidem. They also showed prolonged loss of righting reflex induced by propofol and pentobarbital. Autoradiography indicated no change in GABA_A receptor ligand binding levels. These altered behavioral responses to GABAergic drugs suggest functional up-regulation of 2β2/32 and 3β2/32 receptor subtypes in TASK-1 KO mice. In addition, female, but not male, TASK-1 KO mice were more sensitive to gaboxadol, suggesting an increased influence of 4β or 6β subtypes. The benzodiazepine sensitivity of TASK-3 KO mice was marginally increased. Our results underline that TASK-1 channels perform such key functions in the brain that compensation is needed for their absence. Furthermore, because inhalation anesthetics act partially through GABA_A receptors, the up-regulation of GABA_A receptor function in TASK-1 KO mice might mask TASK-1 channel's significance as a target for inhalation anesthetics.