Journal of Pharmacology and Experimental Therapeutics CELLULAR AND MOLECULAR

[CELLULAR AND MOLECULAR] Bortezomib-Induced Survival Signals and Genes in Human Proximal Tubular Cells

2008-11-12

Bortezomib has been introduced recently in the therapy of multiple myeloma (MM), a disease that is frequently associated with progressive renal failure. Because bortezomib-based therapy has been reported to lead to a rapid recovery of kidney function in patients with MM, we decided to study its direct effects in proximal tubular epithelial cells (PTCs) compared with glomerular mesangial cells (GMCs). After 24 h of stimulation, 50 nM bortezomib led to a 6.37-fold induction of apoptosis and markedly activated caspase-9 and -3 in GMCs but not in PTCs. In PTCs but not in GMCs, bortezomib led to a strong time-dependent degradation of IB- and to a long-lasting phosphorylation of both NF-Bp65 and extracellular signal-regulated kinase 1/2. Microarray analysis in bortezomib-treated PTCs revealed a time-dependent predominance of antiapoptotic genes compared with proapoptotic genes. Bortezomib (50 nM) induced heat shock protein (Hsp) 70 mRNA and protein levels in PTCs, whereas basal and bortezomib-stimulated Hsp70 protein expression was much weaker in GMCs. Moreover, bortezomib induced Bcl-2-associated athanogene (BAG) 3 mRNA and protein expression but inhibited BAG5 mRNA levels in PTCs. These data suggest that the reduced susceptibility of PTCs to bortezomib-induced cell apoptosis is because of cell type-specific effects of this compound on apoptosis/survival genes and pathways. The concept of bortezomib representing a blocker of both NF-B activation and cell survival should be carefully examined in particular renal cell types.

[CELLULAR AND MOLECULAR] Distinct Interactions of Human {beta}1- and {beta}2-Adrenoceptors with Isoproterenol, Epinephrine, Norepinephrine, and Dopamine

Weitl, N., Seifert, R. — 2008-11-12

Fluorescence studies with purified human β₂-adrenoceptor (β₂AR) revealed that the endogenous catecholamines, (-)-epinephrine (EPI), (-)-norepinephrine (NE), and dopamine (DOP), stabilize distinct active receptor conformations. However, the functional relevance of these ligand-specific conformations is as yet poorly understood. We addressed this question by studying fusion proteins of the β₁-adrenoceptor (β₁AR) and β₂AR with the short and long splice variants of G_s (G_s_S and G_s_L), respectively. Fusion proteins ensure efficient receptor/G-protein coupling and defined stoichiometry of the coupling partners. EPI, NE, DOP, and the prototypical synthetic βAR agonist, (-)-isoproterenol (ISO), showed marked differences in their efficacies at stabilizing the high-affinity ternary complex at β₁AR-G_s and β₂AR-G_s fusion proteins. Ternary complex formation was more sensitive to disruption by GTP with the β₂AR than with the β₁AR. Generally, in steady-state GTPase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. Exceptionally, at β₁AR-G_s_L, NE was only a partial agonist. Generally, in adenylyl cyclase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. At β₂AR-G_s_L, NE was only a partial agonist. There was no correlation between efficacy at stabilizing the ternary complex and activating GTPase, and there were also dissociations between K_i values for high-affinity agonist binding and EC₅₀ values for GTPase activation. In contrast to synthetic partial agonists, DOP did not exhibit increased efficacy at βAR-G_s_L versus βAR-G_s_S fusion proteins. In conclusion, our data with βAR-G_s fusion proteins show that endogenous catecholamines and ISO stabilize distinct conformations in the β₁AR and β₂AR.

[CELLULAR AND MOLECULAR] Identification and Characterization of Novel Small-Molecule Protease-Activated Receptor 2 Agonists

2008-11-12

We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide], each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca²⁺ mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH₂ had similar potency, whereas SLIGRL-NH₂ was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.

[CELLULAR AND MOLECULAR] S32826, A Nanomolar Inhibitor of Autotaxin: Discovery, Synthesis and Applications as a Pharmacological Tool

2008-11-12

Autotaxin catalyzes the transformation of lyso-phosphatidylcholine in lyso-phosphatidic acid (LPA). LPA is a phospholipid possessing a large panel of activity, in particular as a motility factor or as a growth signal, through its G-protein coupled seven transmembrane receptors. Indirect evidence strongly suggests that autotaxin is the main, if not the only source of circulating LPA. Because of its central role in pathologic conditions, such as oncology and diabetes/obesity, the biochemical properties of autotaxin has attracted a lot of attention, but confirmation of its role in pathology remains elusive. One way to validate and/or confirm its central role, is to find potent and selective inhibitors. A systematic screening of several thousand compounds using a colorimetric assay and taking advantage of the phosphodiesterase activity of autotaxin that requires the enzymatic site than for LPA generation, led to the discovery of a potent nanomolar inhibitor, [4-(tetradecanoylamino)benzyl]phosphonic acid (S32826). This compound was inhibitory toward the various autotaxin isoforms, using an assay measuring the [¹⁴C]lyso-phosphatidylcholine conversion into [¹⁴C]LPA. We also evaluated the activity of S32826 in cellular models of diabesity and oncology. Nevertheless, the poor in vivo stability and/or bioavailability of the compound did not permit to use it in animals. S32826 is the first reported inhibitor of autotaxin with an IC₅₀ in the nanomolar range that can be used to validate the role of autotaxin in various pathologies in cellular models.

[CELLULAR AND MOLECULAR] Evidence for an Asialoglycoprotein Receptor on Nonparenchymal Cells for O-Linked Glycoproteins

2008-10-17

B cell-activating factor receptor 3 (BR3)-Fc is an IgG1-receptor dimeric fusion protein that has multiple O-linked glycosylation sites and sialylation levels that can vary in the manufacturing process. Increased sialic acid levels resulted from increased site occupancy with the O-linked N-acetylgalactosamine (GalNAc-Gal), but because the ratio of sialic acid per mole of oligosaccharide remained approximately 1, this led to increased asialo terminal GalNAc. Previous studies have demonstrated an effect of terminal asialo Gal or GalNAc on the clearance of glycoproteins due to uptake and degradation by lectin receptors in the liver. However, the previous studies examined N-linked oligosaccharides, and there are less data regarding O-linked oligosaccharides. The objective of these studies was to determine the effects on the pharmacokinetics and distribution of the asialo terminal GalNAc and varying amounts of sialic acid residues on BR3-Fc. The results of the data presented here suggest that exposed Gal on the desialylated BR3-Fc led to rapid clearance due to uptake and degradation in the liver that was associated with nonparenchymal cells. It is interesting to note that the data indicated a decreased clearance and increased exposure of BR3-Fc as the sialic acid levels increased, even though increased sialic acid was associated with increased asialo GalNAc. Therefore, the exposed GalNAc did not seem to play a role in the clearance of BR3-Fc; although the Gal linked to the hydroxyl group at position 3 may have prevented an interaction. Because we did not see uptake of desialylated BR3-Fc in hepatocytes where the asialoglycoprotein receptor is localized, this nonparenchymal cell lectin may have preference for O-linked glycoproteins.

[CELLULAR AND MOLECULAR] G Protein Coupling and Signaling Pathway Activation by M1 Muscarinic Acetylcholine Receptor Orthosteric and Allosteric Agonists

Thomas, R. L., Mistry, R., Langmead, C. J., Wood, M. D., Challiss, R. A. J. — 2008-10-17

The M₁ muscarinic acetylcholine (mACh) receptor is among a growing number of G protein-coupled receptors that are able to activate multiple signaling cascades. AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine) is an allosteric agonist that can selectively activate the M₁ mACh receptor in the absence of an orthosteric ligand. Allosteric agonists have the potential to stabilize unique receptor conformations, which may in turn cause differential activation of signal transduction pathways. In the present study, we have investigated the signaling pathways activated by AC-42, its analog 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone), and a range of orthosteric muscarinic agonists [oxotremorine-M (oxo-M), arecoline, and pilocarpine] in Chinese hamster ovary cells recombinantly expressing the human M₁ mACh receptor. Each agonist was able to activate G_q/11-dependent signaling, as demonstrated by an increase in guanosine 5'-O-(3-thiotriphosphate) ([³⁵S]GTPS) binding to G_q/11 proteins and total [³H]inositol phosphate accumulation assays in intact cells. All three orthosteric agonists caused significant enhancements in [³⁵S]GTPS binding to G_i1/2 subunits over basal; however, neither allosteric ligand produced a significant response. In contrast, both orthosteric and allosteric agonists are able to couple to the G_s/cAMP pathway, enhancing forskolin-stimulated cAMP accumulation. These data provide support for the concept that allosteric and orthosteric mACh receptor agonists both stabilize receptor conformations associated with G_q/11- and G_s-dependent signaling; however, AC-42 and 77-LH-28-1, unlike oxo-M, arecoline, and pilocarpine, do not seem to promote M₁ mACh receptor-G_i1/2 coupling, suggesting that allosteric agonists have the potential to activate distinct subsets of downstream effectors.

[CELLULAR AND MOLECULAR] Use of Acetylcholine Mustard to Study Allosteric Interactions at the M2 Muscarinic Receptor

Suga, H., Figueroa, K. W., Ehlert, F. J. — 2008-10-17

We explored the interaction of a nitrogen mustard derivative of acetylcholine with the human M₂ muscarinic receptor expressed in Chinese hamster ovary cells using the muscarinic radioligand, [³H]N-methylscopolamine (NMS). Acetylcholine mustard caused a concentration-dependent, first-order loss of [³H]NMS binding at 37°C, with the half-maximal rate constant occurring at 24 µM and a maximal rate constant of 0.16 min^-1. We examined the effects of various ligands on the rate of alkylation of M₂ receptors by acetylcholine mustard. N-methylscopolamine and 4-(trimethylamino)-2-butynyl-(3-chlorophenyl)carbamate (McN-A-343) competitively slowed the rate of alkylation, whereas the inhibition by gallamine reached a plateau at high concentrations, indicating allosteric inhibition. In contrast, 17-β-hydroxy-17--ethynyl-5--androstano[3,2-β]-pyrimido[1,2-]benzimidazole (WIN 51708) had no effect. We also measured the inhibition of [³H]NMS binding by acetylcholine mustard at 0°C, conditions under which there is little or no detectable covalent binding. In these experiments, the dissociation constant of the aziridinium ion of acetylcholine mustard was estimated to be 12.3 µM. In contrast, the parent mustard and alcoholic hydrolysis product of acetylcholine mustard were without effect. Our results show that measurement of the effects of ligands on the rate of inactivation of the orthosteric site by a small site-directed electrophile is a powerful method for discriminating competitive inhibition from allosterism.

[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.

[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.

[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.

[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.

[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.