Functional reconstitution of the human chemokine receptor CXCR4 with Gi/Go-proteins in Sf9 insect cells

The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) binds to the chemokine receptor CXCR4 that couples to pertussis toxin-sensitive G-proteins of the G(i)/G(o)-family. CXCR4 plays a role in the pathogenesis of autoimmune diseases, human immunodeficiency virus infection and various tumors, fetal development as well as endothelial progenitor and T-cell recruitment. To this end, most CXCR4 studies have focused on the cellular level. The aim of this study was to establish a reconstitution system for the human CXCR4 that allows for the analysis of receptor/G-protein coupling at the membrane level. We wished to study specifically constitutive CXCR4 activity and the G-protein-specificity of CXCR4. We co-expressed N- and C-terminally epitope-tagged human CXCR4 with various G(i)/G(o)-proteins and regulator of G-protein signaling (RGS)-proteins in Sf9 insect cells. Expression of CXCR4, G-proteins, and RGS-proteins was verified by immunoblotting. CXCR4 coupled more effectively to Galpha(i1) and Galpha(i2) than to Galpha(i3) and Galpha(o) and insect cell G-proteins as assessed by SDF-1alpha-stimulated high-affinity steady-state GTP hydrolysis. The RGS-proteins RGS4 and GAIP enhanced SDF-1alpha-stimulated GTP hydrolysis. SDF-1alpha stimulated [(35)S]guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) binding to Galpha(i2). RGS4 did not enhance GTPgammaS binding. Na(+) salts of halides did not reduce basal GTPase activity. The bicyclam, 1-[[1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100), acted as CXCR4 antagonist but was devoid of inverse agonistic activity. Halides reduced the maximum SDF-1alpha-stimulated GTP hydrolysis in the order of efficacy I(-) > Br(-) > Cl(-). In addition, salts reduced the potency of SDF-1alpha at activating GTP hydrolysis. From our data, we conclude the following: (1) Sf9 cells are a suitable system for expression of functionally intact human CXCR4; (2) Human CXCR4 couples effectively to Galpha(i1) and Galpha(i2); (3) There is no evidence for constitutive activity of CXCR4; (4) RGS-proteins enhance agonist-stimulated GTP hydrolysis, showing that GTP hydrolysis becomes rate-limiting in the presence of SDF-1alpha; (5) By analogy to previous observations made for the beta(2)-adrenoceptor coupled to G(s), the inhibitory effects of halides on agonist-stimulated GTP hydrolysis may be due to increased GDP-affinity of G(i)-proteins, reducing the efficacy of CXCR4 at stimulating nucleotide exchange.     

Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes

The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.Ca²⁺-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H₃ agonist immepip. Best-fit estimates were: maximum inhibition 60±10% and IC₅₀ 68±10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H₃ antagonist thioperamide in a concentration-dependent manner (EC₅₀ 23 nM, K_i 4 nM).In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) evoked by 4-AP-induced depolarisation (resting level 167±14 nM; Δ[Ca²⁺]_i 88±15 nM) was modestly, but significantly reduced (29±5% inhibition) by 300 nM immepip. The action of the H₃ agonist on depolarisation-induced changes in [Ca²⁺]_i was reversed by 100 nM thioperamide.Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels appears to account for the effect of H₃ receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.     

Application of genomics to drug design: the example of the histamine H3 receptor

The histamine H₃ receptor was characterized in the 1980s as an autoreceptor regulating histamine release in brain. Since then, selective drugs have been designed, many of them displaying a high potency in vivo, and used in many studies to delineate the implications of cerebral histaminergic systems in physiological functions such as arousal or cognitive functions. The recent cloning of the H₃ receptor, more than 15 years later, has allowed to start molecular studies that led to important findings for optimization of drug design. In agreement some ligands display distinct affinities for the recombinant rat and human H₃ receptors, a difference that we assign to two amino acids in the third transmembrane domain. In addition, H₃ autoreceptors present in the brain display high constitutive activity including in vivo. As a consequence, inverse agonists enhance histamine neuron activity and constitute a novel potential therapeutic approach to schizophrenia and Alzheimer’s disease.     

H4 receptor antagonism exhibits anti-nociceptive effects in inflammatory and neuropathic pain models in rats

The histamine H₄ receptor (H₄R) is expressed primarily on cells involved in inflammation and immune responses. To determine the potential role of H₄R in pain transmission, the effects of JNJ7777120, a potent and selective H₄ antagonist, were characterized in preclinical pain models. Administration of JNJ7777120 fully blocked neutrophil influx observed in a mouse zymosan-induced peritonitis model (ED₅₀ = 17 mg/kg s.c., 95% CI = 8.5-26) in a mast cell-dependent manner. JNJ7777120 potently reversed thermal hyperalgesia observed following intraplantar carrageenan injection of acute inflammatory pain (ED₅₀ = 22 mg/kg i.p., 95% CI = 10-35) in rats and significantly decreased the myeloperoxide activity in the carrageenan-injected paw. In contrast, no effects were produced by either H₁R antagonist diphenhydramine, H₂R antagonists ranitidine, or H₃R antagonist ABT-239. JNJ7777120 also exhibited robust anti-nociceptive activity in persistent inflammatory (CFA) pain with an ED₅₀ of 29 mg/kg i.p. (95% CI = 19-40) and effectively reversed monoiodoacetate (MIA)-induced osteoarthritic joint pain. This compound also produced dose-dependent anti-allodynic effects in the spinal nerve ligation (ED₅₀ = 60 mg/kg) and sciatic nerve constriction injury (ED₅₀ = 88 mg/kg) models of chronic neuropathic pain, as well as in a skin-incision model of acute post-operative pain (ED₅₀ = 68 mg/kg). In addition, the analgesic effects of JNJ7777120 were maintained following repeated administration and were evident at the doses that did not cause neurologic deficits in rotarod test. Our results demonstrate that selective blockade of H₄ receptors in vivo produces significant anti-nociception in animal models of inflammatory and neuropathic pain.     

Histamine H3 and dopamine D2 receptor-mediated [35S]GTPgamma[S] binding in rat striatum: evidence for additive effects but lack of interactions

The interactions in the rat striatum between H(3) receptors (H(3)Rs) and D(2) receptors (D(2)Rs) were investigated with the [(35)S]GTPgamma[S] binding assay. The H(3)R agonist (R)alpha-methylhistamine increased [(35)S]GTPgamma[S] binding to striatal membranes with an EC(50)=14+/-5 nM and a maximal effect of +19+/-1%. This effect was inhibited by the H(3)R antagonist ciproxifan with a K(i)=1.0+/-0.3 nM. The D(2)R agonist quinpirole increased [(35)S]GTPgamma[S] binding to the same membranes with an EC(50)=1.5+/-0.5 microM and a maximal effect of +28+/-2%. Its effect was blocked by haloperidol with a K(i)=0.3+/-0.1 nM. The maximal effects of the H(3)R and D(2)R agonists were additive (+46+/-3%). However, D(2)R ligands did not modify the effects of H(3)R ligands and vice versa. Ciproxifan behaved as an H(3)R inverse agonist and decreased [(35)S]GTPgamma[S] binding. Haloperidol had no effect and did not change the inverse agonist effect of ciproxifan. Administrations for 10 days of ciproxifan (1.5mg/kg/day) or haloperidol (0.5mg/kg/day) did not change the effects of quinpirole and (R)alpha-methylhistamine, respectively. These data suggest that striatal H(3)Rs and D(2)Rs do not interact through their coupling to G-proteins. However, a hyperactivity of histaminergic and dopaminergic neurons being observed in schizophrenia, the additive activations of H(3)Rs and D(2)Rs suggest that they cooperate to generate some schizophrenic symptoms. Such a postsynaptic mechanism may underlie the antipsychotic-like effects of H(3)R inverse agonists and supports their therapeutic interest, alone or as adjunctive treatment with neuroleptics.     

Characterization of the cloned guinea pig leukotriene B4 receptor: comparison to its human orthologue

A cDNA clone coding for the guinea pig leukotriene B₄ (BLT) receptor has been isolated from a lung cDNA library. The guinea pig BLT receptor has an open reading frame corresponding to 348 amino acids and shares 73% and 70% identity with human and mouse BLT receptors, respectively. Scatchard analysis of membranes prepared from guinea pig and human BLT receptor-transfected human embryonic kidney (HEK) 293 EBNA (Epstein–Bar Virus Nuclear Antigen) cells showed that both receptors displayed high affinity for leukotriene B₄ (K_d value of 0.4 nM) and were expressed at high levels (B_max values ranging from 9 to 12 pmol/mg protein). The rank order of potency for leukotrienes and related analogs in competition for [³H]leukotriene B₄ specific binding at the recombinant guinea pig BLT receptor is leukotriene B₄>20-OH-leukotriene B₄>12(R)-HETE ((5Z,8Z,10E,12(R)14Z)-12-hydroxyeicosatetraen-1-oic acid)>12(S)-HETE ((5Z,8Z,10E,12(S)14Z)-12-Hydroxyeicosatetraen-1-oic acid)>20-COOH-leukotriene B₄>U75302 (6-(6-(3-hydroxy-1E,5Z-undecadienyl)-2-pyridinyl)-1,5-hexanediol)leukotriene C₄=leukotriene D₄=leukotriene E₄. For the human receptor the rank order of 12(S)-HETE, 20-COOH-leukotriene B₄ and U75302 was reversed. Xenopus melanophore and HEK aequorin-based reporter gene assays were used to demonstrate that the guinea pig and human BLT receptors can couple to both the cAMP inhibitory and intracellular Ca²⁺ mobilization signaling pathways. However, in the case of the aequorin-expressing HEK cells (designated AEQ17-293) transfected with either the guinea pig or human BLT receptor, expression of G_α16 was required to achieve a robust Ca²⁺ driven response. Leukotriene B₄ was a potent agonist in functional assays of both the guinea pig and human BLT receptors. U-75302 a leukotriene B₄ analogue which possesses both agonistic and antagonistic properties behaved as a full agonist of the guinea pig and human BLT receptors in AEQ17-293 cells and not as an antagonist. The recombinant guinea pig BLT receptor will permit the comparison of the intrinsic potencies of leukotriene B₄ receptor antagonists used in guinea pig in vivo models of allergic and inflammatory disorders.     

Increased responsiveness to JNK1/2 mediates the enhanced H2O2-induced stimulation of Cl/HCO3 exchanger activity in immortalized renal proximal tubular epithelial cells from the SHR

We have previously demonstrated that exogenous H₂O₂ stimulates Cl⁻/HCO₃⁻ exchanger activity in immortalized renal proximal tubular epithelial (PTE) cells from both the Wistar-Kyoto (WKY) rat and the spontaneously hypertensive rat (SHR), this effect being more pronounced in SHR cells. The aim of the present study was to examine the mechanism of H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity in WKY and SHR cells. It is now reported that the SHR PTE cells were endowed with an enhanced capacity to produce H₂O₂, comparatively with WKY cells and this was accompanied by a decreased expression of SOD2, SOD3, and catalase in SHR PTE cells. The stimulatory effect of H₂O₂ on the exchanger activity was blocked by SP600125 (JNK inhibitor), but not by U0126 (MEK1/2 inhibitor) or SB203580 (p38 inhibitor) in both cell lines. Basal JNK1 and JNK2 protein expression was higher in SHR PTE cells than in WKY PTE cells. H₂O₂ had no effect on p-JNK1/2 in WKY PTE cells over time. By contrast, H₂O₂ treatment resulted in a rapid and sustained increase in JNK1/2 phosphorylation in SHR PTE cells, which was completely abolished by apocynin. Treatment of SHR PTE cells with apocynin significantly decreased the H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity. It is concluded that H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity is regulated by JNK1/2, particularly by JNK2, in SHR PTE cells. The imbalance between oxidant and antioxidant mechanisms in SHR PTE cells enhances the response of JNK1/2 to H₂O₂, which contributes to their increased sensitivity to H₂O₂.     

Characterization of [H]LUF5834: A novel non-ribose high-affinity agonist radioligand for the adenosine A1 receptor

The adenosine A₁ receptor is a promising therapeutic target for neurological disorders such as cognition deficits and is involved in cardiovascular preconditioning. Classically adenosine receptor agonists were all derivatives of adenosine, and thought to require a D-ribose moiety. More recently, however, the discovery of non-adenosine agonists for the human adenosine A₁ receptor (hA₁R) has challenged this dogma (Beukers et al., 2004). In this study we characterize the tritiated form of one of these compounds, [³H]LUF5834, as the first non-ribose partial agonist radioligand with nanomolar affinity for the hA₁R. Due to its partial agonist efficacy, [³H]LUF5834 labeled both G protein-coupled and uncoupled receptors with a similar high affinity. Using [³H]LUF5834 we performed competition binding experiments to characterize a range of A₁R ligands varying in efficacy from the full agonist CPA to the inverse agonist DPCPX. Surprisingly, in the control condition both agonists and inverse agonists displayed biphasic isotherms. With the addition of 1 mM GTP the high affinity isotherm of agonists or the low affinity isotherm of inverse agonists was lost revealing the mechanism of action of such inverse agonists at the A₁R. Consequently, [³H]LUF5834 represents a novel high affinity radioligand for the A₁R and may prove a useful tool to provide estimates of inverse agonist efficacy at this receptor.     

Generation of adenosine A3 receptor functionally humanized mice for the evaluation of the human antagonists

Although the adenosine A(3) receptor (A3AR), which is a G(i/o) protein-coupled receptor, has attracted considerable interest as a potential target for drugs against asthma or inflammation, the in vivo evaluation of the antagonists using rodents in the first step of drug development has been hampered by the lack of highly potent antagonists for the rodent A3AR. To evaluate the pharmacological effects of human A3AR antagonists in mice, we previously generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. However, the human A3AR did not lead to the phosphoinositide 3-kinase (PI3K) gamma-signaling pathway such as IgE/antigen-dependent mast cell degranulation, probably due to the uncoupling of the mouse G(i/o) protein(s). To overcome the uncoupling, we here generated A3AR functionally humanized mice by replacing the mouse A3AR gene with a human/mouse chimeric A3AR sequence in which whole intracellular regions of the human A3AR were substituted for the corresponding regions of the mouse A3AR. The chimeric A3AR led to intracellular Ca(2+) elevation and activation of the PI3Kgamma-signaling pathway, which are equivalent to the actions induced by A3AR in wild-type mice. The human A3AR antagonist had the same binding affinities for the chimeric A3AR as the human A3AR and completely antagonized this potentiation. This is the first direct evidence that the uncoupling of mouse G protein(s) to the human A3AR is due to a sequence difference in the intracellular regions of A3AR. The A3AR functionally humanized mice can be widely employed for pharmacological evaluations of the human A3AR antagonists.