Selectivity and potency of agonists for the three subtypes of cloned human beta-adrenoceptors expressed in Chinese hamster ovary cells

The selectivities, potencies and efficacies of beta3-adrenoceptor (beta3-AR) agonists on human three beta-AR subtypes expressed in Chinese hamster ovary (CHO) cells were investigated using radioligand binding assay and cyclic AMP (cAMP) accumulation assay. The three beta-AR subtypes showed the nature of G protein-coupled receptors with the constitutive activity. BRL37344, CL-316,243 and a newly synthesized beta3-AR agonist N-5984, 6-[2-(R)-[[2-(R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-2,3-dihydro-1,4-benzodioxine-2-(R)-carboxylic acid, were compared for the potency and selectivity for the beta3-AR. In the radioligand binding assay, the affinity of N-5984 for beta3-ARs was 14, 70 and 220 times more potent than those of BRL37344, isoproterenol and CL-316,243, respectively. N-5984 had higher selectivity than BRL37344 for human beta3-ARs compared with either for beta1-ARs or beta2-ARs. N-5984 showed higher potency and intrinsic activity of cAMP production than BRL37344 in CHO cells expressing the beta3-ARs. CL-316,243 had almost no activity of cAMP production in CHO cells expressing any subtype of beta-ARs. These results indicate that N-5984 is the most potent and selective agonist for human beta3-ARs than any other agonists tested.     

Antagonism by antimuscarinic and neuroleptic compounds at the five cloned human muscarinic cholinergic receptors expressed in Chinese hamster ovary cells.

We determined the affinity and selectivity of binding for 24 compounds: nine antimuscarinics (including some antiparkinson drugs) and 15 neuroleptics (including the atypical compounds clozapine, fluperlapine, melperone, rilapine, risperidone, tenilapine, tiosperone and zotepine) at the five human muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Equilibrium dissociation constants (Kd) were obtained from competitive radioligand binding studies with [3H]quinuclidinyl benzilate and membranal preparations of these cells. As expected, QNB had the highest affinity of the compounds studied at the five receptor subtypes and was not selective (Kd ranged from 0.027-0.088 nM). Benztropine had the next highest affinity of the antimuscarinic compounds and thioridazine had the highest affinity of the neuroleptics. Among the antiparkinson drugs, biperiden was the only one selective for the m1 subtype; and among the neuroleptics, the atypical drug clozapine was also selective for the m1 subtype. This selectivity may explain clozapine's unusual efficacy in refractory schizophrenic patients and its low incidence of extrapyramidal side effects. However, because most other atypical neuroleptics studied lacked high affinity and selectivity at muscarinic receptor subtypes, it is likely that other mechanisms are involved as well.     

Effect of O6-benzylguanine on nitrogen mustard-induced toxicity,apoptosis, and mutagenicity in Chinese hamster ovary cells

O6-Benzylguanine (BG) inactivates O6-alkylguanine-DNA alkyltransferase (AGT), resulting in an increase in the sensitivity of cells to the toxic effects of O6-alkylating agents. BG significantly enhances the cytotoxicity and decreases the mutagenicity of nitrogen mustards [i.e., phosphoramide mustard (PM), melphalan, and chlorambucil], a group of alkylating agents not known to produce O6-adducts in DNA. The enhancement is observed in cells irrespective of AGT activity. Exposure of Chinese hamster ovary cells to 100 microM BG results in enhancement in the cytotoxicity of PM (300 microM), chlorambucil (40 microM), and melphalan (10 microM) by 9-, 7-, and 18-fold, respectively. In contrast, mutation frequency after treatment with 300 microM PM is decreased from 259 mutants/10(6) cells to 22 mutants/10(6) cells when cells are pretreated with BG. The enhancement of toxicity of these bis-alkylating agents appears to involve cross-link formation, because neither cytotoxicity nor mutagenicity of a monoalkylating PM analogue is significantly altered when combined with BG. Enhanced cytotoxicity and decreased mutagenicity is concomitant with a dramatic increase in the number of cells undergoing apoptosis when BG is combined with PM, melphalan, or chlorambucil at 72-94 h after treatment. Cell cycle analysis demonstrates that BG alone or combined with nitrogen mustards arrests cells in G1 phase of the cell cycle. At 16 h after treatment, 11 and 57% of cells treated with PM alone or with BG plus PM are in G1 phase, respectively. Our data suggest that treatment with BG causes G1 arrest and drives noncycling cells treated with nitrogen mustards into apoptosis, thus protecting against mutagenic DNA damage introduced by nitrogen mustards.     

Non‐competitive interaction between raclopride and spiperone on human D2L‐receptors in intact Chinese hamster ovary cells

We recently investigated the binding properties of the antagonists [³H]‐raclopride and [³H]‐spiperone to intact Chinese hamster ovary cells expressing recombinant human D_2long‐dopamine receptors (CHO‐D_2L cells). Compared with saturation binding with [³H]‐raclopride, raclopride reduced [³H]‐spiperone binding with to low potency in competition binding experiments. The present findings illustrate the ability of spiperone to inhibit [³H]‐raclopride binding non‐competitively. While raclopride only decreases the apparent K_D of [³H]‐raclopride in saturation binding experiments, spiperone only decreases the number of sites to which [³H]‐raclopride binds with high affinity. Also, while the IC₅₀ of raclopride depends on the concentration of [³H]‐raclopride in competition experiments, this is not the case for spiperone. Kinetic studies reveal that the binding of raclopride at its high affinity sites does not affect the association of subsequently added [³H]‐spiperone nor the rebinding of freshly dissociated [³H]‐spiperone to the same or surrounding receptors. Yet, spiperone does not affect the dissociation rate of [³H]‐raclopride and raclopride does not affect the (genuine) dissociation rate of [³H]‐spiperone. The easiest way to interpret the present findings in molecular terms is to assume that D_2L‐receptors or their dimeric complexes possess two distinct binding sites: one with high affinity/accessibility for [³H]‐raclopride and the other one with high affinity/accessibility for [³H]‐spiperone. The ability of bound spiperone to inhibit high affinity raclopride binding while the reverse is not the case suggests for the occurrence of non‐reciprocal allosteric interactions. These new findings could point at the occurrence of allosteric interactions between different classes of D₂‐receptor antagonists.     

Comparison of the ligand binding and signaling properties of human dopamine D(2) and D(3) receptors in Chinese hamster ovary cells.

Human dopamine D(2) (hD(2)) and D(3) (hD(3)) receptors were expressed at similar, high expression levels in Chinese hamster ovary (CHO) cells, and their coupling to G proteins and further signal transduction pathways were compared. In competition radioligand-binding experiments, guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) treatment of hD(2S)- or hD(3)-CHO cell membranes induced a rightward shift and steeping of the dopamine inhibition curve. This effect was pronounced for hD(2) receptors and small for hD(3) receptors. Activation of G proteins was investigated in [(35)S]GTPgammaS-binding assays. Dopamine stimulated [(35)S]GTPgammaS binding 330 and 70% over basal levels on hD(2)-CHO and hD(3)-CHO cell membranes, respectively. (+)-7-(Dipropylamino)-5, 6,7,8-tetrahydro-2-naphthalenol and PD128907 were partial agonists for both receptors. Haloperidol, risperidone, raclopride, and nemonapride inhibited dopamine-stimulated [(35)S]GTPgammaS binding with potencies comparable to their binding affinities for hD(2) and hD(3) receptors in CHO cell membranes; inverse agonism could not be detected with this assay. Receptor stimulation by dopamine inhibited forskolin-induced cyclic AMP formation in hD(2)-CHO and hD(3)-CHO cells by 70%. Furthermore, the extracellular acidification rate increased when hD(2)-CHO and hD(3)-CHO cells were stimulated by dopamine; this effect was abolished by pertussis toxin pretreatment. In this study, we could demonstrate clear functional effects at different levels of the signaling cascade of hD(2) and hD(3) receptors in CHO cells when expressed at high levels. High-affinity agonist binding to hD(2) and hD(3) receptors was still present, but effects of receptor-G protein uncoupling at hD(3) receptors were small, indicating that hD(3) receptors maintain relatively high-affinity agonist binding in the absence of G proteins.     

A novel surface antigen expressed by a subset of human CD3- CD16+ natural killer cells. Role in cell activation and regulation of cytolytic function

The GL183 mAb was obtained by immunizing BALB/c mice with the E57 clone (CD7+CD2+CD3-CD16+CD56+) derived from human peripheral blood NK cells. In human peripheral blood, GL183-reactive cells ranged between 2 and 12% (mean 6.5%) in 10 different donors. Double fluorescence and FACS analysis showed that GL183+ cells were consistently included in the CD56+ or CD16+ cell populations. Moreover, since only a fraction of CD56+ or CD16+ cells (approximately 40%) coexpressed GL183 surface antigen, reactivity with GL183 mAb appears to define two subsets within the CD3- lymphocyte population expressing NK cell markers. Although, the majority of GL183+ cells were CD3-, approximately 1% expressed CD3 surface antigens. As shown by clonal analysis, these infrequent CD3+GL183+ cells coexpressed CD56 and CD16 antigens. Cloning of CD3-GL183+ or CD3-GL183- cell populations under limiting dilution conditions yielded clonal progenies that maintained their original surface phenotype. Therefore, expression or lack of expression of GL183 surface antigens represents a stable phenotypic property of a subset of human CD3- NK cells. Immunoprecipitation experiments and two-dimensional PAGE analysis indicated that GL183-reactive molecules were represented in different clones either by a single 58-kD chain or, more frequently, by two chains of approximately 55 and approximately 58 kD, respectively. Analysis of GL183+ or GL183- NK clones for their ability to lyse human (IGROV I) or murine (P815) tumor target cells indicated that GL183- clones were, on average, fivefold more efficient in inducing target cell lysis. GL183+ and GL183- clones produced comparable levels of TNF-alpha in response to PHA plus PMA or anti-CD16 mAb plus PMA. Importantly, production of TNF-alpha was also induced by stimulation of GL183+ clones with GL183 mAb plus PMA. These data indicated that GL183 antigen could mediate cell triggering. This concept was confirmed by the analysis of Ca2+ mobilization, as GL183 mAb induced (in GL183+ clones) increments of [Ca2+]i comparable with those induced by PHA. Moreover, GL183 mAb, or its F(ab')2 fragments, strongly enhanced the cytolytic activity of GL183+ clones against a panel of human tumor target cells, including U937, Raji, IGROV I, M14, and A549. In contrast, GL183 mAb, but not the F(ab')2 fragments, sharply inhibited the cytolytic activity of the same clones against P815, M12, and P3U1 murine target cells. In this case, the effect of GL183 mAb (inhibition) was opposite that of PHA or of stimulatory anti-CD2 or anti-CD16 mAbs, which consistently enhanced the target cell lysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Overexpression of the partially activated αIIbβ3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet–like extensions in Chinese hamster ovary cells

Summary. Background: We have recently reported a novel mutation in the β₃ subunit of the platelet fibrinogen receptor (α_IIbβ₃D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen‐dependent, microtubule‐driven proplatelet‐like cell extensions. Results: Here we demonstrate that the partially activated α_IIbβ₃D723H or α_IIbβ₃D723A salt bridge mutants, but not fully activated α_IIbβ₃ mutants, cause this phenotype. Time‐lapse videomicroscopy clearly differentiated these stable microtubule‐driven and nocodazole‐sensitive extensions from common dynamic actin‐driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of α_IIbβ₃, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34⁺‐derived megakaryocyte proplatelets. Mutant α_IIbβ₃D723H signaling was independent of Src, protein kinase C or phosphoinositide 3‐kinase, but correlated with decreased RhoA activity as compared with wild‐type α_IIbβ₃ signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO α_IIbβ₃D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO α_IIbβ₃D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin‐dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule‐driven formation of cytoplasmic extensions.     

Determination of [35S]guanosine-5'-O-(3-thio)triphosphate binding mediated by cholinergic muscarinic receptors in membranes from Chinese hamster ovary cells and rat striatum using an anti-G protein scintillation proximity assay

An assay for measuring agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate (GTPgamma35S) binding to heterotrimeric GTP binding proteins was developed for use in 96-well format using commercially available anti-G protein antibodies captured by anti-IgG-coated scintillation proximity assay beads. Use of an anti-Galphaq/11 antibody to measure GTPgamma35S binding mediated by M1, M3, and M5 receptors stably expressed in Chinese hamster ovary (CHO) cells resulted in a marked increase in agonist-stimulated/basal binding ratio compared with whole membrane binding. Pertussis toxin (PTX) treatment of CHO M1 cells before membrane preparation resulted in a marked reduction in agonist-stimulated GTPgamma35S binding to whole membranes. Direct coupling of M1 receptors in CHO cells to inhibitory G proteins was demonstrated using an anti-Galphai(1-3) antibody, and this binding was inhibited by 76% following PTX treatment. However, PTX had no effect on M1-mediated binding determined using anti-Galphaq/11. CHO M2 receptors mediated robust agonist-stimulated GTPgamma35S binding measured with anti-Galphai(1-3), but coupled only weakly to Galphaq/11. Using membranes from rat striatum, GTPgamma35S binding stimulated by oxotremorine M was demonstrated using anti-Galphaq/11, anti-Galphai(1-3), and anti-Galphao antibodies. Agonist-stimulated binding to striatal membranes showed a marked antibody-dependent GDP requirement with robust signals obtained using 0.1 microM GDP for anti-Galphaq/11 compared with 50 microM GDP for anti-Galphai(1-3) and anti-Galphao. The potencies observed for pirenzepine and AFDX 116 blockade of agonist-stimulated GTPgamma35S binding to striatal membranes determined with anti-Galphaq/11 and anti-Galphao suggested mediation of these responses primarily by M1 and M4 receptors, respectively. Antibody capture GTPgamma35S binding using scintillation proximity assay technology provides a convenient, productive alternative to immunoprecipitation for exploration of receptor-G protein interaction in cells and tissues.