High throughput fluorescence assays for the measurement of mitochondrial activity in intact human neuroblastoma cells.

The mitochondrial permeability transition event is implicated in the activation phase of apoptosis and necrosis, and is therefore postulated to play a role in many disease states. Mitochondrial permeability transition is therefore of increasing pharmaceutical interest. Drug discovery requires the rapid screening of compound libraries to identify functionally active ligands. We report the development of two fluorescence-based approaches for screening compound libraries for effects on mitochondrial function. These assays use the fluorometric imaging plate reader in 96-well format, and two commercially available dyes: JC-1 and calcein-AM. We show here that a JC-1 assay proved highly amenable to HTS implementation. By combining this with a calcein-based assay, these approaches gave complementary information: JC-1 facilitates the discovery of modulators of mitochondrial polarization from a library of approximately 100,000 compounds screened at 8 microM, and the calcein assay identifies permeability transition pore-specific inhibitors.

Retinoic acid evoked-differentiation of neuroblastoma cells predominates over growth factor stimulation: an automated image capture and quantitation approach to neuritogenesis.

To facilitate the characterization of compounds that have positive growth factor mimetic effects on neuritogenesis, we have implemented a high-throughput functional assay which measures, in a multiparametric manner, the proliferation and differentiation characteristics of cells in a microtiter plate. Conditions were established using chronic incubation of SH-SY5Y human neuroblastoma cells with retinoic acid (RA) and/or nerve growth factor (NGF) in which discernible alterations in proliferation, growth, and differentiation of cells were induced. SH-SY5Y cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on Cellomics ArrayScanII was utilized to quantify the effects of these treatments on cell characteristics. NGF and retinoic acid were found to increase multiple parameters of SH-SY5Y differentiation, including an increased proportion of cells having neurites and increased extent of branching. However, marked differences in the effects of these compounds on SH-SY5Y growth and differentiation were also detected: whereas NGF increased cell number, RA treatment decreased cell number, and RA but not NGF caused significant elongation of neurites. This study quantifies and characterizes the effects of differentiating and proliferating agents on a human-derived neuroblastoma cell line. The high-content, rapid-throughput nature of this assay makes it ideal for functional identification and characterization of compounds regulating cell behavior.

Pharmacology of recombinant human GABA(A) receptor subtypes measured using a novel pH-based high-throughput functional efficacy assay.

To facilitate the discovery of novel compounds that modulate human GABA(A) receptor function, we have developed a high throughput functional assay using a fluorescence imaging system. L(tk-) cells expressing combinations of human GABA(A) receptor subunits were incubated with the pH-sensitive dye 2',7'bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, then washed and placed in a 96-well real-time fluorescence plate reader. In buffer adjusted to pH 6.9 there was a robust and persisting acidification response to addition of GABA, which was antagonised by the GABA(A) receptor antagonist bicuculline. The concentration-response relationship for GABA was modulated by allosteric ligands, including benzodiazepine (BZ) site agonists and inverse agonists. The effects of BZ site ligands on the pH response to GABA for receptors containing alpha1beta3gamma2, alpha3beta3gamma2 or alpha5beta3gamma2 subunits were well correlated with results from electrophysiological studies on the same receptor subunit combinations expressed in Xenopus oocytes. Most modulatory compounds tested were found to be relatively unselective across the three subunit combinations tested; however, some showed subtype-dependent efficacy, such as diazepam, which had highest agonist effects on the alpha3beta3gamma2 subtype, substantial but lesser agonism on alpha1beta3gamma2 and still substantial but the least agonism on alpha5beta3gamma2. This indicates that the alpha subunit within the recombinant receptor expressed in L(tk-) cells can affect the efficacy of the response to some BZ compounds. Inhibitors of Na(+)/Cl(-) cotransport, anion/anion exchange and the gastric type of H(+)/K(+) ATPase potently inhibited GABA-evoked acidification, indicating that multiple transporters are involved in the GABA-evoked pH change. This novel fluorescence-based high throughput functional assay allows the rapid characterization of allosteric ligands acting on human GABA(A) receptors.

Functional characterization of bradykinin analogues on recombinant human bradykinin B(1) and B(2) receptors.

We have examined the activity of a range of kinins on recombinant human bradykinin receptors, using a high throughput functional assay which measures intracellular Ca(2+) responses. The most potent agonist for Chinese hamster ovary (CHO) cells stably expressing recombinant human bradykinin B(1) receptors were Des-Arg(9)-bradykinin (EC(50)=7.9 nM) and Des-Arg(10)-kallidin (EC(50)=8.6 nM), while the most potent agonist for CHO cells expressing human bradykinin B(2) receptors was bradykinin (EC(50)=2.0 nM). These findings confirm the validity of the recombinant system and the microtitre plate imaging-based characterization system when compared to known agonist properties of the native receptors. The concentration-response relationship for bradykinin at bradykinin B(2) receptors was potently inhibited by [D-Arg(0),Hyp(3), beta-(2-thienyl)-Ala(5),D-Tic(7),Oic(8)]-bradykinin (Hoe140) (IC(50)=71 nM), which was 500-fold more potent against the B(2)-expressing cells than the B(1) cells. Bradykinin B(1) receptor-mediated responses activated by Des-Arg(10)-kallidin were fully antagonized by Des-Arg(9)-[Leu(8)]bradykinin (IC(50)=59 nM), Des-Arg(10)-Hoe140 (IC(50)=211 nM) and most potently by Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic (B9858) (IC(50)=14 nM), none of which displayed any activity against the bradykinin B(2) receptor cell line up to 3 microM. None of the antagonists displayed partial agonism activity in these cell lines. All bradykinin B(1) and B(2) receptor antagonists tested acted in an apparently non-competitive manner that is likely to be due in part to their kinetics and to the nature of the functional assay used.