Julie Kerby

Pharmacological characterization of a novel cell line expressing human α4β3δ GABAA receptors

The pharmacology of the stable cell line expressing human α4β3δ GABAA receptor was investigated using whole‐cell patch‐clamp techniques. α4β3δ receptors exhibited increased sensitivity to GABA when compared to α4β3γ2 receptors, with EC50s of 0.50 (0.46, 0.53) μM and 2.6 (2.5, 2.6) μM respectively. Additionally, the GABA partial agonists piperidine‐4‐sulphonate (P4S) and 4,5,6,7‐tetrahydroisothiazolo‐[5,4‐c]pyridin‐3‐ol (THIP) displayed markedly higher efficacy at α4β3δ receptors, indeed THIP demonstrated greater efficacy than GABA at these receptors. The δ subunit conferred slow desensitization to GABA, with rate constants of 4.8±0.5 s for α4β3δ and 2.5±0.2 s for α4β3γ2. However, both P4S and THIP demonstrated similar levels of desensitization on both receptor subtypes suggesting this effect is agonist specific. α4β3δ and α4β3γ2 demonstrated equal sensitivity to inhibition by the cation zinc (2–3 μM IC50). However, α4β3δ receptors demonstrated greater sensitivity to inhibition by lanthanum. The IC50 for GABA antagonists SR‐95531 and picrotoxin, was similar for α4β3δ and α4β3γ2. Likewise, inhibition was observed on both subtypes at high and low pH. α4β3δ receptors were insensitive to modulation by benzodiazepine ligands. In contrast Ro15‐4513 and bretazenil potentiated GABA responses on α4β3γ2 cells, and the inverse agonist DMCM showed allosteric inhibition of α4β3γ2 receptors. The efficacy of neurosteroids at α4β3δ receptors was greatly enhanced over that observed at α4β3γ2 receptors. The greatest effect was observed using THDOC with 524±71.6% potentiation at α4β3δ and 297.9±49.7% at α4β3γ2 receptors. Inhibition by the steroid pregnenolone sulphate however, showed no subtype selectivity. The efficacy of both pentobarbitone and propofol was slightly augmented and etomidate greatly enhanced at α4β3δ receptors versus α4β3γ2 receptors. We show that the α4β3δ receptor has a distinct pharmacology and kinetic profile. With its restricted distribution within the brain and unique pharmacology this receptor may play an important role in the action of neurosteroids and anaesthetics.

α4β3δ GABAAReceptors Characterized by Fluorescence Resonance Energy Transfer-derived Measurements of Membrane Potential

Selective modulators of γ-aminobutyric acid, type A (GABAA) receptors containing α4subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(−tk) cells, we stably expressed the native GABAA receptor subunit combinations α3β3γ2,α4β3γ2, and, for the first time, α4β3δ and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABAA receptor antagonists and, for α3β3γ2and α4β3γ2 receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with α4 and β3, δ subunits, compared with γ2, conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. α4β3δ responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3–7-fold more efficacious at α4β3δ compared with α4β3γ2. The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABAA receptor. For GABAA receptors containing α4 subunits, our experiments reveal that inclusion of δ instead of γ2subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of α4β3δ receptors, consistent with a central role for this subunit combination in premenstrual syndrome.

Development of an intact cell reporter gene β-lactamase assay for G protein-coupled receptors for high-throughput screening

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets. Here, we describe the development and characterization of a beta-lactamase reporter gene assay as a functional readout for the ligand-induced activation of the human bradykinin B1 receptor, expressed recombinantly in CHO cells. The beta-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous beta-lactamase activity in mammalian cells. The cell-permeable fluorogenic substrate allows single-cell cloning of cells expressing functional BK1 receptors. Pharmacological characterization reveals comparable sensitivity and potency of known BK1 receptor agonists and antagonists between the beta-lactamase assay, competition-binding assay, and other direct measurements of second messengers. The beta-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity. This CHO-hBK1-beta-lactamase assay is well suited to automation and miniaturization required for high-throughput screening.

Phase 1 clinical study of an embryonic stem cell–derived retinal pigment epithelium patch in age-related macular degeneration

Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)–derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD.

The JAK3 inhibitor WHI-P154 prevents PDGF-evoked process outgrowth in human neural precursor cells.

The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits offers a novel mechanism for treating a variety of CNS disorders. The aim of this study was to investigate pathways controlling neurite outgrowth in human neural precursor cells, in particular in response to platelet‐derived growth factor (PDGF). PDGF‐AA, ‐AB and ‐BB were found to initiate calcium signalling and produce robust increases in neurite outgrowth. PDGF‐induced outgrowth of Tuj1‐positive precursors was abolished by the addition of EGTA, suggesting that calcium entry is a critical part of the signalling pathway. Wortmannin and PD098059 failed to inhibit PDGF‐induced outgrowth. Clostridium Toxin B increased the amount of PDGF‐induced neurite branching but had no effect on basal levels. In contrast, WHI‐P154, an inhibitor of Janus protein tyrosine kinase (JAK3), Hck and Syk, prevented PDGF‐induced neurite outgrowth. PDGF activates multiple signalling pathways with considerable potential for cross‐talk. This study has highlighted the complexity of the pathways leading to neurite outgrowth in human neural precursors, and provided initial evidence to suggest that calcium entry is critical in producing the morphological changes observed.

Native and Recombinant Human Edg4 Receptor-Mediated Ca2+ Signalling

We have developed an assay system suitable for assessment of compound action on the Edg4 subtype of the widely expressed lysophosphatidic acid (LPA)-responsive Edg receptor family. Edg4 was stably overexpressed in the rat hepatoma cell line Rh 7777, and a Ca(2+)-based FLIPR assay developed for measurement of functional responses. In order to investigate the mechanisms linking Edg4 activation to cytosolic Ca(2+) elevation, we have also studied LPA signalling in a human neuroblastoma cell line that endogenously expresses Edg4. LPA responses displayed similar kinetics and potency in the two cell lines. The Ca(2+) signal generated by activation of LPA-sensitive receptors in these cells is mediated primarily by endoplasmic reticulum. However, there is a substantial inhibition of the LPA response by FCCP, indicating that mitochondria also play a key role in the LPA response. Partial inhibition of the response by cyclosporin A could indicate an active Ca(2+) release role for mitochondria in the LPA response. The inositol 1,4,5-triphosphate receptor antagonist 2-aminoethyl diphenyl borate markedly inhibits, but does not abolish, the Ca(2+) response to LPA, suggesting further complexity to the signalling pathways activated by Edg receptors. In comparing Edg signalling in recombinant and native cells, there is a striking overall similarity in receptor expression pattern, agonist potency, and the effect of modulators on the Ca(2+) response. This indicates that the Edg4-overexpressing Rh7777 cell line is a very useful model system for studying receptor pharmacology and signalling mechanisms, and for investigating the Edg4 receptors downstream effects.