Quantitative high-throughput assay to measure MC4R-induced intracellular calcium

The melanocortin-4 receptor (MC4R), a critical G-protein-coupled receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca2+]i). However, very little is known about the physiological significance of MC4R-induced [Ca2+]i since few studies measure MC4R-induced [Ca2+]i. High-throughput, read-out assays for [Ca2+]i have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca2+]i. Therefore, we developed, optimised, and validated a robust quantitative high-throughput assay using Fura-2 ratio-metric calcium dye and HEK293 cells stably transfected with MC4R. The quantitation enables direct comparisons between assays and even between different research laboratories. Assay conditions were optimised step-by-step to eliminate interference from stretch-activated receptor increases in [Ca2+]i and to maximise ligand-activated MC4R-induced [Ca2+]i. Calcium imaging was performed using a PheraStar FS multi-well plate reader. Probenecid, included in the buffers to prevent extrusion of Fura-2 dye from cells, was found to interfere with the EGTA-chelation of calcium, required to determine Rmin for quantitation of [Ca2+]i. Therefore, we developed a method to determine Rmin in specific wells without probenecid, which was run in parallel with each assay. The validation of the assay was shown by reproducible α-melanocyte-stimulating hormone (α-MSH) concentration-dependent activation of the stably expressed human MC4R (hMC4R) and mouse MC4R (mMC4R), inducing increases in [Ca2+]i, for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca2+]i in vitro has potential to advance the development of therapeutic drugs and understanding of MC4R signalling associated with human obesity.

Mechanism of a methylxanthine drug theophylline-induced Ca2+ signaling and cytotoxicity in AML12 mouse hepatocytes

Theophylline is a methylxanthine drug used in therapy for respiratory diseases. However, the impact of theophylline on Ca2+ signaling has not been explored in liver cells. This study examined whether theophylline affected Ca2+ homeostasis and its related cytotoxicity in AML12 mouse hepatocytes. Cell viability was measured by the cell viability reagent (WST-1). Cytosolic Ca2+ concentration ([Ca2+]i) was measured by the Ca2+-sensitive fluorescent dye fura-2. Theophylline (25–125 μM) induced [Ca2+]i rises and cause cytotoxicity in AML12 cells. This cytotoxic response was reversed by chelation of cytosolic Ca2+ with BAPTA/AM. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished theophylline-induced [Ca2+]i rises. Conversely, treatment with theophylline also abolished thapsigargin-induced [Ca2+]i rises. However, inhibition of PLC failed to alter theophylline-evoked [Ca2+]i rises. In Ca2+-containing medium, modulators of store-operated Ca2+ channels inhibited 30% of the [Ca2+]i rises, whereas the PKC modulators had no effect. Furthermore, theophylline-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. Together, in AML12 cells, theophylline caused Ca2+-associated cytotoxicity and induced Ca2+ entry through PLC-independent Ca2+ release from the endoplasmic reticulum and PKC-insensitive store-operated Ca2+ channels. BAPTA-AM with its protective effects may be a potential compound for prevention of theophylline-induced cytotoxicity.

Abstract 17415: Mitochondrial Ca2+/Calmodulin-Dependent Kinase II Inhibition Changes the Calcium Homeostasis in the Endothelium and Decreases the Vascular Relaxation in Mesenteric Arteries

Introduction: The Ca2+/Calmodulin-dependent Kinase II (CaMKII) is present in mitochondria and cytosol. In mitochondria, it regulates the mitochondrial Ca 2+ uptake via the mitochondrial Ca2+ uniporter. Since endothelial nitric oxide synthase activity is regulated by intracellular [Ca2+], we hypothesized that it affects cytosolic Ca2+, NO production and ACh-dependent vasodilation. Hypothesis: Inhibition of mitochondrial CaMKII in endothelium increases the cytosolic [Ca2+], and decreases vasorelaxation by Acetylcholine. Methods: CaMKII in mitochondria was inhibited through expression of the mitochondria-targeted CaMKII inhibitor peptide (mito-CaMKIIN) in a novel transgenic mouse model (endo-mtCaMKIIN) in endothelial cells only or delivered by adenoviral transduction (Ad-mtCaMKIIN) in human Aortic Endothelium cells (HAEC). In HAEC, cytosolic Ca2+ levels (by FURA-2 AM), eNOS activation and NOx levels were measured. Results: The basal Ca2+ levels were higher in the cytosol of mitoCaMKIIN cells (1.08 ± 0.02 Fura-2 ratio normalized by control, p<0.05). Thapsigargin-induced ER Ca 2+ release was significantly higher with mitoCaMKIIN (AUC 0.252 ± 0.027 versus 0.112 ± 0.01275, p<0.05), whereas cytosolic Ca 2+ levels after ACh were reduced (AUC 0.191 ± 0.025 versus 0.435 ± 0.054). Higher levels of phosphorylation of eNOS at Ser1177 and Thr495 sites were seen at baseline. The concentration-response curve of vascular relaxation to acetylcholine and SNP shifted to the right (p<0.05) in mesenteric resistance artery of mitoCaMKIIN mice. Conclusions: The inhibition of mitochondrial CaMKII in the endothelium increases the cytosolic levels, endoplasmic reticulum storage of calcium and eNOS phosphorylation. However, there are lower calcium release and lower sensitivity to acetylcholine and SNP.

Using FluoZin-3 and fura-2 to monitor acute accumulation of free intracellular Cd2+ in a pancreatic beta cell line

The understanding of cellular Cd2+ accumulation and toxicity is hampered by a lack of fluorescent indicators selective for intracellular free Cd2+ ([Cd2+]i). In this study, we used depolarized MIN6 mouse pancreatic beta cells as a model for evaluating [Cd2+]i detection with commercially available fluorescent probes, most of which have been traditionally used to visualize [Ca2+]i and [Zn2+]i. We trialed a panel of 12 probes including fura-2, FluoZin-3, Leadmium Green, Rhod-5N, indo-1, Fluo-5N, and others. We found that the [Zn2+]i probe FluoZin-3 and the traditional [Ca2+]i probe fura-2 responded most consistently and robustly to [Cd2+]i accumulation mediated by voltage-gated calcium channels. While selective detection of [Cd2+]i by fura-2 required the omission of Ca2+ from extracellular buffers, FluoZin-3 responded to [Cd2+]i similarly in the presence or absence of extracellular Ca2+. Furthermore, we showed that FluoZin-3 and fura-2 can be used together for simultaneous monitoring of [Ca2+]i and [Cd2+]i in the same cells. None of the other fluorophores tested were effective [Cd2+]i detectors in this model.