Genetically targeted fluorescent probes reveal dynamic calcium responses to adrenergic signaling in multiple cardiomyocyte compartments.

Calcium (Ca), an important second messenger, regulates many cellular activities and varies spatiotemporally within the cell. Conventional methods to monitor Ca changes, such as synthetic Ca indicators, are not targetable, while genetically encoded Ca indicators (GECI) can be precisely directed to cellular compartments. GECIs are chimeric proteins composed of calmodulin (or other proteins that change conformation on Ca binding) coupled with two fluorescent proteins that come closer together after an increase in [Ca], and enhance Förster resonance energy transfer (FRET) that allows for ratiometric [Ca] assessment.

Impairment of PARK14-dependent Ca(2+) signalling is a novel determinant of Parkinson's disease.

The etiology of idiopathic Parkinson's disease (idPD) remains enigmatic despite recent successes in identification of genes (PARKs) that underlie familial PD. To find new keys to this incurable neurodegenerative disorder we focused on the poorly understood PARK14 disease locus (Pla2g6 gene) and the store-operated Ca(2+) signalling pathway. Analysis of the cells from idPD patients reveals a significant deficiency in store-operated PLA2g6-dependent Ca(2+) signalling, which we can mimic in a novel B6.

Proximal events in 7,12-dimethylbenz[a]anthracene-induced, stromal cell-dependent bone marrow B cell apoptosis: stromal cell-B cell communication and apoptosis signaling.

Intercellular communication is an essential process in stimulating lymphocyte development and in activating and shaping an immune response. B cell development requires cell-to-cell contact with and cytokine production by bone marrow stromal cells. However, this intimate relationship also may be responsible for the transfer of death-inducing molecules to the B cells.

Modulation of Ca(2+) release through ryanodine receptors in vascular smooth muscle by protein kinase Calpha.

The role of protein kinase C (PKC) in Ca(2+) release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca(2+)](i) transients.

Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane, augment triglyceride formation, and protect cells from lipotoxicity.

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec-topic expression of caveolin-3 also results in enhanced FA sequestration.

Hypoxia influences CD40-CD40L mediated inflammation in endothelial and monocytic cells.

The interaction between CD40 and its ligand (CD40L) has been implicated in the pathogenesis of atherosclerosis and is recognized as a central event in the development of immuno-inflammatory processes. Our previous studies have shown that the CD40-CD40L interaction modulates platelet, neutrophil, and endothelial reactive oxygen species (ROS) generation. Hypoxia, known to be associated with tissue ischemia and inflammation, also influences the ROS production and changes the cellular redox state.

Regulation of ROS signal transduction by NADPH oxidase 4 localization.

Reactive oxygen species (ROS) function as intracellular signaling molecules in a diverse range of biological processes. However, it is unclear how freely diffusible ROS dictate specific cellular responses. In this study, we demonstrate that nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (Nox4), a major Nox isoform expressed in nonphagocytic cells, including vascular endothelium, is localized to the endoplasmic reticulum (ER).

Multifocal multiphoton microscopy (MMM) at a frame rate beyond 600 Hz.

We introduce a multiphoton microscope for high-speed three-dimensional (3D) fluorescence imaging. The system combines parallel illumination by a multifocal multiphoton microscope (MMM) with parallel detection via a segmented high-sensitivity charge-couple device (CCD) camera. The instrument consists of a Ti-sapphire laser illuminating a microlens array that projects 36 foci onto the focal plane.

Adiponectin modulates inflammatory reactions via calreticulin receptor-dependent clearance of early apoptotic bodies.

Obesity and type 2 diabetes are associated with chronic inflammation. Adiponectin is an adipocyte-derived hormone with antidiabetic and antiinflammatory actions. Here, we demonstrate what we believe to be a previously undocumented activity of adiponectin, facilitating the uptake of early apoptotic cells by macrophages, an essential feature of immune system function.

H(2)O(2): a mediator of esophagitis-induced damage to calcium-release mechanisms in cat lower esophageal sphincter.

We previously reported that induction of acute experimental esophagitis by repeated perfusion of HCl may affect release of intracellular Ca(2+) stores. We therefore measured cytosolic Ca(2+) in response to a maximally effective dose of ACh in fura 2-AM-loaded lower esophageal sphincter (LES) circular muscle cells and examined the contribution of H(2)O(2) to the reduction in Ca(2+) signal. In normal cells, the ACh-induced Ca(2+) increase was the same in normal-Ca(2+) and Ca(2+)-free medium and was abolished by the phosphatidylinositol 4,5-bisphosphate-specific phospholipase C inhibitor U-73122, confirming that the initial ACh-induced contraction depends on Ca(2+) release from intracellular stores through production of inositol trisphosphate.

Hydrogen peroxide contributes to motor dysfunction in ulcerative colitis.

Ulcerative colitis (UC) affects colonic motor function, but the mechanism responsible for this motor dysfunction is not well understood. We have shown that neurokinin A (NKA) may be an endogenous neurotransmitter mediating contraction of human sigmoid colonic circular muscle (HSCCM). To elucidate factors responsible for UC motor dysfunction, we examined the role of hydrogen peroxide (H(2)O(2)) in the decrease of NKA-induced response of HSCCM.

Quantitative assessment of tyrosine nitration of manganese superoxide dismutase in angiotensin II-infused rat kidney.

Hypertension caused by angiotensin II is characterized by an increase in tissue oxidant stress as evidenced by increased quantities of reactive oxygen and nitrogen species. Manganese superoxide dismutase (MnSOD) is a key mitochondrial antioxidant enzyme that is inactivated in conditions of oxidant stress by reacting with peroxynitrite to form 3-nitrotyrosine in its active site. The increase in 3-nitrotyrosine content in MnSOD in the kidney of angiotensin II-infused rats was assessed in this study by immunohistochemistry, Western blotting, immunoprecipitation, and HPLC with UV detection (HPLC-UV).

Antioxidant improves smooth muscle sarco/endoplasmic reticulum Ca(2+)-ATPase function and lowers tyrosine nitration in hypercholesterolemia and improves nitric oxide-induced relaxation.

Antioxidants improve endothelial function in hypercholesterolemia (HC); however, whether this includes improvement of the vascular smooth muscle response to NO is unknown. NO relaxes arteries, in part, by stimulating Ca(2+) uptake via sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) in aortic smooth muscle, and HC impairs SERCA function and the response to NO. HC induces oxidative stress, which could impair SERCA function.