CRACing the cluster: functionally active Orai1 channels in the absence of clustering with STIM1

CRACing the cluster: functionally active Orai1 channels in the absence of clustering with STIM

Temperature-Controlled Reversible Localized Surface Plasmon Resonance Response of Polymer-Functionalized Gold Nanoprisms in the Solid State

Solid-state temperature responsive localized surface plasmon resonance (LSPR)-based nanosensors were constructed by functionalizing the glass substrate-attached gold nanoprisms with the thermoresponsive polymer poly­(allylamine hydrochloride)-<i>co</i>-poly­(<i>N</i>-isopropylacrylamide). The robustness of the sensor was enhanced by chemically attaching polymer to the nanoprism surface through an amide coupling reaction versus simple physisorption of polymer onto nanoprism. The highest sensitivity of this kind of solid sensing platform was obtained by employing chemically synthesized gold nanoprisms for fabrication. The surface ligand chemistry significantly influenced the swelling and shrinking transition of the polymer during the temperature variation, which resulted in the alteration of the local dielectric environment of the nanoprisms, modulation of their LSPR properties, and enhancement of sensing efficiency of the nanosensors. Importantly, we have shown for the first time that the dimension of the nanostructure plays an important role in achieving the highest sensitivity for these types of sensors. For example, the edge length of the nanoprisms plays a critical role in the temperature sensitivity of the nanosensors where nanoprisms with ∼28 and ∼40 nm edge lengths displayed ∼10.9 and ∼18.2 nm LSPR dipole peak red-shift, respectively, as the solution temperature increased from 18 to 56 °C. We believe the higher temperature sensitivity for larger edge-length nanoprisms was achieved due to their larger sensing volume. The nanosensors were found to be very stable and displayed high reversibility, which suggests that our temperature-dependent nanosensors can potentially be used as a reversible thermal switch

Pathogenic Role of Store-Operated and Receptor-Operated Ca2+ Channels in Pulmonary Arterial Hypertension

Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling, in situ thrombosis, and increased pulmonary vascular stiffness are the major causes for the elevated pulmonary vascular resistance (PVR) in patients with PAH. The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca2+ signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca2+]cyt in PASMC is increased by Ca2+ influx through Ca2+ channels in the plasma membrane and by Ca2+ release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca2+ entry pathways, voltage-dependent Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) and voltage-independent Ca2+ influx through store-operated Ca2+ channels (SOC) and receptor-operated Ca2+ channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH

Ultrasensitive Photoreversible Molecular Sensors of Azobenzene-Functionalized Plasmonic Nanoantennas

This Letter describes an unprecedentedly large and photoreversible localized surface plasmon resonance (LSPR) wavelength shift caused by photoisomerization of azobenzenes attached to gold nanoprisms that act as nanoantennas. The blue light-induced cis to trans azobenzene conformational change occurs in the solid state and controls the optical properties of the nanoprisms shifting their LSPR peak up to 21 nm toward longer wavelengths. This shift is consistent with the increase in thickness of the local dielectric environment (0.6 nm) surrounding the nanoprism and perhaps a contribution from plasmonic energy transfer between the nanoprism and azobenzenes. The effects of the azobenzene conformational change and its photoreversibility were also probed through surface-enhanced Raman spectroscopy (SERS) showing that the electronic interaction between the nanoprisms and bound azobenzenes in their cis conformation significantly enhances the intensity of the Raman bands of the azobenzenes. The SERS data suggests that the isomerization is controlled by first-order kinetics with a rate constant of 1.0 × 10^–4 s^–1. Our demonstration of light-induced photoreversibility of this type of molecular machine is the first-step toward removing present limitations on detection of molecular motion in solid-state devices using LSPR spectroscopy with nanoprisms. Modulating the LSPR peak position and controlling energy transfer across the nanostructure–organic molecule interface are very important for the fabrication of plasmonic-based nanoscale devices

Enhanced Ca2+-sensing Receptor Function in Idiopathic Pulmonary Arterial Hypertension

Rationale: A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca2+]cyt and enhanced Ca2+ influx have been implicated in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH). Objective: We examined whether the extracellular Ca2+-sensing receptor (CaSR) is involved in the enhanced Ca2+ influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension. Methods and Results: In normal PASMC superfused with Ca2+-free solution, addition of 2.2 mM Ca2+ to the perfusate had little effect on [Ca2+]cyt. In IPAH-PASMC, however, restoration of extracellular Ca2+ induced a significant increase in [Ca2+]cyt. Extracellular application of spermine also markedly raised [Ca2+]cyt in IPAH-PASMC, but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143 attenuated, the extracellular Ca2+-induced [Ca2+]cyt rise in IPAH-PASMC. Furthermore, the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNA attenuated the extracellular Ca2+-mediated [Ca2+]cyt increase and inhibited IPAH-PASMC proliferation. Using animal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhanced in PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia. Conclusions: The extracellular Ca2+-induced increase in [Ca2+]cyt due to upregulated CaSR is a novel pathogenic mechanism contributing to the augmented Ca2+ influx and excessive PASMC proliferation in patients and animals with pulmonary arterial hypertension

Inhibition of LOX downregulates PCNA (a marker for cell proliferation) and Bcl–2 (an anti-apoptotic protein) in human PASMC during hypoxia.

<p>A: Western blot analysis on PCNA and Bcl–2 in hypoxic PASMC treated with (+) or without (-) the irreversible LOX inhibitor βAPN (for 48 hrs). B: Summarized data (mean±SE) showing PCNA (left panel) and Bcl–2 (right panel) protein levels in control PASMC (open bars) and PASMC treated with βAPN (solid bars). **<i>P</i><0.01 vs. Control.</p

Chelation of Cu and knockdown of CTR1 both decrease Bcl-2 expression in human PASMC during hypoxia.

<p>A: Western blot analysis on PCNA (a marker for cell proliferation) and Bcl-2 (an anti-apoptotic protein) in PASMC transfected with scrambled siRNA (Cont) or human CTR1-siRNA (siRNA) and PASMC treated with the Cu chelator BCS. B: Summarized data (mean±SE) showing PCNA (left panel) and Bcl-2 (right panel) protein levels in PASMC transfected with Control-siRNA or hCTR1-siRNA and PASMC treated with BCS. *<i>P</i><0.05 vs. Control-siRNA.</p

Upregulated pro-LOX expression in PASMC isolated from patients with idiopathic pulmonary arterial hypertension (IPAH) is associated with increased stiffness.

<p>A: Western blot analysis on pro-LOX in PASMC from normal subjects (Normal) and IPAH patients (IPAH). B: Time courses of the membrane deformation in normal PASMC (open circles) and IPAH-PASMC (closed circles) cultured on uncoated cover slips. Microaspiration was used to determine the membrane deformation which is inversely related to membrane stiffness. <i>P</i><0.01 between the two curves (using two-way ANOVA). C: Time courses of the membrane deformation in normal PASMC and IPAH-PASMC cultured on collagen-coated cover slips. <i>P</i><0.01 between the two curves (using two-way ANOVA). D: Time courses of the membrane deformation in IPAH-PASMC treated with (IPAH+βAPN) or without (IPAH) the LOX inhibitor βAPN. <i>P</i><0.01 between the two curves (using two-way ANOVA).</p

The mRNA expression level of Cu transporters (CTR1 and ATP7A) and lysyl oxidase (LOX) is increased in whole-lung and pulmonary artery (PA) tissues of mice with chronically hypoxia-induced pulmonary hypertension (HPH).

<p>Whole lung tissues and isolated PA tissue from normoxic (Nor, 21% O₂) and hypoxic (Hyp, 10% O₂ for 5 weeks) mice were homogenized and their mRNA transcripts evaluated by RT-PCR utilizing primers specific for ATOX1, ATP7A, CCS, CTR1, LOX, GAPDH or 18s rRNA (internal controls). A: RT-PCR products from whole-lung tissues were separated on 2% agarose gels (upper panel) and the band intensities quantitated by ImageJ, normalized to intensity of GAPDH, and graphed relative to Nor (n = 4 Nor mouse lungs; n = 8 Hyp mouse lungs). B: PA dissected from Nor and Hyp mice were used for RNA extraction (n = 5) and analyzed by quantitative PCR. Real-time PCR reaction was set with primers specific for the indicated genes. The cycle threshold C(t) values were normalized to 18s rRNA to obtain ΔC(t)_, quantified relative to normoxic control for each of the indicated genes (ΔΔC(t)), and graphed as % of normoxic control. C: Representative records of right ventricular pressure (RVP, left panel) and summarized data (mean±SE) showing RV systolic pressure (RVSP) in Nor (n = 6) and Hyp (n = 13) mice. D: Representative records (left panel) and summarized data (right panel, mean±SE) of right ventricular contractility (RV-±dp/dt_max) in Nor and Hyp mice. E: Summarized data (mean±SE) showing the ratio of right ventricle (RV) weight to left ventricle (LV) and septum (S) weight [RV/(LV+S)] in Nor (n = 7) and Hyp (n = 7) mice. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. Nor.</p

Hypoxia-mediated upregulation of mRNA expression of Cu transporters (CTR1, ATP7A) and lysyl oxidase (LOX) is associated with an increase in Cu transportation in human pulmonary arterial smooth muscle cells (PASMC).

<p>A: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and ⁶⁴Cu uptake (mean±SE) in human PASMC exposed to normoxia (Nor) and hypoxia (Hyp, 3% O₂ for 48 hrs, n = 3; right panel). B: Real-time RT-PCR analysis on ATP7A, CTR1, and LOX (left panel) and ⁶⁴Cu uptake (mean±SE, right panel) in human PASMC treated with vehicle (Cont) and CoCl₂ (100 µM for 48 hrs, n = 3; right pane). Lactate dehydrogenase (LDH) and erythropoietin (EPO) were used as positive controls. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. Hyp or CoCl₂.</p