ResearchPad - membrane-physiology https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Ion currents, action potentials, and noradrenergic responses in rat pulmonary vein and left atrial cardiomyocytes]]> https://www.researchpad.co/article/elastic_article_7931 Effects of noradrenaline on action potential (AP) repolarisation in PV cardiomyocytes. A Representative AP recording from a PV cardiomyocyte in control solution and in the presence of 1 μM noradrenaline (NA). B Representative AP recording from another PV cardiomyocyte in control solution and in the presence of 1 μM NA showing lack of AP prolongation. C Before‐and‐after plot of APD30 in PV cardiomyocytes in control solution and after superfusion with 1 μM NA. D Noradrenaline‐induced fold‐changes in APD30 in LA and PV cardiomyocytes.

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<![CDATA[Membrane stabilizer Poloxamer 188 improves yield of primary isolated rat cardiomyocytes without impairing function]]> https://www.researchpad.co/article/Nbc7f16ee-111b-498c-a299-3e01086b303d

Abstract

Intact cardiomyocytes are used to investigate cardiac contractility and evaluate the efficacy of new therapeutic compounds. Primary enzymatic isolation of adult rodent cardiomyocytes has limitations, including low cardiomyocyte survival, which is likely due to ischemic conditions and/or membrane damage. The addition of Poloxamer 188 (P188) has been used to reduce ischemia‐ and membrane‐related damage in ischemia–reperfusion and muscular dystrophy studies. P188 stabilizes membranes, reducing cell death. Cardiomyocytes were isolated from rats, under three conditions: (1) using standard isolation solutions, (2) with P188 added during cannulation (ischemic event), and (3) with P188 added during cannulation, enzymatic digestion, and trituration. Cell survival was assessed by quantifying the number of rod‐shaped versus contracted cells on the day of isolation and up to 3 days post‐isolation. Adding P188 only during cannulation yielded improved survival on the day of isolation. Little difference in survival was seen among the three conditions in the days post‐isolation. Cardiomyocyte function was assessed by measuring calcium transients and unloaded sarcomere lengths for up to 2 days post‐isolation. P188 did not consistently alter calcium handling or sarcomere shortening in the isolated cardiomyocytes. We conclude that the addition of P188 to the cannulation (e.g., wash) of the isolated heart may improve initial survival of cardiomyocytes upon primary enzymatic isolation.

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<![CDATA[Role of sodium‐dependent Pi transporter/Npt2c on Pi homeostasis in klotho knockout mice different properties between juvenile and adult stages]]> https://www.researchpad.co/article/N1b0c87a0-6340-43f5-84b9-f8529f1e5e90

Abstract

SLC34A3/NPT2c/NaPi‐2c/Npt2c is a growth‐related NaPi cotransporter that mediates the uptake of renal sodium‐dependent phosphate (Pi). Mutation of human NPT2c causes hereditary hypophosphatemic rickets with hypercalciuria. Mice with Npt2c knockout, however, exhibit normal Pi metabolism. To investigate the role of Npt2c in Pi homeostasis, we generated α‐klotho−/−/Npt2c−/− (KL2cDKO) mice and analyzed Pi homeostasis. α‐Klotho−/− (KLKO) mice exhibit hyperphosphatemia and markedly increased kidney Npt2c protein levels. Genetic disruption of Npt2c extended the lifespan of KLKO mice similar to that of α‐Klotho−/−/Npt2a−/− mice. Adult KL2cDKO mice had hyperphosphatemia, but analysis of Pi metabolism revealed significantly decreased intestinal and renal Pi (re)absorption compared with KLKO mice. The 1,25‐dihydroxy vitamin D3 concentration was not reduced in KL2cDKO mice compared with that in KLKO mice. The KL2cDKO mice had less severe soft tissue and vascular calcification compared with KLKO mice. Juvenile KL2cDKO mice had significantly reduced plasma Pi levels, but Pi metabolism was not changed. In Npt2cKO mice, plasma Pi levels began to decrease around the age of 15 days and significant hypophosphatemia developed within 21 days. The findings of the present study suggest that Npt2c contributes to regulating plasma Pi levels in the juvenile stage and affects Pi retention in the soft and vascular tissues in KLKO mice.

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<![CDATA[Alteration of renal Na,K‐ATPase in rats following the mediastinal γ‐irradiation]]> https://www.researchpad.co/article/5c82b4d4d5eed0c484e5d62e

Abstract

Na,K‐ATPase represents the key enzyme that maintains the homeostasis of sodium and potassium ions in the cells. It was documented that in directly irradiated organs the activity of this enzyme is decreased. The aim of present study was to clarify the remote effect of irradiation in mediastinal area on the activity of the Na,K‐ATPase in kidneys in rats. Ionizing radiation in single dose 25 Gy resulted in consequent decrease of the body weight gain as well as the size of kidneys in Wistar rats. In addition, radiation induced alterations in the oxidative status of blood plasma. Irradiation also decreased the activity of renal Na,K‐ATPase. Measurements of enzyme kinetics that were dependent on the concentration of energy substrate ATP or cofactor Na+ indicated that the lowered enzyme activity is probably a consequence of decreased number of active molecules of the enzyme, as suggested by lowered V max values. Immunoblot analysis confirmed the lowered expression of the catalytic alpha subunit together with decreased content of the glycosylated form of beta subunit in the renal tissue of irradiated rats. The ability of the enzyme to bind the substrate ATP, as well as Na+ was not affected, as shown by unaltered values of K m and KN a. Irradiation of the body in the mediastinal area despite protection of kidneys by lead plates during application of X‐ray was followed by significant decline of activity of the renal Na,K‐ATPase, what may result in deteriorated homeostasis in the organism.

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<![CDATA[ TRPM7‐mediated spontaneous Ca2+ entry regulates the proliferation and differentiation of human leukemia cell line K562]]> https://www.researchpad.co/article/5c08b870d5eed0c484150010

Abstract

Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real‐time RTPCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2‐APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (I spont) contributive to basal [Ca2+]i was identified by the nystatin‐perforated whole‐cell recording. I spont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA‐mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin‐induced γ‐globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.

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<![CDATA[ AKT and ERK1/2 activation via remote ischemic preconditioning prevents Kcne2‐dependent sudden cardiac death]]> https://www.researchpad.co/article/5c8011cfd5eed0c484a99ac0

Abstract

Sudden cardiac death (SCD) is the leading global cause of mortality. SCD often arises from cardiac ischemia reperfusion (IR) injury, pathologic sequence variants within ion channel genes, or a combination of the two. Alternative approaches are needed to prevent or ameliorate ventricular arrhythmias linked to SCD. Here, we investigated the efficacy of remote ischemic preconditioning (RIPC) of the limb versus the liver in reducing ventricular arrhythmias in a mouse model of SCD. Mice lacking the Kcne2 gene, which encodes a potassium channel β subunit associated with acquired Long QT syndrome were exposed to IR injury via coronary ligation. This resulted in ventricular arrhythmias in all mice (15/15) and SCD in 5/15 mice during reperfusion. Strikingly, prior RIPC (limb or liver) greatly reduced the incidence and severity of all ventricular arrhythmias and completely prevented SCD. Biochemical and pharmacological analysis demonstrated that RIPC cardioprotection required ERK1/2 and/or AKT phosphorylation. A lack of alteration in GSK‐3β phosphorylation suggested against conventional reperfusion injury salvage kinase (RISK) signaling pathway protection. If replicated in human studies, limb RIPC could represent a noninvasive, nonpharmacological approach to limit dangerous ventricular arrhythmias associated with ischemia and/or channelopathy‐linked SCD.

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<![CDATA[Potassium channels contribute to activity‐dependent regulation of dendritic inhibition]]> https://www.researchpad.co/article/5b5b2483463d7e17a9ed4f88

Abstract

GABAergic inhibition plays a critical role in the regulation of neuronal activity. In the neocortex, inhibitory interneurons that target the dendrites of pyramidal cells influence both electrical and biochemical postsynaptic signaling. Voltage‐gated ion channels strongly shape dendritic excitability and the integration of excitatory inputs, but their contribution to GABAergic signaling is less well understood. By combining 2‐photon calcium imaging and focal GABA uncaging, we show that voltage‐gated potassium channels normally suppress the GABAergic inhibition of calcium signals evoked by back‐propagating action potentials in dendritic spines and shafts of cortical pyramidal neurons. Moreover, the voltage‐dependent inactivation of these channels leads to enhancement of dendritic calcium inhibition following somatic spiking. Computational modeling reveals that the enhancement of calcium inhibition involves an increase in action potential depolarization coupled with the nonlinear relationship between membrane voltage and calcium channel activation. Overall, our findings highlight the interaction between intrinsic and synaptic properties and reveal a novel mechanism for the activity‐dependent regulation of GABAergic inhibition.

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