scholarly journals Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells

2012 ◽  
Vol 303 (12) ◽  
pp. C1260-C1268 ◽  
Author(s):  
Marina Dobrivojević ◽  
Aleksandra Sinđić ◽  
Bayram Edemir ◽  
Stefanie Kalweit ◽  
Wolf-Georg Forssmann ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Natriuretic Peptide ◽  
Natriuretic Peptides ◽  
Specific Inhibitor ◽  
Rgs Proteins ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intracellular Ca

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential ( Vm) and intracellular Ca2+ using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B2R) and Ca2+-dependent Cl− channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 μM). The BK-induced Ca2+ signal was blocked by the B2R inhibitor HOE 140. [Des-Arg9]-bradykinin, an activator of B1R, had no effect on intracellular Ca2+. NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K+ channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca2+ signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 μM) completely inhibited the BK-induced Ca2+ signaling. UTP, another activator of the PLC-mediated Ca2+ signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B2R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

Molecular identification of a TTX-sensitive Ca2+current

2001 ◽  
Vol 280 (5) ◽  
pp. C1327-C1339 ◽  
Author(s):  
Silvia Guatimosim ◽  
Eric A. Sobie ◽  
Jader dos Santos Cruz ◽  
Laura A. Martin ◽  
W. J. Lederer
Keyword(s):  
Cardiac Myocytes ◽  
Ion Selectivity ◽  
Inactivation Kinetics ◽  
Experimental Conditions ◽  
Guinea Pig Heart ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intracellular Ca ◽  
Rat Myocytes

The TTX-sensitive Ca2+ current [ I Ca(TTX)] observed in cardiac myocytes under Na+-free conditions was investigated using patch-clamp and Ca2+-imaging methods. Cs+ and Ca2+were found to contribute to I Ca(TTX), but TEA+ and N-methyl-d-glucamine (NMDG+) did not. HEK-293 cells transfected with cardiac Na+ channels exhibited a current that resembled I Ca(TTX) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na+ channel itself gives rise to I Ca(TTX). Furthermore, repeated activation of I Ca(TTX) led to a 60% increase in intracellular Ca2+ concentration, confirming Ca2+ entry through this current. Ba2+ permeation of I Ca(TTX), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na+channels under our experimental conditions. The report of block of I Ca(TTX) in guinea pig heart by mibefradil (10 μM) was supported in transfected HEK-293 cells, but Na+current was also blocked (half-block at 0.45 μM). We conclude that I Ca(TTX) reflects current through cardiac Na+ channels in Na+-free (or “null”) conditions. We suggest that the current be renamed I Na(null) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between I Na(null)and Ca2+ flux through slip-mode conductance of cardiac Na+ channels is discussed in the context of ion channel biophysics and “permeation plasticity.”

Extracellular ATP dissociates nonmuscle myosin from P2X7complex: this dissociation regulates P2X7pore formation

2009 ◽  
Vol 297 (2) ◽  
pp. C430-C439 ◽  
Author(s):  
Ben J. Gu ◽  
Catherine Rathsam ◽  
Leanne Stokes ◽  
Andrew B. McGeachie ◽  
James S. Wiley
Keyword(s):  
Specific Inhibitor ◽  
Life Time ◽  
Cell Types ◽  
Interferon Γ ◽  
Extracellular Atp ◽  
Nonmuscle Myosin ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Myosin Va

The P2X7receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X7channel and massive K+efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X7were isolated by using anti-P2X7monoclonal antibody-coated Dynabeads from both interferon-γ plus LPS-stimulated monocytic THP-1 cells and P2X7-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X7in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X7receptor by ATP caused dissociation of P2X7from nonmuscle myosin in both cell types. The interaction of P2X7and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X7pore function without any increase in surface expression or ion channel function of P2X7receptors. S- l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X7-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X7and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X7channel to a pore.

Cells expressing unique Na+/Ca2+ exchange (NCX1) splice variants exhibit different susceptibilities to Ca2+ overload

2006 ◽  
Vol 290 (5) ◽  
pp. H2155-H2162 ◽  
Author(s):  
Cecilia Hurtado ◽  
Michele Prociuk ◽  
Thane G. Maddaford ◽  
Elena Dibrov ◽  
Nasrin Mesaeli ◽  
...  
Keyword(s):  
Splice Variant ◽  
Cardiac Glycosides ◽  
Splice Variants ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
Neonatal Cardiomyocytes ◽  
Simulated Ischemia ◽  
293 Cells ◽  
Intracellular Ca ◽  
Specific Alternative

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 μM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.

scholarly journals Influence of intracellular Ca2+ and alternative splicing on the pharmacological profile of ANO1 channels

2016 ◽  
Vol 311 (3) ◽  
pp. C437-C451 ◽  
Author(s):  
Tae Sik Sung ◽  
Kate O'Driscoll ◽  
Haifeng Zheng ◽  
Nicholas J. Yapp ◽  
Normand Leblanc ◽  
...  
Keyword(s):  
Splice Variants ◽  
Channel Blockers ◽  
Anoctamin 1 ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
Naturally Occurring ◽  
293 Cells ◽  
Intracellular Ca ◽  
Inside Out

Anoctamin-1 (ANO1) is a Ca2+-activated Cl− channel expressed in many types of cells. Splice variants of ANO1 have been shown to influence the biophysical properties of conductance. It has been suggested that several new antagonists of ANO1 with relatively high affinity and selectivity might be useful for experimental and, potentially, therapeutic purposes. We investigated the effects of intracellular Ca2+ concentration ([Ca2+]i) at 100-1,000 nM, a concentration range that might be achieved in cells during physiological activation of ANO1 channels, on blockade of ANO1 channels expressed in HEK-293 cells. Whole cell and excised patch configurations of the patch-clamp technique were used to perform tests on a variety of naturally occurring splice variants of ANO1. Blockade of ANO1 currents with aminophenylthiazole (T16Ainh-A01) was highly dependent on [Ca2+]i. Increasing [Ca2+]i reduced the potency of this blocker. Similar Ca2+-dependent effects were also observed with benzbromarone. Experiments on excised, inside-out patches showed that the diminished potency of the blockers caused by intracellular Ca2+ might involve a competitive interaction for a common binding site or repulsion of the blocking drugs by electrostatic forces at the cytoplasmic surface of the channels. The degree of interaction between the channel blockers and [Ca2+]i depends on the splice variant expressed. These experiments demonstrate that the efficacy of ANO1 antagonists depends on [Ca2+]i, suggesting a need for caution when ANO1 blockers are used to determine the role of ANO1 in physiological functions and in their use as therapeutic agents.

scholarly journals Processing of Pro–B-Type Natriuretic Peptide: Furin and Corin as Candidate Convertases2

2010 ◽  
Vol 56 (7) ◽  
pp. 1166-1176 ◽  
Author(s):  
Alexander G Semenov ◽  
Natalia N Tamm ◽  
Karina R Seferian ◽  
Alexander B Postnikov ◽  
Natalia S Karpova ◽  
...  
Keyword(s):  
Heart Failure ◽  
Natriuretic Peptide ◽  
Spectrometric Analysis ◽  
Precursor Molecule ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Lovo Cells ◽  
Interfering Rna

Abstract Background: B-type natriuretic peptide (BNP) and its N-terminal fragment (NT-proBNP) are the products of the enzyme-mediated cleavage of their precursor molecule, proBNP. The clinical significance of proBNP-derived peptides as biomarkers of heart failure has been explored thoroughly, whereas little is known about the mechanisms of proBNP processing. We investigated the role of 2 candidate convertases, furin and corin, in human proBNP processing. Methods: We measured proBNP expression in HEK 293 and furin-deficient LoVo cells. We used a furin inhibitor and a furin-specific small interfering RNA (siRNA) to explore the implication of furin in proBNP processing. Recombinant proBNPs were incubated with HEK 293 cells transfected with the corin-expressing plasmid. We applied mass spectrometry to analyze the products of furin- and corin-mediated cleavage. Results: Reduction of furin activity significantly impaired proBNP processing in HEK 293 cells. Furin-deficient LoVo cells were unable to process proBNP, whereas coexpression with furin resulted in effective proBNP processing. Mass spectrometric analysis revealed that the furin-mediated cleavage of proBNP resulted in BNP 1–32, whereas corin-mediated cleavage led to the production of BNP 4–32. Some portion of proBNP in the plasma of heart failure patients was not glycosylated in the cleavage site region and was susceptible to furin-mediated cleavage. Conclusions: Both furin and corin are involved in the proBNP processing pathway, giving rise to distinct BNP forms. The significance of the presence of unprocessed proBNP in circulation that could be cleaved by the endogenous convertases should be further investigated for better understanding BNP physiology.

Olive compounds attenuate oxidative damage induced in HEK-293 cells via MAPK signaling pathway

2017 ◽  
Vol 39 ◽  
pp. 18-27 ◽  
Author(s):  
Amina Maalej ◽  
Maurizio Forte ◽  
Zouhaier Bouallagui ◽  
Stella Donato ◽  
Luigi Mita ◽  
...  
Keyword(s):  
Oxidative Damage ◽  
Signaling Pathway ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Expression and Secretion of an Atrial Natriuretic Peptide in Beige-Like 3T3-L1 Adipocytes

2019 ◽  
Vol 20 (24) ◽  
pp. 6128 ◽  
Author(s):  
In-Seon Bae ◽  
Sang Hoon Kim
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Peptide Hormone ◽  
Brown Adipocyte ◽  
Omega 3 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Beige Adipocytes ◽  
Atrial Natriuretic

The browning of white adipose tissue (beige adipocytes) stimulates energy expenditure. Omega-3 fatty acids have been shown to induce thermogenic action in adipocytes via G-protein coupled receptor 120 (GPR120). Atrial natriuretic peptide (ANP) is a peptide hormone that plays the role of maintaining normal blood pressure in kidneys to inhibit Na+ reuptake. Recently, ANP was found to induce adipocyte browning by binding to NPR1, an ANP receptor. However, the expression of ANP in adipocytes has not yet been studied. Therefore, in this study, we investigate the expression of ANP in beige-like adipocytes induced by docosahexaenoic acids (DHA), T3, or a PPAR agonist, rosiglitazone. First, we found that brown adipocyte-specific genes were upregulated in beige-like adipocytes. DHA promoted ANP expression in beige-like cells, whereas DHA-induced ANP expression was abolished by GPR120 knockout. ANP secretion of beige-like adipocytes was increased via PKC/ERK1/2 signaling in the GPR120 pathway. Furthermore, ANP secreted from beige-like adipocytes acted on HEK-293 cells, the recipient cells, leading to increased cGMP activity. After the NPR1 knockdown of HEK-293 cells, cGMP activity was not changed. Taken together, our findings indicate that beige-like adipocytes induce ANP secretion, which may contribute to improving obesity-associated metabolic disease.

scholarly journals An Activating Deletion Variant in the Submembrane Region of Natriuretic Peptide Receptor-B Causes Tall Stature

2020 ◽  
Vol 105 (7) ◽  
pp. 2354-2366 ◽  
Author(s):  
Peter Lauffer ◽  
Erick Miranda-Laferte ◽  
Hermine A van Duyvenvoorde ◽  
Arie van Haeringen ◽  
Franziska Werner ◽  
...  
Keyword(s):  
Natriuretic Peptide ◽  
Skin Fibroblasts ◽  
Growth Disorders ◽  
Tall Stature ◽  
Natriuretic Peptide Receptor ◽  
Hek 293 ◽  
Peptide Receptor ◽  
Cgmp Production ◽  
Hek 293 Cells ◽  
293 Cells

Abstract Context C-type natriuretic peptide (CNP) is critically involved in endochondral bone growth. Variants in the genes encoding CNP or its cyclic guanosine monophosphate (cGMP)-forming receptor (natriuretic peptide receptor-B [NPR-B], gene NPR2) cause monogenic growth disorders. Here we describe a novel gain-of-function variant of NPR-B associated with tall stature and macrodactyly of the great toes (epiphyseal chondrodysplasia, Miura type). Design History and clinical characteristics of 3 family members were collected. NPR2 was selected for sequencing. Skin fibroblasts and transfected HEK-293 cells were used to compare mutant versus wild-type NPR-B activities. Homology modeling was applied to understand the molecular consequences of the variant. Results Mother’s height was +2.77 standard deviation scores (SDS). The heights of her 2 daughters were +1.96 SDS at 7 years and +1.30 SDS at 4 years of age. Skeletal surveys showed macrodactyly of the great toes and pseudo-epiphyses of the mid- and proximal phalanges. Sequencing identified a novel heterozygous variant c.1444_1449delATGCTG in exon 8 of NPR2, predicted to result in deletion of 2 amino acids Met482-Leu483 within the submembrane region of NPR-B. In proband’s skin fibroblasts, basal cGMP levels and CNP-stimulated cGMP production were markedly increased compared with controls. Consistently, assays with transfected HEK-293 cells showed markedly augmented baseline and ligand-dependent activity of mutant NPR-B. Conclusions We report the second activating variant within the intracellular submembrane region of NPR-B resulting in tall stature and macrodactyly. Our functional and modeling studies suggest that this domain plays a critical role in the baseline conformation and ligand-dependent structural rearrangement of NPR-B required for cGMP production.

scholarly journals Bupivacaine inhibits a small conductance calcium-activated potassium type 2 channel (SK2) in HEK 293 cells

2019 ◽  
Author(s):  
Hongfei Chen ◽  
Fangfang Xia ◽  
Zhousheng Jin ◽  
Yuting He ◽  
Zhengjie Chen ◽  
...  
Keyword(s):  
Inhibitory Effect ◽  
Heart Cells ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Ic50 Value ◽  
Small Conductance

Abstract Background: Bupivacaine blocks many ion channels in the heart muscle, which could cause severe cardiotoxicity. Small conductance calcium-activated potassium type 2 channels (SK2 channels) are widely distributed in the heart cells and are involved in relevant physiological functions. However, whether bupivacaine can inhibit SK2 channels is still unknown. This study investigated the effect of bupivacaine on SK2 channels. Methods: The SK2 channel gene was transfected into human embryonic kidney 293 cells (HEK-293 cells) with Lipofectamine 2000. The whole-cell patch clamp technique was used to study the effect of bupivacaine on SK2 channels. The inhibitory effect of various concentrations of bupivacaine on SK2 currents exhibited a non-linear relation, and the half-maximal inhibitory concentration (IC50) value was determined. Results: Bupivacaine inhibited the SK2 channels reversibly in a dose-dependent manner. The IC50 value of bupivacaine, ropivacaine and lidocaine on the SK2 current was 133.7, 189.3, and 885.8 µM, respectively. The degree of SK2 current inhibition by bupivacaine was dependent on the intracellular concentration of free calcium. Conclusions: The results of this study suggested a new inhibitory effect of bupivacaine on SK2 channels. Future studies should be concerned with the effects of SK2 on bupivacaine cardiotoxicity. Keywords: Bupivacaine, SK2 channel, inhibition, cardiotoxicity, HEK 293.

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels

2003 ◽  
Vol 285 (2) ◽  
pp. C467-C479 ◽  
Author(s):  
Mu-Lan He ◽  
Hana Zemkova ◽  
Taka-aki Koshimizu ◽  
Melanija Tomić ◽  
Stanko S. Stojilkovic
Keyword(s):  
Purinergic Receptors ◽  
Host Cells ◽  
P2x Receptor ◽  
Wild Type ◽  
Hek 293 ◽  
Human Embryonic Kidney Cells ◽  
Voltage Gated ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Intracellular Ca

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.

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