Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc kinase-dependent connexin43 phosphorylation

2017 ◽  
Vol 312 (4) ◽  
pp. C385-C397 ◽  
Author(s):  
Lise Hangaard ◽  
Elena V. Bouzinova ◽  
Christian Staehr ◽  
Vibeke S. Dam ◽  
Sukhan Kim ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Tyrosine Phosphorylation ◽  
Intercellular Communication ◽  
Arterial Wall ◽  
Electrical Coupling ◽  
Input Resistance ◽  
Vascular Wall ◽  
Oscillatory Activity ◽  
A7r5 Cells

Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na-K-ATPase. The Na-K-ATPase is therefore important for synchronized VSMC oscillatory activity, i.e., vasomotion. The signaling between the Na-K-ATPase and gap junctions is unknown. We tested here the hypothesis that this signaling involves cSrc kinase. Intercellular communication was assessed by membrane capacitance measurements of electrically coupled VSMCs. Vasomotion in isometric myograph, input resistance, and synchronized [Ca2+]i transients were used as readout for intercellular coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semiquantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca2+ transients in VSMC in the arterial wall. Ouabain also increased input resistance in the arterial wall. These effects of ouabain were antagonized by inhibition of tyrosine phosphorylation with genistein, PP2, and by an inhibitor of the Na-K-ATPase-dependent cSrc activation, pNaKtide. Moreover, inhibition of cSrc phosphorylation increased vasomotion amplitude and decreased the resistance between cells in the vascular wall. Ouabain inhibited the electrical coupling between A7r5 cells, but pNaKtide restored the electrical coupling. Ouabain increased cSrc autophosphorylation of tyrosine 418 (Y418) required for full catalytic activity whereas pNaKtide antagonized it. This cSrc activation was associated with Cx43 phosphorylation of tyrosine 265 (Y265). Our findings demonstrate that Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc-dependent Cx43 tyrosine phosphorylation.

The α2 isoform of the Na,K-pump is important for intercellular communication, agonist-induced contraction, and EDHF-like response in rat mesenteric arteries

2012 ◽  
Vol 303 (1) ◽  
pp. H36-H46 ◽  
Author(s):  
Vladimir V. Matchkov ◽  
Nina Moeller-Nielsen ◽  
Vibeke Secher Dam ◽  
Zahra Nourian ◽  
Donna M. Briggs Boedtkjer ◽  
...  
Keyword(s):  
Intercellular Communication ◽  
Electrical Coupling ◽  
Vascular Wall ◽  
Mesenteric Arteries ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Small Arteries ◽  
Development In Vitro

The specific role of different isoforms of the Na,K-pump in the vascular wall is still under debate. We have previously suggested that the α2 isoform of the Na,K-pump (α2), Na+, Ca2+-exchange (NCX), and connexin43 form a regulatory microdomain in smooth muscle cells (SMCs), which controls intercellular communication and contractile properties of the vascular wall. We have tested this hypothesis by downregulating α2 in cultured SMCs and in small arteries with siRNA in vivo. Intercellular communication was assessed by using membrane capacitance measurements. Arteries transfected in vivo were tested for isometric and isobaric force development in vitro; [Ca2+]i was measured simultaneously. Cultured rat SMCs were well-coupled electrically, but 10 μM ouabain uncoupled them. Downregulation of α2 reduced electrical coupling between SMCs and made them insensitive to ouabain. Downregulation of α2 in small arteries was accompanied with significant reduction in NCX expression. Acetylcholine-induced relaxation was not different between the groups, but the endothelium-dependent hyperpolarizing factor-like component of the response was significantly diminished in α2-downregulated arteries. Micromolar ouabain reduced in a concentration-dependent manner the amplitude of norepinephrine (NE)-induced vasomotion. Sixty percent of the α2-downregulated arteries did not have vasomotion, and vasomotion in the remaining 40% was ouabain insensitive. Although ouabain increased the sensitivity to NE in the control arteries, it had no effect on α2-downregulated arteries. In the presence of a low NE concentration the α2-downregulated arteries had higher [Ca2+]i and tone. However, the NE EC50 was reduced under isometric conditions, and maximal contraction was reduced under isometric and isobaric conditions. The latter was caused by a reduced Ca2+-sensitivity. The α2-downregulated arteries also had reduced contraction to vasopressin, whereas the contractile response to high K+ was not affected. Our results demonstrate the importance of α2 for intercellular coupling in the vascular wall and its involvement in the regulation of vascular tone.

Cellular and Network Mechanisms of Slow Oscillatory Activity (<1 Hz) and Wave Propagations in a Cortical Network Model

2003 ◽  
Vol 89 (5) ◽  
pp. 2707-2725 ◽  
Author(s):  
Albert Compte ◽  
Maria V. Sanchez-Vives ◽  
David A. McCormick ◽  
Xiao-Jing Wang
Keyword(s):  
Network Model ◽  
Slow Wave Sleep ◽  
Model Neuron ◽  
Input Resistance ◽  
Oscillatory Activity ◽  
Recurrent Excitation ◽  
Slow Adaptation ◽  
Pharmacological Manipulations

Slow oscillatory activity (<1 Hz) is observed in vivo in the cortex during slow-wave sleep or under anesthesia and in vitro when the bath solution is chosen to more closely mimic cerebrospinal fluid. Here we present a biophysical network model for the slow oscillations observed in vitro that reproduces the single neuron behaviors and collective network firing patterns in control as well as under pharmacological manipulations. The membrane potential of a neuron oscillates slowly (at <1 Hz) between a down state and an up state; the up state is maintained by strong recurrent excitation balanced by inhibition, and the transition to the down state is due to a slow adaptation current (Na+-dependent K+ current). Consistent with in vivo data, the input resistance of a model neuron, on average, is the largest at the end of the down state and the smallest during the initial phase of the up state. An activity wave is initiated by spontaneous spike discharges in a minority of neurons, and propagates across the network at a speed of 3–8 mm/s in control and 20–50 mm/s with inhibition block. Our work suggests that long-range excitatory patchy connections contribute significantly to this wave propagation. Finally, we show with this model that various known physiological effects of neuromodulation can switch the network to tonic firing, thus simulating a transition to the waking state.

scholarly journals Proteoglycans and glycosaminoglycans induce gap junction synthesis and function in primary liver cultures.

1987 ◽  
Vol 105 (1) ◽  
pp. 541-551 ◽  
Author(s):  
D C Spray ◽  
M Fujita ◽  
J C Saez ◽  
H Choi ◽  
T Watanabe ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Electrical Coupling ◽  
Sulfated Polysaccharides ◽  
Sulfate Proteoglycan ◽  
Dye Coupling ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
In Cells

Intercellular communication via gap junctions, as measured by dye and electrical coupling, disappears within 12 h in primary rat hepatocytes cultured in serum-supplemented media or within 24 h in cells in a serum-free, hormonally defined medium (HDM) designed for hepatocytes. Glucagon and linoleic acid/BSA were the primary factors in the HDM responsible for the extended life span of the electrical coupling. After 24 h of culture, no hormone or growth factor tested could restore the expression of gap junctions. After 4-5 d of culture, the incidence of coupling was undetectable in a serum-supplemented medium and was only 4-5% in HDM alone. However, treatment with glycosaminoglycans or proteoglycans of 24-h cultures, having no detectable gap junction protein, resulted in synthesis of gap junction protein and of reexpression of electrical and dye coupling within 48 h. Most glycosaminoglycans were inactive (heparan sulfates, chondroitin-6 sulfates) or only weakly active (dermatan sulfates, chondroitin 4-sulfates, hyaluronates), the weakly active group increasing the incidence of coupling to 10-30% with the addition of 50-100 micrograms/ml of the factor. Treatment of the cells with 50-100 micrograms/ml of heparins derived from lung or intestine resulted in cells with intermediate levels of coupling (30-50%). By contrast, 10-20 micrograms/ml of chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, or liver-derived heparin resulted in dye coupling in 80-100% of the cells, with numerous cells showing dye spread from a single injected cell. Sulfated polysaccharides of glucose (dextran sulfates) or of galactose (carrageenans) were inactive or only weakly active except for lambda-carrageenan, which induced up to 70% coupling (albeit no multiple coupling in the cultures). The abundance of mRNA (Northern blots) encoding gap junction protein and the amounts of the 27-kD gap junction polypeptide (Western blots) correlated with the degree of electrical and dye coupling indicating that the active glycosaminoglycans and proteoglycans are inducing synthesis and expression of gap junctions. Thus, proteoglycans and glycosaminoglycans, especially those found in abundance in the extracellular matrix of liver cells, are important in the regulation of expression of gap junctions and, thereby, in the regulation of intercellular communication in the liver. The relative potencies of heparins from different tissue sources at inducing gap junction expression are suggestive of functional tissue specificity for these glycosaminoglycans.

scholarly journals Circulating Extracellular Vesicles from Patients with Sickle Cell Disease Progressively Disrupt Different Types of Endothelial Intercellular Junctions

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4823-4823
Author(s):  
Gabrielle Lapping-Carr ◽  
Joanna Gemel ◽  
Yifan Mao ◽  
Eric C. Beyer
Keyword(s):  
Plasma Membrane ◽  
Sickle Cell Disease ◽  
Gap Junctions ◽  
Extracellular Vesicles ◽  
Sickle Cell ◽  
Intercellular Communication ◽  
Cell Disease ◽  
Endothelial Monolayer ◽  
Cell Cell

Introduction: Aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of crises in sickle cell disease (SCD), including acute chest syndrome (ACS). We previously demonstrated that the plasma of SCD patients contains circulating small extracellular vesicles (EVs) and that those vesicles can disrupt endothelial integrity in vitro, including a decrease in VE-cadherin. The current study was designed to examine the effects of those EVs on additional components of the endothelial junctions including tight (zonula occludens 1, ZO-1) and gap junctions (connexin43, Cx43) and to test the hypothesis that the junctions would be more severely affected by EVs isolated from patients during an episode of ACS than by those isolated from the same patient at baseline. Methods: We identified subjects with SCD in our biobank who had plasma isolated at baseline and at the beginning of an admission for ACS (prior to transfusion). Samples were considered baseline if the patient was more than 4 weeks since transfusion and had no new health-related complaints. ACS was defined by the presence of an infiltrate on chest x-ray combined with fever, pain, hypoxia or cough. EVs were isolated from plasma using established methodologies. To determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with EVs for 48 h. Cells were fixed and studied by fluorescence microscopy (after immunolocalization of Cx43, ZO-1 and/or VE-cadherin and staining of nuclei with DAPI). Proteins were detected and quantified by immunoblotting. mRNA expression was determined by RT-qPCR. Gap junction mediated intercellular communication was assessed following microinjection of Lucifer yellow and neurobiotin. Results: Microscopy confirmed our previous observation that EVs isolated from subjects with SCD caused in vitro disruption of endothelial monolayers and that damage is significantly worse when EVs are isolated during an episode of ACS. The distribution and abundance of VE-cadherin and ZO-1 at the plasma membrane of undisturbed cells were minimally affected by SCD EVs. While baseline EVs did not detectably affect the distribution of Cx43, EVs isolated during ACS caused a loss of Cx43 from the plasma membrane. The integrated intensity of Cx43 membrane staining was decreased by ~20% following treatment with ACS EVs. Cx43 protein decreased on average by 32 % and Cx43 mRNA levels by 21% in cells treated with ACS EVs compared to baseline from the same patient. EVs isolated during ACS caused significant disruption in intercellular transfer compared to EVs isolated at baseline (67-94% reduction) (Figure 1). Conclusions: Our results show that subjects with SCD produce small EVs that cause disruption of the endothelial monolayer in vitro. Gap junctions composed of Cx43 are the most sensitive of the cell-cell junctions in this setting, since their abundance and function are reduced by ACS EVs even when the endothelial monolayer appears intact. Disruption of endothelial intercellular communication mediated by Cx43 appears to be an early and sensitive event in the endothelial disturbance caused by EVs in SCD patients. Disclosures No relevant conflicts of interest to declare.

Abstract 2779: Two- Dimentional Imaging Of Lipids Deposited In The Coronary Plaques By Near-infrared Fluorescence Angioscopy

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Yasumi Uchida
Keyword(s):  
Coronary Artery ◽  
Arterial Wall ◽  
Near Infrared ◽  
In Vitro Study ◽  
Vascular Wall ◽  
List Type ◽  
Lipid Deposition ◽  
Two Dimensional ◽  
Deep Layers

Background: The lipids deposited deep in the vascular wall are hardly detectable by any available imaging modalities including IVUS and OCT. Since near-infrared (NIR) light penetrates relatively deeply into the tissues, we devised a near-infrared fluorescence angioscope (NIRFA) for two dimensional imaging of lipids deposited even in deep layers of vascular wall. Aim: To examine feasibility of NIRFA for imaging of lipids in coronary arterial wall in man. Methods: NIRFA is composed of a quartz fiberscope incorporated in 5F balloon catheter, fluorescence exciter, fluorescence emitter, ICCD and recorder. NIR exciter filter of 710nm and emitter filter of 780nm were employed since among the major 30 substances composing atherosclerotic plaques, free cholesterol and cholesteryl esters alone exhibit NIR autofluorescence while oxLDL and TG not. In vitro study: Coronary artery removed from human cadaver was perfused with saline and an angioscope was introduced into it for imaging of lipids and the obtained NIR images were compared with histology. Clinical study: During routine coronary angiography, lipid deposition in the coronary artery was surveyed by NIRFA in 7 patients with coronary artery disease. Results: In vitro study. Coronary segments without lipid deposition by histology did not exhibit autofluorescence. Those with lipid deposition by histology, the lipids deposited within 700μm in depth from luminal surface exhibited autofluorescence. Deposited lipids not forming lipid pool exhibited strong and homogenous autofluorescence. Lipid pool exhibited no or weak autofluorescence surrounded by strong ones, indicating it is filled with oxLDL and/or TG. Clinical study. Autofluorescence was frequently detected not only in yellow plaques but also in white plaques by conventional angioscopy and hard plaques by IVUS. Also, autofluorescence was frequently detected in apparently normal coronary segments. Conclusion: The results indicate that this NIRFA is feasible for two dimensional imaging of lipids deposited even in deep layers of coronary arterial wall which are not detectable by conventional angioscopy, OCT and IVUS.

scholarly journals Osteoporosis-decreased extracellular matrix stiffness impairs connexin 43-mediated gap junction intercellular communication in osteocytes

2020 ◽  
Vol 52 (5) ◽  
pp. 517-526
Author(s):  
Demao Zhang ◽  
Xin Li ◽  
Caixia Pi ◽  
Linyi Cai ◽  
Yang Liu ◽  
...  
Keyword(s):  
Mechanical Property ◽  
Extracellular Matrix ◽  
Mouse Model ◽  
Gap Junctions ◽  
Gap Junction ◽  
Intercellular Communication ◽  
Cell Communication ◽  
Matrix Stiffness ◽  
Gap Junction Intercellular Communication

Abstract Osteocytes are the main sensitive and responsive cells for mechanical stimuli in bone. The connexin family enables them to communicate with each other via forming functional gap junctions. However, how osteoporosis-impaired extracellular mechanical property modulates gap junction intercellular communication in osteocytes remains elusive. In this study, we established an ovariectomy (OVX)-induced osteoporosis mouse model in vivo and a polydimethylsiloxane (PDMS)-based cell culture substrate model in vitro to explore the influence of extracellular matrix (ECM) stiffness on cell-to-cell communication in osteocytes. Firstly, we established an OVX-induced osteoporosis mouse model by characterizing the changes in radiography, morphology and histochemistry of femurs. Our results showed that osteoporosis decreased the bone matrix stiffness together with the changes including the loss of osteocytes and the decrease of protein markers. Meanwhile, the dendritic process interconnection and channel-forming protein, Cx43, were reduced in osteoporosis mice. Next we mimicked ECM stiffness changes in vitro by using PDMS substrates at ratios 1:5 for normal stiffness and 1:45 for osteoporosis stiffness. Our results showed that the decreased ECM stiffness reduced the number of dendritic processes in a single cell and gap junctions between adjacent osteocytes. We further detected the decreased expression of Cx43, in the substrate with decreased stiffness. Finally, we found that gap junction-based intercellular communication was reduced in living osteocytes in the substrate with decreased stiffness. This study demonstrates the correlation between ECM mechanical property and cell-to-cell communication in osteocytes and might pave the way for further exploration of osteoporosis in terms of biomechanics.

scholarly journals Epigenetic Targets for Oligonucleotide Therapies of Pulmonary Arterial Hypertension

2020 ◽  
Vol 21 (23) ◽  
pp. 9222
Author(s):  
William Gerthoffer
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Small Molecules ◽  
Vascular Remodeling ◽  
Arterial Wall ◽  
Vascular Wall ◽  
Wall Structure ◽  
Wall Thickening ◽  
Pulmonary Arterial

Arterial wall remodeling underlies increased pulmonary vascular resistance and right heart failure in pulmonary arterial hypertension (PAH). None of the established vasodilator drug therapies for PAH prevents or reverse established arterial wall thickening, stiffening, and hypercontractility. Therefore, new approaches are needed to achieve long-acting prevention and reversal of occlusive pulmonary vascular remodeling. Several promising new drug classes are emerging from a better understanding of pulmonary vascular gene expression programs. In this review, potential epigenetic targets for small molecules and oligonucleotides will be described. Most are in preclinical studies aimed at modifying the growth of vascular wall cells in vitro or normalizing vascular remodeling in PAH animal models. Initial success with lung-directed delivery of oligonucleotides targeting microRNAs suggests other epigenetic mechanisms might also be suitable drug targets. Those targets include DNA methylation, proteins of the chromatin remodeling machinery, and long noncoding RNAs, all of which act as epigenetic regulators of vascular wall structure and function. The progress in testing small molecules and oligonucleotide-based drugs in PAH models is summarized.

ROLE OF DIRECT INTERCELLULAR COMMUNICATION THROUGH GAP JUNCTIONS IN LYMPHOCYTE-MACROPHAGE INTERACTIONS IN VITRO

2004 ◽  
Vol 13 (3) ◽  
pp. 41
Author(s):  
Ernesto Oviedo-Orta ◽  
Sharada Karanam ◽  
Ulrike Benbow ◽  
W Howard Evans ◽  
Andrew C Newby
Keyword(s):  
Gap Junctions ◽  
Intercellular Communication

A model of smooth muscle cell synchronization in the arterial wall

2007 ◽  
Vol 293 (1) ◽  
pp. H229-H237 ◽  
Author(s):  
Jens Christian Brings Jacobsen ◽  
Christian Aalkjær ◽  
Holger Nilsson ◽  
Vladimir V. Matchkov ◽  
Jacob Freiberg ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Sarcoplasmic Reticulum ◽  
Gap Junctions ◽  
Electrical Coupling ◽  
Vascular Wall ◽  
Whole Cell ◽  
Calcium Dependent ◽  
Cellular Processes ◽  
The Individual ◽  
Number Of Cells

Vasomotion is a rhythmic variation in microvascular diameter. Although known for more than 150 years, the cellular processes underlying the initiation of vasomotion are not fully understood. In the present study a model of a single cell is extended by coupling a number of cells into a tube. The simulated results point to a permissive role of cGMP in establishing intercellular synchronization. In sufficient concentration, cGMP may activate a cGMP-sensitive calcium-dependent chloride channel, causing a tight spatiotemporal coupling between release of sarcoplasmic reticulum calcium, membrane depolarization, and influx of extracellular calcium. Low [cGMP] is associated only with unsynchronized waves. At intermediate concentrations, cells display either waves or whole cell oscillations, but these remain unsynchronized between cells. Whole cell oscillations are associated with rhythmic variation in membrane potential and flow of current through gap junctions. The amplitude of these oscillations in potential grows with increasing [cGMP], and, past a certain threshold, they become strong enough to entrain all cells in the vascular wall, thereby initiating sustained vasomotion. In this state there is a rhythmic flow of calcium through voltage-sensitive calcium channels into the cytoplasm, making the frequency of established vasomotion sensitive to membrane potential. It is concluded that electrical coupling through gap junctions is likely to be responsible for the rapid synchronization across a large number of cells. Gap-junctional current between cells is due to the appearance of oscillations in the membrane potential that again depends on the entrainment of sarcoplasmic reticulum and plasma membrane within the individual cell.

Characterization of gap junctions between pairs of Leydig cells from mouse testis

1994 ◽  
Vol 267 (2) ◽  
pp. C570-C580 ◽  
Author(s):  
E. M. Perez-Armendariz ◽  
M. C. Romano ◽  
J. Luna ◽  
C. Miranda ◽  
M. V. Bennett ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Leydig Cells ◽  
Connexin 43 ◽  
Electrical Coupling ◽  
Cell Voltage ◽  
Mouse Testis ◽  
Coupling Coefficients ◽  
Saline Solutions

Leydig cells are coupled in vivo by numerous gap junctions. In vivo and in vitro cells were immunolabeled by connexin 43 (Cx43) but not by Cx26 or Cx32 antibodies; immunoblotting confirmed specificity of Cx43 labeling. Pairs of Leydig cells dissociated from mouse testis were studied by dual whole cell voltage clamp, and a high incidence of dye (n = 20) and electrical coupling (n = 60; > 90%) was found. Coupling coefficients were near 1 and junctional conductance (gj) averaged 7.2 +/- 1.2 nS (SE, n = 40). Large transjunctional voltage (Vj) decreased gj; currents decayed exponentially with time constants of seconds that decreased at greater Vj. The residual conductance at large Vj was at least approximately 40% of the initial conductance. Exposure of cell pairs to saline solutions saturated with CO2 (n = 15) or containing 2 mM halothane (n = 15) or 3.5 mM heptanol (n = 15) rapidly and reversibly reduced gj. In eight cell pairs, gating of single junctional channels was observed during halothane-induced reduction in gj. Most gating events at Vj < 40 mV were fit by a Gaussian distribution with a mean of approximately 100 pS. With Vj > 40 mV, smaller transitions of approximately 30 pS were also recorded, and the frequency and duration of the approximately 100-pS transitions decreased. Also, approximately 70-pS transitions between 30- and 100-pS conductances were observed in the absence of 70-pS transitions to or from the baseline, indicating that the 30-pS conductance was a substate induced by large Vj.

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