Mechanical stimulation initiates cell-to-cell calcium signaling in ovine lens epithelial cells

1996 ◽  
Vol 109 (2) ◽  
pp. 355-365 ◽  
Author(s):  
G.C. Churchill ◽  
M.M. Atkinson ◽  
C.F. Louis
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Gap Junction ◽  
Mechanical Stimulation ◽  
Primary Cultures ◽  
Channel Blockers ◽  
Extracellular Medium ◽  
Gap Junction Channels ◽  
Cell Propagation ◽  
Cytosolic Factor

Although abnormalities in calcium regulation have been implicated in the development of most forms of cataract, the mechanisms by which Ca2+ is regulated in the cells of the ocular lens remain poorly defined. Cell-to-cell Ca2+ signaling was investigated in primary cultures of ovine epithelial cells using the Ca(2+)-reporter dye fura-2 and fluorescence microscopy. Mechanical stimulation of a single cell with a micropipette initiated a propagated increase in cytosolic free Ca2+ that spread from the stimulated cell through 2–8 tiers of surrounding cells. During this intercellular Ca2+ wave, cytosolic Ca2+ increased 2- to 12-fold from resting levels of approximately 100 nM. Nanomolar extracellular Ca2+ did not affect the cell-to-cell propagation of the Ca2+ wave, but reduced the magnitude of the cytosolic Ca2+ increases, which was most evident in the mechanically-stimulated cell. Depletion of intracellular Ca2+ stores with thapsigargin eliminated the propagated intercellular Ca2+ wave, but did not prevent the cytosolic Ca2+ increase in the mechanically-stimulated cell, which required extracellular Ca2+ and was attenuated by the addition of the Ca2+ channel blockers Ni2+, Gd3+ and La3+ to the medium. These results are most easily explained by a mechanically-activated channel in the plasma membrane of the stimulated cell. The propagated increase in cytosolic Ca2+ appeared to be communicated to adjacent cells by the passage of an intracellular messenger other than Ca2+ through gap junction channels. However, if the plasma membrane of the mechanically-stimulated cell was ruptured such that there was loss of cytosolic contents, the increase in cytosolic Ca2+ in the surrounding cells was elicited by both a messenger passing through gap junction channels and by a cytosolic factor(s) diffusing through the extracellular medium. These results demonstrate the existence of intercellular Ca2+ signaling in lens cells, which may play a role in regulating cytosolic Ca2+ in the intact lens.

Sharing signals: connecting lung epithelial cells with gap junction channels

2002 ◽  
Vol 283 (5) ◽  
pp. L875-L893 ◽  
Author(s):  
Michael Koval
Keyword(s):  
Epithelial Cells ◽  
Gap Junctions ◽  
Gap Junction ◽  
Alveolar Epithelial Cells ◽  
Lung Epithelial Cells ◽  
Cell Phenotype ◽  
Connexin Expression ◽  
Gap Junction Channels ◽  
Alveolar Epithelial ◽  
Junctional Communication

Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.

A role for Ca(2+)-conducting ion channels in mechanically-induced signal transduction of airway epithelial cells

1994 ◽  
Vol 107 (11) ◽  
pp. 3037-3044 ◽  
Author(s):  
S. Boitano ◽  
M.J. Sanderson ◽  
E.R. Dirksen
Keyword(s):  
Epithelial Cells ◽  
Mechanical Stimulation ◽  
Airway Epithelial Cells ◽  
Effective Concentration ◽  
Channel Blockers ◽  
Airway Epithelial ◽  
Free Medium ◽  
Half Maximal Effective Concentration ◽  
Induced Signal ◽  
Stimulation Of

Mechanical stimulation of a single cell in a cultured monolayer of airway epithelial cells initiates an intercellularly communicated increase in intracellular Ca2+ concentration ([Ca2+]i) that propagates radically through adjacent cells via gap junctions, forming an intercellular Ca2+ wave. Mechanically-induced intercellular Ca2+ waves also occur in the absence of extracellular Ca2+. However, in Ca(2+)-free medium an increase in [Ca2+]i of the stimulated cell does not occur. Thus, mechanically-induced [Ca2+]i changes in the stimulated cell are influenced by the extracellular Ca2+ concentration. To investigate if a channel-mediated Ca2+ flux across the plasma membrane contributes to the elevation of [Ca2+]i in the stimulated cell we used digital image microscopy to measure mechanically-induced [Ca2+]i changes in the presence of Ca2+ channel blockers. In Ca(2+)-free medium containing Gd3+ (20 microM) mechanical stimulation resulted in an [Ca2+]i increase in the stimulated cell. The delay time between mechanical stimulation and increase in [Ca2+]i of the stimulated cell was dependent on extracellular [Gd3+], with a half-maximal effective concentration of approximately 40 microM. Mechanical stimulation in Ca(2+)-free medium containing La3+ (10 microM) or Ni2+ (100 microM) gave similar results. Mechanical stimulation in Ca(2+)-free medium containing the dihydropyridine Ca2+ channel blockers nifedipine (10 microM) and nimodipine (10 microM) also resulted in an increase of [Ca2+]i of the stimulated cell. Mechanical stimulation of cells treated with thapsigargin to deplete intracellular Ca2+ stores, in the presence of 1.3 mM extracellular Ca2+, results in an increase in [Ca2+]i of the stimulated cell without the propagation of an intercellular Ca2+ wave.(ABSTRACT TRUNCATED AT 250 WORDS)

New Roles for Connexons

Physiology ◽  
2003 ◽  
Vol 18 (3) ◽  
pp. 100-103 ◽  
Author(s):  
Lisa Ebihara
Keyword(s):  
Plasma Membrane ◽  
Ion Channels ◽  
Gap Junction ◽  
Paracrine Signaling ◽  
Gap Junction Channels ◽  
Cellular Processes ◽  
Gap Junction Hemichannels

Connexons or gap junction hemichannels are large, nonselective ion channels that reside in the nonjunctional plasma membrane before their assembly into gap junction channels. Increasing evidence suggests that these channels can open under certain conditions and may participate in a number of cellular processes, including the release of small metabolites such as ATP and NAD+, which are involved in paracrine signaling.

scholarly journals Synchronization Temperature Determines the Location of RSV Fusion During Entry in Cultured Cells

2021 ◽  
Author(s):  
Christian Cadena-Cruz ◽  
Marcio De-Avila-Arias ◽  
Heather M. Costello ◽  
Leidy Hurtado-Gomez ◽  
Walter Martínez-De-La-Rosa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Bronchial Epithelial Cells ◽  
Viral Gene ◽  
Virus Species ◽  
Human Bronchial Epithelial Cells ◽  
Animal Cells ◽  
Bronchial Epithelial

AbstractRespiratory syncytial virus (RSV) is the most frequent cause of bronchiolitis in children under five years of age. No vaccines against this virus are currently available. RSV infection of a cell is initiated by fusion between the virion membrane and a cellular membrane, but it is not clear if the fusion process takes place at the plasma membrane or within an endosome. Most such experiments have been initiated at the traditional synchronization temperature of 4°C, an abnormal temperature for animal cells and one at which cellular homeostasis may be negatively affected. We have compared two synchronization temperatures (4°C and 22°C) to determine the kinetics of RSV entry into human bronchial epithelial cells. Following inoculation, virus entry was halted at different times by the addition of neutralizing antibody or temperature reduction to 4°C. We engineered a virus that encodes an extra viral gene, beta-lactamase fused to the viral phosphoprotein (P), to enable rapid detection after infection initiation. We found that the synchronization temperature used during inoculation determines the site of fusion. Transition from 4°C to 37°C resulted in RSV entry via the endosomal pathway but also induced F-actin disruption and plasma membrane blebbing, whether the cells were inoculated with RSV or not. Transition from 22°C to 37°C resulted in RSV entry by fusion at the plasma membrane and without the F-actin and plasma membrane disruptions. These results suggest that RSV normally enters cells by fusion at the plasma membrane and that the induction of endocytosis by infection synchronization at 4°C may be an artefact caused by distortion of the plasma membrane-supporting cytoskeleton.Author SummaryIn order to understand the overall mechanism driving infection, it is important to determine how the virus enters cells. The pathway that RSV uses to infect cells is unclear. It is a common practice to attach the virions at 4°C to synchronize the viral infection. In this report, we found that warming up primary cultures of undifferentiated normal human bronchial epithelial cells to 37°C from 4°C triggered dramatic changes in their cell membrane and cytoskeleton totally unrelated to the presence of the virions. The assessment of viral content delivery to the cytoplasm using RSV engineered to express BlaM allowed us to find that the virions attached at 4°C or 22°C fused their envelope with endosome or plasma membrane, respectively. Consequently, the entry via endosome after attachment at 4°C is an experimental artefact and RSV infects by fusing its envelope with the plasma membrane. The implications go beyond RSV since the entry of several virus species have been explored by synchronizing the infection after attachment at 4°C.

Evidence for voltage-sensitive, calcium-conducting channels in airway epithelial cells

1995 ◽  
Vol 269 (6) ◽  
pp. C1547-C1556 ◽  
Author(s):  
S. Boitano ◽  
M. L. Woodruff ◽  
E. R. Dirksen
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mechanical Stimulation ◽  
Transmembrane Potential ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
High K ◽  
Epithelial Cultures ◽  
K Concentration ◽  
Fluorescence Imaging Microscopy

In airways epithelial cultures, mechanical stimulation induces intracellular Ca2+ concentration ([Ca2+]i) changes by causing Ca2+ entry and intracellular Ca2+ release. Mechanically induced Ca2+ fluxes across the plasma membrane are blocked by Ni2+ (Boitano, S., M. J. Sanderson, and E. R. Dirksen. J. Cell. Sci. 107: 3037-3044, 1994). In this report we use fluorescence imaging microscopy with fura 2 and intracellular recording of the transmembrane potential to further characterize Ca2+ flux in the plasma membrane of these cells. Mechanically induced Ca2+ influx is blocked by nifedipine. Addition of the dihydropyridine agonist BAY K8644 (2 microM) leads to a delayed increase of [Ca2+]i that is dependent on extracellular Ca2+. Switching to high extracellular K+ concentration ([K+]o) causes depolarization of the plasma membrane and a transient increase in [Ca2+]i. The number of cells that respond to high [K+]o is significantly decreased by Ni2+ (1 mM) or nifedipine (10 microM). Mechanical stimulation causes a rapid depolarization of the stimulated cell that can be suppressed by the K+ ionophore valinomycin. Valinomycin treatment also blocks mechanically induced Ca2+ dux. These results suggest that voltage-sensitive Ca(2+)-conducting channels exist in airway epithelial cells, and these channels contribute to the [Ca2+]i changes observed after mechanical stimulation or depolarization of the plasma membrane.

scholarly journals Heterocellular gap junctional communication between alveolar epithelial cells

2001 ◽  
Vol 280 (6) ◽  
pp. L1085-L1093 ◽  
Author(s):  
Valsamma Abraham ◽  
Michael L. Chou ◽  
Philip George ◽  
Patricia Pooler ◽  
Aisha Zaman ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Gap Junctions ◽  
Gap Junction ◽  
Alveolar Epithelial Cells ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Gap Junction Channels ◽  
Alveolar Epithelial ◽  
Junction Protein

We analyzed the pattern of gap junction protein (connexin) expression in vivo by indirect immunofluorescence. In normal rat lung sections, connexin (Cx)32 was expressed by type II cells, whereas Cx43 was more ubiquitously expressed and Cx46 was expressed by occasional alveolar epithelial cells. In response to bleomycin-induced lung injury, Cx46 was upregulated by alveolar epithelial cells, whereas Cx32 and Cx43 expression were largely unchanged. Given that Cx46 may form gap junction channels with either Cx43 or Cx32, we examined the ability of primary alveolar epithelial cells cultured for 6 days, which express Cx43 and Cx46, to form heterocellular gap junctions with cells expressing other connexins. Day 6 alveolar epithelial cells formed functional gap junctions with other day 6 cells or with HeLa cells transfected with Cx43 (HeLa/Cx43), but they did not communicate with HeLa/Cx32 cells. Furthermore, day 6alveolar epithelial cells formed functional gap junction channels with freshly isolated type II cells. Taken together, these data are consistent with the notion that type I and type II alveolar epithelial cells communicate through gap junctions compatible with Cx43.

Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions

2003 ◽  
Vol 83 (4) ◽  
pp. 1359-1400 ◽  
Author(s):  
JUAN C. SÁEZ ◽  
VIVIANA M. BERTHOUD ◽  
MARÍA C. BRAÑES ◽  
AGUSTÍN D. MARTÍNEZ ◽  
ERIC C. BEYER
Keyword(s):  
Plasma Membrane ◽  
Gap Junction ◽  
Protein Trafficking ◽  
Genetic Diseases ◽  
Physiological Role ◽  
Channel Structure ◽  
Membrane Channels ◽  
Intercellular Signaling ◽  
Gap Junction Channels ◽  
Extracellular Milieu

Sáez, Juan C., Viviana M. Berthoud, María C. Brañes, Agustín D. Martínez, and Eric C. Beyer. Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions. Physiol Rev 83: 1359-1400, 2003; 10.1152/physrev.00007.2003.—Members of the connexin gene family are integral membrane proteins that form hexamers called connexons. Most cells express two or more connexins. Open connexons found at the nonjunctional plasma membrane connect the cell interior with the extracellular milieu. They have been implicated in physiological functions including paracrine intercellular signaling and in induction of cell death under pathological conditions. Gap junction channels are formed by docking of two connexons and are found at cell-cell appositions. Gap junction channels are responsible for direct intercellular transfer of ions and small molecules including propagation of inositol trisphosphate-dependent calcium waves. They are involved in coordinating the electrical and metabolic responses of heterogeneous cells. New approaches have expanded our knowledge of channel structure and connexin biochemistry (e.g., protein trafficking/assembly, phosphorylation, and interactions with other connexins or other proteins). The physiological role of gap junctions in several tissues has been elucidated by the discovery of mutant connexins associated with genetic diseases and by the generation of mice with targeted ablation of specific connexin genes. The observed phenotypes range from specific tissue dysfunction to embryonic lethality.

scholarly journals Gap Junction Channels of Innexins and Connexins: Relations and Computational Perspectives

2019 ◽  
Vol 20 (10) ◽  
pp. 2476 ◽  
Author(s):  
Alejandro Sánchez ◽  
Carlos Castro ◽  
Dora-Luz Flores ◽  
Everardo Gutiérrez ◽  
Pierre Baldi
Keyword(s):  
Gap Junction ◽  
Cell Communication ◽  
Machine Learning Techniques ◽  
Computational Techniques ◽  
Tissue Formation ◽  
Clinical Tool ◽  
Mediated Communication ◽  
Gap Junction Channels ◽  
Learning Techniques ◽  
Cell Propagation

Gap junction (GJ) channels in invertebrates have been used to understand cell-to-cell communication in vertebrates. GJs are a common form of intercellular communication channels which connect the cytoplasm of adjacent cells. Dysregulation and structural alteration of the gap junction-mediated communication have been proven to be associated with a myriad of symptoms and tissue-specific pathologies. Animal models relying on the invertebrate nervous system have exposed a relationship between GJs and the formation of electrical synapses during embryogenesis and adulthood. The modulation of GJs as a therapeutic and clinical tool may eventually provide an alternative for treating tissue formation-related diseases and cell propagation. This review concerns the similarities between Hirudo medicinalis innexins and human connexins from nucleotide and protein sequence level perspectives. It also sets forth evidence of computational techniques applied to the study of proteins, sequences, and molecular dynamics. Furthermore, we propose machine learning techniques as a method that could be used to study protein structure, gap junction inhibition, metabolism, and drug development.

scholarly journals Porcine aortic endothelial gap junctions: identification and permeation by caged InsP3

1996 ◽  
Vol 109 (7) ◽  
pp. 1765-1773 ◽  
Author(s):  
T.D. Carter ◽  
X.Y. Chen ◽  
G. Carlile ◽  
E. Kalapothakis ◽  
D. Ogden ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Gap Junctions ◽  
Gap Junction ◽  
Primary Cultures ◽  
Western Blots ◽  
Aortic Endothelial Cells ◽  
Gap Junction Channels ◽  
Connexin 37 ◽  
Porcine Aortic Endothelial Cells ◽  
Aortic Endothelial

Gap junction channels permit the direct intercellular transfer of ions and small molecules and allow electrotonic coupling within tissues. Porcine aortic endothelial cells were extensively coupled, as assessed by gap junctional transfer of Lucifer yellow and the fluorescent calcium indicators fluo-3 and furaptra, but were not permeable to rhodamine B isothiocyanate-dextran 10S. The subunit composition of gap junction channels of porcine aortic endothelial cells was characterised using both northern blot analysis and RT-PCR techniques. Messenger RNA encoding connexins 37 and 43, but not 26, 32 or 40, were found in freshly isolated and cultured porcine aortic endothelial cells. Western blots using antipeptide antibodies raised to unique sequences of connexins 37, 40 and 43 showed the presence of connexins 37 and 43, but no connexin 40 was detected. Immunostaining with anticonnexin 43 antibodies showed extensive punctate fluorescent decoration of contacting membranes, whilst antibodies to connexin 37 showed predominantly intracellular staining. Caged InsP3 was found to readily permeate endothelial gap junctions. These results show that primary cultures of porcine aortic endothelial cells express connexin 37 and 43, and provide strong evidence that the second messenger molecule InsP3 can permeate porcine endothelial gap junctions.

scholarly journals ATP Release Mechanisms in Primary Cultures of Epithelia Derived from the Cysts of Polycystic Kidneys

1999 ◽  
Vol 10 (2) ◽  
pp. 218-229
Author(s):  
PATRICIA D. WILSON ◽  
JEFFREY S. HOVATER ◽  
CASH C. CASEY ◽  
JAMES A. FORTENBERRY ◽  
ERIK M. SCHWIEBERT
Keyword(s):  
Epithelial Cells ◽  
Primary Cultures ◽  
Atp Release ◽  
Cyst Fluid ◽  
Extracellular Nucleotides ◽  
Channel Blockers ◽  
Ion Channel Blockers ◽  
Autosomal Dominant Polycystic Kidney ◽  
Cyst Fluids

Abstract. Autosomal dominant polycystic kidney disease (ADPKD) cyst enlargement is exacerbated by accumulation of fluid within the lumen of the cyst. Extracellular nucleotides and nucleosides stimulate fluid and chloride (Cl-) secretion across epithelia and are potent autocrine and paracrine agonists within tissues. This study tests the hypothesis that ATP may be released by ADPKD epithelial cells. Once released, extracellular nucleotides and their metabolites may become “trapped” in the cyst lumen. As a consequence, extracellular ATP may augment ADPKD cyst enlargement through stimulation of salt and water secretion across ADPKD epithelia that encapsulate ADPKD cysts. To test this hypothesis, bioluminescence detection assays of ATP released from primary cultures of human ADPKD epithelial cells were compared with non-ADPKD human epithelial primary cultures. ADPKD cultures release comparable or greater amounts of ATP than non-ADPKD cultures derived from proximal tubule or cortex. ATP release in both ADPKD and non-ADPKD primary epithelial monolayers was directed largely into the apical medium; however, basolateral-directed ATP release under basal and stimulated conditions was also observed. Hypotonicity potentiated ATP release into the apical and basolateral medium in a reversible manner. Reconstitution of isotonic conditions with specific osmoles or inhibition with mechanosensitive ion channel blockers dampened hypotonicity-induced ATP release. “Flashfrozen” cyst fluids from ADPKD cysts, harvested from multiple donor kidneys, were screened by luminometry. A subset of cyst fluids contained as much as 0.5 to 10 μM ATP, doses sufficient to stimulate purinergic receptors. Taken together, these results show that ADPKD and non-ADPKD human epithelial primary cultures release ATP under basal and stimulated conditions and that ATP is released in vitro and into the cyst fluid by cystic epithelial cells in concentrations sufficient to stimulate ATP receptors. It is hypothesized that extracellular nucleotide release and signaling may contribute detrimentally to the gradual expansion of cyst fluid volume that is a hallmark of ADPKD.

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