Potassium channels in primary cultures of seawater fish gill cells. II. Channel activation by hypotonic shock

2000 ◽  
Vol 279 (5) ◽  
pp. R1659-R1670 ◽  
Author(s):  
C. Duranton ◽  
E. Mikulovic ◽  
M. Tauc ◽  
M. Avella ◽  
P. Poujeol
Keyword(s):  
Cellular Localization ◽  
Present Report ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Scorpion Venom ◽  
Convincing Evidence ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Hypotonic Shock ◽  
Apical Side

Previous studies performed on apical membranes of seawater fish gills in primary culture have demonstrated the existence of stretch-activated K+channels with a conductance of 122 pS. The present report examines the involvement of K+ channels in ion transport mechanisms and cell swelling. In the whole cell patch-clamp configuration, K+ currents were produced by exposing cells to a hypotonic solution or to 1 μM ionomycin. These K+ currents were inhibited by the addition of quinidine and charybdotoxin to the bath solution. Isotopic efflux measurements were performed on cells grown on permeable supports using 86Rb+ as a tracer to indicate potassium movements. Apical and basolateral membrane86Rb effluxes were stimulated by the exposure of cells to a hypotonic medium. During the hypotonic shock, the stimulation of86Rb efflux on the apical side of the monolayer was inhibited by 500 μM quinidine or 100 μM gadolinium but was insensitive to scorpion venom [ Leirus quinquestriatus hebraeus (LQH)]. An increased 86Rb efflux across the basolateral membrane was also reduced by the addition of quinidine and LQH venom but was not modified by gadolinium. Moreover, basolateral and apical membrane 86Rb effluxes were not modified by bumetanide or thapsigargin. There is convincing evidence for two different populations of K+ channels activated by hypotonic shock. These populations can be separated according to their cellular localization (apical or basolateral membrane) and as a function of their kinetic behavior and pharmacology.

Water-permeable and -impermeable barriers of snake distal tubules

1984 ◽  
Vol 246 (3) ◽  
pp. F290-F299 ◽  
Author(s):  
K. W. Beyenbach
Keyword(s):  
Epithelial Cell ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Cell Swelling ◽  
Transepithelial Transport ◽  
Pressure Gradients ◽  
Transport Pathway ◽  
Luminal Membrane ◽  
Apical Side ◽  
Distal Tubules

Isolated perfused snake distal tubules transport Na from lumen bath via an amiloride-sensitive transport pathway. Elevation of the luminal Na concentration from 16 to 150 mM leads to non-steady-state electrical behavior and cell swelling. To elucidate the mechanism of cell swelling, the water permeabilities of the epithelium and its apical and basolateral membrane were assessed. Distal tubules were found to be virtually impermeable to transepithelial water flow. Hydraulic conductivity measured 1.2 X 10(-7) cm3 X s-1 X cm-2 X atm-1 in the absence or presence of vasopressin. Effects of transepithelial osmotic pressure gradients on epithelial cell volume revealed the luminal membrane as the water-impermeable cellular barrier and the basolateral membrane as a barrier that is freely permeable to water. Epithelial cell swelling was blocked during perfusion with 150 mM Na when the perfusate also contained amiloride (10(-5) M). These results support the hypothesis that, in the case of transepithelial transport in the presence of high luminal Na concentrations, Na entry across the apical membrane exceeds Na extrusion across the basolateral membrane. Hence, the cells accumulate solute: Na from the apical side and some anion from the apical and/or serosal side. Concomitantly, the epithelial cells swell as water enters across the highly permeable basolateral membrane.

Potassium channels in primary cultures of seawater fish gill cells. I. Stretch-activated K+ channels

2000 ◽  
Vol 279 (5) ◽  
pp. R1647-R1658 ◽  
Author(s):  
C. Duranton ◽  
E. Mikulovic ◽  
M. Tauc ◽  
M. Avella ◽  
P. Poujeol
Keyword(s):  
Potassium Channels ◽  
Primary Cultures ◽  
Dicentrarchus Labrax ◽  
Sea Bass ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Ionic Substitution ◽  
Gill Cells

Previous studies using the patch-clamp technique demonstrated the presence of a small conductance Cl− channel in the apical membrane of respiratory gill cells in primary culture originating from sea bass Dicentrarchus labrax. We used the same technique here to characterize potassium channels in this model. A K+ channel of 123 ± 3 pS was identified in the cell-attached configuration with 140 mM KCl in the bath and in the pipette. The activity of the channel declined rapidly with time and could be restored by the application of a negative pressure to the pipette (suction) or by substitution of the bath solution with a hypotonic solution (cell swelling). In the excised patch inside-out configuration, ionic substitution demonstrated a high selectivity of this channel for K+ over Na+ and Ca2+. The mechanosensitivity of this channel to membrane stretching via suction was also observed in this configuration. Pharmacological studies demonstrated that this channel was inhibited by barium (5 mM), quinidine (500 μM), and gadolinium (500 μM). Channel activity decreased when cytoplasmic pH was decreased from 7.7 to 6.8. The effect of membrane distension by suction and exposure to hypotonic solutions on K+ channel activity is consistent with the hypothesis that stretch-activated K+ channels could mediate an increase in K+ conductance during cell swelling.

Basolateral chloride current in human airway epithelia

2007 ◽  
Vol 293 (4) ◽  
pp. L991-L999 ◽  
Author(s):  
Omar A. Itani ◽  
Fred S. Lamb ◽  
James E. Melvin ◽  
Michael J. Welsh
Keyword(s):  
Cystic Fibrosis ◽  
Signaling Pathways ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Airway Epithelia ◽  
Human Airway ◽  
Well Differentiated ◽  
Human Airway Epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl− absorption. However, the pathways for basolateral Cl− exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl− conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl− conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl− current that was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl− > Br− >> I−. Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca2+ and inducing cell swelling had no effect. The basolateral Cl− current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl− currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl− conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl− conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl− movement.

Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium

2005 ◽  
Vol 288 (4) ◽  
pp. G705-G717 ◽  
Author(s):  
Xing-He Weng ◽  
Klaus W. Beyenbach ◽  
Andrea Quaroni
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Immunofluorescent Staining ◽  
Normal Epithelium ◽  
Ussing Chambers ◽  
Apical Side ◽  
Structural And Functional Properties

The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by RT-PCR, Western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes, and tight junctions expressing zonula occludens-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone, and insulin, monolayer transepithelial voltage was −6.8 mV (apical side), transepithelial resistance was 1,050 Ω·cm2, and short-circuit current ( Isc) was 8.1 μA/cm2. Transcellular and paracellular resistances were estimated as 14.8 and 1.1 kΩ·cm2, respectively. Serosal ouabain reduced voltage and current toward zero, as did apical amiloride. The presence of mRNA of α-epithelial Na+ channel (ENaC) was confirmed. Na-d-glucose cotransport was identified with an antibody to Na+-glucose cotransporter (SGLT) 1. The unidirectional mucosa-to-serosa Na+ flux (19 nmol·min−1·cm−2) was two times as large as the reverse flux, and net transepithelial Na+ flux was nearly double the amiloride-sensitive Isc. In plain Ringer solution, the amiloride-sensitive Isc went toward zero. Under these conditions plus mucosal amiloride, serosal dibutyryl-cAMP elicited a Cl−-dependent Isc consistent with the stimulation of transepithelial Cl− secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na+ absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl− secretion activated by cAMP.

Inhibition of Na transport by 2-chloroadenosine: dissociation from production of cyclic nucleotides

1990 ◽  
Vol 68 (10) ◽  
pp. 1357-1362
Author(s):  
Russell F. Husted ◽  
Gerard P. Clancy ◽  
Abigail Adams-Brotherton ◽  
John B. Stokes
Keyword(s):  
Adenosine Receptors ◽  
Cyclic Nucleotides ◽  
Cell Function ◽  
Apical Membrane ◽  
Second Messengers ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Good Evidence ◽  
Camp Content ◽  
Adenosine Transport

The adenosine analogue 2-chloroadenosine (2-CA) is often used to determine the biologic effects of adenosine because 2-CA is less susceptible to degradation than adenosine. We studied the effects of 2-CA on primary cultures of rat inner medullary collecting ducts because there is good evidence that adenosine can influence cell function through its effects on second messengers. 2-CA inhibited Na+ transport across the apical membrane and increased cAMP content of the cells. The major adenosine receptors in these cells appear to be the stimulatory (A2) type. Stimulation of cAMP by 2-CA was more potent when applied to the apical membrane than to the basolateral membrane, an effect opposite to that of vasopressin. These results imply that adenosine receptors are more numerous or more effective on the apical membrane than on the basolateral membrane. Inhibition of Na+ transport was probably not mediated by an adenosine receptor as evidenced by (i) a lack of effect of adenosine and other adenosine analogues on Na+ transport; (ii) a lack of effect of nonmetabolizable cyclic nucleotides on Na+ transport; and (iii) a clear discrepancy in the temporal course of 2-CA effects on a second messenger system (cAMP) and 2-CA inhibition of Na+ transport. Dipyridimole, an inhibitor of adenosine transport, also reduced Na+ transport. Taken together, the data suggest that 2-CA inhibits Na+ transport by interfering with adenosine transport or metabolism.Key words: cAMP, cGMP, 2-chloroadenosine, vasopressin, Na+ transport, dipyridimole, adenosine metabolism.

scholarly journals Medicinal Leech CNS as a Model for Exosome Studies in the Crosstalk between Microglia and Neurons

2018 ◽  
Vol 19 (12) ◽  
pp. 4124 ◽  
Author(s):  
Antonella Raffo-Romero ◽  
Tanina Arab ◽  
Issa Al-Amri ◽  
Francoise Le Marrec-Croq ◽  
Christelle Van Camp ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Present Report ◽  
Primary Cultures ◽  
Cell Communication ◽  
Medicinal Leech ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
A Cell

In healthy or pathological brains, the neuroinflammatory state is supported by a strong communication involving microglia and neurons. Recent studies indicate that extracellular vesicles (EVs), including exosomes and microvesicles, play a key role in the physiological interactions between cells allowing central nervous system (CNS) development and/or integrity. The present report used medicinal leech CNS to investigate microglia/neuron crosstalk from ex vivo approaches as well as primary cultures. The results demonstrated a large production of exosomes from microglia. Their incubation to primary neuronal cultures showed a strong interaction with neurites. In addition, neurite outgrowth assays demonstrated microglia exosomes to exhibit significant neurotrophic activities using at least a Transforming Growth Factor beta (TGF-β) family member, called nGDF (nervous Growth/Differentiation Factor). Of interest, the results also showed an EV-mediated dialog between leech microglia and rat cells highlighting this communication to be more a matter of molecules than of species. Taken together, the present report brings a new insight into the microglia/neuron crosstalk in CNS and would help deciphering the molecular evolution of such a cell communication in brain.

Abstract 99: Hypotonic Swelling Promotes Nitric Oxide Release in Cardiac Ventricular Myocytes

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Luis Gonano ◽  
Malena Morell ◽  
Juan I Burgos ◽  
Martin Vila Petroff
Keyword(s):  
Nitric Oxide ◽  
Cardiac Myocytes ◽  
Cardiac Myocyte ◽  
Contractile Function ◽  
Cell Swelling ◽  
Contractile Dysfunction ◽  
Ischemia And Reperfusion ◽  
Hypotonic Solution ◽  
No Release ◽  
Hypotonic Swelling

Cardiac myocyte swelling occurs in multiple pathological situations and in particular contributes to the deleterious effects of ischemia and reperfusion by promoting contractile dysfunction. We investigated whether hypotonic swelling promotes nitric oxide (NO) release in cardiac myocytes and if so, whether it impacts on swelling induced contractile dysfunction. Perfusing rat cardiac myocytes, loaded with the NO sensor DAF-FM, with a hypotonic solution (HS; 217 mOsm), increased cell volume, reduced myocyte contraction and Ca2+ transient amplitude and significantly increased DAF-FM fluorescence. When cells were exposed to the HS supplemented with 2.5 mM of the NO synthase inhibitor L-NAME, cell swelling occurred in the absence of NO release. Swelling-induced NO release was also prevented by the NOS1 inhibitor, Nitroguanidine. In addition, Colchicine (an inhibitor of microtubule polymerization) prevented the increase in DAF-FM fluorescence induced by HS indicating that microtubule integrity is necessary for swelling-induced NO release. The swelling-induced negative inotropic effect was exacerbated in the presence of either L-NAME, Nitroguandine or the guanylate cyclase inhibitor, ODQ, suggesting that NOS1-derived NO provides contractile support via a GMP-dependent mechanism. Indeed, ODQ reduced Ca2+ wave velocity and the HS-induced increment in ryanodine receptor (RyR2) phosphorylation at site Ser2808 suggesting that in the context of hypotonic swelling, cGMP may contribute to preserve contractile function by enhancing SR Ca2+ release. Our findings suggest a novel mechanism for NO release in cardiac myocytes with putative pathophysiological relevance in the context of ischemia and reperfusion, where it may be cardioprotective by reducing the extent of contractile dysfunction associated with hypotonic swelling.

Convergence of apical and basolateral endocytic pathways at apical late endosomes in absorptive cells of suckling rat ileum in vivo

1990 ◽  
Vol 97 (2) ◽  
pp. 385-394
Author(s):  
M. Fujita ◽  
F. Reinhart ◽  
M. Neutra
Keyword(s):  
Basolateral Membrane ◽  
Hydrolytic Enzymes ◽  
Membrane Domains ◽  
Rat Ileum ◽  
Absorptive Cells ◽  
Apical Side ◽  
Late Endosomes ◽  
Membrane Components ◽  
Endocytic Pathways

Absorptive cells of the intestinal epithelium endocytose proteins from both apical and basolateral membrane domains. In absorptive cells of suckling rat ileum, luminal protein tracers first enter an apical tubulovesicular endosomal system, then enter larger apical endosomal vesicles and multivesicular bodies (MVB), and finally are delivered to a giant supranuclear lysosomal vacuole. To determine whether proteins endocytosed from the basolateral domain in vivo enter the same endosomal or lysosomal compartments as those taken up from the apical side, we simultaneously applied cationized ferritin (CF) apically (by intra-luminal injection) and horseradish peroxidase (HRP) basally (by intravenous injection), and examined absorptive cells after 3 min to 60 min using light, electron and fluorescence microscopy. At early times, CF and HRP entered separate endosomal compartments at apical and basolateral poles. At no time did HRP enter the apical tubulovesicular system, and CF never entered early basolateral endosomes. After 15 min, however, both tracers appeared together in large late endosomes and MVB located apically, above the giant vacuole. From 15 to 60 min both tracers accumulated in the giant vacuole. Membranes of some apical late endosomes, all apical MVB, the giant vacuole, and occasional sub-nuclear vesicles contained immunoreactive Igp120, a glycoprotein specific to late compartments of the endosome-lysosome system. These results show that highly polarized intestinal epithelial cells have separate apical and basolateral early endosomal compartments, presumably to maintain distinct membrane domains while allowing endocytosis and recycling of membrane from both surfaces. Apical and basolateral endocytic pathways, and presumably vesicles delivering hydrolytic enzymes and lysosomal membrane components, converge at the apical late endosome.

Abstract 423: Caveolin-3 Enriches and Dynamically Interacts With Swell1

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Daniel G Turner ◽  
Leonid Tyan ◽  
Sami Stroebel ◽  
Frank Deguire ◽  
Di Lang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Hek293 Cells ◽  
Scaffolding Protein ◽  
Cell Swelling ◽  
Hypotonic Solution ◽  
Hek 293 ◽  
Dynamic Relationship ◽  
293 Cells ◽  
Fret Efficiency ◽  
Oxide Synthase

Caveolae are small (50-100nm) membrane invaginations formed by caveolin proteins enriched with cholesterol and lipids. Caveolae play a crucial role in mechanoprotection and mechano-electrical transduction by buffering membrane tension and facilitating activation of mechanosensitive ion channels, including a recently discovered swelling-activated chloride channel SWELL1 (also known as LRRC8A). However, the dynamic relationship between the muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) and SWELL1 is poorly understood. The objective of this study was to determine how Cav3 interacts with SWELL1 channels and modulates their activity during mechano-electrical transduction. In HEK 293 cells transfected with Cav3, co-immunoprecipitation analysis recapitulated cardiac data showing association between SWELL1 and Cav3. Using transiently expressed Cav3-GFP and SWELL1-mCherry fusion proteins in HEK293 cells, we observed a high FRET efficiency between the two proteins in an isotonic (1T) solution, confirming their close (<5nm) proximity. In a hypotonic solution (0.7T, mimicking cell stretch), FRET efficiency decreased two-fold. Furthermore, FRET efficiency decreased two-fold to control levels when incubated with methyl-beta cyclodextrin, a cholesterol solubilizer. These data suggest that the relationship between Cav3 and SWELL1 is dependent on membrane mechanical tension and caveolae lipid raft integrity. Interestingly, in transfected cells, SWELL1 protein expression and whole cell swelling-activated chloride current ( I Cl,swell ) were increased four-fold and two-fold, respectively, while mRNA expression was reduced two-fold. This may indicate that caveolae formed by Cav3 expression enrich for SWELL1 and increase their half-life, thus requiring lower mRNA availability despite higher protein expression. Our findings indicate a close dynamic interplay between Cav3 and SWELL1, with a strong regulatory action of Cav3 on I Cl,swell activity. Given that I Cl,swell increases and interprotein FRET efficiency decreases in hypotonic solution, it is likely that Cav3 inhibits the activation of SWELL1 similarly to its known inhibition of nitric oxide synthase.

Sign in / Sign up

Export Citation Format

Share Document