Development of Gap Junctions in Hippocampal Astrocytes: Evidence That Whole Cell Electrophysiological Phenotype Is an Intrinsic Property of the Individual Cell

2006 ◽  
Vol 96 (3) ◽  
pp. 1383-1392 ◽  
Author(s):  
Gary P. Schools ◽  
Min Zhou ◽  
Harold K. Kimelberg
Keyword(s):  
Gap Junctions ◽  
Ion Channel ◽  
Gap Junction ◽  
Time Course ◽  
Expression Profiles ◽  
Dye Coupling ◽  
Cell Coupling ◽  
Whole Cell ◽  
Channel Expression ◽  
Mature Astrocyte

Gap junction communication between astrocytes is prevalent and has been proposed to be involved in several astrocyte functions. One such proposal involves gap junctions in potassium spatial buffering. However, little is known about the developmental time course of gap junction coupling and how much the syncytium affects whole cell measurements of ion currents. Our previous work described three types of hippocampal astrocyte, each with a distinct electrophysiological profile when recorded in whole cell voltage-clamp mode. In the current study we correlated post–whole cell recording immunohistochemistry for GLAST and the spread of injected dye from the recorded cell with the measured electrophysiological phenotype to quantify cell coupling of astrocytes and the type of astrocyte coupled, in the rat hippocampus. We found that passive astrocytes, which predominate after 3 wk postnatally, have much lower membrane resistances ( Rm) and are more frequently dye coupled and to more cells, than outwardly and variably rectifying astrocytes that predominate in early postnatal development. Dye coupling in GLAST(+) cells was first detected in the first postnatal week and the degree of coupling peaked before the complete transition to the low Rm, passive electrophysiological type. Also, the degree of dye coupling did not correlate with the passive electrophysiological phenotype. Passive cells were also detected after pretreatment with a gap junction inhibitor. Further evidence that cell coupling does not contribute to the mature astrocyte electrophysiological phenotype came from recording of excised membrane patches, which predominantly corresponded to the ion channel expression profiles of their cells of origin. These findings imply that in the hippocampus, interastrocyte cell coupling likely contributes little to the overall whole cell current profile of diverse glia, and the electrophysiological passivity reflects the intrinsic ion channel expression of the mature astrocyte.

scholarly journals Gap junction structures after experimental alteration of junctional channel conductance.

1985 ◽  
Vol 101 (5) ◽  
pp. 1741-1748 ◽  
Author(s):  
T M Miller ◽  
D A Goodenough
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Electron Micrographs ◽  
Time Course ◽  
Structural Changes ◽  
Lucifer Yellow ◽  
Freeze Fracture ◽  
Channel Conductance ◽  
Experimental Alteration ◽  
Quick Freezing

Gap junctions are known to present a variety of different morphologies in electron micrographs and x-ray diffraction patterns. This variation in structure is not only seen between gap junctions in different tissues and organisms, but also within a given tissue. In an attempt to understand the physiological meaning of some aspects of this variability, gap junction structure was studied following experimental manipulation of junctional channel conductance. Both physiological and morphological experiments were performed on gap junctions joining stage 20-23 chick embryo lens epithelial cells. Channel conductance was experimentally altered by using five different experimental manipulations, and assayed for conductance changes by observing the intercellular diffusion of Lucifer Yellow CH. All structural measurements were made on electron micrographs of freeze-fracture replicas after quick-freezing of specimens from the living state; for comparison, aldehyde-fixed specimens were measured as well. Analysis of the data generated as a result of this study revealed no common statistically significant changes in the intrajunctional packing of connexons in the membrane plane as a result of experimental alteration of junctional channel conductance, although some of the experimental manipulations used to alter junctional conductance did produce significant structural changes. Aldehyde fixation caused a dramatic condensation of connexon packing, a result not observed with any of the five experimental uncoupling conditions over the 40-min time course of the experiments.

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 Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions

2001 ◽  
Vol 154 (1) ◽  
pp. 217-230 ◽  
Author(s):  
X. Xu ◽  
W.E.I. Li ◽  
G.Y. Huang ◽  
R. Meyer ◽  
T. Chen ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Neural Crest ◽  
Cell Motility ◽  
Gap Junction ◽  
Connexin 43 ◽  
Neural Crest Cell ◽  
Neural Crest Cells ◽  
Functional Coupling ◽  
Dye Coupling ◽  
Crest Cell

Connexin 43 (Cx43α1) gap junction has been shown to have an essential role in mediating functional coupling of neural crest cells and in modulating neural crest cell migration. Here, we showed that N-cadherin and wnt1 are required for efficient dye coupling but not for the expression of Cx43α1 gap junctions in neural crest cells. Cell motility was found to be altered in the N-cadherin–deficient neural crest cells, but the alterations were different from that elicited by Cx43α1 deficiency. In contrast, wnt1-deficient neural crest cells showed no discernible change in cell motility. These observations suggest that dye coupling may not be a good measure of gap junction communication relevant to motility. Alternatively, Cx43α1 may serve a novel function in motility. We observed that p120 catenin (p120ctn), an Armadillo protein known to modulate cell motility, is colocalized not only with N-cadherin but also with Cx43α1. Moreover, the subcellular distribution of p120ctn was altered with N-cadherin or Cx43α1 deficiency. Based on these findings, we propose a model in which Cx43α1 and N-cadherin may modulate neural crest cell motility by engaging in a dynamic cross-talk with the cell's locomotory apparatus through p120ctn signaling.

Electrophysiological Characteristics of Reactive Astrocytes in Experimental Cortical Dysplasia

2001 ◽  
Vol 85 (4) ◽  
pp. 1719-1731 ◽  
Author(s):  
A. Bordey ◽  
S. A. Lyons ◽  
J. J. Hablitz ◽  
H. Sontheimer
Keyword(s):  
Gap Junction ◽  
Cortical Layer ◽  
Reactive Gliosis ◽  
Delayed Rectifier ◽  
Nuclear Staining ◽  
Dye Coupling ◽  
Biochemical Alterations ◽  
Gap Junction Coupling ◽  
Channel Expression ◽  
Junction Coupling

Neocortical freeze lesions have been widely used to study neuronal mechanisms underlying hyperexcitability in dysplastic cortex. Comparatively little attention has been given to biophysical changes in the surrounding astrocytes that show profound morphological and biochemical alterations, often referred to as reactive gliosis. Astrocytes are thought to aid normal neuronal function by buffering extracellular K+. Compromised astrocytic K+ buffering has been proposed to contribute to neuronal dysfunction. Astrocytic K+ buffering is mediated, partially, by the activity of inwardly rectifying K+ channels ( K IR) and may involve intracellular redistribution of K+ through gap-junctions. We characterized K+ channel expression and gap-junction coupling between astrocytes in freeze-lesion-induced dysplastic neocortex. Whole cell patch-clamp recordings were obtained from astrocytes in slices from postnatal day (P) 16–P24 rats that had received a freeze-lesion on P1. A marked increase in glial fibrillary acidic protein immunoreactivity was observed along the entire length of the freeze lesion. Clusters of proliferative (bromo-deoxyuridine nuclear staining, BrdU+) astrocytes were seen near the depth of the microsulcus. Astrocytes in cortical layer I surrounding the lesion were characterized by a significant reduction in KIR. BrdU-positive astrocytes near the depth of the microsulcus showed essentially no expression of K IRchannels but markedly enhanced expression of delayed rectifier K+ ( K DR) channels. These proliferative cells showed virtually no dye coupling, whereas astrocytes in the hyperexcitable zone adjacent to the microsulcus displayed prominent dye-coupling as well as large K IR and outward K+ currents. These findings suggest that reactive gliosis is accompanied by a loss of K IR currents and reduced gap junction coupling, which in turn suggests a compromised K+buffering capacity.

VOCCs and TREK-1 ion channel expression in human tenocytes

2007 ◽  
Vol 292 (3) ◽  
pp. C1053-C1060 ◽  
Author(s):  
Merzesh Magra ◽  
Steven Hughes ◽  
Alicia J. El Haj ◽  
Nicola Maffulli
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Ionic Currents ◽  
Cell Types ◽  
Functional Expression ◽  
Tissue Cell ◽  
Pharmacological Management ◽  
Whole Cell ◽  
Voltage Gated ◽  
Channel Expression

Mechanosensitive and voltage-gated ion channels are known to perform important roles in mechanotransduction in a number of connective tissues, including bone and muscle. It is hypothesized that voltage-gated and mechanosensitive ion channels also may play a key role in some or all initial responses of human tenocytes to mechanical stimulation. However, to date there has been no direct investigation of ion channel expression by human tenocytes. Human tenocytes were cultured from patellar tendon samples harvested from five patients undergoing routine total knee replacement surgery (mean age: 66 yr; range: 63–73 yr). RT-PCR, Western blotting, and whole cell electrophysiological studies were performed to investigate the expression of different classes of ion channels within tenocytes. Human tenocytes expressed mRNA and protein encoding voltage-operated calcium channel (VOCC) subunits (Ca α1A, Ca α1C, Ca α1D, Ca α2δ1) and the mechanosensitive tandem pore domain potassium channel (2PK+) TREK-1. They exhibit whole cell currents consistent with the functional expression of these channels. In addition, other ionic currents were detected within tenocytes consistent with the expression of a diverse array of other ion channels. VOCCs and TREK channels have been implicated in mechanotransduction signaling pathways in numerous connective tissue cell types. These mechanisms may be present in human tenocytes. In addition, human tenocytes may express other channel currents. Ion channels may represent potential targets for the pharmacological management of chronic tendinopathies.

scholarly journals Remodeling of Cardiac Gap Junctional Cell–Cell Coupling

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2422
Author(s):  
Stefan Dhein ◽  
Aida Salameh
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Conduction System ◽  
Cell Coupling ◽  
Connexin Expression ◽  
Gap Junction Channels ◽  
Developmental States ◽  
Gap Junction Channel ◽  
Arrhythmogenic Substrate ◽  
Cell Cell

The heart works as a functional syncytium, which is realized via cell-cell coupling maintained by gap junction channels. These channels connect two adjacent cells, so that action potentials can be transferred. Each cell contributes a hexameric hemichannel (=connexon), formed by protein subuntis named connexins. These hemichannels dock to each other and form the gap junction channel. This channel works as a low ohmic resistor also allowing the passage of small molecules up to 1000 Dalton. Connexins are a protein family comprising of 21 isoforms in humans. In the heart, the main isoforms are Cx43 (the 43 kDa connexin; ubiquitous), Cx40 (mostly in atrium and specific conduction system), and Cx45 (in early developmental states, in the conduction system, and between fibroblasts and cardiomyocytes). These gap junction channels are mainly located at the polar region of the cardiomyocytes and thus contribute to the anisotropic pattern of cardiac electrical conductivity. While in the beginning the cell–cell coupling was considered to be static, similar to an anatomically defined structure, we have learned in the past decades that gap junctions are also subject to cardiac remodeling processes in cardiac disease such as atrial fibrillation, myocardial infarction, or cardiomyopathy. The underlying remodeling processes include the modulation of connexin expression by e.g., angiotensin, endothelin, or catecholamines, as well as the modulation of the localization of the gap junctions e.g., by the direction and strength of local mechanical forces. A reduction in connexin expression can result in a reduced conduction velocity. The alteration of gap junction localization has been shown to result in altered pathways of conduction and altered anisotropy. In particular, it can produce or contribute to non-uniformity of anisotropy, and thereby can pre-form an arrhythmogenic substrate. Interestingly, these remodeling processes seem to be susceptible to certain pharmacological treatment.

scholarly journals Are there functional gap junctions or junctional hemichannels in macrophages?

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 328-334 ◽  
Author(s):  
LA Alves ◽  
R Coutinho-Silva ◽  
PM Persechini ◽  
DC Spray ◽  
W Savino ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Gap Junctions ◽  
Cell Line ◽  
Connexin 43 ◽  
Lucifer Yellow ◽  
Dye Coupling ◽  
Dye Uptake ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

Abstract The existence of functional gap junctions in migratory cells of the immune system is a controversial issue. In this report, we have focused on one particular cell type, namely the macrophages, because connexin- 43, a protein that forms gap junctions, has been described in peritoneal macrophages and a macrophage cell line (J774), by Northern and Western blot analysis. To test whether these cell types expressed functional gap junctions, we assayed dye coupling by intracellular injection of Lucifer Yellow. We observed that nonstimulated macrophages are not coupled among themselves and did not form functional gap junctions with an epithelial cell line, which expresses functional gap junctions formed by connexin-43. Dye coupling was also not detected between macrophages previously activated by lipopolysaccharide or interferon-gamma. We further examined the presence of functional coupling using the more sensitive technique of dual whole cell patch- clamp, and again, did not find electrical coupling between macrophages, consistent with the dye microinjection data. We also examined the possible presence of hemigap junction channels activated by extracellular adenosine triphosphate (ATP) using a dye uptake assay and the whole cell patch-clamp technique. Conditions expected to close gap junction hemichannels (exposure to octanol and low intracellular pH) did not decrease ATP-induced Lucifer Yellow uptake, whereas conditions expected to increase hemichannel opening either did not affect ATP permeabilization (dibutyryl adenosine monophosphate) or decreased it (zero extracellular CA+2). Finally, in experiments using resident macrophages derived from conexin-43 knockout mice, we observed ATP induced dye uptake. Our experimental data thus indicate that macrophages in vitro do not form functional gap junctions and that the permeability pathway activated by extracellular ATP is not formed by a hemigap junction channel.

scholarly journals Precise control of ion channel and gap junction expression is required for patterning of the regenerating axolotl limb

2020 ◽  
Vol 64 (10-11-12) ◽  
pp. 485-494
Author(s):  
Konstantinos Sousounis ◽  
Burcu Erdogan ◽  
Michael Levin ◽  
Jessica L. Whited
Keyword(s):  
Ion Channels ◽  
Gap Junctions ◽  
Ion Channel ◽  
Gap Junction ◽  
Regulation Of Gene Expression ◽  
Transcriptional Networks ◽  
Channel Proteins ◽  
Junction Protein ◽  
Ion Channel Proteins ◽  
Voltage Gradients

Axolotls and other salamanders have the capacity to regenerate lost tissue after an amputation or injury. Growth and morphogenesis are coordinated within cell groups in many contexts by the interplay of transcriptional networks and biophysical properties such as ion flows and voltage gradients. It is not, however, known whether regulators of a cell’s ionic state are involved in limb patterning at later stages of regeneration. Here we manipulated expression and activities of ion channels and gap junctions in vivo, in axolotl limb blastema cells. Limb amputations followed by retroviral infections were performed to drive expression of a human gap junction protein Connexin 26 (Cx26), potassium (Kir2.1-Y242F and Kv1.5) and sodium (NeoNav1.5) ion channel proteins along with EGFP control. Skeletal preparation revealed that overexpressing Cx26 caused syndactyly, while overexpression of ion channel proteins resulted in digit loss and structural abnormalities compared to EGFP expressing control limbs. Additionally, we showed that exposing limbs to the gap junction inhibitor lindane during the regeneration process caused digit loss. Our data reveal that manipulating native ion channel and gap junction function in blastema cells results in patterning defects involving the number and structure of the regenerated digits. Gap junctions and ion channels have been shown to mediate ion flows that control the endogenous voltage gradients which are tightly associated with the regulation of gene expression, cell cycle progression, migration, and other cellular behaviors. Therefore, we postulate that mis-expression of these channels may have disturbed this regulation causing uncoordinated cell behavior which results in morphological defects.

cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture

1989 ◽  
Vol 257 (1) ◽  
pp. C1-C11 ◽  
Author(s):  
J. C. Saez ◽  
W. A. Gregory ◽  
T. Watanabe ◽  
R. Dermietzel ◽  
E. L. Hertzberg ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Spherical Shape ◽  
Rat Hepatocytes ◽  
Dye Coupling ◽  
Cyclic Monophosphate ◽  
Junction Protein ◽  
Gap Junction Protein ◽  
Alpha Amanitin ◽  
Cells Cultured

Freshly isolated adult rat hepatocytes were found to be coupled through gap junctions, but coupling decreased abruptly 5-8 h after plating the cells on plastic culture dishes in physiological saline containing insulin and fetal calf serum. Loss of intercellular coupling was associated with disappearance of 27-kDa gap junction protein and of gap junctions seen by electron microscopy or immunocytochemistry. This disappearance of coupling was delayed approximately 8 h by treatment of the cultures with membrane permeant adenosine 3',5'-cyclic monophosphate (cAMP) [but not guanosine 3',5'-cyclic monophosphate (cGMP)] derivatives. Levels of gap junction protein and anatomically identified junctions were also maintained by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Level of mRNA encoding the gap junction protein was maintained longer in cells treated with 8-BrcAMP than in untreated cells, but 8-BrcAMP did not detectably increase the transcription rate. Thus prolongation of coupling must result at least partially from extension of the lifetime of gap junction mRNA, allowing translation of message and assembly of channels for a longer period after plating. Treatment of cells with mRNA or protein synthesis inhibitors (alpha-amanitin and cycloheximide) prolonged coupling to a similar extent as did treatment with 8-BrcAMP. alpha-Amanitin blocked transcription of gap junction mRNA, but levels of cytoplasmic mRNA encoding the 27-kDa gap junction protein were maintained, presumably by block of transcription of an mRNA degrading factor. The factor is probably a protein, since a similar effect on mRNA level was produced in cycloheximide-treated cells. Cells cultured in control medium were also observed to flatten as they became uncoupled, whereas cells cultured for as long as 16 h in elevated 8-BrcAMP remained round and well coupled. The correlation between shape and coupling strength was not obtained after treatment with the microtubule stabilizing agent, taxol, which maintained the spherical shape of the cells but did not delay the disappearance of dye coupling. Nocodazole, which blocks microtubule formation, also maintained the spherical shape of the cells but delayed the disappearance of dye coupling. In addition to gating by covalent modification or other mechanisms, hormones and drugs that alter the intracellular cAMP concentration may affect intercellular communication by changing the lifetime of the mRNA encoding the main gap junction protein, thereby decreasing or increasing its synthesis. In addition, cAMP may act by decreasing removal of junctions from appositional membranes.

Dye coupling in horizontal cells of developing rabbit retina

2000 ◽  
Vol 17 (2) ◽  
pp. 255-262 ◽  
Author(s):  
DIANNA A. JOHNSON ◽  
STEPHEN L. MILLS ◽  
MICHAEL F. HABERECHT ◽  
STEPHEN C. MASSEY
Keyword(s):  
Gap Junctions ◽  
Lucifer Yellow ◽  
Horizontal Cell ◽  
Plexiform Layer ◽  
Cell Types ◽  
Outer Plexiform Layer ◽  
Horizontal Cells ◽  
Rabbit Retina ◽  
Dye Coupling ◽  
Cell Coupling

In the mature rabbit retina, two classes of horizontal cells, A type and B type, provide lateral inhibition in the outer plexiform layer (OPL) and spatially modify the activation of bipolar cells by photoreceptors. Gap junctions connecting homologous horizontal cells determine the extent to which this inhibitory activity spreads laterally across the OPL. Little is currently known about the expression of gap junctions in horizontal cells during postnatal development or how cell–cell coupling might contribute to subsequent maturational events. We have examined the morphological attributes and coupling properties of developing A and B type horizontal cells in neonatal rabbit retina using intracellular injections of Lucifer Yellow and Neurobiotin. Prelabeling with DAPI permitted the targeting of horizontal cell bodies for intracellular injection in perfused preparations of isolated retina. A and B type horizontal cells were identifiable at birth although their dendritic field sizes had not reached adult proportions and their synaptic contacts in the OPL were minimal. Both cell types exhibited homologous dye coupling at birth. Similar to that seen in the adult, no heterologous coupling was observed, and homologous coupling among A type cells was stronger than that observed among B type cells. The spread of tracer compounds through gap junctions of morphologically immature horizontal cells suggests that ions and other small, bioactive compounds may likewise spread through coupled, horizontal networks to coordinate the subsequent maturational of emerging outer plexiform layer pathways.

Sign in / Sign up

Export Citation Format

Share Document