Relationship between Na+ current expression and cell-cell coupling in astrocytes cultured from rat hippocampus

1991 ◽  
Vol 65 (4) ◽  
pp. 989-1002 ◽  
Author(s):  
H. Sontheimer ◽  
S. G. Waxman ◽  
B. R. Ransom
Keyword(s):  
Lucifer Yellow ◽  
Na Channel ◽  
Dye Coupling ◽  
Cell Coupling ◽  
Na Current ◽  
Na Currents ◽  
Coupled Cells ◽  
Hippocampal Astrocytes ◽  
Cell Cell

1. Cell-cell coupling between hippocampal astrocytes in culture was studied by following the intracellular spread of the low molecular weight fluorescent dye Lucifer yellow (LY). Dye coupling appeared as early as 24 h after plating, at which time approximately 20% of all astrocytes that physically contacted neighboring cells showed dye coupling. 2. The percentage of coupled cells increased with time in culture and peaked after 10 days in vitro (DIV) when approximately 50% of astrocytes showed coupling. Further time in culture, up to 20 DIV, did not increase the percentage of coupled cells. Thus, coupled and noncoupled astrocytes coexist in hippocampal cultures in approximately equal numbers. 3. Na+ currents were expressed in a subpopulation of hippocampal astrocytes and changed characteristics during in vitro development. A "neuronal type" of Na+ current, so called because of an h alpha curve that had a midpoint near -60 mV, was observed within the first 5 days post-plating. A "glial type" of Na+ current, characterized by a -25 mV shift in its h alpha curve, was only expressed after 6 days in culture. 4. Na+ current expression was restricted to hippocampal astrocytes that did not exhibit dye coupling; astrocytes that exhibited dye coupling (n = 39) did not show measurable Na+ currents. 5. The failure to see Na+ currents in coupled astrocytes cannot be explained by insufficient space-clamp since astrocytes acutely uncoupled with octanol (10 microM) did not reveal Na+ current expression. Control experiments showed that low concentrations of octanol (i.e., 10-100 microM) did not block Na+ currents; blockage of Na+ currents by octanol was only observed at high concentrations (e.g., 50-fold the concentration used for uncoupling). These observations support the idea that Na(+)-channel expression was restricted to noncoupled astrocytes. 6. The time courses for the development of cell coupling and Na+ current expression appeared to be inversely correlated and suggested a gradual increase in cell coupling in concert with a loss in Na+ current expression with time in culture.

Na(+)-current expression in rat hippocampal astrocytes in vitro: alterations during development

1991 ◽  
Vol 65 (1) ◽  
pp. 3-19 ◽  
Author(s):  
H. Sontheimer ◽  
B. R. Ransom ◽  
A. H. Cornell-Bell ◽  
J. A. Black ◽  
S. G. Waxman
Keyword(s):  
Steady State ◽  
Time Constant ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Na Current ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Current Densities ◽  
Hippocampal Astrocytes

1. With the use of whole-cell patch-clamp recording. Na(+)-current expression was studied in hippocampal astrocytes in vitro, individually identified by filling with Lucifer yellow (LY) and staining for glial fibrillary acidic protein (GFAP) and vimentin. 2. The proportion of astrocytes that express Na+ currents in rat hippocampal cultures changes during development in vitro and decreases from approximately 75% at day 1 to approximately 30% after 10 days in culture. 3. The sodium currents expressed in astrocytes can be differentiated into two types on the basis of kinetics. At early times in culture the time course of Na+ currents is fast in both onset and decay with an average decay time constant of 1.27 ms, whereas after 6 days Na+ currents become comparatively slow and decayed with an average time constant of 1.86 ms. 4. As with the time-course of Na+ currents, the two age groups of astrocytes (i.e., days 1-5 and day 6 and older) differ with respect to their steady-state inactivation characteristics. Early after plating and up to day 5, the midpoint of the steady-state inactivation curve is close to -60 mV, as also observed in hippocampal neurons of various ages; in contrast, after 6 days in culture the curve is shifted by approximately 25 mV toward more hyperpolarized potentials with a midpoint close to -85 mV. 5. In contrast to h infinity-curves, current-voltage (I-V) curves of Na(+)-current activation were identical in all astrocytes studied and did not change with time in culture. 6. In astrocytes expressing Na+ currents, current densities (average of 35 pA/pF on day 1) decreased throughout the first 5 days and were almost abolished around days 4 and 5 in culture. Beginning on day 6, however, current densities increased again and maintained a steady level (average of 14 pA/pF) for the duration of the time period in culture (20 days). This biphasic time course closely correlates with the time course of changes in Na(+)-current kinetics and steady-state inactivation. 7. These data suggest that Na+ currents in cultured hippocampal astrocytes show characteristic changes with increasing time in culture. During the first 4–5 days in culture, hippocampal astrocytes display Na+ currents with properties similar to those of hippocampal neurons. Our data further suggest that Na+ currents with distinctive, “glial-type” characteristics are only expressed in hippocampal astrocytes after 6 days in culture.

scholarly journals Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve.

1985 ◽  
Vol 86 (5) ◽  
pp. 739-762 ◽  
Author(s):  
G K Wang ◽  
G Strichartz
Keyword(s):  
Steady State ◽  
Ionic Currents ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Current ◽  
Toxin Concentration ◽  
Toxin Molecule ◽  
Channel Inactivation ◽  
Na Currents ◽  
Voltage Dependent

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.

Gap-junctional communication in a communication-defective and in a communication-competent teratocarcinoma cell line

1985 ◽  
Vol 76 (1) ◽  
pp. 85-95
Author(s):  
C.W. Lo ◽  
D. Fang ◽  
M.L. Hooper
Keyword(s):  
Cell Line ◽  
Coupling Coefficient ◽  
Dye Coupling ◽  
Cell Coupling ◽  
Teratocarcinoma Cell ◽  
Gap Junctional Communication ◽  
Junctional Communication ◽  
Gap Junctional ◽  
Ionic Coupling ◽  
Cell Cell

We examined the gap-junctional communication properties of a communication-defective cell line R5/3 and its communication-competent revertant H2T12. For these studies, we carried out microelectrode impalements to monitor ionic coupling and dye coupling. Our dye-injection experiments revealed that the H2T12 cells are much more efficient in dye coupling than the R5/3 cells. This latter observation is in agreement with the previous finding that the H2T12 cells are much better metabolically coupled than the R5/3 cells. With ionic coupling measurements, however, both cell lines exhibited similar levels of cell-cell coupling. The R5/3 cells demonstrated an ionic coupling coefficient of 0.19 +/− 0.011 (S.E.M.) and H2T12 a coupling coefficient of 0.25 +/− 0.009 (S.E.M.). These results in conjunction with observations from other studies indicate that the different experimental approaches for monitoring gap-junctional communication may have different levels of sensitivity for detecting as opposed to measuring the level of cell-cell coupling.

scholarly journals TGF-β1 facilitates cell–cell communication in osteocytes via connexin43- and pannexin1-dependent gap junctions

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Wenjing Liu ◽  
Demao Zhang ◽  
Xin Li ◽  
Liwei Zheng ◽  
Chen Cui ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Lucifer Yellow ◽  
Cell Communication ◽  
Deep Understanding ◽  
Gap Junctional Intercellular Communication ◽  
Tgf Β1 ◽  
Cell Cell

Abstract Connexins and pannexins are two families of channel forming proteins that are able to pass small molecules to achieve communication between cells. While connexins have been recognized to mediate gap junctional intercellular communication (GJIC), pannexins are far less known. Our previous study reported the potential role of TGF-β1 in mediating of connexins in osteocytes in vitro. Herein, we aimed to elucidate the influence of TGF-β1 on cell–cell communication based on gap junctions assembled by connexins and pannexins in vitro and ex vivo. We first showed that TGF-β1 positively affected the elongation of dendritic processes of osteocytes. Our data indicated that TGF-β1 increased expressions of connexin43 (Cx43) and pannexin1 (panx1), which are indispensable for hemichannel formation in gap junctions, in osteocytes in vitro and ex vivo. TGF-β1 enhanced gap junction formation and impacted cell–cell communication in living osteocytes, as indicated by the scrape loading and Lucifer yellow transfer assays. TGF-β1 enhanced the expressions of Cx43 and panx1 via activation of ERK1/2 and Smad3/4 signalling. The TGF-β1-restored expressions of Cx43 and panx1 in osteocytes in the presence of an ERK inhibitor, U0126, further demonstrated the direct participation of Smad3/4 signalling. TGF-β1 increased the accumulation of Smad3 in the nuclear region (immunofluorescence assay) and promoted the enrichment of Smad3 at the binding sites of the promoters of Gja1 (Cx43) and Panx1 (ChIP assay), thereby initiating the enhanced gene expression. These results provide a deep understanding of the molecular mechanisms involved in the modulation of cell–cell communication in osteocytes induced by TGF-β1.

Altered sodium current response to intracellular fatty acids in halothane-hypersensitive skeletal muscle

1996 ◽  
Vol 271 (1) ◽  
pp. C347-C353 ◽  
Author(s):  
S. J. Wieland ◽  
Q. H. Gong ◽  
J. E. Fletcher ◽  
H. Rosenberg
Keyword(s):  
Skeletal Muscle ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Sodium Current ◽  
Primary Cultures ◽  
Na Channel ◽  
Current Response ◽  
Whole Cell ◽  
Na Currents

Biopsies of human skeletal muscle were analyzed by an in vitro contracture test (IVCT) for responsiveness to a halothane challenge: noncontracting (nonresponsive; IVCT-) and contracting (IVCT+). A muscle biopsy that is IVCT+ indicates potential malignant hyperthermia (MH) susceptibility. Primary cultures were grown from portions of the skeletal muscle biopsies, and voltage-activated currents were measured by whole cell recording in the presence or absence of 2-5 microM intracellular arachidonic or oleic acids. In untreated IVCT- cells, Na+ currents were predominantly tetrodotoxin (TTX) insensitive, indicating that most of the current was carried through the embryonic SkM2 isoform of the Na+ channel. Inclusion of fatty acids in the recording pipette of IVCT- cells produced an increase in voltage-activated Na+ currents during 20 min of recording. Approximately 70% of currents in fatty acid-treated cells were TTX sensitive, indicating activation of the adult SkM1 isoform of the Na+ channel. In contrast to IVCT- cells, IVCT+ cells expressed Na+ currents that were predominantly TTX sensitive even in the absence of added fatty acid, thus showing a relatively large baseline functional expression of SkM1 channels. Addition of fatty acids to the recording pipette produced little further change in the magnitude or TTX sensitivity of the whole cell currents in IVCT+ cells, suggesting altered functional regulation of Na+ channels in MH muscle.

Gap junctions in human umbilical cord endothelial cells contain multiple connexins

1997 ◽  
Vol 272 (1) ◽  
pp. C117-C130 ◽  
Author(s):  
H. Van Rijen ◽  
M. J. van Kempen ◽  
L. J. Analbers ◽  
M. B. Rook ◽  
A. C. van Ginneken ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lucifer Yellow ◽  
Umbilical Vein ◽  
Electrical Conductance ◽  
Distribution Patterns ◽  
Human Umbilical Cord ◽  
Dye Coupling ◽  
Gap Junctional

We investigated the expression pattern of gap junctional proteins (connexins, Cx) in situ and in vitro and their functional characteristics in cultured human umbilical vein endothelial cells (HUVEC) and cultured human umbilical artery endothelial cells (HUAEC). In both arteries and veins, Cx37, Cx40, and Cx43 could be detected in situ and in vitro (passages 2-4). Distribution patterns of Cx40 and Cx43 were homogeneous in situ but more heterogeneous in vitro. Cx37 is heterogeneously expressed both in situ and in vitro. Among most cells, no Cx37 staining could be detected; when present, it was found as bright spots between some clusters of cells. Cx40 was more abundant in cultured arterial endothelium than in cultured venous endothelium. Dye-coupling experiments with Lucifer yellow CH revealed extensive dye spread in HUVEC (15.2 +/- 0.4, mean +/- SE, n = 110) but was significantly restricted in HUAEC (9.8 +/- 0.3, n = 110). Electrophysiological gap junctional characteristics were determined in cultured HUVEC and HUAEC pairs by use of the dual voltage-clamp technique. In contrast to the dye-coupling experiments, mean macroscopic electrical conductance was significantly larger for HUAEC pairs (31.4 +/- 6.0 nS, n = 12) than for HUVEC pairs (16.6 +/- 2.8, n = 18). In HUVEC, we measured multiple single gap junctional channel conductances in the range of 19-75 pS. Interestingly, additional conductances of 80-200 pS were measured in HUAEC, possibly partially reflecting activity of channels formed of Cx40, which are more abundant in the cultured arterial endothelial cells.

scholarly journals Na channel distribution in vertebrate skeletal muscle.

1986 ◽  
Vol 87 (6) ◽  
pp. 907-932 ◽  
Author(s):  
J H Caldwell ◽  
D T Campbell ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Current Density ◽  
Sharp Decrease ◽  
Na Channel ◽  
Na Current ◽  
Na Currents ◽  
Current Densities ◽  
Kinetics Of ◽  
A Current ◽  
Vertebrate Skeletal Muscle

The loose patch voltage clamp has been used to map Na current density along the length of snake and rat skeletal muscle fibers. Na currents have been recorded from (a) endplate membrane exposed by removal of the nerve terminal, (b) membrane near the endplate, (c) extrajunctional membrane far from both the endplate and the tendon, and (d) membrane near the tendon. Na current densities recorded directly on the endplate were extremely high, exceeding 400 mA/cm2 in some patches. The membrane adjacent to the endplate has a current density about fivefold lower than that of the endplate, but about fivefold higher than the membrane 100-200 micron from the endplate. Small local variations in Na current density are recorded in extrajunctional membrane. A sharp decrease in Na current density occurs over the last few hundred micrometers from the tendon. We tested the ability of tetrodotoxin to block Na current in regions close to and far from the endplate and found no evidence for toxin-resistant channels in either region. There was also no obvious difference in the kinetics of Na current in the two regions. On the basis of the Na current densities measured with the loose patch clamp, we conclude that Na channels are abundant in the endplate and near-endplate membrane and are sparse close to the tendon. The current density at the endplate is two to three orders of magnitude higher than at the tendon.

Microfluidic Device for Spatial Control of Cell Seeding in Engineered Tissues

2013 ◽  
Author(s):  
Zaw Win ◽  
Geoffrey D. Vrla ◽  
Emily N. Sevcik ◽  
Patrick W. Alford
Keyword(s):  
Cell Shape ◽  
Microcontact Printing ◽  
Contractile Function ◽  
Cell Coupling ◽  
Engineered Tissues ◽  
Cell Organization ◽  
Microfabrication Techniques ◽  
Cell Cell

In vivo tissues have finely controlled hierarchical structure that is often difficult to mimic in vitro. Microfabrication techniques, such as microcontact printing, can be used to reproduce tissue structure in vitro by controlling cell shape and orientation [1]. Several recent results suggest that cellular organization and structure can influence tissue function in engineered tissues [2–4]. For example, using microcontact printing and muscular thin film technology, we recently demonstrated that engineered vascular tissues whose smooth muscle cells possessed more elongated spindle-like geometries, similar to in vivo structure, exhibited more physiological contractile function [5]. In these studies, cells were seeded using traditional imprecise seeding methods. But recent results have shown that cell-cell coupling plays a significant role in functional contractility [6], suggesting that not only cellular geometry, but cell-cell organization, within a tissue is important to reproduce in engineered tissues to mimic in vivo function.

Ion channels in spinal cord astrocytes in vitro. II. Biophysical and pharmacological analysis of two Na+ current types

1992 ◽  
Vol 68 (4) ◽  
pp. 1001-1011 ◽  
Author(s):  
H. Sontheimer ◽  
S. G. Waxman
Keyword(s):  
Spinal Cord ◽  
Steady State ◽  
Stellate Cells ◽  
Cultured Neurons ◽  
Na Current ◽  
Patch Clamp Recording ◽  
H Infinity ◽  
Na Currents ◽  
Current Activation

1. Na+ currents expressed in astrocytes cultured from spinal cord were studied by whole cell patch-clamp recording. Two subtypes of astrocytes, pancake and stellate cells, were morphologically differentiated and showed expression of Na+ channels at densities that are unusually high for glial cells (2-8 channels/microns2) and comparable to cultured neurons. 2. Na+ currents in stellate and pancake astrocytes were comparable to neuronal Na+ currents with regard to Na(+)-current activation (tau m) and inactivation (tau h) time constants, which were equally fast in both astrocyte types. However, they differed with respect to voltage dependence of activation, and current-voltage (I-V) curves were approximately 10 mV more positive in stellate cells (-11.1 +/- 5.6 mV, mean +/- SD) than in pancake cells (19.7 +/- 4.5 mV). Steady-state activation (m infinity curves) was 16 mV more negative in pancake (mean V1/2 = -48.8 mV) than in stellate cells (mean V1/2 = -32.7 mV). 3. Steady-state inactivation (h infinity curves) of Na+ currents was distinctly different in the two astrocyte types. In stellate astrocytes h infinity curves had midpoints close to -65 mV (-64.6 +/- 6.5 mV), similar to most cultured neurons. In pancake astrocytes h infinity-curves were approximately 25 mV more negative, with midpoints close to -85 mV (84.5 +/- 9.5 mV). 4. The two forms of Na+ currents were additionally distinguishable by their sensitivity to tetrodotoxin (TTX). Na+ currents in stellate astrocytes were highly TTX sensitive [half-maximal inhibition (Kd) = 5.7 nM] whereas Na+ currents in pancake astrocytes were relatively TTX resistant, requiring 100- to 1,000-fold higher concentrations for blockage (Kd = 1,007 nM). 5. Na+ currents were fit by the Hodgkin-Huxley (HH) model. In pancake astrocytes, as in squid gigant axons, Na(+)-current kinetics could be well described with an m3h model, whereas in stellate astrocytes Na+ currents were better described with higher-order power terms for activation (m). On average, best fits were obtained using an m4h model. 6. Pancake astrocytes were capable of generating action-potential (AP)-like responses under current clamp whereas stellate astrocytes were not. The h infinity curve for APs shows that membrane potentials more negative than -70 mV are required to allow these responses to occur.(ABSTRACT TRUNCATED AT 400 WORDS)

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