Regulation of conductive Cl- transport in human fibroblasts

1988 ◽  
Vol 255 (4) ◽  
pp. C552-C558 ◽  
Author(s):  
P. Lin ◽  
M. Ahluwalia ◽  
E. Gruenstein
Keyword(s):  
Human Fibroblasts ◽  
Normal Growth ◽  
Growth Conditions ◽  
Disulfonic Acid ◽  
Ionophore A23187 ◽  
Simultaneous Exposure ◽  
Electrically Conductive ◽  
Cl Channels ◽  
The One ◽  
Cl Conductance

Under normal growth conditions, approximately 20% of the efflux of Cl- from human fibroblasts occurs via an electrically conductive pathway or Cl- channel. This basal Cl- conductance is insensitive to the Cl- -anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and to the Cl- -cation cotransport inhibitor bumetanide. Exposure of the cells to dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) for 15 min increases the electrically conductive component of Cl- efflux by approximately 20%. Unlike the basal Cl- conductance, the cAMP-activated channel is DIDS sensitive, indicating that cAMP activates a different Cl- pathway from the one responsible for the basal Cl- conductance. Elevation of intracellular Ca2+ by addition of the ionophore A23187 also stimulates Cl- efflux via a DIDS inhibitable, electrically conductive Cl- pathway. That the cAMP- and Ca2+-stimulated pathways are different is suggested by the observation that simultaneous exposure of cells to optimal levels of dibutyryl cAMP and A23187 results in an increased Cl- efflux equal to the sum of the two factors acting independently. Prostaglandin E1, a known activator of adenylate cyclase, also elevates the levels of intracellular free Ca2+ in these cells and concomitantly activates both the cAMP- and the Ca2+-stimulated Cl- channels. Although regulated, Cl- channels are known to function in the modulation of nerve and muscle excitability, their role in fibroblast function is not clear.

Functional identification of a sarcolemmal chloride channel from bovine tracheal smooth muscle

1996 ◽  
Vol 271 (5) ◽  
pp. C1716-C1724 ◽  
Author(s):  
D. Salvail ◽  
A. Alioua ◽  
E. Rousseau
Keyword(s):  
Smooth Muscle ◽  
Lipid Bilayers ◽  
Reversal Potential ◽  
Tracheal Smooth Muscle ◽  
Disulfonic Acid ◽  
Equilibrium Potential ◽  
Functional Identification ◽  
Cl Channels ◽  
Cl Conductance

The biophysical and pharmacological characteristics of unitary Cl- currents from bovine tracheal smooth muscle cells were studied after reconstitution of microsomal vesicles into planar lipid bilayers. Two types of currents were recorded simultaneously in KCl buffer: the well-defined Ca(2+)-dependent K+ conductance [GK(Ca)] and a much smaller Cl- current, indicating that the Cl- channels under scrutiny originate from the same membrane as the GK(Ca)-type channels, the plasma membrane of airway smooth muscle (ASM) cells. The GK(Ca) activities were eliminated by the use of CsCl buffer. The average unitary Cl- conductance measured in 50 mM trans-250 mM cis CsCl was 77 +/- 6 pS (n = 21), and the reversal potential measured in various CsCl gradients followed the Cl- equilibrium potential as determined from the Nernst equation. In contrast with the previous reports describing the Ca2+ sensitivity of macroscopic ASM Cl- currents, this channel was found to be insensitive to cytoplasmic and extracellular Ca2+ levels. Phosphorylation cocktails, including protein kinases A, G, or C, did not alter the activity of the channel nor did changes in pH. Among a series of Cl- channel inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid [50% effective concentration (EC50) = 30 microM] and 5-nitro-2-(3-phenylpropylamino) benzoic acid (EC50 = 130 microM) were the most potent blockers of the current examined. The exact role of this surface Cl- conductance remains unclear, and its involvement in cellular activity needs further investigation.

scholarly journals Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium.

1992 ◽  
Vol 99 (2) ◽  
pp. 263-290 ◽  
Author(s):  
D P Joseph ◽  
S S Miller
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Membrane Voltage ◽  
Disulfonic Acid ◽  
Equilibrium Potential ◽  
Ionophore A23187 ◽  
Conductance Changes ◽  
Cl Conductance

Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)-choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina-facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o-aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP-induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology.

Chloride channels in apical and basolateral membranes of CCD cells (RCCT-28A) in culture

1992 ◽  
Vol 263 (2) ◽  
pp. F243-F250 ◽  
Author(s):  
P. Dietl ◽  
B. A. Stanton
Keyword(s):  
Chloride Channels ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Disulfonic Acid ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Cl Channel ◽  
Cl Channels ◽  
Inside Out ◽  
Cl Conductance

Previously, we found that isoproterenol activates whole cell Cl- conductance by a pathway involving adenosine 3',5'-cyclic monophosphate and protein kinase A (PKA) in a renal cell line (RCCT-28A) derived from the cortical collecting duct. The goal of the present study was to determine whether PKA activates Cl- channels in the apical and/or basolateral membrane. Using the patch clamp technique we found a 305-pS Cl- channel, described previously (22), located exclusively in the apical membrane and an outwardly rectifying Cl- channel (13/96 pS) located exclusively in the basolateral membrane. The outward rectifier was highly selective to Cl- versus cations, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, but was not regulated by cytoplasmic pH or Ca2+. Neither isoproterenol nor PKA activated the 305-pS Cl- channel. In contrast, PKA activated a subset of outwardly rectifying channels in inside-out patches. In another subset of outwardly rectifying channels, formation of the inside-out configuration increased channel activity. These channels, however, were not sensitive to PKA. In conclusion, these experiments show that isoproterenol increases the Cl- conductance of RCCT-28A cells by activating a subset of outwardly rectifying Cl- channels located in the basolateral membrane.

Pathways of Cl- transport in human fibroblasts

1988 ◽  
Vol 255 (1) ◽  
pp. C112-C122 ◽  
Author(s):  
P. Lin ◽  
E. Gruenstein
Keyword(s):  
Carboxylic Acid ◽  
Anion Exchange ◽  
Human Fibroblasts ◽  
Disulfonic Acid ◽  
Simultaneous Presence ◽  
Extracellular Medium ◽  
Electrically Conductive ◽  
Cl Transport ◽  
Conductive Pathway ◽  
Normal Human

Three pathways of Cl- efflux were identified in normal human fibroblasts. Twenty percent of the total Cl- efflux is via an electrically conductive pathway with an efflux constant of 0.016 min-1. This pathway is insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and bumetanide but is partially inhibited by anthracene-9-carboxylic acid. Twenty-five percent of the Cl- efflux occurs via Cl- with cation cotransport having an efflux constant of 0.020 min-1. This pathway is inhibited by bumetanide and is dependent on the simultaneous presence of Na+, K+, and Cl-. Under basal conditions, the energetics of this pathway indicate that it is operating close to equilibrium. Fifty percent of the Cl- efflux occurs via an anion exchange pathway having an efflux constant of 0.040 min-1 that is inhibited by DIDS or by removal of Cl- from the extracellular medium. Together these pathways account for 95% of the total Cl- efflux.

scholarly journals Versatile Physiological Functions of Plant GSK3-Like Kinases

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 697
Author(s):  
Juan Mao ◽  
Wenxin Li ◽  
Jing Liu ◽  
Jianming Li
Keyword(s):  
Stress Responses ◽  
Glycogen Synthase ◽  
Plant Stress ◽  
Normal Growth ◽  
Growth Conditions ◽  
Genetic Studies ◽  
Physiological Functions ◽  
Environmental Signals ◽  
Plant Stress Responses ◽  
Diverse Plant

The plant glycogen synthase kinase 3 (GSK3)-like kinases are highly conserved protein serine/threonine kinases that are grouped into four subfamilies. Similar to their mammalian homologs, these kinases are constitutively active under normal growth conditions but become inactivated in response to diverse developmental and environmental signals. Since their initial discoveries in the early 1990s, many biochemical and genetic studies were performed to investigate their physiological functions in various plant species. These studies have demonstrated that the plant GSK3-like kinases are multifunctional kinases involved not only in a wide variety of plant growth and developmental processes but also in diverse plant stress responses. Here we summarize our current understanding of the versatile physiological functions of the plant GSK3-like kinases along with their confirmed and potential substrates.

scholarly journals Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.

1989 ◽  
Vol 9 (6) ◽  
pp. 2615-2626 ◽  
Author(s):  
E Hickey ◽  
S E Brandon ◽  
G Smale ◽  
D Lloyd ◽  
L A Weber
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Normal Growth ◽  
Gene Families ◽  
Growth Conditions ◽  
Complete Sequence ◽  
Start Codon ◽  
Hybridization Probe ◽  
Reading Frame ◽  
Alpha Gene

Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

Human primary fibroblasts in vitro express a purinergic P2X7 receptor coupled to ion fluxes, microvesicle formation and IL-6 release

1999 ◽  
Vol 112 (3) ◽  
pp. 297-305
Author(s):  
A. Solini ◽  
P. Chiozzi ◽  
A. Morelli ◽  
R. Fellin ◽  
F. Di Virgilio
Keyword(s):  
Purinergic Receptor ◽  
Morphological Changes ◽  
Human Fibroblasts ◽  
Ion Fluxes ◽  
Disulfonic Acid ◽  
Triggered Release ◽  
Primary Fibroblasts ◽  
Purinergic P2x7 Receptor ◽  
Skin Biopsies

We have investigated reponses to extracellular ATP in human fibroblasts obtained by skin biopsies. Our data show that these cells express a P2X7 purinergic receptor, as judged by (1) RT-PCR with specific primers, (2) reactivity with a specific anti-P2X7 antiserum, (3) activation by the selective P2X agonist benzoylbenzoylATP and (4) stimulation of transmembrane ion fluxes. Stimulation with benzoylbenzoylATP, and to a lesser extent with ATP, also caused striking morphological changes and increased formation of cytoplasmic microvesicles. These changes were fully reversible upon nucleotide removal. Two known blockers of P2X receptors, oxidised ATP and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, inhibited the morphological changes fully and the ion fluxes partially. The residual rise in intracellular Ca2+ levels and membrane depolarization observed in the presence of the inhibitors were dependent upon activation of a P2Y-type receptor exhibiting a peculiar pharmacological profile, in that CTP was the preferred agonist. ATP stimulation triggered release of the pro-inflammatory cytokine IL-6 in fibroblasts pre-treated with PMA and bacterial endotoxin. These observations reveal a novel pathway for fibroblast activation and for their recruitment in the inflammatory response.

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences

1988 ◽  
Vol 8 (9) ◽  
pp. 3827-3836
Author(s):  
N P Williams ◽  
P P Mueller ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Normal Growth ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Yeast Genes ◽  
Reading Frames

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

Cl- fluxes related to fluid secretion by the rat parotid: involvement of Cl(-)-HCO3- exchange

1992 ◽  
Vol 262 (3) ◽  
pp. G393-G398 ◽  
Author(s):  
J. E. Melvin ◽  
R. J. Turner
Keyword(s):  
Channel Blocker ◽  
Kinetic Studies ◽  
Fluid Secretion ◽  
Disulfonic Acid ◽  
K Channel ◽  
Maximal Effect ◽  
Partial Recovery ◽  
Free Medium ◽  
Cl Channels ◽  
Rat Parotid

Muscarinic-induced 36Cl- and 86Rb+ (K+ substitute) fluxes were studied in rat parotid acini. Stimulation resulted in a rapid [half time (t1/2) less than 30 s] decrease in both Cl- and Rb+ content (approximately 50 and 30%, respectively) followed by a slower partial recovery (t1/2 approximately 3-4 min) to approximately 80% of resting levels for both ions. Cl- loss was inhibited by the venom of Leiurus quinquestriatus, which contains the maxi-K+ channel blocker charybdotoxin. Cl- recovery was blunted in the presence of bumetanide, an inhibitor of Na(+)-K(+)-Cl- cotransport, or on HCO3- removal and was completely blocked in the presence of bumetanide and 4,4' diisothiocyanostilbene-2,2' disulfonic acid (DIDS), an inhibitor of Cl(-)-HCO3- exchange. In HCO3(-)-containing medium a rapid (t1/2 less than 1 min), DIDS-inhibitable cytoplasmic alkalinization (approximately 0.4 pH unit) was observed in acini switched to a Cl(-)-free solution. This alkalinization was not seen in HCO3(-)-free medium but persisted in the absence of Na+, consistent with the presence of a potent Na(+)-independent Cl(-)-HCO3- exchanger. Kinetic studies indicated that the half-maximal effect of this exchanger for extracellular Cl- was approximately 18 mM. These results are consistent with the hypothesis that secretagogue-induced KCl loss by salivary acinar cells occurs via electrically coupled K+ and Cl- channels. In addition, they provide strong evidence that Cl- entry into, and thus fluid secretion by, these cells is mediated by both Cl(-)-HCO3- exchange and Na(+)-K(+)-Cl- cotransport.

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