Cholesterol Depletion Facilitates Recovery from Hypotonic Cell Swelling in CHO Cells

The technique of variable-angle total internal reflection fluorescence microscopy (TIRFM) and its application to nanotomography of cell surfaces are described. Present applications include (1) 3D imaging of chromosomes in their metaphase to demonstrate axial resolution in the nanometre range, (2) measurements of cell-substrate topology, which upon cholesterol depletion shows some loosening of cell-substrate contacts, and (3) measurements of cell topology upon photodynamic therapy (PDT), which demonstrate cell swelling and maintenance of focal contacts. The potential of the method for in vitro diagnostics, but also some requirements and limitations are discussed.

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Ca2+ uptake in GH3 cells during hypotonic swelling: the sensory role of stretch-activated ion channels

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.6.c1790 ◽

1996 ◽

Vol 270 (6) ◽

pp. C1790-C1798 ◽

Cited By ~ 46

Author(s):

Y. Chen ◽

S. M. Simasko ◽

J. Niggel ◽

W. J. Sigurdson ◽

F. Sachs

Keyword(s):

Volume Regulation ◽

Cell Swelling ◽

Gh3 Cells ◽

Membrane Tension ◽

Hypotonic Cell Swelling ◽

Stretch Activated Channels ◽

Ca2 Channels ◽

Hypotonic Swelling ◽

Volume Decrease

Hypotonic cell swelling triggers an increase in intracellular Ca2+ concentration that is deemed responsible for the subsequent regulated volume decrease in many cells. To understand the mechanisms underlying this increase, we have studied the Ca2+ sources that contribute to hypotonic cell swelling-induced Ca2+ increase (HICI) in GH3 cells. Fura 2 fluorescence of cell populations revealed that extracellular, but not intracellular, stores of Ca2+ were required. HICI was abolished by nifedipine, a blocker of L-type Ca2+ channels, and Gd3+, a nonspecific blocker of stretch-activated channels (SACs), suggesting two components for the Ca2+ membrane pathway: L-type Ca2+ channels and SACs. Using HICI as an assay, we found that venom from the spider Grammostola spatulata could block HICI without blocking L-type Ca2+ channels. The venom did, however, block SAC activity. This suggests that Ca(2+)-permeable SACs, rather than L-type Ca2+ channels, are the sensing elements for HICI. These results support the model for volume regulation in which SACs, activated by an increase of the membrane tension during hypotonic cell swelling, trigger HICI, leading to a volume decrease.

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Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.2.c328 ◽

1999 ◽

Vol 276 (2) ◽

pp. C328-C336 ◽

Cited By ~ 89

Author(s):

Christopher M. Gillen ◽

Bliss Forbush

Keyword(s):

Cell Swelling ◽

Primary Role ◽

Functional Interaction ◽

Hek 293 ◽

Hek 293 Cells ◽

Hek Cells ◽

293 Cells ◽

Hypotonic Cell Swelling ◽

The Relationship ◽

Fold Activation

We have studied the regulation of the K-Cl cotransporter KCC1 and its functional interaction with the Na-K-Cl cotransporter. K-Cl cotransporter activity was substantially activated in HEK-293 cells overexpressing KCC1 (KCC1-HEK) by hypotonic cell swelling, 50 mM external K, and pretreatment with N-ethylmaleimide (NEM). Bumetanide inhibited 86Rb efflux in KCC1-HEK cells after cell swelling [inhibition constant ( K i) ∼190 μM] and pretreatment with NEM ( K i ∼60 μM). Thus regulation of KCC1 is consistent with properties of the red cell K-Cl cotransporter. To investigate functional interactions between K-Cl and Na-K-Cl cotransporters, we studied the relationship between Na-K-Cl cotransporter activation and intracellular Cl concentration ([Cl]i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activity than controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells was activated <2-fold by low-Cl hypotonic prestimulation, compared with 10-fold activation in HEK-293 cells and >20-fold activation in cells overexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cells had lower resting [Cl]i than HEK-293 cells; cell volume was not different among cell lines. We found a steep relationship between [Cl]i and Na-K-Cl cotransport activity within the physiological range, supporting a primary role for [Cl]iin activation of Na-K-Cl cotransport and in apical-basolateral cross talk in ion-transporting epithelia.

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Small-conductance Cl? channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP

Pflügers Archiv - European Journal of Physiology ◽

10.1007/bf00374833 ◽

1992 ◽

Vol 421 (2-3) ◽

pp. 238-246 ◽

Cited By ~ 48

Author(s):

K. Kunzelmann ◽

R. Kubitz ◽

M. Grolik ◽

R. Warth ◽

R. Greger

Keyword(s):

Cell Swelling ◽

Ht29 Cells ◽

Cl Channels ◽

Hypotonic Cell Swelling ◽

Small Conductance

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Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP, Ca2+ and hypotonic cell swelling

Pflügers Archiv - European Journal of Physiology ◽

10.1007/bf00374754 ◽

1994 ◽

Vol 428 (1) ◽

pp. 76-83 ◽

Cited By ~ 14

Author(s):

K. Kunzelmann ◽

N. Allert ◽

R. Kubitz ◽

W. V. Breuer ◽

Z. I. Cabantchik ◽

...

Keyword(s):

Chloride Conductance ◽

Cell Swelling ◽

Ht29 Cells ◽

Hypotonic Cell Swelling

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Preferential potentiation by hypotonic cell swelling of muscarinic cation current in guinea pig ileum

AJP Cell Physiology ◽

10.1152/ajpcell.1997.272.1.c240 ◽

1997 ◽

Vol 272 (1) ◽

pp. C240-C253 ◽

Cited By ~ 25

Author(s):

Y. Waniishi ◽

R. Inoue ◽

Y. Ito

Keyword(s):

Smooth Muscle ◽

Guinea Pig ◽

Muscarinic Receptor ◽

Time Course ◽

Cell Swelling ◽

Patch Clamp Technique ◽

Cationic Current ◽

Whole Cell Patch Clamp ◽

Voltage Dependent ◽

Hypotonic Cell Swelling

The effects of hypotonic cell swelling (HCS) on muscarinic receptor-activated cationic current in guinea pig ileal smooth muscle were investigated by the whole cell patch-clamp technique. With nystatin-perforated recording, reduced external tonicity from 312 to 262 mosM caused cell swelling but hardly affected the membrane currents activated by depolarization, such as outward-rectifying K and voltage-dependent Ca currents. In contrast, the inward current evoked by carbachol at -60 mV was greatly increased (approximately 50%) by the same extent of hypotonicity. This effect is likely to occur through potentiation of nonselective cation channels coupled to the muscarinic receptor (mNSCCs) and probably does not involve elevated intracellular Ca2+ concentration ([Ca2+]i), since neither removal of external Ca2+ nor [Ca2+]i buffering with 10 mM 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid significantly affected the results. Furthermore, the time course and degree of this potentiation closely matched those of video-microscopically monitored HCS. These results support the view that mechanosensitive modulation may be a powerful mechanism to regulate mNSCCs activity in gut smooth muscle, together with membrane potential and [Ca2+]i.

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Membrane Reserves and Hypotonic Cell Swelling

The Journal of Membrane Biology ◽

10.1007/s00232-006-0080-8 ◽

2006 ◽

Vol 214 (1-2) ◽

pp. 43-56 ◽

Cited By ~ 82

Author(s):

Nicolas Groulx ◽

Francis Boudreault ◽

Sergei N. Orlov ◽

Ryszard Grygorczyk

Keyword(s):

Cell Swelling ◽

Hypotonic Cell Swelling

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Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis

Biochemical Journal ◽

10.1042/bj20042082 ◽

2005 ◽

Vol 390 (1) ◽

pp. 273-283 ◽

Cited By ~ 57

Author(s):

Riikka Hynynen ◽

Saara Laitinen ◽

Reijo Käkelä ◽

Kimmo Tanhuanpää ◽

Sari Lusa ◽

...

Keyword(s):

Hela Cells ◽

Cho Cells ◽

Cholesterol Metabolism ◽

Reductase Activity ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Receptor Expression ◽

Cholesterol Depletion ◽

Related Protein ◽

Cellular Cholesterol

ORP2 [OSBP (oxysterol-binding protein)-related protein 2] belongs to the 12-member mammalian ORP gene/protein family. We characterize in the present study the effects of inducible ORP2 overexpression on cellular cholesterol metabolism in HeLa cells and compare the results with those obtained for CHO cells (Chinese-hamster ovary cells) that express ORP2 constitutively. In both cell systems, the prominent phenotype is enhancement of [14C]cholesterol efflux to all extracellular acceptors, which results in a reduction of cellular free cholesterol. No change was observed in the plasma membrane cholesterol content or distribution between raft and non-raft domains upon ORP2 expression. However, elevated HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase activity and LDL (low-density lipoprotein) receptor expression, as well as enhanced transport of newly synthesized cholesterol to a cyclodextrin-accessible pool, suggest that the ORP2 expression stimulates transport of cholesterol out of the endoplasmic reticulum. In contrast with ORP2/CHO cells, the inducible ORP2/HeLa cells do not show down-regulation of cholesterol esterification, suggesting that this effect represents an adaptive response to long-term cholesterol depletion in the CHO cell model. Finally, we provide evidence that ORP2 binds PtdIns(3,4,5)P3 and enhances endocytosis, phenomena that are probably interconnected. Our results suggest a function of ORP2 in both cholesterol trafficking and control of endocytic membrane transport.

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