A Structural Model for the Mass Action Kinetic Analysis of P-gp Mediated Transport Through Confluent Cell Monolayers

The epithelia lining the epididymides of many species consists of several cell types. We have provided evidence that the basal cells are essential to the integrated functions of the epithelium. Basal cells, but not principal cells, and other cells in the epididymis express TRPC3 and COX-1. We have isolated basal cells from intact rat epididymis using antibody-coated Dynabeads and subjected them to whole-cell patch-clamp measurement of nonselective cation channel activity, a feature of TRPC3 protein, and Fluo-3 fluorescence measurement of intracellular Ca2+ concentration. The results show that a nonselective cation current blockable by La3+ (0.1 mM), Gd3+ (0.1 mM), or SKF96365 (20 μM) could be activated by lysylbradykinin (200 nM). In cells loaded with Fluo-3, addition of lysylbradykinin (100 nM) caused a sustained increase of intracellular Ca2+. This effect was blocked by Gd3+ (0.1 mM) or SKF96365 (20 μM) and was not observed in Fluo-3–loaded principal cells. Stimulation of basal cell/principal cell cocultures with lysylbradykinin (200 nM) evoked in principal cells a current with CFTR-Cl− channel characteristics. Isolated principal cells in the absence of basal cells did not respond to lysylbradykinin but responded to PGE2 (100 nM) with activation of a CFTR-like current. Basal cells, but not principal cells, released prostaglandin E2 when stimulated with lysylbradykinin (100 nM). The release was blocked by SKF96365 (20 μM) and BAPTA-AM (0.05 or 0.1 mM). Confluent cell monolayers harvested from a mixture of disaggregated principal cells and basal cells responded to lysylbradykinin (100 nM) and PGE2 (500 nM) with an increase in electrogenic anion secretion. The former response was dependent on prostaglandin synthesis as piroxicam blocked the response. However, cell cultures obtained from principal cells alone responded to PGE2 but not to bradykinin. These results support the notion that basal cells regulate principal cells through a Ca2+ and COX signaling pathway.

Download Full-text

Expression of epithelial Na channels in Xenopus oocytes.

The Journal of General Physiology ◽

10.1085/jgp.96.1.23 ◽

1990 ◽

Vol 96 (1) ◽

pp. 23-46 ◽

Cited By ~ 29

Author(s):

L G Palmer ◽

I Corthesy-Theulaz ◽

H P Gaeggeler ◽

J P Kraehenbuhl ◽

B Rossier

Keyword(s):

Sedimentation Coefficient ◽

High Sensitivity ◽

Sucrose Density Gradient ◽

Na Channel ◽

Channel Activity ◽

Membrane Expression ◽

Cell Monolayers ◽

A6 Cells ◽

Epithelial Na Channels ◽

Confluent Cell

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride-sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.

Download Full-text

Temperature-responsive intelligent interfaces for biomolecular separation and cell sheet engineering

Journal of The Royal Society Interface ◽

10.1098/rsif.2008.0499.focus ◽

2009 ◽

Vol 6 (suppl_3) ◽

Cited By ~ 181

Author(s):

Kenichi Nagase ◽

Jun Kobayashi ◽

Teruo Okano

Keyword(s):

Cell Sheet ◽

Copolymer Composition ◽

Cell Sheets ◽

Chromatographic Separations ◽

Cell Sheet Engineering ◽

External Temperature ◽

Cell Monolayers ◽

Temperature Responsive ◽

Confluent Cell ◽

Dependent Cell

Temperature-responsive intelligent surfaces, prepared by the modification of an interface with poly( N -isopropylacrylamide) and its derivatives, have been used for biomedical applications. Such surfaces exhibit temperature-responsive hydrophilic/hydrophobic alterations with external temperature changes, which, in turn, result in thermally modulated interactions with biomolecules and cells. In this review, we focus on the application of these intelligent surfaces to chromatographic separation and cell cultures. Chromatographic separations using several types of intelligent surfaces are mentioned briefly, and various effects related to the separation of bioactive compounds are discussed, including wettability, copolymer composition and graft polymer architecture. Similarly, we also summarize temperature-responsive cell culture substrates that allow the recovery of confluent cell monolayers as contiguous living cell sheets for tissue-engineering applications. The key factors in temperature-dependent cell adhesion/detachment control are discussed from the viewpoint of grafting temperature-responsive polymers, and new methodologies for effective cell sheet culturing and the construction of thick tissues are summarized.

Download Full-text

Effect of high extracellular K+ on Na-K-ATPase in cultured canine kidney cells

AJP Renal Physiology ◽

10.1152/ajprenal.1990.259.2.f227 ◽

1990 ◽

Vol 259 (2) ◽

pp. F227-F232 ◽

Cited By ~ 1

Author(s):

M. A. Manuli ◽

I. S. Edelman

Keyword(s):

Atpase Activity ◽

Northern Analysis ◽

Beta Subunits ◽

Cell Monolayers ◽

High K ◽

Confluent Cell ◽

K Transport ◽

Darby Canine Kidney ◽

Canine Kidney ◽

Specific Mrna

The Madin-Darby canine kidney (MDCK) cell line was used to evaluate the influence of high extracellular K+, independent of hormonal effects, on renal Na-K-adenosinetriphosphatase (ATPase) activity and abundance. Confluent cell monolayers were incubated in control (5 mM) or high K+ (7.5 mM) medium for 24 h. Exposure to high K+ elicited a 46% rise in Na-K-ATPase activity and a 55% increase in ouabain-sensitive 86Rb uptake. Na-K-ATPase abundance, estimated from the number of ouabain-binding sites, also increased 63% over control in cells exposed to 7.5 mM K+, and as a consequence there was no statistically significant change in the catalytic turnover number. Northern blot analysis using rat cDNA probes for the alpha 1- and beta-subunits showed no corresponding changes in subunit-specific mRNA abundances at 24 h. We conclude that chronic exposure to high extracellular K+ produces a rise in renal epithelial Na-K-ATPase activity and active K+ transport, independent of changes in aldosterone, renal blood flow, or extracellular Na+ concentration. This effect is due to an increase in enzyme abundance rather than a change in catalytic turnover rate. The results of Northern analysis suggest that regulation of Na-K-ATPase activity and abundance by high K+ may involve translational or posttranslational mechanisms, but further study with cDNA probes of canine origin is needed to resolve this issue.

Download Full-text

A chip device to determine surface charge properties of confluent cell monolayers by measuring streaming potential

Lab on a Chip ◽

10.1039/d0lc00558d ◽

2020 ◽

Vol 20 (20) ◽

pp. 3792-3805 ◽

Cited By ~ 1

Author(s):

András Kincses ◽

Ana R. Santa-Maria ◽

Fruzsina R. Walter ◽

László Dér ◽

Nóra Horányi ◽

...

Keyword(s):

Cell Surface ◽

Surface Charge ◽

Streaming Potential ◽

Cell Monolayers ◽

Biological Barriers ◽

Cell Surface Charge ◽

Cell Layers ◽

Confluent Cell

Chip device to monitor streaming potential of confluent cell layers reflecting cell surface charge important for the function of biological barriers.

Download Full-text

Three-dimensional stochastic model of actin–myosin binding in the sarcomere lattice

The Journal of General Physiology ◽

10.1085/jgp.201611608 ◽

2016 ◽

Vol 148 (6) ◽

pp. 459-488 ◽

Cited By ~ 32

Author(s):

Srboljub M. Mijailovich ◽

Oliver Kayser-Herold ◽

Boban Stojanovic ◽

Djordje Nedic ◽

Thomas C. Irving ◽

...

Keyword(s):

Kinetic Models ◽

Striated Muscle ◽

Three Dimensional ◽

Limited Range ◽

Mass Action ◽

Myosin Filament ◽

Mass Action Kinetic ◽

Cross Bridge ◽

Myosin Binding ◽

Cross Bridges

The effect of molecule tethering in three-dimensional (3-D) space on bimolecular binding kinetics is rarely addressed and only occasionally incorporated into models of cell motility. The simplest system that can quantitatively determine this effect is the 3-D sarcomere lattice of the striated muscle, where tethered myosin in thick filaments can only bind to a relatively small number of available sites on the actin filament, positioned within a limited range of thermal movement of the myosin head. Here we implement spatially explicit actomyosin interactions into the multiscale Monte Carlo platform MUSICO, specifically defining how geometrical constraints on tethered myosins can modulate state transition rates in the actomyosin cycle. The simulations provide the distribution of myosin bound to sites on actin, ensure conservation of the number of interacting myosins and actin monomers, and most importantly, the departure in behavior of tethered myosin molecules from unconstrained myosin interactions with actin. In addition, MUSICO determines the number of cross-bridges in each actomyosin cycle state, the force and number of attached cross-bridges per myosin filament, the range of cross-bridge forces and accounts for energy consumption. At the macroscopic scale, MUSICO simulations show large differences in predicted force-velocity curves and in the response during early force recovery phase after a step change in length comparing to the two simplest mass action kinetic models. The origin of these differences is rooted in the different fluxes of myosin binding and corresponding instantaneous cross-bridge distributions and quantitatively reflects a major flaw of the mathematical description in all mass action kinetic models. Consequently, this new approach shows that accurate recapitulation of experimental data requires significantly different binding rates, number of actomyosin states, and cross-bridge elasticity than typically used in mass action kinetic models to correctly describe the biochemical reactions of tethered molecules and their interaction energetics.

Download Full-text