Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling

We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na(+) that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ～330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens' circulation of Na(+) was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium.     

Connections between connexins, calcium, and cataracts in the lens

There is a good deal of evidence that the lens generates an internal micro circulatory system, which brings metabolites, like glucose, and antioxidants, like ascorbate, into the lens along the extracellular spaces between cells. Calcium also ought to be carried into the lens by this system. If so, the only path for Ca2+ to get out of the lens is to move down its electrochemical gradient into fiber cells, and then move by electrodiffusion from cell to cell through gap junctions to surface cells, where Ca-ATPase activity and Na/Ca exchange can transport it back into the aqueous or vitreous humors. The purpose of the present study was to test this calcium circulation hypothesis by studying calcium homeostasis in connexin (Cx46) knockout and (Cx46 for Cx50) knockin mouse lenses, which have different degrees of gap junction coupling. To measure intracellular calcium, FURA2 was injected into fiber cells, and the gradient in calcium concentration from center to surface was mapped in each type of lens. In wild-type lenses the coupling conductance of the mature fibers was approximately 0.5 S/cm2 of cell to cell contact, and the best fit to the calcium concentration data varied from 700 nM in the center to 300 nM at the surface. In the knockin lenses, the coupling conductance was approximately 1.0 S/cm2 and calcium varied from approximately 500 nM at the center to 300 nM at the surface. Thus, when the coupling conductance doubled, the concentration gradient halved, as predicted by the model. In knockout lenses, the coupling conductance was zero, hence the efflux path was knocked out and calcium accumulated to approximately 2 microM in central fibers. Knockout lenses also had a dense central cataract that extended from the center to about half the radius. Others have previously shown that this cataract involves activation of a calcium-dependent protease, Lp82. We can now expand on this finding to provide a hypothesis on each step that leads to cataract formation: knockout of Cx46 causes loss of coupling of mature fiber cells; the efflux path for calcium is therefore blocked; calcium accumulates in the central cells; at concentrations above approximately 1 microM (from the center to about half way out of a 3-wk-old lens) Lp82 is activated; Lp82 cleaves cytoplasmic proteins (crystallins) in central cells; and the cleaved proteins aggregate and scatter light.     

Osmotic regulation in the marine alga, Codium decorticatum. I. Regulation of turgor pressure by control of ionic composition.

Codium decorticatum regulates its internal ionic composition and osmotic pressure in response to changes in external salinity. Over a salinity range of 23 to 37% (675 to 1120 mosmol/kg) Codium maintains a constant turgor pressure of 95 mosmol/kg (2.3 atm), observed as a constant difference between internal and external osmotic pressures. The changes in internal osmotic pressure are due to changes in intracellular inorganic ions. At 30 0/00 salinity the major intracellular ions are present in the following concentrations (mmol/kg cell H20): K+, 295; Na+, 255; Cl-, 450. At different salinities intracellular ion concentrations remain in constant proportion to the external ion concentrations, and thus the equilibrium potentials are approximately constant. The potential difference between the vacuole and seawater (-76 mV), whici is predominantly a K+ diffusion potential, is also constant with changing salinity. Comparison of the equilibrium potentials with the vacuole potential suggests that Cl- is actively absorbed and Na+ actively extruded, whereas K+ may be passively distributed between the vacuole and seawater. Turgor pressure does not change with environmental hydrostatic pressure, and increasing the external osmotic pressure with raffinose elicits a response similar to that obtained by increasing the salinity. These two results suggest that the stimulus for turgor regulation is a change in turgor pressure rather than a change in internal hydrostatic pressure or ion concentrations.     

Differential effects of hydrostatic pressure on cation transport pathways of isolated articular chondrocytes

Articular cartilages are exposed to significant loads in vivo, which by their effects on chondrocyte metabolism can alter the mechanical properties of the extracellular matrix. The mechanism(s) by which chondrocytes sense and respond to load are not well understood. One component of load, hydrostatic pressure, can be studied independently of the other factors that change during load. In this study, the effects of pressure have been investigated on three K transport pathways in isolated bovine articular chondrocytes. Pressure inhibited the Na/K pump (ouabain-sensitive), Na/K/2Cl cotransporter (bumetanide-sensitive), and residual (ouabain- and bumetanide-insensitive) pathways; however, the response of each system was different. Both pressure level and duration were important in determining the extent of inhibition. There was marked suppression of the Na/K pump, particularly when pressure (2.5-50 MPa) was maintained for the full incubation period (usually 10 min). The Na/K/2Cl cotransporter was more pressure-sensitive, with only a short application (20 sec) of a low pressure (7.5 MPa) being sufficient for inhibition. Over the higher range (20-50 MPa), pressure had little further effect. The inhibitory action on the Na/K pump was dependent on the [Na]i. Thus, when the [Na]i was set to values above or below those normally present, the inhibitory effect was reduced or abolished. The suppressive effect of pressure on Na/K pump and residual pathways was reversed at atmospheric pressure. The pressure dependence of inhibition of the K flux through the residual pathway was similar to that reported for lipid bilayers. These results indicate that hydrostatic pressure may act directly on chondrocyte membrane transporters. Alterations to matrix synthesis resulting from the application of load might therefore result in part from variations to the intracellular ionic/osmotic composition of chondrocytes arising from changes to the activity of membrane transport pathways.     

Noradrenergic β-Adrenoceptor-Mediated Intracellular Molecular Mechanism of Na–K ATPase Subunit Expression in C6 Cells

Rapid eye movement sleep deprivation-associated elevated noradrenaline increases and decreases neuronal and glial Na–K ATPase activity, respectively. In this study, using C6 cell-line as a model, we investigated the possible intracellular molecular mechanism of noradrenaline-induced decreased glial Na–K ATPase activity. The cells were treated with noradrenaline in the presence or absence of adrenoceptor antagonists, modulators of extra- and intracellular Ca⁺⁺ and modulators of intracellular signalling pathways. We observed that noradrenaline acting on β-adrenoceptor decreased Na–K ATPase activity and mRNA expression of the catalytic α2-Na–K ATPase subunit in the C6 cells. Further, cAMP and protein kinase-A mediated release of intracellular Ca⁺⁺ played a critical role in such decreased α2-Na–K ATPase expression. In contrast, noradrenaline acting on β-adrenoceptor up-regulated the expression of regulatory β2-Na–K ATPase subunit, which although was cAMP and Ca⁺⁺ dependent, was independent of protein kinase-A and protein kinase-C. Combining these with previous findings (including ours) we have proposed a working model for noradrenaline-induced suppression of glial Na–K ATPase activity and alteration in its subunit expression. The findings help understanding noradrenaline-associated maintenance of brain excitability during health and altered states, particularly in relation to rapid eye movement sleep and its deprivation when the noradrenaline level is naturally altered.     

Intracellular trafficking of FXYD1 (phospholemman) and FXYD7 proteins in Xenopus oocytes and mammalian cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.     

Interaction of intracellular ion buffering with transmembrane-coupled ion transport

The role of the Na/Ca exchanger in the control of cellular excitability and tension development is a subject of current interest in cardiac physiology. It has been suggested that this coupled transporter is responsible for rapid changes in intracellular calcium activity during single beats, generation of plateau currents, which control action potential duration, and control of intracellular sodium during Na/K pump suppression, which may occur during terminal states of ischemia. The actual behavior of this exchanger is likely to be complex for several reasons. First, the exchanger transports two ionic species and thus its instantaneous flux rate depends on both intracellular sodium and calcium activity. Secondly, the alteration in intracellular calcium activity, which is caused by a given transmembrane calcium flux, and which controls the subsequent exchanger rate, is a complex function of available intracellular calcium buffering. The buffers convert the ongoing transmembrane calcium fluxes into changes in activity that are a small and variable fraction of the change in total calcium concentration. Using a number of simple assumptions, we model changes in intracellular calcium and sodium concentration under the influence of Na/Ca exchange, Na/K ATPase and Ca-ATPase pumps, and passive sodium and calcium currents during periods of suppression and reactivation of the Na/K ATPase pump. The goal is to see whether and to what extent general notions of the role of the Na/Ca exchanger used in planning and interpreting experimental studies are consistent with its function as derived from current mechanistic assumptions about the exchanger. We find, for example, that based on even very high estimates of intracellular calcium buffering, it is unlikely that Na/Ca exchange alone can control intracellular sodium during prolonged Na/K pump blockade. It is also shown that Na/Ca exchange can contaminate measurements of Na/K pump currents under a variety of experimental conditions. The way in which these and other functions are affected by the dissociation constants and total capacity of the intracellular calcium buffers are also explored in detail.     

Effects of high hydrostatic pressures on the activity of the membrane ATPases of some organs implicated in hydromineral regulation.

1. The effects of high hydrostatic pressures have been studied on the ATPases extracted from tissues implicated in iono- and osmoregulation of a frog and various teleostean fishes. Pressure affects enzyme activity in the same qualitative way, whatever the tissue and the species considered. 2. The Mg2+ ATPase activity is maximally enhanced at 250 kg/cm2. A slight inhibition is observed at higher pressures up to 1000 kg/cm2. 3. The (Na+ + K+)ATPase is little affected by low pressures but strongly inhibited at 500 kg/cm2 and more. 4. The results are discussed in terms of pressure effects on the recently described protein-lipid interaction linked to ATPase activity.     

Na+/K+ATPase as a signaling molecule during bovine sperm capacitation

A heteromeric integral membrane protein, Na+/K+ATPase is composed of two polypeptides, alpha and beta, and is active in many cell types, including testis and spermatozoa. It is a well-known ion transporter, but binding of ouabain, a specific inhibitor of Na+/K+ATPase, to Na+/K+ATPase in somatic cells initiates responses that are similar to signaling events associated with bovine sperm capacitation. The objectives of the present study were to demonstrate the presence of Na+/K+ATPase in bovine sperm and to investigate its role in the regulation of bovine sperm capacitation. The presence of Na+/K+ATPase in sperm from mature Holstein bulls was demonstrated by immunoblotting and immunocytochemistry using a monoclonal antibody developed in mouse against the beta 1 polypeptide of Na+/K+ATPase. Binding of ouabain to Na+/K+ATPase inhibited motility (decreased progressive motility, average path velocity, and curvilinear velocity) and induced tyrosine phosphorylation and capacitation but did not increase intracellular calcium levels in spermatozoa. Furthermore, binding of ouabain to Na+/K+ATPase induced depolarization of sperm plasma membrane. Therefore, binding of ouabain to Na+/K+ATPase induced sperm capacitation through depolarization of sperm plasma membrane and signaling via the tyrosine phosphorylation pathway without an appreciable increase in intracellular calcium. To our knowledge, this is the first report concerning the signaling role of Na+/K+ATPase in mammalian sperm capacitation.     

Polarization variations induced by high hydrostatic pressures in the isolated frog skin as related to the effects on passive ionic permeability and active Na+ transport.

The effects of a wide range of hydrostatic pressures (from 50 to 1000 kg/cm2) have been investigated on the spontaneous potential difference (PD), the short-circuit current (SCC) and the activity of the membrane ATPases of the isolated abdominal skin from the frog Rana temporaria L. Two types of variations in PD are induced by pressure changes: short and transient potential variations which appear to be related to the pressure change (compression and decompression) and lasting variations which persist as long as pressure is applied and whose nature appears to be related to the pressure magnitude. Long-lasting potential changes have particularly been investigated. At pressures lower than 500 kg/cm2, the skin potential increases while a pressure over 500-600 kg/cm2 induces a depolarization. Both variations consecutively occur at 500 +/- 100 kg/cm2. These effects of pressure have been shown to be reversible up to about 800 kg/cm2. The question of the origin of the potential changes is discussed and it is proposed that the lasting hyperpolarization results from an effect on the passive permeabilities to Na+, K+ and Cl- ions inducing in turm a secondary readjustment (stimulation) of the Na+ active transport while the depolarization at high pressures reflects a direct inhibition of the Na+ pump. These interpretations are supported by experimental data on the effects of pressure on the short-circuit current and on the activity of the skin (Na+ + K+) ATPase.     

Human and dog erythrocytes: relationship between cellular ATP levels, ATP consumption and potassium concentrations.

The intracellular K+/Na+ ratio of various mammalian cell types are known to differ remarkably. Particularly noteworthy is the fact that erythrocytes of different mammalian species contain entirely different potassium and sodium concentrations. The human erythrocyte is an example of the supposedly "normal" high potassium cell, while the dog erythrocyte contains ten times more sodium than potassium ions (Table I). Furthermore, this difference is sustained despite the plasma sodium and potassium concentrations being almost identical in both species (high Na+ and low K+). In spite of these inorganic ion differences, both human and dog erythrocytes contain 33% dry material (mostly Hb) and 67% water. Conventional cell theory would couple cellular volume regulation with Na+ and K+ dependent ATPase activity which is believed to control intracellular Na+/K+ concentrations. Since the high Na+ and low K+ contents of dog erythrocytes are believed to be due to the lack of the postulated Na/K-ATPase enzyme, they must presumably have an alternative mechanism of volume regulation, otherwise current ideas of membrane ATPase activity coupled volume regulation need serious reconsideration. The object of our investigation was to explore the relationship between ATPase activity, ATP levels and the Na+/K+ concentrations in human and dog erythrocytes. Our results indicate that the intracellular ATP level in erythrocytes correspond with their K+, Na+ content. They are discussed in relation to conventional membrane transport theory and also to Ling's "association-induction hypothesis", the latter proving to be a more useful basis on which to interpret results.     

TSH induces co‐localization of TSH receptor and Na/K‐ATPase in human erythrocytes

Thyroid stimulating hormone (TSH) binds to a specific TSH receptor (TSHR) which activates adenylate cyclase and increases cAMP levels in thyroidal cells. Recent studies have reported the presence of TSH receptor in several extra‐thyroidal cell types, including erythrocytes. We have previously suggested that TSH is able to influence the erythrocyte Na/K‐ATPase ouabain binding properties through a receptor mediated mechanism. The direct interaction of TSH receptor with the Na/K‐pump and a functional role of TSHR in erythrocytes was not demonstrated. The interaction of TSH receptor with Na/K‐pump and a TSHR functional role are not yet demonstrated in erythrocytes. In this study, we examined the interaction between the two receptors after TSH treatment using immunofluorescence coupled to confocal microscopy and a co‐immunoprecipitation technique. The cAMP dependent signalling after TSH treatment was measured to verify TSHR functionality. We found that TSH receptor and Na/K‐ATPase are localized on the membranes of both erythrocytes and erythrocyte ghosts; TSH receptor responds to TSH treatment by increasing intracellular cAMP levels from two to tenfold. In ghost membranes TSH treatment enhances up to three fold co‐localization of TSHR with Na/K‐ATPase and co‐immunoprecipitation confirms their direct physical interaction. In conclusion our results are compatible with the existence, in erythrocytes, of a functional TSHR that interacts with Na/K‐ATPase after TSH treatment, thus suggesting a novel cell signalling pathway, potentially active in local circulatory control. Copyright © 2009 John Wiley & Sons, Ltd.     

K+ activity and regulation of intracellular pH in the sea urchin egg during fertilization

Fertilization of the sea urchin egg is accompanied by changes in intracellular ion activities and transmembrane fluxes, which regulate the sequence of biochemical events of metabolic derepression. Changes in intracellular K+ activity during fertilization have been controversial and here we report our measurements using intracellular K+-sensitive microelectrodes. A small, but statistically significant, transient rise in internal K+ activity was detected during the first 10 min of fertilization. Since this change in K+ activity was ouabain sensitive, intracellular K+ activity in the fertilized egg appears to be regulated by the increased Na+, K+ ATPase activity, rather than the previously suggested K+ decompartmentalization. Increasing external K+ concentration was found to stimulate ouabain-sensitive alkalinization in the fertilized egg. The data are consistent with the possibility that Na+, K+ ATPase may regulate cytoplasmic pH by recycling Na+ that enters the cell through Na+-H+ antiport.     

Changes in plant–soil hydraulic pressure gradients of soybean in response to soil drying

Plant gas‐exchange response to drying soil in many instances tracks a common pattern when expressed as a function of fraction of transpirable soil water (FTSW). There is little decrease in gas exchange until FTSW decreases to a value in the range of about 0.3–0.45, then with further drying gas exchange declines approximately linearly. This unique pattern is hypothesised to reflect mainly changes in the water potential gradient between bulk soil and plant. The primary objective was to directly document the basis of this response by measuring the hydrostatic pressure gradient required in the soil to maintain leaf xylem at zero potential with decreasing FTSW. Pots in which soybean (Glycine max) plants were grown were placed in a pressure chamber and the pressure adjusted to maintain zero water potential in a leaf petiolule. These results showed a small, relatively constant hydrostatic pressure had to be applied to the soil to maintain zero leaf xylem water potential until FTSW decreased to approximately 0.3–0.45. Thereafter, the required hydrostatic pressure gradient increased as FTSW continued to decrease. Hydraulic conductance was calculated to be relatively stable early in the drying cycle, and then decrease as the soil dried to comparatively high FTSW of 0.5–0.7.     

Sodium and proton transport in Mycoplasma gallisepticum.

When washed cells of Mycoplasma gallisepticum were incubated at 37 degrees C in 250 mM 22NaCl, the intracellular Na+ increased, and the K+ decreased. The addition of glucose to these Na+-loaded cells caused Na+ efflux and K+ uptake (both ions moving against concentration gradients). This effect of glucose was blocked by the ATPase inhibitor dicyclohexylcarbodiimide, which prevents the generation of a proton motive force in these cells. In additional experiments, Na+ extrusion was studied by diluting the 22Na+-loaded cells into Na+-free media and following the loss of 22Na+ from the cells. Glucose stimulated 22Na+ extrusion in such cells by a dicyclohexylcarbodiimide-sensitive mechanism. Proton movement was studied by measuring the pH gradient across the cell membrane with the 9-aminoacridine fluorescence technique. Glucose addition to cells preincubated with cations other than Na+ resulted in cell alkalinization (which was prevented by dicyclohexylcarbodiimide). This observation is consistent with the operation of a proton-extruding ATPase. When glucose was added to Na+-loaded cells and diluted into Na+-free media, intracellular acidification was observed, followed several minutes later by a dicyclohexylcarbodiimide-sensitive alkalinization process. The initial acidification was probably due to the operation of an Na+-H+ antiport, since Na+ exit was occurring simultaneously with H+ entry. When Na+-loaded cells were diluted into Na+-containing media, the subsequent addition of glucose resulted in a weak acidification, presumably due to H+ entry in exchange for Na+ (driven by the ATPase) plus a continuous passive influx of Na+. All of the data presented are consistent with the combined operation of an ATP-driven proton pump and an Na+ -H+ exchange reaction.     

Adenosine triphosphatase activity associated with purified cholinergic synaptic vesicles of Torpedo marmorata.

A rapid method for purifying Torpedo electric organ vesicles is described, which employs an isoosmotic continuous sucrose-glycine gradient followed by chromagography on CPG-10-3000 porous glass beads. The synaptic vesicles have a buoyant density of 1.057 g/ml. The purified vesicles are free of cholinesterase, lactate dehydrogenase and Na+, K+-stimulated ATPase activity. They contain a ouabaininsensitive, Na+, K+-inhibited, Mg2+, Ca2+-stimulated ATPase activity. This is further stimulated by acetylcholine but not by choline.     

Regulation of (Na++K+)-ATPase by inorganic phosphate: pH dependence and physiological implications

Inhibition of Na++K+-dependent ATPase activity by Pi was maximal in the pH range of 6.1-7, but decreased with increasing pH in the range of 7-8.5. Ki of Pi was 2.8 mM at pH 7.1, and 12 mM at pH 7.8. K+-dependent phosphorylation of the enzyme by Pi, which is thought to be responsible for inhibition of ATPase activity, also decreased with increasing pH. The data suggest that (a) previously observed requirement of high Pi concentrations for inhibition of ATPase activity and associated pump fluxes may have been due to high pH of the assays; (b) at normal values of intracellular pH the pump may be partially inhibited by intracellular Pi; and (c) this effect of Pi may be amplified or dampened with alterations in intracellular pH and ATP/Pi ratio.     

Steady-state voltages, ion fluxes, and volume regulation in syncytial tissues.

Equations are developed that describe the steady-state relationships among ion fluxes, solute fluxes, water flow, voltage, concentration of solute, and hydrostatic pressure in a spherically symmetrical syncytial tissue. Each cell of the syncytium is assumed to have membrane channels for Na, K, and Cl, a membrane pump for Na/K, and some concentration of intracellular protein of net negative charge. However, the surface cells and inner cells of the tissue are assumed to have different distributions of membrane transport properties, hence there is a radial circulation of fluxes and a radial distribution of forces. Some reasonable approximations are made that allow analytic solutions of the nonlinear differential equations. These solutions are used to analyze data from the frog lens and are shown to account for the known steady-state properties of this tissue. Moreover, these solutions are used to make predictions on other steady-state properties, which have not been directly measured, and graphical results on the circulation of water, ions and solute through the frog lens are presented.     

Tissue distribution of an endogenous ligand to the Na, K ATPase molecule

A variety of evidence indicates the presence of a circulating ligand to the Na, K ATPase molecule that is involved in the regulation of extracellular sodium metabolism. To examine the potential role of endogenous ligands to the Na, K ATPase molecule in the regulation of intracellular sodium metabolism, the tissue distribution of digitalis-like activity was quantitated in several brain regions and peripheral organs. The digitalis-like activity of desalted and delipidated extracts of tissue was widely distributed and produced a displacement of tritiated ouabain that was parallel to the displacement produced by cold ouabain. These results suggest that tissue contains an endogenous ligand to the Na, K ATPase molecule and that this ligand may regulate intracellular sodium metabolism in an autocoid-like manner.     

A method for controlling body wall turgor during electrophysiological recording from central neurons in ecdysing insects

A method for obtaining intracellular recordings from central neurons of insects during the performance of ecdysial behaviors is described. Body wall turgor must be restored to high levels in order to provide the CNS with appropriate sensory feedback required for the generation of motor output. This is accomplished by installing the insect in a decompression chamber so that an opening in the animal's ventral surface communicates with the atmosphere. Reduction of chamber pressure causes expansion of the insect's body, and access to thoracic ganglia is provided by the ventral opening in the animal's body wall. It is suggested that adaptations of this technique to invertebrates with hydrostatic skeletons is possible and would permit electrophysiological studies of behaviors which normally require high blood pressures for their performance.