Maintenance Of Cell Volume Research Articles

Altered taurine release following hypotonic stress in astrocytes from mice deficient for GFAP and vimentin

Astrocytes maintain their volume in response to changes in osmotic pressure in their environment by an afflux/influx of ions and organic osmoequivalents. The initial swelling of an astrocyte transferred to a hypoosmotic medium is thus reversed within minutes. The mechanisms which trigger this process as well as the sensors for cell volume are largely unknown, however, the cytoskeleton appears to be involved. We have addressed the role of one component of the cytoskeleton, the intermediate filaments, in the maintenance of astrocytic cell volume. Astrocytes from wild type mice were compared with cells from mice deficient for either glial fibrillary acidic protein (GFAP–/–) or vimentin (vimentin–/–) and with astrocytes from mice deficient for both proteins (GFAP–/–vim–/–). Whereas GFAP–/– and vimentin–/– cultured or reactive astrocytes retain intermediate filaments, the GFAP–/–vim–/– astrocytes are completely devoid of these structures. The rate of efflux of the preloaded osmoequivalent 3 H -taurine from primary and passaged cultures of astrocytes was monitored. A reduction of NaCl (25 mM) in the perfusion medium led to a 400–900% increase of 3 H -taurine afflux in astrocytes from wild type mice. The stimulated efflux was not significantly affected in astrocytes from GFAP–/– or vimentin–/– mice. However, the efflux from astrocytes from GFAP–/–vim–/– mice was 25–46% lower than the wild type levels. The results strengthen the role of the cytoskeleton in astrocyte volume regulation and suggest an involvement of intermediate filaments in the process.

Stimulation of Na+-K+-2Cl- cotransporter in neuronal cells by excitatory neurotransmitter glutamate.

Na+-K+-2Cl- cotransporters are important in renal salt reabsorption and in salt secretion by epithelia. They are also essential in maintenance and regulation of ion gradients and cell volume in both epithelial and nonepithelial cells. Expression of Na+-K+-2Cl- cotransporters in brain tissues is high; however, little is known about their function and regulation in neurons. In this study, we examined regulation of the Na+-K+-2Cl- cotransporter by the excitatory neurotransmitter glutamate. The cotransporter activity in human neuroblastoma SH-SY5Y cells was assessed by bumetanide-sensitive K+ influx, and protein expression was evaluated by Western blot analysis. Glutamate was found to induce a dose- and time-dependent stimulation of Na+-K+-2Cl- cotransporter activity in SH-SY5Y cells. Moreover, both the glutamate ionotropic receptor agonist N-methyl-D-aspartic acid (NMDA) and the metabotropic receptor agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) significantly stimulated the cotransport activity in these cells. NMDA-mediated stimulation of the Na+-K+-2Cl- cotransporter was abolished by the selective NMDA-receptor antagonist (+)-MK-801 hydrogen maleate. trans-ACPD-mediated effect on the cotransporter was blocked by the metabotropic receptor antagonist (+)-alpha-methyl-(4-carboxyphenyl)glycine. The results demonstrate that Na+-K+-2Cl- cotransporters in neurons are regulated by activation of both ionotropic and metabotropic glutamate receptors.

Comparison of Na-K-Cl Cotransporters: NKCC1, NKCC2, AND THE HEK CELL Na-K-Cl COTRANSPORTER

The Na-K-Cl cotransporter (NKCC) mediates the coupled movement of ions into most animal cells, playing important roles in maintenance of cell volume and in epithelial Cl transport. Two forms of NKCC have been described: NKCC1, the "housekeeping" isoform that is also responsible for Cl accumulation in secretory epithelial cells, and NKCC2, which mediates apical Na+K+Cl entry into renal epithelial cells. Here we examine the kinetic properties of NKCC1, NKCC2, and the endogenous HEK-293 cell cotransporter. Stable expression of rabbit NKCC2A was obtained in HEK-293 cells utilizing a chimera (h1r2A0.7) in which the 5'-untranslated region and cDNA encoding 104 amino acids of the N terminus are replaced by the corresponding sequence of NKCC1. h1r2A0.7 exhibits Na and Cl affinities near those of NKCC1, but it has a 4-fold lower Rb affinity, and a 3-fold higher affinity for the inhibitor bumetanide. The activity of h1r2A0.7 is increased on incubation in low [Cl] media as is NKCC1, but the resting level of activity is higher in h1r2A0.7 and activation is more rapid. h1r2A0.7 exhibits an appropriate volume response, unlike NKCC1 for which concomitant changes in [Cl]i appear to be the overriding factor. These results support a model in which apical NKCC2 activity is matched to basolateral Cl exit through changes in [Cl]i. Reverse transcriptase-polymerase chain reaction of HEK-293 cell mRNA is positive with NKCC1 primers and negative with NKCC2 primers. Surprisingly, we found that the behavior of the endogenous HEK cell Na-K-Cl cotransporter is unlike either of the two forms which have been described: compared with NKCC1, HEK cell cotransporter has a 2.5-fold lower Na affinity, an 8-fold lower Rb affinity, and a 4-fold higher bumetanide affinity. These results suggest the presence of a novel isoform of NKCC in HEK-293 cells.

Beta-agonist-induced activation of Na+ absorption and KCl release in rat fetal distal lung epithelium: a study of cell volume regulation.

The effects of terbutaline (a selective beta 2-adrenoceptor agonist) on cell volume and ion transport in rat fetal distal lung epithelial (FDLE) cells were studied. In FDLE cells, benzamil (1 microM) induced cell shrinkage, while cell volume was increased by 1 mM quinine or 2 mM Ba2+ but was not affected by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 20 microM). Terbutaline (10 nM) induced transient cell swelling, which was suppressed by benzamil, while 10 microM terbutaline induced initial rapid cell shrinkage followed by delayed slow cell shrinkage, which was suppressed by 1 mM quinine or 20 microM NPPB but not by 2 mM Ba2+. Application of benzamil enhanced the cell shrinkage induced by 10 microM terbutaline. These observations suggest that: (1) benzamil-blockable Na(+)-permeable channels and quinine- and Ba(2+)-blockable K+ channels contribute to maintenance of cell volume of FDLE cells; (2) terbutaline at both 10 nM and 10 microM activates benzamil-blockable Na(+)-permeable channels; (3) quinine-blockable K+ channels are activated by 10 microM terbutaline; and (4) NPPB-blockable Cl- channels are responsible for Cl- movement in cell volume changes induced by terbutaline. The present study demonstrates that the cellular mechanisms of volume change induced by terbutaline are closely related to activation of Na+ absorption and KCl release.

Rapid and transient up-regulation of Na+/myo-inositol cotransporter transcription in the brain of acute hypernatremic rats.

The osmoregulatory system is well developed in the brain. Osmolytes contribute to maintenance of cell volume and cellular functions without changing intracellular ionic composition. Myo-inositol is regarded as one of the major osmolytes in the brain. In the present study, we investigated the changes in expressions of sodium myo-inositol cotransporter (SMIT) mRNA in the brain of acute hypernatremic rats by in-situ hybridization and Northern blot methods. Under moderate acute hypernatremic conditions, SMIT mRNA level increased markedly at 1 h and returned to almost control levels at 3 h, in accordance with plasma Na+ concentrations. Especially, distinct increases in SMIT mRNA expression were observed in the granule cells and glial cells in the cerebellum. These findings indicate that SMIT plays an important role in osmoregulation, especially in the early stages of acute hypernatremia in the brain.

Rapid and transient up-regulation of Na+/myo-inositol cotransporter transcription in the brain of acute hypernatremic rats

The osmoregulatory system is well developed in the brain. Osmolytes contribute to maintenance of cell volume and cellular functions without changing intracellular ionic composition. Myo-inositol is regarded as one of the major osmolytes in the brain. In the present study, we investigated the changes in expressions of sodium myo-inositol cotransporter (SMIT) mRNA in the brain of acute hypernatremic rats by in-situ hybridization and Northern blot methods. Under moderate acute hypernatremic conditions, SMIT mRNA level increased markedly at 1 h and returned to almost control levels at 3 h, in accordance with plasma Na+ concentrations. Especially, distinct increases in SMIT mRNA expression were observed in the granule cells and glial cells in the cerebellum. These findings indicate that SMIT plays an important role in osmoregulation, especially in the early stages of acute hypernatremia in the brain.

Expression of Na+/myo-inositol cotransporter mRNA in the inner ear of the rat.

We have demonstrated the cellular localization of Na+/myo-inositol cotransporter (SMIT) mRNA in the rat inner ear by in situ hybridization. In the cochlea, the most intense SMIT mRNA signals were observed in fibrocytes of the spiral ligament, moderate signals were found in the spiral limbus, inner hair cells and spiral ganglion cells, while the hybridization signals were almost undetectable in the marginal cells of the stria vascularis and outer hair cells. In the vestibular system, moderate hybridization signals were found in the sensory epithelium, fibrocytes and vestibular ganglion cells. These findings suggest that SMIT plays an important role in maintenance of intracellular ionic balance and cell volume in the inner ear, especially in the fibrocytes associated with generation of the ion gradients between the endolymph and perilymph.

Hypo-osmotic challenge stimulates transepithelial K+ secretion and activates apical IsK channel in vestibular dark cells.

Volume regulation of vestibular dark cells from the gerbilline inner ear in response to a hypo-osmotic challenge depends on the presence of cytosolic K+ and Cl-. The present study addresses the questions: (i) whether and by what mechanism K+ is released during volume regulation, (ii) whether the osmolarity of the basolateral medium has an effect on the steady-state rate of transepithelial K+ transport and (iii) whether there is cross-talk between the basolateral membrane responsible for K+ uptake and the apical membrane responsible for K+ release. K+ secretion (JK+,probe) and current density (Isc,probe) were measured with vibrating probes in the vicinity of the apical membrane and the transepithelial potential (Vt) and resistance (Rt) were measured in a micro-Ussing chamber. The equivalent short-circuit current (Isc) was calculated. The current (IIsK), conductance (gIsK) and inactivation time constant (tau IsK) of the IsK channel and the apparent reversal potential of the apical membrane (Vr) were obtained with the cell-attached macropatch technique. Vr was corrected (Vrc) for the membrane voltage (Vm) measured separately with microelectrodes. A hypo-osmotic challenge (294 to 154 mosM by removal of 150 mM mannitol) on the basolateral side of the epithelium increased JK+,probe and Isc,probe by a factor of 2.7 and 1.6. When this hypo-osmotic challenge was applied to both sides of the epithelium Vt and Isc increased from 5 to 14 mV and from 189 to 824 microA/cm2 whereas Rt decreased from 27 to 19 omega-cm2. With 3.6 mM K+ in the pipette IIsK was outwardly directed, tau IsK was 267 msec and the hypo-osmotic challenge caused IIsK and gIsK to increase from 14 to 37 pA and from 292 to 732 pS. Vrc hyperpolarized from -44 to -76 mV. With 150 mM K+ in the pipette IIsK was inwardly directed, tau IsK was 208 msec and the hypo-osmotic challenge caused IIsK and gIsK to increase in magnitude from 0 to -21 pA and from 107 to 1101 pS. Vrc remained unchanged (-2 vs. 1 mV). These data demonstrate that a hypo-osmotic challenge stimulates transepithelial K+ secretion and activates the apical IsK channel. The hypo-osmotically-induced increase in K+ secretion exceeded the estimated amount of K+ release necessary for the maintenance of constant cell volume, suggesting that the rate of basolateral K+ uptake was upregulated in the presence of the hypo-osmotic challenge and that cross-talk exists between the apical membrane and the basolateral membrane.

Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells.

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of approximately 30% in suspended (i.e., round) cells and approximately 60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with BAPTA-AM and by depletion of Cl-, indicating the role of calcium-regulated K+ and Cl- efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (approximately 0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na(+)-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl(-)-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl- that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.

Effect of axoplasmic transport on the mechanism responsible for maintenance of cell volume in rat skeletal muscles

The effect of colchicine, a drug impairing fast axoplasmic flow, on the ability of muscle fibers to recover their volume after dehydration in a hypertonic medium, was studied in experiments on rat diaphragm. It was found that the ability of the muscle fibers to maintain their volume in a hyperosmolar medium depends on the effect of “trophic factors” transferred to the muscle by the axonic flow. Elimination of this effect by blocking the axoplasmic transport with colchicine impairs the ability of active chloride transport to be enhanced following an increase in the muscle osmotic pressure, which results in the loss of the muscle fiber's capability to maintain stable content of intracellular water in a hypertonic medium.

The Na-K-Cl cotransporters

The Na-K-Cl cotransporters are a class of membrane proteins that transport Na, K, and Cl ions into and out of cells in an electrically neutral manner, in most cases with a stoichiometry of 1Na:1K:2Cl. Na-K-Cl cotransporters are present in a wide variety of cells and tissues, including reabsorptive and secretory epithelia, nerve and muscle cells, endothelial cells, fibroblasts, and blood cells. Na-K-Cl cotransport plays a vital role in renal salt reabsorption and in salt secretion by intestinal, airway, salivary gland, and other secretory epithelia. Cotransport function also appears to be important in the maintenance and regulation of cell volume and of ion gradients by both epithelial and nonepithelial cells. Na-K-Cl cotransport activity is inhibited by "loop" diuretics, including the clinically efficacious agents bumetanide and furosemide. The regulation of Na-K-Cl cotransport is mediated, at least in some cases, through direct phosphorylation of the cotransport protein. Cotransporter regulation is highly tissue specific, perhaps in part related to the presence of different Na-K-Cl cotransporter isoforms. In epithelia, both absorptive (kidney-specific) and secretory isoforms have been identified by cDNA cloning and sequencing and Northern blot analysis; alternatively spliced variants of the kidney-specific isoform have also been identified. The absorptive and secretory isoforms exhibit approximately 60% identity at the amino acid sequence level; these sequences in turn show approximately 45% overall homology with those of thiazide-sensitive, bumetanide-insensitive, Na-Cl cotransport proteins of winter flounder urinary bladder and mammalian kidney. This review focuses on recent developments in the identification of Na-K-Cl cotransport proteins in epithelial and on the regulation of epithelial Na-K-Cl cotransporter function at cellular and molecular levels.

Cell volume and ion transport regulation

The cell membrane of virtually all animal cells is highly permeable to water, and hence cell volume is determined by the cellular content of osmotically active solutes, and by the osmolarity of the extracellular fluid. Maintenance of cell volume is thus a fundamental physiological process and a requirement for cell survival. In most animal cells total volume and water content are closely regulated (Gilles, 1978; Ma&night, 1988). Most animal cells defend themselves from colloid-osmotic swelling by the operation of ion pumps, which transport action ions from the intracellular to the extracellular compartment, therefore tending to reduce the intracellular concentration of permeant ions, and compensating for the excess concentration of impermeant solutes. This mechanism, referred to as the pumpleak hypothesis was first described by Wilson (1954) and then further proposed and substantiated by Leaf (1956) and Tosteson and Hoffman (1960). According to this hypothesis a constant cellular volume is achieved because the dominant extracellular diffusible cation, sodium, is effectively extruded from the cells through the activity of a sodium pump. The obligatory extracellular location of sodium effectively offsets the tendency for cells to swell as a consequence of the colloid osmotic force generated by the impermeant cell solutes. As originally proposed by Leaf (1956), the properties of

Regulation of mammalian brain cell volume.

Maintenance of brain cell volume is of crucial importance for normal central nervous system (CNS) function. This review considers volume regulation primarily in response to disturbances of body fluid osmolality. Brain cells counter the tendency to swell or shrink by appropriate adjustment of their internal osmotic potential. This is achieved by loss or uptake of inorganic ions and low molecular weight organic solutes (osmolytes). The latter comprise mainly amino acids, myoinositol, choline, and methylamines. Taurine may be of particular importance in volume control, especially in young animals. Brain cell volume regulation, however, is only one contributory factor to maintenance of constant brain volume (water content), and operates in parallel with important alterations in bulk fluid and electrolyte movement across the blood-brain barrier and between the interstitium and cerebrospinal fluid, which themselves moderate the requirement for transient alteration in cell volume during acute osmotic imbalance. Although altered cerebral content of inorganic ions and osmolytes are usually regarded as responses, respectively, to acute and chronic osmotic disturbances, osmolytes (especially taurine) may also participate in short-term cell volume regulation.

Influence of Rising Atmospheric CO2 Concentrations and Temperature on Growth, Yield and Grain Quality of Cereal Crops

A possible scenario for the end of the 21st century is that the atmospheric CO2 concentration will be in the range of 510-760 μL L-1 and that the mean global temperature will be 1.5-4.5�C higher. Further, there may be greater incidences of extreme climatic events, which together with the CO2 and temperature changes will influence development, growth and grain yield of cereals such as rice and wheat. For these C3 plants, the driving force for the growth response to elevated CO2 is higher leaf CO2 assimilation rates (A). However, the response of A to CO2 depends on temperature with maximum absolute increases occuring at temperatures which do not cause flower abortion, while negligible increases are observed at low temperatures. At high temperatures, where A is reduced because of partial inactivation of photosynthetic enzymes, the increase in A due to CO2 enrichment is still observed. Other factors, such as changes in shoot water relations or hormone concentrations, may influence growth at elevated CO2 concentrations. Wheat and rice development is accelerated by high temperature and consequently grain yield is reduced because there is less time for radiation to be intercepted during the vegetative phase. Although high CO2 also accelerates development in rice and, to a lesser extent in wheat, the extra carbohydrate produced by increases in A results in at least a 40% increase in grain yield at temperatures which do not cause flower abortion. This is due mainly to increased tiller numbers rather than increases in the number or weight of individual grains. However, the yield enhancement due to high CO2 will not necessarily compensate for decreases in yield caused by accelerated development at high temperatures. As predicted by the response of A to high CO2, the relative increase in yield, due to rising CO2 concentrations, is smaller at lower temperatures. Elevated atmospheric CO2 may improve the tolerance of plants to heat-induced drought stress by facilitating the maintenance of cell volume and photosynthetic function in the leaves. Increased carbohydrate storage in the stems may also be an advantage during grain filling if the flag leaves senesce prematurely. However, it is unlikely that the effect of very high temperatures on flower abortion will be ameliorated by high CO2. For bread making, the quality of flour produced from grain developed at high temperatures is poorer. High CO2 may also have an effect through a reduction in the protein content of wheat grain. For rice, the amylose content of the grain, a major determinant of cooking quality is increased under elevated CO2.

Independence of Na:K pump activity and basolateral membrane conductance in bullfrog corneal epithelium

1. 1. The effects of inhibition of the Na:K pump on membrane conductances in the isolated bullfrog corneal epithelium were assessed on the basis of measurements of DC and AC electrical parameters in conjunction with morphological analyses. 2. 2. Inhibition of the Na:K pump by either 0.1 mM ouabain or 5 mM BaCl 2 increased the epithelial thickness and constricted the intercellular spaces. Initially, ouabain did not change either the apical or basolateral membrane conductance whereas barium selectively decreased the latter by 35%. 3. 3. Therefore, a decrease in Na:K pump activity has no direct effect on membrane conductance. Nevertheless, its activity is linked to cell volume maintenance.

Maintenance of cell volume in the central nervous system.

Maintenance of the ionic and osmotic composition and volume of intra- and extracellular fluids in the brain is crucial for normal functioning of the central nervous system (CNS). Osmoregulation in the CNS is mediated by solute and water transport across the blood-brain barrier, choroid plexus and plasma membrane of glial cells and neurons. Despite its clinical and physiological significance, however, little is known about the underlying cellular and molecular mechanisms by which CNS osmotic and ionic balance is maintained. In this review, I will discuss our current understanding of cell volume regulation in the CNS and how it relates to various disease processes, such as hyponatremia, renal failure and hypernatremia. A detailed understanding of brain osmoregulatory processes represents a fundamental physiological problem and is required for the treatment of numerous disease states, particularly those encountered in the practice of nephrology.

Na+/K+ pump inhibition induces cell shrinkage in cultured chick cardiac myocytes.

Myocardial cell swelling occurs in ischemia and in reperfusion injury before the onset of irreversible injury. Swelling has been attributed to failure of the Na+/K+ pump and the accumulation of intracellular Na+. To evaluate the role of the pump-leak model of cell volume maintenance, short term changes in cell volume in response to Na+/K+ pump inhibition were studied in aggregates of cultured embryonic chick cardiac myocytes using optical and biochemical methods. Exposure to 100 microM ouabain over 20 min induced cell shrinkage of approximately 10%. Cell water was also decreased by Na+/K+ pump inhibition; incubation for 1 hr either in the presence of 100 microM ouabain or in K(+)-free solution reduced cell water by 18.4% and 28.4% respectively. When exposed to ouabain in the absence of extracellular Ca2+, the aggregates swelled by approximately 15%, indicating that extracellular Ca2+ was required for the ouabain-induced shrinkage to occur. Ouabain still caused shrinkage, however, in the presence of the Ca2+ channel blockers verapamil (10 microM) and nifedipine (10 microM), suggesting that Na+/Ca2+ exchange, rather than Ca2+ channels, is the route for Ca2+ influx during Na+/K+ pump inhibition. Efflux of amino acids (taurine, aspartate, glutamate, glycine and alanine) from confluent monolayers of chick heart cells exposed to ouabain for 20 min was nearly double that observed in control solution. These results suggest that, during Na+/K+ pump inhibition, chick heart cells can limit accumulation of intracellular sodium by means of Na+/Ca2+ exchange, and that a rise in intracellular [Ca2+], also mediated by Na+/Ca2+ exchange, promotes the loss of amino acids and ions to cause cell shrinkage. Therefore, swelling during ischemic injury may not result from Na+/K+ pump failure alone, but may reflect the exhaustion of alternative volume regulatory transport mechanisms.

Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: dependence on medium osmolality and regulation by signalling systems.

To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and 86Rb and 22Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 microliters per 10(6) cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P less than 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by approximately 7%, P less than 0.05) and by ethylisopropyl amiloride (by approximately 13%, P less than 0.005). Ouabain-sensitive 86Rb influx was decreased by 30-40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an approximately eightfold increase in bumetanide-inhibited 86Rb influx and 86Rb efflux. The (ouabain and bumetanide)-insensitive component of 86Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive 86Rb efflux was increased by approximately 1.5-2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited 22Na influx was increased by approximately sixfold following osmotic-shrinkage of VSMC. The data show that both Na+/H+ exchange and Na+/K+/2Cl- cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na+/K+/2Cl- cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro-4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na+/K+/2Cl- cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na+/H+ exchange activity. NaF increased Na+/H+ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na+/K+/2Cl- cotransporter and the Na+/H+ exchanger.

Photodynamic treatment of yeast cells with the dye Toluidine blue: all-or-none loss of plasma membrane barrier properties

Photodynamic treatment of Kluyveromyces marxianus with the sensitizer Toluidine blue leads to the loss of colony forming capacity. In this paper, the influence of this treatment on the barrier properties of the plasma membrane has been studied. Photodynamic treatment with the dye Toluidine blue resulted in efflux of potassium ions and E 260-absorbing material. Moreover, cells became stainable with erythrosine. It is concluded that the permeability change induced by photodynamic treatment proceeds in an all-or-none fashion. Treatment of this yeast strain, with the dye and light, also induced a diminution of the cell volume. This process is most likely not coupled to the cellular potassium content, but rather to the integrity of the vacuole. These data suggest that the vacuole has an important function in the maintenance of cell volume. Finally, it was observed that the loss of cell viability was not induced by the all-or-none loss of barrier properties.

Efflux and accumulation of amino nitrogen in relation to the volume of rat renal inner medullary cells exposed to media of variable osmolality

The rate of efflux of 2-amino[ 14C]isobutyric acid (AIB) from pre-loaded slices of rat renal inner medulla has been followed during incubation in media whose osmolality was varied between 350 and 2500 mosmol/kg H 2O by adjustment of NaCl and urea concentrations. Efflux was biphasic, and it was assumed that the second, slower phase represented mainly cellular loss of AIB. As a function of cell volume (water content) the mean net rate of 2nd phase efflux declined far more abruptly (−36%) during an increase in external osmolality from 350 to 720 mosmol/kg than during further increase to 2500 mosmol/kg, over which range the rate of efflux fell by only a further 12%. Conversely, relative decrements of steady-state cell water contents during these two transitions were −17% and −37%, respectively. It is probable that in strongly hyperosmolal media (above 720 mosmol/kg) reduction in the rate of amino acid catabolism, with resultant cellular accumulation, becomes more important than passive efflux as a determinant of cell amino nitrogen content, and that this is caused by the enzyme-destabilizing effect of high intracellular concentrations of permeant urea. This interpretation is supported by the finding in the present study that trimethylamine N-oxide, which is known to counteract the destabilzing effect of urea, completely inhibited the accumulation of amino nitrogen (ninhydrin-positive substances) in media stronger than 720 mosmol/kg, as well as leading to further reduction of steady-state cell water contents, but was without effect on either variable in more dilute media. It is proposed that, under the conditions of this investigation, amino acids contribute to cell volume maintenance mainly by efflux and by metabolic accumulation under mildly and strongly hyperosmolal conditions, respectively, and that this interpretation is consistent with recent findings on the fluctuations in medullary levels of Na +, urea and total amino nitrogen in the intact kidneys of rats during acute water diuresis and oliguria (Law, R.O. (1991) Pflügers Arch. 418, 442–446).