Energy-dependent volume regulation in primary cultured cerebral astrocytes

Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo.     

Fluoxetine Inhibits K+ Transport Pathways (K+ Efflux, Na+-K+-2Cl− Cotransport, and Na+ Pump) Underlying Volume Regulation in Corneal Endothelial Cells

We have studied regulatory volume responses of cultured bovine corneal endothelial cells (CBCEC) using light scattering. We assessed the contributions of fluoxetine (Prozac) and bumetanide-sensitive membrane ion transport pathways to such responses by determining K⁺ efflux and influx. Cells swollen by a 20% hypo-osmotic solution underwent a regulatory volume decrease (RVD) response, which after 6 min restored relative cell volume by 98%. Fluoxetine inhibited RVD recovery; 20 μm by 26%, and 50 μm totally. Fluoxetine had a triphasic effect on K⁺ efflux; from 20 to 100 μm it inhibited efflux 2-fold, whereas at higher concentrations the efflux first increased to 1.5-fold above the control value, and then decreased again. Cells shrunk by a 20% hyperosmotic solution underwent a regulatory volume increase (RVI) which also after 6 min restored the cell volume by 99%. Fluoxetine inhibited RVI; 20 μm by 25%, and 50 μm completely. Bumetanide (1 μm) inhibited RVI by 43%. In a Cl⁻-free medium, fluoxetine (50–500 μm) progressively inhibited bumetanide-insensitive K⁺ influx. The inhibitions of RVI and K⁺ influx induced by fluoxetine 20 to 50 μm were similar to those induced by 1 μm bumetanide and by Cl⁻-free medium. A computer simulation suggests that fluoxetine can interact with the selectivity filter of K⁺ channels. The data suggest that CBCEC can mediate RVD and RVI in part through increases in K⁺ efflux and Na-K-2Cl cotransport (NKCC) activity. Interestingly, the data also suggest that fluoxetine at 20 to 50 μm inhibits NKCC, and at 100–1000 μm inhibits the Na⁺ pump. One possible explanation for these findings is that fluoxetine could interact with K⁺-selective sites in K⁺ channels, the NKC cotransporter and the Na⁺ pump.     

Volume regulation in intertidalProcephalothrix spiralis (Nemertina) andClitellio arenarius (Oligochaeta)

Summary Intertidal worms such as the nemertine,P. spiralis, and the oligochaete,C. arenarius, are exposed to fluctuating salinity conditions in their natural habitat. Both species are volume regulators. To determine the influence of neurosecretory and neuroglandular mechanisms on regulatory volume increase (RVI) in hyperosmotic media, control and decerebrated worms of both species were (1) exposed to 70% SW followed by return to 100% SW [70% SW (2, 12 or 24 h) 100% SW (12 h)] or (2) exposed to a fluctuating salinity regimen [70% SW (2 h) 100% SW (12 h) repeat]. The effects of fluctuating salinity on regulatory volume decrease (RVD), in hypoosmotic media, were examined simultaneously.RVI was manifested either as a shrinkage limitation or a net volume gain. In both cases it was accompanied by increases in Na and Cl but not K content (moles/gram solute free dry weight=g s.f.d.w.). Repeated return to full-strength seawater potentiated the RVI response in controlC. arenarius only. Whole animal volume control is a result of extracellular as well as intracellular volume regulation. InC. arenarius, decerebration prevented RVI by apparently inhibiting extracellular volume control through reduction in ion uptake. It also resulted in reduced capacity to maintain a hyperosmotic gradient across the body wall which further supports an effect of decerebration on ion uptake at the integument or gut. InP. spiralis, in which whole animal volume regulation is primarily an intracellular phenomenon, decerebration did not affect RVI.RVD (swelling limitation and net volume loss) was, in all worms, accompanied by a decrease in Na and Cl but not K content (moles/g s.f.d.w.). Repeated exposure to 70% SW reduced the RVD response inP. spiralis and in controlC. arenarius. Decerebration eliminated extracellular swelling limitation and delayed net volume loss inC. arenarius but had no measurable effect onP. spiralis (corroborating previous results; Ferraris and Schmidt-Nielsen 1982a, b).It appears that neuroendocrine factors primarily stimulate extracellular volume regulation in RVI as well as in RVD, but have no effect on intracellular volume regulation in these worms. When the worms are exposed to repeated changes in medium osmolality RVD is reduced in both species, while RVI is enhanced in controlC. arenarius only. Thus, while both extracellular and intracellular RVD are reduced, only extracellular RVI is potentiated.Abbreviations g s.f.d.w. gram solute free dry weight - NPS ninhydrin positive substances - RVD regulatory volume decrease - RVI regulatory volume increase - SW seawater     

Involvement of integrins in osmosensing and signaling toward autophagic proteolysis in rat liver

Inhibition of autophagic proteolysis by hypoosmotic or amino acid-induced hepatocyte swelling requires osmosignaling toward p38MAPK; however, the upstream osmosensing and signaling events are unknown. These were studied in the intact perfused rat liver with a preserved in situ environment of hepatocytes. It was found that hypoosmotic hepatocyte swelling led to an activation of Src (but not FAK), Erks, and p38MAPK, which was prevented by the integrin inhibitory hexapeptide GRGDSP, but not its inactive analogue GRGESP. Src inhibition by PP-2 prevented hypoosmotic MAP kinase activation, indicating that the integrin/Src system is located upstream in the osmosignaling toward p38MAPK and Erks. Inhibition of the integrin/Src system by the RGD motif-containing peptide or PP-2 also prevented the inhibition of proteolysis and the decrease in autophagic vacuole volume, which is otherwise observed in response to hypoosmotic or glutamine/glycine-induced hepatocyte swelling. These inhibitors, however, did not affect swelling-independent proteolysis inhibition by phenylalanine. In line with a role of p38MAPK in triggering the volume regulatory decrease (RVD), PP-2 and the RGD peptide blunted RVD in response to hypoosmotic cell swelling. The data identify integrins and Src as upstream events in the osmosignaling toward MAP kinases, proteolysis, and RVD. They further point to a role of integrins as osmo- and mechanosensors in the intact liver, which may provide a link between cell volume and cell function.     

Regulatory volume decrease in nematocytes isolated from acontia of Aiptasia diaphana.

Regulatory volume decrease (RVD) and the mechanisms of its regulation were investigated in microbasic mastigophore nematocytes isolated from the acontia of Aiptasia diaphana (Coelenterates, Cnidaria), a marine species that can be exposed to considerable changes in osmotic pressure. Exposure of isolated cells to a 35% hypoosmotic shock lead to the expected osmotic swelling followed by a rapid RVD. RVD was blocked if Ca2+ influx was prevented either by applying a Ca2+-free medium or by treating the cells with Gd3+. Furthermore, the calmodulin action inhibitor trifluoperazine (TFP), prevented RVD and also caused a larger swelling than that induced by preventing Ca2+ influx. Treatment of nematocytes with quinine completely blocked the RVD. Such an effect was prevented by gramicidine. A partial inhibition of RVD was caused by treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). It is concluded that: i) the nematocytes regulate volume under hypoosmotic shock; ii) the regulatory mechanisms consist mainly in increased conductance to K+, and consequently, of Cl-, and, to a lesser extent, in H+/K+-Cl-/HCO3- exchange, and iii) the ionic fluxes are triggered by increased [Ca2+]i with the possible involvement of calmodulin.     

Volume regulation in leukocytes: Requirement for an intact cytoskeleton

Animal cells regulate their volume by controlling the flux of ions across their plasma membrane. Recent evidence suggests that ion channels and pumps are physically associated with, and may be regulated by components of the cytoskeleton. To elucidate the role of elements of the cytoskeleton in volume regulation, we studied the effects of cytoskeletal disrupting agents on regulatory volume decrease (RVD) in three different leukocyte types: Jurkat lymphoma cells, HL-60 cells, and human peripheral blood neutrophils. Cell volume was measured in two ways: (i) electronically with a Coulter counter and (ii) by forward light scattering in a flow cytometer. Exposure of all leukocyte types to hypotonic medium (200 mOsm) resulted in an immediate increase in cell volume followed by a regulatory decrease to baseline by 20 min. In the presence of the microtubule disrupting agents, colchicine and nocodazole, RVD was totally inhibited which corresponded to loss of microtubules as determined by immunofluorescence. Similarly, RVD was inhibited in Jurkat cells incubated with the actin binding agents, cytochalasin B (CB) or D (CD). In contrast, in HL-60 cells and human neutrophils, RVD was unaffected by treatment with either CB or CD. While cytochalasins are generally thought of as microfilament disrupting agents, their primary action is to prevent F-actin polymerization. The extent of ensuing microfilament disruption depends in part on the rate of filament turnover. In an attempt to understand the differential effects of the cytochalasins on RVD, the F-actin content of the different cells was determined by NBD-phallacidin staining and flow cytometry. Pretreatment with CB or CD resulted in profound actin disassembly in Jurkat cells (relative fluorescence index RFI: 1.0 control vs. 0.21 ± 0.01 for CB and 0.48 ± 0.02 for CD). However, the cytochalasins did not induce net disassembly in either HL-60 cells or human neutrophils. To study the effects of an increase in F-actin on volume regulation, neutrophils were treated with the chemoattractant f-Met-Leu-Phe or with an antibody (Ab) to β2 integrins followed by a cross-linking secondary Ab. Despite an increase in F-actin in both circumstances, RVD remained intact. Taken together, these results suggest that both microtubules and microfilaments are important in volume regulation. © 1995 Wiley-Liss, Inc.     

Potentiation of Regulatory Volume Decrease by a P2-Like Receptor and Arachidonic Acid in American Alligator Erythrocytes

This study examined the role of a P2 receptor and arachidonic acid (AA) in regulatory volume decrease (RVD) by American alligator red blood cells (RBCs). Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy. Gadolinium (50 μM), hexokinase (2.5 U/ml), and suramin (100 μM) increased osmotic fragility, blocked volume recovery after hypotonic shock, and prevented a rise in intracellular Ca²⁺ that normally occurs during cell swelling. The P2X antagonists PPADS (50 μM) and TNP-ATP (10 μM) also increased fragility and inhibited volume recovery. In contrast, ATPγS (10 μM), α,β-methylene-ATP (50 μM) and Bz-ATP (50 μM) had the opposite effect, whereas 2-methylthio-ATP (50 μM) and UTP (10 μM) had no effect. In addition, the phospholipase A₂ (PLA₂) inhibitors ONO-RS-082 (10 μM), chlorpromazine (10 μM), and isotetrandrine (10 μM) increased osmotic fragility and blocked volume recovery, whereas AA (10 μM) and its nonhydrolyzable analog eicosatetraynoic acid (ETYA, 10 μM) had the reverse effect. Further, AA (10 μM), but not ATPγS (10 μM), prevented the inhibitory effect of a low Ca²⁺-EGTA Ringer on RVD, whereas both AA (10 μM) and ATPγS (10 μM) caused cell shrinkage under isosmotic conditions. In conclusion, our results are consistent with the presence of a P2-like receptor whose activation stimulated RVD. In addition, AA also was important for volume recovery.     

Regulatory volume decrease in cultured kidney cells (A6): role of amino acids

Volume regulation was studied in A6 epithelia grown on permeable supports by measuring cell thickness (Tc) while simultaneously recording short circuit current (ISC) and transepithelial conductance (Gt). Lowering the tonicity of the basolateral solution (pi b) from 250 or 215 to 140 mOsm/kg elicited a rapid rise in Tc followed by a regulation of the cell volume towards control. This decrease in Tc displays the characteristics of the regulatory volume decrease (RVD). Upon restoring the isoosmotic conditions, Tc decreased rapidly below its control value. A post RVD regulatory volume increase (RVI) as described for other cell types was not observed. The subsequent reduction of the basolateral osmolality increased Tc to the level recorded at the end of the first hypoosmotic pulse. Because cell content was not altered during the isoosmotic period the second hypoosmotic challenge was isotonic with the cell and did therefore not evoke an RVD. However, the cell did not lose its ability to volume regulate since an RVD could be elicited by further reduction of pi b from 140 to 100 mOsm/kg. The possibility of an involvement of amino acids in the RVD was tested. The amount of amino acids in the cell as well as excreted in the bath was determined by amino acid analysis. Millimolar concentrations of threonine, serine, alanine, glutamate, glycine and aspartate were found in the cell extract. The cellular amino acid concentration was 28.8 +/- 0.4 mM. The amounts of glycine, aspartate and glutamate excreted from the cell during the hypotonic treatment were significantly larger than in control conditions. The excretion of these amino acids during hypotonicity decreased the cellular amino acid concentration by 8.4 +/- 0.2 mM. This quantity cannot completely account for the RVD during the first hypotonic challenge. The addition of glycine, aspartate and glutamate to the bathing solutions, although used at concentrations higher than intracellularly, did not reduce RVD. On the contrary, this maneuver increased the amplitude of the RVD following both hypoosmotic pulses. This result suggests a stimulatory role of the amino acids on the processes responsible for the RVD.     

Kinetics of ATP release and cell volume regulation of hyposmotically challenged goldfish hepatocytes

In most animal cells, hypotonic swelling is followed by a regulatory volume decrease (RVD) thought to prevent cell death. In contrast, goldfish hepatocytes challenged with hypotonic medium (180 mosM, HYPO) increase their volume 1.7 times but remain swollen and viable for at least 5 h. Incubation with ATPgammaS (an ATP analog) in HYPO triggers a 42% volume decrease. This effect is concentration dependent (K(1/2) = 760 nM) and partially abolished by P2 receptor antagonists (64% inhibition). A similar induction of RVD is observed with ATP, UTP, and UDP, whereas adenosine inhibits RVD. Goldfish hepatocytes release more than 500 nM ATP during the first minutes of HYPO with no induction of RVD. The fact that similar concentrations of ATPgammaS did trigger RVD could be explained by showing that ATPgammaS induced ATP release. Finally, we observed that in a very small extracellular volume, hepatocytes do show a 56% RVD. This response was diminished by P2 receptor antagonists (73%) and increased (73%) when the extracellular ATP hydrolysis was inhibited 72%. Using a mathematical model, we predict that during the first 2 min of HYPO exposure the extracellular [ATP] is mainly governed by ATP diffusion and by both nonlytic and lytic ATP release, with almost no contribution from ecto-ATPase activity. We show that goldfish hepatocytes under standard HYPO (large volume) do not display RVD unless this is triggered by the addition of micromolar concentrations of nucleotides. However, under very low assay volumes, sufficient endogenous extracellular [ATP] can build up to induce RVD.     

Volume regulation of Chinese hamster ovary cells in anisoosmotic media

Chinese hamster ovary (CHO) cells when suspended in anisoosmotic media regulate their volumes by the activation of specific ion transport pathways. In hypoosmotic media the cells first swell and then return to their isoosmotic volumes by the loss of cellular KCl and osmotically obliged water. This regulatory volume decrease (RVD) is insensitive to ouabain or bumetanide but is blocked by quinine, cetiedil and oligomycin C. Based on cell volume and membrane potential measurements under various experimental conditions, we conclude that hypoosmotic shock activates independent, conductive transport pathways for K+ and for Cl-, respectively. The anion pathway can also transport NO3- and SCN- but not gluconate- anions. Osmotic shrinkage of CHO cells does not produce a regulatory volume increase (RVI) unless the cells have previously undergone a cycle of RVD. RVI is a Na+-dependent, amiloride-sensitive, but ouabain- and oligomycin-insensitive process, probably involving a Na+-H+ exchange system. Internal acidification of isoosmotic cells by addition of a permeable weak acid also activates an amiloride-sensitive Na+-H+ exchange, producing a volume increase. Both RVD and RVI in CHO cells seem to involve molecular mechanisms similar to those described for the volume regulation of lymphocytes, indicating the prevalence of these phenomena in nucleated mammalian cells. Cultured CHO cell lines may provide a basis for a genetic characterization of the volume-regulatory transport pathways.     

Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential

Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K⁺ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (V_m), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in V_m. Cell volume and V_m changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by V_m depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K⁺ or Cl⁻ channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations –all leading to changes in V_m– define the success of RVD.     

Toxicity of organotins towards cyanobacterial photosynthesis and nitrogen fixation

Abstract Inhibition of photosynthesis by a range of organotin compounds in Plectonema boryanum was concentration-dependent and decreased in the order tributyltin (Bu₃SnCl) > tripropyltin (Pr₃SnCl) ≥ dibutyltin (Bu₂SnCl₂) ≥ triphenyltin (Ph₃SnCl) > triethyltin (Et₃SnCl) > trimethyltin (Me₃SnCl) > monobutyltin (BuSnCl₃). IC₅₀ values were determined for the most toxic organotin species and varied from approximately 1.2 μM for Bu₃SnCl to approximately 13 μM for Ph₃SnCl. A similar order of inhibition of photosynthesis was observed in Anabaena cylindrica , although here IC₅₀ values were slightly lower (e.g. approximately 1 μM for Bu₃SnCl and 5 μM for Ph₃SnCl).Nitrogenase activity was generally more sensitive to inhibition by organotin compounds than photosynthesis in A. cylindrica and this was particularlyy evident for Bu₂SnCl₂; approximate IC₅₀ values for Bu₂SnCl₂ were 3 and 9 μM, as estimated by nitrogenase activity and photosynthesis, respectively. These results indicate that organotin compounds have the potential to inhibit cyanobacterial metabolism in aquatic systems.     

Extracellular signal regulated MAP-kinase signalling in osmotically stressed trout hepatocytes

Activation of the extracellular signal-regulated MAP-kinase (ERK) by anisoosmotic conditions, the underlying signalling pathways, and the role of protein kinases in cell volume regulation were investigated in trout hepatocytes. While hyperosmolarity left phosphorylated ERK (pERK) levels unaffected, hypoosmolarity caused a significant increase of pERK within 2 min which peaked at around 30 min. Chelating extracellular Ca2+ to prevent the influx of Ca2+ associated with swelling reduced iso- and abolished hypoosmotic ERK activation. Similarly, inhibiting the ERK activator MEK, tyrosine kinases, or PKC inhibited the increase of pERK. In contrast, exposing cells to chelerytrine or staurosporine, PKC inhibitors of little specificity, increased pERK independently from osmotic conditions. Blocking PI3 kinase, application of 8-Br-cAMP, exposure to a P-receptor antagonist, and inhibition of p38 MAP-kinase had no effect on ERK activity. A significant reduction of regulatory volume decrease (RVD) after hypoosmotic swelling caused by MEK-inhibition and an even more pronounced reduction due to p38 inhibition indicates a role for MAP-kinases in volume regulation, but a lack of correlation between the impact of protein kinase inhibitors on pERK levels and on RVD suggests that ERK may merely modulate volume recovery. Immunocytochemical detection of pERK indicated cytoplasmic activation, but no nuclear accumulation within 30 min, supporting the notion that ERK exerts non-genomic effects. Overall, our data underscore the complexity of hypoosmotic ERK signalling and suggest a role of ERK and p38 in acute cell volume regulation.     

Osmolytes responsible for volume reduction under isosmotic or hypoosmotic conditions in Barnacle muscle cells.

In numerous animal cells, experimental manipulations that increase the intracellular free Ca2+ concentration induce cell volume reduction. This may occur under isosmotic conditions, e.g. when external Ca2+ (Ca(o)) is replaced by Mg2+ (42) or during exposure to hypoosmotic conditions (i.e. regulatory volume decrease, RVD) in the presence of Ca(o). We determined the osmolytes responsible for volume reduction under isosmotic and hypoosmotic conditions in barnacle muscle cells. Organic osmolytes (i.e. free amino acids and methylamines) and inorganic ions accounted for approximately 78% and 22% of the intracellular isosmotic activity, respectively. Isosmotic Ca(o) removal induced a net loss of KCI (with a ratio of 1K:1Cl) and free amino acids (FAA, mainly glycine and taurine). During RVD. the same ions (but in a proportion of 2K:1Cl) and FAA were lost. Since RVD was accompanied by extracellular alkalinization, the 2K:1Cl loss may be explained by the presence of a K+/H+ exchanger (or K+-OH- co-transporter) or Cl-/OH- exchanger. The lack of RVD in the absence of Ca(o) cannot be attributed to the loss of intracellular osmolytes during isosmotic Ca(o) removal because addition of Ca(o) during cell swelling promoted RVD.     

Calcium-dependent control of volume regulation in renal proximal tubule cells: II. Roles of dihydropyridine-sensitive and-insensitive Ca2+ entry pathways

Summary The Ca^2– entry pathways in the basolateral plasma membrane of the isolated, nonperfused proximal straight tubule (PST) of rabbit kidney were investigated using fura-2 fluorescence microscopy. Under isotonic conditions, reduction of bath [Ca^2–] from 1 mM to 1 M caused intracellular free calcium concentration ([Ca²⁺]_i) to fall close to zero. Treatment with 10 M verapamil, a calcium channel blocker, had a similar effect. Treatment with verapamil or low Ca²⁺ also induced fluctuations in cell volume. However, isotonic treatment with 10 M nifedipine, a dihydropyridine (DHP)-type calcium channel blocker, did not affect [Ca²⁺]_i or cell volume, indicating that the endogenous Ca²⁺ entry pathway is verapamil-sensitive but DHP-insensitive. When cells were exposed to hypotonic solutions in the presence of 1 mM Ca²⁺, they swelled and underwent normal RVD while [Ca²⁺]_i increased transiently to a peak before decreasing to a late phase plateau level above the baseline level (see McCarty, N.A., O'Neil, R.G. 1991.J. Membrane Biol. 123:149–160). When cells were swollen in the presence of verapamil or low bath [Ca²⁺], RVD was abolished and [Ca²⁺]_i fell well below the baseline during the late phase response. In contrast, when cells were swollen in the presence of nifedipine, RVD and the late phase rise in [Ca²⁺]_i were abolished, but [Ca²⁺]_i did not fall below the baseline level in the late phase, indicating that nifedipine inhibited the swelling-induced Ca²⁺ entry but that Ca²⁺ entry by another pathway was undisturbed. It was concluded that PST cells are characterized by two Ca²⁺ permeability pathways in the basolateral membrane. Under both isotonic and hypotonic conditions, Ca²⁺ entry occurs at a slow rate via a verapamil-sensitive, DHP-insensitive baseline Ca²⁺ entry pathway. Cell swelling activates a separate DHP-sensitive, verapamil-sensitive Ca²⁺ entry pathway, which is responsible for the supply of Ca ions to the Ca²⁺-dependent mechanism by which cell volume regulation is achieved.     

Membrane trafficking factors are involved in the hypotonic activation of the taurine channel in the little skate (Raja erinacea) red blood cell

In response to volume expansion, red blood cells of the little skate (Raja erinacea) initially swell and then release small organic compounds and osmotically obligated water in what is called a regulatory volume decrease (RVD) to restore cell volume. One of the major intracellular solutes lost during this process is the non-metabolized beta amino acid taurine. This hypoosmotic-induced increase in cell taurine permeability requires the anion exchanger, skAE1. The abundance of this transporter increases on the surface plasma membrane by a process of exocytosis. The second-messenger pathways involved in exocytosis of skAE1 were investigated with the use of inhibitors which affect membrane trafficking. Hypoosmotic-stimulated taurine uptake was significantly decreased by 42% with wortmannin, a phosphatidylinositol 3-kinase (PI3 kinase) inhibitor. Additional evidence for the involvement of PI3K was obtained with a second inhibitor, LY294002, which decreased the hypoosmotic-stimulated taurine uptake by 28%. The state of actin is also involved, as the actin filament depolymerizer latrunculin B decreased hypoosmotic-stimulated taurine uptake by approximately 40%. Although hypoosmotic conditions did not stimulate changes in the distribution of actin between filamentous and globular forms, latrunculin stimulated a decrease in filamentous actin and increase in globular actin in both isoosmotic and hypoosmotic conditions. Disruptors of other potential cytoskeletal factors (myosin, kinesin, dynein, and microtubules) did not affect taurine uptake. The present results suggest that the exocytosis of skAE1 stimulated by hyposmotic-induced cell volume expansion requires activation of PI3 kinase and is regulated by the state of actin filaments.     

Calcium-dependent control of volume regulation in renal proximal tubule cells: I. Swelling-Activated Ca2+ entry and release

Summary The mechanism of Ca²⁺-dependent control of hypotonic cell volume regulation was investigated in the isolated, nonperfused renal proximal straight tubule. When proximal tubules were exposed to hypotonic solution with 1 mM Ca²⁺, cells swelled rapidly and then underwent regulatory volume decrease (RVD). This treatment resulted in an increase in intracellular free calcium concentration ([Ca²⁺]_i) by a mechanism that had two phases: the first was a transient increase from baseline (136 nM) to a peak (413 nM) that occurred in the first 15–20 sec, but was followed by a rapid decay toward the pre-swelling levels. The second phase was characterized by a sustained elevation of [Ca²⁺]_i above the baseline (269 nM), which was maintained over several minutes. The dependence of these two phases on extracellular Ca²⁺ was determined. Reduction of bath [Ca²⁺] to 10 or 1 M partially diminished the transient phase, but abolished the sustained phase completely, such that [Ca²⁺]_i fell below the base-line levels during RVD. It was concluded that the transient increase resulted predominantly from swelling-activated release of intracellular Ca²⁺ stores and that the sustained phase was due to swelling-activated Ca²⁺ entry across the plasma membrane. Ca²⁺ entry probably also contributed to the transient increase in [Ca²⁺]_i. The time dependence of swelling-activated Ca²⁺ entry was also investigated, since it was previously shown that RVD was characterized by a calcium window period (<60 sec). during which extracellular Ca²⁺ was required. Outside of this time period, RVD would inactivate and could not be reactivated by subsequent addition of Ca²⁺. It was found that the Ca²⁺ permeability did not inactivate over several minutes, indicating that the temporal dependence of RVD on extracellular Ca²⁺ is not due to the transient activation of a Ca²⁺ entry pathway.     

5-Lipoxygenase Metabolites of Arachidonic Acid Regulate Volume Decrease by Mudpuppy Red Blood Cells

We examined whether metabolites of arachidonic acid (AA) regulate K⁺ efflux during regulatory volume decrease (RVD) by mudpuppy red blood cells (RBCs). Volume regulation was inhibited by the phospholipase A₂ antagonists mepacrine (10 μm) and ONO-RS-082 (10 μm); the inhibitory effect of ONO-RS-082 was reversed by gramicidin (5 μm). Eicosatetraynoic acid (ETYA, 100 μm), a general antagonist of AA metabolism, also blocked RVD. In addition, volume regulation was inhibited by the lipoxygenase pathway antagonist nordihydroguaiaretic acid (NDGA, 10 μm), the 5 lipoxygenase antagonists AA-861 (5 μm) and curcumin (20 μm), and by the 5-lipoxygenase activating protein inhibitor L-655,298 (5 μm). Inhibition by all four of these agents was reversed with gramicidin. In contrast, the 12- and 15-lipoxygenase pathway inhibitor ethyl-3,4-dihydroxy-benzylidene-cyanoacetate (EDBCA, 1 μm) and the cytochrome P-450 monooxygenase pathway blocker ketoconazole (20 μm) had no effect. On the other hand, the cyclooxygenase pathway inhibitor aspirin (100 μm) slightly enhanced RVD. Consistent with these findings, a K⁺-selective whole cell conductance responsible for K⁺ efflux during cell swelling was inhibited by ONO-RS-082 (10 μm), NDGA (10 μm), AA-861 (5 μm), curcumin (20 μm), and l-655,298 (5 μm). In contrast, EDBCA (1 μm), ketoconazole (20 μm), and indomethacin (10 μm) did not block this whole cell conductance. These results indicate that a channel mediating K⁺ loss during RVD is regulated by a 5-lipoxygenase metabolite of arachidonic acid. Received: 12 December 1996/Revised: 28 February 1997     

Regulatory volume decrease in Leishmania mexicana: effect of anti-microtubule drugs

The trypanosomatid cytoskeleton is responsible for the parasite''s shape and it is modulated throughout the different stages of the parasite''s life cycle. When parasites are exposed to media with reduced osmolarity, they initially swell, but subsequently undergo compensatory shrinking referred to as regulatory volume decrease (RVD). We studied the effects of anti-microtubule (Mt) drugs on the proliferation of Leishmania mexicana promastigotes and their capacity to undergo RVD. All of the drugs tested exerted antiproliferative effects of varying magnitudes [ansamitocin P3 (AP3)> trifluoperazine > taxol > rhizoxin > chlorpromazine]. No direct relationship was found between antiproliferative drug treatment and RVD. Similarly, Mt stability was not affected by drug treatment. Ansamitocin P3, which is effective at nanomolar concentrations, blocked amastigote-promastigote differentiation and was the only drug that impeded RVD, as measured by light dispersion. AP3 induced 2 kinetoplasts (Kt) 1 nucleus cells that had numerous flagella-associated Kts throughout the cell. These results suggest that the dramatic morphological changes induced by AP3 alter the spatial organisation and directionality of the Mts that are necessary for the parasite''s hypotonic stress-induced shape change, as well as its recovery.