Hyposmotic Shock Research Articles

Cellular osmoregulation of the ark clam (Anadara kagoshimensis) hemocytes to hyposmotic media.

Many bivalve species are considered to be euryhaline organisms due to effective adaptation to fluctuations of environmental salinity. Cellular mechanisms responsible for tolerance to salinity changes remain unclear for bivalves despite this question being critically important for commercially cultured species frequently introduced into regions differing from natural habitat by salinity regime. In the present work laser diffraction method was used for the analysis of volume changes in hemoglobin-containing ark clam (Anadara kagoshimensis) hemocytes following hyposmotic stimulation. Hemocytes responded to hyposmotic shock (decrease of media osmolarity from 461to 216 mОsm/L) by a rapid swelling up to 171.5 ± 15.2% of control level. At normal osmotic conditions (osmolarity 461 mOsm/L), hemocyte mean cellular volume (MCV) was 354.0 ± 24.4 fl and maximum MCV of hyposmotically swollen cells prior lysis was 555.5 ± 57.4 fl (at the osmolarity 194 mOsm/L). Ark clam hemocytes demonstrated volume recovery response following hyposmotic swelling. Regulatory volume decrease (RVD) reaction did not depend on hemoglobin confirmation status. Final MCV of swollen hemocytes at the end of experimental period of RVD in oxygenated and deoxygenated suspensions did not significantly differ.

Ectoine bio-milking in methanotrophs: A step further towards methane-based bio-refineries into high added-value products

This communication showed for the first time that the methanotrophic strain Methylomicrobium alcaliphilum 20Z (M. alcaliphilum 20Z) can efficiently synthesize and excrete (through a tailored bio-milking process) ectoine under continuous mode using methane (CH4) as the sole energy and carbon source. First, three consecutive 50h fed batch fermentations consisting of alternating high salinity (6% NaCl for 24h) and low salinity (0% NaCl for 24h) cultivation stages were carried out in triplicate to determine the influence of sudden modifications in media salinity on ectoine synthesis and excretion. The results demonstrated that M. alcaliphilum 20Z exhibited a rapid response to osmotic shocks, which resulted in the release of the accumulated ectoine under hyposmotic shocks and the immediate uptake of the previously excreted ectoine during hyperosmotic shocks. A second experiment was carried out under continuous cultivation mode in two sequential stirred tank reactors operated at NaCl concentrations of 0 and 6%. Cells exhibited a constant intra-cellular ectoine concentration of 70.4±14.3mggbiomass−1 along the entire operation period when cultivated at a NaCl concentration of 6%. The centrifugation of the cultivation broth followed by a hyposmotic shock resulted in the excretion of ∼70% of the total intra-cellular ectoine. In brief, this research shows the feasibility of the continuous bioconversion of diluted CH4 emissions into high added-value products such as ectoine, which can turn greenhouse gas (GHG) abatement into a sustainable and profitable process.

Volume regulation of intestinal cells of echinoderms: Putative role of ion transporters (Na+/K+-ATPase and NKCC)

Echinoderms are exclusively marine osmoconformer invertebrates. Some species occupy the challenging intertidal region. Upon salinity changes, the extracellular osmotic concentration of these animals also varies, exposing tissues and cells to osmotic challenges. Cells and tissues may then respond with volume regulation mechanisms, which involve transport of ions and water into and/or out of the cells, through ion transporters, such as the Na+/K+-ATPase and NKCC. The goal of this study was to relate the cell volume regulation capacity of echinoderm intestinal cells Na+/K+-ATPase and NKCC activities, in three echinoderm species: Holothuria grisea, Arbacia lixula, and Echinometra lucunter. Isolated cells of these species displayed some control of their cell volume upon exposure to anisosmotic media (isolated intestinal cells, calcein fluorescence as indicator of volume change), with a distinct higher capacity shown by H. grisea, which did not swell even upon 50% hyposmotic shock. The holothuroid cells showed indirect evidence (effect of furosemide) of the participation of NKCC in this process, with a secretory function, and of a secondary role by the NKA (effect of ouabain). Other mechanisms are probably responsible for this function in the urchins. Variable expression of these transporters, and others not examined here, may to some extent account for the variability in cell volume regulation capacity in echinoderm cells.

Cadmium affects osmotic phase and regulatory volume decrease in cultured human embryonic kidney cells

The present investigation aims to verify whether cadmium (Cd<sup>2+</sup>), a metal possibly accumulated in body tissues from air and food, affects cell volume regulation capability in cultured human embryonic kidney (HEK 293 Phoenix) cells. The osmotic phase (OP), which is the expected cell swelling due to aquaporins involvement after hyposmotic challenge, and regulatory volume decrease (RVD), bringing cell volume back to control values through Ca<sup>2+</sup>-dependent ion efflux (K+ and Cl–), have been monitored in HEK 293 cells treated with Cd<sup>2+</sup> (1-10-100 μM) for different time intervals (30 min, 3 h, overnight) and then submitted to 15 % hyposmotic shock. The results show that both 1 and 10 μM Cd<sup>2+</sup> significantly reduced OP, whereas 100 μM impaired Cd<sup>2+</sup> RVD mechanisms. The use of glutathione (GSH, 200 μM) confirmed that Cd<sup>2+</sup> elicited its effect <em>via</em> oxidative damage, being RVD inhibition after Cd<sup>2+</sup> treatment prevented by this antioxidant compound. Our findings show that: i) HEK 293 cells are a suitable model to assay the effect of xenobiotics on cell homeostasis; ii) Cd<sup>2+</sup>, depending on its concentration, affects cell homeostasis at different levels, <em>i.e.</em> water and ion permeability, responsible for, respectively, OP and RVD mechanism, adding thus more information to the knowledge of Cd<sup>2+</sup> toxicology.

Small membrane permeable molecules protect against osmotically induced sealing of t-tubules in mouse ventricular myocytes.

Cardiac t-tubules are critical for efficient excitation-contraction coupling but become significantly remodeled during various stress conditions. However, the mechanisms by which t-tubule remodeling occur are poorly understood. Recently, we demonstrated that recovery of mouse ventricular myocytes after hyposmotic shock is associated with t-tubule sealing. In this study, we found that the application of Small Membrane Permeable Molecules (SMPM) such as DMSO, formamide and acetamide upon washout of hyposmotic solution significantly reduced the amount of extracellular dextran trapped within sealed t-tubules. The SMPM protection displayed sharp biphasic concentration dependence that peaks at ∼140 mM leading to >3- to 4-fold reduction in dextran trapping. Consistent with these data, detailed analysis of the effects of DMSO showed that the magnitude of normalized inward rectifier tail current (IK1,tail), an electrophysiological marker of t-tubular integrity, was increased ∼2-fold when hyposmotic stress was removed in the presence of 1% DMSO (∼140 mM). Analysis of dynamics of cardiomyocytes shrinking during resolution of hyposmotic stress revealed only minor increase in shrinking rate in the presence of 1% DMSO, and cell dimensions returned fully to prestress values in both control and DMSO groups. Application and withdrawal of 10% DMSO in the absence of preceding hyposmotic shock induced classical t-tubule sealing. This suggests that the biphasic concentration dependence originated from an increase in secondary t-tubule sealing when high SMPM concentrations are removed. Overall, the data suggest that SMPM protect against sealing of t-tubules following hyposmotic stress, likely through membrane modification and essentially independent of their osmotic effects.

Cell Membrane Integrity Examined by Nonlinear Light Scattering

Mechanosensitive (MS) channels are responsible for preserving the integrity of the cytoplasmic membrane (CM) in the face of environmental shifts in osmolarity in a variety of cell types. Our understanding of the spatial and temporal activity of MS channels can be improved by applying new techniques capable of in vivo monitoring of their activity. Previously, we have shown that second-harmonic light scattering (SHS) provides real-time information regarding molecular uptake at biological membranes and characterizes permeability changes induced in cellular membranes1-2. Herein, we use SHS to examine changes in the physical integrity of the E. coli CM induced by hypo-osmotic shock. Given that the MS channels in E. coli are likely to have open pore diameters of >1.8 nm they can easily allow passive diffusion of low molecular weight species. We characterize the molecular uptake kinetics of malachite green cation, MG+ (ca. 1 nm diameter) under different osmotic gradients. These include; 1) cells treated with PBS, 2) cells treated in distilled water, and 3) cells re-suspended in PBS after treatment with distilled water. Comparing the adsorption and transport rates of MG+ at the CM in these conditions reveals that MS channels contribute considerably to CM permeability under hyp-osmotic shock. Our results are consistent with previous reports that MS channels in E. coli can function as a conduit for ion entry (Ca2+, H+) and not just osmolyte efflux3. Interestingly the majority of the cells exposed to high hypo-osmotic pressure (ca. 6 atm.) preserve their integrity even after ∼ 20 minutes. We suggest that SHS may represent a new experimental paradigm for not only investigating cell membrane integrity in real-time but also for examining MS channel activators.REFERENCES1. Wilhelm., Chem. Phys. Lett. (2014).2. Wilhelm, Biochem. (2015).3. Cox, Nat. Commun. (2013).

Different abilities to regulate tissue hydration upon osmotic challenge in vitro, in the cephalopods Octopus vulgaris and O. insularis

The spatial distribution of cephalopods is influenced by salinity and temperature. In marine osmoconformers, one reason for different spatial distributions maybe the putative distinct capacity to regulate tissue hydration. Adult Octopus vulgaris (n = 6) and O. insularis (n = 6) were obtained by divers from sites along the southern and northeastern Brazilian coasts, respectively. In the laboratory, octopuses were acclimated to full-strength seawater (salinity 34), for 5–10 days. They were then anesthetized (10 min in 4 °C seawater), a portion of the second right arm was removed, and cut into three portions. Tissues were placed in control isosmotic saline (osmolality 1072 mOsm/kg H2O). Fragments were weighed and transferred to either isosmotic, hyposmotic (50%), or hyperosmotic (150%) salines, and their weight was observed for 120 min. Tissue from both species maintained their hydration/weight following the hyposmotic shock, but differed in their response to the hyperosmotic challenge, where hydration remained unchanged in O. insularis, but was significantly reduced in O. vulgaris. This result could partially explain why O. insularis is more abundant than O. vulgaris throughout the shallow reefs and tide pools in the warmer and salty tropical waters of the northeastern Brazilian coast.

DMSO Protects against Stress-Induced Sealing of Cardiac T-Tubules

Cardiac t-tubules are critical for efficient excitation-contraction coupling and undergo significant remodeling during various experimental and clinical stress conditions including heart failure. Recently, we have shown that recovery of ventricular myocytes after a brief hyposmotic shock is associated with significant sealing of t-tubules. Dimethyl Sulfoxide (DMSO) is a commonly used solvent and has been reported to have cardioprotective properties although the mechanisms underlying these protective effects remain largely unknown. Therefore, we tested whether the cardioprotective effects of DMSO are mediated by its action on t-tubular remodeling. We found that application of DMSO at the time of resolution of hyposmotic stress, when t-tubule sealing occurs, reduced the amount of fluorescent dextran trapped within sealed t-tubules. The effect of DMSO displayed sharp biphasic concentration dependence with 1% being the most effective dose (∼4-fold reduction of dextran trapping; p<0.01) while 10% DMSO was ineffective in preventing t-tubule sealing. The data were corroborated by measuring IK1 tail current which reflects the amount of ttubular membrane. IK1 tail was recorded in cardiomyocytes that were previously detubulated using the hyposmotic stress protocol. Consistent with reduced dextran trapping in the presence of 1% DMSO, normalized IK1 tail was significantly larger in cardiomyocytes that had undergone detubulation with 1% DMSO (19.8 ± 2.4 % vs 7.9 ± 2.4 %, p<0.01). Image analysis of cardiomyocyte dimensions during the osmotic stress protocol did not reveal any significant differences in cells treated with 1% DMSO upon resolution of hyposmotic stress. In particular, cell dimensions returned fully to pre-stress values in both control and DMSO groups. The data suggest that DMSO prevents t-tubule remodeling independent of its osmotic effects.

Ca2 + homeostasis in sealed t-tubules of mouse ventricular myocytes

We have recently shown that in mouse ventricular myocytes, t-tubules can be quickly and tightly sealed during the resolution of hyposmotic shock of physiologically relevant magnitude. Sealing of t-tubules is associated with trapping extracellular solution inside the myocytes but the ionic homeostasis of sealed t-tubules and the consequences of potential transtubular ion fluxes remain unknown. In this study we investigated the dynamics of Ca2+ movements associated with sealing of t-tubules. The data show that under normal conditions sealed t-tubules contain Ca2+ at concentrations below 100μM. However, blockade of voltage-dependent Ca2+ channels with 10μM nicardipine, or increasing extracellular concentration of K+ from 5.4mM to 20mM led to several fold increase in concentration of t-tubular Ca2+. Alternatively, the release of Ca2+ from sarcoplasmic reticulum using 10mM caffeine led to the restoration of t-tubular Ca2+ towards extracellular levels within few seconds. Sealing of t-tubules in the presence of extracellular 1.5mM Ca2+ and 5.4mM extracellular K+ led to occasional and sporadic intracellular Ca2+ transients. In contrast, sealing of t-tubules in the presence of 10mM caffeine was characterized by a significant long lasting increase in intracellular Ca2+. The effect was completely abolished in the absence of extracellular Ca2+ and significantly reduced in pre-detubulated myocytes but was essentially preserved in the presence of mitochondrial decoupler dinitrophenol. This study shows that sealed t-tubules are capable of highly regulated transport of Ca2+ and present a major route for Ca2+ influx into the cytosol during sealing process.

Letter to the editor: “The origin of free glutamate in milk: a role for anionic amino acid transporters”

Letter to the editor: “The origin of free glutamate in milk: a role for anionic amino acid transporters”

Regulation of Muscle Hydration Upon Hypo‐ or Hyper‐Osmotic Shocks: Differences Related to Invasion of the Freshwater Habitat by Decapod Crustaceans

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages.

Sea Water Acidification Affects Osmotic Swelling, Regulatory Volume Decrease and Discharge in Nematocytes of the Jellyfish Pelagia noctiluca

Background: Increased acidification/P_CO2 of sea water is a threat to the environment and affects the homeostasis of marine animals. In this study, the effect of sea water pH changes on the osmotic phase (OP), regulatory volume decrease (RVD) and discharge of the jellyfish Pelagia noctiluca (Cnidaria, Scyphozoa) nematocytes, collected from the Strait of Messina (Italy), was assessed. Methods: Isolated nematocytes, suspended in artificial sea water (ASW) with pH 7.65, 6.5 and 4.5, were exposed to hyposmotic ASW of the same pH values and their osmotic response and RVD measured optically in a special flow through chamber. Nematocyte discharge was analyzed in situ in ASW at all three pH values. Results: At normal pH (7.65), nematocytes subjected to hyposmotic shock first expanded osmotically and then regulated their cell volume within 15 min. Exposure to hyposmotic ASW pH 6.5 and 4.5 compromised the OP and reduced or totally abrogated the ensuing RVD, respectively. Acidic pH also significantly reduced the nematocyte discharge response. Conclusion: Data indicate that the homeostasis and function of Cnidarians may be altered by environmental changes such as sea water acidification, thereby validating their use as novel bioindicators for the quality of the marine environment.

Direct relationship between osmotic and ionic conforming behavior and tissue water regulatory capacity in echinoids

Echinoderms are considered marine osmoconforming invertebrates. However, many are intertidal or live next to estuaries, tolerating salinity changes and showing extracellular gradients to dilute seawater. Three species of echinoids – Lytechinus variegatus, which can occur next to estuarine areas, the rocky intertidal Echinometra lucunter, and the mostly subtidal Arbacia lixula – were submitted to a protocol of stepwise (rate of 2–3psu/h) dilution, down to 15psu, or concentration, up to 45psu, of control seawater (35psu). Coelomic fluid samples were obtained every hour. The seawater dilution experiment lasted 8h, while the seawater concentration experiment lasted 6h. Significant gradients (40–90% above value in 15psu seawater) for osmolality, sodium, magnesium, and potassium were shown by L. variegatus and E. lucunter. A. lixula showed the smallest gradients, displaying the strongest conforming behavior. The esophagus of the three species was challenged in vitro with 20 and 50% osmotic shocks (hypo- and hyperosmotic). A. lixula, the most “conforming” species, showed the highest capacity to avoid swelling of its tissues upon the −50% hyposmotic shock, and was also the species less affected by salinity changes concerning the observation of spines and ambulacral feet movement in the whole-animal experiments. Thus, the most conforming species (A. lixula) displayed the highest capacity to regulate tissue water/volume, and was also the most euryhaline among the three studied species. In addition, tissues from all three species swelled much more than they shrank under osmotic shocks of same magnitude. This distinct trend to gain water, despite the capacity to hold some gradients upon seawater dilution, helps to explain why echinoderms cannot be fully estuarine, or ever enter fresh water.

Digestive cells from Mytilus galloprovincialis show a partial regulatory volume decrease following acute hypotonic stress through mechanisms involving inorganic ions

The response of isolated digestive cells of the digestive gland of Mytilus galloprovincialis to hypotonic shock was studied using videometric methods. The isolated cells exposed to a rapid change (from 1100 to 800 mosmol kg(-1) ) of the bathing solution osmolality swelled but thereafter underwent a regulatory volume decrease (RVD), tending to recover the original size. When the hypotonic stress was applied in the presence of quinine and glibenclamide, known inhibitors of swelling activated ion channels, the cells did not exhibit an RVD response; in addition, they showed a larger increase in size in respect to control cells. These observations suggest that the digestive cells of the digestive gland have the machinery to cope with the hyposmotic shock allowing them to exhibit a small but significant RVD preventing an excessive increase in cell size. The pharmacological treatment of digestive cells during the RVD experiments suggests that cell volume is regulated by K(+) and Cl(-) efflux followed by an obliged water efflux from the cell. The involvement of organic osmolytes such as taurine and betaine seems to be excluded by NMR measurement on digestive cells.

Lead hampers gill cell volume regulation in marine crabs: Stronger effect in a weak osmoregulator than in an osmoconformer

Hepatus pudibundus is a strictly marine osmoconformer crab, while Callinectes ornatus inhabits estuarine areas, behaving as a weak hyper-osmoregulator in diluted seawater. Osmoconformers are expected to have higher capacity for cell volume regulation, but gill cells of a regulator are expected to display ion transporters to a higher degree. The influence of lead nitrate (10 μM) on the ability of isolated gill cells from both species to volume regulate under isosmotic and hyposmotic conditions were here evaluated. Without lead, under a 25% hyposmotic shock, the gill cells of both species were quite capable of cell volume maintenance. Cells of C. ornatus, however, had a little swelling (5%) during the hyposmotic shock of greater intensity (50%), while cells of H. pudibundus were still capable of volume regulation. In the presence of lead, even under isosmoticity, the gill cells of both species showed about 10% volume reduction, indicating that lead promotes the loss of water by the cells. When lead was associated with 25% and 50% hyposmotic shock, C. ornatus cells lost more volume (15%), when compared to isosmotic conditions, while H. pudibundus cells showed volume regulation. We then analyzed the possible ways of action of lead on the mechanisms of cell volume regulation in the two species. Verapamil (100 μM) was used to inhibit Ca 2+ channels, ouabain (100 μM) to inhibit Na +/K +-ATPase, and HgCl 2 (100 μM) to inhibit aquaporins. Our results suggest that: (1) Ca 2+ channels are candidates for lead entry into gill cells of H. pudibundus and C. ornatus, being the target of lead action in these cells; (2) aquaporins are much more relevant for water flux in H. pudibundus; and (3) the Na +/K +-ATPase is much more relevant for volume regulation in C. ornatus. Osmoregulators may be more susceptible to metal contamination than osmoconformers, especially in situations of reduced salinity, for two basic reasons: (1) lower capacity of volume regulation and (2) putative higher uptake of Pb 2+ through ionic pathways that operate in salt absorption, such as, for example, the Na +/K +-ATPase.

Hyposmotic stress triggers ATP release from porcine lens via connexin and pannexin hemichannels

AbstractPurpose Purinergic receptors in the lens suggest function can be altered by agonists present in aqueous humor. Some agonists may originate from the lens itself. Here, we examine the ability of osmotic shock to trigger ATP release from the intact lens.Methods Porcine lenses were exposed to hyposmotic (200 mOsm) or hyperosmotic (500mOsm) solution and ATP in the bathing medium was measured by luciferase assay. Because hemichannels are permeable to large solutes, the ability of propidium iodide (PI) (MW 668) to enter the epithelium was examined.Results ATP release into the bathing medium was stimulated when lenses were exposed to hyposmotic solution. Hyperosmotic solution did not detectably increase ATP release. Hyposmotic solution‐induced release of ATP was partially suppressed by the connexin hemichannel inhibitor, 18α glycyrrhetinic acid (AGA) or by probenecid, a pannexin hemichannel blocker, and was abolished by AGA and probenecid added together. Consistent with opening of connexin and/or pannexin hemichannels, lenses exposed to hyposmotic solution displayed a ~4 fold increase in the ability of PI to enter the epithelium. In parallel studies, hyposmotic solution was shown to activate a Src family tyrosine kinase (SFK) and cause an SFK‐dependent increase of Na,K‐ATPase activity in the epithelium.Conclusion Hemichannels contribute to ATP release when the intact lens is subjected to hyposmotic shock. The amount of ATP release appeared sufficient to activate purinergic receptors that cause tyrosine kinase‐dependent stimulation of active Na‐K transport. The responses could perhaps signify an autoregulatory loop initiated by mechanical stress or osmotic swelling.

Distinct patterns of water and osmolyte control between intertidal (Bunodosoma caissarum) and subtidal (Anemonia sargassensis) sea anemones

Anemones are frequently found in rocky intertidal coasts. As they have highly permeable body surfaces, exposure to the air or to salinity variations inside tidal pools can represent intense osmotic and ionic challenges. The intertidal Bunodosoma caissarum has been compared with the subtidal Anemonia sargassensis concerning their response to air exposure or salinity changes. B. caissarum maintains tissue hydration through mucus production and dome-shape formation when challenged with air exposure or extreme salinities (fresh water or hypersaline seawater, 45psu) for 1–2h. Upon exposure to mild osmotic shocks for 6h (hyposmotic: 25psu, or hyperosmotic: 37psu), B. caissarum was able to maintain its coelenteron fluid (CF) osmolality stable, but only in 25psu. A. sargassensis CF osmolality followed the external medium in both salinities. Isolated cells of the pedal disc of B. caissarum showed full capacity for calcium-dependent regulatory volume decrease (RVD) upon 20% hyposmotic shock, at least partially involving the release of KCl via K+–Cl− cotransport, and also of organic osmolytes. Aquaporins (HgCl2-inhibited) likely participate in this process. Cells of A. sargassensis showed partial RVD, after 20min. Cells from both species were not capable of regulatory volume increase upon hyperosmotic shock (20%). Whole organism and cellular mechanisms allow B. caissarum to live in the challenging intertidal habitat, frequently facing air exposure and seawater dilution.

Specificity of the volume-activated amino acid efflux pathway in cultured human breast cancer cells

It has been shown that cell swelling stimulates the efflux of taurine from MCF-7 and MDA-MB-231 cells via a pathway which has channel-like properties. The purpose of this study was to examine the specificity of the volume-activated taurine efflux pathway in both cell lines. A hyposmotic shock increased the efflux of glycine, L-alanine, AIB (α-aminoisobutyric acid), D-aspartate but not L-leucine from MDA-MB-231 and MCF-7 cells. It was evident that the time course of activation/inactivation of those amino acids whose efflux was affected by cell swelling was similar to that of volume-activated taurine efflux. The effect of exogenous ATP on swelling-induced glycine, AIB and D-aspartate efflux from MDA-MB-231 cells was similar to that found on taurine efflux. In addition, volume-activated AIB efflux from MDA-MB-231 cells, like that of swelling-induced taurine efflux, was inhibited by diiodosalicylate. Tamoxifen inhibited volume-activated taurine release from both MDA-MB-231 and MCF-7 cells. The results suggest that neutral and anionic α-amino acids are able to utilize the volume-activated taurine efflux pathway in both cell lines. The effect of tamoxifen on breast cancer growth may, in part, be related to perturbations in cell volume regulation.

Evidence for Aquaporin-mediated Water Transport in Nematocytes of the Jellyfish Pelagia noctiluca

Nematocytes, the stinging cells of Cnidarians, have a cytoplasm confined to a thin rim. The main cell body is occupied by an organoid, the nematocyst, containing the stinging tubule and venom. Exposed to hypotonic shock, nematocytes initially swell during an osmotic phase (OP) and then undergo regulatory volume decrease (RVD) driven by K⁺, Cl^- and obligatory water extrusion mechanisms. The purpose of this report is to characterize the OP. Nematocytes were isolated by the NaSCN/Ca²⁺ method from tentacles of the jellyfish Pelagia noctiluca, collected in the Strait of Messina, Italy. Isolated nematocytes were subjected to hyposmotic shock in 65% artificial seawater (ASW) for 15 min. The selective aquaporin water channel inhibitor HgCl₂ (0.1-25 µM) applied prior to osmotic shock prevented the OP and thus RVD. These effects were attenuated in the presence of 1mM dithiothreitol (DTT), a mercaptide bond reducing agent. AgNO₃ (1 µM) and TEA (tetraethylammonium, 100 µM), also reported to inhibit water transport, did not alter the OP but significantly diminished RVD, suggesting different modes of action for the inhibitors tested. Based on estimates of the nematocyte surface area and volume, and OP duration, a relative water permeability of ∼10^-7 cm/sec was calculated and the number of putative aquaporin molecules mediating the OP was estimated. This water permeability is 3-4 orders of magnitude lower in comparison to higher order animals and may constitute an evolutionary advantage for Cnidarian survival.

AQP1 mediates water transport in the carotid body

In this study, we explored the presence of aquaporins (AQPs), a family of membrane water channel proteins, in carotid body (CB) type I chemoreceptor cells. The CB is a polymodal chemoreceptor whose major function is to detect changes in arterial O2 tension to elicit hyperventilation during hypoxia. The CB has also been proposed to function as a systemic osmoreceptor, thus we hypothesized that the presence of AQPs in type I cell membrane may confer higher sensitivity to osmolarity changes and hence accelerate the activation of chemoreceptor cells. We detected the expression of AQP1, AQP7, and AQP8 in the CB and confirmed the location of AQP1 in type I cells. We have also shown that inhibition of AQP1 expression clearly reduced type I cell swelling after a hyposmotic shock, demonstrating that AQP1 has a major contribution in transmembrane water movement in these chemoreceptor cells. Interestingly, CB AQP1 expression levels change during postnatal development, increasing during the first postnatal weeks as the organ matures. In conclusion, in this study, we report the novel observation that AQPs are expressed in the CB. We also show that AQP1 mediates water transport across the cell membrane of type I cells, supporting the contribution of this protein to the osmoreception function of the CB.