Hyposmotic Conditions Research Articles

BIOENERGETICA DEL LANGOSTINO Macrobrachium tenellum (SMITH, 1871) EN FUNCION DE LA SALINIDAD DEL AGUA

<p><strong>Background. </strong>The <em>Macrobrachium tenellum</em> prawn is captured and commercialized by local fishermen along its distribution interval. It is considered an euryhaline palemonid prawn. If its cultivation is wanted, the best conditions for its growth and reproduction should be investigated, including proper intervals of temperature, salinity, density and dissolved oxygen. <strong>Objective.</strong> To determine the effect of two different salinities (0 and 15PSU) on the physiological rates that balance the energy equation in <em>M. tenellum</em> adults and to determine if energy use is more efficient under isosmotic conditions. <strong>Methodology</strong>. Specimens were collected in Guasave, Sinaloa, Mexico, in July 2018 and taken to the Aquaculture Laboratory at UAdeO. The energy balance was calculated in hyposmotic (0 PSU) and isosmotic (15 PSU) conditions at preferred temperature (28.5 ± 1 ° C). The energy invested in respiration and excretion, the feces lost and the ingested food and the growth potential was calculated using a semi-open respirometer. <strong>Results.</strong> The highest energy expenditure occurred in the respiratory rate (routine metabolism), then feces production and finally ammonia production in both salinities. The highest total energy expenditure was observed in fresh water. The highest amount of energy directed to the growth or reproduction was observed in brackish water (15 PSU), as well as the highest rate of food ingestion. <strong>Implications.</strong> Knowledge on the use of energy in the feed in the best saline condition for its cultivation useful for culture purposes. <strong>Conclusion.</strong> The saline condition in where more energy is channeled for the growth of <em>M. tenellum</em> is 15UPS (isosmotic point) and in it is also in where less energy is spent.</p>

The neuropeptide RhoprCCHamide2 inhibits serotonin-stimulated transcellular Na+ transport across the anterior midgut of the vector of Chagas disease, Rhodnius prolixus.

Rhodnius prolixus is a blood-feeding insect vector of Trypanosoma cruzi, a protozoan parasite that causes Chagas disease. During each blood meal, the animals ingest large volumes of blood, that may be up to 12 times the unfed body mass. These blood meals impose a significant osmotic stress for the animals due to the hyposmotic condition of the ingested blood compared with the insect's hemolymph. Thus the insect undergoes a massive postprandial diuresis that allows for the excretion of the plasma fraction of the blood in less than two hours. Diuresis is performed by the excretory system, consisting of the Malpighian tubules and gut, under the control of diuretic and anti-diuretic factors. We investigated the ion transport machinery triggered by stimulation with the diuretic factor serotonin in the anterior midgut (i.e.crop) and the effect of the diuretic modulator RhoprCCHamide2. Ussing chamber assays revealed that serotonin-stimulated increase in transepithelial short-circuit current (Isc) was more sensitive to the blockage with amiloride than 5-N-ethyl-N-isopropyl amiloride (EIPA), suggesting the involvement of Na+ channels. Incubation in Na+-free, but not Cl--free saline, blocked the effect of serotonin on Isc. Moreover, treatment with Na+-K+-2Cl- cotransporter (NKCC) and Na+-Cl- cotransporter (NCC) blockers had no effect on fluid secretion but was blocked by amiloride. Blockage of Na+/K+-ATPase with ouabain inhibited Isc but the H+-ATPase inhibitor bafilomycin had no effect. The neuropeptide RhoprCCHamide2 diminished serotonin-stimulated Isc across the crop. The results suggest that Na+ undergoes active transport via an apical amiloride-sensitive Na+ channel and a basolateral ouabain-sensitive Na+/K+-ATPase, while Cl- is transported through a passive paracellular pathway.

639 Regulatory function of TRPV4 on the dermal component of inflammatory skin conditions

Several skin-expressed Transient Receptor Potential (TRP) channels act as sensors of temperature, mechanical, and chemical stimuli. In the skin, TRPV4 is expressed in many nonneuronal cell populations including keratinocytes, immune cells and on skin appendages. This thermo- and osmosensitive protein has a high impact on the epidermal barrier, regulation of hair growth and sebum production and the secretion of inflammatory cytokines. Since we have less information about the role of TRPV4 in the dermis we aimed to investigate the functional presence of TRPV4 on human dermal fibroblasts (FB). First, we showed that TRPV4 is expressed on FBs both on mRNA and protein levels. We found that both osmotic- (using hyposmotic conditions) and pharmacological activation - by the specific agonist GSK1016790A - of TRPV4 resulted an increased Ca2+[IC] level on human FBs. This effect was inhibited by the specific antagonist of TRPV4, HC-067047. Next we examined the effect of TRPV4 activation on the expression of different regulatory genes which are implicated in collagen synthesis, inflammatory processes (interleukins and growth factors) and extracellular matrix remodelling. We found that TRPV4 significantly influenced the expression profile of these genes. Of particular note GSK1016790A exerted remarkable proinflammatory effects by inducing elevated interleukin-8 (IL-8) cytokine release. Since IL-8 is a chemoattractant we performed an invasion assay on FBs and the activation of TRPV4 resulted in higher migration activity of FBs, moreover the collected supernatant from these FBs increased the human keratinocyte proliferation. In all cases these effects were abrogated by HC-067047. These findings indicate that TRPV4 may be a key regulator of FBs biological processes. Therefore, pharmacological modulation of TRPV4 could be a promising tool for the management of wound healing, skin remodelling, fibrosis and skin-related inflammatory conditions.

Disturbance in Na+ regulation in cells rich in mitochondria isolated from gills of the yellow clam Mesodesma mactroides exposed to copper under different osmotic conditions

Cells rich in mitochondria were isolated from gills of the seawater clam Mesodesma mactroides, incubated in isosmotic saline solution (840 mOsmol/kg H2O), and exposed (3 h) to environmentally realistic Cu concentrations (nominally: 0, 5, 9 and 20 μg/L). In cells exposed to 20 μg Cu/L, Cu accumulation, Na+ content reduction and carbonic anhydrase (CA) activity inhibition were observed, without significant changes in cell viability and Na+,K+-ATPase (NKA) activity. In the absence of Cu, cell viability and Cu content were reduced in hyposmotic media respect with the control, without changes in Na+ content and enzyme (CA and NKA) activities. In the presence of 5 or 9 μg/L Cu, cell Cu content was increased, especially at 670 mOsmol/kg H2O. Cell Na+ content and NKA activity were reduced after exposure to 20 μg/L Cu at 670 mOsmol/kg H2O. In turn, CA activity was dependent on Cu concentration, being significantly reduced in cells exposed to 9 and 20 μg/L Cu in both hyposmotic conditions. These findings indicate that Cu also negatively affects Na+ regulation in gill cells of the seawater clam M. mactroides, with Cu toxicity increasing at hyposmotic conditions. Also, they indicate that physiology is more important than water chemistry in predicting Cu toxicity in environments of changing salinity, pointing out CA activity as a potential biomarker of Cu exposure.

Mesodesma mactroides Gill Cells Exposed to Copper: Does Hyposmotic Saline Increase Cytotoxicity or Cellular Defenses?

Gill cells of filter feeding mollusks have cellular defense mechanisms, such as multixenobiotic resistance (MXR), that allow them to extrude possible contaminants. To analyze the cytotoxicity and cellular defenses of gills in the clam Mesodesma mactroides, gill cells were exposed to copper in both iso- and hyposmotic solutions. Analysis of MXR activity by fluorescence microscopy showed that hyposmotic saline activated defenses, whereas the presence of copper in isosmotic solution inhibited the activation of defenses. Cell viability was decreased in cells exposed to copper in isosmotic saline, but not in cells exposed to hyposmotic saline. We conclude that when cells cannot defend themselves due to decreased MXR, cell death occurs. In addition, gill cells under hyposmotic conditions have a greater capacity for defense and a lower rate of cellular mortality than when they are maintained under isosmotic conditions.

Novel roles for the lens in preserving overall ocular health

Outside the traditional roles of the lens as an important refractive element and a UV filter, it was David Beebe’s group that first demonstrated that the lens acts an oxygen sink that protects the tissues of the anterior segment of the eye from oxygen or oxygen metabolites. In this review, we follow on from this work, and present new evidence from our laboratory to demonstrate that the lens serves as a reservoir for the release of the antioxidant glutathione (GSH) into the aqueous humor to provide a source of GSH and/or its precursor amino acids to nearby tissues that interface with the aqueous humor, or to remove toxic metabolites from the eye via the aqueous outflow pathway. In addition to GSH release, our laboratory and others have shown that ATP is released from the lens under hyposmotic conditions to activate purinergic signalling pathways in an autocrine manner to alter lens function. In this review, we raise the idea that ATP and/or its subsequent degradation product adenosine may exert a paracrine function and influence purinergic signalling systems in other tissues to alter aqueous humor outflow. These new secondary roles indicate that the lens is not just a passive optical element, but a highly dynamic and active tissue that interacts with its neighbouring tissues, through modifying the environments in which these tissues function. We believe that the lens actively contributes to the ocular environment and as a consequence, removal of the lens would alter the functionality of neighbouring tissues. We speculate that a long term effect of lens removal may be to inadvertently increase the exposure of anterior tissues of the eye to oxidative stress due to elevated oxygen levels and a reduction in the availability of GSH and purinergic signalling molecules in the aqueous humor. Since cataract surgery is now being performed on younger patients due to our increasing diabetic population, over time, we predict these changes may increase the susceptibility of these tissues to oxidative stress and the incidence of subsequent ocular pathologies. If our view of the lens is correct, the actual loss of the biological lens may have longer term consequences for overall ocular health than currently appreciated.

Chaperone roles for TMAO and HSP70 during hyposmotic stress in the spiny dogfish shark (Squalus acanthias).

Salinity decreases are experienced by many marine elasmobranchs. To understand how these fishes cope with hyposmotic stress on a cellular level, we used the spiny dogfish shark (Squalus acanthias) as a model to test whether a reciprocal relationship exists between the cell's two primary protein protection mechanisms, the chemical (e.g., trimethylamine oxide, TMAO) and molecular (e.g., heat shock protein 70, HSP70) chaperone systems. This relationship is interesting given that many elasmobranchs are expected to gain water and lose osmolytes, chemical chaperones, and ions as they osmoconform to new, lowered salinity. Dogfish were cannulated for repeated blood sampling and exposed to 70% seawater (SW) for 48 h. These hyposmotic conditions had no effect on red blood cell (RBC) and white muscle TMAO concentrations, and did not result in HSP70 induction or signs of protein damage (i.e., increased ubiquitin), suggesting that TMAO levels were sufficiently protective in these tissues. However, in the gill, we observed a significant decrease in TMAO concentration and a significant induction of HSP70 as well as signs of protein damage. In the face of this cellular stress response, gill Na(+)/K(+)-ATPase (NKA) activity significantly increased during hyposmotic conditions, as expected. We suggest that this functional preservation in the gill is partly the result of HSP70 induction with lowered salinity. We conclude a reciprocal relationship between TMAO and HSP70 in the gills of dogfish as a result of in vivo hyposmotic stress. When osmotically induced protein damage surpasses the protective capacity of remaining TMAO, HSP70 is induced to preserve tissue and organismal function.

Calcium entry via connexin hemichannels in lens epithelium

Exposure to hyposmotic solution causes release of ATP from lens cells via hemichannels. Because hemichannel opening feasibly could swamp the cells with calcium, we carried out studies to measure the magnitude of the increase in cytoplasmic calcium concentration caused by hemichannel opening. In studies on porcine lens epithelial cells in primary culture, propidium iodide (PI) uptake was measured as an index of hemichannel opening. PI uptake was increased significantly in cells exposed to hyposmotic solution. The PI increase under hyposmotic conditions was suppressed by GAP 27, a connexin inhibitor peptide. In studies on cells loaded with Fura-2, continuous exposure to hyposmotic solution caused a cytoplasmic calcium concentration increase that peaked within ∼30 s then remained elevated at or below the peak response for more than 60 min. The peak calcium concentration was 186 ± 2.3 nM compared to a baseline value of 98.0 ± 1.4 nM. The calcium concentration increased a lot further in cells exposed to A23187 (2.5 μM) or the sodium–calcium exchange inhibitor SN-6 (10 μM) added after the onset of the calcium rise in hyposmotic solution. The cytoplasmic calcium increase in hyposmotic solution was abolished by GAP 27. Calcium returned to baseline in cells exposed to hyposmotic solution then treated with GAP 27 starting 2 min after the onset of the calcium rise. The calcium increase in hyposmotic solution did not occur when calcium was eliminated from the bathing medium. The responses to hyposmotic and hyperosmotic stress were different. There was no detectable increase in calcium or PI entry in cells exposed to hyperosmotic solution (500mOsm). In summary, GAP 27-sensitive accumulation of PI by cultured lens epithelium points to connexin hemichannel opening and associated calcium entry. Even though connexins form channels with a large carrying capacity, calcium entry does not increase the cytoplasmic calcium concentration beyond a tolerable physiological range.

211 Concanavalin A-Induced Hepatitis in Mice Is Prevented by Depletion of Gut Bacteria Using Antibiotics

L or hyposmotic conditions, as measured by LDH and HMGB1 release. Upon hyposmotic stress of hepatocytes, cell death occurred in the absence of caspase activation. Similar to primary hepatocytes, K18-DE mice treated with Fas-L had elevated serum necrosis markers (LDH and HMGB1) as compared to their WT counterparts. K18-DE mutation renders the K18 obligate heteropolymer, K8, less susceptible to phosphorylation at its p38 stress kinase site K8 S79, without altering the binding of K8/K18 to p38 kinase. Notably, K8 S79 phosphorylation plays an important role in mediating keratin filament reorganization upon stress. Conclusions: Caspase digestion of K18 plays an essential role in promoting apoptosisassociated keratin filament collapse in the liver. Keratin phosphorylation-mediated filament reorganization appears to be critical in protecting hepatocytes from necroapoptosis, and provides a mechanism for susceptibility to tissue injury in human IF mutations that involve caspase cleavage sites.

Metabolism of amino acids during hyposmotic adaptation in the whiteleg shrimp, Litopenaeus vannamei

The penaeid prawn, Litopenaeus vannamei, was employed to investigate intracellular isosmotic regulation in situations where invertebrates encounter hyposmosis. Hemolymph osmolality was first analyzed to confirm osmoregulatory conditions in the experimental animals, followed by analysis of amino acids in muscle and hemolymph using high-performance liquid chromatography. Total muscle amino acid levels decreased when hemolymph osmolality was extremely low, whereas glycine and L-serine levels increased in the hemolymph. These results suggest that tissue amino acids were released into the hemolymph to lower the osmolality of the tissues for purposes of low-salinity adaptation. Next, oxygen consumption and ammonia excretion rates were examined, and the O/N ratio was determined. Oxygen consumption levels and ammonia excretion rates increased, and the O/N ratio decreased when the animals were exposed to low salinity. These results suggest that amino acids were abundantly consumed as an energy source when animals were exposed to low salinity. To confirm the consumption of particular amino acids, the specific activity of L-serine ammonia lyase was also examined. Specific activity was highest when L-serine levels in the hemolymph were highest. Thus, it appears that L-serine levels increased under hyposmotic conditions due to the consumption of L-serine as an energy source. It was concluded that particular amino acids as osmolytes are likely metabolized as energy sources and consumed for purposes of hyposmotic adaptation.

Effects of copper on osmoregulation in sheepshead minnow, Cyprinodon variegatus acclimated to different salinities

The sheepshead minnow, Cyprinodon variegatus is a euryhaline fish that inhabits estuaries and coastal marshes where it encounters a wide range of salinities. Many of these areas also have elevated levels of contaminants, creating the potential for toxic ions to interfere with the uptake of ions for osmoregulation. To determine whether the effect of copper on osmoregulatory activity is dependent on the osmotic conditions that individuals have been living at, fish were acclimated for 14 days to 2.5, 10.5 or 18.5ppt seawater and then exposed to a fixed free cupric ion level (14.6μM Cu2+) for 6h. Plasma Na, plasma Cl, wet/dry weight ratio, transepithelial potential difference (TEPD) and branchial Na+/K+-ATPase activity were determined before and after copper exposure. We also computed Na and Cl equilibrium potentials. Following the salinity acclimation (in fish not yet exposed to copper), fish from the low salinity group (2.5ppt) had lower TEPD, lower plasma Na levels and higher branchial Na+/K+-ATPase activity compared to the fish acclimated to higher salinities. No differences in plasma Cl and wet/dry weight ratio were detected. Copper exposure caused a significant decrease in plasma Na levels and Na+/K+-ATPase activity and an increase in wet/dry weight ratio, but these changes were limited to the 2.5ppt salinity group. No significant changes in plasma Cl were detected. Copper treatment resulted in a small decrease in TEPD for all except the lowest salinity acclimation group. A comparison of equilibrium potentials with TEPD showed evidence of active transport of both Na and Cl in 2.5ppt acclimated fish but not for the 10.5 or the 18.5ppt acclimated fish. Our results show that effects of copper on osmoregulation are dependent on the fish’ past salinity regime, and that these effects tend to be more pronounced for euryhaline fish that have been living under hyposmotic conditions.

Identification of differentially transcribed genes in shrimp Litopenaeus vannamei exposed to osmotic stress and challenged with WSSV virus

The effects of hyposmotic stress and white spot syndrome virus (WSSV) challenge in expression was studied in the marine shrimp Litopenaeus vannamei. Messenger RNA from gills of shrimp submitted to osmotic stress was isolated to identify genes differentially expressed through the suppressive subtractive hybridization (SSH) method. Two subtractive libraries forward and two reverse were constructed to identify up and down-regulated genes under these conditions. About 192 clones were sequenced, of which 46 genes were identified. These genes encode proteins corresponding to a wide range of biological roles, including defense, cell signaling, electron transfer, cell proliferation and differentiation, apoptosis, intermediary metabolism, cytoskeleton and digestion. Among the identified genes, 19 were up-regulated and 27 were down-regulated in the animals kept at a lower ion concentration. We evaluated the expression of eight genes by RT-qPCR in shrimp submitted to hyposmotic conditions with and without WSSV challenge. The SSH enabled the identification of genes that are influenced by hyposmotic stress. A significant up-regulation was observed in lectin-C, QM, TGF beta inducible nuclear protein 1, ciclophilin, malate dehydrogenase, mitochondrial ATP synthase F chain and ATP synthase subunit 9 precursor transcripts. However, the expression of these genes in L. vannamei was not affected by WSSV infection both at isosmotic and hyposmotic conditions.

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.

Capacitance increases of dissociated tilapia prolactin cells in response to hyposmotic and depolarizing stimuli

Prolactin (PRL) is the major hormonal mediator of adaptation to hyposmotic conditions. In tilapia ( Oreochromis mossambicus), PRL cells are segregated to the rostral pars distalis of the anterior pituitary facilitating the nearly pure culture of dissociated PRL cells. Membrane capacitance ( C m) was recorded at 1 Hz or higher for tens of minutes as a surrogate monitor of PRL secretion by exocytosis from cells under perforated patch clamp. The study compares secretory responses to trains of depolarizing clamps (100 at 2.5 Hz, from −70 to +10 mV for 100 ms) to the physiological stimulus, exposure to hyposmotic medium, here a switch from 350 to 300 mOsm saline ([Ca 2+] 15 mM). Two-thirds of cells tested with each stimulus responded. In response to depolarizing clamps, C m increased linearly at an average rate of 7.2 fF/s. The increase was also linear in response to hyposmotic perfusion, but the average rate was 0.68 fF/s. Response to depolarization was reversibly blocked in Ca 2+-omitted saline, or in saline with 30 μM Cd 2+. It was unaffected by 0.1 μM tetrodotoxin. By contrast, responses were reduced but not absent during perfusion of hyposmotic saline with Ca 2+-omitted; 30 μM Cd 2+ appeared to enhance the hyposmotic response. BAPTA-AM eliminated responses to both stimuli, confirming that secretion was dependent on increases of intracellular [Ca 2+]. Together with previous observations from this laboratory of [Ca 2+] i with simultaneous collection and immunoassay of perfusate for PRL, we conclude that depolarization and hyposmotic stimuli initiate secretion by independent mechanisms.

Regulatory volume decrease and P receptor signaling in fish cells: mechanisms, physiology, and modeling approaches

For animal cell plasma membranes, the permeability of water is much higher than that of ions and other solutes, and exposure to hyposmotic conditions almost invariably causes rapid water influx and cell swelling. In this situation, cells deploy regulatory mechanisms to preserve membrane integrity and avoid lysis. The phenomenon of regulatory volume decrease, the partial or full restoration of cell volume following cell swelling, is well-studied in mammals, with uncountable investigations yielding details on the signaling network and the effector mechanisms involved in the process. In comparison, cells from other vertebrates and from invertebrates received little attention, despite of the fact that e.g. fish cells could present rewarding model systems given the diversity in ecology and lifestyle of this animal group that may be reflected by an equal diversity of physiological adaptive mechanisms, including those related to cell volume regulation. In this review, we therefore present an overview on the most relevant aspects known on hypotonic volume regulation presently known in fish, summarizing transporters and signaling pathways described so far, and then focus on an aspect we have particularly studied over the past years using fish cell models, i.e. the role of extracellular nucleotides in mediating cell volume recovery of swollen cells. We, furthermore, present diverse modeling approaches developed on the basis of data derived from studies with fish and other models and discuss their potential use for gaining insight into the theoretical framework of volume regulation.

Muscarinic Receptor Stimulation of D‐aspartate Uptake Into Human SH‐SY5Y Neuroblastoma Cells Is Attenuated By Hyposmolarity

In the present study, the ability of muscarinic cholinergic receptors (mAChRs) to regulate the uptake of glutamate (monitored as D‐aspartate) into human SH‐SY5Y neuroblastoma cells under isotonic or hypotonic conditions has been examined. In isotonic media, agonist activation of mAChRs resulted in a significant increase (250–300% of control) in the uptake of D‐aspartate and concurrently, a cellular redistribution of the excitatory amino acid transporter 3 (EAAT3) to the plasma membrane. In hypotonic media, the ability of mAChR activation to facilitate D‐aspartate uptake was significantly attenuated (40–50%) and the cellular distribution of EAAT3 was disrupted. Reduction of mAChR‐stimulated D‐aspartate uptake under hyposmotic conditions could be fully reversed upon re‐exposure of the cells to isotonic media. Under both isotonic and hypotonic conditions, mAChR‐mediated increases in D‐aspartate uptake were dependent upon cytoskeletal integrity, protein kinase C (PKC) and phosphatidylinositol 3‐kinase (PI3K) activities and the availability of intracellular Ca2+. In contrast, a dependence on extracellular Ca2+ was only observed under isotonic conditions. The results suggest that although the uptake of D‐aspartate into SH‐SY5Y cells is enhanced following mAChR activation, this process is markedly attenuated by hyposmolarity. Supported by GM007767 (DJF).

Involvement of volume‐sensitive Cl− channels in the proliferation of human subcutaneous pre‐adipocytes

1. Obesity is a significant challenge in terms of public health and preventive medicine. Inhibition of pre-adipocyte proliferation is believed to be important in the proposed anti-obesity mechanism. The aim of the present study was to examine the interplay between Cl(-) channels and their possible involvement in the proliferation of undifferentiated human pre-adipocytes. 2. Pre-adipocytes were isolated from human abdominal subcutaneous adipose tissue. Membrane ion currents were recorded using the whole-cell patch-clamp technique. Expression of the Cl(-) channel ClC-3 gene and protein was determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot, respectively. Cell proliferation was evaluated using the [(3)H]-thymidine incorporation assay. 3. Electrophysiological recordings revealed a volume-sensitive Cl(-) current (I(Cl.vol)) expressed in pre-adipocytes that was activated under hyposmotic conditions (external osmolarity decreased by 80%) and inhibited by the Cl(-) channel blocker tamoxifen. 4. Expression of the ClC-3 channel gene and protein was confirmed by RT-PCR and western blot analysis. Blocking I(Cl.vol) with tamoxifen supressed the proliferation of pre-adipocytes in a concentration-dependent manner. 5. Collectively, the results of the present study indicate that the volume-sensitive Cl(-) channel participates in regulation of the proliferation of human subcutaneous pre-adipocytes.

Caveolae Act as Membrane Reserves Which Limit Mechanosensitive ICl,swell Channel Activation during Swelling in the Rat Ventricular Myocyte

BackgroundMany ion channels are preferentially located in caveolae where compartmentalisation/scaffolding with signal transduction components regulates their activity. Channels that are mechanosensitive may be additionally dependent on caveolar control of the mechanical state of the membrane. Here we test which mechanism underlies caveolar-regulation of the mechanosensitive I Cl,swell channel in the adult cardiac myocyte.Methodology/Principal FindingsRat ventricular myocytes were exposed to solution of 0.02 tonicity (T; until lysis), 0.64T for 10–15 min (swelling), and/or methyl-β-cyclodextrin (MBCD; to disrupt caveolae). MBCD and 0.64T swelling reduced the number of caveolae visualised by electron microscopy by 75 and 50% respectively. MBCD stimulated translocation of caveolin 3 from caveolae-enriched buoyant membrane fractions, but both caveolin 1 and 3 remained in buoyant fractions after swelling. I Cl,swell inhibition in control cells decreased time to half-maximal volume (t 0.5,vol; 0.64T), consistent with a role for I Cl,swell in volume regulation. MBCD-treated cells showed reduced time to lysis (0.02T) and t 0.5,vol (0.64T) compared with controls. The negative inotropic response to swelling (an index of I Cl,swell activation) was enhanced by MBCD.Conclusions/SignificanceThese data show that disrupting caveolae removes essential membrane reserves, which speeds swelling in hyposmotic conditions, and thereby promotes activation of I Cl,swell. They illustrate a general principle whereby caveolae as a membrane reserve limit increases in membrane tension during stretch/swelling thereby restricting mechanosensitive channel activation.

KV7/M Channels Mediate Osmotic Modulation of Intrinsic Neuronal Excitability

Modest decreases in extracellular osmolarity induce brain hyperexcitability that may culminate in epileptic seizures. At the cellular level, moderate hyposmolarity markedly potentiates the intrinsic neuronal excitability of principal cortical neurons without significantly affecting their volume. The most conspicuous cellular effect of hyposmolarity is converting regular firing neurons to burst-firing mode. This effect is underlain by hyposmotic facilitation of the spike afterdepolarization (ADP), but its ionic mechanism is unknown. Because blockers of K(V)7 (KCNQ) channels underlying neuronal M-type K(+) currents (K(V)7/M channels) also cause spike ADP facilitation and bursting, we hypothesized that lowering osmolarity inhibits these channels. Using current- and voltage-clamp recordings in CA1 pyramidal cells in situ, we have confirmed this hypothesis. Furthermore, we show that hyposmotic inhibition of K(V)7/M channels is mediated by an increase in intracellular Ca(2+) concentration via release from internal stores but not via influx of extracellular Ca(2+). Finally, we show that interfering with internal Ca(2+)-mediated inhibition of K(V)7/M channels entirely protects against hyposmotic ADP facilitation and bursting, indicating the exclusivity of this novel mechanism in producing intrinsic neuronal hyperexcitability in hyposmotic conditions.

Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses

The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosine phosphorylation as measured by western blotting techniques; however, a similar increase was not detected when spermatozoa were incubated under hyposmotic conditions. The general protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine inhibited ( P < 0.05) tyrosine phosphorylation in samples from cells under hyperosmotic conditions. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) also inhibited ( P < 0.05) protein tyrosine phosphorylation in cells under hyperosmotic conditions. In summary, these data indicate that incubation of equine spermatozoa under both hyposmotic and hyperosmotic conditions can increase superoxide anion generation. Under hyperosmotic conditions, this increased generation of superoxide anion was accompanied by increased protein tyrosine phosphorylation.