Increase In Extracellular Osmolality Research Articles

Suero salino hipertónico e insuficiencia cardiaca: ¿«sodio-centrista» o «cloro-centrista»?

Up to 50% of patients admitted for heart failure have congestion at discharge despite diagnostic and therapeutic advances. Both persistent congestion and diuretic resistance are associated with worse prognosis. The combination of hypertonic saline and loop diuretic has shown promising results in different studies. However, it has not yet achieved a standardized use, partly because of the great heterogeneity in the concentration of sodium chloride, the dose of diuretic or the amount of sodium in the diet. Classically, the movement of water from the intracellular space due to an increase in extracellular osmolarity has been postulated as the main mechanism involved. However, chloride deficit is postulated as the main up-regulator of plasma volume changes, and its correction may be the main mechanism involved. This ‘chloride centric’ approach to heart failure opens the door to therapeutic strategies that would include diuretics to correct hypochloremia, as well as sodium free chloride supplementation.

Osmolality Effects on CHO Cell Growth, Cell Volume, Antibody Productivity and Glycosylation.

The addition of nutrients and accumulation of metabolites in a fed-batch culture of Chinese hamster ovary (CHO) cells leads to an increase in extracellular osmolality in late stage culture. Herein, we explore the effect of osmolality on CHO cell growth, specific monoclonal antibody (mAb) productivity and glycosylation achieved with the addition of NaCl or the supplementation of a commercial feed. Although both methods lead to an increase in specific antibody productivity, they have different effects on cell growth and antibody production. Osmolality modulation using NaCl up to 470 mOsm kg−1 had a consistently positive effect on specific antibody productivity and titre. The addition of the commercial feed achieved variable results: specific mAb productivity was increased, yet cell growth rate was significantly compromised at high osmolality values. As a result, Feed C addition to 410 mOsm kg−1 was the only condition that achieved a significantly higher mAb titre compared to the control. Additionally, Feed C supplementation resulted in a significant reduction in galactosylated antibody structures. Cell volume was found to be positively correlated to osmolality; however, osmolality alone could not account for observed changes in average cell diameter without considering cell cycle variations. These results help delineate the overall effect of osmolality on titre and highlight the potentially negative effect of overfeeding on cell growth.

Nitric oxide acutely modulates hypothalamic and neurohypophyseal carbon monoxide and hydrogen sulphide production to control vasopressin, oxytocin and atrial natriuretic peptide release in rats.

Nitric oxide (NO) negatively modulates the secretion of vasopressin (AVP), oxytocin (OT) and atrial natriuretic peptide (ANP) induced by the increase in extracellular osmolality, whereas carbon monoxide (CO) and hydrogen sulphide (H2 S) act to potentiate it; however, little information is available for the osmotic challenge model about whether and how such gaseous systems modulate each other. Therefore, using an acute ex vivo model of hypothalamic and neurohypophyseal explants (obtained from male 6/7-week-old Wistar rats) under conditions of extracellular iso- and hypertonicity, we determined the effects of NO (600μmolL-1 sodium nitroprusside), CO (100μmolL-1 tricarbonylchloro[glycinato]ruthenium [II]) and H2 S (10mmolL-1 sodium sulphide) donors and nitric oxide synthase (NOS) (300μmolL-1 Nω -methyl-l-arginine [LNMMA]), haeme oxygenase (HO) (200μmolL-1 Zn(II) deuteroporphyrin IX 2,4-bis-ethylene glycol [ZnDPBG]) and cystathionine β-synthase (CBS) (100μmolL-1 aminooxyacetate [AOA]) inhibitors on the release of hypothalamic ANP and hypothalamic and neurohypophyseal AVP and OT, as well as on the activities of NOS, HO and CBS. LNMMA reversed hyperosmolality-induced NOS activity, and enhanced hormonal release by the hypothalamus and neurohypophysis, in addition to increasing CBS and hypothalamic HO activity. AOA decreased hypothalamic and neurohypophyseal CBS activity and hormonal release, whereas ZnDPBG inhibited HO activity and hypothalamic hormone release; however, in both cases, AOA did not modulate NOS and HO activity and ZnDPBG did not affect NOS and CBS activity. Thus, our data indicate that, although endogenous CO and H2 S positively modulate AVP, OT and ANP release, only NO plays a concomitant role of modulator of hormonal release and CBS activity in the hypothalamus and neurohypophysis and that of HO activity in the hypothalamus during an acute osmotic stimulus, which suggests that NO is a key gaseous controller of the neuroendocrine system.

Mannitol decreases neocortical epileptiform activity during early brain development via cotransport of chloride and water

Seizures and brain injury lead to water and Cl− accumulation in neurons. The increase in intraneuronal Cl− concentration ([Cl−]i) depolarizes the GABAA reversal potential (EGABA) and worsens seizure activity. Neocortical neuronal membranes have a low water permeability due to the lack of aquaporins necessary to move free water. Instead, neurons use cotransport of ions including Cl− to move water. Thus, increasing the extracellular osmolarity during seizures should result in an outward movement of water and salt, reducing [Cl−]i and improving GABAA receptor-mediated inhibition. We tested the effects of hyperosmotic therapy with a clinically relevant dose of mannitol (20 mM) on epileptiform activity, spontaneous multiunit activity, spontaneous inhibitory post-synaptic currents (sIPSCs), [Cl−]i, and neuronal volume in layer IV/V of the developing neocortex of C57BL/6 and Clomeleon mice. Using electrophysiological techniques and multiphoton imaging in acute brain slices (post-natal day 7–12) and organotypic neocortical slice cultures (post-natal day 14), we observed that mannitol: 1) decreased epileptiform activity, 2) decreased neuronal volume and [Cl−]i through CCCs, 3) decreased spontaneous multi-unit activity frequency but not amplitude, and 4) restored the anticonvulsant efficacy of the GABAA receptor modulator diazepam. Increasing extracellular osmolarity by 20 mOsm with hypertonic saline did not decrease epileptiform activity. We conclude that an increase in extracellular osmolarity by mannitol mediates the efflux of [Cl−]i and water through CCCs, which results in a decrease in epileptiform activity and enhances benzodiazepine actions in the developing neocortex in vitro. Novel treatments aimed to decrease neuronal volume may concomitantly decrease [Cl−]i and improve seizure control.

A comparison of anticonvulsant efficacy and action mechanism of Mannitol vs Phenytoin in adult rat neocortical slices

We show that, in adult rat neocortical slices, an anticonvulsant effect comparable to that of Phenytoin can be obtained through a Mannitol-induced increase in extracellular osmolarity of only 30mOsm/L. The anticonvulsant action of extracellular hyperosmolarity has been known for decades but has not found a feasible therapeutic application, yet. A 30mOsm/L increase in extracellular osmolarity is already utilized in neurocritical care though not as an anticonvulsant agent: the data suggest a possible effective anticonvulsant use, too, in this setting.We used multiple electrode arrays to characterize and compare the anticonvulsant mechanisms of Mannitol and Phenytoin. Phenytoin decreased the voltage, duration and spatial spread of rhythmic repetitive, ictal-like activity. In contrast, Mannitol did not significantly affect voltage, duration and spatial spread of rhythmic repetitive, ictal-like activity but rather it inhibited the rate of epileptiform discharges.

A comparison of freezing-damage during isochoric and isobaric freezing of the potato.

BackgroundFreezing is commonly used for food preservation. It is usually done under constant atmospheric pressure (isobaric). While extending the life of the produce, isobaric freezing has detrimental effects. It causes loss of food weight and changes in food quality. Using thermodynamic analysis, we have developed a theoretical model of the process of freezing in a constant volume system (isochoric). The mathematical model suggests that the detrimental effects associated with isobaric freezing may be reduced in an isochoric freezing system. To explore this hypothesis, we performed a preliminary study on the isochoric freezing of a produce with which our group has experience, the potato.MethodExperiments were performed in an isochoric freezing device we designed. The device is robust and has no moving parts. For comparison, we used a geometrically identical isobaric freezing device. Following freezing and thawing, the samples were weighed, examined with colorimetry, and examined with microscopy.ResultsIt was found that potatoes frozen to −5 °C in an isochoric system experienced no weight loss and limited enzymatic browning. In contrast the −5 °C isobaric frozen potato experienced substantial weight loss and substantial enzymatic browning. Microscopic analysis shows that the structural integrity of the potato is maintained after freezing in the isochoric system and impaired after freezing in the isobaric system.DiscussionTissue damage during isobaric freezing is caused by the increase in extracellular osmolality and the mechanical damage by ice crystals. Our thermodynamic analysis predicts that during isochoric freezing the intracellular osmolality remains comparable to the extracellular osmolality and that isochoric systems can be designed to eliminate the mechanical damage by ice. The results of this preliminary study seem to confirm the theoretical predictions.ConclusionThis is a preliminary exploratory study on isochoric freezing of food. We have shown that the quality of a food product preserved by isochoric freezing is better than the quality of food preserved to the same temperature in isobaric conditions. Obviously, more extensive research remains to be done to extend this study to lower freezing temperatures and other food items.

Isochoric and isobaric freezing of fish muscle

We have recently shown that, a living organism, which succumbs to freezing to −4 °C in an isobaric thermodynamic system (constant atmospheric pressure), can survive freezing to −4 °C in an isochoric thermodynamic system (constant volume). It is known that the mechanism of cell damage in an isobaric system is the freezing caused increase in extracellular osmolality, and, the consequent cell dehydration. An explanation for the observed survival during isochoric freezing is the thermodynamic modeling supported hypothesis that, in the isochoric frozen solution the extracellular osmolality is comparable to the cell intracellular osmolality. Therefore, cells in the isochoric frozen organism do not dehydrate, and the tissue maintains its morphological integrity. Comparing the histology of: a) fresh fish white muscle, b) fresh muscle frozen to −5 °C in an isobaric system and c) fresh muscle frozen to −5 °C I in an isochoric system, we find convincing evidence of the mechanism of cell dehydration during isobaric freezing. In contrast, the muscle tissue frozen to −5 °C in an isochoric system appears morphologically identical to fresh tissue, with no evidence of dehydration. This is the first experimental evidence in support of the hypothesis that in isochoric freezing there is no cellular dehydration and therefore the morphology of the frozen tissue remains intact.

Protein Kinase R Mediates the Inflammatory Response Induced by Hyperosmotic Stress.

High extracellular osmolarity results in a switch from an adaptive to an inflammatory gene expression program. We show that hyperosmotic stress activates the protein kinase R (PKR) independently of its RNA-binding domain. In turn, PKR stimulates nuclear accumulation of nuclear factor κB (NF-κB) p65 species phosphorylated at serine-536, which is paralleled by the induction of a subset of inflammatory NF-κB p65-responsive genes, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and IL-1β. The PKR-mediated hyperinduction of iNOS decreases cell survival in mouse embryonic fibroblasts via mechanisms involving nitric oxide (NO) synthesis and posttranslational modification of proteins. Moreover, we demonstrate that the PKR inhibitor C16 ameliorates both iNOS amplification and disease-induced phenotypic breakdown of the intestinal epithelial barrier caused by an increase in extracellular osmolarity induced by dextran sodium sulfate (DSS) in vivo Collectively, these findings indicate that PKR activation is an essential part of the molecular switch from adaptation to inflammation in response to hyperosmotic stress.

Coordination of Gene Expression and Cell Cycle Progression in Response to Stress; a Novel Checkpoint in S Phase

Exposure of cells to increases in extracellular osmolarity results in the activation of the Hog1 stress‐activated protein kinase (SAPK). Activation of Hog1 is required to generate a set of osmoadaptive responses essential for survival under high osmolarity. Adaptation to osmostress requires the induction of a large number of genes, which indicates the necessity to regulate several aspects of the cell physiology. In addition to gene expression, the SAPK also controls cell cycle. Here, we showed that the SAPK is able to modulate cell cycle delay in different phases of the cell cycle including S phase. During S phase, Hog1 controls Mrc1, a protein of the replication complex, to control DNA replication (Duch et al., 2013). The control of replication through Mrc1 is required to coordinate stress‐responsive gene induction with duplication of DNA upon osmostress. Remarkably, we have recently found that Mrc1 is also important to delay replication in response to other environmental stresses suggesting a conserved mechanism of S phase regulation upon transcriptional outbursts.Support or Funding InformationThis work was supported by grants from the Spanish Government (BFU2012‐33503 to F.P., BFU2014 52333 to E.d.N.) and the Fundación Marcelino Botín to F.P. F.P. and E.d.N. are recipients of an ICREA Academia (Generalitat de Catalunya).

Heparin-induced hyperkalaemia in a patient with type 2 diabetes: a case report

Introduction: Heparin is extensively used amongst different medical and surgical specialities for prophylaxis and treatment of venous thromboembolism and suspected coronary syndromes. Hyperkalaemia is known to occur as an effect of heparin treatment through its action on the renin-angiotensin system, especially when used at treatment doses. Hyperkalaemia is a serious electrolyte disturbance, which may precipitate potentially fatal cardiac arrhythmias. We describe a case of a patient developing hyperkalaemia secondary to a prophylactic dose of low-molecular weight heparin. Case report: A 65-year-old man with ischaemic heart disease, type 2 diabetes and peripheral vascular disease was admitted under the vascular surgical team with ischaemic right leg. He was commenced on prophylactic low-molecular weight heparin. Four days into his hospital stay he became hyperkalaemic with serum potassium of 6.1 mmol/l (normal 4.5–5.3 mmol/l). The potassium level remained persistently elevated in spite of stopping the ACE inhibitor and spironolactone, which he was on for several years. He responded only briefly to insulin–dextrose infusions. The hyperkalaemia resolved gradually on discontinuing the prophylactic heparin. Discussion: Compared to normal population, hyperkalaemia is more prevalent among diabetics. Hyperglycaemia causes an increase in extracellular osmolality, which then leads to elevated potassium levels. Hyperkalaemia will be prolonged if duration of heparin therapy is prolonged before discontinuation. Conclusion: This case illustrates the potentially harmful effect of low dose low-molecular weight heparin. Although there are warnings in the British National Formulary about the risks of hyperkalaemia with heparin use, awareness of this side effect amongst medical and surgical staff appears to be low. With increasing use of prophylactic heparin amongst medical patients, heparin-associated hyperkalaemia may well be seen more frequently.

Extreme Hypernatriämie

The case of a female patient with a suprasellar optic glioma is reported, who was admitted to the intensive care unit due to decompensated diabetes insipidus with hypernatremia of 194mmol/l. The sodium concentration was reduced slowly over 4 days and the patient recovered without sequelae. Based on this case the article deals with the pathophysiology and therapy of hypernatremia. An increase in extracellular osmolarity leads to augmented production of intracellular osmolytes in order to maintain the cell volume constant. Due to this counterregulation correction of the sodium concentration must be done with caution.

Taurine Biosynthesis by Neurons and Astrocytes

The physiological roles of taurine, a product of cysteine degradation and one of the most abundant amino acids in the body, remain elusive. Taurine deficiency leads to heart dysfunction, brain development abnormalities, retinal degradation, and other pathologies. The taurine synthetic pathway is proposed to be incomplete in astrocytes and neurons, and metabolic cooperation between these cell types is reportedly needed to complete the pathway. In this study, we analyzed taurine synthesis capability as reported by incorporation of radioactivity from [(35)S]cysteine into taurine, in primary murine astrocytes and neurons, and in several transformed cell lines (human (SH-SY5Y) and murine (N1E-115) neuroblastoma, human astrocytoma (U-87 MG and 1321 N1), and rat glioma (C6)). Extensive incorporation of radioactivity from [(35)S]cysteine into taurine was observed in rat glioma cells as well as in primary mouse astrocytes and neurons, establishing the presence of an intact taurine synthesis pathway in these cells. Interestingly, exposure of cells to cysteine or cysteamine resulted in elevated intracellular hypotaurine without a corresponding increase in taurine levels, suggesting that oxidation of hypotaurine limits taurine synthesis in cells. Consistent with its role as an organic osmolyte, taurine synthesis was stimulated under hypertonic conditions in neurons.

Volume regulation in mammalian skeletal muscle: the role of sodium–potassium–chloride cotransporters during exposure to hypertonic solutions

Controversy exists as to whether mammalian skeletal muscle is capable of volume regulation in response to changes in extracellular osmolarity despite evidence that muscle fibres have the required ion transport mechanisms to transport solute and water in situ. We addressed this issue by studying the ability of skeletal muscle to regulate volume during periods of induced hyperosmotic stress using single, mouse extensor digitorum longus (EDL) muscle fibres and intact muscle (soleus and EDL). Fibres and intact muscles were loaded with the fluorophore, calcein, and the change in muscle fluorescence and width (single fibres only) used as a metric of volume change. We hypothesized that skeletal muscle exposed to increased extracellular osmolarity would elicit initial cellular shrinkage followed by a regulatory volume increase (RVI) with the RVI dependent on the sodium–potassium–chloride cotransporter (NKCC). We found that single fibres exposed to a 35% increase in extracellular osmolarity demonstrated a rapid, initial 27–32% decrease in cell volume followed by a RVI which took 10-20 min and returned cell volume to 90–110% of pre-stimulus values. Within intact muscle, exposure to increased extracellular osmolarity of varying degrees also induced a rapid, initial shrinkage followed by a gradual RVI, with a greater rate of initial cell shrinkage and a longer time for RVI to occur with increasing extracellular tonicities. Furthermore, RVI was significantly faster in slow-twitch soleus than fast-twitch EDL. Pre-treatment of muscle with bumetanide (NKCC inhibitor) or ouabain (Na+,K+-ATPase inhibitor), increased the initial volume loss and impaired the RVI response to increased extracellular osmolarity indicating that the NKCC is a primary contributor to volume regulation in skeletal muscle. It is concluded that mouse skeletal muscle initially loses volume then exhibits a RVI when exposed to increases in extracellular osmolarity. The rate of RVI is dependent on the degree of change in extracellular osmolarity, is muscle specific, and is dependent on the functioning of the NKCC and Na+, K+-ATPase.

Stimulation of regulatory volume increase (RVI) in avian articular chondrocytes by gadolinium chloride

Chondrocytes, the resident cell-type of articular cartilage, are responsible for the regulation of the extracellular matrix (ECM) in response to their physico-chemical environment. Due to the nature of cartilage loading, chondrocytes are exposed to constant changes in extracellular osmolality with a gradual increase throughout the day. As an increase in osmolality attenuates matrix synthesis, we have studied cell volume regulation (regulatory volume increase (RVI)) after hypertonic challenge and the regulation of RVI by the actin cytoskeleton. Using freshly isolated avian articular chondrocytes, changes in actin organisation were studied by confocal laser scanning microscopy following a 43% increase in extracellular osmolality. Using calcein-loading chondrocytes, the capacity for RVI was determined and the rate of volume recovery (t1/2) mathematically extrapolated. Following an increase in extracellular osmolality there was a significant increase (p < 0.05) in cortical actin, inhibited by the removal of extracellular calcium EGTA or by the addition of 100 micromol.L-1 gadolinium chloride. Most cells exhibited slow RVI (t1/2 = 55.5 +/- 5.5 min), whereby inhibition of actin polymerisation by gadolinium chloride or the removal of extracellular calcium significantly increased the rate of volume recovery via a bumetanide-sensitive pathway (t1/2 of 29.6 +/- 6.5 min and 13.8 +/- 3.1 min, respectively). These data suggest the Na+-K+-2Cl(-) (NKCC) co-transporter regulated by the actin cytoskeleton is involved in avian chondrocyte RVI.

Skeletal Muscle Regulatory Responses to Increased Extracellular Lactate

Mammalian skeletal muscle cells exhibit cell shrinkage followed by regulatory volume increase (RVI) in response to increases in extracellular osmolarity caused by addition of concentrated NaCl or sucrose. We hypothesized that increased extracellular sodium lactate would similarly result in a transient loss of cell volume, followed by RVI, and that the RVI would be more rapid and pronounced than that seen with NaCl due to entry of lactate into the cell. The present study measured the time course of change single fibre width in response to varied increases in extracellular NaCl or Na‐lactate. Adult mouse EDL single fibres were isolated using collagenase and incubated in DMEM prior to and during experimentation. Fibre images were obtained at 3–6 s intervals for up to 40 min. Fibre images were analyzed for width at 2 sites. Increased osmolarity resulted in a rapid decrease in fibre width, followed by RVI. The volume recovery was faster and more complete when [lactate] ≥20 mmol/L. There was a linear relationship between increases extracellular [lactate], cell shrinkage, and magnitude of RVI. It appears that the inward transport of lactate into skeletal muscle contributes, together with increased NKCC activity, to the RVI in mammalian skeletal muscle.Supported by NSERC of Canada.

How Yeast Responds to Change

The origin of the rapid adaptive response of yeast cells to changes in environmental osmolarity has been unclear. Mettetal et al. (see the Perspective by Lipan) now show that increases in extracellular osmolarity activate the high-osmolarity glycerol signaling pathway, which changes transcription of particular target genes. By measuring the cellular response to pulses of medium with increased ionic strength, the authors were able to develop a predictive model of the dynamics of this regulatory system. Rather than changes in gene expression, which have often been suggested to be at the core of the response to osmotic shock, the fast response is actually dominated by a nontranscriptional response that probably involves altered glycerol transport. J. T. Mettetal, D. Muzzey, C. Gómez-Uribe, A. van Oudenaarden, The frequency dependence of osmo-adaptation in Saccharomyces cerevisiae . Science 319 , 482-484 (2008). [Abstract] [Full Text] O. Lipan, Enlightening rhythms. Science 319 , 417-418 (2008). [Summary] [Full Text]

Suicidal Erythrocyte Death Following Cellular K+ Loss

Hallmarks of apoptosis include cell shrinkage, which is at least partially due to cellular K⁺ loss. The decline of cellular K⁺ concentration has been suggested to participate in the triggering of apoptosis. Suicidal erythrocyte death or eryptosis is triggered by increased cytosolic Ca²⁺ activity leading to activation of Ca²⁺-sensitive K⁺ channels with subsequent cellular K⁺ loss and cell shrinkage, and to Ca²⁺-sensitive scambling of the cell membrane with subsequent phosphatidylserine (PS) exposure at the cell surface. Phosphatidylserine exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. The present study explored whether cellular loss of K⁺ and/or cell shrinkage actively participate in the triggering of cell membrane phospholipid scrambling. Cellular K⁺ loss was achieved by treatment of human erythrocytes with the K⁺ ionophore valinomycin (1 nM) at different extracellular K⁺ concentrations (5-125 mM) and osmolarities (300-550m Osm). Cell volume was estimated from forward scatter and PS exposure from annexin V binding in FACS analysis. Treatment with 1 nM valinomycin indeed decreased forward scatter and increased annexin V binding. The effect was significantly blunted in the presence of staurosporine (1 µM). Increase of extracellular K⁺ concentration gradually blunted the decrease of forward scatter but inhibited annexin V binding only at extracellular K⁺ concentrations ??75 mM. An increase of extracellular osmolarity (+150 mM or 250 mM sucrose) reversed the protective effect of 75 mM KCl during valinomycin treatment. A correlation between forward scatter and annexin binding at different osmolarities and K⁺ concentrations suggests that the cellular K⁺ content determines the rate of suicidal erythrocyte death primarily through its influence on cell volume.

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

Hyperosmolarity Causes BKCa-Dependent Vasodilatations in Rat Skeletal Muscle Arteries

The release of different metabolites during skeletal muscle contraction causes a pronounced increase in extracellular osmolarity (hyperosmolarity (HO)). HO has been considered as a possible mediator of the exercise hyperemia. In the present study, we investigated the vasodilatory effect of physiologically relevant increases in the extracellular osmolarity in isolated rat gluteal muscle arterioles. In addition, we analyzed the underlying mechanisms of the HO-induced vasodilatations. Rat gluteal arteries were isolated and mounted in an organ bath for isometric tension recording. After precontraction with norepinephrine, 20, 40, or 60 mmol x L(-1) sucrose, mannitol, or urea was added in control conditions, after removal of the endothelium or in the presence of inhibitors. Application of sucrose or mannitol induced large and fast concentration-dependent vasodilatations (up to 46.15% with 60 mmol x L(-1) sucrose). Removal of the vascular endothelium had no effect on this relaxation. Inhibition of the Na+/K+ pumps with ouabain, the Kir IR channels with Ba2+ and the K ATP channels with glibenclamide did not alter the HO-induced relaxations. Incubation with the KCa channel blockers charybdotoxin and apamin significantly inhibited sucrose-induced vasodilatations. In addition, application of the specific BK Ca channel blocker iberiotoxin significantly decreased the HO-induced vasodilatations. The present study shows that an increase in the extracellular osmolarity elicits strong, fast, and long-lasting relaxations of rat skeletal muscle arterioles, suggesting an important role both at the onset and during the steady-state phase of an exercise bout. Vascular smooth muscle BK Ca channels seem to play a crucial role in the HO-induced vasorelaxations.

Volume sensitivity of the bestrophin family of chloride channels

Bestrophins are a newly identified family of Cl(-) channels. Mutations in the founding member of the family, human bestrophin-1 (hBest1), are responsible for a form of early onset macular degeneration called Best vitelliform macular dystrophy. The link between dysfunction of hBest1 and macular degeneration remains unknown. Because retinal pigmented epithelium (RPE) cells may be subjected to varying osmotic pressure due to light-dependent changes in the ionic composition of the subretinal space and because RPE cells may undergo large volume changes during phagocytosis of shed photoreceptor discs, we investigated whether bestrophin currents were affected by cell volume. When hBest1 and mBest2 were overexpressed in HEK 293, HeLa, and ARPE-19 cells, a new Ca(2+)-activated Cl(-) current appeared. This current was very sensitive to cell volume. A 20% increase in extracellular osmolarity caused cell shrinkage and a approximately 70-80% reduction in bestrophin current. Decreases in extracellular osmolarity increased the bestrophin currents slightly, but this was difficult to quantify due to simultaneous activation of endogenous volume-regulated anion channel (VRAC) current. To determine whether a similar current was present in mouse RPE cells, the effect of hyperosmotic solutions on isolated mouse RPE cells was examined. Mouse RPE cells exhibited an endogenous Cl(-) current that resembled the expressed hBest1 in that it was decreased by hypertonic solution. We conclude that bestrophins are volume sensitive and that they could play a novel role in cell volume regulation of RPE cells.