Cellular osmoregulation: beyond ion transport and cell volume

All cells are characterized by the expression of osmoregulatory mechanisms, although the degree of this expression is highly variable in different cell types even within a single organism. Cellular osmoregulatory mechanisms constitute a conserved set of adaptations that offset antagonistic effects of altered extracellular osmolality/environmental salinity on cell integrity and function. Cellular osmoregulation includes the regulation of cell volume and ion transport but it does not stop there. We know that organic osmolyte concentration, protein structure, cell turnover, and other cellular parameters are osmoregulated as well. In this brief review two important aspects of cellular osmoregulation are emphasized: 1) maintenance of genomic integrity, and 2) the central role of protein phosphorylation. Novel insight into these two aspects of cellular osmoregulation is illustrated based on two cell models, mammalian kidney inner medullary cells and teleost gill epithelial cells. Both cell types are highly hypertonicity stress-resistant and, therefore, well suited for the investigation of osmoregulatory mechanisms. Damage to the genome is discussed as a newly discovered aspect of hypertonic threat to cells and recent insights on how mammalian kidney cells deal with such threat are presented. Furthermore, the importance of protein phosphorylation as a core mechanism of osmosensory signal transduction is emphasized. In this regard, the potential roles of the 14-3-3 family of phospho-protein adaptor molecules for cellular osmoregulation are highlighted primarily based on work with fish gill epithelial cells. These examples were chosen for the reader to appreciate the numerous and highly specific interactions between stressor-specific and non-specific pathways that form an extensive cellular signaling network giving rise to adaptive compensation of hypertonicity. Furthermore, the example of 14-3-3 proteins illustrates that a single protein may participate in several pathways that are non-specific with regard to the type of stress and, at the same time, in stress-specific pathways to promote cell integrity and function during hypertonicity.     

Responses of respiration and photosynthesis of Scenedesmus protuberans Fritsch to gradual and steep salinity increases

The effect of an increase in salinity on the physiology of thehalotolerant chlorophyte Scenedesmus protuberans was studiedin light-limited continuous cultures. It was observed that agradual, as well as a steep increase in salinity resulted inlower biomass. However, the mechanisms by which this was achievedwere different. In the culture that was exposed to a gradualsalinity increase, respiration and the cellular protein contentof the culture were initially unaffected. However, this culturewas not able to maintain its cellular chlorophyll content and,consequently, gross and net photosynthesis decreased. The culturethat was exposed to a steep salinity increase rapidly reactedby increasing its respiration and cellular protein content,which is ascribed to an induction of osmoregulation. This culturewas able to maintain its gross photosynthesis rate. It is speculatedthat, in this species, a steep salinity increase induces a nearlyimmediate osmoregulatory response, allowing growth to continue.If the cells are exposed to a gradual salinity increase, inductionof osmoregulation lags behind and, consequently, photosynthesisand growth rate will be^* affected.     

The physical state of osmoregulatory solutes in unicellular algae. A natural-abundance carbon-13 nuclear-magnetic-resonance relaxation study.

Natural-abundance 13C n.m.r. spin-lattice relaxation-time measurements have been carried out on intact cells of the unicellular blue--green alga Synechococcus sp. and the unicellular green alga Dunaliella salina, with the aim of characterizing the environments of the organic osmoregulatory solutes in these salt-tolerant organisms. In Synechococcus sp., all of the major organic osmoregulatory solute, 2-O-alpha-D-glucopyranosylglycerol, is visible in spectra of intact cells. Its rotational motion in the cell is slower by a factor of approx. 2.4 than in aqueous solution, but the molecule is still freely mobile and therefore able to contribute to the osmotic balance. In D. salina, only about 60% of the osmoregulatory solute glycerol is visible in spectra of intact cells. The rotational mobility of this observable fraction is approximately half that found in aqueous solution, but the data also indicate that there is a significant concentration of some paramagnetic species in D. salina which contributes to the overall spin-lattice relaxation of the glycerol carbon atoms. The non-observable fraction, which must correspond to glycerol molecules that have very broad 13C resonances and that are in slow exchange with bulk glycerol, has not been properly characterized as yet, but may represent glycerol in the chloroplast. The implications of these findings in relation to the physical state of the cytoplasm and the mechanism of osmoregulation in these cells are discussed.     

Identification and characterization of an ATP binding cassette L-carnitine transporter in Listeria monocytogenes

We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated proteins presumed to form the permease (OpuCB and OpuCD). The operon is preceded by a potential SigB-dependent promoter. An opuC-defective mutant was generated by the insertional inactivation of the opuCA gene. The mutant was impaired for growth at high osmolarity in brain heart infusion broth and failed to grow in a defined medium. Supplementation of the defined medium with peptone restored the growth of the mutant in this medium. The mutant was found to accumulate the compatible solutes glycine betaine and choline to same extent as the parent strain but was defective in the uptake of L-carnitine. We conclude that the opuC operon in L. monocytogenes encodes an ABC compatible solute transporter which is capable of transporting L-carnitine and which plays an important role in osmoregulation in this pathogen.     

Mechanisms of response to salinity in halotolerant microalgae

Summary A limited number of organic solutes are used by microalgae to adjust their internal osmotic pressure in response to changing external salinities. Glycerol and proline are used by the most extremely halotolerant algae. Only glycerol allows growth at salinities approaching saturation. In addition to organic osmoregulatory solutes, inorganic ions also play an important role in osmoregulation. The ability of microalgae to maintain intracellular ions at levels compatible with metabolic functions may set upper limits for their salt tolerance. Requirements for NaCl in the external medium for nutrient transport may define the lower salinity limits for growth observed for some euryhaline algae.Osmotic upshocks generally cause severe temporary inhibition of photosynthesis in euryhaline microalgae. Extensive osmotic downshocks have little effect on photosynthesis in microalgae with strong cell walls, while wall-less species appear to be more sensitive. Rapid glycerol synthesis takes place in response to increased external salinity inChlamydomonas pulsatilla both in light and dark. Starch supplies carbon for glycerol synthesis in the dark and also during the initial periods of inhibition of photosynthesis in the light. Turnover of osmoregulatory solutes such as glycerol and isofloridoside may be an important aspect of the osmoregulatory mechanism.At salinities beyond the growth limit for the green flagellateChlamydomonas pulsatilla, resting spores are formed that enable this alga to survive extreme salinities.     

Osmoregulatory capacity of the Cladocera

Within the order Cladocera are found almost all varieties of osmotic regulation, which make it possible for them to live in waters of a wide range of salt concentrations. Many Cladocera are very powerful osmoregulators and are comparable to the teleosts and decapod crustaceans in their abilities. The variety of osmoregulatory capacities within the cladocerans are illustrated and discussed. The function of mitochondrion-rich ion transporting cells found in nuchal glands or on epipodites are diseussed and the physiological mechanisms involved in osmoregulation are compared with similar mechanisms in other crustaceans and in teleosts. Data is provided on osinotic regulation in eggs and embryos in open and elosed brood chambers. Other topics include the occurrence of physiological races in some species, recent changes in osmoregulatory abilities following man-induced changes in salinity, the effects of temperature on osmoregulation and the effects of pH.     

Energetics of osmoregulation: II. Water flux and osmoregulatory work in the euryhaline fish, Fundulus heteroclitus

Teleost fish experience passive osmotic water influx in fresh water (FW) and water outflux in salt water, which is normally compensated by water flow driven by active ion transport mechanisms. Euryhaline fish may also minimize osmotic energy demand by "behavioral osmoregulation", seeking a medium isotonic with their body fluids. Our goal was to evaluate the energy requirement for osmoregulation by the euryhaline fish Fundulus heteroclitus, to determine whether it is of sufficient magnitude to favor behavioral osmoregulation. We have developed a method of weighing small fish repetitively for long periods without apparent damage, which was used to assess changes in water content following changes in external salinity. We found that cold (4 degrees C) inhibits osmoregulatory active transport mechanisms in fish acclimated to warmer temperatures, leading to a net passive water flux which is reversed by rewarming the fish. A sudden change of salinity at room temperature triggers a transient change in water content and the initial slope can be used to measure the minimum passive flux at that temperature. With some reasonable assumptions as to the stoichiometry of the ion transport and ATP-generating processes, we can calculate the amount of respiration required for ion transport and compare it to the oxygen uptake measured previously under the same conditions. We conclude that osmoregulation in sea water requires from 6% to 10% of the total energy budget in sea water, with smaller percentages in FW, and that this fraction is probably sufficient to be a significant selective driving force favoring behavioral osmoregulation under some circumstances.     

盐分和水分胁迫对芦荟幼苗渗透调节和渗调物质积累的影响

用不同浓度NaCl和等渗聚乙二醇(PEG 6000)处理芦荟(Aloe vera L.)幼苗,10 d后测定叶片相对生长速率和厚度、叶片中主要有机溶质、无机离子含量及渗透调节能力.结果表明,-0.44、-0.88 MPa NaCl和PEG处理使芦荟叶片的相对生长速率和叶片厚度明显下降,且盐胁迫对幼苗生长的抑制和叶片含水量降低的效应明显高于等渗的水分胁迫,其叶片渗透调节能力随处理渗透势的降低而增加, -0.88 MPa PEG胁迫的芦荟幼苗的渗透调节能力高于等渗盐分胁迫.在主要渗透调节物质可溶性糖、有机酸、K 、Ca2 和Cl-中,-0.88 MPa PEG处理下含量比相同渗透势的NaCl处理下显著增加的是有机溶质,因此推断有机溶质含量高是PEG胁迫下渗透调节能力较强的主要因素.     

Cyclic di‐AMP targets the cystathionine beta‐synthase domain of the osmolyte transporter OpuC

Cellular turgor is of fundamental importance to bacterial growth and survival. Changes in external osmolarity as a consequence of fluctuating environmental conditions and colonization of diverse environments can significantly impact cytoplasmic water content, resulting in cellular lysis or plasmolysis. To ensure maintenance of appropriate cellular turgor, bacteria import ions and small organic osmolytes, deemed compatible solutes, to equilibrate cytoplasmic osmolarity with the extracellular environment. Here, we show that elevated levels of c‐di‐AMP, a ubiquitous second messenger among bacteria, result in significant susceptibility to elevated osmotic stress in the bacterial pathogen Listeria monocytogenes. We found that levels of import of the compatible solute carnitine show an inverse correlation with intracellular c‐di‐AMP content and that c‐di‐AMP directly binds to the CBS domain of the ATPase subunit of the carnitine importer OpuC. Biochemical and structural studies identify conserved residues required for this interaction and transport activity in bacterial cells. Overall, these studies reveal a role for c‐di‐AMP mediated regulation of compatible solute import and provide new insight into the molecular mechanisms by which this essential second messenger impacts bacterial physiology and adaptation to changing environmental conditions.     

Osmoadaptation of haloalkaliphilic bacteria: Role of osmoregulators and their possible practical application

The review discusses osmoadaptation of haloalkaliphilic bacteria from diverse taxonomic and physiological groups, inhabiting soda lakes. Our experimental research has confirmed the similarity of the osmoregulation strategies in neutrophilic and alkaliphilic halophiles, independent of their pH homeostasis mechanism. The external osmotic pressure is equilibrated either due to accumulation of ions from the environment, or by accumulation or synthesis of cytoplasmic osmoregulatory compounds. The alkaliphiles following the “compatible solutes” strategy contain low or moderate concentrations of salts in their cytoplasm; their proteins do not require adaptation to salts. Those that follow the “salt-in” strategy do not synthesize osmoregulators; they accumulate high levels of salts within the cell and thus equilibrate the osmotic pressures of the cell and the environment. The proteins of these bacteria contain more acidic amino acid residues compared to the proteins of neutrophiles. The functions of bacterial organic osmoregulatory compounds are discussed, as well as their characteristics of possible practical value. Applications for ectoine and betaine are discussed based on the published data.     

The unusually strong stabilizing effects of glycine betaine on the structure and function of the oxygen-evolving Photosystem II complex

Natural osmoregulatory substances (osmolytes) allow a wide variety of organisms to adjust to environments with high salt and/or low water content. In addition to their role in osmoregulation, some osmolytes protect proteins from denaturation and deactivation by, for example, elevated temperature and chaotropic compounds. A ubiquitous protein-stabilizing osmolyte is glycine betaine (N-trimethyl glycine). Its presence has been reported in bacteria, in particular cyanobacteria, in animals and in plants from higher plants to algae. In the present review we describe the experimental evidence related to the ability of glycine betaine to enhance and stabilize the oxygen-evolving activity of the Photosystem II protein complexes of higher plants and cyanobacteria. The osmolyte protects the Photosystem II complex against dissociation of the regulatory extrinsic proteins (the 18 kD, 23 kD and 33 kD proteins of higher plants and the 9 kD protein of cyanobacteria) from the intrinsic components of the Photosystem II complex, and it also stabilizes the coordination of the Mn cluster to the protein cleft. By contrast, glycine betaine has no stabilizing effect on partial photosynthetic processes that do not involve the oxygen-evolving site of the Photosystem II complex. It is suggested that glycine betaine might act, in part, as a solute that is excluded from charged surface domains of proteins and also as a contact solute at hydrophobic surface domains.     

Ontogeny of osmoregulation in postembryonic fish: a review

Salinity and its variations are among the key factors that affect survival, metabolism and distribution during the fish development. The successful establishment of a fish species in a given habitat depends on the ability of each developmental stage to cope with salinity through osmoregulation. It is well established that adult teleosts maintain their blood osmolality close to 300 mosM kg(-1) due to ion and water regulation effected at several sites: tegument, gut, branchial chambers, urinary organs. But fewer data are available in developing fish. We propose a review on the ontogeny of osmoregulation based on studies conducted in different species. Most teleost prelarvae are able to osmoregulate at hatch, and their ability increases in later stages. Before the occurrence of gills, the prelarval tegument where a high density of ionocytes (displaying high contents of Na+/K+-ATPase) is located appears temporarily as the main osmoregulatory site. Gills develop gradually during the prelarval stage along with the numerous ionocytes they support. The tegument and gill Na+/K+-ATPase activity varies ontogenetically. During the larval phase, the osmoregulatory function shifts from the skin to the gills, which become the main osmoregulatory site. The drinking rate normalized to body weight tends to decrease throughout development. The kidney and urinary bladder develop progressively during ontogeny and the capacity to produce hypotonic urine at low salinity increases accordingly. The development of the osmoregulatory functions is hormonally controlled. These events are inter-related and are correlated with changes in salinity tolerance, which often increases markedly at the metamorphic transition from larva to juvenile. In summary, the ability of ontogenetical stages of fish to tolerate salinity through osmoregulation relies on integumental ionocytes, then digestive tract development and drinking rate, developing branchial chambers and urinary organs. The physiological changes leading to variations in salinity tolerance are one of the main basis of the ontogenetical migrations or movements between habitats of different salinity regimes.     

Osmoadaptation of haloalkaliphilic bacteria: role of osmoregulators and their possible practical application

The review discusses osmoadaptation of haloalkaliphilic bacteria from diverse taxonomic and physiological groups, inhabiting soda lakes. Our experimental research has confirmed the similarity of the osmoregulation strategies in neutrophilic and alkaliphilic halophiles, independent of their pH homeostasis mechanism. The external osmotic pressure is equilibrated either due to accumulation of ions from the environment, or by accumulation or synthesis of cytoplasmic osmoregulatory compounds. The alkaliphiles following the "compatible solutes" strategy contain low or moderate concentrations of salts in their cytoplasm; their proteins do not require adaptation to salts. Those that follow the "salt-in" strategy do not synthesize osmoregulators: they accumulate high levels of salts within the cell and thus equilibrate the osmotic pressures of the cell and the environment. The proteins of these bacteria contain more acidic amino acid residues compared to the proteins of neutrophiles. The functions of bacterial organic osmoregulatory compounds are discussed, as well as their characteristics of possible practical value. Applications for ectoine and betaine are discussed based on the published data.     

Metamorphosis and early larval development of the flatfishes (Pleuronectiformes): an osmoregulatory perspective

Flatfish (Pleuronectiformes) distribution in the environment is influenced by salinity, and varies among species and with developmental stage. Osmoregulatory ability likely plays an important role in defining species and developmental stage-specific distribution. Although the mechanisms of osmoregulation in adult and juvenile teleosts have been widely addressed, far less is known about their larval osmoregulatory physiology. Much of our current understanding of larval fish ion-regulation stems from studies using flatfishes, and this article reviews advances in this field, primarily from the point of view of the developing flatfishes. Addressed here are the ontogeny of salinity tolerance, the development of several important osmoregulatory tissues (the skin, gut, and gill), and the influence of the endocrine system on osmoregulation during early larval development and metamorphosis.     

Coincident detection of CSF Na+ and osmotic pressure in osmoregulatory neurons of the supraoptic nucleus

Behavioral and neuroendocrine responses underlying systemic osmoregulation are under the concerted control of centrally located osmoreceptors and cerebrospinal fluid (CSF) Na+ concentration ([Na+]) detectors. Although the process underlying osmoreception is understood, the mechanism by which [Na+] is detected and integrated with cellular information derived from osmoreceptors is unknown. Here, we show that shifts in extracellular [Na+] ([Na+]0) cause proportional changes in the relative Na+ permeability of mechanosensitive cation channels responsible for signal transduction in the osmosensory neurons of the supraoptic nucleus. This effect causes the generation of Na+ specific receptor potentials under isotonic conditions and modulates osmoreceptor potentials and electrical responsiveness during osmotic perturbation. These results provide a cellular basis for Na+-sensing and for the coordinated detection of CSF [Na+] and osmolality in central osmoregulatory neurons.     

Gluconeotrehalose is the principal organic solute in the psychrotolerant bacterium <Emphasis Type="Italic">Carnobacterium</Emphasis> strain 17-4

A high proportion of microorganisms that colonise cold environments originate from marine sites; hence, they must combine adaptation to low temperature with osmoregulation. However, little or nothing is known about the nature of compatible solutes used by cold-adapted organisms to balance the osmotic pressure of the external medium. We studied the intracellular accumulation of small organic solutes in the Arctic isolate Carnobacterium strain 17-4 as a function of the growth temperature and the NaCl concentration in the medium. Data on 16S rDNA sequence and DNA–DNA hybridisation tests corroborate the assignment of this isolate as a new species of the bacterial genus Carnobacterium. The growth profiles displayed maximal specific growth rate at 30°C in medium without NaCl, and maximal values of final biomass at growth temperatures between 10 and 20°C. Therefore, Carnobacterium strain 17-4 exhibits halotolerant and psychrotolerant behaviours. The solute pool contained glycine-betaine, the main solute used for osmoregulation, and an unknown compound whose structure was identified as α-glucopyranosyl-(1-3)-β-glucopyranosyl-(1-1)-α-glucopyranose (abbreviated as gluconeotrehalose), using nuclear magnetic resonance and mass spectrometry. This unusual solute consistently accumulated to high levels (0.35 ± 0.05 mg/mg cell protein) regardless of the growth temperature or salinity. The efficiency of gluconeotrehalose in the stabilisation of four model enzymes against heat damage was also assessed, and the effects were highly protein dependent. The lack of variation in the gluconeotrehalose content observed under heat stress, osmotic stress, and starvation provides no clue for the physiological role of this rare solute.     

Effects of salinity on growth and content of intracellular solutes in Heterococcus sp. (Tribonennatales, Xanthophyceae) from Antarctica

The growth and osmoregulation of Heterococcus sp. isolated from dry soil collected in Antarctica were investigated in relation to salinity. This alga was able to grow over a wide range of saline conditions from 0 to 0.8 mol L^-1 NaCI, although growth was suppressed in proportion to the increase in extracellular concentration of NaCI. This indicates that the alga is halotolerant. Using gas-liquid chromatography-mass spectroscopy, it was found to contain mannitol as an osmoticum. The intracellular content of mannitol increased with increase in concentration of extracellular NaCI, suggesting that mannitol acts as an osmoregulatory substance.     

Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes

Microorganisms respond to osmotic stress mostly by accumulating compatible solutes, either by uptake from the medium or by de novo synthesis. These osmotically active molecules preserve the positive turgor pressure required for cell division. The diversity of compatible solutes is large but falls into a few major chemical categories; they are usually small organic molecules such as amino acids or their derivatives, and carbohydrates or their derivatives. Some are widely distributed in nature while others seem to be exclusively present in specific groups of organisms. This review discusses the diversity and distribution of known classes of compatible solutes found in prokaryotes as well as the increasing knowledge of the genes and pathways involved in their synthesis. The alternative roles of some archetypal compatible solutes not subject to osmoregulatory constraints are also discussed.