Extracellular NaCl Concentration Research Articles

Multi-barrelled ion-selective microelectrodes as tools for the investigation of volume regulation mechanisms in invertebrate nerve cells under hyperosmotic conditions

Changes in cell volume can be recorded by a potentiometric method based on ion-selective microelectrodes filled with the classical K + exchanger Corning 477317, which is very sensitive to large quaternary ammonium ions (PNAS 82 (1985) 6014; Electrochim. Acta 47 (2001) 309). We investigated the effect of hyperosmotic bathing solutions on cell volume, the intracellular concentrations of Na +, Cl −, and K + ([Na +] i, [Cl −] i, [K +] i) as well as on the membrane potential of leech Retzius neurones by using multi-barrelled ion-selective microelectrodes. We found that an increase in the extracellular NaCl concentration induced a shrinkage of the cells as well as an increase in [Na +] i, [Cl −] i and [K +] i. Provided that the change in osmolality was moderate (≤+40%), cell shrinkage and the changes in [Na +] i, [Cl −] i and [K +] i were proportional to the increase in the extracellular osmolality, i.e. the cells behaved like ideal osmometers. When the bath osmolality was increased more strongly, significant differences from the ideal behaviour were observed. In particular, the changes in [Na +] i and [Cl −] i were larger than expected from the changes in cell volume. The results show that leech neurones raise their intracellular osmolyte content in response to severe osmotic stress, thereby reducing changes in their cellular volume.

Expression and regulation of the Na +/K +/2Cl − cotransporter NKCC1 in rat liver and human HuH-7 hepatoma cells

The expression of sodium potassium chloride cotransporter 1 (NKCC1) was studied in different liver cell types. NKCC1 was found in rat liver parenchymal and sinusoidal endothelial cells and in human HuH-7 hepatoma cells. NKCC1 expression in rat hepatic stellate cells increased during culture-induced transformation in the myofibroblast-like phenotype. NKCC1 inhibition by bumetanide increased α 1-smooth muscle actin expression in 2-day-cultured hepatic stellate cells but was without effect on basal and platelet-derived-growth-factor-induced proliferation of the 14-day-old cells. In perfused rat liver the NKCC1 made a major contribution to volume-regulatory K + uptake induced by hyperosmolarity. Long-term hyperosmotic treatment of HuH-7 cells by elevation of extracellular NaCl or raffinose concentration but not hyperosmotic urea or mannitol profoundly induced NKCC1 mRNA and protein expression. This was antagonized by the compatible organic osmolytes betaine or taurine. The data suggest a role of NKCC1 in stellate cell transformation, hepatic volume regulation, and long-term adaption to dehydrating conditions.

Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration.

Hypertonicity-induced binding of the transcription factor TonEBP/OREBP to its cognate DNA element, ORE/TonE, is associated with increased transcription of several osmotically regulated genes. Previously, it was found that hypertonicity rapidly causes nuclear translocation and phosphorylation of TonEBP/OREBP and, more slowly, increases TonEBP/OREBP abundance. Also, the C terminus of TonEBP/OREBP was found to contain a transactivation domain (TAD). We have now tested for tonicity dependence of the TAD activity of the 983 C-terminal amino acids of TonEBP/OREBP. HepG2 cells were cotransfected with a reporter construct and one of several TAD expression vector constructs. The reporter construct contained GAL4 DNA binding elements, a minimal promoter, and the Photinus luciferase gene. TAD expression vectors generate chimeras comprised of the GAL4 DNA binding domain fused to (i) the 983 C-terminal amino acids of TonEBP/OREBP, (ii) 17 glutamine residues, (iii) the TAD of c-Jun, or (iv) no TAD. All TAD-containing chimeras were functional at normal extracellular osmolality (300 mosmol/kg), but the activity only of the chimera containing the 983 C-terminal amino acids of TonEBP/OREBP varied with extracellular NaCl concentration, decreasing by >80% at 200 mosmol/kg and increasing 8-fold at 500 mosmol/kg. The chimera containing the 983 C-terminal amino acids of TonEBP/OREBP was constitutively localized to the nucleus and showed tonicity-dependent posttranslational modification consistent with phosphorylation. The activity at 500 mosmol/kg was reduced by herbimycin, a tyrosine kinase inhibitor and by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a protein kinase CK2 inhibitor. Thus, the 983 C-terminal amino acids of TonEBP/OREBP contain a TAD that is regulated osmotically, apparently by tonicity-dependent phosphorylation.

Regulated overexpression of heat shock protein 72 protects Madin-Darby canine kidney cells from the detrimental effects of high urea concentrations.

Exposure of renal medullary cells to elevated extracellular NaCl concentrations is associated with increased heat shock protein 72 (HSP72) expression and improved resistance to subsequent exposure to a high urea concentration (600 mM). To establish a causal relationship between HSP72 expression and protection against high urea concentrations, HSP72 was inducibly overexpressed in Madin-Darby canine kidney (MDCK) cells, in the absence of hypertonic stress before urea exposure. For this purpose, the human stress-inducible HSP72 gene was cloned downstream from a dexamethasone (DEX)-inducible promoter in the eukaryotic expression vector pLKneo. This construct allowed robust induction of HSP72 by exposure of stably transfected MDCK cells (MDCK-LK72) to 0.1 microM DEX. Increased HSP72 abundance significantly improved survival rates after 24-h exposure of the cells to medium containing 600 mM urea (14 versus 43%). In mock-transfected or wild-type cells, DEX had no significant effect on HSP72 abundance or urea resistance. In accordance with those findings, lactate dehydrogenase activity in the supernatant was significantly reduced, compared with appropriate control samples, only in MDCK-LK72 cells overexpressing HSP72. Labeling with annexin V-FITC and propidium iodide, followed by flow cytometry, revealed that overexpression of HSP72 was associated with a reduction in the number of apoptotic-lysed cells, a concomitant retardation of apoptosis, and an increase in the number of viable cells. These data support the view that HSP72, which is very abundant in the renal inner medulla, is an important component of the defense mechanism of medullary cells against extreme concentrations of urea.

A novel low-affinity H+-Cl- co-transporter in yeast: characterization by patch clamp

We have used the patch clamp technique to characterize a novel low affinity H+ coupled Cl- transporter in the plasma membrane of Saccharomyces cerevisiae. Cl- -induced inward currents were observed upon the addition of millimolar concentrations of extracellular Cl-. These currents were independent of the cation species present but were dependent on the membrane voltage and the extracellular pH such that Cl--induced inward currents were reduced as pH was increased from 5.5 to 8.0. These data are consistent with an electrogenic co-transport of nH+ and Cl- where n > 1. In accord with the results from patch clamp experiments, Cl- uptake into intact yeast cells was pH-dependent with an apparent Km of 93 mM. Furthermore, Cl- uptake was up-regulated by pre-incubation in elevated extracellular NaCl concentrations, but was insensitive to a pre-incubation in sorbitol. A possible role for the nH+/Cl- symport in equimolar osmoregulation during salt stress is discussed.

H-sgk Serine-Threonine Protein Kinase as Transcriptional Target of p38/MAP Kinase pathway in HepG2 Human Hepatoma Cells

The human serum and glucocorticoid dependent serine/threonine kinase h-sgk has previously been discovered as cell volume regulated gene. The present study has been performed to elucidate the involvement of p38-kinase in the transcriptional control of h-sgk by osmotic cell shrinkage. The p38-kinase has previously been cloned as the mammalian homologue of HOG1 kinase, which constitutes a part of the osmosensor in the yeast Saccharomyces cerevisiae. Phosphorylated (active) p38-kinase has been estimated with Western blotting, transcription of hsgk using Northern blotting. Both, increase of extracellular NaCl concentration by 50 mmol/l and addition of 10 μmol/l anisomycin increase phosphorylation of the p38-kinase within 5 to 10 minutes. h-sgk transcription is upregulated by addition of 50 mmol/l NaCl and by anisomycin (10 μmol/l), effects completely inhibited by the specific p38-kinase inhibitor, SB 203580 (10 μmol/l). In conclusion, the stimulation of h-sgk transcription by osmotic cell shrinkage is mediated by p38-kinase.

Two mechanisms that raise free intracellular calcium in rat hippocampal neurons during hypoosmotic and low NaCl treatment.

Previous studies have shown that exposing hippocampal slices to low osmolarity (pi(o)) or to low extracellular NaCl concentration ([NaCl](o)) enhances synaptic transmission and also causes interstitial calcium ([Ca(2+)](o)) to decrease. Reduction of [Ca(2+)](o) suggests cellular uptake and could explain the potentiation of synaptic transmission. We measured intracellular calcium activity ([Ca(2+)](i)) using fluorescent indicator dyes. In CA1 hippocampal pyramidal neurons in tissue slices, lowering pi(o) by approximately 70 mOsm caused "resting" [Ca(2+)](i) as well as synaptically or directly stimulated transient increases of calcium activity (Delta[Ca(2+)](i)) to transiently decrease and then to increase. In dissociated cells, lowering pi(o) by approximately 70 mOsm caused [Ca(2+)](i) to almost double on average from 83 to 155 nM. The increase of [Ca(2+)](i) was not significantly correlated with hypotonic cell swelling. Isoosmotic (mannitol- or sucrose-substituted) lowering of [NaCl](o), which did not cause cell swelling, also raised [Ca(2+)](i). Substituting NaCl with choline-Cl or Na-methyl-sulfate did not affect [Ca(2+)](i). In neurons bathed in calcium-free medium, lowering pi(o) caused a milder increase of [Ca(2+)](i), which was correlated with cell swelling, but in the absence of external Ca(2+), isotonic lowering of [NaCl](o) triggered only a brief, transient response. We conclude that decrease of extracellular ionic strength (i.e., in both low pi(o) and low [NaCl](o)) causes a net influx of Ca(2+) from the extracellular medium whereas cell swelling, or the increase in membrane tension, is a signal for the release of Ca(2+) from intracellular stores.

Inhibition of NaCl-induced heat shock protein 72 expression renders MDCK cells susceptible to high urea concentrations.

Exposure of Madin-Darby canine kidney (MDCK) cells to elevated extracellular NaCl concentrations is associated with increased heat shock protein 72 (HSP72) expression and improved survival of these pretreated cells upon exposure to an additional 600 mM urea in the medium. To establish a causal relationship between HSP72 expression and cell protection against high urea concentrations, two approaches to inhibit NaCl-induced HSP72 synthesis prior to exposure to 600 mM urea were employed. First, the highly specific p38 kinase inhibitor SB203580 was added (100 microM) to the hypertonic medium (600 mosm/kg H2O by NaCl addition, 2 days of exposure), which significantly reduced HSP72 mRNA abundance and HSP72 content. Survival of these cells after a 24-h urea treatment (600 mM) was markedly curtailed compared with appropriate controls. Second, a pcDNA3-based construct, containing 322 bases of the HSP72 open reading frame in antisense orientation and the geneticine resistance gene, was transfected into MDCK cells. Clones with strong inhibition of HSP72 synthesis and others which express the protein at normal levels (comparable to nontransfected MDCK cells) after heat shock treatment or hypertonic stress were established. When these transformants were subjected to hypertonic stress for 2 days prior to exposure to an additional 600 mM urea for 24 h, cell survival was significantly reduced in those clones in which HSP72 expression was strongly inhibited. These results provide further evidence for the protective function of HSP72 against high urea concentrations in renal epithelial cells.

Biophysical Characteristics of Swelling-Activated Cl − Channels in Human Tracheal 9HTEo-Cells

The question of whether a single molecule can account for every observed swelling-activated Cl- current deserves to be addressed and biophysical description seems to be an adequate criterion to classify these channels. We studied the biophysical properties of swelling-activated Cl- currents in 9HTEo-cells using whole-cell and outside-out patch clamp recordings. Hypotonic shock activated outwardly rectifying currents that inactivated at potentials higher than 20 mV. The decay phase of the current was well fitted by two exponential functions and both time constants were voltage-dependent. Two voltage-dependent time constants were also necessary to describe reactivation. The midpoint of current inactivation was 54 mV. The voltage dependence of kinetics did not significantly change by modifying the extracellular NaCl concentration while the inactivation midpoint slightly shifted. In conclusion, our results indicate that the voltage-dependent properties of the swelling-activated Cl- currents in 9HTEo- cells are largely independent from the extracellular ionic strength and the extracellular Cl- concentration. Excised patches from cells exposed to hypotonic shock showed single channel currents that inactivated at positive membrane potentials and displayed chord conductance of approximately 60 pS at 100 mV and of approximately 20 pS at -80 mV. The permeability sequence for the single channel was I- > Br- > Cl- > gluconate and currents were blocked by Reactive blue 2. These properties indicate that intermediate conductance outwardly rectifying channels are responsible for the macroscopic swelling-activated current.

Xanthine affects [Ca2+]i and contractile responses of ventricular cardiocytes to electrical stimulation.

Xanthine, a major purine by-product of ATP, accumulates during myocardial ischemia. In the present study, we show that xanthine (0.5-1 mM) impaired the occurrence of cytosolic Ca2+ concentration ([Ca2+]i) transients, visualized in fura 2-loaded cells, and twitches of contraction in ventricular cardiocytes in response to electrical stimulation. This effect of xanthine was independent of superoxide anion production. That it was a result of decreased membrane excitability was supported by the following: 1) it was reversed by increasing either the amplitude of the stimulus voltage required to stimulate cardiocytes or the extracellular concentration of NaCl; and 2) xanthine reversed the depolarization following electrical stimulation in cardiocytes loaded with the voltage-sensitive dye bis-oxonol. P2 purinergic-agonists, including ATP (10 microM), but not P1 purinergic agonists reproduced the effects seen with xanthine. In addition, a lack of additivity between xanthine and ATP at maximal concentrations was observed. We conclude that xanthine, through activation of a P2 purinoceptor, may contribute to myocardial arrhythmia occurring during ischemia-reperfusion injury.

Syk-dependent and -independent Signaling Cascades in B Cells Elicited by Osmotic and Oxidative Stress

It was found that Syk protein-tyrosine kinase is rapidly activated in B cells after H2O2 treatment (oxidative stress) or increased extracellular NaCl concentration (osmotic stress) as well as in response to B cell receptor activation. In this study we examined the involvement of Syk in responses elicited by these types of extracellular stress, particularly Ca2+ responses and c-Jun amino-terminal kinase (JNK) activation, using a chicken B cell line, DT40, as well as the DT40-derived mutant DT40/Syk(-), which does not express Syk. Osmotic stress evokes increases in [Ca2+]i by stimulating an extracellular Ca2+ influx in both DT40 and DT40/Syk(-) cells. In comparison, oxidative stress elicits an increase in [Ca2+]i by stimulating both an extracellular Ca2+ influx and Ca2+ release from internal stores in DT40 cells, but this Ca2+ response is partially abolished in DT40/Syk(-) cells, indicating that the oxidative stress-induced Ca2+ response is at least partly dependent on Syk. Interestingly, the depletion of Ca2+ results in a significantly decreased level of Syk activation in DT40 cells stimulated by oxidative but not osmotic stress. Furthermore, JNK is activated to different extents by these two types of stress. The extent of JNK activation in DT40/Syk(-) cells in response to osmotic stress is comparable to that observed in DT40 cells. Intriguingly, oxidative stress-induced JNK activation is significantly compromised in DT40/Syk(-) cells. Collectively, these results indicate that both the Ca2+ response and JNK activity induced by oxidative stress are partly dependent on Syk, whereas those induced by osmotic stress are independent of Syk.

Peanut rhizobia under salt stress: role of trehalose accumulation in strain ATCC 51466

Strain ATCC 51466, a motile peanut Rhizobium sp., showed patterns of utilization of diverse carbon sources characteristic of fast growers. Bacteria had periplasmic neutral glucans with molecular weight close to 3000. When the extracellular concentration of NaCl was raised to 400 mM, the lag phase of the culture was prolonged about threefold and the generation time was increased almost twice. The changes in growth behavior of salt-stressed bacteria were accompanied by the full suppression of periplasmic oligoglucans and the accumulation of cellular trehalose. Almost identical changes in cell-associated oligoglucans were observed after exposing peanut Rhizobium sp. strain ATCC 10317 to hypersalinity. When the osmotic pressure of the medium was augmented by the addition of either 200 mM mannitol or 16% (w/v) polyethylene glycol, cells of strain ATCC 51466 contained decreased levels of oligoglucans and accumulated trehalose. On the other hand, the content of cellular trehalose increased throughout logarithmic and stationary phases of growth of strain ATCC 51466 in a medium supplemented with 400 mM NaCl. When bacterial cultures were shifted from hypersaline to basal media, oligoglucans were the only oligosaccharides detected. The addition of 10 mM proline to bacteria grown under hypersalinity led to a 50% decrease in the level of trehalose and to the accumulation of oligoglucans. The addition of 10 mM glycine betaine to bacteria grown under hypersalinity also produced accumulation of oligoglucans, but the level of trehalose did not decrease. The results presented here are consistent with a role for trehalose as a compatible solute in peanut Rhizobium ATCC 51466, and they suggest that exogenously added proline may act as a compatible solute in preference to trehalose.Key words: periplasmic glucans, trehalose, peanut Rhizobium, osmotic stress.

23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli.

23Na NMR spectroscopy was used to determine free Na+ concentrations in a halotolerant bacterium, Brevibacterium sp., and Escherichia coli. The internal Na+ concentration of both strains depended little on the growth phases and was unchanged after 5 d storage at 2 degrees C. In Brevibacterium sp. the level of intracellular sodium increased gradually at higher extracellular NaCl concentrations in both the presence and absence of yeast extract in the growth medium. E. coli cells accumulated a higher concentration of free Na+ than those of Brevibacterium sp. The change of Na+ concentration in both strains was inverse to that of growth rate. When appropriate amounts of osmoprotectants (proline, glycine betaine, or gamma-aminobutyrate) were added with the NaCl, internal free Na+ levels in Brevibacterium sp. were lowered, but those of E. coli were unchanged. While addition of KCl to medium containing NaCl increased the intracellular level of free Na+, the total sodium concentration in the cells remained unchanged, indicating that sodium that had been bound or attached was made free in the cytosol. In Brevibacterium sp. grown in the presence of 0.5 M NaCl, free and bound sodium concentrations in the cytosol were estimated to be 0.14 and 0.23 mumol (mg protein)-1, respectively. As a result, visibility by 23Na NMR was 38%.

Osmotic regulation of synthesis of glycerophosphocholine from phosphatidylcholine in MDCK cells

Glycerophosphocholine (GPC) is osmotically regulated in renal medullary cells and in cultured Madin-Darby canine kidney (MDCK) cells. Previously, it was shown that a high extracellular concentration of urea or NaCl causes these cells to accumulate large amounts of GPC. GPC is known to be a product of phosphatidylcholine (PC) catabolism. The purpose of the present experiments was to examine the role of changes in the rate of GPC synthesis from PC in hyperosmotically induced GPC accumulation. When 1-palmitoyl-2-lysophosphatidyl-[methyl-3H]choline ([3H]LPC) is added to the medium, it is taken up by the cells and most of it is rapidly converted to PC. During a chase, 3H lost from PC appears almost exclusively in GPC and sphingomyelin. The rate of catabolism of PC is twofold greater in cells exposed to high NaCl (200 mosmol/kgH2O, added for 2 days) than in control or high-urea medium. Increased PC catabolism in NaCl-treated cells is associated with a 2.6-fold increase in GPC synthesis from PC; sphingomyelin synthesis decreases, and total cell PC does not change. Also, neither total mass nor specific radioactivity of lysophosphatidylcholine changes. PC catabolism is unaffected by short (2 h) exposure to high NaCl or urea. To investigate the enzymatic basis for the increased PC catabolism in response to high NaCl, phospholipase activity was measured in cell homogenates with 1-palmitoyl-2-[1-14C]palmitoyl-PC as a substrate. Exposure of cells to high NaCl for 2 days (but not 2 h) increases activity 2.8-fold compared with control or high-urea medium. Lysophospholipase activity (measured with [3H]LPC as the substrate) is unchanged. The increased phospholipase activity occurs with dipalmitoyl PC, but not sn-2-arachidonyl PC, as a substrate. Collectively, these data suggest a role for a phospholipase, unrelated to the arachidonyl-selective enzyme, in the regulation of PC catabolism during accumulation of GPC induced by prolonged exposure to high extracellular NaCl.

Characterization of a glycerol/H+ symport in the halotolerant yeast Pichia sorbitophila

Pichia sorbitophila is a halotolerant yeast capable of surviving to extracellular NaCl concentrations up to 4 M in mineral medium when glucose or glycerol are the only carbon and energy sources. Evidence is presented here that glycerol, the main compatible solute this yeast accumulates so as to maintain osmotic balance, is actively co-transported with protons. This transport system was shown to be constitutive, not needing induction by either glycerol or salt, and was not repressible by glucose. In glucose- or glycerol-grown cells, a simple diffusion was detectable, and iterative calculations were performed to calculate kinetic parameters, in the presence and in the absence of NaCl. At 25 degrees C, pH 5.0, in glucose-grown cells these were: Km = 0.81 +/- 0.11 mM and Vmax = 634.2 +/- 164.8 mumol h-1 per g (glycerol); Km = 1.28 +/- 0.60 mM and Vmax = 558.6 +/- 100.6 mumol h-1 per g (protons). Correspondent stoichiometry was approximately 1, either for these conditions or in the presence of 1 M-NaCl. An increase in accumulation capacity was evident when different concentrations of NaCl were present. This capacity was shown to be dependent on delta pH and membrane potential, consistently with an electrogenic character. We suggest that the main role of this system is in osmoregulation, by keeping glycerol accumulated inside the cells, compensating for leakage, due to its liposoluble character.

Intracellular pH and Its Regulation in Pelvetia Zygotes

We have investigated the role of intracellular pH (pH i) in early plant development using ion-selective microelectrodes to record from eggs and embryos of the brown alga Pelvetia. Temporal changes in pH i were investigated by recording from zygotes at all stages of the first cell cycle. pH i was 7.57 ± 0.09 in recently fertilized eggs, but decreased by approximately 0.2 units a few hours postfertilization. Proton motive force (pmf) was also monitored and found to be less than -50 mV throughout the first cell cycle. Because of the low pmf values, we suggest that secondary active transport is probably not coupled to H +, and instead we propose that solute transport is driven by the Na + electrochemical potential. Zygotes strictly regulated pH i over a wide range of extracellular pH (pH o); pH i varied by less than 0.2 units over pH o values from 6.2 to 9.2. Inhibitor studies were conducted to investigate the mechanism of regulation. Agents known to inhibit the H +-ATPase (antimycin A, KCN, carbonylcyanide m -chlorophenylhydrazone, N,N′-dicyclohexylcarbodiimide, and erythrosin B) caused marked cytoplasmic acidification. Addition of amiloride, an inhibitor of Na +/H + antiport, also resulted in acidification, as did removal of NaCl from the medium. By contrast, increasing extracellular NaCl concentration caused transient alkalinization of the cytoplasm. Taken together, these results indicate that proton pumping by an H +-ATPase and Na +/H + antiporter contribute to pH regulation.

Effects of bradykinin on cell volume and intracellular pH in NIH 3T3 fibroblasts expressing the ras oncogene

BCECF fluorescence has been applied to determine intracellular pH (pH i) in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ras) and otherwise identical cells not expressing the oncogene (−ras). In +ras cells, pH i is significantly more alkaline (6.79 ± 0.03 n = 12) than in −ras cells (6.64 ± 0.02, n = 8). Bradykinin (100 nmol/1) leads to intracellular alkalinization in both +ras (to 6.96 ± 0.04, n = 12) and −ras cells (to 6.85 ± 0.02, n = 8). The effect or bradykinin is completely abolished in the presence of dimelhylamiloride (100μmol/1), which does not modify pH i in the absence or bradykinin. Similar to bradykinin, cell shrinkage by addition of 15 mmol/1 NaCl to the extracellular fluid leads to intracellular alkalinization (by 0.08 ± 0.01, n = 15). Cell volume is significantly greater in +ras cells (2.7 ± 0.4 pl, n = 15) than in −ras calls (2.2 ± 0.4 pl, n = 15), Bradykinin leads to cell shrinkage in both +ras cells (by 7 ± 1%, n = 17) and −ras cells (by 5 ± 1%, n = 15). The effect of bradykinin on cell volume can be reversed by the reduction or extracellular NaCl concentration by 15 mmol/1 NaCl in +ras cells and by 7 mmol/1 NaCl in −rus cells. This maneuver completely abolishes (in −ras cells) or blunts (in +ras cells) the alkalinizing effect of bradykinin. In conclusion, +ras cells are more alkaline than −ras cells. Bradykinin leads to further intracellular alkalinization by activation of the Na +/H +-exchanger, at least in part secondary to hormone-induced cell shrinkage.

Renal medullary organic osmolytes

Sorbitol, inositol, GPC, and betaine are the predominant organic osmolytes in renal medullary cells. They protect the cells from harmful effects of the high interstitial NaCl and urea concentrations that occur normally in the renal medulla with operation of the urinary concentrating mechanism. Their levels correlate with extracellular NaCl concentration and, in the case of GPC, also with urea. Sorbitol is synthesized from glucose in a reaction catalyzed by aldose reductase. Inositol and betaine are transported into the cell. Glycerophosphorylcholine synthesis is dependent on choline. The transcription of aldose reductase and the transport of betaine and inositol are regulated, dependent on the degree of hypertonicity. Normal organic osmolyte regulation contributes to the survival and growth of medullary cells in their hyperosmolal environment, and defective regulation can damage them.

Intracellular betaine substitutes for sorbitol in protecting renal medullary cells from hypertonicity

Renal medullary cells are normally exposed to a variably high extracellular NaCl concentration. They compensate by accumulating large amounts of organic osmolytes, including sorbitol and betaine. The sorbitol is synthesized from glucose, catalyzed by aldose reductase. Previously, inhibition of aldose reductase activity was noted to greatly reduce renal medullary cell survival and growth (measured by cloning efficiency) in tissue cultures of renal medullary cells in hypertonic medium. In contrast, inhibition of aldose reductase and renal medullary sorbitol accumulation is not associated with kidney damage in vivo. In the present experiments we find that addition of betaine to the medium, and its resultant uptake by the cells, largely replaces the decrease in sorbitol caused by aldose reductase inhibitors and restores the cloning efficiency. We presume that in vivo uptake of betaine by renal medullary cells similarly protects them from harm when aldose reductase inhibitors lower sorbitol. The results also demonstrate that one organic osmolyte can substitute for another in protecting cells from hypertonicity, consistent with the compatible osmolytes hypothesis.

Importance of organic osmolytes for osmoregulation by renal medullary cells.

The cells in the renal medulla protect themselves from the extracellular hypertonicity in that region of the kidney by accumulating large amounts of sorbitol, inositol, glycerophosphorylcholine, and betaine. The system is uniquely active in this part of the body, but it represents a throwback to primitive mechanisms by which cells in virtually all organisms, including bacteria, yeasts, plants, and lower animals counteract water stress. In this brief review, we summarize how these "compatible organic osmolytes" help the renal medullary cells to survive, the mechanisms by which the organic osmolytes are accumulated, and how the accumulation is controlled to adjust for changing extracellular NaCl and urea concentrations. The compatible organic osmolytes are all intermediates in important biochemical pathways, and although the medical consequences are not yet fully worked out, it is already apparent that inappropriate accumulation of these solutes has major pathophysiological consequences.