Stabilization and swelling of hagfish slime mucin vesicles.

When agitated, Atlantic hagfish (Myxine glutinosa) produce large quantities of slime that consists of hydrated bundles of protein filaments and membrane-bound mucin vesicles from numerous slime glands. When the slime exudate contacts seawater, the thread bundles unravel and the mucin vesicles swell and rupture. Little is known about the mechanisms of vesicle rupture in seawater and stabilization within the gland, although it is believed that the vesicle membrane is permeable to most ions except polyvalent anions. We hypothesized that the most abundant compounds within the slime gland exudate have a stabilizing effect on the mucin vesicles. To test this hypothesis, we measured the chemical composition of the fluid component of hagfish slime exudate and conducted functional assays with these solutes to test their ability to keep the vesicles in a condensed state. We found K(+) concentrations that were elevated relative to plasma, and Na(+), Cl(-) and Ca(2+) concentrations that were considerably lower. Our analysis also revealed high levels of methylamines such as trimethylamine oxide (TMAO), betaine and dimethylglycine, which had a combined concentration of 388 mmol l(-1) in the glandular fluid. In vitro rupture assays demonstrated that both TMAO and betaine had a significant effect on rupture, but neither was capable of completely abolishing mucin swelling and rupture, even at high concentrations. This suggests that some other mechanism such as the chemical microenvironment within gland mucous cells, or hydrostatic pressure is responsible for stabilization of the vesicles within the gland.

Trimethylamine oxide, betaine and other osmolytes in deep-sea animals: depth trends and effects on enzymes under hydrostatic pressure.

Most shallow teleosts have low organic osmolyte contents, e.g. 70 mmol/kg or less of trimethylamine oxide (TMAO). Our previous work showed that TMAO contents increase with depth in muscles of several Pacific families of teleost fishes, to about 180 mmol/kg wet wt at 2.9 km depth in grenadiers. We now report that abyssal grenadiers (Coryphaenoides armatus, Macrouridae) from the Atlantic at 4.8 km depth contain 261 mmol/kg wet wt in muscle tissue. This precisely fits a linear trend extrapolated from the earlier data. We also found that anemones show a trend of increasing contents of methylamines (TMAO, betaine) and scyllo-inositol with increasing depth. Previously we found that TMAO counteracts the inhibitory effects of hydrostatic pressure on a variety of proteins. We now report that TMAO and, to a lesser extent, betaine, are generally better stabilizers than other common osmolytes (myo-inositol, taurine and glycine), in terms of counteracting the effects of pressure on NADH Km of grenadier lactate dehydrogenase and ADP Km of anemone and rabbit pyruvate kinase.

Dogmas and controversies in the handling of nitrogenous wastes: osmoregulation during early embryonic development in the marine little skate Raja erinacea; response to changes in external salinity.

Marine elasmobranchs retain relatively high levels of urea to counterbalance the osmotic strength of seawater. Oviparous species, such as the little skate Raja erinacea, release encapsulated embryos that hatch after about 9 months on the seafloor. To study the ureosmotic capability of skate embryos, we measured a variety of possible osmolytes and ornithine-urea cycle (OUC) enzyme activities in little skate embryos, and determined their physiological response to dilute seawater (75% SW) exposure relative to controls (100% SW). The urea:trimethylamine oxide (TMAO) + other osmolytes ratio was 2.3-2.7:1. At the earliest stage of development investigated (4 months), there were significant levels of the key OUC enzyme, carbamoyl phosphate synthetase III, as well as ornithine transcarbamoylase, arginase and glutamine synthetase, providing evidence for a functional OUC. Embryos (4 and 8 months) survived and recovered from exposure to 5 days of 75% SW. There was a significant increase in the rate of urea excretion (five- to tenfold), no change in OUC enzyme activities, and significant decreases in the tissue content of urea, TMAO and other osmolytes in embryos exposed to 75% SW compared to 100% SW. Taken together, the data indicate that little skate embryos synthesize and retain urea, as well as a suite of other osmolytes, in order to regulate osmotic balance with the external environment. Interestingly, these ureosmotic mechanisms are in place as early as 4 months, around the time at which the egg capsule opens and the embryo is in more direct contact with the external environment.

The effects of hypoosmotic infusion on the composition of renal tissue of the Australian brush-tailed possum Trichosurus vulpecula.

Although the occurrence of organic osmolytes in the inner medulla of the marsupial kidney has been recently reported [Comp. Biochem. Physiol. (2002) 132B 635-644], changes in these substances, in response to water loading in vivo, has not been studied. Adult Trichosurus vulpecula, the Australian brush-tailed possum, were subjected to water deprivation for 48 h. Following anaesthesia and unilateral nephrectomy, the animals were perfused with hypo-osmotic saline (80 mmol l(-1); 1.5 ml min(-1)) for 60 min. This resulted in a rapid increase in urine volume and a corresponding fall in urine osmolality. At the end of the infusion the animals were killed and the second kidney removed. Analysis of the renal tissue revealed that water content of cortical, outer and inner medullary regions of the kidney increased slightly following infusion, while sodium, and chloride contents of all three regions fell. Potassium contents, on the other hand, were barely changed. Of the organic osmolytes determined, very significant decreases in the inner medulla, following infusion, were found for sorbitol (from 397+/-79 to 266+/-49 mmol kg(-1) protein), inositol (247+/-23 to 190+/-25 mmol kg(-1) protein), and betaine (464+/-70 to 356+/-21 mmol kg(-1) protein), while only inositol was significantly decreased in the outer medulla (197+/-22 to 150+/-16 mmol kg(-1) protein). Glycerophosphorylcholine levels were low throughout the kidney and were not significantly affected by the infusion. It was concluded that inositol and sorbitol play a significant role as compatible organic osmolytes in the possum kidney, while betaine functions as the principal counteracting osmolyte. Amino acid levels in the cortex and outer medulla showed no overall change in amount following infusion, although there were highly significant changes in individual amino acids. In the inner medulla there was a highly significant reduction in total amino acids with infusion, largely due to a fall in amounts of taurine (104+/-4 to 75+/-17 mmol kg(-1) protein), and glycine (97+/-15 to 71+/-18 mmol kg(-1) protein). A fall in free amino acid levels in the inner medulla appears to significantly contribute to the process of intracellular osmotic adjustment during an induced diuresis.

Trimethylamine oxide counteracts effects of hydrostatic pressure on proteins of deep-sea teleosts.

In shallow marine teleost fishes, the osmolyte trimethylamine oxide (TMAO) is typically found at <70 mmol/kg wet weight. Recently we found deep-sea teleosts have up to 288 mmol/kg, increasing in the order shallow < bathyal < abyssal. We hypothesized that this protein stabilizer counteracts inhibition of proteins by hydrostatic pressure, and showed that, for lactate dehydrogenases (LDH), 250 mM TMAO fully offset an increase in NADH K(m) at physiological pressure, and partly reversed pressure-enhanced losses of activity at supranormal pressures. In this study, we examined other effects of pressure and TMAO on proteins of teleosts that live from 2000-5000 m (200-500 atmospheres [atm]). First, for LDH from a grenadier (Coryphaenoides leptolepis) at 500 atm for 8 hr, there was a significant 15% loss in activity (P < 0.05 relative to 1 atm control) that was reduced with 250 mM TMAO to an insignificant loss. Second, for pyruvate kinase from a morid cod (Antimora microlepis) at 200 atm, there was 73% increase in ADP K(m) without TMAO (P < 0.01 relative to K(m) at 1 atm) but only a 29% increase with 300 mM TMAO. Third, for G-actin from a grenadier (C. armatus) at 500 atm for 16 hr, there was a significant reduction of F-actin polymerization (P < 0.01 compared to polymerization at 1 atm) that was fully counteracted by 250 mM TMAO, but was unchanged in 250 mM glycine. These findings support the hypothesis. J. Exp. Zool. 289:172-176, 2001.

Hypotaurine, N-methyltaurine, taurine, and glycine betaine as dominant osmolytes of vestimentiferan tubeworms from hydrothermal vents and cold seeps.

Organic osmolytes, solutes that regulate cell volume, occur at high levels in marine invertebrates. These are mostly free amino acids such as taurine, which are "compatible" with cell macromolecules, and methylamines such as trimethylamine oxide, which may have a nonosmotic role as a protein stabilizer, and which is higher in many deep-sea animals. To better understand nonosmotic roles of osmolytes, we used high-performance liquid chromatography and (1)H-nuclear magnetic resonance (NMR) to analyze vestimentiferans (vestimentum tissue) from unusual marine habitats. Species from deep hydrothermal vents were Riftia pachyptila of the East Pacific Rise (2,636 m) and Ridgeia piscesae of the Juan de Fuca Ridge (2,200 m). Species from cold hydrocarbon seeps were Lamellibrachia sp. and an unnamed escarpid species from subtidal sediment seeps (540 m) off Louisiana and Lamellibrachia barhami from bathyal tectonic seeps (1,800-2,000 m) off Oregon. Riftia were dominated by hypotaurine (152 mmol/kg wet wt), an antioxidant, and an unidentified solute with an NMR spectrum consistent with a methylamine. Ridgeia were dominated by betaine (N-trimethylglycine; 109 mmol/kg), hypotaurine (64 mmol/kg), and taurine (61 mmol/kg). The escarpids were dominated by taurine (138 mmol/kg) and hypotaurine (69 mmol/kg). Both Lamellibrachia populations were dominated by N-methyltaurine (209-252 mmol/kg), not previously reported as a major osmolyte, which may be involved in methane and sulfate metabolism. Trunk and plume tissue of the Oregon Lamellibrachia were nearly identical to vestimentum in osmolyte composition. The methylamines may also stabilize proteins against pressure; they were significantly higher in the three deeper-dwelling groups.

Developmental changes in organic osmolytes in prenatal and postnatal rat tissues.

At high osmotic pressures, mammalian kidney medulla, heart, lens, and brain utilize organic osmolytes to regulate cell volume. However the types and proportions of these solutes vary among tissues in patterns and for non-osmotic roles not fully elucidated. To clarify these, we analyzed osmolyte-type solute contents in rat tissues at 7 and 2 days prenatal and at 0, 7, 14, 21 (weaning), 35 (juvenile) and 77 (adult) days postnatal. Placentas were dominated by betaine, taurine, and creatine, which decreased between the prenatal times. Fetuses were dominated by glutamate and taurine, which increased between the times. In cerebrum, hindbrain and diencephalon, taurine dominated at early stages, but dropped after postnatal day 7, while myo-inositol, glutamine, creatine and glutamate increased after birth, with the latter two dominating in adults. In olfactory bulb, taurine content declined gradually with age and was equal to glutamate in adults. In all brain regions, glycerophosphorylcholine (GPC) reached a peak in juveniles. In postnatal renal medulla, urea, sodium, GPC, betaine, and taurine increased sharply at day 21. Thereafter, most increased, but taurine decreased. In heart, taurine dominated, and increased with age along with creatine and glutamine, while glutamate decreased after postnatal day 7. In lens, taurine dominated and declined in adults. These patterns are discussed in light of hypotheses on non-osmotic and pathological roles of these solutes.

Trimethylamine oxide stabilizes teleost and mammalian lactate dehydrogenases against inactivation by hydrostatic pressure and trypsinolysis.

Trimethylamine N-oxide (TMAO) is an organic osmolyte present at high levels in elasmobranchs, in which it counteracts the deleterious effects of urea on proteins, and is also accumulated by deep-living invertebrates and teleost fishes. To test the hypothesis that TMAO may compensate for the adverse effects of elevated pressure on protein structure in deep-sea species, we studied the efficacy of TMAO in preventing denaturation and enhanced proteolysis by hydrostatic pressure. TMAO was compared to a common 'compatible' osmolyte, glycine, using muscle-type lactate dehydrogenase (A(4)-LDH) homologs from three scorpaenid teleost fish species and from a mammal, the cow. Test conditions lasted 1 h and were: (1) no addition, (2) 250 mmol l(-)(1) TMAO and (3) 250 mmol l(-)(1) glycine, in the absence and presence of trypsin. Comparisons were made at 0. 1 and 101.3 MPa for the deeper occurring Sebastolobus altivelis, 0.1, 50.7 and 101.3 MPa for the moderate-depth congener S. alascanus, 0. 1 and 25.3 MPa for shallow-living Sebastes melanops and 0.1 and 50.7 MPa for Bos taurus. Susceptibility to denaturation was determined by the residual LDH activity. For all the species and pressures tested, 250 mmol l(-)(1) TMAO reduced trypsinolysis significantly. For all except S. altivelis, which was minimally affected by 101.3 MPa pressure, TMAO stabilized the LDH homologs and reduced pressure denaturation significantly. Glycine, in contrast, showed no ability to reduce pressure denaturation alone, and little or no ability to reduce the rate of proteolysis.

High contents of trimethylamine oxide correlating with depth in deep-sea teleost fishes, skates, and decapod crustaceans.

In muscles of shallow-living marine animals, the osmolyte trimethylamine N-oxide (TMAO) is reportedly found (in millimoles of TMAO per kilogram of tissue wet weight) at 30-90 in shrimp, 5-50 in crabs, 61-181 in skates, and 10-70 in most teleost fish. Recently our laboratory reported higher levels (83-211 mmol/kg), correlating with habitat depth, in deep-sea gadiform teleosts. We now report the same trend in muscles of other animals, collected off the coast of Oregon from bathyal (1800-2000 m) and abyssal plain (2850 m) sites. TMAO contents (mmol/kg +/- SD) were as follows: zoarcid teleosts, 103 +/- 9 (bathyal) and 197 +/- 2 (abyssal); scorpaenid teleosts, 32 +/- 0 (shallow) and 141 +/- 16 (bathyal); rajid skates, 215 +/- 13 (bathyal) and 244 +/- 23 (abyssal); caridean shrimp, 76 +/- 16 (shallow), 203 +/- 35 (bathyal), and 299 +/- 28 (abyssal); Chionoecetes crabs, 22 +/- 2 (shallow) and 164 +/- 15 (bathyal). Deep squid, clams, and anemones also had higher contents than shallow species. Osmoconformers showed compensation between TMAO and other osmolytes. Urea contents (typically 300 mmol/kg in shallow elasmobranchs) in skates were 214 +/- 5 (bathyal) and 136 +/- 9 (abyssal). Glycine contents in shrimp were 188 +/- 17 (shallow) and 52 +/- 20 (abyssal). High TMAO contents may reflect diet, reduce osmoregulatory costs, increase buoyancy, or counteract destabilization of proteins by pressure.

Effects of urea on M4-lactate dehydrogenase from elasmobranchs and urea-accumulating Australian desert frogs.

We measured the effect of urea on M4-lactate dehydrogenase (M4-LDH) from elasmobranchs and Australian desert frogs (urea accumulators) and from two animals that do not accumulate urea, the axolotl and the rabbit. An analysis of the effect of urea on the Kd(NADH), V, V/K(m(prr)) and V/K(m(NADH)) shows that in all cases the major effect of urea was on the binding of pyruvate, which fits with data in the literature that show that urea acts as a competitive inhibitor of LDH. The characteristics of the elasmobranch enzymes are consistent with a proposed adaptation model, but the situation for the enzymes from the aestivating frogs is equivocal. Urea (400 mM) had less effect on the K(m(prr)) of M4-LDH from the urea accumulators than it did on the non-accumulators, suggesting a general adaptation and that the enzyme produced by the aestivating frogs (urea accumulators) is kinetically different from that of non-aestivating frogs (non-accumulators). A new approach is used to characterize the overall pattern of adaptation to urea. The pattern is similar in an enzyme from an elasmobranch and an aestivating frog despite the temporary presence of urea in the latter and the phylogenetic difference between these animals.

Cerebral cell volume regulation during hypernatremia in developing rats.

Cell volume regulation is a vital biological function in all species. Maintenance of cerebral cell size in the face of osmotic stress is especially important because the brain is contained in the non-complaint skull. The developmental aspects of this adaptive process are not known. Therefore, we evaluated cerebral cell volume regulation during hypernatremia in pre-weaning and adult rats. Hypernatremia was induced by injections of 1 M NaCl for 48 h. Brain water, electrolyte, and organic osmolyte contents were measured in hypernatremia and sham injected littermate control rats at the following ages: 12, 18 and 20 days and adults. In normonatremic rats, there was a steady decline in brain water content during development that was paralleled by a gradual fall in the cerebral levels of Na+, K+, and all organic osmolytes. The change in brain water content correlated most closely with the decrease in cerebral taurine content. In the face of equivalent elevations in serum Na+ concentration, there was comparable brain cell shrinkage and similar increases in total cerebral electrolyte and organic osmolyte content in rats at all 4 ages studied. Taurine was the predominant organic osmolyte prior to weaning, constituting 16-49% of the increment in nonperturbing solute content in hypernatremic animals between 12-20 days of age; in contrast, taurine contributed only 10% to the cerebral organic osmolyte pool in adult rats. We conclude that the capacity of brain cells to accumulate inorganic electrolytes and organic osmolytes during adaptation to hypernatremia is adequately expressed in developing rats, aged 12 days or older. Moreover, we speculate that the immature animal behaves as if it has an elevated 'set point' to protect the higher brain water content that is present earlier in development.

Time-dependent aspects of osmolyte changes in rat kidney, urine, blood and lens with sorbinil and galactose feeding.

Sorbitol plus myo-inositol, betaine and glycerophosphorylcholine (GPC) are cellular osmolytes in the mammalian renal medulla. Galactosemia and hyperglycemia can cause excessive levels of galactitol or sorbitol in several organs via aldose reductase (AR) catalysis. AR inhibitors can reduce these polyols. To examine osmolyte responses to polyol perturbations, male Wistar rats were fed normal diet, the AR inhibitor sorbinil (at 40 mg/kg/d), 25% galactose, or a combination, for 10, 21 and 42 days. All animals at 21 days had higher apparent renal AR activity than at 10 or 42 days, possibly providing resistance to sorbinil. Sorbinil feeding alone tended to increase urinary, plasma and renal urea levels. It reduced AR activity and sorbitol contents in renal inner medulla, though less so at 21 days; other renal osmolytes, especially betaine, were elevated. Galactose feeding caused little change in renal AR activity, and resulted in high galactose and galactitol contents in renal medulla, urine, blood and lens (and higher renal Na+ contents at 10 days). Renal sorbitol, inositol and GPC decreased, while betaine contents trended higher at all times. Sorbinilgalactose feeding reduced renal AR activities and galactitol contents (again less so at 21 days), urine, blood and lens galactitol, and further reduced renal sorbitol contents. At 10 and 21 days it tended to raise renal betaine more, and restore inositol (but not GPC) contents to control levels. At 42 days it reduced renal and urinary Na+ and galactose, and decreased renal betaine to control levels. Under most conditions, total renal (non-urea) organic osmolyte contents (presumed to be mostly intracellular) and Na+ plus galactose contents (presumed mostly extracellular) changed together such that cell volumes may have been maintained. The exception was 10 days on galactose, where total osmolytes appeared too low. In galactose-fed animals, urine/plasma ratios suggest some renal galactitol efflux, and cellular galactitol probably helps maintain osmotic balance rather than cause swelling.

Effects on rat renal osmolytes of extended treatment with an aldose reductase inhibitor.

1. The mammalian renal medulla uses sorbitol, myo-inositol, betaine and glycerophosphorylcholine as intracellular osmolytes. 2. Sorbitol synthesis was inhibited by feeding male Wistar rats the aldose reductase inhibitor sorbinil at 40 mg/kg/day for 71 d, and renal inner medullas were extracted for analysis. 3. Aldose reductase activities and sorbitol contents were greatly reduced in sorbinil-treated animals, while betaine contents increased significantly (with no other osmolytes changing). 4. The betaine increase compensated for the sorbitol decrease such that the total organic osmolytes maintained the same ratio to sodium contents as controls. 5. These results are identical to the pattern previously reported for sorbinil treatment of rats for 10 d, but not for 21 d.

Effects of dietary protein and salt on rat renal osmolytes: covariation in urea and GPC contents.

Renal medullary cells contain high levels of (glycine) betaine, glycerophosphorylcholine (GPC), myo-inositol, and sorbitol. Two functions of these have been proposed: 1) that they are compatible osmolytes which regulate cell volume (against high external NaCl) without inhibiting proteins and 2) that methylamines (GPC and betaine) are counteracting osmolytes which stabilize proteins against perturbation from high renal urea. As a test of the latter, osmolyte contents in kidney medullas were measured in rats subjected to three types of dietary manipulation: 1) diets with protein and NaCl contents varied oppositely, 2) diets with a constant low NaCl and varied protein content, and 3) a low-calorie diet. With low-protein and low-calorie diets, only renal contents of urea, GPC, and inositol decreased; betaine and sorbitol contents increased such that contents of total nonurea organic osmolytes remained constant. With high-protein diets, only renal contents of sodium, urea, and GPC increased, with the latter giving total organic osmolytes a consistent correlation to sodium. Across all diets, the only consistent (linear) correlations were 1) between urea and GPC contents, supporting previous suggestions that GPC is the major counteractant to urea, and 2) between total organic osmolytes and sodium (but not urea) contents, as predicted by the compatible osmolytes hypothesis.

Effects of an aldose reductase inhibitor on organic osmotic effectors in rat renal medulla.

Renal medullary cells use sorbitol, betaine, and other organic compounds as osmotic effectors (osmolytes) to balance high extracellular NaCl. Excess sorbitol is also implicated in diabetes complications in several organs including kidneys. To study regulation of renal sorbitol, male Wistar rats were given an aldose reductase inhibitor, sorbinil, at 40 mg.kg-1.day-1 in food to block sorbitol formation from glucose. Inner medullas of kidneys were analyzed for osmolytes by high-performance liquid chromatography and atomic absorption. Animals on sorbinil had significantly reduced medullary sorbitol contents in a group on ad libitum water for 10 days (2.7 mmol/kg wet wt compared with 4.8 in controls) and in an antidiuretic group kept 7 days and an additional 3 days without water (3.8 mmol/kg wet wt compared with 7.2 in antidiuretic controls). In both groups, betaine contents were significantly elevated (9.2 mmol/kg wet wt compared with 5.5 in ad libitum water controls: 6.4 mmol/kg wet wt compared with 4.2 in antidiuretic controls). No other osmolytes differed. Total contents of nonurea organic osmolytes maintained a constant ratio to sodium contents; thus increased betaine concomitant with decreased sorbitol may have maintained constant cell volume. In contrast, in animals kept 21 days on sorbinil, there were significant decreases in urea and inositol contents. However, there were no significant differences in sorbitol or betaine compared with controls, suggesting a compensating increase in sorbitol production or in sorbinil removal.

Counteracting effects of urea and betaine in mammalian cells in culture.

Urea and methylamines, such as betaine, are among the major organic osmotic effectors accumulated by organisms under hyperosmotic (high NaCl) stress; the mammalian renal medulla also accumulates such compounds in antidiuresis. Studies on isolated proteins show that urea generally destabilizes protein structure, whereas methylamines are generally stabilizers capable of offsetting the effects of urea. The counteracting-osmolytes hypothesis predicts that cells exposed to high urea concentrations require methylamines for optimal function. In this study, urea, betaine, and other solutes (NaCl, glycerol, sorbitol) were added to growth medium of cultured mammalian cells under conditions in which most solutes entered the cells. For two renal [Madin-Darby canine kidney (MDCK) and PAP-HT25] and one nonrenal (Chinese hamster ovary) cell line, urea (greater than 100 mM) or betaine (greater than 50-100 mM) alone inhibited cell growth and survival, measured as colony-forming efficiency. However, the addition of betaine (up to 120 mM) to media with urea (50-300 mM) greatly increased colony-forming efficiency above that with urea alone. A similar, although less marked effect, was seen on colony sizes with MDCK cells. These results support the counteracting-osmolytes hypothesis.

Effects of NaCl, glucose, and aldose reductase inhibitors on cloning efficiency of renal medullary cells.

To analyze the effects of sorbitol accumulation on the survival and growth of epithelial cells from rabbit renal inner medulla, cloning efficiency (an index of cell viability) was measured at normal and high glucose and NaCl concentrations and when sorbitol accumulation was prevented by Tolrestat and Sorbinil, which inhibit aldose reductase. With PAP-HT25 cells grown to near confluence, high NaCl increases aldose reductase activity, causing enough rise in cell sorbitol concentration to balance most of the increased osmolality of the high extracellular NaCl. Inhibition of aldose reductase prevents both the increased enzyme activity and sorbitol accumulation in a dose-related manner. Paralleling this, colony-forming efficiency is not affected by the inhibitors at a normal NaCl concentration but is greatly reduced when extracellular NaCl is high. On the other hand, high glucose levels, as occur in diabetes, increase sorbitol content well above the concentration required for osmotic balance and inhibit colony-forming efficiency. Under those conditions, aldose reductase inhibitors lower cell sorbitol and reverse (at 300-350 mosmol/kgH2O) or reduce (at 500-550 mosmol/kgH2O) the decrease in colony-forming efficiency caused by high glucose. Thus sorbitol accumulation is necessary for osmoregulation when induced by high osmolality but is harmful when induced by high glucose.

Distribution of major organic osmolytes in rabbit kidneys in diuresis and antidiuresis.

Sorbitol, glycerophosphorylcholine (GPC), inositol, and betaine are organic osmolytes that accumulate in renal medullary cells. Two roles have been proposed for them: 1) that all four are "compatible osmolytes" that help regulate cell volume without disturbing function, and 2) that the methylamines (GPC and betaine) are "counteracting osmolytes," i.e., stabilizers that offset the perturbing effects of the high urea concentration. To test these hypotheses we have measured the osmolyte gradients in diuresis and antidiuresis in rabbit kidneys cut in 7 sections along the corticopapillary axis. In both antidiuresis and diuresis, inositol was highest in the outer medulla and decreased toward the tip of the inner medulla. In antidiuresis, contents of sodium, urea, sorbitol, GPC, and betaine increased monotonically toward the tip of the inner medulla. All osmolytes were significantly lower in diuresis compared with antidiuresis in two or more kidney sections. Urea, GPC, and sorbitol had the greatest differences between the two states. The sum of the four (mainly intracellular) compatible osmolytes showed a strong linear correlation with Na (presumably mostly extracellular), with a similar slope in both states, consistent with the compatible osmolytes hypothesis. Considering the osmolytes individually, only two linear correlations were highly significant and had similar slopes in both diuresis and antidiuresis: betaine with Na and GPC with urea. The latter is consistent with the counteracting osmolytes hypothesis, suggesting that GPC is the main agent stabilizing against urea in the renal medulla.

A simple HPLC method for quantitating major organic solutes of renal medulla.

A simple high performance liquid chromatography (HPLC) method of separating and quantitating the predominant organic solutes of the renal medulla is described. These organic solutes include myo-inositol, glycerophosphorylcholine, sorbitol, betaine, and urea. Other physiologically significant solutes, including glucose and mannitol, can be separated and quantitated concurrently with this method. With the use of this technique, the organic solutes of the rabbit kidney were determined. No new organic compounds were detected by HPLC that could significantly contribute to intracellular osmolality of the medulla. The values for the organic solutes already described were similar to those obtained by more complicated and limited approaches such as classical enzymatic techniques, ion electrodes, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectroscopy.

Osmotic effectors in kidneys of xeric and mesic rodents: corticomedullary distributions and changes with water availability.

Urea, sodium, the methylamines glycine betaine and glycerophosphorylcholine (GPC), and the polyols sorbitol and myo-inositol are reported to be the major osmolytes in kidneys of laboratory mammals. These were measured (millimoles per kilogram wet weight) in kidney regions and urines of three species of wild rodents with different dehydration tolerances: the pocket mouse Perognathus parvus (xeric), vole Microtus montanus (mesic), and deer mouse Peromyscus m. gambeli (intermediate). In animals kept without water for 4-6 days, sodium, urea, betaine and GPC + choline were found in gradients increasing from cortex to outer to inner medulla in all species, with Perognathus having the highest levels. Sorbitol was high in the inner medulla but low in the cortex and outer medulla; inositol was highest in the outer medulla. Totals of methylamines and methylamines plus polyols in the medulla showed high linear correlations (positive) with urea and with sodium values. Whole medullae were analyzed at several time points in Microtus and Peromyscus subject to water diuresis followed by antidiuresis. In 102 h diuresis in Microtus, all osmolytes decreased except inositol; however, only urea, sodium and sorbitol reached new steady states within 24 h. Urea returned to initial values in 18 h antidiuresis, while other osmolytes required up to 90 h. In Peromyscus, all osmolytes except the polyols declined in diuresis (max. 78 h test period). During antidiuresis, urea and GPC + choline rose to initial values in 18 h, with sodium and betaine requiring more time. In plots of both species combined, total methylamines + polyols correlated linearly (positive) with sodium, and GPC + choline with urea. Estimates of tissue concentrations suggest that total methylamines + polyols can account for intracellular osmotic balance in all species in antidiuresis and that sufficient concentrations of methylamines may be present to counteract perturbing effects of urea on proteins.