Medium Tonicity Research Articles

Two modes of stimulation by ammonia of taurine release from cultured rabbit Müller cells

A previous study revealed that a 10-min (`acute') treatment of cultured Müller glia with ammonium ions (further referred to as `ammonia') at 0.5–5 mM concentration stimulated the release of newly loaded taurine (Tau) by a cAMP-dependent, osmoresistant mechanism. Here we showed that a 24 h treatment of the cells with 1 mM ammonia increased both Tau release and intracellular cAMP content in a degree similar to acute treatment with 5 mM ammonia, and the effects were similarly resistant to an increase of medium tonicity by addition of 50 mM sucrose. A 65 min superfusion of the cells with a guanylate cyclase inhibitor [methylene blue (MB)], a protein kinase inhibitor (H7) and a calcium-free buffer containing 10 mM Mg 2+ (OCa-10Mg) also increased Tau release and cAMP level in the cells. Acute treatment with 5 mM ammonia of cells pretreated for 24 h with 1 mM ammonia or for 65 min with MB, H7 or OCa-10Mg produced additional significant stimulation of Tau release, without further increasing the cAMP level in the cells. By contrast, a 10-min treatment with 65 mM KCl, which is a potent, cAMP-independent stimulus of Tau release in untreated Müller glia, produced no further enhancement of Tau release in ammonia-, MB-, H7 or 0Ca-10Mg-pretreated cells. The results indicate that acute treatment with ammonia, on top of treatments that evoke Tau release associated with an increase of cAMP, produces an extra Tau release that is cAMP-independent. Tau released by this extra ammonia treatment possibly originates from a different pool than Tau liberated by the pretreatments or 65 mM KCl.

Inducible Expression of Na+/Myo-inositol Cotransporter mRNA in Anterior Epithelium of Bovine Lens: Affiliation With Hypertonicity and Cell Proliferation

Bovine lenses were pretreated with physiological (314±6 mosm) or hypertonic medium (>450 mosm) for 20 hours prior to introduction of the lenses into modified Ussing chambers (providing a leakproof seal at the lens equator). The anterior (epithelial-containing) and posterior (cell-free) aspects of the mounted intact lens could thus be independently bathed with myo-[3H]inositol. Myo-inositol uptake as a function of concentration across the anterior aspect was indicative of an active carrier-mediated component and at high concentration, significant myo-inositol influx due to passive diffusion. Myo-inositol uptake was markedly stimulated across the anterior aspect and inhibitable by ouabain but not stimulated across the posterior aspect, when lenses were exposed to hyperosmotic medium by the addition of raffinose or sodium chloride. Myo-inositol rapidly desaturates across both aspects of the lens consistent with its efflux being largely a function of passive diffusional leakout from the extracellular space. In order to corroborate the apparent hypertonic-induced upregulation of gene expression inferred by kinetic analysis, the level of Na+/myo-inositol cotransporter mRNA was determined by reverse transcription and quantitative PCR using harvested anterior epithelial cells from intact lenses exposed to physiologic or hypertonic medium for 20 hours. RNA samples were microextracted from both the central and equatorial regions of the lenticular epithelium of the anterior lens capsule. The abundance of Na+/myo-inositol cotransporter mRNA was similar from epithelium of the central zone irrespective of medium tonicity but was markedly elevated from the equatorial zone of osmotically-stressed lenses. While both the quiescent epithelial cells of the central region and actively dividing epithelial cells of the equatorial zone of the intact lens express Na+/myo-inositol cotransporter mRNA, only the equatorial epithelium responds to hypertonic insult with enhanced uptake of myo-inositol due to increased transcription of the Na+/myo-inositol cotransporter gene, suggesting a possible role for the state of cell proliferation.

Molecular dissection of gating in the ClC-2 chloride channel.

The ClC-2 chloride channel is probably involved in the regulation of cell volume and of neuronal excitability. Site-directed mutagenesis was used to understand ClC-2 activation in response to cell swelling, hyperpolarization and acidic extracellular pH. Similar to equivalent mutations in ClC-0, neutralizing Lys566 at the end of the transmembrane domains results in outward rectification and a shift in voltage dependence, but leaves the basic gating mechanism, including swelling activation, intact. In contrast, mutations in the cytoplasmic loop between transmembrane domains D7 and D8 abolish all three modes of activation by constitutively opening the channel without changing its pore properties. These effects resemble those observed with deletions of an amino-terminal inactivation domain, and suggest that it may act as its receptor. Such a 'ball-and-chain' type mechanism may act as a final pathway in the activation of ClC-2 elicited by several stimuli.

Ammonia-induced taurine release from cultured rabbit Müller cells is an osmoresistant process mediated by intracellular accumulation of cyclic AMP.

A previous study demonstrated the release of newly loaded radiolabelled taurine (Tau) from cultured rabbit Müller glia not only following typical cell volume-increasing treatments with high (65 mM) potassium ions or hypotonic media, but also with ammonium chloride (further referred to as ammonia), in a dose-dependent manner, at doses ranging from physiological (0.25 mM) to those accompanying hyperammonemic coma (5 mM) (Faff-Michalak et al., Glia 10:114-120, 1994). Stimulation of Tau release by ammonia, but not by 65 mM potassium, was correlated with a dose-dependent increase of intracellular cAMP levels. The release, as measured at 5 mM ammonia, was abolished by compounds that prevented cAMP increase: an adenylate cyclase inhibitor, miconazole, a protein kinase A inhibitor HA 1004, an anion channel blocker, niflumic acid, and a Tau transport site agonist, beta-alanine. The release by ammonia differed from potassium-induced release in its resistance to 1) increase of medium tonicity by addition of 50 mM sucrose; 2) addition of the anion/cation cotransport blocker, furosemide; and 3) removal of calcium from the superfusion medium. The results suggest that ammonia-induced Tau release is mediated by intracellular accumulation of cAMP and may occur either via an osmoresistant, cAMP-controlled channel or a cAMP-activated Tau transporter. The release observed at the physiological concentration of ammonium chloride suggest a role for ammonia as a signal molecule.

The cultured human cervical epithelium: a new model for studying paracellular transport.

Human cervical cells form confluent cultures on filters with a relatively high degree of paracellular permeability. We sought to determine the extent to which acute and reversible changes in permeability in responses to modulators of paracellular resistance affect the resistance of the lateral intercellular space (RLIS), or of the tight junctions (RTJ). The question was approached experimentally by measuring the effects of combinations of physical/chemical conditions whose actions on the paracellular resistance are relatively well understood (e.g., changes in hydrostatic gradients, medium tonicity, and medium calcium concentration). Changes in paracellular permeability were determined relative to changes in transepithelial electrical resistance (TER) and to the epithelial permeability to the trisulfonic acid pyranine (Ppyr). Changes in RTJ were determined relative to changes in the transepithelial cation selectivity, as determined from changes in dilution potential. 1) Low extracellular calcium abrogates the TER and decrease transepithelial cation selectivity (i.e., decreases RTJ). 2) Changes in TER induced by changes in hydrostatic gradients and medium tonicity modulate TER but have no effect on cation selectivity (i.e., the effect was on RLIS). 3) Low-calcium-related decreases in TER are additive to those of hydrostatic and hypertonic gradients and hypotonic conditions. 4) Subluminal to luminal hydrostatic and hypertonic gradients decrease TER, but the effects are nonadditive. These results indicate that RLIS and RTJ can be selectively modulated. Based on quantitative analysis of the data it is predicated that RLIS and RTJ constitute the TER of the cultured cervical epithelium, and contribute about 25 and 75%, respectively, to the total resistance.

Effect of medium tonicity on transepithelial H(+)-HCO3-fluxes in rat proximal tubule.

1. The effect of luminal and capillary perfusion with hypotonic or hypertonic solutions containing 25 mM NaHCO3 or NaH2PO4 plus NaCl, K+, Ca2+, Mg2+ and acetate at an osmolality of 100 or 500 mosmol kg-1 on rat proximal H+ secretion was estimated by monitoring luminal pH with Sb microelectrodes. The results were compared to perfusions with the same ionic concentration in which tonicity was adjusted to 300 mosmol kg-1 with raffinose. 2. The kinetics of acidification of luminally injected bicarbonate buffer permits evaluations of H(+)-HCO3-fluxes as well as stationary pH gradients; the kinetics of alkalinization of luminally injected acid phosphate buffer indicates H(+)-HCO3-backfluxes from blood to lumen. 3. In alkalinization experiments, luminal perfusion with hypotonic solution during presence of blood in capillaries or hypotonic capillary perfusion leads to a decrease of stationary pH, an increase of alkalinization half-time and consequently a decrease of passive H(+)-HCO3-backflux. 4. In alkalinization experiments, during luminal and/or capillary perfusions with hypertonic solutions, no significant differences in the stationary pH, alkalinization half-time and H(+)-HCO3-backflux were found. 5. During acidification experiments, with both hypo- and hypertonic perfusions, no significant differences in stationary pH, acidification half-time and H(+)-HCO3-flux were observed. 6. Luminal perfusion with hypotonic solution increases specific epithelial resistance in the presence of blood in capillaries. Luminal perfusion with hypertonic solution does not change this parameter. 7. Volume changes, measured by the split-drop method, are slow during the first 30 s and do not explain the increased alkalinization half-time during luminal perfusion with hypotonic solution, since this is the period of fastest pH change. 8. Luminal perfusion with hypotonic solution decreases apparent H+ permeability in the presence of blood or hypotonic solution in capillaries. Hypertonic solutions in all experimental conditions had no significant effect on this parameter. 9. The data indicate that decrease of tonicity of fluids in contact with proximal tubule epithelium affects passive H(+)-HCO3-backflux, which proceeds in part through the shunt path, while acidification (H+ secretion), which is transcellular, is not affected by extracellular tonicity.

Role of calcium in organic osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium.

Madin-Darby canine kidney (MDCK) cells accumulate the nonperturbing osmolytes myo-inositol and betaine when grown in hypertonic medium. When returned to isotonic conditions, there is a transient basolateral efflux of these osmolytes, contributing to regulatory volume decrease. Using fura-2 fluorescence, we estimated intracellular calcium concentrations after switching MDCK cells from 500 to 300 mosM medium. Cell calcium increased 565 +/- 93 nM within 5 min. Lowering extracellular calcium inhibited the increase in cell calcium and osmolyte efflux when cells were shifted from 500 to 300 mosM medium. The calcium channel blockers lanthanum and nifedipine also inhibited osmolyte efflux after the shift from 500 to 300 mosM. In the absence of change in medium tonicity, increasing cell calcium by exposure to 1 microM ionomycin did not alter osmolyte efflux. As in PAP-HT25 cells, the cytochrome P-450 inhibitors ketoconazole and SKF-525A inhibited the efflux of both osmolytes caused by a reduction in osmolarity. Thus an early rise in cell calcium that is dependent on extra-cellular calcium and a pathway blocked by inhibitors of cytochrome P-450 oxidase are critical in regulation of osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium.

Sulfate concentrations and transport in human bronchial epithelial cells.

Inorganic sulfate concentrations in the cytoplasm of human bronchial epithelial cells exceeded levels in the bathing medium under all circumstances tested. Cell sulfate concentrations were directly related to medium sulfate concentrations and inversely related to medium chloride concentrations. In physiological media there was a sulfate compartment of approximately 0.3 mM that exchanged very slowly with extracellular sulfate. In media lacking chloride, sulfate was accumulated by the cells to a level as high as 2 mM. Sulfate uptake was markedly inhibited by external chloride and by stilbene sulfonic acid derivatives but was not affected by sodium in the medium. Efflux of 35SO4(2-) was stimulated by both chloride and sulfate in the bathing medium but inhibited by stilbenes. The following compounds had no effect on sulfate movements: phorbol esters, adenosine 3',5'-cyclic monophosphate derivatives, and okadaic acid. Changes in medium tonicity were likewise without effect. Our results suggest that human bronchial epithelial cells maintain a steady-state disequilibrium for inorganic sulfate. Furthermore, sulfate appears to exist in at least two compartments in the cells: one that is slowly exchangeable with sulfate in the medium and another exchangeable compartment that is of negligible size in physiological media but that becomes very large in media lacking chloride. Sulfate is transported by an anion exchanger of broad specificity that is not influenced by substances known to modulate chloride channels.

Medium tonicity regulates expression of the Na(+)- and Cl(-)-dependent betaine transporter in Madin-Darby canine kidney cells by increasing transcription of the transporter gene.

Betaine is one of the major compatible osmolytes accumulated by kidney derived Madin-Darby canine kidney cells cultured in hypertonic medium. Betaine is accumulated by Na(+)- and Cl(-)-dependent uptake from the medium. To gain insight into the mechanism by which hypertonicity evokes an increase in the Vmax of the betaine transporter in Madin-Darby canine kidney cells, we measured the relative abundance of mRNA for the transporter in cells shifted to a hypertonic medium and found parallel increases in mRNA abundance and cotransporter activity. The increase in mRNA levels preceded the increase in transporter activity slightly. Transcription of the gene for the transporter rose rapidly and to the same relative extent as mRNA abundance in cells shifted to hypertonic medium, indicating that transcription of the gene for the cotransporter plays a major role in regulating the accumulation of betaine in response to hypertonicity.

Regions involved in the opening of CIC-2 chloride channel by voltage and cell volume.

Regulation of cell volume is essential for every cell and is accomplished by the regulated loss or gain of intracellular ions or other osmolytes. Regulatory volume decrease often involves the parallel activation of potassium and chloride channels. Overexpression of P-glycoprotein leads to volume-activated Cl- currents but its physiological importance for volume regulation is unclear. CIC-2 is a ubiquitously expressed Cl- channel activatable by non-physiologically strong hyperpolarization. We now show that CIC-2 can be activated by extracellular hypotonicity, which suggests that it has a widespread role in volume regulation. Domains necessary for activation by both voltage and volume are localized to the amino terminus. Mutations in an 'essential' region lead to constitutively open channels unresponsive to medium tonicity, whereas deletions in a 'modulating' region produce partially opened channels responsive to both hypo- and hypertonicity. These domains can be transplanted to different regions of the protein without loss of function.

Hypotonicity and cell volume regulation in shark rectal gland: role of organic osmolytes and F-actin.

Hypotonic stress (reduction of external tonicity from approximately 900 mosM and 295 mM NaCl to approximately 600 mosM and 135 mM NaCl) produced a relatively slow regulatory volume decrease (RVD) in dogfish shark (Squalus acanthias) rectal gland cells. During the 5-h experiment, cell K+ content remained unchanged; cell content of Na+ and Cl- dropped in the initial swelling phase by some 50% (reflecting the corresponding reduction in medium NaCl), and then remained unchanged during volume recovery phase. Also, cellular fluxes of 86Rb+ and urea were not affected by hypotonic stress. However, hypotonicity enhanced 10- to 20-fold the efflux of organic cell osmolytes taurine, betaine, and trimethyloxamine, and this accounted for the loss of osmotically obliged water during RVD. Enhancement of osmolyte efflux by hypotonic stress was abolished by readjusting the low-Na+ saline to isotonicity (approximately 900 mosM) with innocuous cations (choline+, Li+, or N-methylglucamine+). The results suggest that reduction of medium tonicity may be the determinant for the RVD response to hypotonic stress. The above properties of the observed RVD were also displayed when studying changes on cell F-actin at the basolateral cell face; hypotonic stress (medium with 135 mM NaCl) produced a rapid disappearance of fluorescence related to this cytoskeletal component, whereas no such changes were seen in low-Na+ salines made isotonic with choline or N-methylglucamine chloride nor in a saline made hyposmolar by omitting urea. Hence, hypotonicity is required to affect F-actin organization (depolymerization?). These changes of F-actin fluorescence are transient; they were completed within 5-10 min of hypotonic stress, and afterwards a gradual reconstitution of cell F-actin organization was seen. The above observations are consistent with the assumption that, in shark rectal gland cells, transient loss of cytoskeleton (F-actin) organization at the basolateral cell face, induced by hypotonicity, brings about a selective efflux of organic osmolytes, thus producing the observed RVD.

Renal Na-myo-inositol cotransporter mRNA expression in Xenopus oocytes: regulation by hypertonicity

Canine renal cells in culture (MDCK cells) accumulate organic osmolytes, including myo-inositol (MI), in response to hypertonic stress. When medium tonicity is increased, intracellular concentration of MI rises because hypertonicity elicits increased uptake of MI via Na-MI cotransporter(s). To study the mechanism for this increase in cotransporter activity, poly(A)+ RNA isolated from MDCK cells maintained in hypertonic or isotonic medium was injected into Xenopus oocytes, and Na-dependent MI uptake was measured 3-5 days later. Poly(A)+ RNA from hypertonic cells induced clear expression of the cotransporter. In contrast, oocytes injected with poly(A)+ RNA isolated from MDCK cells maintained in isotonic medium exhibited cotransporter activity like oocytes injected with water. Upon size fractionation of RNA, peak activity appeared in a fraction that contained poly(A)+ RNA with median size of approximately 4 kilobases. Na-dependent MI uptake by poly(A)+ RNA-injected oocytes was inhibited by both phlorizin and phloretin. We suggest that hypertonicity-induced upregulation of the Na-MI cotransporter involves an increase in mRNA and synthesis of cotransporter protein(s).

Effect of solute permeability in determination of elastic modulus using the vesicular swelling method

The modulus of elasticity of artificial and biological membranes can be determined in membrane vesicles by monitoring the limitation of vesicular swelling during a slow decrease in medium tonicity. The higher the elastic modulus of the membrane, the more effectively the vesicles will resist swelling. This method assumes that the solutes in the system are impermeant, so that the final volume of the vesicles is determined solely by a balance of osmotic and hydrostatic forces. In this paper, we present the results of computer simulation of vesicular swelling in which the solute permeability of the membrane was varied. We find that even a small permeability will lead to a loss of solute from the vesicle that will retard the increase in vesicular volume during dilution of the medium, and thereby cause the apparent modulus of elasticity to be much greater than the true value. For example, if one takes the mannitol permeability in brush border membrane vesicles from small intestine to be 0.004 micron/s (a reasonable estimate), one finds that a vesicular swelling study using mannitol as the principal solute will show the apparent elastic modulus of the vesicles to be greater than 10 times larger than the true value. With higher permeabilities, the effect is even more dramatic. We conclude that determination of impermeance of solutes is a critical prerequisite for making valid determinations of membrane elastic modulus using the vesicular swelling method.

Stimulation of calcium-sodium exchange in dog red blood cells by hemolysis and resealing

Osmotic hemolysis and resealing greatly increase calcium influx in dog red blood cells. The resealed ghosts show a saturable calcium entry pathway with complex kinetics. As expected for a calcium-sodium exchanger, calcium uptake is stimulated by internal sodium and inhibited by external sodium. Compared to fresh, intact red cells the resealed ghost calcium-sodium exchanger is less responsive to quinidine and to alterations in medium tonicity. The differences in calcium uptake rate among cells from different donors are minimized in the ghost preparation. There are several ways to stimulate sodium-dependent calcium movements in these cells, of which hemolysis-resealing is the most potent. The results of these and previous studies suggest that dog red blood cells have a latent capacity for calcium-sodium exchange.

Osmotic Effects on Amino Acid Oxidation in Skate Liver Mitochondria

The mechanism responsible for the osmotic sensitivity of sarcosine oxidation by liver mitochondria from the little skate, Raja erinacea Mitchill, is examined. Assay medium tonicity, rather than a solute effect (urea or trimethylamine oxide), is probably responsible for the inverse relationship between osmolarity and the rate of oxidation of sarcosine by these mitochondria. Sarcosine oxidation proceeds through the flavin-linked sarcosine oxidase with the resultant glycine catabolized in the NAD-linked glycine cleavage system. The tonicity-sensitive component of the sarcosine oxidative pathway is not the glycine cleavage system. Sarcosine oxidation in the presence of rotenone is sensitive to medium tonicity. Oxidation of serine, which is also catabolized through the glycine cleavage system, is not as sensitive to tonicity as is sarcosine oxidation. Mitochondrial volume changes also appear to affect the transport of glycine. Although sarcosine does not appear to share the glycine transporter, it is possible that sarcosine transport is similarly sensitive to medium tonicity. The effects of osmolarity on the oxidation of dimethylglycine appear to support this hypothesis. Tonicity effects on sarcosine oxidase cannot yet be eliminated.

Effect of anisotonic media on cell volume and electrolyte fluxes in slices of the dogfish (Squalus acanthias) rectal gland

Osmotic responses of slices of dogfish rectal gland to hypotonic (urea-free) and hypertonic media were studied. Transfer of tissue from isotonic (890 mosM) to hypotonic (550 mosM) saline produced an osmotic swelling associated with a slow net uptake of cell K+ (and Cl−) and a slow, two-component efflux of urea. Media made hypertonic (1180 mosM) by addition of urea or mannitol produced osmotic shrinkage with a net loss of KCl. The cell osmotic responses in hypotonic media were lower than predicted for an ideal osmometer. No volume regulatory responses were seen subsequent to the initial osmotic effects. The cation influx in hypotonic media lacked specificity: in the presence of 0.5 mM ouabain or in K+-free media a net influx of Na+ was found. At steady state, the cell membrane potential evaluated from the Nernst potentials of K+ and triphenylmethyl phosphonium+, was independent of medium tonicity, suggesting the membrane potential as a determinant in the cellular osmotic response. Zero-time86Rb+ fluxes were measured:86Rb+ influx was not affected by hypotonicity, implying an unchanged operation of the Na+−K+-ATPase. On the other hand,86Rb+ efflux was significantly reduced at hypotonicity; this effect was transient, the efflux returning to the control value once the new steady state of cell volume had been reached. A controlled efflux system is therefore involved in the cell osmotic response. The absence of the volume regulatory phenomenon suggests that the cells are not equipped with a volume-sensing mechanism.

On the membrane potential independent mechanism of the sodium pump-induced inhibition of spontaneous electrical activity of japanese land snail neurons

1. 1. The dependence of excitability on pump activity of bursting pacemaker of Japanese land snail neurons was studied. 2. 2. The incubation of neurons in potassium-free solution, in spite of the fact that the membrane was hyperpolarized, the frequency of bursting activity was increased and sometimes the interburst hyperpolarization fully disappeared. 3. 3. The 4 mM potassium or rubidium pump activation after preincubation of the neurons in potassiumfree solution leads to transient inhibition of bursting activity independent of the membrane potential level. 4. 4. Adrenaline has a potentiation effect on the potassium-induced inhibition of electrical activity after preincubation of the neurons in the potassium free solutions. This effect of adrenaline was absent in ouabain containing medium. 5. 5. Increasing the external medium tonicity has an inhibitory effect on electrical activity of neurons. 6. 6. It was suggested that the pump-induced cell volume changes could be one of the membrane potential independent mechanisms by which Na-K pump could modulate the membrane excitability.

Hyperosmotic injury in mammalian cells. 3. Volume and alkali cation alterations of CHO cells in unprotected and DMSO-treated cultures

Correlated studies on volume distributions and cation (Na + and K +) content of CHO cells in suspension were carried out after various exposures to hypertonic NaCl or sucrose (500–7550 m Osm in both the presence and absence of DMSO (5–20%; w v ). The effects superimposed by ouabain (10 −2–10 −4 m), amphotericin B (6–18 μg/ml), and glutaraldehyde (1.25%) on the above-mentioned parameters were also investigated. Volumetric analysis of CHO cells with the Coulter Channelyzer indicated a biphasic dose-dependent response to hypertonic media, the duration of the TABLE 2 Correlation between Volume, Survival, and Cation Content of CHO Cells Exposed to Hypertonic Media in Suspension Osmolality m Osm Exposure (min) Hypertonic agent NaCl Sucrose V a Na + K + S b V Na + K + S 1000 60 or less Small High High High Normal Low High High 1500–2000 60 or less Small High Low Low Normal Low High High 2000 or over 60 or more Small or large c High Very low Very low Small Very low Very low Very low a V, modal volume. b S, surviving fraction [data from Ref. (18)]. c Time dependency visible during a 2-hr observation period. shrinkage- re-expansion cycle being inversely proportional to medium osmolality. In hypertonic sucrose, CHO cells apparently fail to shrink up to approximately 1000 m Osm. In both hypertonic sucrose and NaCl there is a constant loss of intracellular K + at velocities which depend primarily on medium tonicity and also on exposure length. K + loss is accompanied by increased levels of intracellular Na + only in cells exposed to hypertonic salt; in hypertonic (1–2 m) sucrose intracellular Na + shows a progressive diminution. In NaCl-DMSO experiments the amount of K + lost from the cells is less in comparison to that observed in “unprotected” cell suspensions. DMSO also mitigates excessive intracellular accumulations of Na +. In contrast to ouabain and amphotericin B, DMSO was found to slow down considerably the early volume re-expansion phase. Glutaraldehyde on the other hand totally suppressed the ability of cells to readjust their volume in hypertonic solutions. By correlating the present data with several earlier published results on CHO cells' posthypertonic survival, it appears that intracellular K + plays a key role in the chain of reactions that enable nucleated mammalian cells to sustain hypertonic stresses. Whether intracellular K + is equally important for the survival of cells at subzero temperatures remains to be determined.

Influence of medium tonicity on respiration by suspensions of human platelets.

We describe the use of graded decrements of medium osmolarity to progressively unmask respiratory capacities of whole human platelets in suspension. This departure led to the first demonstration that human platelet mitochondria are capable of tightly coupled respiration that responds to addition of mitochondrial substrates, ADP, and inhibitors in a way that other mammalian mitochondria are expected to behave. In 300 mosM media added alpha-glycerophosphate (G3P), succinate, or ADP effected only slight stimulation of base-line O2 consumption. At 180 mosM O2 consumption peaked and was not significantly affected by succinate or ADP. At 80 mosM base-line O2 consumption fell precipitously and was restored by G3P or succinate prior to being raised to its highest levels by ADP. Added NADH had no effect on O2 consumption at 80 mosM but sharply stimulated it when platelet suspensions were exposed to 60 mosM media by pretreatment with distilled water. At 80 mosM, selected compounds that inhibit or uncouple oxidative phosphorylation of isolated mammalian mitochondria from a variety of cells exerted similar influences on while platelets.

Effect of guanosine 3′:5′-monophosphate on the hydroosmotic activity of adenosine 3′:5′-monophosphate in toad urinary bladder

Guanosine 3′:5′-monophosphate has a slight hydroosmotic effect on toad urinary bladder. Furthermore, this nucleotide strongly inhibits the responses to 3′:5′-adenosine monophosphate and oxytocin. The response to an increase in medium tonicity is not modified by the guanosine nucleotide. A role for guanosine 3′:5′-monophosphate in the regulation of water permeability in toad urinary bladder is proposed.