Different patterns of cell volume regulation in hyposmotic media between attached and suspended HeLa cells.

Both attached and suspended HeLa cells swelled in a medium of a hypotonic osmolality of 235 mosmol/kg H2O. When the osmolality was further decreased to 166 mosmol/kg H2O, attached cells instantly swelled and then rapidly lost water and K+, followed by slow gains of them. Suspended cells instantly swelled and then K+ loss and regulatory volume decrease (RVD) occurred. Neither 0.1 mM ouabain nor 10 mM TEA changed the water loss of attached cells, whereas ouabain inhibited RVD of suspended cells. Quinine (1 mM) inhibited water losses from both cells and comparison of the losses implies stronger activation of K+ channel in attached cells than in suspended cells. Omission of medium Ca2+ or addition of 10 mM BaCl2 inhibited RVD in part. These results suggest that hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca(2+)-dependent K+ channel. Suspended cells osmotically swell, followed by RVD with K+ and Na+ releases through the K+ channel and Na(+)-pump, respectively. The different patterns of volume changes may relate to the difference of activity or time of activation of the K+ channel between both cells.     

Effects of L-alanine on membrane potential, potassium (86Rb) permeability and cell volume in hepatocytes from Raja erinacea

Isolated hepatocytes from the elasmobranch Raja erinacea were examined for their regulatory responses to a solute load following electrogenic uptake of L-alanine. The transmembrane potential (Vm) was measured with glass microelectrodes filled with 0.5 M KCl (75 to 208 M omega in elasmobranch Ringer's solution) and averaged -61 +/- 16 mV (S.D.; n = 68). L-Alanine decreased (depolarized) Vm by 7 +/- 3 and 18 +/- 2 mV at concentrations of 1 and 10 mM, respectively. Vm did not repolarize to control values during the 5-10 min impalements, unless the amino acid was washed away from the hepatocytes. The depolarizing effect of L-alanine was dependent on external Na+, and was specific for the L-isomer of alanine, as D- and beta-alanine had no effect. Hepatocyte Vm also depolarized on addition of KCN or ouabain, or when external K+ was increased. Rates of 86Rb+ uptake and efflux were measured to assess the effects of L-alanine on Na+/K+-ATPase activity and K+ permeability, respectively. Greater than 80% of the 86Rb+ uptake was inhibited by 2 mM ouabain, or by substitution of choline+ for Na+ in the incubation media. L-Alanine (10 mM) increased 86Rb+ uptake by 18-49%, consistent with an increase in Na+/K+ pump activity, but had no effect on rubidium efflux. L-Alanine, at concentrations up to 20 mM, also had no measurable effect on cell volume as determined by 3H2O and [14C]inulin distribution. These results indicate that Na+-coupled uptake of L-alanine by skate hepatocytes is rheogenic, as previously observed in other cell systems. However, in contrast to mammalian hepatocytes, Vm does not repolarize for at least 10 min after the administration of L-alanine, and changes in cell volume and potassium permeability are also not observed.     

The effects of osmotic stress on the electrical properties of the axons of a marine osmoconformer (Mala squinado. brachyura: crustacea).

In contrast to the depolarization observed in hyperosmotic media, exposure of peripheral nerve to hyposmotic conditions induced pronounced axonal hyperpolarization. It is suggested that this hyperpolarization resulted from increased potassium and chloride permeabilities which could assist axonal volume regulation in hyposmotic conditions. The hyperpolarization was readily reversible, but the spike-generating mechanism suffered irreversible damage at hyposmotic concentrations below 665 m-osmoles. It is suggested that this axonal damage contributes to the lethal effects of hyposmotic stress in this crustacean osmoconformer and, possibly, in some euryhaline osmoregulators.     

Effects of hyperosmotic medium on hepatocyte volume, transmembrane potential and intracellular K+ activity

Hepatocyte transmembrane potential (Vm) behaves as an osmometer and varies with changes in extracellular osmotic pressure created by altering the NaCl concentration in the external medium (Howard, L.D. and Wondergem, R. (1987) J. Membr. Biol. 100, 53). We now have demonstrated similar effects on Vm by increasing external osmolality with added sucrose and not altering ionic strength. We also have demonstrated that hyperosmotic stress-induced depolarization of Vm results from changes in membrane K+ conductance, gK, rather than from changes in the K+ equilibrium potential. Vm and aKi of hepatocytes in liver slices were measured by conventional and ion-sensitive microelectrodes, respectively. Cell water vols. were estimated by differences in wet and dry weights of liver slices after 10-min incubations. Effect of hyperosmotic medium on membrane transference number for K+, tK, was measured by effects on Vm of step-changes in external [K+]. Hepatocyte Vm decreased 34, 52 and 54% when tissue was superfused with medium made hyperosmotic with added sucrose (50, 100 and 150 mM). Correspondingly, aKi increased 10, 18 and 29% with this hyperosmotic stress of added sucrose. Tissue water of 2.92 +/- 0.10 kg H2O/kg dry weight in control solution decreased to 2.60 +/- 0.05, 2.25 +/- 0.06 and 2.22 +/- 0.05 kg H2O/kg dry weight with additions to medium of 50, 100 and 150 mM sucrose, respectively. Adding 50 mM sucrose to medium decreased tK from 0.20 +/- 0.01 to 0.05 +/- 0.01. Depolarization by 50% with hyperosmotic stress (100 mM sucrose) also occurred in Cl-free medium where Cl- was substituted with gluconate. We conclude that hepatocytes shrink during hyperosmotic stress, and the aKi increases. The accompanying decrease in Vm is opposite to that expected by an increase in aKi, and at least in part results from a concomitant decrease in gK. Changes in membrane Cl- conductance most likely do not contribute to osmotic stress-induced depolarization, since equivalent decreases in Vm occurred with added sucrose in cells depleted of Cl- by superfusing tissue with Cl-free medium.     

Efflux of potassium (86Rb+) attenuates the volume-restorative effect of sodium-amino acid cotransport in rat renal inner medullary cells shrunken by exposure to hyperosmotic media.

When the osmolality of the bathing medium was increased from 710 to 2000 mosmol/kg H2O, cells in incubated slices of rat renal inner medulla lost water and K+, and the rate of efflux of preloaded 86Rb+ (a tracer for K+) was significantly depressed. Addition of 2-aminoisobutyric acid (AIB, 10 mmol/l) partly restored cell water content but without re-accumulation of K+; the rate of 86Rb+ efflux was greatly increased. The presence of Ba2+ (1 mmol/l) or trifluoperazine (50 mumol/l) led to complete recovery of cell volume and K+ contents, with markedly reduced efflux of 86Rb+. Neither additive had any significant effect upon these variables in the absence of AIB or in media of 710 mosmol/kg. Efflux of 86Rb+ was pH-sensitive within the physiological range, and was depressed when external AIB was reduced below approx. 5 mmol/l. When external Na+ was increased from 145 to 500 mmol/l (total osmolality 350 to 2500 mosmol/kg) efflux was retarded only slightly if AIB was present, but markedly if AIB was omitted. Inner medullary cells may contain a class of Ba(2+)-inhibitable, calmodulin-dependent K+ conductive pathway which is activated in strongly hyperosmotic media by the operation of an inwardly-directed Na(+)-amino acid symport (cf. Law, R.O. (1988) Pflügers Arch. 413, 43-50) and which serves to moderate the volume-restorative effect of this membrane mechanism.     

Swelling-activated K+ efflux and regulatory volume decrease efficiency in human bronchial epithelial cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.     

Effects of oral contraceptives on body fluid regulation.

To test the hypothesis that estrogen reduces the operating point for osmoregulation of arginine vasopressin (AVP), thirst, and body water balance, we studied nine women (25 +/- 1 yr) during 150 min of dehydrating exercise followed by 180 min of ad libitum rehydration. Subjects were tested six different times, during the early-follicular (twice) and midluteal (twice) menstrual phases and after 4 wk of combined [estradiol-norethindrone (progestin), OC E + P] and 4 wk of norethindrone (progestin only, OC P) oral contraceptive administration, in a randomized crossover design. Basal plasma osmolality (P(osm)) was lower in the luteal phase (281 +/- 1 mosmol/kgH(2)O, combined means, P < 0.05), OC E + P (281 +/- 1 mosmol/kgH(2)O, P < 0.05), and OC P (282 +/- 1 mosmol/kgH(2)O, P < 0. 05) than in the follicular phase (286 +/- 1 mosmol/kgH(2)O, combined means). High plasma estradiol concentration lowered the P(osm) threshold for AVP release during the luteal phase and during OC E + P [x-intercepts, 282 +/- 2, 278 +/- 2, 276 +/- 2, and 280 +/- 2 mosmol/kgH(2)O, for follicular, luteal (combined means), OC E + P, and OC P, respectively; P < 0.05, luteal phase and OC E + P vs. follicular phase] during exercise dehydration, and 17beta-estradiol administration lowered the P(osm) threshold for thirst stimulation [x-intercepts, 280 +/- 2, 279 +/- 2, 276 +/- 2, and 280 +/- 2 mosmol/kgH(2)O for follicular, luteal, OC E + P, and OC P, respectively; P < 0.05, OC E + P vs. follicular phase], without affecting body fluid balance. When plasma 17beta-estradiol concentration was high, P(osm) was low throughout rest, exercise, and rehydration, but plasma arginine vasopressin concentration, thirst, and body fluid retention were unchanged, indicating a lowering of the osmotic operating point for body fluid regulation.     

Transmembrane Na+ and Ca2+ electrochemical gradients in cardiac muscle and their relationship to force development

Na+- and CA2+-sensitive microelectrodes were used to measure intracellular Na+ and Ca2+ activities (alpha iCa) of sheep ventricular muscle and Purkinje strands to study the interrelationship between Na+ and Ca2+ electrochemical gradients (delta muNa and delta muCa) under various conditions. In ventricular muscle, alpha iNa was 6.4 +/- 1.2 mM and alpha iCa was 87 +/- 20 nM ([Ca/+] = 272 nM). A graded decrease of external Na+ activity (alpha oNa) resulted in decrease of alpha iNa, and increase of alpha iCa. There was increase of twitch tension in low- alpha oNa solutions, and occasional increase of resting tension in 40% alpha oNa. Increase of external Ca2+ (alpha oCa) resulted in increase of alpha iCa and decrease of alpha iNa. Decrease of alpha oCa resulted in decrease of alpha iCa and increase of alpha iNa. The apparent resting Na-Ca energy ratio (delta muCa/delta muNa) was between 2.43 and 2.63. When the membrane potential (Vm) was depolarized by 50 mM K+ in ventricular muscle, Vm depolarized by 50 mV, alpha iNa decreased, and alpha iCa increased, with the development of a contracture. The apparent energy coupling ratio did not change with depolarization. 5 x 10(-6) M ouabain induced a large increase in alpha iNa ad alpha iCa, accompanied by an increase in twitch and resting tension. Under the conditions we have studied, delta muNa and delta muCa appeared to be coupled and n was nearly constant at 2.5, as would be expected if the Na-Ca exchange system was able to set the steady level of alpha iCa. Tension threshold was about 230 nM alpha iCa. The magnitude of twitch tension was directly related to alpha iCa.     

Effects of high altitude and water deprivation on arginine vasopressin release in men

High-altitude exposure changes the distribution of body water and electrolytes. Arginine vasopressin (AVP) may influence these alterations. The purpose of this study was to examine the effect of a 24-h water deprivation trial (WDT) on AVP release after differing altitude exposures. Seven healthy males (age 22 +/- 1 yr, height 176 +/- 2 cm, mass 75.3 +/- 1.8 kg) completed three WDTs: at sea level (SL), after acute altitude exposure (2 days) to 4,300 m (AA), and after prolonged altitude exposure (20 days) to 4,300 m (PA). Body mass, standing and supine blood pressures, plasma osmolality (Posm), and plasma AVP (PAVP) were measured at 0, 12, 16, and 24 h of each WDT. Urine volume was measured at each void throughout testing. Baseline Posm increased from SL to altitude (SL 291.7 +/- 0.8 mosmol/kgH2O, AA 299.6 +/- 2.2 mosmol/kgH2O, PA 302.3 +/- 1.5 mosmol/kgH2O, P < 0.05); however, baseline PAVP measurements were similar. Despite similar Posm values, the maximal PAVP response during the WDT (at 16 h) was greater at altitude than at SL (SL 1.7 +/- 0.5 pg/ml, AA 6.4 +/- 0.7 pg/ml, PA 8.7 +/- 0.9 pg/ml, P < 0.05). In conclusion, hypoxia appeared to alter AVP regulation by raising the osmotic threshold and increasing AVP responsiveness above that threshold.     

Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.     

Volume regulation mechanisms in Rana castebeiana cardiac tissue under hyperosmotic stress

Volume changes of cardiac tissue under hyperosmotic stress in Rana catesbeiana were characterized by the identification of the osmolytes involved and the possible regulatory processes activated by both abrupt and gradual changes in media osmolality (from 220 to 280mosmol/kg H(2)O). Slices of R. catesbeiana cardiac tissue were subjected to hyperosmotic shock, and total tissue Na(+), K(+), Cl(-) and ninhydrin-positive substances were measured. Volume changes were also induced in the presence of transport inhibitors to identify osmolyte pathways. The results show a maximum volume loss to 90.86+/-0.73% of the original volume (measured as 9% decrease in wet weight) during abrupt hyperosmotic shock. However, during a gradual osmotic challenge the volume was never significantly different from that of the control. During both types of hyperosmotic shock, we observed an increase in Na(+) but no significant change in Cl(-) contents. Additionally, we found no change in ninhydrin-positive substances during any osmotic challenge. Pharmacological analyses suggest the involvement of the Na(+)/H(+) exchanger, and perhaps the HCO(3)(-)/Cl(-) exchanger. There is indirect evidence for decrease in Na(+)/K(+)-ATPase activity. The Na(+) fluxes seem to result from Mg(2+) signaling, as saline rich in Mg(2+) enhances the regulatory volume increase, followed by a higher intracellular Na(+) content. The volume maintenance mechanisms activated during the gradual osmotic change are similar to that activated by abrupt osmotic shock.     

A slow outward current and a hypoosmolality induced anion conductance in embryonic chicken osteoclasts

In this paper we report on a hypoosmolality induced current, I(osmo), in embryonic chicken osteoclasts, which could only be studied when blocking a simultaneously active, unidentified slow outward current, I(slo). I(slo) was observed in all of the examined cells when both the intracellular and extracellular solutions contained sodium as the major cation and no potassium. The current was outwardly rectifying and activated at membrane potentials more positive than -44 +/- 12 mV (n = 31). The time to half activation of the current was also voltage dependent and was 350 ms at Vm = +80 mV, and 78 ms at Vm = +120 mV. The current did not inactivate during periods up to 5 s. Extracellular 4-AP (5 mM), TEA (5 mM) and Ba2+ (1 mM), blockers of K+ conductances in chicken osteoclasts, did not influence I(slo). However, I(slo) was inhibited by 50 microM extracellular verapamil, which allowed us to study I(osmo) in isolation. Exposure of the osteoclasts to hypotonic solution resulted in the development of a depolarization activated I(osmo). It developed after a 1-min delay and reached its maximum within 10 minutes. Half-maximal activation occurred after 4.4 +/- 0.9 min (n = 9). The current activated within a few ms upon depolarization and did not inactivate during at least 5 sec. I(osmo) reversed around the calculated Nernst potential for Cl- (E(Cl) = +7.3 mV and V(rev) = +5.4 +/- 3.6 mV, n = 9). The underlying conductance, G(osmo) exhibited moderate outward rectification around 0 mV in symmetrical Cl- solutions. Ion substitution experiments showed that G(osmo) is an anion conductance with P(Cl) approximately = P(F) > P(gluc) > P(Na). I(osmo) was blocked by 0.5 mM SITS but 50 microM verapamil, 5 mM TEA, 5 mM 4-AP, 1 mM Ba2+, 50 microM cytochalasin D and 0.5 mM alendronate did not have any effect on the current. Cl- currents have been implicated in charge neutralization during osteoclastic acid secretion for bone resorption. The present results imply that osmolality may be a factor controlling this charge neutralization.     

Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of beta-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs-Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l beta-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that beta-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that beta-alanine exerted its protective effects by interfering with Na+ accumulation induced by hypoxia. The addition of gamma-amino-butyric acid, which interfered with beta-alanine uptake by the hepatocytes or of Na+/H+ ionophore monensin, reverted beta-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving beta-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K+ efflux from hepatocyte with BaCl(2) (1mmol/l). Altogether these results indicated that beta-alanine protected against hypoxic liver injury by preventing Na+ overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.     

Inward rectification in rat olfactory receptor neurons.

Inwardly rectifying currents in enzymically dissociated olfactory receptor neurons of rat were studied by using patch-clamp techniques. Upon hyperpolarization to membrane potentials more negative than -100 mV, small inward-current relaxations were observed. Activation was described by a single exponential with a time constant that decreased e-fold for a 21 mV hyperpolarization. The current was not reduced by the external application of 5 mM Ba2+, but was abolished by the addition of 5 mM Cs+ to the bath solution. Increasing the external K+ concentration ([K+]o) to 25 mM dramatically enhanced the current without affecting the voltage range or the kinetics of activation. In 25 mM [K+]o, tail currents reversed at -26 mV, significantly more positive than the K+ equilibrium potential of -44 mV. These characteristics are consistent with those of a mixed Na+/K+ inward rectification that has been reported in several types of neuronal, cardiac and smooth muscle cells. The current may contribute to controlling cell excitability during the response to some odorants.     

Na+-H+ exchange in isolated renal brush-border membrane vesicles in response to metabolic acidosis. Kinetic effects

Chronic metabolic acidosis increased the Na+-H+ exchange activity in isolated renal brush-border membrane vesicles. Treatment altered the initial rate of Na+ uptake by increasing Vm (acidotic, 15.3 +/- 0.7 nmol of Na+ X mg-1 X 2 s-1; normal, 11.3 +/- 0.9 nmol of Na+ X mg-1 X 2 s-1), and not the apparent affinity KNa+ (acidotic, 10.2 +/- 0.5 mM; normal 10.2 +/- 0.6 mM). Metabolic acidosis resulted in the proportional increase in 1 mM Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and acidotic rats. When the data were analyzed by the Hill equation, it was found that metabolic acidosis did not change the n (acidotic, 1.33 +/- 0.13; normal, 1.43 +/- 0.07) or the K'H+ (acidotic, 0.27 +/- 0.05 microM; normal, 0.28 +/- 0.06 microM), but increased the apparent Vm (acidotic, 1.10 +/- 0.08 nmol of Na+ X mg-1 X 2 s-1; normal, 0.81 +/- 0.07 nmol of Na+ X mg-1 X 2 s-1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and acidotic animals was not influenced by membrane potential. We conclude that metabolic acidosis leads to either an increase in the number of functioning exchangers or an increase in the turnover rate of the limiting step in the exchange.     

Ion transport in a human lens epithelial cell line exposed to hyposmotic and apoptotic stress

Membrane transport changes in human lens epithelial (HLE‐B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium content, K_i, uptake of the K congener rubidium, Rb_i, and water content were measured after hyposmotic stress induced by hypotonicity, and apoptotic stress by the protein‐kinase inhibitor staurosporine (STP). Cell water increased in hyposmotic (150 mOsm) as compared to isosmotic (300 mOsm) balanced salt solution (BSS) by >2‐fold at 5 min and decreased within 15 min to baseline values accompanied by a 40% K_i loss commensurate with cell swelling and subsequent cell shrinkage likely due to regulatory volume decrease (RVD). Loss of K_i, and accompanying water, and Rb_i uptake in hyposmotic BSS were prevented by clotrimazole (CTZ) suggesting water shifts associated with K and Rb flux via intermediate conductance K (IK) channels, also detected at the mRNA and protein level. In contrast, 2 h after 2 µM STP exposure, the cells lost ～40% water and ～60% K_i, respectively, consistent with apoptotic volume decrease (AVD). Indeed, water and K_i loss was at least fivefold greater after hyposmotic than after apoptotic stress. High extracellular K and 2 mM 4‐aminopyridine (4‐AP) but not CTZ significantly reduced apoptosis. Annexin labeling phosphatidylserine (PS) at 15 min suggested loss of lipid asymmetry. Quantitative PCR revealed significant IK channel expression during prolonged hyposmotic stress. Results suggest in HLE‐B3 cells, IK channels likely partook in and were down regulated after RVD, whereas pro‐apoptotic STP‐activation of 4‐AP‐sensitive voltage‐gated K channels preceded or accompanied PS externalization before subsequent apoptosis. J. Cell. Physiol. 223: 110–122, 2010. © 2009 Wiley‐Liss, Inc.     

The value of urine osmolality as an index of stress in the ovine fetus

In ovine fetuses, during 100-130 days of gestation, urine osmolalities less than 175 mosmol/kg water were associated with plasma immunoreactive adrenocorticotrophin (ACTH) concentrations below 40 pg/ml in 40/41 samples. In 18/29 fetuses with urine osmolalities greater than 175 mosmol/kg water plasma ACTH was significantly elevated. In 38 samples of fetal blood there was a significant correlation between plasma ADH and ACTH concentrations. By least squares regression the equation to the line was [ACTH] = 5.06 + 3.70 [ADH] (r = 0.62, P less than 0.001). In 50 samples from fetuses of gestational ages 100-140 days, with urine osmolalities of 302 +/- 86 mosmol/kg (mean +/- SD) the blood pH, pO2 and pCO2 values were not significantly different from those in 50 samples from fetuses with urine osmolalities of 125 +/- 22 mosmol/kg. It is proposed that the measurement of fetal urine osmolality provides a good index of fetal stress. A fetus with a urine osmolality less than 175 mosmol/kg is almost invariably in the optimum, unstressed condition.     

Electrophysiological examination of transmitter release in non-quantal form in the mouse diaphragm and the activity of membrane ATP-ase.

The subsynaptic area of mouse diaphragm fibres was hyperpolarized by 1--2 mV during local curarization of the junctional zone in the presence of the reversible anticholinesteraze prostigmine (6 X 10(-6) M), or after treatment of the muscle with organophosphate cholinesterase inhibitor Soman. In a solution containing 5 mM K+ the mean hyperpolarization was 1.1 +/- 0.27 mV at mean resting potential--70 mV. After adding 2 X 10(-5) M ouabain the hyperpolarization increased to 1.5 +/- 0.25 mV. Removal of potassium ions from the bathing medium also increased curare induced hyperpolarization to 1.80 +/- 0.40 mV. Reactivation of membrane ATP-ase by addition of K+ after a period in K+-free medium reduced the hyperpolarization to zero, where measurements were performed 10--20 min after the readdition. It was concluded that spontaneous non-quantal leakage of acetylcholine occurs at the mouse neuromuscular junction, as it does in the frog (ref. Katz and Miledi 1977). Conditions which block the Na+-K+-dependent ATP-ase of nerve terminals increased the continuous leakage of ACh and activation of the pump decreased it.     

Cell volume affects glycogen phosphorylase activity in fish hepatocytes

The activity of glycogen phosphorylase (GPase) in the active a-form (GPase a) is dependent on the hydration state of hepatocytes. We establish that GPase a catalysis in catfish (Ameiurus nebulosus) hepatocytes is a function of medium osmolarity and that a linear relationship exists between GPase a activity and osmolarity between 254 mosmol l^–1 and 478 mosmol l^–1. Exposure of isolated hepatocytes to hyperosmotic media increases enzyme activity up to 7-fold, indicative of covalent phosphorylation. GPase activation associated with cell shrinkage peaks within 10 min of exposure. The average degree of activation (2.7-fold-increase of GPase a) is only slightly less than in hepatocytes exposed to glucagon (3.1-fold-increase) under isosmotic conditions; with glucagon, the maximum is reached within 2 min. Phosphorylation status remains elevated during the entire 40 min experimental period; cells do not undergo regulatory volume increase (RVI) during this period and do not regain pre-exposure volume. We interpret the increased GPase a activity as an inherent response to hyperosmotic stress, likely brought about by molecular crowding. Activation of the enzyme results in increased glucose production from endogenous glycogen. Glucose is not retained in the liver cells, but may act as an oxidative substrate in extrahepatic tissues for the increased metabolic demand of ion regulation. Protein kinase A or intracellular Ca²⁺ make apparently small contributions to the activation of GPase, leaving us to speculate on alternate routes of enzyme activation. Conversely, hepatocyte swelling in hyposmotic medium leads to significant decreases in GPase a activity and curtailed glucose output. A minimum is attained in 10 min, and pre-insult rates are re-established within 40 min, somewhat lagging behind readjustment in cell volume by regulatory volume decrease (RVD). We conclude that cell swelling and subsequent RVD do not signify stress to the cells and metabolic demand may be decreased under cell swelling conditions. Alteration of GPase phosphorylation with extracellular osmolarity appears to be a general phenomenon, since we also find it in hepatocytes of another freshwater catfish (Clarias batrachus) and a marine scorpaenid (Sebastes caurinus).Abbreviations BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid - BSA bovine serum albumin - cAMP adenosine 3',5-cyclic monophosphate - GPase glycogen phosphorylase - MDH malate dehydrogenase - MHM modified Hanks medium - PKA c-AMP dependent protein kinase A - 8-Br-Rp-cAMPS 8-Bromo-Rp-3',5'-cyclic adenosine monophosphorothioate - RT room temperature - RVD regulatory volume decrease - RVI regulatory volume increaseCommunicated by L.C.-H. Wang     

Effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum (T-strain mycoplasma).

The effect of ammonium ion concentration and osmotic pressure on growth of Ureaplasma urealyticum type VIII was determined by using a well-buffered broth medium containing 10 mM urea. The addition of NH4Cl to the medium at concentrations up to 10 mM did not affect growth; however, addition of larger quantities progressively decreased both the specific growth rate (mu) and the maximum yield of the culture, with concentrations of 80 mM completely inhibiting growth. Addition of either 150 mM KCl or NaCl to the medium did not inhibit growth, indicating that the growth-inhibitory effect was specific to NH4+ and was neither a result of increased Cl- concentration nor increased osmotic pressure. Concentrations of NH4Cl as high as 100 mM did not affect growth of either Acholeplasma laidlawii or Mycoplasma hominis. U. urealyticum was more sensitive to osmotic pressure: osmotic pressures of 710 to 780 mosmol/kg (with KCl, NaCl, or sucrose) resulted in both a substantially lower growth rate and a 5- to 10-fold lower peak yield of organisms. Both A laidlawii and M. hominis were less sensitive to increased osmotic pressure.