On the role of ATP release, ectoATPase activity, and extracellular ADP in the regulatory volume decrease of Huh-7 human hepatoma cells

Hypotonicity triggered in human hepatoma cells (Huh-7) the release of ATP and cell swelling, followed by volume regulatory decrease (RVD). We analyzed how the interaction between those processes modulates cell volume. Cells exposed to hypotonic medium swelled 1.5 times their basal volume. Swelling was followed by 41% RVD40 (extent of RVD after 40 min of maximum), whereas the concentration of extracellular ATP (ATPe) increased 10 times to a maximum value at 15 min. Exogenous apyrase (which removes di- and trinucleotides) did not alter RVD, whereas exogenous Na+-K+-ATPase (which converts ATP to ADP in the extracellular medium) enhanced RVD40 by 2.6 times, suggesting that hypotonic treatment alone produced a basal RVD, whereas extracellular ADP activated RVD to achieve complete volume regulation (i.e., RVD40 ≈100%). Under hypotonicity, addition of 2-(methylthio)adenosine 5′-diphosphate (2MetSADP; ADP analog) increased RVD to the same extent as exposure to Na+-K+-ATPase and the same analog did not stimulate RVD when coincubated with MRS2211, a blocker of ADP receptor P2Y13. RT-PCR and Western blot analysis confirmed the presence of P2Y13. Cells exhibited significant ectoATPase activity, which according to RT-PCR analysis can be assigned to ENTPDase2. Both carbenoxolone, a blocker of conductive ATP release, and brefeldin A, an inhibitor of exocytosis, were able to partially decrease ATPe accumulation, pointing to the presence of at least two mechanisms for ATP release. Thus, in Huh-7 cells, hypotonic treatment triggered the release of ATP. Conversion of ATPe to ADPe by ENTPDase 2 activity facilitates the accumulated ADPe to activate P2Y13 receptors, which mediate complete RVD.

Sulfhydryl oxidation and activation of red cell K(+)-Cl- cotransport in the transgenic SAD mouse

The SAD mouse is characterized by the expression of human SAD hemoglobin (Hb), a super S Hb with a higher tendency to polymerize than HbS due to the presence of two additional mutations, Antilles beta 23Ile and D Punjab beta 121Glu. Monovalent cation transport was studied in erythrocytes from SAD-1 (Hb SAD = 19%) and beta-thal/SAD-1 (Hb SAD = 26%) mice. Erythrocytes containing Hb SAD exhibited dehydration, increased maximal rate of Na(+)-K+ pump, unchanged Rb+ flux via the Gardos channel, and increased K(+)-Cl- cotransport. K(+)-Cl- cotransport was defined as Cl(-)-dependent (substitution with sulfamate or methanesulfonate) okadaic acid-sensitive K+ efflux. Volume regulatory decrease via K(+)-Cl- cotransport was also increased in swollen SAD erythrocytes compared with controls. K(+)-Cl- cotransport was stimulated by staurosporine in all mouse strains, but the extent of stimulation was reduced in beta-thal/SAD-1 mice. Treatment with dithiothreitol reduced K(+)-Cl- cotransport activity in SAD-1 and beta-thal/SAD-1 mice to levels similar to that of control strains, indicating that reversible sulfhydryl oxidation contributes to the activated state of K(+)-Cl- cotransport in mouse erythrocytes that express transgenic human Hb SAD.

Effect of basolateral or apical hyposmolarity on apical maxi K channels of everted rat collecting tubule

We are able to evert and perfuse rat cortical collecting tubules (CCT) at 37 degrees C. Patch-clamp techniques were used to study high-conductance potassium channels (maxi K) on the apical membrane. Under control conditions (150 mM Na+ and 5 mM K+ in pipette and bathing solutions), the slope conductance averaged 109.8 +/- 6.6 pS (12 channels), and reversal potential (expressed as pipette voltage) was +26.3 +/- 2.4 mV. The percent of time the channel spends in the open state and unitary current when voltage was clamped to 0 mV were 1.4 +/- 0.7% and 3.12 +/- 0.42 pA, respectively. In six patches voltage clamped to 0 mV, the isosmotic solution perfused through the everted tubule (basolateral surface) was exchanged for one made 70 mosmol/kgH2O hyposmotic to the control saline. Open probability increased from 0.019 to 0.258, an increase of 0.239 +/- 0.065 (P ' 0.005). In four patches where a maxi K channel was evident, no increase in open probability was observed when a hyposmotic saline was placed on the apical surface. However, when vasopressin was present on the basolateral surface, apical application of hyposmotic saline resulted in a series of bursts of channel activity. The average increase in open probability during bursts was (0.055 +/- 0.017, P < 0.005). We conclude that one function of the maxi K channel located in the apical membrane of the rat CCT may be to release intracellular solute (potassium) during a volume regulatory decrease induced by placing a dilute solution on the basolateral surface or when the apical osmolarity is reduced in the presence of vasopressin. These data are consistent with the hypothesis that the physiological role of the channel is to regulate cell volume during water reabsorption.

Hypotonicity increases basolateral taurine permeability in rabbit proximal convoluted tubule

The permeabilities of the basolateral membrane of rabbit proximal convoluted tubule (PCT) to taurine (PTau) and glucose (PGlc) were estimated under control and hypotonic conditions using the initial rate of increase in cellular volume (CV) induced on isotonic replacement of 40 mM mannitol by one or the other of these substrates. Under control conditions, addition of taurine led to an increase in CV at an initial rate of 7.1 +/- 1.7%/min, leading to a cell swelling of 30.2 +/- 4.8% after 5 min (n = 6). Addition of glucose led to an increase in CV at an initial rate of 30.0 +/- 3.8%/min, leading to a cell swelling of 25.7 +/- 3.1% after 5 min (n = 7). After a period of recovery of 5 min in the absence of taurine or glucose, a 40 mosmol/kg hypotonic shock induced a cell swelling of 14.2 +/- 1.3 and 16.1 +/- 5.2%, respectively, followed by an almost complete volume regulatory decrease after 5 min. At that time, addition of taurine under continuous hypotonicity induced an increase in CV at an initial rate 2.57 +/- 0.17 times larger than that observed under the isotonic condition (P < 0.005), while addition of glucose induced an initial increase in CV identical to that observed under the isotonic condition. The increases in CV observed on addition of taurine were completely abolished in the absence of sodium under both isotonic and hypotonic conditions. The permeability to K+ was also estimated, in the absence of sodium, using the initial rate of increase in CV induced on isotonic replacement of 40 mM N-methyl-D-glucamine by K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Volume-activated Rb+ transport in astrocytes in culture

The involvement of K+ on the volume regulatory process in astrocytes was investigated by characterizing the hyposmolarity-induced efflux of K+ using 86Rb as a tracer. About 70 and 30% of the intracellular content of 86Rb was released after reductions in osmolarity from 320 to 160 or 220 mosM, respectively, during the time in which cells exhibit a volume regulatory response subsequent to swelling. No significant increase in 86Rb efflux was observed with lower reductions in osmolarity. The 86Rb efflux was Ca2+ independent and insensitive to temperature. It was inhibited by furosemide but not by bumetanide and was unaffected when nitrate, but not gluconate, replaced intracellular Cl-. The efflux was markedly inhibited by quinidine and by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Quinidine also prevented the volume regulatory decrease of cells, and this effect was overcome when a large cation permeability was imposed by gramicidin. In isosmotic conditions 86Rb efflux was not activated by N-ethylmaleimide, but this drug strongly inhibited the hyposmolarity-activated release. These findings suggest that 86Rb efflux from astrocytes associated to cell swelling is not mediated by an electroneutral cotransporter and rather favor the implication of a conductive exit pathway that may be a Ca(2+)-independent K+ channel.

Amino acid loss during volume regulatory decrease in cultured chick heart cells

Mechanisms of volume regulation in hyposomotically treated cultured chick heart cell preparations were studied using optical, biochemical, and nuclear magnetic resonance methods. This approach afforded the resolution of time-dependent responses that might ordinarily be obscured by the complex morphology of intact cardiac muscle preparations. In hyposmotic solutions, cells swelled to a peak volume within 3 min and slowly regulated toward original volume (regulatory volume decrease, RVD). Upon return of the cells to isosmotic solution following hyposmotic treatment, the cells shrank to a steady-state volume that was substantially less than the initial volume in control solution. A vigorous RVD could also be elicited by hyposmotic swelling under Cl(-)-free conditions. Measurement of both inorganic cation loss via atomic absorption spectroscopy and organic solute loss via 1H-nuclear magnetic resonance and high-pressure liquid chromatographic techniques revealed that the RVD observed following exposure to hyposomotic solutions was mediated in part by a substantial loss of taurine, glutamate, aspartate, and glycine as well as loss of inorganic ions (Na+,K+). The hyposmotically activated transport of amino acids was also associated with the production of glutamate and aspartate. The volume regulatory release and production of amino acids have significant implications for the metabolic and functional integrity of cardiac cells.

Oxygenation-activated K fluxes in trout red blood cells

The effect of oxygenation on the dissipative fluxes of K in trout red blood cells has been determined. Unidirectional influx under low oxygen tension (PO2 = 1 kPa) was 0.56 +/- 0.07 mmol.l-1 packed cells.h-1. Within a few minutes of equilibration with high oxygen tension (PO2 = 120 kPa), influx was increased 14-fold, and this was associated with a progressive loss of KCl and a cell shrinkage. K influx progressively declined over the following 3 h to levels close to those characteristic of cells at low oxygen tension. Replacement of medium Cl by NO3- or methane sulfonate inhibited the stimulation due to high oxygen as did furosemide and low extracellular pH. The oxygenation-stimulated influx was highly volume sensitive, being increased by up to 100% by osmotic swelling and decreased by osmotic shrinkage. By contrast, the small influx under low oxygen tension was unaffected by either Cl replacement or by shrinkage and increased only with extreme swelling. Thus high oxygen tension activated a Cl-dependent and furosemide-sensitive K flux. Once activated, the mechanism was rapidly deactivated on transfer back to low oxygen tension but slowly deactivated when maintained at high PO2. The oxygenation-stimulated flux mechanism promotes a rapid and more complete volume regulatory decrease than in cells at low oxygen tension.

Involvement and source of calcium in volume regulatory decrease of collapsed proximal convoluted tubule

We examined the role of Ca2+ in the volume regulatory decrease (VRD) of rabbit collapsed proximal tubules. Reduction of bath osmolality by 125 mosmol/kgH2O led to an initial cell swelling of 62.3 +/- 7.5% followed by a partial regulatory phase bringing cell volume to a value of 13.3 +/- 2.9% above control (n = 5). This swelling was accompanied by a transient intracellular Ca2+ ([Ca2+]i) increase from 174 +/- 33 to 306 +/- 67 nM (P < 0.05, n = 8). In the same condition, but in absence of extracellular Ca2+ ([Ca2+]e) [1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], VRD following hypotonic shock was identical to that observed in presence of [Ca2+]e (n = 5), and [Ca2+]i increased transiently from 136 +/- 29 to 161 +/- 31 nM (P < 0.05, n = 5). Addition of 100 microM 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an agent known to inhibit Ca2+ release from intracellular stores, did not affect the initial cell swelling (63.4 +/- 4.2%), and VRD occurred to the same extent (25.0 +/- 7.1%, n = 4), although at a lower rate. In these conditions, [Ca2+]i, which was 113 +/- 30 nM in the isotonic solution, decreased progressively to 81 +/- 20 nM over the 5-min hypotonic period (n = 5). Mere preincubation with 100 microM TMB-8 before hypotonic shock led to a VRD identical to that observed in presence of Ca2+ and absence of TMB-8 while still blocking the Ca2+ release, with cell Ca2+ decreasing progressively from 179 +/- 32 to 87 +/- 21 nM (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)