Pablo J. Schwarzbaum

Coupling of Energy Supply and Energy Demand in Isolated Goldfish Hepatocytes

The allocation of metabolic energy under fluctuating conditions of supply and demand was studied in isolated goldfish hepatocytes. Inhibition of glycolytic ATP production resulted in a decrease in the activity of one of the major ATP consuming components of cellular energy budgets, the sodium pump ((Na⁺, K⁺)-ATPase), because of the removal of substrates for the trichloroacetic acid (TCA) cycle. This was confirmed through restoration of activity to control levels by adding pyruvate and malate to the suspension. Ouabain-sensitive oxygen consumption constituted a fairly constant fraction of 15% to 25% of total oxygen consumption, whereas the relative load exerted by the pump on anaerobic energy metabolism (on the basis of measurements of Rb⁺ flux and lactate production) was much higher, ranging from 90% to 50% during the experimental period. Moreover, when pump activity was inhibited by the addition of ouabain, oxidative energy output decreased immediately, whereas under the restricted conditions of glycolytic energy supply a corresponding ouabain-sensitive component of lactate production was not observed. Such an apparent mismatch between supply and demand ofATP is interpreted as reflecting the flexibility of energy allocation under energy-limiting conditions. When ATP production was reduced by inhibiting either glycolysis or oxidative phosphorylation, the concentration of ATP in the hepatocytes dropped within 30 min from the control steady state to a new, lower, steady state that was maintained for at least 60 min.

Contrasting effects of temperature acclimation on mechanisms of ionic regulation in a eurythermic and a stenothermic species of freshwater fish (Rutilus rutilus and salvelinus alpinus)

Abstract 1. 1. Effects of temperature on aspects of ionic regulation in kidney and gills of Rutilus rutilus and Salvelinus alpinus were studied by a variety of techniques. 2. 2. In the kidney tissue of cold acclimated R. rutilus, the maximal number of binding sites per mg protein (Bmax) and the activity of Na+, K+-ATPase, as well as tissue respiration increased significantly, whereas in S. alpinus no changes in these variables were observed. On the other hand, 86Rb+ efflux from kidney tissue was unaffected by temperature in R. rutilus, whereas it was about 60% lower in 5. alpinus acclimated and measured at 5°C, than in specimens acclimated and measured at 15°C. 3. 3. In both species, the activity of gill Na+, K+-ATPase was 1.5- to 2-fold higher in cold-acclimated than in warm-acclimated fish although Bmax decreased in R. rutilus, but remained constant in S. alpinus. 4. 4. Problems of ionic regulation are discussed with regard to differences in temperature sensitivity of passive flows and primary active transport of ions in the tissues of poikilothermic animals.

Homeostasis of extracellular ATP in human erythrocytes.

We explored the intra- and extracellular processes governing the kinetics of extracellular ATP (ATPe) in human erythrocytes stimulated with agents that increase cAMP. Using the luciferin-luciferase reaction in off-line luminometry we found both direct adenylyl cyclase activation by forskolin and indirect activation through β-adrenergic stimulation with isoproterenol-enhanced [ATP]e in a concentration-dependent manner. A mixture (3V) containing a combination of these agents and the phosphodiesterase inhibitor papaverine activated ATP release, leading to a 3-fold increase in [ATP]e, and caused increases in cAMP concentration (3-fold for forskolin + papaverine, and 10-fold for 3V). The pannexin 1 inhibitor carbenoxolone and a pannexin 1 blocking peptide (10Panx1) decreased [ATP]e by 75–84%. The residual efflux of ATP resulted from unavoidable mechanical perturbations stimulating a novel, carbenoxolone-insensitive pathway. In real-time luminometry experiments using soluble luciferase, addition of 3V led to an acute increase in [ATP]e to a constant value of ∼1 pmol × (106 cells)−1. A similar treatment using a surface attached luciferase (proA-luc) triggered a rapid accumulation of surface ATP levels to a peak concentration of 2.4 pmol × (106 cells)−1, followed by a slower exponential decay (t½ = 3.7 min) to a constant value of 1.3 pmol × (106 cells)−1. Both for soluble luciferase and proA-luc, ATP efflux was fully blocked by carbenoxolone, pointing to a 3V-induced mechanism of ATP release mediated by pannexin 1. Ecto-ATPase activity was extremely low (∼28 fmol × (106 cells min)−1), but nevertheless physiologically relevant considering the high density of erythrocytes in human blood.

Effects of Temperature on the (Na⁺ + K⁺)-ATPase and Oxygen Consumption in Hepatocytes of Two Species of Freshwater Fish, Roach (Rutilus rutilus) and Brook Trout (Salvelinus fontinalis)

Effects of temperature on the (Na+ + K+)-ATPase were studied in isolated hepatocytes of two species of freshwater fish acclimated to different temperatures. Binding of 3H+-ouabain to hepatocytes was used to determine the density of (Na+ + K+)-ATPase, and oxygen consumption was measured to estimate the cost of cation pumping. Intracellular sodium concentration was manipulated by incubating hepatocytes at 20°C or 0°-4°C before the measurement of oxygen consumption. In hepatocytes of Rutilus rutilus, the maximal number of ouabain binding sites per 10⁶ cells (Bmax) was 1.86 times higher in the 5° C- acclimated than in the 20° C-acclimated group. The equilibrium dissociation constants (Kds) in both acclimation groups were not statistically different. In Salvelinus fontinalis, Bmax and Kd values did not change with temperature acclimation. Ouabain-sensitive oxygen consumption (OS) in hepatocytes of R. rutilus was 1.60-1. 73 times higher in the cold- than in the warm-acclimated group. In S. fontinalis, acclimation temperature did not affect OS. On the other hand, cold preincubation of hepatocytes significantly increased OS in both acclimation groups and species. Results are discussed with regard to species-specific differences in the adaptation to temperature change at the cellular level, the main point being that in response to cold-acclimation R. rutilus adjusts the activity of the (Na+ + K+)-ATPase in a compensatory fashion, whereas S. fontinalis modulates passive ion leakage.

Immunocytochemical localization of NTPDases1 and 2 in the neural retina of mouse and zebrafish

Ectonucleoside triphosphate diphosphohydrolases (E‐NTPDases) are a family of membrane‐bound enzymes that hydrolyze extracellular di‐ and triphosphate nucleosides. E‐NTPDases have been proposed to control extracellular nucleotide levels that mediate intercellular communication by binding to specific membrane receptors. Here we show a detailed immunocytochemical localization of two enzymes of the E‐NTPDase family in the retinal layers of two vertebrate species, namely, the mouse and the zebrafish. In the mouse retina, NTPDase2 was chiefly localized in Müller glia and ganglion cell processes. NTPDase1 was located on neurons as well, since it was expressed by horizontal and ganglion cell processes, suggesting that nucleotides such as ATP and ADP can be hydrolyzed at the surface of these cells. NTPDase1 was also detected in intraretinal blood vessels of the mouse. Regarding zebrafish, NTPDases1 and 2 seem to be differentially localized in horizontal cell processes, photoreceptor segments, and ganglion cell dendrites and axons, but absent from Müller glia. Moreover, NTPDases1 and 2 appear to be expressed within the germinal margin of the zebrafish retina that contains proliferative and differentiating cells. Retinal homogenates from both species exhibited ecto‐ATPase activity which might be attributed at least to NTPDases1 and 2, whose expression is described in this report. Our results suggest a compartmentalized regulation of extracellular nucleotide/nucleoside concentration in the retinal layers, supporting a relevant role for extracellular nucleotide mediated‐signaling in vertebrate retinas. Synapse 63:291–307, 2009.

Staurosporine-induced cell death in salmonid cells: the role of apoptotic volume decrease, ion fluxes and MAP kinase signaling

Apoptotic cell death in mammalian models is frequently associated with cell shrinkage. Inhibition of apoptotic volume decrease (AVD) is cytoprotective, suggesting that cell shrinkage is an important early event in apoptosis. In salmonid hepatoma and gill cells staurosporine induced apoptosis, as assessed by activation of effector caspases, nuclear condensation, and a decrease of mitochondrial membrane potential (MMP), and these changes were accompanied by cell shrinkage. The Cl− transport inhibitor DIDS and the K+ channel inhibitor quinidine prevented AVD, but only DIDS inhibited apoptosis. Other Cl− flux inhibitors, as well as a pan-caspase inhibitor, did not prevent cell shrinkage, but still prevented caspase activation. Furthermore, regulatory volume decrease (RVD) under hypotonic conditions was not facilitated, but diminished in apoptotic cells. Since all transport inhibitors used blocked RVD, but only DIDS and quinidine inhibited AVD, the ion transporters involved in both processes are apparently not identical. In addition, our data indicate that inhibition of Cl− fluxes rather than blocking cell shrinkage or K+ fluxes is important for preventing apoptosis. In line with this, inhibition of MAP kinases reduced RVD and not AVD, but still diminished caspase activation. Finally, we observed that MAP kinases were activated upon staurosporine treatment and that at least activation of ERK was prevented when AVD was inhibited.

An unexpected effect of ATP on the ratio between activity and phosphoenzyme level of Na+/K(+)-ATPase in steady state.

According to the Albers-Post model the hydrolysis of ATP catalyzed by the Na+/K(+)-ATPase requires the sequential formation of at least two conformers of a phosphoenzyme (E1P and E2P), followed by the K(+)-stimulated hydrolysis of E2P. In this paper we show that this model is a particular case of a more general class of models in all of which the ratio between ATPase activity (v) and total phosphoenzyme level (EP) in steady state is determined solely by the rate constants of interconversion between phosphoconformers and of dephosphorylation. Since these are thought to be unaffected by ATP, the substrate curves for ATPase activity and EP should be identical in shape so that the ratio v/EP ought to be independent of the concentration of ATP. We tested this prediction by parallel measurements of v and EP as a function of [ATP] in the absence or presence of non-limiting concentrations of K+, Rb+ or NH+4. In the absence of K+ or its congeners, both curves followed Michaelis-Menten kinetics, with almost identical Km values (0.16 microM) so that v/EP remained independent of [ATP]. In the presence of either K+, Rb+ or NH+4, v and EP increased with [ATP] along the sum of two Michaelis-Menten equations. The biphasic response of v is well known but, to the best of our knowledge, our results are the first demonstration that the response of EP to [ATP] is also biphasic. Under these conditions, the ratio v/EP increased with [ATP] from 19.8 to 40.1 s-1 along a hyperbola that was half-maximal at 9.5 microM. To preserve the validity of the current model it seems necessary to assume that ATP acts on the E1P <--> E2P transition and/or on the rate of hydrolysis of E2P. The latter possibility was ruled out. We also found that to fit the Albers-Post model to our data, the rate constant for K+ deocclussion from E2 has to be about 10-times higher than that reported from measurements of partial reactions. The results indicate that the Albers-Post model quantitatively predicts the experimental behavior of the Na(+)-ATPase activity but is unable to do this for the Na+/K(+)-ATPase activity, unless additional and yet unproved hypothesis are included.

Volumetric and ionic responses of goldfish hepatocytes to anisotonic exposure and energetic limitation

SUMMARY The relationship between cell volume and K+ transmembrane fluxes of goldfish (Carassius auratus) hepatocytes exposed to anisotonic conditions or energetic limitation was studied and compared with the response of hepatocytes from trout (Oncorhynchus mykiss) and rat (Rattus rattus). Cell volume was studied by video- and fluorescence microscopy, while K+ fluxes were assessed by measuring unidirectional 86Rb+ fluxes. In trout and rat hepatocytes, hyposmotic (180 mosmoll-1) exposure at pH 7.45 caused cell swelling followed by a regulatory volume decrease (RVD), a response reported to be mediated by net efflux of KCl and osmotically obliged water. By contrast, goldfish hepatocytes swelled but showed no RVD under these conditions. Although in goldfish hepatocytes a net (86Rb+)K+ efflux could be activated by N-ethylmaleimide, this flux was not, or only partially, activated by hyposmotic swelling (120-180 mosmoll-1). Blockage of glycolysis by iodoacetic acid (IAA) did not alter cell volume in goldfish hepatocytes, whereas in the presence of cyanide (CN-), an inhibitor of oxidative phosphorylation, or CN- plus IAA (CN-+IAA), cell volume decreased by 3-7%. Although in goldfish hepatocytes, energetic limitation had no effect on (86Rb+)K+ efflux, (86Rb+)K+ influx decreased by 57-66% in the presence of CN- and CN-+IAA but was not significantly altered by IAA alone. Intracellular K+ loss after 20 min of exposure to CN- and CN-+IAA amounted to only 3% of the total intracellular K+. Collectively, these observations suggest that goldfish hepatocytes, unlike hepatocytes of anoxia-intolerant species, avoid a decoupling of transmembrane K+ fluxes in response to an osmotic challenge. This may underlie both the inability of swollen cells to undergo RVD but also the capability of anoxic cells to maintain intracellular K+ concentrations that are almost unaltered, thereby prolonging cell survival.

Regulatory volume decrease and P receptor signaling in fish cells: mechanisms, physiology, and modeling approaches

For animal cell plasma membranes, the permeability of water is much higher than that of ions and other solutes, and exposure to hyposmotic conditions almost invariably causes rapid water influx and cell swelling. In this situation, cells deploy regulatory mechanisms to preserve membrane integrity and avoid lysis. The phenomenon of regulatory volume decrease, the partial or full restoration of cell volume following cell swelling, is well-studied in mammals, with uncountable investigations yielding details on the signaling network and the effector mechanisms involved in the process. In comparison, cells from other vertebrates and from invertebrates received little attention, despite of the fact that e.g. fish cells could present rewarding model systems given the diversity in ecology and lifestyle of this animal group that may be reflected by an equal diversity of physiological adaptive mechanisms, including those related to cell volume regulation. In this review, we therefore present an overview on the most relevant aspects known on hypotonic volume regulation presently known in fish, summarizing transporters and signaling pathways described so far, and then focus on an aspect we have particularly studied over the past years using fish cell models, i.e. the role of extracellular nucleotides in mediating cell volume recovery of swollen cells. We, furthermore, present diverse modeling approaches developed on the basis of data derived from studies with fish and other models and discuss their potential use for gaining insight into the theoretical framework of volume regulation.

Metabolic and ionic responses of trout hepatocytes to anisosmotic exposure.

SUMMARY Trout hepatocytes exposed to hypo- or hyperosmotic conditions respond by swelling and shrinking, respectively, followed by regulatory volume changes that almost, although not completely, restore cell volume. These anisosmotic conditions have a significant impact on metabolic functions. In hyposmotic medium, oxygen consumption (V̇O2) and glucose production rates were significantly reduced, whereas lactate accumulation was not significantly affected. By contrast, hyperosmotic conditions did not affect V̇O2 and lactate production but caused a sustained reduction in glucose production. Volume changes were also accompanied by alterations in intracellular free calcium ([Ca2+]i). At the cell population level, hyposmotic exposure evoked a moderate and slowly developing increase in [Ca2+]i, whereas hyperosmolarity caused a pronounced and sustained increase, which peaked at the time of maximum cell shrinkage but clearly exceeded a mere concentration effect due to volume reduction. Responses of individual cells were highly variable in hyposmotic medium, with only 60% showing a clear increase in [Ca2+]i, while in hyperosmotic conditions all cells displayed elevated [Ca2+]i levels. A decrease in intracellular pH (pHi) observed in hyposmotic medium was insensitive to EIPA, an inhibitor of Na+/H+ exchange, and SITS, an inhibitor of Cl–/HCO3– exchange, but was prevented in Cl–-free medium. In hyperosmotic medium, pHi increased. This alkalinization did not occur under conditions of blocked Na+/H+ exchange and was significantly diminished upon inhibition of Cl–/HCO3– exchange, suggesting an important role of these ion transporters in regulatory volume increase of trout hepatocytes.