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P J Schwarzbaum

Publications and source records attributed to P J Schwarzbaum.

At least 19 recordsLinked to original sources
PubMed2005

Potassium transmembrane fluxes in anoxic hepatocytes from goldfish (Carassius auratus L.).

Despite the fact that anoxic goldfish hepatocytes can maintain the transmembrane gradients of Na(+), H(+) and Ca(2+), cyanide (CN) intoxication leads to a rapid breakdown of K(+) homeostasis. In this study, [(86)Rb(+)] K(+) fluxes across the plasma membrane of goldfish hepatocytes were studied in order to identify the possible causes of this imbalance. Four minutes of cyanide incubation induced an acute and stable 61% decrease of K(+) influx (mostly driven by Na,K-ATPase activity), whereas K(+) efflux increased by 24.3%, this imbalance yielding a net K(+) efflux of 0.279+/-0.024 nmol 10(-6) cells(-1) min(-1). This uncoupling was not observed when glycolytic ATP production was inhibited with iodoacetic acid. Although the CN-induced decrease of K(+) influx was fully reversible upon washout of the inhibitor, it could not be prevented by any of the following treatments: (1) addition of 2% bovine serum albumin, which binds extracellular fatty acids known to activate specific K(+) channels; (2) addition of ascorbate, which acts as a radical scavenger; (3) inclusion of 5 mM glucose as an extracellular carbon source; and (4) removal of medium oxygen (obtained by nitrogen bubbling). Regarding the elevation of K(+) efflux in the presence of CN, neither ATP-dependent K(+) channels nor the KCl cotransporter appeared to be activated, whereas BaCl(2), an inhibitor of voltage-gated K(+) channels, decreased K(+) efflux of CN-intoxicated cells to control levels. In summary, these results indicate that, in goldfish hepatocytes, the CN-induced K(+) imbalance results from acute Na,K-ATPase inhibition together with the activation of voltage-dependent K(+) channels, the latter probably resulting from transient membrane depolarization.

Animals↗
PubMed2004

Effects of extracellular nucleotides and their hydrolysis products on regulatory volume decrease of trout hepatocytes.

In trout hepatocytes, hypotonic swelling is followed by a compensatory shrinkage called regulatory volume decrease (RVD). It has been postulated that extracellular ATP and other nucleotides may interact with type 2 receptors (P(2)) to modulate this response. In addition, specific ectoenzymes hydrolyze ATP sequentially down to adenosine, which may bind to type 1 receptors (P(1)) and also influence RVD. Accordingly, in this study, we assessed the role of extracellular nucleoside 5'-tri- and diphosphates and of adenosine on RVD of trout hepatocytes. The extent of RVD after 40 min of maximum swelling was denoted as RVD(40), whereas the initial rate of RVD was called v(RVD). In the presence of hypotonic medium (60% of isotonic), hepatocytes swelled 1.6 times followed by v(RVD) of 1.7 min(-1) and RVD(40) of 60.2%. ATP, UTP, UDP, or ATPgammaS (P(2) agonists; 5 microM) increased v(RVD) 1.5-2 times, whereas no changes were observed in the values of RVD(40). Addition of 100 microM suramin or cibacron blue (P(2) antagonists) to the hypotonic medium produced no effect on v(RVD) but a 53-58% inhibition of RVD(40). Incubation of hepatocytes in the presence of either 5 microM [gamma-(32)P]ATP or [alpha-(32)P]ATP induced the extracellular release of [gamma-(32)P]P(i) (0.21 nmol.10(-6) cells(-1).min(-1)) and [alpha-(32)P]P(i) ( approximately 8 x 10(-3) nmol.10(-6) cells(-1).min(-1)), suggesting the presence of ectoenzymes capable of fully dephosphorylating ATP. Concerning the effect of P(1) activation on RVD, 5 microM adenosine, both in the presence and absence of 100 microM S-(4-nitrobenzil)-6-tioinosine (a blocker of adenosine uptake), decreased RVD(40) by 37-44%, whereas 8-phenyl theophylline, a P(1) antagonist, increased RVD(40) by 15%. Overall, results indicate that ATP, UTP, and UDP, acting via P(2), are important factors promoting RVD of trout hepatocytes, whereas adenosine binding to P(1) inhibits this process.

Adenosine↗
PubMed2003

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

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 (86)Rb(+) 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 ((86)Rb(+))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 ((86)Rb(+))K(+) efflux, ((86)Rb(+))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.

Anaerobiosis↗
PubMed2002

Identification of two distinct E-NTPDases in liver of goldfish (Carassius auratus L.).

We have recently reported the existence of ATPase activity capable of hydrolyzing extracellular ATP and localized at the external cell membrane of goldfish hepatocytes [Am. J. Physiol. (1998) 274 R1031]. In the present study, we investigated whether one or more enzymes of the ATP diphosphohydrolase family (called E-NTPDases) are responsible for the hydrolysis of extracellular ATP and other nucleotides. Using soluble extracts from goldfish liver, enzyme activity was detected in the presence of ATP (32.1+/-4.0 nmol Pi liberated mg protein(-1) min(-1)), ADP (20.7+/-3.3 nmol Pi liberated mg protein(-1) min(-1)) and UTP (20.7+/-1.2 nmol Pi liberated mg protein(-1) min(-1)). In line with the presence of this hydrolytic activity, liver samples separated by non-denaturing gel electrophoresis and subsequently exposed to either ATP, ADP or UTP yielded a single band with enzyme activity and similar electrophoretic mobility. Subsequent SDS-PAGE electrophoresis of the active bands resulted in the appearance of two protein bands with molecular masses of 70 and 64 kDa. Immunoblotting of soluble extracts and microsomes obtained from goldfish liver, using a monoclonal antibody against CD39 (a well-known E-NTPDase), detected a single 97-kDa protein. The enzyme activity measured in solution and in native gels, together with structural information from denaturing gels plus immunoblots, points to the existence, in goldfish liver, of at least two different E-NTPDases.

Acid Anhydride Hydrolases↗
PubMed2001

Metabolic responses to epinephrine stimulation in goldfish hepatocytes: evidence for the presence of alpha-adrenoceptors.

The effect of epinephrine on various aspects of cellular metabolism was studied in hepatocytes from the goldfish Carassius auratus. Epinephrine increased cytosolic free calcium ([Ca2+](i)) from a baseline value of 108 +/- 22 nM to a peak value of 577 +/- 127 nM in suspensions of hepatocytes. Responses of single cells ranged from a single spike (66% of hepatocytes) to variable oscillatory patterns (34%). The increase in [Ca(2+)](i) was independent of the presence of extracellular Ca2+ and was prevented by the alpha-adrenergic antagonist phentolamine. Cellular glucose release induced by epinephrine (1.7- to 3.2-fold) was significantly reduced in Ca2+-depleted cells and in the presence of phentolamine, providing evidence for the co-occurrence of alpha-adrenoceptors and a Ca2+-independent, presumably beta-adrenergic, system in these cells. Furthermore, epinephrine stimulated oxygen consumption in a Ca2+-dependent manner, which was not due to stimulated Na(+) pump activity. An increased rate of acid secretion of 50%, evoked by epinephrine, appears to be mediated by enhanced Na(+)/H(+) exchange but did not result in intracellular alkalization.

Acids↗
PubMed2001

Regulation of intracellular pH in anoxia-tolerant and anoxia-intolerant teleost hepatocytes.

Mechanisms of intracellular pH (pHi) regulation were investigated in anoxia-tolerant hepatocytes from goldfish Carassius auratus, and compared to the situation in the anoxia-intolerant hepatocytes from trout Oncorhynchus mykiss. Under normoxic conditions, the pHi of goldfish hepatocytes was regulated by a Na(+)/H(+) exchanger and a Na(+)-independent Cl(-)/HCO(3)(-) exchanger, the latter being activated only after acidification of the cells. Mechanisms of acid secretion appear to be fuelled, at least in part, by lactate formation under fully aerobic conditions, as inhibition of glycolysis caused a drastic reduction of steady state proton release. In trout hepatocytes both a Na(+)/H(+) exchanger and a Cl(-)/HCO(3)(-) exchanger were found to be tonically active, as described previously. During chemical anoxia a constant pHi was maintained in goldfish hepatocytes, whereas it was reversibly reduced by 0.3 units in the trout cells. Under these conditions a reversible increase in the rate of acid secretion was induced in the cells from both species. In the goldfish cells this was based on a SITS-sensitive transporter, possibly involving export of lactate, with no contribution from Na(+)/H(+) exchange. By contrast, in hepatocytes from trout, CN-induced acid secretion was dominated by the activity of the Na(+)/H(+) exchanger. Brief exposure to extracellular acidosis had no dramatic effects on the energetics of hepatocytes from either species.

Adenosine Triphosphate↗
PubMed2000

Oxygen-dependent energetics of anoxia-tolerant and anoxia-intolerant hepatocytes.

The oxygen-dependence of cellular energetics was investigated in hepatocytes from goldfish Carassius auratus (anoxia-tolerant) and rainbow trout Oncorhynchus mykiss (anoxia-intolerant). In goldfish hepatocytes, an approximately 50 % reduction in the rate of oxygen consumption was observed in response to both acute and prolonged hypoxia, the latter treatment shifting the threshold for this reduction to a higher oxygen level. A concomitant increase in the rate of lactate production did not compensate for the decreased aerobic ATP supply, resulting in an overall metabolic depression of 26 % during acute hypoxia and of 42 % during prolonged hypoxia. Trout hepatocytes showed a similar suppression of cellular respiration after prolonged hypoxia but were unresponsive to acute hypoxia. Similarly, the rate of lactate production was unaltered during acute hypoxia but was increased during prolonged hypoxia, metabolic depression amounting to 7 % during acute hypoxia and 30 % during prolonged hypoxia. In both species, the affinity of hepatocytes for oxygen decreased during hypoxia, but this alteration was not sufficient in absolute terms to account for the observed decrease in aerobic ATP supply. Protein synthesis was suppressed in both cell types under hypoxia, whereas Na(+)/K(+)-ATPase activity decreased in trout but not in goldfish hepatocytes, emphasising the importance of membrane function in these cells during conditions of limited energy supply.

Adenosine Triphosphate↗
PubMed1999

Are the states that occlude rubidium obligatory intermediates of the Na(+)/K(+)-ATPase reaction?

In the Albers-Post model, occlusion of K(+) in the E(2) conformer of the enzyme (E) is an obligatory step of Na(+)/K(+)-ATPase reaction. If this were so the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na(+)/K(+)-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K(+) congener Rb(+). Assuming that always two Rb(+) are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb(+)]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb(+)] <10 mM. This points to the presence of an extra ATP hydrolysis that is not Na(+)-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb(+) increases ATP hydrolysis without occlusion.

Adenosine Triphosphate↗
PubMed1999

Energetics of trout hepatocytes during A23187-induced disruption of Ca2+ homeostasis.

The impact of an increase of intracellular Ca2+ i on the energy metabolism of trout hepatocytes was assessed by applying the Ca2+ ionophore A23187 and studying the consequences of the ensuing elevation of Ca2+ i on various metabolic parameters. After application of A23187 no loss of viability occurred for 2 h, but glutathione content decreased by 46%. A concomitant decrease of [ATP] as well as of Na,K-ATPase activity by over 50% could be prevented by incubating the cells in a Ca2+-free medium. Upon addition of the ionophore cellular oxygen consumption more than doubled in a strictly Ca2+-dependent manner, with half of this increase being sensitive to ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter. This increase in oxygen consumption was transient in nature and at its peak it was similar in magnitude to that induced by 2,4-dinitrophenol. Similarly, oxygen consumption sensitive to the protein synthesis inhibitor cycloheximide was transiently increased by A23187, but returned to control levels within 30 min of incubation. These results suggest that elevation of intracellular Ca2+ leads to an energetic imbalance not related to stimulation of ATP consuming processes, but mainly due to impairment of mitochondrial function, possibly by the decoupling of oxidative phosphorylation and by inducing dissipative Ca2+ cycling.

Adenosine Triphosphate↗
PubMed1998

Loss of K+ homeostasis in trout hepatocytes during chemical anoxia: a screening study for potential causes and mechanisms.

In isolated trout hepatocytes intoxication with CN- (chemical anoxia) leads to a rapid breakdown of K+ homeostasis. In the present study an attempt has been made to identify the causes and mechanisms underlying this phenomenon. Our results indicate that neither Ca2+ elevation nor cell swelling, both of which occurred during chemical anoxia and could be prevented by exposure to Ca2+ chelating agents or to hyperosmotic conditions, respectively, is solely responsible for the breakdown of K+ homeostasis. From a number of inhibitors of dissipative K+ fluxes tested, only BaCl2, an inhibitor of voltage-gated K+ channels, proved to be effective in significantly reducing K+ efflux during chemical anoxia. The KCl cotransporter known to be involved in regulatory volume decrease after hypoosmotic shock does not seem to be activated during CN(-)-induced cell swelling.

Anaerobiosis↗
PubMed1998

Functional role of ecto-ATPase activity in goldfish hepatocytes.

Extracellular [gamma-32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus gamma-32Pi due to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 +/- 0.4 nmol P(i).(10(6) cells)-1.min-1 and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 +/- 123 microM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 microM [alpha-32P]ATP to the hepatocyte suspension induced the extracellular release of alpha-32P(i) [8.2 pmol.(10(6) cells)-1.min-1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 microM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol.(10(6) cells)-1.min-1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+ (Ca2+i). This effect could be partially mimicked by adenosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of Ca2+i and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

Adenosine↗
PubMed1997

Acute and chronic effects of temperature, and of nutritional state, on ion homeostasis and energy metabolism in teleost hepatocytes.

Short- and long-term effects of temperature on ion flux and energy turnover were studied in hepatocytes from thermally acclimated trout and roach. In trout hepatocytes K+ efflux was insensitive towards acute exposure to low temperature but was downregulated during cold acclimation of the fish so as to balance the uncompensated decreased K+ (Rb+) uptake of the cells. In contrast, both K+ (Rb+) uptake and K+ efflux of roach hepatocytes were temperature sensitive in the short term. These acute effects, however, were offset during cold acclimation by a near perfect compensation of both fluxes leading to re-establishment of ion flux homeostasis at the original level. Our findings, based on a new method permitting the simultaneous monitoring of K+ efflux and uptake in the same cell population, provide experimental verification of two of the three possible strategies, recently discussed by Cossins et al. (1995), by which the ionic steady state of fish cells may adjust to acute and chronic temperature change. By comparing hepatocytes from two groups of trout, one kept on a maintenance diet (ration I), the other fed ad libitum (ration II), we discovered striking effects of nutritional state on the absolute levels as well as on the temperature relationships of K+ uptake and protein synthetic activity. Both of these functions in the hepatocytes increased in the ration II fed as compared to the ration I fed trouts, but the increase of protein synthetic activity was greater and more uniform at the three experimental temperatures than that of K+ uptake. Moreover, protein synthetic activity proved to be considerably more temperature sensitive than K+ uptake and, in contrast to the latter, showed a compensatory response after cold acclimation.

Acclimatization↗
PubMed1997

Effects of energy limitation on Ca2+ and K+ homeostasis in anoxia-tolerant and anoxia-intolerant hepatocytes.

To gain more insight into the mechanistic basis of anoxia tolerance and intolerance, a comparative study was conducted on calcium homeostasis in goldfish and trout hepatocytes subjected to different forms of energy limitation. Using the fluorescent Ca2+ indicator fura 2, we observed that both chemical anoxia and true anoxia led to an increase of the concentration of cytosolic free calcium (Ca2+i) in the anoxia-sensitive hepatocytes of rainbow trout, whereas Ca2+i was maintained at control levels in the anoxia-tolerant hepatocytes of goldfish. Various lines of evidence suggest an intracellular origin of the Ca2+ increase observed in trout cells. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition pore in mammalian cells, was ineffective in preventing the Ca2+ increase, whereas a high dose of fructose depressed the Ca2+ surge by approximately 50%. The latter effect was not accompanied by improvement of the energetic state of the cells. A comparison of chemical anoxia with true (physiological) anoxia revealed that both treatments affected energy metabolism to a similar degree in trout hepatocytes, whereas the decrease of ATP seen in goldfish hepatocytes during chemical anoxia was absent during true anoxia. Elevation of Ca2+i with the calcium ionophore A-23187 led to a decoupling of unidirectional K+ fluxes in both normoxic and anoxic trout cells, whereas in goldfish hepatocytes the coupling of K+ fluxes was not affected by the rise of Ca2+i.

Adenosine Triphosphate↗
PubMed1996

Membrane-metabolic coupling and ion homeostasis in anoxia-tolerant and anoxia-intolerant hepatocytes.

The relationship between membrane function and energy metabolism was studied in rainbow trout hepatocytes, an anoxia-intolerant cell system, and compared with the situation in hepatocytes from the goldfish, a typical anoxia-tolerant species. In trout hepatocytes, under normoxia and under chemical anoxia, inhibition of ATP consumption by the Na+ pump induced a decrease in ATP production of the same magnitude. In response to chemical anoxia, total ATP production was reduced to 15% and Na+ pump activity to 22% of the control rate under normoxia. Measurement of the cellular ATP content under these conditions revealed that, despite the reduction in Na+ pump activity, the cells became rapidly depleted of ATP, with the time course of this process resembling that observed in the anoxic rat hepatocyte. This is in contrast to the responses of goldfish hepatocytes, where, during chemical anoxia, 1) inhibition of the Na+ pump did not lead to a corresponding reduction in ATP production and 2) ATP levels, after a transient decrease, stabilized at a new steady state. To investigate the consequences of chemical anoxia on ion homeostasis, efflux and uptake rates of K+ were determined simultaneously. In the trout cells, chemical anoxia led to a decoupling of influx and efflux rates, the latter exceeding the former three- to eightfold. In contrast, goldfish hepatocytes were able to preserve ion homeostasis by a concerted decrease in Rb+ uptake and K+ efflux, so that the net flux of K+ was always close to zero. In neither species did chemical anoxia induce a change in pump density. Other potential control mechanisms are briefly discussed.

Adenosine Triphosphate↗
PubMed1995

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.

Adenosine Triphosphatases↗