Increased Pump Activity Research Articles

The calcium sensing receptor modulates H+-ATPase activity in intercalated cells.

Previous studies found that calcium sensing receptor (CaSR) it's expressed in intercalated cells of the collecting duct and that its activation by calcium in the luminal membrane promotes acidification of urine. Therefore, the aim of the study was to analyze the effects of CaSR stimulus on the biochemical activity of the vacuolar H+-ATPase in a cellular model of intercalated cells, MDCK-C11 cells. Biochemical activity of H+-ATPase was performed using cell homogenates and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium ([Ca2+]i) were also determined using Fluo-4. A significant increase of vacuolar H+-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd3+ (300 μM), neomycin (200 μM) and by the calcimimetic R-568 (1 μM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca2+ concentration ([Ca2+]o) between 10-2 and 2 mM. The calciolytic NPS 2143 (150 nM) significantly reduced the vacuolar H+-ATPase activity observed with 2 mM [Ca2+]o. Inhibition of phospholipase C (PLC) activity with U73122 (5 x 10-7 M) reversed the increase in pump activity observed in the presence of Gd3+. Activation of CaSR by the specific CaSR agonist R-568 produced a sustained rise of [Ca2+]i, an effect that disappears when extracellular calcium was removed in the presence of thapsigargin. In summary, CaSR stimulation induces an increase in the vacuolar H+-ATPase activity of MDCK-C11 cells, an effect that involves an increase in [Ca2+]i and require PLC activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially reducing the formation of calcium phosphate stones.

α1-Na/K-ATPase inhibition rescues aberrant dendritic calcium dynamics and memory deficits in the hippocampus of an Angelman syndrome mouse model

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of function of the maternal copy of the UBE3A gene. Previous studies reported an increase in α1-Na/K-ATPase (α1-NaKA) expression in the AS hippocampus at the age of 2 weeks as the initial and isolated molecular alteration. This increase was further implied upon actuating much of the hippocampal-related deficits in an AS mouse model, although the underlying mechanism was never investigated. Here, we showed that enhanced α1-NaKA expression resulted in increased pump activity that reduced activity-dependent dendritic Ca2+ dynamics in the AS hippocampus, as well as selective inhibition of α1-NaKA by marinobufagenin (MBG) to normalize these aberrant Ca2+ dynamics. In addition, we demonstrated that selective α1-NaKA inhibition corrected impaired hippocampal synaptic plasticity and hippocampal-dependent cognitive deficits. Furthermore, we showed that the isolated increase in hippocampal α1-NaKA expression in AS mice at 2 weeks of age was accompanied by an unexpected enhancement in excitability. Altogether, our study implicates the modification of Ca2+ dynamics as one of the major underlying mechanisms by which enhanced α1-NaKA expression induces deleterious effects in the hippocampus of AS model mice. Finally, we propose a therapeutic approach for AS and possibly other neurodevelopmental disorders that entail aberrant NaKA expression or abnormal Ca2+ dynamics.

Impact of Farnesol as a Modulator of Efflux Pumps in a Fluconazole-Resistant Strain of Candida albicans.

Aim: This work studied the impact of the quorum-sensing molecule, farnesol (FAR), on fluconazole (FLC)-resistant Candida albicans isolate CY 1123 compared with the susceptible standard strain C. albicans SC5314. The genes encoding efflux pumps belonging to the ATP-binding cassette (ABC) and major facilitator superfamilies, together with overexpression or point mutation of the ERG11 gene, are the main resistance mechanisms to azole antifungal drugs. Results: The upregulation of genes coding for CDR1, CDR2, and MDR1 were confirmed by qPCR with respect to the housekeeping gene ACT1 in the resistant strain. The contribution of the ERG11 gene was also observed. Markedly, increased pump activity (Cdr1 and/or Cdr2) in the CY 1123 strain was confirmed using diS-C3(3) assay. However, the addition of FAR to the yeasts diminished the difference in staining levels between the SC5314 and CY 1123 strains, demonstrating the concentration-dependent character that could be caused by an effective modulation of Cdr pumps. FAR (60 and 100 μM) was also able to decrease the minimal inhibitory concentrations (MIC50), denoting the inhibition of planktonic cells by 50%, from 8 to 4 μg/mL of FLC when the resistant strain CY 1123 was not cultivated with FLC. However, when it was exposed to 64 μg/mL of FLC, the MIC50 shifted from 64 to 8 μg/mL. Conclusion: Besides the many other effects of FAR on eukaryotic and prokaryotic cells, it also affects ABC efflux transporters, resulting in changes in resistance to azoles in C. albicans isolates. However, this effect is dependent on FAR concentrations.

Regulation of the cardiac sodium pump

In cardiac muscle, the sarcolemmal sodium/potassium ATPase is the principal quantitative means of active transport at the myocyte cell surface, and its activity is essential for maintaining the trans-sarcolemmal sodium gradient that drives ion exchange and transport processes that are critical for cardiac function. The 72-residue phosphoprotein phospholemman regulates the sodium pump in the heart: unphosphorylated phospholemman inhibits the pump, and phospholemman phosphorylation increases pump activity. Phospholemman is subject to a remarkable plethora of post-translational modifications for such a small protein: the combination of three phosphorylation sites, two palmitoylation sites, and one glutathionylation site means that phospholemman integrates multiple signaling events to control the cardiac sodium pump. Since misregulation of cytosolic sodium contributes to contractile and metabolic dysfunction during cardiac failure, a complete understanding of the mechanisms that control the cardiac sodium pump is vital. This review explores our current understanding of these mechanisms.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1134-y) contains supplementary material, which is available to authorized users.

Nanomolar concentrations of ouabain stimulate Na‐K ATPase through sodium hydrogen exchanger‐1 (NHE1) dependent mechanism.

Recent investigations have shown increased Na‐H exchanger activity and plasma levels of endogenous cardiotonic glycosides, such as ouabain in spontaneously hypertensive rats. At high concentrations, ouabain inhibits pump activity, and at lower concentrations, it increases pump activity and induces cell proliferation. Stimulation of NHE1 and Na‐K ATPase activity involve protein kinase B (AKT), suggesting that both NHE1 and Na‐K ATPase are mediated by the same proteins. We hypothesize that the activity of NHE1 and Na‐K ATPase are linked. In the present study we aim to 1) determine the role of activation of NHE1 in ouabain‐sensitive stimulation of Na‐K ATPase, 2) determine if NHE1 and Na‐K ATPase association increases when cells were treated with NH4Cl or ouabain in various kidney proximal tubule cell lines (OK, HK2, HK5, and HK11). Our data suggests that NH4Cl stimulation of NHE1 increases ouabain sensitive 86Rb uptake in all cell lines examined.Treatment with ethylisopropylamiloride (EIPA) a NHE1 specific inhibitor decreased Na‐K ATPase activity in HK5 and HK11 cells and prevented nanomolar ouabain‐stimulated Na‐K ATPase activity in OK cells. Immunoblot analysis indicated that NH4Cl increased membrane expression of NHE1 and Na‐K ATPase in OK and HK11 cells. Surprisingly, treatment with EIPA alone increased membrane expression of NHE1 and Na‐K ATPase. The reason for increased presence of these proteins in the membrane is unknown, however, tyrosine phosphorylation of the catalytic subunit of Na‐K ATPase, believed to be an indication of Na‐K ATPase activation, is decreased under these same conditions. These results suggest that activation of NHE1 is critical for ouabain stimulated increase in Na‐K ATPase activity, and that upon activation, NHE1 and Na‐K ATPase associate, perhaps to form a signalosome.

Involvement of the cytoskeleton in the effect of PGE 2 on ion transport in the rat distal colon

This work aimed at studying the effect of PGE 2 on water and chloride absorption from the rat distal colon and at investigating the involvement of the cytoskeleton in the modulation of colonic transporters. PGE 2 increased significantly net water and chloride absorption. It increased also the activity of the Na +K +-ATPase and the expression of the Na +K +2Cl − cotransporter. The increase in pump activity was ascribed to its phosphorylation by PKA or PKC when activated upon binding of PGE 2 to its receptors, and was deemed responsible for the increase in Cl − absorption. Cytochalasin B (CytoB), a disrupter of microfilaments, decreased net water and chloride absorption in presence or absence of PGE 2. Furthermore it down-regulated both pump and cotransporter, and lowered Na +K +-ATPase activity. It was suggested that an intact actin cytoskeleton is required for the basal and the PGE 2-elicited trafficking of both transporters. On the other hand, colchicine, an inhibitor of microtubule polymerization, had no effect on the absorption of water and chloride but abrogated the stimulatory effect of PGE 2. Colchicine exerted a similar effect to that of cytochlasin on the expression of both pump and cotransporter in presence or absence of PGE 2 except for the basal activity of the pump which was not altered by microtubule disruption. It was concluded that both microfilament and microtubular networks are involved in the basal and PGE 2-elicited increase in colonic ion absorption.

Cys-113 and Cys-422 Form a High Affinity Metalloid Binding Site in the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg(2+)-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.

Interaction with the Na,K-ATPase and Tissue Distribution of FXYD5 (Related to Ion Channel)

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the alpha subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates alpha. Co-expressing FXYD5 with the alpha1 and beta1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive (86)Rb(+) uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.

Activity of yeast multidrug resistance pumps during growth is controlled by carbon source and the composition of growth-depleted medium: DiS-C 3(3) fluorescence assay

Like other tested wild-type strains (DTXII and IL-125-2B), exponential glucose- and/or fructose-grown cells of Saccharomyces cerevisiae BY4742 exhibit the previously described high activity of Pdr5p and Snq2p pumps (measured as export of the potentiometric fluorescent probe diS-C 3(3)). Upon saccharide depletion from the medium the pump activity in these cells, which differ from other strains in having a lower membrane potential, sharply drops to a very low level similar to that found in cells grown on ethanol or glycerol. This negligible pump activity in respiring cells thus appears to have a universal character. Addition of glucose or fructose to respiring BY4742 cells grown to low culture densities restores multidrug resistance pump activity due partly to pump synthesis in pre-existing cells and partly to the high pump activity of newly grown cells; no such pump activity boost occurs when the sugar is added to high-density cultures of ethanol-grown or post-diauxic glucose-grown cells, even if these cultures are diluted to low density by their original growth-depleted medium. A strong sugar-induced increase in pump activity is found solely if respiring cells from high-density cultures are resuspended in fresh YPD or YPE medium before sugar addition. Its absence in respiring cells suspended in growth-depleted medium reflects an as yet unidentified effect of the composition of the growth-exhausted medium (depletion of some components and/or accumulation of extracellular metabolites during yeast growth) on sugar-induced pump activity rise.

Ang II attenuates IGF-1-stimulated Na+, K+-ATPase activity via PI3K/Akt pathway in vascular smooth muscle cells

We have investigated the role of phosphatidylinositol 3-kinase (PI3K) and serine/threonine protein kinase B (Akt) in mediating vascular smooth muscle cells (VSMC) sodium pump (Na+, K(+)-ATPase) regulatory interactions between insulin-like growth factor-1 (IGF-1) and angiotensin II (Ang II). Treatment with IGF-1 (100 nM) for 30 min or Ang II (100 nM) for 10 min increased sodium pump activity. Pretreatment with Ang II for 10 min, abolished IGF-1 increased sodium pump activity. Given separately for 6 h, Ang II and IGF-1 stimulated alpha1 mRNA accumulation. Phosphorylation on Ser473 of Akt was increased after treatment with both IGF-1 and Ang II. Pretreatment with 100 nM of PI3K inhibitor Wortmannin (WT) for 30 min decreased: IGF-1 and Ang II-stimulated pump activity, phosphorylation of Akt and PI3K protein expression. Pretreatment with Ang II attenuated IGF-1-stimulated sodium pump activity, phosphorylation of Akt and PI3K protein expression. IGF-1 increased the association between IRS-1 and p85, and Ang II as well as PI3K inhibition decreased this IGF-1 effect. These results suggest that Ang II, which increases pump activity alone, reduces the IGF-1 stimulation of sodium pump activity by attenuating PI3K/Akt signaling. These results implicate PI3K/Akt pathways in Ang II/IGF-1 regulation of the sodium pump in VSMC.

Modulation of corneal endothelial hydration control mechanisms by Rolipram.

Corneal stromal hydration is maintained by an active HCO3- transport mechanism located in the corneal endothelium. Whilst modulation of transport activity by changes in intracellular cAMP concentration have been noted, the site of effect is undefined. To resolve this question, the effects of Rolipram, a cAMP phosphodiesterase inhibitor, on endothelial physiology were determined. Addition of 0.1 mM Rolipram caused a threefold increase in intracellular cAMP with no change in cGMP. Associated with the increase in cAMP was a transient whole corneal thinning and a similarly transient increase in trans-endothelial potential difference, short-circuit current and resistance. The membrane potential hyperpolarized and the intracellular Na+ concentration decreased. The decreased intracellular Na+ was associated with an increased rate of Na+ extrusion between the endothelial cell and extracellular space. It is concluded that Rolipram increases the concentration of cAMP which activates the basolateral membrane Na+/K+-ATPase activity and increases net HCO3- transport. In addition there is a reduction in endothelial permeability which combined with the increase in pump activity may jointly explain the observed stromal thinning. The duplicity of responses indicates that if cAMP has a physiological role in regulating corneal hydration then it may operate on both the endothelial pump and the endothelial permeability.

Proteolipids: small hydrophobic peptides in the field of sodium tolerance

High soil salinity that develops in areas surrounding deserts is an increasing threat for agriculture. It has thus become an important goal for basic and applied research to identify the pathways regulating salt sensitivity in plants. Recently, major progress has led to the identification of plant genes encoding sodium transporters. The AtNHX1 protein mediates sodium detoxification by sequestration in the vacuole. The SOS1 protein has been proposed to function as Na+/H+ antiporter leading to Na+ efflux from plant cells and the AtHKT1 potassium transporter could also mediate the uptake of sodium in plants.Catherine Navarre and Andre Goffeau1xMembrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane. Navarre, C. and Goffeau, A. EMBO J. 2000; 19: 2515–2524Crossref | PubMedSee all References1 have identified PMP3 as a new determinant of sodium tolerance in yeast. Unlike AtHKT1, AtNHX1 and SOS1, whose molecular architectures are typical for membrane transporters, PMP3 belongs to the much less characterized class of small highly hydrophobic peptides called proteolipids. In their study, Navarre and Goffeau show that, in yeast, PMP3 disruption leads to a strong increase in salt sensitivity. This phenotype is not linked to a regulation of sodium efflux pumps by PMP3 because the increase of salt sensitivity as a result of PMP3 disruption or efflux pump disruption are additive. Furthermore, PMP3 disruption restores the growth of yeast potassium transport mutants. Finally, the authors identify membrane hyperpolarization as the central mechanism underlying the increase of K+ and Na+ uptake. This effect on membrane polarization is not linked to an increase in pump activity. Therefore PMP3, a membrane protein, probably mediates or upregulates an ion leakage pathway modulating yeast membrane potential.Gene families encoding proteolipids have now been identified in bacteria, fungi, plants and animals. In plants, proteolipid genes have been found in Lophopyrum elongatum, wheat, barley and Arabidopsis. Three lines of argument suggest that they could also be involved in the determination of plant sodium sensitivity. First, they show high sequence similarity with yeast PMP3. Second, Navarre and Goffeau demonstrate that RCI2A, one of the PMP3 homologues from Arabidopsis, can complement the salt hypersensitivity phenotype of a yeast mutant with disrupted PMP3. Third, in L. elongatum, the PMP3 homologue ESI3 has been shown to be rapidly and transiently induced after exposure to high sodium concentrations, suggesting that it provides early protection against salt stress. Altogether, the data presented by Navarre and Goffeau suggest that an unexpected class of protein, the proteolipids, represent new molecular determinants for salt resistance in yeast and plants.

Molecular Dissection of the C-Terminal Regulatory Domain of the Plant Plasma Membrane H+-ATPase AHA2: Mapping of Residues that When Altered Give Rise to an Activated Enzyme

The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants. The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues that when altered lead to increased pump activity group together in two regions of the C-terminus. One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases. Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins.

Short-term regulation of endothelial Na(+)-K(+)-pump activity by cGMP: a 133Cs magnetic resonance study.

The effect of nitric oxide radicals (NO) on the activity of porcine aortic endothelial Na(+)-K(+)-ATPase is reported. Measurements were made using an in vitro cell system and 133Cs magnetic resonance (NMR). It is shown that NO, through stimulation of guanylate cyclase, results in a reduction of pump activity. Similar observations were made using 8-Br-cGMP. Measurement of the cytosolic volume indicated no changes in volume during incubation with 8-Br-cGMP. Our measurements indicate a continuous regulation of endothelial Na(+)-K(+)-ATPase activity by endogenous NO. This regulation could be removed by L-NAME, resulting in a small increase in pump activity.

Effects of veratridine on sodium currents and fluxes.

Veratridine causes Na+ channels to stay open during a sustained membrane depolarization by abolishing inactivation. The consequential Na+ influx, either by itself or by causing a maintained depolarization, leads to many secondary effects such as increasing pump activity, Ca2+ influx, and in turn exocytosis. If the membrane is voltage clamped in the presence of the alkaloid, a lasting depolarizing impulse induces, following the "normal" transient current, another much more slowly developing Na+ current that reaches a constant level after a few seconds. Repolarization then is followed by an inward tail current that slowly subsides. Development of these slow currents is enhanced by additional treatment with agents that inhibit inactivation. Most of these phenomena can be satisfactorily explained by assuming that Na+ channels must open before veratridine binds to them, and that the slow current changes reflect the kinetics of binding and unbinding. It is unclear, however, where the alkaloid stays when it is not bound. Although the effect sets in promptly, once this pool is filled, access to it from outside must be impeded since in most preparations veratridine can only partially be washed out. Cooling acts as if the available concentration is reduced, but this reversible "reduction" takes much longer to develop than the cold-induced changes in kinetics. Several authors assume that the binding site, site 2, is accessed from the lipid phase of the membrane. Considerations of this kind are often based on experiments with batrachotoxin, the widely used site-2 ligand which has a much higher affinity and acts as a full agonist in contrast to the partial agonist veratridine. Batrachotoxin thus lends itself to binding studies using radiolabeled derivatives. Such experiments may eventually lead to the characterization of neurotoxin site 2; the first promising steps have been taken. Modern techniques of molecular biology will almost certainly be successful, and one hopes for point-mutated channels with distinctly different reactions also to veratridine. A considerable amount of research is still required to clarify the structural basis for the numerous allosteric interactions with other sites, the mechanism of the very large potential shift of activation, the reduced single-channel conductance and selectivity, and the chemical nature of the different affinities of the site-2 toxins. Note Added in Proof. A report on point mutations with effects on neurotoxin site 2 (see Sect. 8) has just appeared: Wang S-Y, Wang GK (1988) Point mutations in segment I-S6 render voltage-gated Na+ channels resistant to batrachotoxin. Proc Natl Acad USA 95:2653-2658. In microliter muscle Na+ channels expressed in mammalian cells, mutation Asn434Lys leads to complete, Asn434Ala to partial insensitivity to 5 mM batrachotoxin. (Asn434 corresponds to Asn419 of Trainer et al. 1996). The mutant channel displays almost normal current kinetics and in the presence of veratridine little, if any, slow tail current. However, veratridine inhibits peak Na+ currents in the mutant which may point to a complex structure of site 2.

Na, K-ATPase pump in activated human lymphocytes: on the mechanisms of rapid and long-term increase in K influxes during the initiation of phytohemagglutinin-induced proliferation

Functional expression of Na, K-ATPase pump as determined by ouabain-sensitive Rb influxes has been investigated in human peripheral blood lymphocytes, activated by phytohemagglutinin (PHA) from resting state to proliferation. It is found that a rapid twofold elevation of ouabain-sensitive Rb influx in response to PHA is followed by a long-term increase in pump activity, which precedes the DNA synthesis and is temporally related to the growth phase of mitogenic response. Unlike the early pump activation, the late enhanced pump activity is not the result of elevated cell Na content, it is inhibited by cycloheximide and requires new protein synthesis. Actinomycin D and α-amanitin, in doses, which suppress the PHA-induced increase in the RNA synthesis, do not abolish the elevated Rb influx until 20–24 h of mitogenic activation and inhibit the late, growth-associated increase in Rb influx. It is concluded that (1) in mitogen-activated cells both short- and long-term control is involved in the enhanced pump activity, and (2) translational and transcriptional mechanisms may contribute to the long-term up-regulation of Na, K-ATPase pump during blast transformation of human lymphocytes.

Pump-leak parallelism in sodium-absorbing epithelia: the role of ATP-regulated potassium channels.

In all Na(+)-absorbing and Cl(-)-secreting epithelia, an increase in the activity of the Na+,K(+)-pump at the basolateral membrane is accompanied by an increase in the K+ conductance of that barrier and vice versa. We have recently identified an ATP-regulated K+ channel, K(ATP), in basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells that could be responsible for this parallelism between pump activity and leak. Thus, an increase in pump activity would result in a decrease in local ATP activity and an increase in local ADP activity and, in turn, an increase in the open-probability of the channel whereas a decrease in in pump activity would have the opposite effect. Further, the likelihood that the number of pumps far exceeds the number of leaks per unit area of membrane suggests that the ATP and ADP activities that influence K(ATP) channel activity may differ markedly from the "bulk" cytoplasmic values.

IGF-I regulation of Na(+)-K(+)-ATPase in rat arterial smooth muscle.

Insulin-like growth factor I (IGF-I) is vasodilatory and mitogenic for vascular smooth muscle cells (VSMC). Alteration in VSMC Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity is hypothesized to underlie abnormal vascular tone and growth in hypertension and diabetes. Therefore, we investigated effects of IGF-I on Na(+)-K(+)-ATPase activity in rat aortic VSMC. IGF-I increases pump activity in a dose- and time-dependent manner: the minimal dose required was 10(-10) M, and the minimal time required was 20 min (at 10(-8) M) to increase activity. Similar effects persisted through 12 h. In Na(+)-loaded cells, IGF-I does not further stimulate activity. Blockade of Na+/H+ exchange attenuates IGF-I-induced increases in activity after 30 min but has no effect after 12 h. Northern blot analyses reveal that expression of the alpha 1- and the alpha 2-subunits of the pump were unaffected by IGF-I. Plasma membrane alpha 1- and alpha 2-protein were also unaffected, suggesting translocation of preformed pools was not responsible for the increases. Inhibitors revealed that neither tyrosine kinase activity, RNA transcription, protein synthesis, nitric oxide synthase activity, or protein kinase C activity mediated this IGF-I effect. Therefore, IGF-I regulates Na pump activity in the short term by an Na+/H+ exchange-dependent but transcription/translocation-independent mechanism. These data suggest that IGF-I, known to be produced by VSMC, may regulate tone and growth responses abnormal in disease states such as hypertension and diabetes.

Phosphorylation site-specific antibodies to cardiac phospholamban.

The majority of Ca2+ required for cardiac contraction is supplied hy the sarcoplasmic reticulum (SR), an intracellular store. Ca2+ released during contraction is returned to the SR by the activity of the enzyme Ca2+-Mg2+ ATPase, thus facilitating muscle relaxation. The sequestration of Ca2+ by the calcium pump is regulated hy ii second cardiac protein phospholamban (PLB), which in response to p-adrenergic stimulation of the heart is phosphorylated at two sites, Ser16 by the CAMP protein dependent kinasc (c-PKA), and Thr17 by Ca2+/calmodulin (CaM) dependent kinase I I [ I ] . This leads to increased pump activity resulting in an ahbrcviation of the Ca2+ transient, increased loading o l the SR which produces a larger Ca2+ transient at the next contraction. The contrihution o l each site of phosphorylation o f PLB to this process remains unknown, although hoth appear important in vivo 121. I t has hcen suggested that each signalling cascade controls a distinct region 0 1 thc SR 131. In this study we have prepared antihodics wholly specific for each of the two phosphorylated l'omis of PLB, which we aim to use to study the spatial distrihution of site-specific phosphorylation in cardiac muscle cells. Rabbits were immuniscd with a peptide derived from the phosphorylation sequence (if PLB (RSAIRRASTIEY amide [4]) following phosphorylation of either Ser16 or ThrI7. The spccilicity of antibodies so produced was examined, firstly by considering the PLB phosphorylation-site specificity of each antihody and then hy examining the discrimination between PLB and other muscle phosphoproteins. Phospholanihan was presented to these antibodies in three forms, unphosphorylated, SerI6 and ThrI7 phosphorylated as descrihed elsewhere [ S ] . Antiserum PSI6 (produced by immunisation with Ser16 phosphorylated peptide) detected Ser16 phosphorylated PLB exclusively. Similarly, antiserum PT17 only detected Thr I7 phosphorylated PLB. Thus antihodies totally specific for each phosphorylated form of phospholamban were produced. Figure I indicates the specilicity of the PS-16 and PT-17 antibodies when presented with phosphorylated cardiac tissue honiogenatcs. Panel A shows an autoradiograph of the cardiac muscle phosphoproteins generated by CAMP and Ca2+/CaM dependent interventions in vitro. PLB represents one of a number of phosphoproteins, which is identified by its distinctive reduction to monomer on hoiling (arrow). The PSI6 antiserum proves to be completely specific for serine phosphorylated PLB in rat cardiac honiogenate, identifying no other components ( C ) . The hinding of the PS16 antiserum with serinc phosphoylated PLB is completely aholished when competed with the serine phosphorylated peptide (D). Thc PTI7 antiserum specifically recognises the threonine phosphorylated PLB (E), which is completely aholished when competed with the threoninc phosphorylated peptide (F). The PTI7 antiserum does crossreact with one additional component (4XkDa) which is unelfected hy competition with the threonine phosphorylated peptide and thus considered non-specific. We are currently raising monoclonal antibodies to hoth phosphorylation sites o f PLB in order to overcome the detection of this contminat ing component hy the PTI7 antihody. Fip. 1. Antibodv soecificitv in the Dresence of cardiac muscle protcins. Rat heart homogenates were phosphorylated in the presence of CAMP (lanes 1,2) or CaC12 and CaM (lanes 3,4) (7). Samples were solubilised at 30'C (lanes 1, 3) for 30 minutes o r at l00'C (lanes 2, 4) for 5 minutes in Laemmli sample buffer. 100pg protein loaded per track onto SDS-PAGE gels (7-20% linear acrylamide gradient). Proteins were transferred to PVDF membrane and probed with PS-16 (1 : lO 000; panel C); PS-16 ( I : 10 000) plus 1pM Ser16-phosphorylated peptide (panel D); PT-17 (I:S000; panel E); PT-17 (1:SOOO) plus 1pM Thr17 phosphopeptide (panel F). The immunohlots were developed using a peroxidase based chemilumincscent detection kit which we estimate is 100 times more sensitive than methoxy-naphthol based peroxidase detection. An autoradiograph (10 day exposure) oC a representative memhrane is shown (panel A). Arrows indicate phospholamban, pentameric and monomeric forms. Asterisk indicates 48kDa component recognised hy antiserum PT17.

Hydrogen peroxide stimulates sodium-potassium pump activity in cultured pulmonary arterial endothelial cells.

Oxidant injury to pulmonary vascular endothelium is an important factor in the pathogenesis of acute lung injury. Oxidant injury to other cell types has been reported to alter the function of Na-K-adenosinetriphophatase (ATPase) an enzyme important in maintenance of cellular ionic homeostasis and in transport of ions across biological membranes. We investigated the effect of H2O2 (0.001-10 mM) or xanthine (X) (15.2 micrograms/ml) plus xanthine oxidase (XO) (0.0153 U/ml) on the Na-K pump activity of cultured bovine pulmonary arterial endothelial cells (PAECs). We used a functional assay, using 86RbCl as a tracer for K+ and expressing Na-K pump activity as ouabain-inhibitable K+ uptake. Our results demonstrate that H2O2 and X/XO stimulate Na-K pump activity of bovine PAECs, an effect prevented by catalase. In addition, we assessed the affinity, number, and turnover of [3H]ouabain binding sites on intact endothelial monolayers and found that H2O2 increased affinity to [3H]ouabain, decreased the number of binding sites, and increased the rate of pump turnover. Influx of 22Na increased in response to a nonlytic concentration of H2O2. Cell injury, as assessed by 51Cr release, adherent cell number, and phase-microscopic morphology, was not observed after 30-min incubations with the lowest dose (1 mM) of H2O2 effective in stimulating Na-K pump activity, or after incubation with X/XO. Na-K pump inhibition by ouabain significantly increased the 51Cr release caused by H2O2 or by X/XO, suggesting that the increase in Na-K pump activity may be a compensatory response to the cellular alterations produced by H2O2. Incubation with H2O2 decreased cell ATP content, an effect which was not prevented by coincubation with ouabain. In summary, these results show that H2O2 increases Na-K pump activity of PAECs, an effect mediated, at least in part, by increased intracellular [Na] and by an increased rate of pump turnover. It is possible that the increased pump activity may be an early marker of endothelial cell perturbation.