Activation of sodium transport in human erythrocytes by beta-adrenoceptor stimulation in vivo

Beta-adrenoceptor stimulation in vivo shifts potassium into the cells. To examine whether human erythrocytes participate in this process, we measured, along with serum or plasma potassium, the concentrations of potassium and sodium in erythrocytes. Beta-adrenoceptor stimulation was obtained by infusion of either fenoterol or hexoprenaline into 6 volunteers at rest or by endogenous amines provoked in 14 volunteers during ergometric exercise. Metabolic effects were followed at rest on serum insulin, C-peptide, and growth hormone levels, and during exercise on pH on lactate concentration in blood. The potassium concentration (mean +/- S.E.M.) dropped (p less than 0.01) in serum from 4.64 +/- 0.37 to 3.19 +/- 0.43 mmol x l-1 in the first hour at rest and in plasma from 5.70 +/- 0.93 to 4.63 +/- 0.45 in 90 sec directly after exercise. The concentration of erythrocyte sodium dropped (p less than 0.001) from 9.68 +/- 0.73 to 8.81 +/- 0.62 mmol x l-1 in cells and from 9.62 +/- 1.16 to 8.55 +/- 1.24 during exercise for 90 s, respectively. Changes in the concentration ratio of cellular sodium to potassium confirmed this sodium shift. An increased sodium transport in erythrocytes due to beta-adrenoceptor stimulation in vivo appears to complement a shift of serum potassium into the cells and may be mediated by the membrane-bound sodium, potassium ATPase.     

Activation of rat liver and rabbit skeletal muscle glycogen synthases by rat liver cytosolic protein phosphatases

To gain more insight into the nature of the substrate specificity of protein phosphatases, four forms of glycogen synthase D were used as substrates for previously characterized protein phosphatases, IA, IB, and II, from rat liver cytosol. The phosphatase activity was measured as the conversion of glycogen synthase D to synthase I. While glycogen synthase isolated from rat liver as the D-form was activated mainly by phosphatase IA, rabbit skeletal muscle glycogen synthase previously phosphorylated in vitro by cyclic AMP-dependent protein kinase or phosphorylase kinase was activated efficiently by phosphatases IA, IB, and II. Glycogen synthase isolated from rabbit skeletal muscle as the D-form, however, was a poor substrate for all three phosphatases. These results suggest that the phosphorylation state as well as the primary structure of synthase D markedly affects the rate of its activation by individual protein phosphatases. A protein phosphatase released from rat liver particulate glycogen, on the other hand, activated all forms of synthase D used here readily and at about the same rate.     

Modified norcantharidins; synthesis, protein phosphatases 1 and 2A inhibition, and anticancer activity

Fourteen modified norcantharidin analogues have been synthesised and screened for their ability to inhibit the serine/threonine protein phosphatases 1 and 2A. The most potent compounds found were 10 (PP1 IC(50)=13+/-5 microM; PP2A IC(50)=7+/-3 microM) and 16 (PP1 IC(50)=18+/-8 microM; PP2A IC(50)=3.2+/-0.4 microM). Overall, only analogues possessing at least one acidic residue at the former anhydride warhead displayed any PP1 or PP2A inhibitory action. The ability of these analogues to inhibit PP1 and PP2A correlates well with their observed anti-cancer activity against a panel of five cancer cell lines: A2780 (human ovarian carcinoma), G401 (human kidney carcinoma), HT29 (human colorectal carcinoma), H460 (human lung carcinoma) and L1210 (murine leukemia).     

Regulation of sodium transport in erythrocytes

The kinetic response of swine erythrocyte (Na + K)-ATPase to Na⁺ concentration was hyperbolic in low KCl (5–25 mm) but became increasingly sigmoidal (n = 2.2) as KCl was increased to 150 mm. The addition of 150 mm LiCl did not cause an increase in sigmoidicity although it decreased the apparent affinity for Na⁺. The dependence of ouabain-inhibited efflux of Na⁺ on internal Na⁺ concentration was measured in intact cells with intracellular cation concentrations altered by incubation in p-ehloromercuriphenyl sulfonate. The response to Na⁺ was sigmoidal (n = 2.2) in cells containing high K⁺ but hyperbolic in preparations in which most of the intracellular K⁺ was replaced by Li⁺, even in the presence of 150 mm external KCl. The data are consistent with a model in which internal K⁺ is an allosteric (feedback) inhibitor of Na⁺ efflux and there are three Na⁺ sites which interact cooperatively.     

A two-site model for sodium transport in human erythrocytes

A kinetic model has been proposed for human erythrocytes to account for the responses of sodium transport to alterations in the extracellular sodium concentration in the presence and absence of potassium. The proposed model characterizes the movement of sodium from the outer surface of the erythrocyte membrane to the inner surface in terms of a carrier which has two sites with differing affinities for sodium ions.     

Glucose transport inhibition in human erythrocytes by calmodulin antagonists

Calmodulin antagonists (tryphtazin, lidocaine, dykain, palmitate) inhibit glucose transport from human erythrocytes. Glucose efflux inhibition is proportional to the concentration of antagonists in the medium and is of uncompetitive character. It is accompanied by a decrease in the maximum transport rate with the unchanged constant of dissociation in the complex: carrier-sugar. Calcium ionophores A23187 and divaleryldibenzo-18-crown-6 eliminated the inhibiting effect of pharmacological agents on glucose transport. The authors think that the glucose transport inhibition under the influence of calmodulin antagonists may be realized through the calmodulin-dependent chain inhibition under the influence of calmodulin antagonists in the carbohydrate transport system.     

Anhydride modified cantharidin analogues: synthesis, inhibition of protein phosphatases 1 and 2A and anticancer activity

Two series of anhydride modified cantharidin analogues were synthesised and screened for their phosphatase inhibition (PP1 and PP2A) and cytotoxicity in various cancer cell lines (Ovarian A2780, ADDP; Osteosarcoma 143B; and Colon HCT116 and HT29). One series was synthesised by a novel, high yielding one-pot hydrogenation-ring-opening-esterification procedure, the other by acid catalysed acetal formation. Analogues 5-7 and 9 displayed moderate PP2A selectivity (ca. 5- to 20-fold) and inhibition typically in the low microM range (comparable, in some cases to cantharidin). The anticancer activity of these analogues varied with the cell line under study; however, many of them showed selective cytotoxicity for the colon tumour cell lines.     

Irreversible inhibition of water transport in erythrocytes by fluoresceinmercuric acetate

Fluoresceinmercuric acetate (FMA) is approximately 5 times more potent than p-chloromercuribenzene sulfonate (PCMBS) in inhibiting water transport across human erythrocyte membranes. Half maximal inhibition occurred at approximately 60 m. While cysteine fully reversed the inhibition induced by p-chloromercuribenzene sulfonate it had no effect on the inhibition induced by fluoresceinmercuric acetate. A comparison of the structures of fluoresceinmercuric acetate and p-chloromercuribenzene sulfonate suggests that either there are two sulfhydryl groups in close proximity or the larger aromatic ring structure of fluoresceinmercuric acetate causes tighter binding to one sulfhydryl group at the active site.     

Sequence homologies between type 1 and type 2A protein phosphatases

The Mr = 33,000 catalytic fragment of rabbit skeletal muscle type 1 protein phosphatase was digested with trypsin after reduction and alkylation. The resulting peptides were isolated, subjected to automated Edman degradation, and their sequences compared to the deduced peptide sequence of the bovine type 2A protein phosphatase cDNA. Of 10 tryptic peptides from the type 1 phosphatase that were sequenced, nine showed a high degree of homology with the type 2A phosphatase. This provides the first direct sequence comparison suggesting that the type 1 and type 2 protein phosphatases, distinguished functionally by their substrate specificities and sensitivity to inhibitors, make up part of a family of closely related gene products with similar structures.     

cAMP regulation of protein phosphatases PP1 and PP2A in brain

Normal functioning of the brain is dependent upon a complex web of communication between numerous cell types. Within neuronal networks, the faithful transmission of information between neurons relies on an equally complex organization of inter- and intra-cellular signaling systems that act to modulate protein activity. In particular, post-translational modifications (PTMs) are responsible for regulating protein activity in response to neurochemical signaling. The key second messenger, cyclic adenosine 3′,5′-monophosphate (cAMP), regulates one of the most ubiquitous and influential PTMs, phosphorylation. While cAMP is canonically viewed as regulating the addition of phosphate groups through its activation of cAMP-dependent protein kinases, it plays an equally critical role in regulating removal of phosphate through indirect control of protein phosphatase activity. This dichotomy of regulation by cAMP places it as one of the key regulators of protein activity in response to neuronal signal transduction throughout the brain. In this review we focus on the role of cAMP in regulation of the serine/threonine phosphatases protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) and the relevance of control of PP1 and PP2A to regulation of brain function and behavior.     

Activation of ERK by sodium tungstate induces protein synthesis and prevents protein degradation in rat L6 myotubes

The balance between the rates of protein synthesis and degradation in muscle is regulated by PI3K/Akt signaling. Here we addressed the effect of ERK activation by sodium tungstate on protein turnover in rat L6 myotubes. Phosphorylation of ERK by this compound increased protein synthesis by activating MTOR and prevented dexamethasone-induced protein degradation by blocking FoxO3a activity, but it did not alter Akt phosphorylation. Thus, activation of ERK by tungstate improves protein turnover in dexamethasone-treated cells. On the basis of our results, we propose that tungstate be considered an alternative to IGF-I and its analogs in the prevention of skeletal muscle atrophy.     

Identification of protein phosphatases 1 and 2B as ribosomal protein S6 phosphatases in vitro and in vivo

Protein phosphatases 1 and 2B from rabbit skeletal muscle were found to catalyze the dephosphorylation of ribosomal protein S6 in vitro. Phosphorylation of protein phosphatase-1 by the transforming protein of Rous sarcoma virus, pp60v-src, abolished S6 dephosphorylation by the purified enzyme. Analysis of the dephosphorylation of phosphorylase a and phosphorylase kinase in Xenopus oocyte extracts and after microinjection indicated the presence of oocyte enzymes similar to protein phosphatases-1 and -2B. Studies with 32P-labeled 40 S ribosomal subunits suggested that these enzymes were functioning as S6 phosphatases in oocytes. These findings support the hypothesis that regulation of protein phosphatase activity may be involved in the increase in S6 phosphorylation observed after mitogenic stimulation.     

Effects on transport of rapidly penetrating,competing substrates: Activation and inhibition of the choline carrier in erythrocytes by imidazole

Summary The properties of the choline transport system are fundamentally altered in saline solution containing 5mm imidazole buffer instead of 5mm phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at thecis side inhibits, and at thetrans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.     

Osteoblastic protein tyrosine phosphatases inhibition and connexin 43 phosphorylation by alendronate

Bisphosphonates (BPs), potent inhibitors of bone resorption which inhibit osteoclasts, have also been shown to act on osteocytes and osteoblasts preventing apoptosis via connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. We previously demonstrated the presence of a saturable, specific and high affinity binding site for alendronate (ALN) in osteoblastic cells which express Cx43. However, cells lacking Cx43 also bound BPs. Herein we show that bound [³H]-alendronate is displaced by phosphatase substrates. Moreover, similar to Na₃VO₄, ALN inhibited the activity of transmembrane and cytoplasmic PTPs, pointing out the catalytic domain of phosphatases as a putative BP target. In addition, anti-phospho-tyrosine immunoblot analysis revealed that ALN stimulates tyrosine phosphorylation of several proteins of whole cell lysates, among which the major targets of the BP could be immunochemically identified as Cx43. Additionally, the transmembrane receptor-like PTPs, RPTPµ and RPTPα, as well as the cytoplasmic PTP1B, are highly expressed in ROS 17/2.8 cells. Furthermore, we evidenced that Cx43 interacts with RPTPµ in ROS 17/2.8 and ALN decreases their association. These results support the hypothesis that BPs bind and inhibit PTPs associated to Cx43 or not, which would lead to the activation of signaling pathways in osteoblasts.     

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.     

The transport of sodium into human erythrocytes in vivo

The relative Na²⁴ specific activity of red cells and plasma was measured at periods up to 30 hours following a single intravenous injection of Na²⁴ in normal healthy young adults. The average specific activity of the red cells relative to that of the plasma at 24 hours and beyond was found to average 0.83 ± 0.05 in a series of five normal individuals, significantly different from 1.0. This indicates that all the intracellular Na is not exchangeable in 24 hours, and confirms earlier in vitro results. The red cell Na concentration in man was shown to be 12.1 ± 1.1 m.eq. Na/liter red cell, as measured in a series of nineteen normal healthy young adults. A theoretical analysis of the data on exchangeable cell Na suggests that the red cell Na (5.3 m.eq. Na/liter blood) is divided into a fast compartment comprising 4.25 m.eq. Na/liter blood, and a slow compartment comprising 1.07 m.eq. Na/liter blood. If these compartments are arranged in parallel, the flux between plasma and fast compartment is 1.32 m.eq. Na/liter blood hour, and that between plasma and slow compartment is 0.016 m.eq. Na/liter blood hour. Results of experiments on two patients with congenital hemolytic jaundice suggest that the fraction of slowly exchanging Na may increase with the age of the red cell.