Functional heterogeneity of polyphosphoinositides in human erythrocytes.

After labelling of erythrocytes with [32P]P1 for 23 h, the specific radioactivities of the phosphomonoester groups of PtdIns4P and of PtdIns(4,5)P2 approached equilibrium values which were close to that of the gamma-phosphate of ATP (78-85%), showing that almost all of these phosphate groups were metabolically active. Phosphoinositidase C (PIC) activation, using Ca2+ and the ionophore A23187, of 32P-prelabelled erythrocytes was used to investigate a possible functional heterogeneity of the phosphoinositides. Hydrolysis of PtdIns(4,5)P2, measured from its radioactivity, decreased as function of the time of prelabelling up to a constant value equal to that measured from its content. In contrast, hydrolysis of PtdIns4P, determined both from radioactivity and from content, was always the same. These data suggest that newly labelled molecules of PtdIns(4,5)P2, initially accessible to PIC, then moved towards a PIC-resistant pool. This was further confirmed by measuring the fraction of labelled PtdIns(4,5)P2 molecules accessible to PIC after a prelabelling period of 5 min and different times of reincubation. Hydrolysis by PIC was also measured in erythrocytes in which the phosphoinositide content had been modified by activation (Mg2+-enriched cells) or inhibition (ATP-depleted cells) of the phosphoinositide kinases. The sizes of the PIC-resistant pools of polyphosphoinositides were not affected by these treatments, indicating that the kinases (and the phosphatases) act on the PIC-sensitive pools. This was also shown by the decrease in the production of Ins(1,4,5)P3 upon PIC activation in ATP-depleted erythrocytes. A model is presented in which the PIC-sensitive pools of polyphosphoinositides are those which are accessible to the kinases and the phosphatases and are rapidly turned over.     

Turnover of phosphomonoester groups and compartmentation of polyphosphoinositides in human erythrocytes.

The turnover of phosphomonoester groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was investigated in human erythrocytes by short-term labelling with [32P]Pi. The procedure applied ensured a quantitative extraction of erythrocyte polyphosphoinositides as well as their reliable separation for the determinations of pool sizes and specific radioactivities. The pool sizes of phosphatidylinositol (PtdIns), PtdIns4P and PtdIns(4,5)P2 are 25, 11 and 44 nmol/ml of cells respectively. Under steady-state conditions, the phosphorylation fluxes from [gamma-32P]ATP into PtdIns4P and PtdIns(4,5)P2 are in the ranges 14-22 and 46-94 nmol X h-1 X ml of cells-1 respectively. Only 25-60% of total PtdIns4P and 6-10% of total PtdIns(4,5)P2 take part in the rapid tracer exchange, i.e. are compartmentalized. In isolated erythrocyte ghosts, the turnover of PtdIns4P approximately corresponds to that in intact erythrocytes, although any compartmentation can be excluded in this preparation. Under the conditions of incubation employed, the turnover of PtdIns(4,5)P2 is more than one order of magnitude smaller in isolated ghosts than that obtained for intact erythrocytes.     

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.     

PIP2 activates TRPV5 and releases its inhibition by intracellular Mg2+

The transient receptor potential type V5 channel (TRPV5) is a Ca2+-selective TRP channel important for epithelial Ca2+ transport. Intracellular Mg2+ causes a fast voltage-dependent block of the TRPV5 channel by binding to the selectivity filter. Here, we report that intracellular Mg2+ binding to the selectivity filter of TRPV5 also causes a slower reversible conformational change leading to channel closure. We further report that PIP2 activates TRPV5. Activation of TRPV5 by PIP2 is independent of Mg2+. Yet, PIP2 decreases sensitivity of the channel to the Mg2+-induced slow inhibition. Mutation of aspartate-542, a critical Mg2+-binding site in the selectivity filter, abolishes Mg2+-induced slow inhibition. PIP2 has no effects on Mg2+-induced voltage-dependent block. Thus, PIP2 prevents the Mg2+-induced conformational change without affecting Mg2+ binding to the selectivity filter. Hydrolysis of PIP2 via receptor activation of phospholipase C sensitizes TRPV5 to the Mg2+-induced slow inhibition. These results provide a novel mechanism for regulation of TRP channels by phospholipase C-activating hormones via alteration of the sensitivity to intracellular Mg2+.     

Phloretin keto-enol tautomerism and inhibition of glucose transport in human erythrocytes (including effects of phloretin on anion transport).

Under various pH conditions phloretin demonstrates keto-enol tautomerism with a pK value of 7.26 +/- 0.06. As Wilbrandt has shown ((1950) Arch. Exp. Pathol. Pharmacol. 212, 9-29) phloretin added to erythrocytes inhibits glucose efflux, but not glucose influx. At pH 6.5 a Ki value of 0.36 and at pH 9 of 22.7 microM was measured; only the ketonic form of phloretin contributes to the inhibition of glucose efflux. This was also the case for inhibition of galactose efflux and anion exchange. The geometry optimization of a large number of conformations of the ketonic and enolic forms of phloretin demonstrates different shapes of the molecules. Only the ketonic form shows several overlapping structures with beta-D-glucopyranose. Considering surplus binding of phloretin under glucose efflux conditions as being equivalent to the number of glucose transporters, a number of about 200,000 molecules was determined. By two independent methods 210,000 and 171,000 molecules per cell were calculated. This result is in close agreement with the number of glucose transporter sites of the erythrocyte.     

Specific inhibition of protein kinase by diazenedicarboxylic acid bis-(N,N-dimethylamide).

Diazenedicarboxylic acid bis (N,N-dimethylamide), commonly called diamide, inhibits purified protein kinase isolated from swine kidney. The extent of inhibition is increased in the presence of cysteine, reduced glutathione and dithiothreitol. The inhibition of protein kinase is reversible and greater than 80% of the activity of an inhibited preparation can be recovered by dialysis. Diamide appears to be specific for the inhibition of protein kinase. The activities of various other kinases acting on low molecular weight substrates were unaffected by diamide. Diamide is the first known low molecular weight inhibitor of protein kinase and may prove to be an extremely useful agent in elucidating the physiological role of this enzyme.     

Fluorescence measurements of fusion between human erythrocytes induced by poly(ethylene glycol).

The kinetics of poly(ethylene glycol) (PEG)-induced fusion between intact human erythrocytes was continuously monitored by a fluorescence lipid mixing method, utilizing the dequenching of the fluorescence probe, 1-oleoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl ] phosphatidylcholine (C12-NBD-PC). The steady-state fluorescence intensity was detected from the surface of cells in a monolayer on an alcian blue-coated glass coverslip. The relief of fluorescence self-quenching after fusion between C12-NBD-PC labeled and unlabeled intact erythrocytes was measured. The extent of fluorescence dequenching was normalized based on the measured concentration of probes in membranes, the projected partial dequenching due both to dilution by intercellular fusion, and the dilution between the inner and outer leaflets of membranes (flip-flop). There was no significant increase in fluorescence intensity during PEG treatment of 5 min, at 4 degrees C. Intensity increased immediately after the dilution of PEG, and reached saturation in 30 min. The efficiency of fusion increased with the increasing of PEG concentrations. Only 4% enhancement of saturated relative fluorescence intensity was detected in 25 wt% PEG-induced cell fusion; 23% enhancement in 30 wt%; and 66% enhancement in 35 wt%. The transfer of fluorescent probes between membrane bilayer leaflets (flip-flop) was also monitored during the fusion process. Flip-flop was monitored in confluent monolayers as well as in isolated cells. There was no significant spontaneous flip-flop within 30 min of dilution. The relative fluorescence intensity enhancement contributed by the dilution of probes between fused labeled and unlabeled cells (at a 1:1 ratio) was found to account for only 39% of the observed final dequenching, whereas the contribution by flip-flop associated with cell fusion was found to account for 9%, and flip-flop without fusion contributed approximately 18%. A portion of the flip-flop is a consequence of hemolysis. Therefore, fluorescence dequenching measurements of fusion of whole cells must be interpreted with caution.     

Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition

MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively. The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.     

Lysis of erythrocytes from stored human blood by phospholipase C (Bacillus cereus).

The ability of phospholipase C (Bacillus cereus) to lyse erythrocytes from human blood that had been stored under Transfusion Service conditions for up to 16 weeks has been examined. When incubated at 20 degrees C with enzyme (0.03 mg/ml, 55 units/ml) for up to 1 h fresh erythrocytes were not lysed. After about 4 weeks of storage a population of very readily lysed erythrocytes appeared. The morphological changes in erythrocytes from blood stored up to 16 weeks were examined by scanning electron microscopy. The proportion of very readily lysed erythrocytes correlated well with the proportion of spheroechinocytes I. This morphological form was shown to be preferentially removed by phospholipase C and before lysis a transient appearance of smooth spheres occurred. The decrease in blood ATP concentrations on storage was measured and found to correlate with the disappearance of discoid erythrocyte forms, but not directly with the increased susceptibility of the erythrocytes to lysis by the enzyme. However, erythrocytes of up to at least 15 weeks of age could be made less susceptible to lysis by pre-incubation in a medium designed to cause intracellular regeneration of ATP. During the lysis of spheroechinocytes I by electrophoretically pure recrystallized phospholipase C a rapid degradation of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphatidylinositol) occurred together with a slower degradation of sphingomyelin.     

Protein kinase C-dependent phosphorylation of profilin is specifically stimulated by phosphatidylinositol bisphosphate (PIP2)

Calf spleen profilin is shown to be an in vitro substrate of purified human placental protein kinase C (PKC), with an apparent Km of 4 microM. Phosphatidylinositol bisphosphate (PIP2) was an effective activator of the profilin phosphorylation by PKC and caused a maximum 13-fold increase of Vmax with a half maximal effect at 40 micrograms/ml. The action of PIP2 was not mimicked by phosphatidylserine, phosphatidic acid or phosphatidylinositol, whereas phosphatidylinositol monophosphate was slightly stimulatory. By contrast, protein kinase C-dependent phosphorylation of histone type III-S, myelin basic protein or lipocortin-I was not affected by PIP. It is suggested that PIP2 modifies the nature of the profilin-PKC interactions.     

Pitavastatin enhanced BMP-2 and osteocalcin expression by inhibition of Rho-associated kinase in human osteoblasts

To clarify the mechanism of the stimulatory effect of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) on bone formation, we investigated the effect of pitavastatin, a newly developed statin, on expression of bone morphogenetic protein-2 (BMP-2) and osteocalcin in primary cultured human osteoblasts. Pitavastatin increased the expression level of mRNA for BMP-2, and much more effectively for osteocalcin. This stimulatory effect was abolished by the addition of geranylgeranyl pyrophosphate, an essential molecule for prenylation of small GTP-binding proteins such as Rho GTPase, but not by inhibitors of nitric oxide synthase and various protein kinases. Pitavastatin suppressed the Rho-associated kinase (Rho-kinase) activity. Hydroxyfasudil, a specific inhibitor of Rho-kinase, increased BMP-2 and osteocalcin expression. These mRNA levels were strongly suppressed by dexamethasone, but restored by co-treatment with hydroxyfasudil. These observations suggest that the Rho-kinase negatively regulates bone formation and the inhibition of Rho and Rho-kinase pathway is the major mechanism of the statin effect on bone. Moreover, a Rho-kinase inhibitor may be a new therapeutic reagent for the treatment of osteoporosis such as glucocorticoid-induced osteoporosis.     

Effect of neomycin and ionophore A23189 on ATP levels and turnover of polyphosphoinositides in human erythrocytes.

The relationship of polyphosphoinositide metabolism to erythrocyte ATP levels was examined. Turnover of polyphosphoinositides was not closely dependent on ATP as it is reported to be in yeast. Neomycin increased 32P incorporation into diphosphoinositide and to a lesser extent into triphosphoinositide without affecting intracellular ATP. Treatment of intact cells with ionophore A23187 resulted in a decrease of at least 80% in polyphosphoinositide levels which followed a decrease in cellular ATP and an increase in phosphatidate. The results indicate that polyphosphoinositide turnover does not regulate energy charge in the erythrocyte. However some of the events which follow treatment of erythrocytes with metabolic inhibitors or calcium and ionophore may be related to the accompanying decrease in polyphosphoinositide levels.     

Regulation of type II PIP kinase by PKD phosphorylation

The type II PIP kinases phosphorylate the poorly understood inositol lipid PtdIns5P, producing the multi-functional lipid product PtdIns(4,5)P(2). To investigate the regulation of these enzymes by phosphorylation, we partially purified a protein kinase from pig platelets that phosphorylated type IIalpha PIP kinase on an activation loop threonine residue, T376. Pharmacological studies suggested this protein kinase was protein kinase D (PKD), and in vitro experiments confirmed this identification. A phospho-specific antibody was developed and used to demonstrate phosphorylation of T376 in living cells, and its enhancement under conditions in which PKD was activated. Although we were unable to determine the effects of phosphorylation on PIP kinase activity directly, mutation of T376 to aspartate significantly inhibited enzyme activity. We conclude that the type II PIP kinases are physiological targets for PKD phosphorylation, and that this modification is likely to regulate inositol lipid turnover by inhibition of these lipid kinases.     

p-(Chloromercuri)benzenesulfonate binding by membrane proteins and the inhibition of water transport in human erythrocytes

The binding of [203Hg]-p-(chloromercuri)benzenesulfonate to the membrane proteins of human erythrocytes and erythrocyte ghosts was examined under conditions where binding to the bulk of membrane sulfhydryl groups was blocked by N-ethylmaleimide. Binding was essentially complete within 90 min when approximately 40 nmol was bound per milligram of membrane protein. This binding was correlated with the inhibition of water transport measured by an NMR technique. Maximal inhibition was observed with the binding of approximately 10 nmol of p-(chloromercuri)benzenesulfonate/mg of membrane protein. Under these conditions, both band 3 and band 4.5 bound 1 mol of inhibitor/mol of protein. In contrast to previous experiments, these results indicate that band 4.5 proteins as well as band 3 have to be considered as playing a role in water transport.     

Studies on PIP2-phosphomonoesterase activity in human platelets

An occurrence of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphomonoesterase in human platelets was demonstrated by analyzing phosphoinositides metabolism. The activity of the enzyme was maximum at pH 7.0. It was active even in the absence of Ca2+ or Mg2+ but it was enhanced in the presence of Mg2+ or NaF. The activity was inhibited by pyrophosphate. The activity was not altered in the presence of Ca2+. Thereby, besides phosphodiesteric cleavage by phospholipase C, the amount of PIP2 in activated platelets may be reduced by the combined effect of PIP2-phosphomonoesterase and suppressed activity of PI-kinase by Ca2+.     

Restricted diffusion of integral membrane proteins and polyphosphoinositides leads to their depletion in microvesicles released from human erythrocytes.

Radioactive PtdIns(3)P was detected in human platelets incubated with [32P]Pi, but remained unaffected by thrombin treatment. In contrast, [32P]PtdIns(3,4)P2 was absent from resting platelets, but was produced by thrombin-activated platelets in a dose- and time-dependent manner. [32P]PtdInsP3 was never found under these conditions. These changes are similar to those elicited in other cells by platelet-derived growth factor or the oncogene product pp60c-src.     

Purification and characterization of pyridoxal kinase from human erythrocytes

Pyridoxal kinase has been purified 50,000-fold from human erythrocytes. The purification procedure included dextran-induced aggregation of red blood cells, ammonium sulphate fractionation of the haemolysate, DEAE-cellulose chromatography, hydroxyapatite chromatography. Sephadex G-100 gel filtration and omega-aminooctyl agarose chromatography. The enzyme preparation migrated as a single protein and activity band on analytical gel electrophoresis. Determination of the Michaelis constants for pyridoxal, pyridoxine and pyridoxamine using a new assay gave comparable values of 33 microM, 16 microM and 6.2 microM respectively. Various amines were shown as competitive inhibitors of pyridoxal kinase with respect to ATP. The inhibition order was: N-dansyl-1,8-diaminooctane greater than 1,8-diaminooctane greater than 1,6-diaminohexane greater than 1,4-diaminobutane greater than gamma-aminobutyric acid, whereas octane, hexane and butane were not inhibitors. Results suggest that the amino groups on the above inhibitors are essential for competitive inhibition at saturating concentrations of pyridoxal. It was also observed that increasing the chain length of the hydrophobic backbone of these competitive inhibitors can facilitate its action.