The birefringence of the human red cell ghosts

The type of birefringence described by Mitchison, which extends some 0.5 µ in from the surface of the human red cell ghost in glycerol and which shows a maximum retardation of about 7 A, is only found in ghosts which are sufficiently well hemoglobinised to be seen with the ordinary microscope. Ghosts from which all hemoglobin has been lost are not visible with the ordinary microscope and are not birefringent, although they are clearly visible with phase contrast. About 90 per cent of the ghosts in glycerol preparations are of the latter type, the exact percentage being a function of time. Mitchison's measurements of birefringence, although reproducible, accordingly apply only to ghosts in which some hemoglobin still remains complexed with the lipoprotein layers of the red cell ultrastructure, and do not enable one to draw conclusions as to the thickness and orientation of the lipoprotein surface layers.     

Cytosolic protein concentration is the primary volume signal for swelling-induced [K-Cl] cotransport in dog red cells.

Chloride-dependent K transport ([K-Cl] cotransport) in dog red cells is activated by cell swelling. Whether the volume signal is generated by a change in cell configuration or by the dilution of some cytosolic constituent is not known. To differentiate between these two alternatives we prepared resealed ghosts that, compared with intact red cells, had the same surface area and similar hemoglobin concentration, but a greatly diminished volume. Swelling-induced [K-Cl] cotransport was activated in the ghosts at a volume (20 fl) well below the activation volume for intact cells (70 fl), but at a similar hemoglobin concentration (30-35 g dry solids per 100 g wet weight). Ghosts made to contain 40% albumin and 60% hemoglobin showed activation of [K-Cl] cotransport at a concentration of cell solids similar to intact cells or ghosts containing only hemoglobin. [K-Cl] cotransport in the resealed ghosts became quiescent at a dry solid concentration close to that at which shrinkage-induced Na/H exchange became activated. These results support the notion that the primary volume sensor in dog red cells is cytosolic protein concentration. We speculate that macromolecular crowding is the mechanism by which cells initiate responses to volume perturbation.     

Some common signal transduction events are not necessary for the elicitor-induced accumulation of silymarin in cell cultures of Silybum marianum

A variety of pharmacological effectors of signal transduction pathways were used to investigate the elicitor-activated sequence of cellular responses by which yeast extract (YE) or methyljasmonate (MeJA) enhanced production of silymarin in cell cultures of Silybum marianum. As we recently showed that inhibition of external and internal calcium fluxes significantly increased flavonolignan production in S. marianum cultures, we examined whether calcium mediates signaling events leading to enhancement of silymarin production upon YE or MeJA elicitation. Pre-treatment of cultures with calcium chelators, calcium blockers or intracellular antagonists enhanced the elicitor effect of YE or MeJA. The increase of intracellular-free Ca(2+) level also promoted the elicitor effect, suggesting that an external source of calcium or alterations in internal calcium fluxes were not required for the elicitation to occur. Activation of phosphorylation/dephosphorylation cascades did not appear to mediate the elicitation mechanism; the increase in silymarin induced by elicitation was not suppressed by inhibitors of protein phosphatases or by protein kinase inhibitors. No H(2)O(2) generation was detected at any time after elicitation. Also, diphenyleneiodonium, a potent inhibitor of NAD(P)H-oxidase, did not block silymarin production in elicited cultures. From these results, we conclude that S. marianum cell cultures do not appear to employ conserved signaling components in the transduction of the elicitor signal to downstream responses such as silymarin production.     

Membrane transport of L-triiodthyronine by human red cell ghosts.

L-T₃ transport has been investigated in human red cell ghosts. Determination of initial T₃ uptake revealed two separate saturable uptake systems, one with a K_m of 1.6 × 10^?8M, the other with a K_m of 3.3 × 10^?6M. Binding experiments resulted in two dissociation constants, 1.4 × 10^?7M.and 2.6 × 10^?6M. Uptake was dependent on the ghost volume, indicating an intravesicular location of T₃. The T₃ was concentrated 6 times by the ghosts. Ouabain reduced the uptake by the low K_m system, but was without effect on the high K_m system. Thus evidence is provided both of binding of T₃ to the ghost membrane and of its uphill transport across the membrane.     

Membrane compartmentalized ATP and its preferential use by the Na, K-ATPase of human red cell ghosts

This paper describes work which begins to define the molecular organization in the region of the membrane that comprises the functional domain of the Na:K pump. The membrane-bound phosphoglycerate kinase (PGK) and Na, K-ATPase appear to be directly linked via a compartmentalized form of ATP. Evidence for the membrane pool of ATP is based on the labeling characteristics of the phosphoproteins by [γ-(32)P]ATP of ghosts incubated under various conditions. Preincubation of ghosts in the presence of ATP at 37 degrees C, but not at 0 degrees C, completely obscures the formation of the Na-phosphoprotein in ghosts washed and subsequently incubated in the presence of [gamma-(32)P]ATP. In contrast to the Na component, the Mg component of phosphorylation is only slightly altered by preincubation with ATP. ATPase activity measured as (32)P(i) liberated during the subsequent incubation at 0 degrees C, reflects completely the differential effects of preincubation with ATP on (32)P incorporation into phosphoprotein. ATP placed within the pool by preincubation can be removed by operating the Na, K-ATPase or the PGK reaction in the reverse direction by use of exogenous substrates. Alternatively, the membrane pool of ATP can be formed also from exogenous substrates by running the PGK reaction in the forward direction. These results, while providing direct support for a membrane compartment of ATP, also indicate the location of this compartment in relation to the PGK and the Na, K-ATPase. In addition, these results also imply that the Mg and Na components are different enzymatic entities since substrate ATP can be derived from separate sources.     

The role of phosphotyrosine phosphatases in haematopoietic cell signal transduction

Phosphotyrosine phosphatases (PTPases) are the enzymes which remove phosphate groups from protein tyrosine residues. An enormous number of phosphatases have been cloned and sequenced during the past decade, many of which are expressed in haematopoietic cells. This review focuses on the biochemistry and cell biology of three phosphatases, the transmembrane CD45 and the cytosolic SH2-domain-containing PTPases SHP-1 and SHP-2, to illustrate the diverse ways in which PTPases regulate receptor signal transduction. The involvement of these and other PTPases has been demonstrated in haematopoietic cell development, apoptosis, activation and non-responsiveness. A common theme in the actions of many haematopoietic cell PTPases is the way in which they modulate the thresholds for receptor signalling, thereby regulating critical events in the positive and negative selection of lymphocytes. There is growing interest in haematopoietic PTPases and their associated regulatory proteins as targets for pharmaceutical intervention and in the involvement of these enzymes in human disease.     

Demonstration of adenylate cyclase activity in human red blood cell ghosts.

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine 3',5'-monophosphate (cyclic AMP) - 2 h-1 - mg-1 protein. This enzymatic activity is strongly stimulated by NaF and 5'-guanylimidodiphosphate, is slightly stimulated by epinephrine, norepinephrine, isoproterenol, and prostaglandin E1 and is inhibited by calcium. The hormone stimulation is not potentiated by 5'-guanylylimidodiphosphate.     

Studies on the cation permeability of human red cell ghosts

Summary Net K movements in reconstituted human red cell ghosts and the resealing of ghosts to cations after osmotic hemolysis of red cells have been studied as functions of the free Ca ion concentration. The Ca-dependent specific increase in K permeability was shown to be mediated by a site close to the internal surface of the membrane with an apparent dissociation constant at pH 7.2 for Ca (K _D1) of 3–5×10^–7 m, for Sr of 7×10^–6 m. Ba and Mg did not increase the K-permeability of the membrane but inhibited the Ca-mediated permeability changes.K _D1 decreased in a nonlinear fashion when the pH was increased from 6.0 to 8.5. Two different pK values of this membrane site were found at pH 8.3 and 6.3. The Ca-activated net K efflux into a K-free medium was almost completely inhibited by an increase in intracellular Na from 4 to 70mm. Extracellular K antagonized this Na effect. Changes in the extracellular Na (0.1–140mm) or K(0.1–6mm) concentrations had little effect and did not changeK _D1. The Ca-stimulated recovery of a low cation permeability in ghost cells appeared to be mediated by a second membrane site which was accessible to divalent cations only during the process of hemolysis in media of low ionic strength. The apparent dissociation constant for Ca at this site (K _D2) varied between 6×10^–7 and 4×10^–6 m at pH 7.2. Mg, Sr, and Ba could replace Ca functionally. The selectivity sequence was Ca>Sr>Ba>Mg.K _D2 was independt on the pH value in the range between 6.0 and 8.0. Hill coefficients of 2 were observed for the interaction of Ca with both membrane sites suggesting that more than one Ca ion is bound per site. The Hill coefficients were affected neither by the ion composition nor by the pH values of the intra- and extracellular media. It is concluded that two different pathways for the permeation of cations across the membrane are controlled by membrane sites with high affinities for Ca: One specific for K, one unspecific with respect to cations. The K-specific channel has properties similar to the K channel in excitable tissues.     

Sodium-phosphate cotransport in human red blood cells. Kinetics and role in membrane metabolism

Orthophosphate (Pi) uptake was examined in human red blood cells at 37 degrees C in media containing physiological concentrations of Pi (1.0- 1.5 mM). Cells were shown to transport Pi by a 4,4'-dinitro stilbene- 2,2'-disulfonate (DNDS) -sensitive pathway (75%), a newly discovered sodium-phosphate (Na/Pi) cotransport pathway (20%), and a pathway linearly dependent on an extracellular phosphate concentration of up to 2.0 mM (5%). Kinetic evaluation of the Na/Pi cotransport pathway determined the K1/2 for activation by extracellular Pi ([Na]o = 140 mM) and extracellular Na [( Pi]o = 1.0 mM) to be 304 +/- 24 microM and 139 +/- 8 mM, respectively. The phosphate influx via the cotransport pathway exhibited a Vmax of 0.63 +/- 0.05 mmol Pi (kg Hb)-1(h)-1 at 140 mM Nao. Activation of Pi uptake by Nao gave Hill coefficients that came close to a value of 1.0. The Vmax of the Na/Pi cotransport varied threefold over the examined pH range (6.90-7.75); however, the Na/Pi stoichiometry of 1.73 +/- 0.15 was constant. The membrane transport inhibitors ouabain, bumetanide, and arsenate had no effect on the magnitude of the Na/Pi cotransport pathway. No difference was found between the rate of incorporation of extracellular Pi into cytosolic orthophosphate and the rate of incorporation into cytosolic nucleotide phosphates, but the rate of incorporation into other cytosolic organic phosphates was significantly slower. Depletion of intracellular total phosphorus inhibited the incorporation of extracellular Pi into the cytosolic nucleotide compartment; and this inhibition was not reversed by repletion of phosphorus to 75% of control levels. Extracellular 32Pi labeled the membrane-associated compounds that migrate on thin-layer chromatography (TLC) with the Rf values of ATP and ADP, but not those of 2,3-bisphosphoglycerate (2,3-DPG), AMP, or Pi. DNDS had no effect on the level of extracellular phosphate incorporation or on the TLC distribution of Pi in the membrane; however, substitution of extracellular sodium with N-methyl-D-glucamine inhibited phosphorylation of the membranes by 90% and markedly altered the chromatographic pattern of the membrane-associated phosphate.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of glucocorticoid hormones in interleukin-1 signal transduction via the sphingomyelin pathway

Interleukin-1 and glucocorticoid hormones are the key transmitters of interaction between the neuroendocrine and immune systems. To study the molecular mechanisms of immunomodulatory effects of Interleukin-1 and glucocorticoid hormones, a search for changes in activity of neutral sphingomyelinase: the main marker of initiation of Interleukin-1beta signal transduction via the sphingomyelin pathway in target cells, was accomplished. The Interleukin-1beta was found to activate neutral sphingomyelinase both in P2 fraction of murine brain cortex and membranes of immune-competent cells. Experimental modifications of endogenous glucocorticoid level in the mouse blood were for the first time shown to induce changes in neutral sphingomyelinase activity in membranes of the cells of the immune and nervous systems. It appears that the sphingomyelinase pathway of Interleukin-1beta signaling might be a possible target for glucocorticoid hormones' immune-modulating effects.     

Unmasking of a potassium leak in resealed human red blood cell ghosts.

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0 degrees C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.     

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts.

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg(2+)-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable Kd of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 microM free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.