Biomedical subjects

B Sarkadi

Publications and source records attributed to B Sarkadi.

At least 73 records · Page 4Linked to original sources

PubMed1989

Volume regulatory mechanisms of human granulocytes in hypoosmotic media.

Human neutrophil granulocytes, when placed into hypoosmotic media, first swell, than show a regulatory volume decrease (RVD) reaction. This RVD is the most effective at pH 7.5 when inosine is used as a metabolic substrate. The RVD reaction is blocked by the inhibitors of the calcium-induced K+ transport, such as quinine, quinidine, trifluoperazine (TFP), as well as by those of the conductive Cl-transport (dipyridamole, SITS, oligomycin C). Removal of external calcium by EGTA, or addition of the calcium channel inhibitor, verapamil, reduce the RVD. Ouabain affects RVD only after long preincubation and furosemide or phloretin have hardly any effect on this process. In a hypoosmotic KCl medium neutrophils show a TFP-, and SITS-sensitive secondary volume increase. All these data indicate the opening of Ca2+-induced K+ channels and of conductive Cl- channels in hypoosmotically shocked neutrophils. Under these conditions, however, direct measurement of cytoplasmic calcium by Indo-1 does not show any major change in the overall cytoplasmic free Ca2+ levels. In neutrophils, the reduced calcium signal evoked by formyl-methionyl-leucyl-phenylalanine (fMLP) after a hypoosmotic shock, suggests that cellular calcium metabolism is altered under these conditions.

Biological Transport, Active↗

PubMed1988

Effects of phosphoinositides on calcium movements in human platelet membrane vesicles.

In a mixed endoplasmic and surface-type membrane vesicle preparation from human platelets the polyphosphoinositides PIP and PIP2, similarly to IP3, were found to induce a rapid calcium release reaction. At physiological (resting) cytoplasmic calcium concentrations (0.1-0.3 microM) the PIP2 and IP3 concentrations producing half-maximum calcium release were similar (0.7 microM) and both agents could mobilize about 30-40% of the intravesicular calcium. However, the phosphodiesteric degradation of PIP2 in the membrane vesicles was found to be negligible and the ion- and drug-sensitivities of the calcium release reactions were different. The IP3-induced calcium release was selectively inhibited by micromolar calcium concentrations and by cinnarizine, while the PIP2-induced release was blocked by magnesium ions and neomycin. The calcium release evoked by either agent was inhibited by low concentrations of lanthanum but, in contrast to the ATP-dependent calcium pump, it was insensitive to vanadate, quercetin and to the lowering of the incubation temperature. When added simultaneously or in a rapid succession, maximum effective IP3 and PIP2 concentrations produced an additive calcium release reaction. Based on these data we suggest that IP3 and PIP2, respectively, induce rapid transmembrane calcium movements involving different transport pathways and/or membrane calcium pools, which are not related to the active calcium transport systems.

Blood Platelets↗

PubMed1988

Characterization of membrane calcium pumps by simultaneous immunoblotting and 32P radiography.

Calcium pumps of various plasma membrane, endoplasmic reticulum and sarcoplasmic reticulum preparations were visualized by simultaneous immunoblotting and autoradiography of the 32P-labelled phosphoenzymes. The pump proteins and their fragments produced by a proteolytic pretreatment of the membranes were selectively phosphorylated by [gamma-32P]ATP, separated on an acidic SDS-polyacrylamide gel, blotted onto nitrocellulose and reacted with polyclonal antibodies raised against the purified human erythrocyte and rat skeletal muscle sarcoplasmic reticulum calcium pumps, respectively. The immuno-reaction was detected by peroxidase staining, while the phosphoproteins were shown by autoradiography of the same blot. An antibody against the erythrocyte calcium pump, reacting on the blot with the 140 kDa erythrocyte calcium pump and its 80 kDa proteolytic fragment, did not show a cross-reaction with the calcium pump of similar molecular mass in rat synaptosome membranes or with any of the endoplasmic- or sarcoplasmic-type calcium pumps. An anti-sarcoplasmic reticulum calcium pump antibody cross reacted with several sarcoplasmic and endoplasmic calcium pump proteins and their proteolytic fragments but with none of the plasma membrane pumps. This sensitive double-labelling method can be applied to study structural relationships and molecular alterations in various ion pump proteins.

Animals↗

PubMed1988

Volume regulatory mechanisms of human platelets.

Platelets were found to defend themselves against hypoosmotic swelling by a regulatory volume decrease mechanism, similar to that of T-lymphocytes. An oligomycin C sensitive Cl- -channel and a Ca2+-dependent, quinine- and trifluoperazine-sensitive K+-channel play a significant role in the process: platelets lose K+ and Cl- (and consequently water) through these. Activation of platelets accelerates the volume regulatory mechanism in spite of the fact that the Na+/H+ exchange process, activated simultaneously, works against volume decrease. The most rapid volume regulation was observed in activated platelets with inhibited Na+/H+ exchange process.

Blood Platelets↗

PubMed1987

Activation of sodium-proton exchange is not a prerequisite for Ca2+ mobilization and aggregation in human platelets.

Recently it has been suggested [(1987) Nature 325, 456-458; (1987) FEBS Lett. 212, 123-126] that the activation of Na+/H+ exchange is a prerequisite for platelet aggregation and the development of the Ca2+ signal. As direct evidence for the role of the Na+/H+-exchange pathway the inhibition of the Ca2+ signal by EIPA, a specific inhibitor of Na+/H+ exchange, was offered. Here we demonstrate that low concentrations of EIPA (below 1 microM) completely block Na+/H+ exchange while EIPA inhibits aggregation or Ca2+ mobilization only in concentrations 100-times greater than 1 microM. Moreover, another amiloride analogue, CBDMB, developed to act predominantly on Na+/Ca2+ exchange, does not affect Na+/H+ exchange in platelets but blocks aggregation and Ca2+ mobilization. We conclude that while Na+/H+ exchange has a fundamental role in platelet functions it is not prerequisite for the development of Ca2+ signal and aggregation.

Amiloride↗

PubMed1987

Conformational changes of the in situ red cell membrane calcium pump affect its proteolysis.

In inside-out red cell membrane vesicles trypsin digestion reduces the molecular mass of the 32P-labeled acyl-phosphate intermediate of the calcium pump from the original 140 kDa to about 80 kDa with a simultaneous activation of the calcium uptake. This process is slightly stimulated by the presence of calcium, as compared to EGTA, or EGTA + vanadate, but the proteolytic pattern is similar under all these conditions. However, trypsin degradation of the 80 kDa polypeptide, resulting in the loss of calcium transport activity and 32P-phosphoenzyme formation, is rapid in the presence of calcium, inhibited by EGTA and almost fully blocked by EGTA + vanadate. In the presence of these latter ligands, probably locking the calcium pump in an E2 conformation, the 80 kDa protein becomes insensitive even to excessive digestion by the non-specific protease, pronase. The data indicate major changes in the molecular arrangement of the calcium pump protein when transformed from a calcium-liganded (E1) to an E2 conformation.

Calcium-Transporting ATPases↗

PubMed1987

The maximal velocity and the calcium affinity of the red cell calcium pump may be regulated independently.

The kinetics of active Ca2+ transport in inside-out red cell membrane vesicles and the Ca2+-ATPase activity of the purified Ca2+ pump were studied and the effects of calmodulin, acidic phospholipids, and controlled trypsinization were compared. In the presence of calmodulin the maximal rate and the apparent affinity of the pump for Ca2+ were greatly increased in both preparations. The lowest value of Km(Ca) was between 0.5 and 0.7 microM depending on the concentration of calmodulin and on the enzyme preparation. Positive cooperativity for Ca2+ activation with a Hill coefficient of 1.6-1.7 was observed in all cases. When acidic phospholipids (phosphatidylinositol 4-phosphate was routinely used) were added to the inside-out vesicles or to the purified enzyme, maximal transport rates equal to those obtained with calmodulin were measured but the Km(Ca) decreased to 0.25 microM and the positive cooperativity disappeared (the Hill coefficient approached 1). Highly active, calmodulin-independent proteolytic fragments of molecular mass of 81 and 76 kDa were produced with controlled trypsinization. When the trypsin treatment was directed to obtain primarily the 81-kDa fragment, the preparation showed characteristics similar to those of the intact Ca2+ pump in the presence of calmodulin; that is, the same Vmax was obtained, the Km(Ca2+) was 0.5-0.6 microM, and the Hill coefficient was about 1.6. Addition of phosphatidylinositol 4-phosphate or allowing further proteolysis to produce the 76-kDa fragment, shifted the Km(Ca) to 0.25 and reduced the Hill coefficient to 1, without changes in the maximal rate. Based on these results it is suggested that the maximal velocity and the Ca2+ affinity on the erythrocyte Ca2+ pump may be regulated independently and that independent polypeptide regions of the enzyme are involved in the regulations.

Biological Transport, Active↗

PubMed1987

Enhancing effect of alpha-interferon on the C2 synthesis by cultured human monocytes.

C2 synthesis by human peripheral blood monocytes cultured in the presence of human alpha-interferon (IFN-alpha) was studied. IFN-alpha was added in different amounts (1-1000 IU/ml) to the cultures on day 3 and was removed on day 7. As control, mock interferon (m-IFN) was also tested. C2 content of the culture supernatants was measured by immunohaemolytic method. IFN-alpha was found to enhance C2 synthesis in a dose-dependent way. The enhancing effect could be observed even after the removal of IFN-alpha. C2 production by the cultured monocytes was increased by m-IFN as well; the extent of enhancement however, was found to be significantly lower than that induced by the corresponding amount of IFN-alpha. In contrast to the enhancing effect on C2 production, IFN-alpha did not influence total protein synthesis in the cultures, suggesting a selective stimulatory action on the C2 gene.

Biological Assay↗

PubMed1986

Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis.

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.

Adenosine Triphosphatases↗

PubMed1986

Demonstration of two distinct calcium pumps in human platelet membrane vesicles.

Membrane vesicles from human platelets were prepared by various disruption and isolation techniques reported in the literature to yield fractions of predominantly surface or intracellular membrane origin. ATP + Mg2+-dependent Ca2+ accumulation and the formation of acylphosphate intermediates of the calcium pump(s) were followed in parallel experiments, and the consequences of a limited proteolysis of the membranes examined. In all types of preparations active Ca2+ uptake had both oxalate-sensitive and insensitive fractions and calmodulin had no effect on the rate of Ca2+ uptake. Limited proteolysis by trypsin eliminated oxalate-sensitive Ca2+ uptake while it had no effect on the oxalate-insensitive fraction. The Ca2+-induced EP complex had an apparent molecular mass of 100-110 kDa in all of the preparations, the EP showing a broad or even duplicated line in most autoradiographies. Mild trypsin digestion resulted in the formation of 80-, 55-, and 35-kDa phosphorylated fragments. The 80-kDa fragment corresponded to the limit polypeptide found in the proteolyzed erythrocyte membrane Ca2+ pump, its phosphorylation was stimulated by lanthanum, and it appeared in a different time course than the smaller fragments. The molecular mass and the formation pattern of the latter species corresponded to the tryptic fragments in the sarcoplasmic reticulum Ca2+ pump. Based on these results we suggest that platelet membrane preparations contain two types of Ca2+ pump proteins, one similar to the sarcoplasmic reticulum-type and the other to the erythrocyte-type enzyme.

Adenosine Triphosphatases↗

PubMed1985

Cation and anion transport pathways in volume regulatory response of human lymphocytes to hyposmotic media.

The regulatory volume decrease of osmotically swollen human peripheral blood lymphocytes can be inhibited by agents acting on volume-activated K+- or Cl--transport pathways. Quinine, cetiedil, and 3,3'-dipropylthiadicarbocyanine were found to block the volume-induced K+ transport by interaction with sites on the outside face of the membrane, perhaps by competition with external K+. Drugs known to influence calmodulin action inhibit both volume-induced K+ and Cl- transport to varying degrees. Those inhibitors, particularly of K+ transport, are correlated with their calmodulin-antagonist activity. Penetrating sulfhydryl (SH) reagents (in contrast to nonpenetrating ones) are potent inhibitors of both volume-induced K+ and Cl- movements, indicating the presence of functionally important SH groups located within the membrane or at the cytoplasmic face. A number of agents, such as dipyridamole and oligomycin C, are specific inhibitors of the volume-activated anion pathway. In all respects studied, the inhibition characteristics of the volume-activated K+ pathway of lymphocytes resemble those of the Ca2+-activated K+ channel of red cells. In contrast, the volume-induced anion permeability differs from the primary anion-transport pathway of red cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1985

Analysis of Ca2+-induced K+-transport of human erythrocytes in propionate media.

A method is described which allows studying specific cation transport pathways of the cell membranes by converting ion fluxes into volume changes. Lipophilic weak electrolytes, such as propionate, rapidly penetrate the cell membranes in their undissociated acid from but not as negatively charged ions. When human red cells are incubated in isoosmotic K-propionate media an intracellular acidification occurs with a limited propionate uptake and volume increase (corresponding to the buffering capacity of the cytoplasm). If both protons and alkali cations are rendered permeable, a rapid salt influx and volume increase is observed. The latter can be quantitatively followed by electronic sizing methods. A detailed characterization of the system is provided through studies with ionophores, inhibitors of the red cell anion exchange system and drugs which activate or inhibit the Ca2+-induced K+ transport. It is demonstrated that in K-propionate media the permeability of the Ca2+-induced K+ pathway can be directly estimated. The method is suitable to observe population (all-or-none) responses in the activation of the K+ pathway under certain experimental conditions. The application of the method in the search for cation-proton exchanger systems is discussed and its use for demonstration purposes by producing selective lysis of red cells is described.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1984

Cyclic AMP-dependent protein kinase stimulates the phosphorylation of phosphatidylinositol to phosphatidylinositol-4-monophosphate in a plasma membrane preparation from pig granulocytes.

Plasma membranes prepared from pig granulocytes were incubated in the presence of [gamma-32P]ATP. The dissociated catalytic subunit of cyclic AMP-dependent protein kinase stimulated the incorporation of 32P into both the protein and lipid fractions of the membrane. The SDS gel-electrophoretic analysis of the 32P-labelled proteins showed that the protein kinase phosphorylated preferentially a 24000-Mr protein, though other 32P-labelled proteins were also detected. 32P-labelled membrane lipids were analysed in two different thin layer chromatographic systems. 32P-labelling was found exclusively in polyphosphoinositides. On addition of the protein kinase the 32P-labelling of both polyphosphoinositides was increased but a higher amount of phosphate was incorporated into phosphatidylinositol-4-phosphate than into phosphatidylinositol-4,5-bisphosphate.

Adenosine Triphosphate↗

PubMed1984

Cyclic AMP-dependent protein kinase and Ca2+-calmodulin stimulate the formation of polyphosphoinositides in a sarcoplasmic reticulum preparation of rabbit heart.

A rabbit heart membrane fraction enriched in sarcoplasmic reticulum was incubated in a reaction mixture containing [gamma-32P]ATP. The catalytic subunit of cyclic AMP-dependent protein kinase enhanced the 32P-labelling of both phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. Ca2 +-calmodulin also increased the 32P-incorporation into both polyphosphoinositides. Upon SDS gel-electrophoretic analysis of the membrane proteins, phospholamban was found to be concurrently phosphorylated by the exogenous catalytic subunit as well as by an endogenous Ca2+-calmodulin-dependent protein kinase.

Animals↗

PubMed1984

Volume regulation of Chinese hamster ovary cells in anisoosmotic media.

Chinese hamster ovary (CHO) cells when suspended in anisoosmotic media regulate their volumes by the activation of specific ion transport pathways. In hypoosmotic media the cells first swell and then return to their isoosmotic volumes by the loss of cellular KCl and osmotically obliged water. This regulatory volume decrease (RVD) is insensitive to ouabain or bumetanide but is blocked by quinine, cetiedil and oligomycin C. Based on cell volume and membrane potential measurements under various experimental conditions, we conclude that hypoosmotic shock activates independent, conductive transport pathways for K+ and for Cl-, respectively. The anion pathway can also transport NO3- and SCN- but not gluconate- anions. Osmotic shrinkage of CHO cells does not produce a regulatory volume increase (RVI) unless the cells have previously undergone a cycle of RVD. RVI is a Na+-dependent, amiloride-sensitive, but ouabain- and oligomycin-insensitive process, probably involving a Na+-H+ exchange system. Internal acidification of isoosmotic cells by addition of a permeable weak acid also activates an amiloride-sensitive Na+-H+ exchange, producing a volume increase. Both RVD and RVI in CHO cells seem to involve molecular mechanisms similar to those described for the volume regulation of lymphocytes, indicating the prevalence of these phenomena in nucleated mammalian cells. Cultured CHO cell lines may provide a basis for a genetic characterization of the volume-regulatory transport pathways.

Animals↗

PubMed1984

Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. I. Distinctions between volume-activated Cl- and K+ conductance pathways.

Human peripheral blood lymphocytes (PBL), when placed into hypotonic media, first swell and then shrink back to their original volumes because of a rapid KCl leakage via volume-activated K+ and anion permeation pathways. By using gramicidin, a cation channel-forming ionophore, cation transport through the cell membrane can be shunted so that the salt fluxes and thus the volume changes are limited by the rate of the net anion movements. The "gramicidin method," supplemented with direct measurements of volume-induced ion fluxes, can be used to assess the effects of drugs and of various treatments on cation and anion permeabilities. It is demonstrated that quinine and cetiedil are much more effective blockers of volume-induced K+ transport than of Cl- transport, while dipyridamole, DIDS, and NIP-taurine inhibit only volume-induced Cl- movement. Oligomycins block both cation and anion transport pathways, oligomycin A being more effective in inhibiting K+ transport and oligomycin C preferentially blocking Cl- movement. Ca depletion of PBL abolishes volume-induced K+ transport but has no effect on Cl- transport. Repletion of cell calcium by ionophore A23187 immediately restores rapid K+ transport without significantly affecting volume-induced Cl- transport. These observations, taken together with other reported information, can be best explained by a model in which cell swelling activates independent Cl- and K+ conductance pathways, the latter being similar in properties to the Ca2+-activated K+ transport observed in various cell membranes.

Azepines↗

PubMed1984

Ionic events during the volume response of human peripheral blood lymphocytes to hypotonic media. II. Volume- and time-dependent activation and inactivation of ion transport pathways.

Hypotonic dilution of human peripheral blood lymphocytes (PBL) induces large conductive permeabilities for K+ and Cl-, associated with the capacity of the cells to regulate their volumes. When rapid cation leakage is assured by the addition of the ionophore gramicidin, the behavior of the anion conductance pathway can be independently examined. Using this technique it is demonstrated that the volume-induced activation of Cl- transport is triggered at a threshold of approximately 1.15 X isotonic cell volume. If the volume of a cell is increased to this level or above, the Cl- transport system is activated, whereas if the volume of a swollen cell is decreased below the threshold value, the Cl- transport is inactivated. Activation and inactivation are independent of the relative volume changes and of the actual cellular Na+, K+, or Cl- concentrations, as well as of the changes in membrane potential in PBL. When net salt movement and thus volume change are inhibited by specific blockers of K+ transport (e.g., quinine, or Ca2+ depletion), volume-induced Cl- conductance shows a time-dependent inactivation, with a half-time of 5-8 min. The Cl- conductance, when activated, appears to involve an all-or-none response. In contrast, volume-induced K+ conductance is a graded response, with the increase in K+ flux being roughly proportional to the hypotonicity-induced increase in cell volume. The data indicate that during lymphocyte volume response in hypotonic media, anion conductance increases by orders of magnitude, exceeding the K+ conductance, so that the rate of the volume decrease (KCl efflux) is determined by a graded alteration in K+ conductance. When the cell volume approaches the isotonic value, it is stabilized by the inactivation of the anion conductance pathway.

Biological Transport↗

PubMed1984

Responses of lymphocytes to anisotonic media: volume-regulating behavior.

The regulatory responses elicited in lymphoid cells suspended in anisotonic media are reviewed. The immediate response approximates osmometric behavior. In addition, in hypotonic media, the initial osmometric swelling is followed by a regulatory volume decrease (RVD), which is associated with KCl loss. The volume-induced effluxes of K+ and Cl- are mediated by two independent conductive pathways. Ca2+-depletion experiments and studies of inhibitor susceptibility suggest that Ca2+ may mediate the activation of the K+ pathway. The responses of the two main lymphocyte subpopulations to hypotonic challenge are different. RVD is much more rapid in T- than in B-cells, regardless of their tissue of origin. Under certain conditions, shrunken lymphocytes will regain their initial volume. This regulatory volume increase (RVI) is due to NaCl uptake, followed by a secondary exchange of Na+ for K+ via the Na+-K+ pump. Na+ is primarily taken up in exchange for H+ through an amiloride-sensitive pathway, whereas Cl- enters in exchange for HCO-3 (or OH-). Anion and cation fluxes responsible for RVI are electroneutral. Some of the volume-sensitive pathways can also be activated in isotonic cells. The conductive K+ pathway is activated by Ca2+ plus ionophore A23187, and the Na+-H+ exchanger can be activated by cytoplasmic acidification. The responses of lymphocytes to anisotonic challenge are compared with those of other cells, and the possible significance of the volume-induced fluxes is discussed.

Animals↗