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Biomedical subjects

A Rothstein

Publications and source records attributed to A Rothstein.

At least 73 records · Page 4Linked to original sources
PubMed1983

Volume restoration in osmotically swollen lymphocytes does not involve changes in free Ca2+ concentration.

An increase in cytoplasmic free [Ca2+], [Ca2+]i, has been suggested as the trigger for the permeability changes that bring about cell volume restoration following exposure to anisotonic media. This idea was directly tested in human peripheral lymphocytes undergoing regulatory volume decrease following a hypotonic dilution of the suspension. [Ca2+]i was measured with the intracellularly trapped fluorescent indicator, quin2, and showed no measureable change on hypotonic swelling or during the subsequent volume decrease. Moreover, even though the incorporated quin2 adds significant Ca-buffering to the cytoplasm, regulatory volume decrease occurred normally in the quin2-loaded cells. It appears that alterations in [Ca2+]i are not involved in these processes of volume regulation. An intracellularly trapped derivative of fluorescein, bis(carboxyethyl)carboxyfluorescein, was used to monitor cytoplasmic pH, which also showed no change during regulatory volume decrease.

Body Fluids↗
PubMed1983

Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes.

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.

Animals↗
PubMed1983

Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification.

After swelling in hypotonic solutions, peripheral blood mononuclear cells (PBM) shrink toward their original volumes. Upon restoration of isotonicity, the cells initially shrink but then regain near-normal size again. This regulatory volume increase (RVI) is abolished by removal of Na+o or Cl-o or by addition of amiloride. RVI is unaffected by removal of K+o or by ouabain and is only partially inhibited by 1 mM furosemide. As a result of increased influx, the cells gain both Na+ and K+ during reswelling. In contrast, only Na+ content increases in the presence of ouabain. Amiloride largely eliminates the changes in the content of both cations. Using diS-C3-(5), no significant membrane potential changes were detected during RVI, which suggests that the fluxes are electroneutral. The cytoplasmic pH of volume-static cells was measured with 5,6-dicarboxyfluorescein. After acid loading, the addition of extracellular Na+ induced an amiloride-inhibitable alkalinization, which is consistent with Na+/H+ exchange. Cytoplasmic pH was not affected by cell shrinkage itself, but an internal alkalinization, which was also amiloride sensitive and Na+ dependent, developed during reswelling. In isotonic lightly buffered solutions without HCO-3, an amiloride-sensitive acidification of the medium was measurable when Na+ was added to shrunken PBM. K+ was unable to mimic this effect. The observations are compatible with the model proposed by Cala (J. Gen. Physiol. 1980. 76:683-708), whereby an electroneutral Na+o/H+i exchange is activated by osmotic shrinking. Cellular volume gain occurs as Cl-o simultaneously exchanges for either HCO-3i or OH-i. Na+i is secondarily replaced by K+ through the pump, but this step is not essential for RVI.

Acid-Base Equilibrium↗
PubMed1983

Volume-induced anion conductance in human B lymphocytes is cation independent.

Peripheral blood T lymphocytes swollen in hypotonic media regain normal size by releasing internal KCl through activation of conductive K+ and Cl- pathways. In contrast, no regulatory volume decrease (RVD) is observed in tonsillar B lymphocytes. The volume-induced K+ permeability is minimal in these cells, but little was known about the effect of swelling on anion transport and its coupling to cation translocation. The induction of an anion-conductive pathway on swelling of B cells was demonstrated in the following way: 1) when an exogenous cation ionophore was added, hypotonic swelling was followed by secondary volume changes, the direction of which was dictated by the electrochemical gradients of the diffusible ions; 2) the rate of 36Cl efflux was markedly increased by swelling; and 3) upon swelling, the membrane potential approached the Cl- equilibrium potential. Quinine, which blocks K+ transport and RVD in swollen T cells, has no effect on volume-induced anion fluxes. Consequently, secondary volume changes could be elicited by gramicidin in cells pretreated with quinine. It can be concluded that in B cells, swelling leads to activation of a conductive Cl- permeability but not of K+ permeability. These and other findings support the view that the Cl- pathway functions independently of the K+ pathway.

Anions↗
PubMed1982

The red cell band 3 protein: its role in anion transport.

Studies of anion transport across the red blood cell membrane fall generally into two categories: (1) those concerned with the operational characterization of the transport system, largely by kinetic analysis and inhibitor studies; and (2) those concerned with the structure of band 3, a transmembrane peptide identified as the transport protein. The kinetics are consistent with a ping-pong model in which positively charged anion-binding sites can alternate between exposure to the inside and outside compartments but can only shift one position to the other when occupied by an anion. The structural studies on band 3 indicate that only 60% of the peptide is essential for transport. That particular portion is in the form of a dimer consisting of an assembly of membrane-crossing strands (each monomer appears to cross at least five times). The assembly presents its hydrophobic residues toward the interior of the bilayer, but its hydrophilic residues provide an aqueous core. The transport involves a small conformational change in which an anion-binding site (involving positively charged residues) can alternate between positions that are topologically in and topologically out.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

Increased anion permeability during volume regulation in human lymphocytes.

Peripheral blood lymphocytes (p.b.ls) readjust their volumes after swelling in hypotonic media. An essential component of the regulatory response is an increase in K+ and Cl- permeability. No evidence was found for a tightly coupled co-transport of K+ and Cl-. The flux of either ion proceeds normally in the virtual absence of the transported counterion. Furthermore, alterations in membrane potential recorded during the phase of volume readjustment can be qualitatively accounted for by an increase in Cl- conductance. In tonsillar lymphocytes, a failure of the K+-permeability is nevertheless increased upon swelling. This further suggests that K+ and Cl- are transported during volume regulation through independent pathways. Cytoplasmic free Ca2+ appears to be involved in regulatory volume decrease. K+ and Cl-. Moreover, swelling and shrinking can be induced in isotonic K+-rich and K+-free media, respectively, by the Ca2+ ionophore. The ion flux and volume changes produced by either swelling or internal Ca2+ can be inhibited by similar concentrations of quinine and phenothiazines. The inhibitory activity of the latter drugs, which are powerful antagonists of calmodulin, suggests the participation of this Ca2+-regulator protein in volume regulation.

Anions↗
PubMed1982

Volume regulation by human lymphocytes. Role of calcium.

Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. 86Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++-containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased 86Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in 86Rb+ efflux. Quinine also inhibited the volume changes and the increased 86Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in 86Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic [Ca++], triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).

Calcimycin↗
PubMed1982

Volume-induced increase of anion permeability in human lymphocytes.

Peripheral blood mononuclear cells (PBM) readjust their volumes after swelling in hypotonic media. This regulatory volume decrease (RVD) is associated with a loss of cellular K+ and is thought to be promoted by an increased permeability to this ion. In contrast, no change in volume was observed when K+ permeability of PBM in isotonic media was increased to comparable or higher levels using valinomycin. Moreover, valinomycin-induced 86Rb+ loss in K+-free medium was considerably slower than in K+-rich medium. These results suggest that anion conductance limits net salt loss in isotonic media. Direct measurements of relative conductance confirmed that in volume-static cells, anion conductance is lower than that of K+. In volume-regulating cells depolarization occurred presumably as a result of increased anion conductance. Accordingly, the efflux of 36Cl from PBM was markedly increased by hypotonic stress. Since both membrane potential and intracellular 36Cl concentration are reduced in hypotonically swollen cells, the increased efflux is probably due to a change in Cl- permeability. Anions and cations seem to move independently through the volume-induced pathways: the initial rate of 86Rb uptake in swollen cells was not affected by replacement of external Cl- by SO=4; conversely, 36Cl fluxes were unaffected by substitution of K+ by Na+. The data indicate that anion conductance is rate-determining in salt and water loss from PBM. An increase in anion conductance is suggested to be the critical step of RVD of human PBM.

Anions↗
PubMed1982

The location of a chymotrypsin cleavage site and of other sites in the primary structure of the 17,000-dalton transmembrane segment of band 3, the anion transport protein of red cell.

A 17,000-dalton transmembrane segment of band 3 protein is further cleaved by chymotrypsin treatment of red blood cell ghosts to 15,000 daltons. The location of this particular chymotrypsin cleavage site was determined by comparing the fragmentation pattern of the 17,000- and 15,000-dalton peptides using cyanogen bromide (CNBr). Each peptide is cleaved at its two methionine residues into three fragments. For each peptide two of the fragments are the same size, 7000 and 4000 daltons, the latter containing, in each case, the binding site of the anion transport inhibitor 4,4'-diisothiocyano-2,2' disulfonic acid (DIDS). The third fragment is 2000 daltons larger in the case of the 17,000-dalton peptide (6000 compared to 4000 daltons). These findings indicate that the chymotrypsin cleavage site is located at the cytoplasmic side of the membrane, 2000 daltons from the N-terminus of the 17,000-dalton peptide. This information allows the mapping of a number of defined sites of the 15,000-dalton segment within the primary structure of band 3. These sites support the suggestion that this peptide segment is folded within the bilayer.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1982

The implications of early psychopathology for the analysability of narcissistic personality disorders.

This paper explores the influences of early psychopathology on questions of analysability in general and upon the analysability of narcissistic personality disorders in particular. Because various theoretical perspectives influence the meanings attributed to genetic and analytic data and may shape the emergence and unfolding of the data, current definitions of analytic process and analysability are reviewed. Work with two unanalysable narcissistic personality disorders is presented to facilitate an inquiry into the influence of their early psychopathology on their egos' limited capacity to become involved in an analytic process. This inquiry is pursued by exploring these subjects' character organizations, life histories and analytic and therapeutic experiences from four complementary perspectives: First, the relationship of their early life experiences to their ego development is examined with particular emphasis on the integration of their self-representations and the development of their capacity for fantasy, self-observation and creative sublimation. Second, the influence of process versus shock trauma in the genesis of their character pathology is delineated. Third, the development of trust in relationship to a variety of alliances with the analyst is considered in relationship to the assessment of analysability. Fourth, the question of their diagnosis is explored.

Adult↗
PubMed1981

Inhibition of anion transport associated with chymotryptic cleavages of red blood cell band 3 protein.

Right-side-out vesicles derived from red blood cells treated with chymotrypsin retain specific anion transport function (defined as transport sensitive to the specific inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), even though the transport protein, band 3, is cleaved into two segments of 60 and 35 kdaltons. In contrast, vesicles derived from alkali-stripped ghosts treated with relatively high concentrations of chymotrypsin retain almost no specific anion function. The loss of function appears to be related to additional cleavages of band 3 protein that occur in treated ghosts, the 60-kdalton segment being reduced first to a 17- and then to a 15-kdalton segment and the 35-kdalton segment being reduced to a 9-kdalton segment plus a carbohydrate containing fragment. The chymotryptic cleavages of band 3 protein of ghosts are preferentially inhibited by high ionic strength, the production of the 9-kdalton segment being somewhat slower than that of the 15-kdalton segment. Vesicles derived from ghosts treated with chymotrypsin at different ionic strengths show a graded reduction in specific anion transport activity, but it was not possible to determine, definitively, which of the additional cleavages was inhibitory. In the light of these data and other information, the functional role of the segments of band 3 is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1981

Effects of quinine on Ca++-induced K+ efflux from human red blood cells.

The Ca++-mediated increase in K+-permeability of intact red blood cells (Gardos effect) was initiated by exposing cells to know concentrations of Ca++ (using EGTA buffers) in the presence of the ionophore A23187. The potency of quinine, an inhibitor of the response, was found to depend on the external K+ concentration. In K+-free solutions the concentration of quinine to achieve 50% inhibition (K50) was 5 microM, but at 5 mM K+ the required concentration was increased 20-fold to 100 microM. An increase in internal Na+ had the opposite effect, allowing a high potency of quinine despite the presence of external K+. Alterations in the internal K+ level, on the other hand, were without effect on the K50, suggesting that the membrane potential is not a factor. This conclusion is supported by the lack of effect on quinine inhibition of substitution of Cl- by NO3-, a considerably more permeant anion. The data are consistent with the hypothesis that quinine inhibits by competitively displacing K+ from an external binding site, the reported K+-activation site for the Ca++-mediated K+-permeability.

Calcimycin↗
PubMed1981

The amino acid conjugate formed by the interaction of the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) with band 3 protein from human red blood cell membranes.

The specific anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and its reduced analog (H2DIDS), when irreversibly bound to band 3 protein of the red blood cell membrane, form amino acid conjugates through interaction with the epsilon-amino group of a particular lysine residue. The specific residue is located in a transmembrane segment of band 3 protein and appears to be a close neighbor of the transport site.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1981

The sulfhydryl groups of the 35,000-dalton C-terminal segment of band 3 are located in a 9000-dalton fragment produced by chymotrypsin treatment of red cell ghosts.

Five sulfhydryl groups of band 3, the anion-transport protein of the red blood cell membrane, can be labeled by N-ethylmaleimide (NEM). Two of these are located in a 35,000-dalton, C-terminal segment produced by chymotrypsin treatment of cells. Extensive treatment of unsealed ghosts with chymotrypsin results in the disappearance of the 35,000-dalton segment, but its two NEM-binding sites area preserved in a 9000-dalton peptide. The latter must therefore be a proteolytic product of the larger segment. Labeling of sulfhydryl groups of band 3 by an impermeant analog of NEM occurs in inside-out, but not in right-side-out vesicles derived from red cell ghosts, supporting the conclusion that NEM-reactive sulfhydryl groups, including those in the 35,000- and 9000-dalton segments, are exposed at the cytoplasmic face of the membrane. These findings support the conclusion that the 35,000-dalton segment crosses the bilayer, and suggest that the 9000-dalton segment may be a membrane-crossing portion of the 35,000-dalton segment.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1981

Mercurials and red cell membranes.

Mercurials influence a large number of protein-mediated functions in membranes including transport phenomena, related enzyme activities and sructural factors such as deformability and phospholipid asymmetry. The sulfhydryl groups that are the targets for mercurials are found in different locations in the membrane, the outer surface, internal compartments, or cytoplasmic surface. Those on the outer surface are immediately influenced even by non-penetrating mercurials, whereas those within the membrane are only accessible after a time delay, to permeating agents. Agents that permeate very rapidly will equilibrate with internal proteins (hemoglobin) producing small transient effects on the membrane, whereas agents that penetrate slowly will produce larger more prolonged effects. The mode of penetration is of importance. Sulfhydryl groups within an intrinsic protein channel will only be affected if the mercurial can penetrate into that channel. Although all mercurials can react with high specificity with sulfhydryl groups, structural factors relating to the membrane architecture and the capacity of different agents to penetrate into the membrane, lead to considerable diversity in their effectiveness against particular functions. Mercurials with different capacities to penetrate can be effective tools in determining the arrangement of functional proteins in the membrane and in determining how they work.

Cell Membrane Permeability↗