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J F Hoffman

Publications and source records attributed to J F Hoffman.

At least 37 records · Page 2Linked to original sources
PubMed1990

Membrane sidedness and the interaction of H+ and K+ on Ca2(+)-activated K+ transport in human red blood cells.

The sided effects of H+ on Ca2(+)-stimulated K+ transport (the Gardos channel) were studied in human red blood cells. Cells were loaded with Ca2+ during energy depletion with the internal pH adjusted to desired levels prior to treatment with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which inhibits pH equilibration across the membrane. This treatment provides a "pH clamp" whereby the internal and external H+ (H+i and H+o) concentrations can be varied separately. Channel activity was evaluated by measuring either net K+ loss or unidirectional 42K+ efflux from cells where SO2(-4) replaced Cl- on both sides of the membrane. When pHi was set at 7.4, decreasing pHo from values of 8.0 to 5.0 inhibited K+ efflux. This effect of H+o could be overcome by increasing K+o at all values of pHo. In addition, this effect of K+o could be separated from its effects on altering the membrane potential, indicating an interaction between K+o and H+o on the channel. A similar interaction was shown to occur between H+i and K+i. K+o is known to be required for activation of Ca2(+)-stimulated K+ transport, since the channel in cells preincubated in the absence of K+o (prior to exposure to Ca+i) becomes refractory to subsequent activation by Ca2+i and K+o. We found that H+o would not substitute for K+o in this regard nor would H+o inhibit the protective effect of K+o; in addition, H+ was not transported inward in exchange for K+i. Thus it would appear that there are two external sites where K+o interacts with the channel. One site is antagonized by H+o, whereas the second site is required for channel activation independent of H+ in the range studied. The inside of the channel would have, by an analogous argument, at least one site where K+i and H+i interact.

Calcium↗
PubMed1990

Anion-coupled Na efflux mediated by the human red blood cell Na/K pump.

The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain-sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS-treated, SO4-equilibrated human red blood cells suspended in HEPES-buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o. Addition of 10 mM Ko to tartrate i,o ghosts, with or without Cli,o, resulted in full activation of Na/K exchange and the pump's electrogenicity. Although it can be concluded that Na efflux in the uncoupled mode occurs by means of a cotransport with cellular anions, the molecular basis for this change in the internal charge structure of the pump and its change in ion selectivity is at present unknown.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

On the mechanism and measurement of shape transformations of constant volume of human red blood cells.

A method is described for measuring the rate of disc-sphere transformations of constant volume by recording changes in the intensity and noise envelope of light transmitted through a stirred suspension of human red blood cells. We used this method to determine the rates and characteristics of disc-sphere transitions induced by various sphering agents (uranyl nitrate, rose bengal, Na glycocholate). It was found that, when sphered by any of these agents, cells spontaneously reverted to discs upon continued incubation in the presence of the agent. The rate of sphering was directly proportional and the rate of spontaneous reversal inversely related to the concentration of sphering agent used. Plasma added before an agent is added prevents subsequent sphering; plasma added after sphering had occurred accelerated reversal. During spontaneous reversal of the spheres to discs, sphering could again be induced prior to subsequent return to discs either by the addition of more agent or, in certain circumstances, by the addition of plasma. Spontaneous reversal of discs to spheres qualitatively correlates with the uptake rate of agent (rose bengal), the cells becoming discs as uptake is completed. Furthermore, establishment of temporary proton gradients across the membrane promotes sphering with return to discs as pH equilibrium takes place. Evidently, the spherical shape is a transient form representing the unequal distribution of agent across the membrane. The spherical form could thus be associated with a change in the relative surface energies between the outer and inner lipid leaflets, such as a difference in their relative surface areas, as hypothesized in the "bilayer couple" models. The molecular mechanism(s) underlying the action of sphering agents and protons may not be the same.

Adult↗
PubMed1986

Effects of altering the ATP/ADP ratio on pump-mediated Na/K and Na/Na exchanges in resealed human red blood cell ghosts.

Resealed human red blood cell ghosts were prepared to contain a range of ADP concentrations at fixed ATP concentrations and vice versa. ATP/ADP ratios ranging from approximately 0.2 to 50 were set and maintained (for up to 45 min) in this system. ATP and ADP concentrations were controlled by the addition of either a phosphoarginine- or phosphocreatine-based regenerating system. Ouabain-sensitive unidirectional Na efflux was determined in the presence and absence of 15 mM external K as a function of the nucleotide composition. Na/K exchange was found to increase to saturation with ATP (K 1/2 approximately equal to 250 microM), whereas Na/Na exchange (measured in K-free solutions) was a saturating function of ADP (K 1/2 approximately equal to 350 microM). The elevation of ATP from approximately 100 to 1,800 microM did not appreciably affect Na/Na exchange. In the presence of external Na and a saturating concentration of external K, increasing the ADP concentration at constant ATP was found to decrease ouabain-sensitive Na/K exchange. The decreased Na/K exchange that still remained when the ADP/ATP ratio was high was stimulated by removal of external Na. Assuming that under normal substrate conditions the reaction cycle of the Na/K pump is rate-limited by the conformational change associated with the release of occluded K [E2 X (K) X ATP----E1 X ATP + K], increasing ADP inhibits the rate of these transformations by competition with ATP for the E2(K) form. A less likely alternative is that inhibition is due to competition with ATP at the high-affinity site (E1). The acceleration of the Na/K pump that occurs upon removing external Na at high levels of ADP evidently results from a shift in the forward direction of the transformation of the intermediates involved with the release of occluded Na from E1P X (Na). Thus, the nucleotide composition and the Na gradient can modulate the rate at which the Na/K pump operates.

Adenosine Diphosphate↗
PubMed1984

Interrelations among Na and K content, cell volume, and buoyant density in human red blood cell populations.

This study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cell's Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na + K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966. Scand. J. Lab. Invest. 18:167-180) cell water and cell volume was found to vary directly with the summed content of Na + K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na + K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.

Body Water↗
PubMed1984

Passive Ca transport in human red blood cell ghosts measured with entrapped arsenazo III.

The rate of Ca influx into ghosts containing arsenazo III changes with time, being most rapid during the first 5 min after Ca is added to the outside and declining thereafter. The rate of Ca influx is a nonlinear function of extracellular Ca and plateaus as the latter is increased above 1 mM. The rate of Ca influx was measured as a function of the transmembrane gradients of Na and K and changes in the permeability of the membrane to K and Cl produced by valinomycin and SITS (4-acetamido-4'-isothiocyano-stilbene-2-2'-disulfonic acid), respectively. Changes in the rate of Ca influx are consistent with expected effects of these treatments on the membrane potential. Oligomycin (10 micrograms/ml) and quinidine (1 mM) inhibit the rate of Ca uptake by inhibiting Ca-induced changes in the K permeability. At constant membrane potential, furosemide produced a slight (15%) consistent increase in Ca uptake. Other experiments show that resealed ghosts are heterogeneous in their passive permeability to Ca and that A23187 can be used to effectively eliminate such differences. The results of this paper show that resealed human red cell ghosts containing arsenazo III can be used to continuously monitor intracellular free Ca and to study the factors that influence the permeability of the red cell membrane to Ca.

Arsenazo III↗
PubMed1984

Ca-induced K transport in human red blood cell ghosts containing arsenazo III. Transmembrane interactions of Na, K, and Ca and the relationship to the functioning Na-K pump.

Increasing free intracellular Ca (Cai) from less than 0.1 microM to 10 microM by means of A23187 activated Ca-stimulated K transport and inhibited the Na-K pump in resealed human red cell ghosts. These ghosts contained 2 mM ATP, which was maintained by a regenerating system, and arsenazo III to measure Cai. Ca-stimulated K transport was activated 50% at 2-3 microM free Cai and the Na-K pump was inhibited 50% by 5-10 microM free Cai. Free Cai from 1 to 8 microM stimulated K efflux before it inhibited the Na-K pump, dissociating the effect of Ca on the two systems. 3 microM trifluoperazine inhibited Ca-stimulated K efflux and 0.5 mM quinidine reduced Na-K pumping by 50%. In other studies, incubating fresh intact cells in solutions containing Ca and 0.5 microM A23187 caused the cells to lose K heterogeneously. Under the same conditions, increasing A23187 to 10 microM initiated a homogeneous loss of K. In ATP-deficient ghosts containing Cai equilibrated with A23187, K transport was activated at the same free Cai as in the ghosts containing 2 mM ATP. Neither Cao nor the presence of an inward Ca gradient altered the effect of free Cai on the permeability to K. In these ghosts, transmembrane interactions of Na and K influenced the rate of Ca-stimulated K efflux independent of Na- and K-induced changes in free Cai or sensitivity to Cai. At constant free Cai, increasing Ko from 0.1 to 3 mM stimulated K efflux, whereas further increasing Ko inhibited it. Increasing Nai at constant Ki and free Cai markedly decreased the rate of efflux at 2 mM Ko, but had no effect when Ko was greater than or equal to 20 mM. These transmembrane interactions indicate that the mechanism underlying Ca-stimulated K transport is mediated. Since these interactions from either side of the membrane are independent of free Cai, activation of the transport mechanism by Cai must be at a site that is independent of those responsible for the interaction of Na and K. In the presence of A23187, this activating site is half-maximally stimulated by approximately 2 microM free Ca and is not influenced by the concentration of ATP. The partial inhibition of Ca-stimulated K efflux by trifluoperazine in ghosts containing ATP suggests that calmodulin could be involved in the activation of K transport by Cai.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1984

Chloride transport properties of human leukemic cell lines K562 and HL60.

The Cl- transport characteristics of the human leukemic cell lines K562 and HL60, with erythroid and granulocytic phenotypic features, respectively, were investigated. Cl- effluxes were measured with 36Cl- under equilibrium conditions in both cell lines and were found to be three orders of magnitude slower than the unidirectional efflux of Cl- in normal erythrocytes. Induction of differentiation of the K562 cell line with hemin does not affect the rate of Cl- transport, while induction of the HL60 cell line with dimethyl sulfoxide results in a small decrease in the rate of Cl- transport. Cl- transport in both cell lines could be divided into two components. One component is inhibited by treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), displays counter-transport characteristics, and has a high energy of activation--all properties characteristic of the human erythrocyte-facilitated anion exchange system. The second component is insensitive to DIDS, is partially inhibited by furosemide, and has a low energy of activation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1984

Membrane electrical parameters of normal human red blood cells.

The studies reported in this chapter indicate that the Na/K pump's contribution to Em of human red blood cells with normal cellular Na is no more than 0.1 to 0.2 mV. Estimates based on this magnitude of the electrogenic component and the pump current (taken as one-third the ouabain-sensitive Na efflux) indicate that Rm in these cells is about 1 to 2 X 10(5) ohm cm2.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1983

Intracellular free Ca and Mg of human red blood cell ghosts measured with entrapped arsenazo III.

Arsenazo III in human red cell ghosts is calibrated to measure intracellular concentrations of free Ca and free Mg. This calibration was established by comparing the absorbance of arsenazo III in ghosts to its absorption in solution at 600, 630, and 655 nm as a function of buffered free Ca (0.4 microM to 70 microM), free Mg (0.05 to 5 mM), and free Ca (4 to 50 microM) at constant free Mg (1.2 mM) at three concentrations of total dye (1.09, 10.9, and 109 microM). In both ghosts and in solution the absorbance of the dye at all three wavelengths could be predicted from dissociation constants and molar extinction coefficients determined for a 1:1 complex with the dye and Mg, another with Ca, and a third complex consisting of two molecules of Ca and two of dye. The absorbance of the dye in ghosts at the same concentrations of free Ca, free Mg, and total dye is equal to that in solution multiplied by the percentage hematocrit and divided by 100, which demonstrates that arsenazo III responds the same inside ghosts as it does free in solution. The results of this paper show that arsenazo III can be used to measure quantitatively and to monitor continuously the concentration of intracellular Ca and Mg in red cell ghosts. Use of this method should facilitate the study of Ca-dependent mechanisms of red blood cells.

Arsenazo III↗
PubMed1980

The link between metabolism and active transport of sodium in human red cell ghosts.

Reconstituted human red blood cell ghosts have been used to assay various metabolic intermediates for their role in driving the Na:K pump. ATP was identified as the primary substrate of the pump. The main evidence was based on 1) the finding that the only requirement for activation of the pump was the presence of ATP, whether incorporated directly or generated by an ATP-yielding reaction; 2) the discriminating effects of various metabolic inhibitors; 3) the competition between the pump and hexokinase reaction for ATP; and 4) the difference in effects of adenosine and inosine in activating the pump in energy-depleted ghosts. ADP was found to affect the Na:K pump due to the presence of an adenylate kinase and perhaps because of an effect on the phosphoryl potential. The sidedness of action of the cardiotonic steroid, strophanthidin, was evaluated and found to inhibit the Na:K pump only from the outside of the membrane. Inhibition of the pump by strophanthidin was also found to spare ATP in reconstituted ghosts provided the nonspecific phosphatase activity was suppressed.

Adenosine Diphosphate↗