The Journal of Membrane Biology

Kilberg, Michael

doi: 10.1007/BF01871236pmid: 6811749

Improvements in the collagenase perfusion techniques have made isolated rat hepatocytes a popular model in which to study hepatic function. Our knowledge of hepatic amino acid transport has been advanced as a result of this methodology. Translocation across the hepatocyte plasma membrane can, in some instances, represent the rate-limiting step in the overall metabolism of certain amino acids. Furthermore, regulation of amino acid uptake by hepatocytes appears to play a role in diabetes, and perhaps in malignant transformation. Comparisons between normal adult hepatocytes and several hepatoma cell lines show basic differences in amino acids transport. There are at least eight distinct systems in normal hepatocytes for transport of the amino acids. One of these, System A, transports the small neutral amino acids most efficiently and responds to a wide variety of hormones. Systems A and N exhibit enhanced uptake rates after the cells have been maintained in the absence of extracellular amino acids, a phenomenon termed adaptive control. Further studies using isolated hepatocytes will increase our basic understanding of membrane transport processes and their regulation.

McDonough, Alicia; Hiatt, Andrew; Edelman, Isidore

doi: 10.1007/BF01871237pmid: 6288956

Antibodies have been produced, in three rabbits, to Na/K-ATPase purified from guinea pig renal outer medulla. Each rabbit produced antibodies to both the α (catalytic) and the β (glycoprotein) subunits of Na/K-ATPase. The titers of the anti-α and anti-β antibodies varied with time and between rabbits. None of the antisera inhibited Na/K-ATPase activity under various preincubation conditions. A method is presented for separating small amounts of anti-α subunit from anti-β subunit antibodies. There was not cross-reactivity of antibodies to one subunit with the other subunit. The α subunit of the Na/K-ATPase was cleaved into a 41,000-dalton peptide (that contains the ATP phosphorylating site) and a 58,000-dalton hydrophobic peptide as described by Castro and Farley (Castro, J., Farley, R.A., 1979,J. Biol. Chem. 254:2221–2228). Anti-α antibodies from all of the rabbits reacted with both proteolytic fragments. The anti-guinea pig Na/K-ATPase antisera (pooled) cross-reacted with the α subunit of Na/K-ATPase from human, cow, dog, rabbit, rat mouse, turtle, and toad; and with the β subunit from human, rat, and mouse. The loci of cross-reactivity were investigated using partially purified canine kidney Na/K-ATPase cleaved with trypsin as described above. The antisera from rabbits 1 and 2 cross-reacted with the 41,000-dalton peptide from the dog but very little with the 58,000-dalton peptide. No cross-reactivity was observed with antiserum from rabbit 3 to either fragment. Guinea pig kidney RNA was translated in a rabbit reticulocyte lysate system followed by immunoprecipitation with the antisera. The molecular weight of the cell-free synthesized α chain was 96,000 daltons. Its identity was established with purified anti-α antibodies and by immunocompetition with purified Na/K-ATPase and Ca-ATPase. Translation of the β subunit was not detected in this system.

Conti, F.; Fioravanti, R.; Segal, J.; Stühmer, W.

doi: 10.1007/BF01871238pmid: 7120361

The effects of hydrostatic pressures up to 62 MPa upon the voltage-clamp currents of intact squid giant axons were measured using mineral oil as the pressure transmitting medium. The membrane resistance and capacitance were not appreciably affected over the whole range of pressures explored. The predominant effect of pressure is to slow the overall kinetics of the voltage-clamp currents. Both the early (Na) currents and the delayed (K) ones were slowed down by approximately the same time scale factor, which was in the range of 2 to 3 when pressure was increased from atmospheric to 62 MPa.

Conti, F.; Fioravanti, R.; Segal, J.; Stühmer, W.

doi: 10.1007/BF01871239pmid: 7120362

The effect of pressure upon the delayed, K, voltage-clamp currents of giant axons from the squidLoligo vulgaris was studied in axons treated with 300nm TTX to block the early, Na, currents. The effect of TTX remained unaltered by pressure. The major change produced by pressures up to 62 MPa is a slowing down of the rising phase of the K currents by a time scaling factor which depends on pressure according to an apparent activation volume, ΔV∓, of 31 cm3/mole at 15°C; ΔV∓ increased to about 42 cm3/mole at 5°C.

Weinstein, Steven; Kostellow, Adele; Ziegler, David; Morrill, Gene

doi: 10.1007/BF01871240pmid: 6288957

Progesterone initiates the resumption of the meiotic divisions in the amphibian oocyte. Depolarization of theRana pipiens oocyte plasma membrane begins 6–10 hr after exposure to progesterone (1–2 hr before nuclear breakdown). The oocyte cytoplasm becomes essentially isopotential with the medium by the end of the first meiotic division (20–22 hr). Voltage-clamp studies indicate that the depolarization coincides with the disappearance of an electrogenic Na+, K+-pump, and other electrophysiological studies indicate a decrease in both K+ and Cl− conductances of the oocyte plasma membrane. Measurement of [3H]-ouabain binding to the plasma-vitelline membrane complex indicates that there are high-affinity (K d-4.2×10−8 m), K+-sensitive ouabain-binding sites on the unstimulated (prophase-arrest) oocyte and that ouabain binding virtually disappears during membrane depolarization. [3H]-Leucine incorporation into the plasma-vitelline membrane complex increased ninefold during depolarization with no significant change in uptake or incorporation into cytoplasmic proteins or acid soluble pool(s). This together with previous findings suggests that progesterone acts at a translational level to produce a cytoplasmic factor(s) that down-regulates the membrane Na+, K+-ATPase and alters the ion permeability and transport properties of both nuclear and plasma membranes.

Shrier, Alvin; Clay, John

doi: 10.1007/BF01871241pmid: 7120363

We have investigated the pacemaker properties of aggregates of cells dissociated from the atria and ventricles of 10 to 14-day-old chick embryonic hearts using a two-microelectrode current and voltage-clamp technique. These preparations usually beat spontaneously and rhythmically in tissue culture medium containing 1.3mm potassium with a beat rate typically in the range of 15–60 beats per minute. The beat rate results show considerable variability, which precludes any statistically significant comparison between the spontaneous activity of atrial and ventricular cell preparations at 10–14 days of development. However, the shapes of pacemaker voltage changes do exhibit differences characteristic of cell type. Spontaneous atrial preparations rapidly depolarize from maximum diastolic potential (∼−90 mV) to a plateau range of pacemaker potentials (−80 to −75 mV). The membrane subsequently depolarizes more gradually until threshold (∼−65 mV) is reached. In contrast, spontaneously beating ventricular cell preparations slowly hyperpolarize after maximum diastolic potential to the −100 to −95 mV range before gradually depolarizing toward threshold. Voltage-clamp analysis reveals a virtual lack of any time-dependent pacemaker current in atrial preparations. These preparations are characterized by an approximately linear background current (I bg) having a slope resistance of ∼100 KΩ cm2. Ventricular preparations have a potassium ion pacemaker current with slow kinetics (I K 2), and a second time-dependent component (I x) which is activated at potentials positive to −65 mV. The background current of these preparations displays inward rectification. Computer simulations of pacemaking reveal that the initial rapid phase of pacemaker depolarization in atrial cells is determined by the membrane time constant, which is the product of membrane capacitance and the slope resistance ofI bg. The hyperpolarization after maximum diastolic potential of ventricular cells is caused byI K 2. The final slow phase of depolarization in both cell types is caused in part by the steady-state amplitude of the fast inward sodium current (I Na). This component has negative slope conductance which effectively increases the slope resistance in the vicinity of threshold compared to TTX-treated preparations. This mechanism is sufficient to produce interbeat intervals several seconds in duration, even in the absence of time-dependent pacemaker current, provided that the background current is at the appropriate level.

Ritchie, Raymond

doi: 10.1007/BF01871242pmid: N/A

The vacuolar equilibrium potential of the lipophilic cation TPMP+ (triphenyl methyl phosphonium) in the giant algaeChara australis andGriffithsia monilis was directly measured. The TPMP+ equilibrium potential was approximately 100mV less negative than the measured vacuolar electrical potential. Thus TPMP+ does not act as a probe of the vacuolar electrical potential and appears to be extruded against an electrochemical gradient. Measurement of the plasmalemma equilibrium potential of TPMP+ showed that extrusion of TPMP+ apparently occurred at both the tonoplast and plasmalemma inChara and at the plasmalemma inGriffithsia. It is concluded that TPMP+ cannot be used as a membrane potential probe inChara orGriffithsia.

Schmalzing, Günther; Kutschera, Petra

doi: 10.1007/BF01871243pmid: 6126596

The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na+K+-ATPase only. Anionic and non-ionic detergents and α-lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.

Gilbert, John; Meissner, Gerhard

doi: 10.1007/BF01871244pmid: 7120364

The Ca2+ permeability of rabbit skeletal muscle sarcolemmal vesicles was investigated by means of radioisotope flux measurements. A membrane vesicle fraction highly enriched in sarcolemma, as revealed by enzymatic markers, was obtained from the 22–27% region of sucrose gradients after isopycnic centrifugation. The ability of sarcolemmal vesicles to exchange Na+ for Ca2+ was investigated by measuring Ca2+ influx into and efflux from sarcolemmal vesicles in the presence and absence of a Na+ gradient. It was found that Ca2+ movements were enhanced in the direction of the higher Na+ concentration. When intra- and extravesicular Na+ concentrations were high, Na+−Na+ exchange predominated and Na+−Ca2+ exchange was low or absent. The presence of the Ca2+ ionophore A23187 in the dilution medium resulted in the rapid release of Ca2+ and the elimination of the Na+-enhanced efflux of Ca2+, suggesting that internal rather than bound external Ca2+ was exchanged with Na+. La3+ abolished Na+−Ca2+ exchange and decreased overall membrane permeability. Na+−Ca2+ exchange was not due to sarcoplasmic reticulum or mitochondrial contaminants. This investigation suggests that skeletal muscle, like cardiac muscle and neurons, is capable of a transmembranous Na+−Ca2+ exchange.