Keiko Hosokawa

Kinetic mechanism of Na+, K+, Cl--cotransport as studied by Rb+ influx into HeLa cells: effects of extracellular monovalent ions.

SummaryOuabain-insensitive, furosemide-sensitive Rb+ influx (JRb) into HeLa cells was examined as functions of the extracellular Rb+, Na+ and Cl− concentrations. Rate equations and kinetic parameters, including the apparent maximumJRb, the apparent values ofKm for the three ions and the apparentKi for K+, were derived. Results suggested that one unit molecule of this transport system has one Na+, one K+ and two Cl− sites with different affinities, one of the Cl− sites related with binding of Na+, and the other with binding of K+(Rb+). A 1∶1 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of22Na+ and Rb+, and a 1∶2 stoichiometry between those of Rb+ and36Cl−. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl− inhibited ouabain-insensitive Rb+ influx, whereas sulfamate and probably also gluconate did not inhibitJRb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na+ and one Cl− bind concurrently to their sites and then one K+ (Rb+) and another Cl− bind concurrently. 2) After completion of ion bindings Na+, K+(Rb−) and Cl− in a ratio of 1∶1∶2 show synchronous transmembrane movements.

Blood flow velocity in the common carotid artery in humans during graded exercise on a treadmill

Cerebral blood volume flow and flow velocity have been reported to increase during dynamic exercise, but whether the two increase in parallel and whether both increases occur as functions of exercise intensity remain unsettled. In this study, blood flow velocity in the common carotid artery was measured using the Doppler ultrasound method in eight healthy male students during graded treadmill exercise. The exercise consisted of stepwise progressive increases and decreases in exercise intensity. The peak intensity corresponded to approximately 85% of maximal oxygen consumption. During this exercise, the heart rate (fc), mean blood pressure (BP) in the brachial artery and mean blood flow velocity (νcc) in the common carotid artery increased as functions of exercise intensity. At the peak exercise intensity, (fc), BP and νcc increased by 134.5%, 20.5% and 51.8% over the control levels before exercise (P < 0.01), respectively. The resistance index (RI) and pulsatility index (PI) were determined from the velocity profile and were expected to reflect the distal cerebral blood flow resistance. The RI and PI increased during the graded exercise, but tended to decrease at the highest levels of exercise intensity. As νcc increased with increases in exercise intensity it would be expected that cerebral blood flow would also increase at these higher intensities. It is also suggested that blood flow velocity in the cerebral artery does not proportionately reflect the cerebral blood flow during dynamic exercise, since the cerebral blood flow resistance changes.

Effects of a time-varying strong magnetic field on transient increase in cytosolic free Ca2+ induced by bradykinin in cultured bovine adrenal chromaffin cells

We tested the effects of exposure to a time‐varying magnetic field changing between 0.07 and 1.7 T at an interval of 3 s on transient increase in intracellular Ca2+ stimulated by bradykinin in bovine adrenal chromaffin cells. Addition of bradykinin induced the increase in intracellular Ca2+ within a few minutes. The exposure to the magnetic field perfectly suppressed the increase in intracellular Ca2+ in Ca2+‐free medium. The inhibition occurred for 15 min when the maximum magnetic flux density was more than 1.4 T. These results suggest that the exposure inhibits Ca2+ release from intracellular Ca2+ stores.

Effects of seven months' exposure to a static 0.2 T magnetic field on growth and glycolytic activity of human gingival fibroblasts

Human gingival fibroblasts in confluent cultures were continuously exposed to a static 0.2 T magnetic field for 6 or 8 months. Culture flasks were not changed during the exposure, but culture medium was renewed. After dilution and mixing of the cultures surviving intact, field-exposed and sham-exposed cultures received further field- or sham-exposure on Sm-Co blocks. Rate of cell proliferation, histogram of the nuclear DNA content, rates of lactate production and glucose consumption and the ATP content were determined and cell morphology was investigated by both light- and electron-microscopy. Results show no marked differences between exposed and control cells.

Effects of nystatin on intracellular contents and membrane transport of alkali cations, and cell volume in HeLa cells

SummaryNystatin (50 μg/ml) had strong influence on the intracellular contents and membrane transports of monovalent ions and water in HeLa cells. The nystatin-induced changes in the intracellular ion content and cell volume were inhibited by sucrose, and Donnan and osmotic equilibria were attained. Using cells under conditions for these equilibria, the concentrations of intracellular impermeant solutes, their mean valence, the differences of their intra- and extracellular osmotic concentrations, and the circumferential tension of the cell membrane were determined. Stimulation by nystatin of the influx of one cation species, e.g. Rb, was inhibited by another cation species, e.g. Na. The stimulatory effect of nystatin on cation fluxes was reversible within 1 hr after ionophore addition, and after 1-hr treatment the intracellular contents of Na and K became proportional to their extracellular concentrations, provided that the sum of these concentrations was constant (300mm). Similar proportionality was also observed in the presence of choline, provided that the choline concentration was less than those of the alkali cations. The implications of these results in relation to the osmotic properties of cultured cells, and the experimental regulation of alkali cations in the cells, are discussed.

Regulatory changes in the K+, Cl− and water contents of HeLa cells incubated in an isosmotic high K+-medium

HeLa cells had their normal medium replaced by an isosmotic medium containing 80 mM K+, 70 mM Na+ and 100 microM ouabain. The cellular contents of K+ first increased and then decreased to the original values, that is, the cells showed a regulatory decrease (RVD) in size. The initial increase was not inhibited by various agents except by substitution of medium Cl- with gluconate. In contrast, the regulatory decrease was inhibited strongly by addition of either 1 mM quinine, 10 microM BAPTA-AM without medium Ca2+, or 0.5 mM DIDS, and partly by either 1 mM EGTA without medium Ca2+, 10 microM trifluoperazine, or substitution of medium Cl- with NO3-. Addition of DIDS to the NO3(-)-substituted medium further suppressed the K+ loss but the effect was incomplete. Intracellular Ca2+ showed a transient increase after the medium replacement. These results suggest that the initial increase in cell K+ is a phenomenon related to osmotic water movement toward Donnan equilibrium, whereas the regulatory K+ decrease is caused by K+ efflux through Ca(2+)-dependent K+ channels. The K+ decrease induced a decrease in cellular water, i.e., RVD. The K+ efflux may be more selectively associated with Cl- efflux through DIDS-sensitive channels than the efflux of other anions.

Effects on Rb(+)(K+) uptake of HeLa cells in a high K(+) medium of exposure to a switched 1.7 Tesla magnetic field.

Effects of a switched, time-varying 1.7 T magnetic field on Rb(+)(K+) uptake by HeLa S3 cells incubated in an isosmotic high K(+) medium were examined. The magnetic flux density was varied intermittently from 0.07-1.7 T at an interval of 3 s. K(+) uptake was activated by replacement of normal medium by high K(+) medium. A membrane-permeable Ca(2+) chelating agent (BAPTA-AM) and Ca(2+)-dependent K(+) channel inhibitors (quinine, charibdotoxin, and iberiotoxin) were found to reduce the Rb(+)(K+) uptake by about 30-40%. Uptake of K(+) that is sensitive to these drugs is possibly mediated by Ca(2+)-dependent K(+) channels. The intermittent magnetic field partly suppress ed the drug-sensitive K(+) uptake by about 30-40% (P < 0.05). To test the mechanism of inhibition by the magnetic fields, intracellular Ca(2+) concentration ([Ca(2+)]c) was measured using Fura 2-AM. When cells were placed in the high K(+) medium, [Ca(2+)]c increased to about 1.4 times the original level, but exposure to the magnetic fields completely suppressed the increase (P < 0.01). Addition of a Ca(2+) ionophore (ionomycin) to the high K(+) medium increased [Ca(2+)]c to the level of control cells, regardless of exposure to the magnetic field. But the inhibition of K(+) uptake by the magnetic fields was not restored by addition of ionomycin. Based on our previous results on magnetic field-induced changes in properties of the cell membrane, these results indicate that exposure to the magnetic fields partly suppresses K(+) influx, which may be mediated by Ca(2+)-dependent K(+) channels. The suppress ion of K(+) fluxes could relate to a change in electric properties of cell surface and an inhibition of Ca(2+) influx mediated by Ca(2+) channels of either the cell plasma membrane or the inner vesicular membrane of intracellular Ca(2+) stores.

Different patterns of cell volume regulation in hyposmotic media between attached and suspended HeLa cells

Both attached and suspended HeLa cells swelled in a medium of a hypotonic osmolality of 235 mosmol/kg H2O. When the osmolality was further decreased to 166 mosmol/kg H2O, attached cells instantly swelled and then rapidly lost water and K+, followed by slow gains of them. Suspended cells instantly swelled and then K+ loss and regulatory volume decrease (RVD) occurred. Neither 0.1 mM ouabain nor 10 mM TEA changed the water loss of attached cells, whereas ouabain inhibited RVD of suspended cells. Quinine (1 mM) inhibited water losses from both cells and comparison of the losses implies stronger activation of K+ channel in attached cells than in suspended cells. Omission of medium Ca2+ or addition of 10 mM BaCl2 inhibited RVD in part. These results suggest that hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca(2+)-dependent K+ channel. Suspended cells osmotically swell, followed by RVD with K+ and Na+ releases through the K+ channel and Na(+)-pump, respectively. The different patterns of volume changes may relate to the difference of activity or time of activation of the K+ channel between both cells.

Kinetic study on the effects of intracellular K++ and Na++ on Na++, K++, Cl+− cotransport of HeLa cells by Rb++ influx determination

SummaryThe effects of intracellular K+ and Na+ (K+c, Na+c) on the Na+,K+,Cl+− cotransport pathway of HeLa cells were studied by measuring ouabain-insensitive, furosemide-sensitive Rb+ influx (JRb) at various intracellular concentrations of K+ and Na+ ([K+]c, [Na+]c). When [K+]c was increased and [Na+]c was decreased, keeping the sums of their concentrations almost constant, JRb as a function of the extracellular Rb+ or Na+ concentration ([Rb+]e, [Na+]e) was stimulated. However, the apparent K0.5 for Rb+e or Na+e remained unchanged and the ratio of the apparent K+0.5 for K+c and the apparent Ki for Na+c was larger than 1. When JRb was increased by hypertonicity by addition of 200 mM mannitol, the apparent maximum JRb increased without change in the apparent K0.5 for Rb+e. These results show that K+c stimulates and Na+c inhibits JRb, without change in the affinities of the pathway for Rb+e and Na+e. The affinity for K+c is slightly lower than that for Na+c. Hypertonicity enhances JRb without any change in the affinity for Rb+e. We derived a kinetic equation for JRb with respect to K+c and Na+c and proposed a general and a special model of the pathway. The special model suggests that, in HeLa cells, JRb takes place when Rb+e binds to the external K+ binding site of the pathway after the binding of K+c to the internal regulatory site.

Properties of Ca2+-dependent K+ Channels of Human Gingival Fibroblasts

Cells in the oral cavity are normally exposed to different temperatures. Ion transport systems are influenced by temperature in other tissues: In particular, changes in intracellular K+ ion can affect cell growth and synthesis of macromolecules. The purpose of this investigation was to identify K+ channels in human gingival fibroblast cells and analyze the effect of temperature on their K+ conduction properties. Ca2+-dependent K+ channels with a large conductance (125 pS in symmetrical K+-rich solutions) were identified in human gingival fibroblasts and studied by the patch-clamp technique. The open probability of the channels varied with membrane potential between +40 and -100 mV. When the bath temperature was decreased from 40 to 4°C, channel conductance was reduced, but the mean open time of the channels was increased. The activation energies for the conductance and the reciprocal of the mean open time were estimated to be 9.1 and 22.9 kj/mol, respectively. These values are lower than those reported for these and other types of channels in cells from other tissues. The open probability of the channels was nearly constant in the temperature range studied. These results suggest that the properties of Ca2+-dependent K+ channels of gingival fibroblasts remain relatively unchanged when the cells are exposed to a wide range of temperatures.