Toshitaka Ikehara

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.

Changes in carotid blood flow and electrocardiogram in humans during and after walking on a treadmill

Blood flow velocity in the common carotid artery and the electrocardiogram were measured simultaneously by telemetry in seven male subjects during 20-min walking on a treadmill at an exercise intensity corresponding to a mean oxygen uptake of 26.0 (SD 2.9) ml · kg −1 · min −1. The mean cardiac cycle was shortened from 0.814 (SD 0.103) s to 0.452 (SD 0.054) s during this exercise. Of this shortening, 73% was due to shortening of the diastolic period and 27% to shortening of the systolic period. In the relatively small shortening of the mean systolic period [from 0.377 (SD 0.043) s to 0.268 (SD 0.029) s], the isovolumetric contraction time was shortened by 56%. During exercise, the heart rate (fc) increased by 79.4% [from 74.3 (SD 9.3) beats · min −1 to 133.3 (SD 14.8) beats · min −1], and the peak blood velocity (S1) in the common carotid artery increased by 56.1% [from 0.82 (SD 0.10) m · s−1 to 1.28 (SD 0.11) m · s−1]. After exercise, the S1 decreased rapidly to the resting level. The fc decreased more slowly, still being higher than the initial resting level 5 min after exercise. The diastolic velocity wave and the end-diastolic foot decreased during exercise. The blood flow rate in the carotid artery increased transiently by 13.5% at the beginning of exercise [from 5.62 (SD 0.63) ml · s−1 to 6.38 (SD 0.85) ml · s−1] and by 26.5% at the end of the exercise period [from 5.62 (SD 0.63) ml · s−1 to 7.11 (SD 1.34) ml · s−1]. The increase of blood flow in the carotid artery at the onset of exercise may have been mainly related to cerebral activation, and partly to an increase of blood flow to the skin of the head. The physiological significance for cerebral function of the increase of blood flow in the artery after the end of exercise is unknown.

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.

Effects of exposure to a time-varying 1.5 T magnetic field on the neurotransmitter-activated increase in intracellular Ca2+ in relation to actin fiber and mitochondrial functions in bovine adrenal chromaffin cells

BACKGROUND It has been reported that exposure to electromagnetic fields influences intracellular signal transduction. We studied the effects of exposure to a time-varying 1.5 T magnetic field on membrane properties, membrane cation transport and intracellular Ca(2+) mobilization in relation to signals. We also studied the mechanism of the effect of exposure to the magnetic field on intracellular Ca(2+) release from Ca(2+) stores in adrenal chromaffin cells. METHODS We measured the physiological functions of ER, actin protein, and mitochondria with respect to a neurotransmitter-induced increase in Ca(2+) in chromaffin cells exposed to the time-varying 1.5 T magnetic field for 2h. RESULTS Exposure to the magnetic field significantly reduced the increase in [Ca(2+)]i. The exposure depolarized the mitochondria membrane and lowered oxygen uptake, but did not reduce the intracellular ATP content. Magnetic field-exposure caused a morphological change in intracellular F-actin. F-actin in exposed cells seemed to be less dense than in control cells, but the decrease was smaller than that in cytochalasin D-treated cells. The increase in G-actin (i.e., the decrease in F-actin) due to exposure was recovered by jasplakinolide, but inhibition of Ca(2+) release by the exposure was unaffected. CONCLUSIONS AND GENERAL SIGNIFICANCE These results suggest that the magnetic field-exposure influenced both the ER and mitochondria, but the inhibition of Ca(2+) release from ER was not due to mitochondria inhibition. The effect of eddy currents induced in the culture medium may indirectly influence intracellular actin and suppress the transient increase in [Ca(2+)]i.

Effects of Electromagnetic Fields On K+(Rb+) Uptake by HeLa Cells

Exposure to strong homogeneous magnetic fields with various magnetic flux densities ofless than 1.6 T had no significant effect on either active or passive Rb+ influxes into HeLa cells at normal or high temperatures. Exposure to a similar magnetic field of 2 T at different temperatures of 10 to 45°C did not cause any change in active or passive Rb+ influx, and no evidence was obtained for the presence of a phase transition point of the cell membrane between 10 and 37°C.

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.

Sterilization Using 365 nm UV-LED

There are several methods used for sterilization. In those methods chlorine, heat and UV rays are traditionally used. In recent years, the UV sterilization is taken notice as a sterilization method that the sterilized object does not change in quality and is environment-friendly. In this paper, an UV-LED is focused because it does not contain harmful substance and has longer operating life. The results have showed that complete germicidal effects for E. coli and Vibrio parahaemolyticus by UV-LED exposure of 30 minutes and 10 minutes, respectively. These results suggest that the UV-LED has sterilization effects. Therefore, UV-LED can be used as a sterilization device.