Calcium mobilizing hormones activate the plasma membrane Ca2+ pump of pancreatic acinar cells

Summary ⁴⁵Ca fluxes and free-cytosolic Ca²⁺ ([Ca²⁺] _i ) measurements were used to study the effect of Ca²⁺-mobilizing hormones on plasma membrane Ca²⁺ permeability and the plasma membrane Ca²⁺ pump of pancreatic acinar cells. We showed before (Pandol, S.J., et al., 1987.J. Biol. Chem. 262:16963–16968) that hormone stimulation of pancreatic acinar cells activated a plasma membrane Ca²⁺ entry pathway, which remains activated for as long as the intracellular stores are not loaded with Ca²⁺. In the present study, we show that activation of this pathway increases the plasma membrane Ca²⁺ permeability by approximately sevenfold. Despite that, the cells reduce [Ca²⁺]i back to near resting levels. To compensate for the increased plasma membrane Ca²⁺ permeability, a plasma membrane Ca²⁺ efflux mechanism is also activated by the hormones. This mechanism is likely to be the plasma membrane Ca²⁺ pump. Activation of the plasma membrane Ca²⁺ pump by the hormones is time dependent and 1.5–2 min of cell stimulation are required for maximal Ca²⁺ pump activation. From the effect of protein kinase inhibitors on hormone-mediated activation of the pump and the effect of the phorbol ester 12-0-tetradecanoyl phorbol, 13-acetate (TPA) on plasma membrane Ca⁺ efflux, it is suggested that stimulation of protein kinase C is required for the hormone-dependent activation of the plasma membrane Ca²⁺ pump.     

The diffusional water permeability in the halotolerant alga Dunaliella as measured by nuclear magnetic resonance

T₁ nuclear relaxation measurements of ¹H and ¹⁷O of water have been applied to study the kinetics of the diffusional transport of water across the cytoplasmic cell membrane of Dunaliella salina and Dunaliella bardawil. The water permeability coefficients at 25°C were found to be 1.5·10⁻³ cm/s and 1.8·10⁻³ cm/s, respectively, with an activation energy of 3.7 kcal/mol. The results indicate that the cell membrane of Dunaliella exhibits high diffusional permeability to water, similar in magnitude to that found for other cells and model membranes, and a relatively low activation energy. This regularity is in contrast to the exceptionally low glycerol permeability of the membrane (Brown, F.F., Sussman, I., Avron, M. and Degani, H. (1982) Biochim. Biophys. Acta 690, 165–173).     

Characterization of H+/OH− currents in phospholipid vesicles

In pure phospholipid vesicles, the conductivity of H⁺/OH^– ions exceeds that for other simple inorganic ions. Protons achieve electrochemical equilibrium across egg phosphatidylcholine vesicles within tens of minutes. When pH gradients are established across vesicles, transmembrane potentials develop. Conversely, the establishment of transmembrane potentials leads to the formation of pH gradients. When the phenomenological permeability of H⁺/OH^– ions in vesicles is estimated, values are obtained that are much greater (six orders of magnitude larger) than those for Na⁺ or K⁺. A wide range in the values for this permeability has been reported; however, much of the discrepancy can be attributed to differences in the vesicle systems and experimental conditions. The H⁺/OH^– current appears to be modulated by changes in membrane dielectric constant. However, the dependence of this current on the pH gradient and on the membrane voltage argues against simple diffusion mechanisms as the source of the H⁺/OH^– current. In addition, in vesicle systems the H⁺/OH^– current shows a surprising invariance to changes in the membrane dipole potential, an observation that argues against the role of simple carriers for H⁺ and OH^– ions.     

Proton transport through membranes induced by weak acids: A study of two substituted benzimidazoles

Summary We report here a kinetic study of the mechanism by which the weak acid TTFB (4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole) transports protons across phospholipid bilayer membranes. A previous kinetic study of the homologous dichloro compound, DTFB, revealed that the rate limiting step for proton translocation was the back diffusion of the neutral, HA, form of the weak acid; we conclude here that this is also the rate limiting step for proton translocation with TTFB. At high concentrations of either DTFB or TTFB the charged permeant species is an HA ₂ ^– complex. The kinetic analysis and independent measurements reveal that the permeability of the membrane to HA and adsorption coefficients of A^– and HA are an order of magnitude higher for TTFB than for DTFB. When either DTFB or TTFB was present in a solution where the pH was less than the pK of the weak acid, an unusual relaxation in the current was noted on application of a voltage step. The amplitude of the relaxation decreased as the voltage was increased. This relaxation is possibly due to a reorientation of the benzimidazole molecules at the membrane-solution interface. We also report experiments performed with DTFB on mitochondria. It was possible to reconcile these results with the bilayer data and, therefore, with the chemiosmotic hypothesis by postulating that the dielectric constant of the mitochondrial membrane is greater than that of a bilayer formed with decane as a solvent. To demonstrate the effect of dielectric constant on permeability, we replaced decane by 1-chlorodecane. This increased the capacitance of the artificial bilayer by a factor of two and the permeability of the bilayer to the A^– form of DTFB by two orders of magnitude.     

Is ubiquinone diffusion rate-limiting for electron transfer?

The different possible dispositions of the electron transfer components in electron transfer chains are discussed: (a) random distribution of complexes and ubiquinone with diffusion-controlled collisions of ubiquinone with the complexes, (b) random distribution as above, but with ubiquinone diffusion not rate-limiting, (c) diffusion and collision of protein complexes carrying bound ubiquinone, and (d) solid-state assembly. Discrimination among these possibilities requires knowledge of the mobility of the electron transfer chain components. The collisional frequency of ubiquinone-10 with the fluorescent probe 12-(9-anthroyl)stearate, investigated by fluorescence quenching, is 2.3 × 10⁹ M^–1 sec^–1 corresponding to a diffusion coefficient in the range of 10^–6 cm²/sec (Fato, R., Battino, M., Degli Esposti, M., Parenti Castelli, G., and Lenaz, G.,Biochemistry,25, 3378–3390, 1986); the long-range diffusion of a short-chain polar Q derivative measured by fluorescence photobleaching recovery (FRAP) (Gupte, S., Wu, E. S., Höchli, L., Höchli, M., Jacobson, K., Sowers, A. E., and Hackenbrock, C. R.,Proc. Natl. Acad. Sci. USA 81, 2606–2610, 1984) is 3×10^–9 cm²/sec. The discrepancy between these results is carefully scrutinized, and is mainly ascribed to the differences in diffusion ranges measured by the two techniques; it is proposed that short-range diffusion, measured by fluorescence quenching, is more meaningful for electron transfer than long-range diffusion measured by FRAP, or microcollisions, which are not sensed by either method. Calculation of the distances traveled by random walk of ubiquinone in the membrane allows a large excess of collisions per turnover of the respiratory chain. Moreover, the second-order rate constants of NADH-ubiquinone reductase and ubiquinol-cytochromec reductase are at least three orders of magnitude lower than the second-order collisional constant calculated from the diffusion of ubiquinone. The activation energies of either the above activities or integrated electron transfer (NADH-cytochromec reductase) are well above that for diffusion (found to be ca. 1 kcal/mol). Cholesterol incorporation in liposomes, increasing bilayer viscosity, lowers the diffusion coefficients of ubiquinone but not ubiquinol-cytochromec reductase or succinate-cytochromec reductase activities. The decrease of activity by ubiquinone dilution in the membrane is explained by its concentration falling below theK _m of the partner enzymes. It is calculated that ubiquinone diffusion is not rate-limiting, favoring a random model of the respiratory chain organization. It is not possible, however, to exclude solid-state assemblies if the rate of dissociation and association of ubiquinone is faster than the turnover of electron transfer.     

Diacylglycerol downregulates junctional membrane permeability. TMB-8 blocks this effect

Summary We tested the question whether junctional cell-to-cell communication is regulated by the diacylglycerol branch of the phosphoinositide transmembrane signal pathway. Cultured epithelial rat liver cells were treated with the synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol, while their junctional permeability was probed with the microinjected 443-dalton fluorescent tracer Lucifer Yellow. The treatment reduced junctional permeability (without affecting Lucifer permeability of nonjunctional cell membrane). The effect was dose dependent, with a threshold of about 25 g diacylglycerol/ml in sparse cultures and about 50 g/ml in confluent cultures. The reduction of junctional permeability began within 3 min of diacylglycerol application, peaked within 20 min, and reversed spontaneously within 90 min. The phorbol ester TPA mimicked the diacylglycerol effect, but the (spontaneous) reversal was slower. We propose that cell-to-cell communication is under dual physiological control: an upregulatory one, as exerted by the cyclic AMP signal route (Loewenstein, W.R., 1985,Biochem. Soc. Symp. London,50: 43–58), and a downregulatory one, by the diacylglycerol signal route.TMB-8 (54–70 m)—a blocker of intracellular Ca²⁺ mobilization-impeded the diacylglycerol action on junctional permeability. It prevented the effect of low diacylglycerol doses completely and it markedly reduced the effect of high doses. (It also counteracted the effect of TPA.) Ca²⁺ thus emerges as a possible candidate for a role in the junctional downregulation by the diacylglycerol signal route. We tentatively advance two models. In one, leaning closely on the Calcium Hypothesis of cell-to-cell channel regulation (Loewenstein, W.R., 1966,Ann. N.Y. Acad. Sci. 137:441–472), Ca²⁺ mediates the action of the route on the channel. In the other, Ca²⁺ acts farther removed from the channel, on protein kinase C.Calmidazolium (5–10 m)—an inhibitor of calmodulin-activated proteins—did not prevent the diacylglycerol-induced reduction of junctional permeability. Nor did sodium orthovanadate (25 or 50 m)—an inhibitor of tyrosyl phosphatase-prevent the reversal of diacylglycerol-induced (or TPA-induced) reduction of junctional permeability.     

Mechanism of action of cholera toxin: Effect of receptor density and multivalent binding on activation of adenylate cyclase

Summary Cl^– influx into cells ofChara corallina is shown to be stimulated by a factor of 2 to 4 by starvation of Cl^–. The time constant for the induction of this effect is about 4.0 ksec and that for its decay when Cl^– is reprovided, 1.7 ksec. Intracellular perfusion of tonoplast-free cells with solutions of varying Cl^– concentration shows that Cl^– influx can be controlled directly by the concentration of Cl^– at the inside of the plasma membrane. Both the time course for the initial stages of induction of the starvation-stimulated flux and its absolute magnitude can be accounted for by assuming cytoplasmic Cl^– concentration to be the only intracellular condition to change during Cl^– starvation. The existence of a feedback loop between cytoplasmic Cl^– and Cl^– influx provides an alternative explanation to observations previously used in support of a Cl^–/OH^– exchange hypothesis (F.A. Smith, 1972,New Phytol. 71:595).     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.     

Studies on the tonoplast action potential ofNitella flexilis

Summary The origins of the two peaks of the action potential inNitella flexilis were analyzed by inserting two microelectrodes. one into the vacuole and the other into the cytoplasm. It was unequivocally demonstrated that the rapid first peak was generated at the plasmalemma and the slow second peak at the tonoplast. MnCl₂ applied in the external medium abolished the second, tonoplast, peak but not the first, plasmalemma, peak, MnCl₂ also inhibited the cessation of the cytoplasmic streaming accompanying the action potential. CaCl₂ added in MnCl₂-containing medium recovered generation of the tonoplast action potential and the streaming cessation. Since it has been established that the cessation of cytoplasmic streaming on membrane excitation is caused by an increase in cytoplasmic free Ca^2– (Williamson, R.E., Ashley, C.C., 1982.Nature (London) 296:647–651: Tominaga, Y., Shimmen, T., Tazawa, M., 1983,Protoplasma 116:75–77), it is suggested that the tonoplast action potential is also induced by an increase in cytoplasmic Ca²⁺ resulting from the plasmalemma excitation. When vacuolar Cl was replaced with SO ₄ ² by vacuolar perfusion, the polarity of the second, slow peak was reversed from vacuolar positive to vacuolar negative with respect to the cytoplasm, supporting the previous report that the tonoplast action potential is caused by increase in Cl permeability (Kikuyama, M., Tazawa, M., 1976.J. Membrane Biol.29:95–110).     

Further evidence for the regulation of the tight junction ion selectivity by cAMP in goldfish intestinal mucosa

Summary It has been reported that cAMP controls the transepithelial Cl^– conductance in fish intestine (Bakker, R., Groot, J.A., 1984,Am. J. Physiol. 246:G213–G217; Krasny, E.J., Madara, J.L., DiBona, D.L., Frizzell, R.A., 1983,Fed. Proc. 42:1100). In both studies, the cAMP effect was interpreted as an increase in tight junction Cl^– conductance, because cAMP did not change the membrane potential or membrane resistance ratio. However, the activation of a Cl^– conductance in the membranes of a subset of the epithelial cells might be difficult to discern from an increase in tight junction Cl^– conductance. Here we report experiments that were designed to distinguish a tight junction Cl^– conductance from a membrane Cl^– conductance in a subpopulation of the epithelial cells. The effect of hypotonicity on the cAMP-induced increase in transepithelial conductance showed that cAMP-induced conductance is located in series with the lateral intercellular spaces. Transepithelial serosa to mucosa direct current caused an increase in resistance due to so-called transport number effects. Forskolin abolished the transport number effects, indicating that cAMP increases the Cl^– conductance of the tight junctions. Increasing cAMP did not change mannitol fluxes, whereas Cl^– fluxes more than doubled. Changes in dilution potential and transepithelial resistance demonstrated that the cAMP-induced conductance is specific for Cl^– and Br^– as opposed to I^–, NO ₃ ^– , SO ₄ ^2– and gluconate^–. In contranst, cytochalasin D also decreased the transepithelial resistance and dilution potential in Nagluconate Ringer's. This demonstrates that cAMP acts on the tight junctions in a more specific manner than cytochalasin D.     

Electrically silent anion transport through bilayer lipid membrane induced by tributylin and triethyllead

Summary The method of the measurement of the nonelectrogenic fluxes of hydrogen (or hydroxyl) ions (J _H) based on the local proton gradients formation in the unstirred layers near a bilayer lipid membrane (BLM) is applied for recording the nonelectrogenic anion/OH^– exchange on BLM induced by tributyltin (TBT) and a novel carrier (Hager, A., Moser, I., & Berthold, W. 1987.Z. Naturforsch.,42C1116–1120), triethyllead (TEL). This method has been used previously for measuring the cation fluxes through BLM. TBT and TEL are shown to be equally efficient in the induction of Cl^–/OH^– exchange.J _H induced by TBT is constant at 4J _H decreases at pH<4 and pH>7. Both ionophores have a transport sequence: I^–> Br^–>Cl^–>F^–. The quatitative measurements reveal that TEL better discriminates these four anions than TBT. It is concluded that this method may prove helpful in a search and study of anion/OH^–-exchangers isolated from natural membranes.     

Size selectivity of aqueous pores in astomatous cuticular membranes isolated from<Emphasis Type="Italic"> Populus canescens</Emphasis> (Aiton) Sm. leaves

Little is known about the permeability of plant cuticles to ionic molecules with hydration shells that render them lipid insoluble and limit their diffusion to narrow aqueous pores. Therefore, the permeation of cuticular membranes to ionised calcium salts with anhydrous molecular weights ranging from 111 to 755 g mol^–1 was studied. Penetration was a first-order process and rate constants (k) (proportional to permeability) decreased exponentially with molecular weight. Plots of log k vs. molecular weight had slopes of –2.11×10^–3 and –2.80×10^–3, respectively, depending on the year in which the cuticular membranes were isolated. This corresponds to decreases in permeability by factors of about 7 to 13 when molecular weight increased from 100 to 500 g mol^–1. This size selectivity is small compared to the dependence on molecular weight of solute mobility in Populus cuticles. A decrease in mobility of neutral molecules by more than 3 orders of magnitude has been reported [A. Buchholz et al. (1998) Planta 206:322–328] for the same range of molecular weights. Hence, discrimination of large ionic species diffusing in aqueous pores (polar pathway) is much smaller than that for neutral solutes diffusing in cutin and waxes (lipophilic pathway). This indicates that formulating large solutes as ionic species would be advantageous.Abbreviation CM Cuticular membrane     

The ionic basis of the resting potential of molluscan unstriated muscle

Summary The membrane potential (Vm) of unstriated, non-spiking fibres from the buccal retractor muscle of the opisthobranch molluscPhiline aperta is primarily determined by the distribution of the potassium ion across the membrane. In salines where potassium is varied and chloride remains constant or nearly so, the membrane potential varied with log external K⁺ with a slope of 50.6 (±2.3) mV per decade. In chloride-free salines the slope was 48.5 mV per decade. The experiments were conducted at temperatures of 18–20° C.A ten-fold reduction in external chloride concentration depolarised the fibres by around 10 mV, indicating that chloride permeability makes some contribution to Vm. In salines where [K]₀·[Cl]₀ is constant the Nernst slope was 55.8 mV per decade compared with the theoretical value of 58 mV.The experimental data suggest that the internal potassium concentration of the fibres is 247±31 mM and pNa/pK is 0.01, giving a predicted value of Vm in sea water of –72 mV. The membrane potential of 90 fibres measured in sea water was –74.2±1.3 mV. The membrane contains an electrogenic sodium pump which contributes 4–5 mV to the membrane potential.     

Voltage dependence of Na/K pump current inXenopus oocytes

Summary Stage V and VI (Dumont, J.N., 1972.J. Morphol. 136:153–180) oocytes ofXenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3mm K Barth's solution and measuring membrane current-voltage (I–V) relationships before and after the addition of 10 m dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membraneI–V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (–40 mV). The resulting membraneI–V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pumpI–V relationship must be free of hysteresis, the presence of hysteresis in DHO differenceI–V curves can be used as a criterion for excluding such data from consideration as a valid measure of the Na/K pumpI–V relationship. DHO differenceI–V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90mm) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0–1.5 A cm^–2, without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pumpI–V relationships do not show a region of negative slope over the voltage range –110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump.     

Proton dissociation dynamics in the aqueous layer of multilamellar phospholipid vesicles

Summary The water layers interspacing between the phospholipid membranes of a multilamellar vesicle are 3–10 water layers across and their width is adjusted by osmotic pressure (Parsegian, V.A., et al., 1986.Methods Enzymol. 127:400–416).In these thin water layers we dissolved pyranine (8 hydroxypyrene 1,3,6 trisulfonate), a compound which, upon photo excitation, ejects it hydroxy proton with time constant of 100 psec. (Gutman, M. 1986.Methods Enzymol. 127:522–538).In the present study we investigated how the width of the aqueous layer, the density of phosphomoieties on the membrane's surface and the activity of water in the layer affect the capacity of protons to diffuse out from the electrostatic cage of the excited anion before it decays to the ground state.Using a combination of steady-state and subnanosecond time-resolved fluorescence measurements we determined the average number of proton excited-anion recombinations before the proton escapes from the Coulomb cage.The probability of recombination in thin water layer is significantly higher than in bulk. The factor contributing most to enhancement of recombination is the diminished water activity of the thin aqueous layer.The time frame for proton escape from an electrostatic trap as big as a membrane-bound protein is 3 orders of magnitude shorter than turnover time of membrane-bound enzymes. Thus the effects of local forces on proton diffusion, at the time scale of physiological processes, is negligible.     

Determination of the inherent kinetics of immobilized glucose oxidase using a diffusion cell

The enzyme glucose oxidase (GO) was covalently immobilized onto a poly(vinyl alcohol) hydrogel, cross-linked with glutardialdehyde and a polyazonium salt. To compare the kinetic parameters of immobilized GO with the known kinetic parameters of soluble GO, the diffusion cell method was used.Between two compartments, containing solutions with different glucose concentrations, a GO-containing hydrogel membrane was placed. Simultaneous diffusion through and enzymatic reaction in the membrane occurred. In this way diffusional effects of the membrane could be eliminated from the effective kinetic parameters to yield the inherent kinetic parameters.It appeared that the enzymatic reaction is independent of the oxygen concentration at oxygen concentrations 0.22 mol m^–3 (Michaelis constant for oxygen < 0.22 mol m^–3). Further, the Michaelis constant for glucose does not change dramatically after immobilizing the enzyme. The maximal reaction rate is depending on the enzyme concentration. As the enzyme concentration in the membrane is not exactly known (mainly due to leakage of enzyme out of the membrane during membrane preparation), only an estimation of the turnover number can be made.The diffusion cell method is easy to carry out. Still, some recommendations can be made on the performance.List of Symbols _g , _0x partition coefficient of glucose and oxygen, respectively - thickness of the wetted membrane (m) - A _m surface area of membrane (m^–2) - C constant (mol² m^–3) - c _g , c _0x concentration of glucose and oxygen, respectively (mol m^–3) - c _g,0 c _g, glucose concentration at the filter-paper/membrane interface next to compartment A and B, respectively (mol m^–3) - c _{g, A} c _{g, B} glucose concentration in compartment A and B, respectively (mol m^–3) - c _GO glucose oxidase concentration (mol m^–3) - D _eff effective diffusion coefficient (m² s^–1) - D _m , D _sl diffusion coefficient in, respectively, the membrane and the solution layer (m² s^–1) - d _dl , d _df , d _sl thickness of, respectively, the diffusion layer, the filter-paper and the solution layer (m) - h _B initial slope of concentration versus time curve of compartment B (mol m^–3 s^–1) - J flux (mol m^–2 s^–1) - J ₀ flux in the membrane at membrane/filter-paper interface next to compartment A and B, respectively (mol m^–2 s^–1) - J _A , J _B flux leaving compartment A and entering compartment B, respectively (mol m^–2 s^–1) - J _m flux through the membrane (mol m^–2 s^–1) - k total mass transfer coefficient (m s^–1) - k ₁ , k ₂ rate constant of a particular reaction step (m³ mol^–1 s^–1) - k_–1, k_–2 rate constant of a particular reaction step (s^–1) - k _cat (intrinsic) catalytic constant of turnover number (s^–1) - k _cat ^* inherent catalytic constant, determined by inserting D _m (s^–1) - k _cat ^** inherent catalytic constant, determined by inserting D _eff (s^–1) - k _m (g) (intrinsic) Michaelis constant for glucose (mol m^–3) - k _m (o) (intrinsic) Michaelis constant for oxygen (mol m^–3) - k _m ^* (g) inherent Michaelis constant for glucose (mol m^–3) - k _m ^* (o) inherent Michaelis constant for oxygen (mol m^–3) - m _GO number of moles of GO present (mol) - P _m permeability of glucose in the mebrane (m s^–1) - P _eff effective permeability (m s^–1) - V volume (m³) - v ₀ initial reaction velocity (mol m^–3 s^–1) - V _max ^** inherent maximal reaction velocity, determined by inserting D_eff (mol m^–3 s^–1) - x distance (m)     

Regulation of the Cell Cycle at the G2/M Boundary in Metastatic Melanoma Cells by 12-O-Tetradecanoyl Phorbol-13-acetate (TPA) by Blocking p34Kinase Activity

12-O-Tetradecanoyl phorbol-13-acetate (TPA) inhibits the growth of most malignant melanoma cells but stimulates the growth of normal human melanocytes. We previously showed that addition of TPA inhibits the growth of the human metastatic melanoma cell line, Demel, by blocking cells at both the G1/S and G2/M cell cycle transitions (D. L. Coppocket al.,1992,Cell Growth Differ.3, 485–494). To examine the G2/M transition, we developed a method to synchronize the cells in early S phase using Lovastatin and mevalonate, followed by treatment with hydroxyurea (HU). TPA (30 nM) was effective in blocking cells from entering mitosis and reentering G1 when added up to the end of G2. These cells arrested in G2. Examination of the levels of cyclins A and B1 demonstrated that the levels of these cyclins were not limiting for entrance into M. However, the addition of TPA blocked the increase in p34^cdc2/cyclin B1 kinase activity. In cells treated with TPA, most p34^cdc2was found in the slowly migrating forms on Western blots, which contained increased levels of phosphotyrosine. In addition, the level of the cyclin-dependent kinase inhibitor p21^Cip1/Waf1, but not of p27^Kip1, was increased. We examined the expression of protein kinase C (PKC) isoforms in Demel cells using Western blots to understand which types were involved in the G2 arrest. Demel cells expressed the PKC α, βI, βII, δ, ε, ι/λ, ζ, and μ isozymes. PKC η and PKC θ were not detected. Addition of TPA did not completely down regulate any PKC isozymes over a 12-h period in these synchronized cells. PKC α, βI, βII, δ, and ε isozymes were translocated to the membrane fraction from the cytosolic fraction when treated with TPA. PKC δ appeared as a doublet and the addition of TPA shifted a majority to the slower migrating form. The level of PKC μ was constant; however, a slow mobility form was observed in TPA-treated cells. This reduced mobility was at least partially due to phosphorylation. Thus, the arrest of growth in G2 appears to be due to the inhibition of the p34^cdc2kinase activity which is associated with the increased expression of p21^Cip1/Waf1and increased phosphorylation on tyrosine of p34^cdc2. This arrest, in turn, is associated with a shift of PKC isozymes PKC α, PKC βI, PKC βII, PKC δ, PKC ε, and PKC μ to the membrane fraction which is induced by addition of TPA.     

Cation selectivity of the resting membrane of squid axon

Summary Permeability constant ratios among monovalent cations were studied in the resting membrane of a giant axon of a Pacific squid,Loligo opalescens, by observing the relationship between the membrane potential and the ion concentration.The average permeability ratios are: Tl, 1.8; K, 1.0; Rb, 0.72; Cs, 0.16; Na, <0.08; Li, <0.08. These permeability ratios suggest that neither valinomycin nor nonactin are adequate models for the sites producing the resting permeability in the axonal membrane.Cyclic polyetherbis(t-butyl cyclohexyl) 18-crown-6 does not increase the permeability ratioP _Cs/P _K except when applied at concentrations (5×10^–5 m) at which the surfactant properties of this molecule may become significant.     

Inhibition of human non-small cell lung cancers with a targeted cytotoxic somatostatin analog, AN-162

Human non-small cell lung cancers (NSCLCs) express receptors for somatostatin. The cytotoxic analog of somatostatin AN-162 (AEZS-124), consisting of doxorubicin linked to a somatostatin analog RC-121 binds to receptors for somatostatin and is targeted to tumors expressing these receptors. The aim of this study was to investigate the effect of targeted cytotoxic somatostatin analog AN-162 on a panel of human NSCLC cell lines (A549, H460, H838, H1299) in vitro (at 0.5–100 μM concentrations) and in vivo on H460 and H1299 NSCLCs xenografted into nude mice (at the dose of 2.5 μmol/kg, i.v., once a week). The expression of mRNA for somatostatin receptor subtypes was investigated by RT-PCR in cell lines and tumor tissues. Somatostatin receptor proteins were also characterized by ligand competition assay and Western blotting. AN-162 significantly decreased cell proliferation in vitro and tumor growth (p < 0.05 vs. all groups) of H460 and H1299 NSCLCs in vivo. Based on real-time PCR array data, AN-162 induced several apoptosis-related genes in vivo in both models. Our results suggest that cytotoxic somatostatin analog AN-162 (AEZS-124) should be considered for the further development of a therapy of patients with NSCLC.     

The mitochondrial megachannel is the permeability transition pore

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca⁺⁺ and inhibited by Mg⁺⁺, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990)Am. J. Physiol. 258, C755–C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.Abbreviations used: PT: permeability transition; PTP: permeability transition pore; MMC: mitochondrial megachannel; IMAC: inner membrane anion channel. P_A: permeability of ion A. CSP: Cyclosporin A.