Showing papers in "The Journal of Membrane Biology in 1986"

Mechanisms of regulation of the Na+/H+ exchanger

Abstract: Exchange of Na + for H + was first proposed as a mechanism for renal acidification almost 40 years ago (Pitts, Ayer & Schiess, 1949). However, it was not until 1976 that the existence of such an exchange system was directly demonstrated in vesicles prepared from brush border membranes of kidney tubules (Muter, Hopfer & Kinne, 1976). From studies in renal systems, it has become clear that the exchanger plays an important role in acid secretion and in transepithelial salt and water transport. More recent studies have demonstrated that the Na+/H + exchanger (antiporter) is not unique to epithelial tissues. It appears to be present in most, if not all animal cells, where it has been implicated in a variety of functions, including the regulation of the cytoplasmic pH and Na + concentration, the control of cell volume and the initiation of growth and proliferation. Given the general interest in these phenomena, the rapidly expanding interest in Na+/ H + exchange is not surprising. Many aspects of its properties and functions have been the subject of recent symposia and review articles (Krulwich, 1983; Ires & Warnock, 1984; Aronson, 1985; Mahnensmith & Aronson, 1985). In many unstimulated nonepithelial cell types the antiport appears to be nearly quiescent when the cytoplasmic pH (pHi) is in the physiological range. However, it can be activated by a wide variety of stimuli including hormones, growth factors, tumor promoters and hypertonic shrinking. An emerging pattern of information reveals striking similarities between the modes of activation of several stimuli and suggests that they may share a common mechanism. It is the intent of this brief and speculative review to summarize the evidence concerning

Permeability of small nonelectrolytes through lipid bilayer membranes

Abstract: Diffusion of small nonelectrolytes through planar lipid bilayer membranes (egg phosphatidylcholine-decane) was examined by correlating the permeability coefficients of 22 solutes with their partition coefficients between water and four organic solvents. High correlations were observed with hexadecane and olive oil (r=0.95 and 0.93), but not octanol and ether (r=0.75 and 0.74). Permeabilities of the seven smallest molecules (mol wt <50) (water, hydrofluoric acid, hydrochloric acid, ammonia, methylamine, formic acid and formamide) were 2- to 15-fold higher than the values predicted by the permeabilities of the larger molecules (50

Role of membrane transport in metabolism and function of glutathione in mammals.

Abstract: Glutathione I occurs in almost all living organisms, and its multifunctional properties have been attracting interest of many biochemists, physiologists, pharmacologists as well as clinical scientists. GSH 1 is a tripeptide with a structure of 7-L-glutamyl-Lcysteinylglycine, which is characterized by the yglutamyl peptide and the reactive thiol group. GSH is synthesized in two steps catalyzed by 7-glutamylcysteine synthetase [EC 6.3.2.2] (reaction 1) and glutathione synthetase [EC 6.3.2.3] (reaction 2), respectively.

Ionic channels formed by Staphylococcus aureus alpha-toxin: Voltage-dependent inhibition by divalent and trivalent cations

Abstract: The interaction of Staphylococcus aureus alpha-toxin with planar lipid membranes results in the formation of ionic channels whose conductance can be directly measured in voltage-clamp experiments. Single-channel conductance depends linearly on the solution conductivity suggesting that the pores are filled with aqueous solution; a rough diameter of 11.4 +/- 0.4 A can be estimated for the pore. The conductance depends asymmetrically on voltage and it is slightly anion selective at pH 7.0, which implies that the channels are asymmetrically oriented into the bilayer and that ion motion is restricted at least in a region of the pore. The pores are usually open in a KCl solution but undergo a dose- and voltage-dependent inactivation in the presence of di- and trivalent cations, which is mediated by open-closed fluctuations at the single-channel level. Hill plots indicate that each channel can bind two to three inactivating cations. The inhibiting efficiency follows the sequence Zn2+ greater than Tb3+ greater than Ca2+ greater than Mg2+ greater than Ba2+, suggesting that carboxyl groups of the protein may be involved in the binding step. A voltage-gated inactivation mechanism is proposed which involves the binding of two polyvalent cations to the channel, one in the open and one in the closed configuration, and which can explain voltage, dose and time dependence of the inactivation.

The role of phosphoinositides in signal transduction.

Abstract: The binding of agonists to protein receptors at the cell surface is well established, but the mechanism by which the \"message\" generated at the receptor is transferred to the inside of the cell is not clearly understood for all types of stimuli. One class of receptors mediates its response through formation of cAMP, catalyzed by adenylate cyclase (a protein present on the cytoplasmic side of the membrane). The coupling of adenylate cyclase to the receptor is further regulated by GTP-binding proteins [186, 258]. It has been suggested that conversion of phosphatidylethanolamine to phosphatidylcholine plays a role in the coupling of receptors to adenylate cyclase [120], although evidence against this hypothesis has also been presented [216; see 202]. Another class of receptors mediates its response through Ca :+ mobilization. Evidence is rapidly mounting that a specific class of phosphotipids, i.e., the phosphoinositides, plays an important role in signal transduction from the receptors at the plasma membrane. There have been numerous recent reviews on the various aspects of phosphoinositide metabolism and its role in signal transduction [see 1, 3, 23, 25, 71, 81, 82, 88, 89, 118, I19, 125, 148, 197,207,211, 220, 237,239]. The emphasis of this review will be to analyze the recent developments ~ and how they relate to earlier observations.

Volume, pH, and ion-content regulation in human red cells: Analysis of transient behavior with an integrated model

Abstract: A basic mathematical model of human red cells is presented which integrates the charge and nonideal osmotic behavior of hemoglobin and of other impermeant cell solutes with the ion transport properties of the red cell membrane. The computing strategy was designed to predict the behavior of all measurable variables in time in ways that optimize comparison with experimentally determined behavior. The need and applications of such a model are illustrated in three separate examples covering different areas of experimentation in the physiology and pathophysiology of red cells.

H+-ATPases from mitochondria, plasma membranes, and vacuoles of fungal cells

Abstract: A remarkable number of cellular activities are directly dependent on ion-pumping ATPases. In addition to the fundamental processes of active transport and ATP synthesis, recent evidence has shown an essential role for ion-pumping ATPases in cell motility, development, ligand-receptor uncoupling, and protein sorting [71, 111,147]. Despite the multiplicity of functions for ATPases, the basic design of these enzymes has been highly conserved. In both eucaryotic and procaryotic organisms, most ionpumping ATPases that have been characterized fit into one of three structural types. In this review we wish to compare these three types of ATPases, drawing upon recent work with fungal cells.

Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine.

Abstract: The osmotic water permeabilityPf of brush border (BBM) and basolateral (BLM) membrane vesicles from rat small intestine and renal cortex was studied by means of stopped-flow spectrophotometry. Scattered light intensity was used to follow vesicular volume changes upon osmotic perturbation with hypertonic mannitol solutions. A theoretical analysis of the relationship of scattered light intensity and vesicular volume justified a simple exponential approximation of the change in scattered light intensity. The rate constants extracted from fits to an exponential function were proportional to the final medium osmolarity as predicted by theory. For intestinal membranes, computer analysis of optical responses fitted well with a single-exponential treatment. For renal membranes a double-exponential treatment was needed, implying two distinct vesicle populations.Pf values for BBM and BLM preparations of small intestine were equal and amount to 60 μm/sec. For renal preparations,Pf values amount to 600 μm/sec for the fast component, BBM as well as BLM, and to 50 (BBM) and 99 (BLM) μm/sec for the slow component. The apparent activation energy for water permeation in intestinal membranes was 13.3±0.6 and in renal membranes, 1.0±0.3 kCal/mole, between 25 and 35°C. The mercurial sulfhydryl reagentpCMBS inhibited completely and reversibly the highPf value in renal brush border preparations. These observations suggest that in intestinal membranes water moves through the lipid matrix but that in renal plasma membranes water channels may be involved. From the highPf values of renal membrane vesicles a transcellular water permeability for proximal tubules can be calculated which amounts to ∼1 cm/sec. This value allows for an entirely transcellular route for water flow during volume reabsorption.

Separate, Ca2+-activated K+ and Cl- transport pathways in Ehrlich ascites tumor cells.

Abstract: The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl− transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl− transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl− conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl− conductance is rate limiting. An A23187-induced activation of42K and36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca2+-activated K+ channel. (ii) unaffected by substitution of NO 3 − or SCN− for Cl−, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl− conductance. The Cl− conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl− transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl− transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl− transport pathway. The activated anion transport pathway is unselective, carrying both Cl−, Br−, NO 3 − , and SCN−. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl− transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by42K flux experiments and indirectly in media where the dominating anion (SCN−) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from42K flux measurements at high external K+, and from net K− flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell.

Mechanisms of aldosterone action in tight epithelia.

Abstract: Since the discovery of aldosterone in the early 1950's (Simpson & Tait, 1952) this adrenocorticosteroid has been recognized as a potent regulator of electrolyte metabolism in all vertebrates (Forman & Mulrow, 1975). In mammals the target tissues for the hormonal action are: the distal segments of the renal tubules, the urinary bladder, the descending colon mucosa, and the salivary and sweat glands. In all of them an aldosterone-induced increase of Na + reabsorption was observed (Crabb6, 1963b; Sharp & Leaf, 1973; Taylor & Palmer, 1982). Most of the present knowledge on the natriferic action of this hormone came, however, from studies on amphibian tight epithelia and in particular the toad urinary bladder. This classical model system for the mammalian distal nephron was chosen by many workers because of its relative histological simplicity and the ease with which its Na + transport can be monitored by short-circuit current recordings (for review see Macknight, DiBona & Leaf, 1980). In biochemical studies, on the other hand, rat and rabbit kidney segments, which provide larger amounts of starting material, were often preferred. An in vitro aldosterone-induced stimulation of Na + transport in toad bladder was first reported by Crabb6 (1961, 1963a). Shortly afterwards it was shown that the hormonal effect involves the induction of protein synthesis and also depends on the availability of metabolic substrates (Edelman, Bogoroch & Porter, 1963; Porter, Bogoroch & Edelman, 1964; Porter & Edelman, 1964; Crabb6 & DeWeer, 1964; Sharp & Leaf, 1964b). Since then research has been carried out in four main areas: 1) Characterization of the cytoplasmic and nuclear aldosterone binding sites.

Voltage-dependent channel formation by rods of helical polypeptides.

Abstract: The voltage-dependence of channel formation by alamethicin and its natural analogues can be described by a dipole flip-flop gating model, based on electric field-induced transbilayer orientational movements of single molecules. These field-induced changes in orientation result from the large permanent dipole moment of alamethicin, which adopts α-helical conformation in hydrophobic medium. It was, therefore, supposed that the only structural requirement for voltage-dependent formation of alamethicin-type channels might be a rigid lipophilic helical segment of minimum length. In order to test this hypothesis we synthesized a family of lipophilic polypeptides—Boc-(Ala-Aib-Ala-Aib-Ala) n -OMe,n=1–4—which adopt α-helical conformation forn=2–4 and studied their interaction with planar lipid bilayers. Surprisingly, despite their large difference in chain length, all four polypeptides showed qualitatively similar behavior. At low field strength of the membrane electric field these polypeptides induce a significant, almost voltage-independent increase of the bilayer conductivity. At high field strength, however, a strongly voltage-dependent conductance increase occurs similar to that observed with alamethicin. It results from the opening of a multitude of ion translocating channels within the membrane phase. The steady-state voltage-dependent conductance depends on the 8th–9th power of polypeptide concentration and involves the transfer of 4–5 formal elementary charges. From the power dependences on polypeptide concentration and applied voltage of the time constants in voltage-jump current-relaxation experiments, it is concluded that channels could be formed from preexisting dodecamer aggregates by the simultaneous reorientation of six formal elementary charges. Channels exhibit large conductance values of several nS, which become larger towards shorter polypeptide chain length. A mean channel diameter of 19 A is estimated corresponding roughly to the lumen diameter of a barrel comprised of 10 α-helical staves. Similar to experiments with the N-terminal Boc-derivative of alamethicin we did not observe the burst sequence of nonintegral conductance steps typical of natural (N-terminal Ac-Aib)-alamethicin. Saturation in current/voltage curves as well as current inactivation in voltage-jump current-relaxation experiments are found. This may be understood by assuming that channels are generated as dodecamers but, while reaching the steady state, reduce their size to that of an octamer or nonamer. We conclude that the overall behavior of these synthetic polypeptides is very similar to that of alamethicin. They exhibit the same concentration and voltage-dependences but lack the stabilizing principle of resolved channel states characteristic of alamethicin.

A cation channel in frog lens epithelia responsive to pressure and calcium

Abstract: Patch-clamp recording from the apical surface of the epithelium of frog lens reveals a cation-selective channel after pressure (about ±30 mm Hg) is applied to the pipette. The open state of this channel has a conductance of some 50 pS near the resting potential (−56.1±2.3 mV) when 107mm NaCl and 10 HEPES (pH 7.3) is outside the channel. The probability of the channel being open depends strongly on pressure but the current-voltage relation of the open state does not. With minimal Ca2+ (55±2 μm) outside the channel, the current-voltage relation is nonlinear even in symmetrical salt solutions, allowing more current to flow into the cell than out. The channel, in minimal Ca2+ solution, is selective among the monovalent cations in the following sequence K+>Rb+>Cs+>Na+>Li+. The conductance depends monotonically on the mole fraction of K+ when the other ion present is Li+ or Na+. The single-channel current is a saturating function of [K+] when K+ is the permeant ion, for [K+]≤214mm. When [Ca2+]=2mm, the currentvoltage relation is linearized and the channel cannot distinguish Na+ and K+.

Ammonium transport in medullary thick ascending limb of rabbit kidney: involvement of the Na+,K+,Cl(-)-cotransporter.

Abstract: In order to investigate the question whether ammonium reabsorption in the thick ascending limb of Henle's loop (TALH) proceeds via the Na+,K+,Cl−-cotransporter, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and the effect of NH 4 + on their transport properties was investigated. It was found that, in the presence of a 78-mmol/liter NaCl gradient, 5 mmol/liter NH 4 + inhibited bumetanide-sensitive rubidium flux by 86%; a similar decrease was observed for 5 mmol/liter, K+. Inhibition of bumetanide-sensitive rubidium uptake by NH 4 + was competitive and an apparentK i of 1.9 mmol/liter was found Bumetanide-sensitive sodium uptake measured in the presence of a 83 mmol/liter KCl gradient was not inhibited by 5 mmol/liter NH 4 + . A 100-mmol/liter NH4Cl gradient was, however, capable of stimulating bumetanide-sensitive sodium uptake to the same extent as a KCl gradient. These data suggest that NH 4 + is accepted by the K+ site of the Na+,K+,Cl-cotransport system and that the transporter can function in a Na+, NH 4 + ,2Cl mode. Since the affinity of the transporter for NH 4 + lies in the concentration range found in the TALH lumen in vivo, it is concluded that Na+, NH 4 + 2Cl-cotransport can contribute to the NH 4 + reabsorption in this tubular segment.

Voltage dependence of the rheogenic Na+/K+ ATPase in the membrane of oocytes of Xenopus laevis.

Abstract: Electrophysiological experiments were performed to analyze the Na+/K+-ATPase in full-grown prophase-arrested oocytes of Xenopus laevis. If the Na+/K+-ATPase is inhibited by dihydroouabain (DHO), the resting potential of the membrane of Na+-loaded oocytes may depolarize by nearly 50 mV. This hyperpolarizing contribution to the resting potential depends on the degree of activation of the Na+/K+-ATPase and varies with intracellular Na+ activity (aiNa) and extracellular K+ (K+o). It is concluded that variations of aiNa among different oocytes are primarily responsible for the variations of resting potentials measured in oocytes of X. laevis. Under voltage-clamp conditions, the DHO-sensitive current also exhibits dependence on aiNa that may be described by a Hill equation with a coefficient of 2. This current will be shown to be identical with the electrogenic current generated by the 3Na+/2K+ pump. The voltage dependence of the pump current was investigated at saturating values of aiNa (33 mmol/liter) and of K+o (3 mmol/liter) in the range from -200 to +100 mV. The current was found to exhibit a characteristic maximum at about +20 mV. This is taken as evidence that in the physiological range at least two steps within the cycle of the pump are voltage dependent and are oppositely affected by the membrane potential.

Bioenergetics of alkalophilic bacteria.

Abstract: The central problem for organisms which grow optimally, and in some cases obligately, at pH values of 10 to 11, is the maintenance of a relatively acidified cytoplasm. A key component of the pH homeostatic mechanism is an electrogenic Na+/H+ antiporter which—by virtue of kinetic properties and/or its concentration in the membrane—catalyzes net proton uptake while the organisms extrude protons during respiration. The antiporter is also capable of maintaining a constant pHin during profound elevations in pHout as long as Na+ entry is facilitated by the presence of solutes which are taken up with Na+. Secondary to the problem of acidifying the interior is the adverse effect of the large pH gradient, acid in, on the total pmf of alkalophile cells. For the purposes of solute uptake and motility, the organisms appear to largely bypass the problem of a low pmf by utilizing a sodium motive force for energization. However, ATP synthesis appears not to resolve the energetics problem by using Na+ or by incorporating the proton-translocating ATPase into intracellular organelles. The current data suggest that effective proton pumping carried out by the alkalophile respiratory chain at high pH may deliver at least some portion of the protons to the proton-utilizing catalysts, i. e., theF1F0-ATPase and the Na+/H+ antiporter, by some localized pathway.

Kinetics of ultrastructural changes during electrically induced fusion of human erythrocytes.

Abstract: The sequence of events during the electrically induced fusion of human erythrocytes was studied by rapid quench freeze-fracture electron microscopy. A single electric field pulse was used to induce fusion of human erythrocytes treated with pronase and closely positioned by dielectrophoresis. The electronic circuit was coupled to a rapid freezing mechanism so that ultrastructural changes of the membrane could be preserved at given time points. Pronase treatment enabled adjacent cells to approach each other within 15 nm during dielectrophoresis. The pulse caused a brief disruption of the aqueous boundaries which separated the cells. Within 100 msec following pulse application, the fracture faces exhibited discontinuous areas which were predominantly free of intramembranous particles. At 2 sec after the pulse, transient point defects attributed to intercellular contact appeared in the same membrane areas and replaced the discontinuous areas as the predominant membrane perturbation. At 10 sec after the pulse, the majority of the discontinuous areas and point defects disappeared as the intercellular distance returned to approximately 15 to 25 nm, except at sites of cytoplasmic bridge formation. Intramembranous particle clearing was observed at 60 sec following pulse application in discrete zones of membrane fusion.

Non-Stokesian nature of transverse diffusion within human red cell membranes.

Abstract: Although much research has recently been carried out on lateral diffusion in biological membranes (see ref. 35 for a recent review), far less attention has been given to the problem of transverse diffusion-diffusion perpendicular to the plane of the membrane. Yet transverse diffusion is of paramount importance not only for understanding the dynamic properties of lipids in membranes but also for predicting the permeabilities of both single cells and epithelia (e.g., kidney, gut, skin and the blood-brain barrier) to physiologically and pharmacologically active agents.

Amiloride-sensitive Na+ transport in human red cells: evidence for a Na/H exchange system.

Abstract: The role of transmembrane pH gradients on the ouabain, bumetanide and phloretin-resistant Na+ transport was studied in human red cells. Proton equilibration through the Jacobs-Stewart cycle was inhibited by the use of DIDS (125 microM) and methazolamide (400 microM). Red cells with different internal pH (pHi = 6.4, 7.0 and 7.8) were prepared and Na+ influx was measured at different external pH (pHo = 6.0, 7.0, 8.0). Na+ influx into acid-loaded cells (pHi = 6.4) markedly increased when pHo was raised from 6.0 to 8.0. Amiloride, a well-known inhibitor of Na+/H+ exchange systems blocked about 60% of the H+-induced Na+ entry, while showing small inhibitory effects in the absence of pH gradients. When pHo was kept at 8.0, the amiloride-sensitive Na+ entry was abolished as pHi was increased from 6.4 to 7.8. Moreover, measurements of H+ efflux into lightly buffered media indicated that the imposition of an inward Na+ gradient stimulated a net H+ efflux which was sensitive to the amiloride analog 5-N-methyl-N-butyl-amiloride. Furthermore, in the absence of a chemical gradient for Na+ (Nai+ = Nao+ = 15 mM, Em = +6.7 mV), an outward H+ gradient (pHi = 6.4, pHo = 8.0) promoted a net amiloride-sensitive Na+ uptake which was abolished at an external pH of 6.0. These findings are consistent with the presence of an amiloride-sensitive Na+/H+ exchange system in human red cells.

Modification of single cardiac Na+ channels by DPI 201-106.

Abstract: In inside-out patches from cultured neonatal rat heart cells, single Na+ channel currents were analyzed under the influence of the cardiotonic compound DPI 201-106 (DPI), a putative novel channel modifier. In absence of DPI, normal cardiac single Na+ channels studied at −30 mV have one open state which is rapidly left with a rate constant of 826.5 sec−1 at 20°C during sustained depolarization., Reconstructed macroscopic currents relax completely with 7 to 10 msec. The current decay fits a single exponential. A considerable percentage of openings may occur during relaxation of the macroscopic current. In patches treated with 3×10−6m DPI in the pipette solution, stepping to −30 mV results in drastically prolonged and usually repetitive openings. This channel activity mostly persists over the whole depolarization (usually 160 msec in duration) but is abruptly terminated on clamping back the patch to the holding potential. Besides these modified events, apparently normal openings occur. The open time distribution of DPI-treated Na+ channels is the sum of two exponentials characterized by time constants of 0.85 msec (which is close to the time constant found in the control patches, 1.21 msec) and 12 msec. Moreover, DPI-modified Na+ channels exhibit a sustained high, time-independent open probability. Similar to normal Na+ channels, the mean number of open DPI-modified Na+ channels is voltage-dependent and increases on shifting the holding potential in the hyperpolarizing direction. These kinetic changes suggest an elimination of Na+ channel inactivation as it may follow from an interaction of DPI with Na+ channels.

Protein kinase C activates the renal apical membrane Na+/H+ exchanger.

Abstract: Studies were performed on purified brush-border membranes from the kidney of the rabbit to examine the relation between protein kinase C and the Na+/H+ exchanger in these membranes. The brush-border membranes were transiently opened by exposure to hypotonic media and the membrane proteins phosphorylated by exposure to ATP and phorbol esters or partially purified protein kinase C. The membranes were resealed and the intravesicular space acidified by incubation in a sodium-free isotonic solution (pH 5.5). The rate of uptake of 1mm 22Na+ (pH 7.5), with and without amiloride (1mm), was assayed and the proton gradient-stimulated, amiloride-inhibitable component of22Na+ taken as a measure of the activity of the Na+/H+ exchanger. 12-0-tetradecanoyl phorbol-13-acetate (TPA) increased the amiloride-sensitive component of22Na+ uptake TPA did not affect the amiloride-insensitive component of22Na+ uptake or the equilibrium concentration of sodium. TPA also did not affect the rate of dissipation of the proton gradient in the absence of sodium or the rate of sodium-dependent or-independent uptake ofd-glucose. Other “active” phorbol esters stimulated the rate of Na+/H+ exchange, but phorbol esters of the 4 α configuration did not. Incubation of the opened membranes in partially purified protein kinase C increased the rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake. The stimulatory effect of TPA and protein kinase C was not additive. In the absence of ATP, neither TPA nor protein kinase C affected Na+/H+ exchange transport. To determine the membrane-bound protein substrates, parallel experiments were conducted with γ-[32P] ATP in the phosphorylating solutions. The reaction was stopped by SDS and the phosphoproteins resolved by PAGE and autoradiography. TPA stimulation of protein kinase C resulted in phosphorylation of approximately 13 membrane-bound proteins ranging in apparent molecule from 15,000 to 140,000 daltons. These studies indicate that activation of endogenous renal brush-border protein kinase C by phorbol esters or exposure of these membranes to exogenous protein kinase C increases the rate of proton gradient-stimulated, amiloride-inhibitable sodium transport. Protein kinase C activation also results in phosphorylation of a finite number of membrane-bound proteins.

Caffeine Inhibition of Calcium Accumulation by the Sarcoplasmic Reticulum in Mammalian Skinned Fibers

Abstract: Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca2+ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca2+ exceeds 0.3 to 0.4 microM, when free ATP exceeds 0.8 to 1 mM, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5 mM and is half-maximal at approximately 0.5 mM. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3 mM. Varying free Mg2+ (0.1 to 2 mM) or MgATP (0.03 to 10 mM) has no effect on inhibition. Average residual uptake rates in the presence of 5 mM caffeine at pCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca2+ concentration dependence of Ca uptake (K0.5, 0.5 to 0.8 microM; Hill n, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.

Evidence for a Na+/K+/Cl− cotransport system in basolateral membrane vesicles from the rabbit parotid

Abstract: Sodium (22Na) transport was studied in a basolateral membrane vesicle preparation from rabbit parotid. Sodium uptake was markedly dependent on the presence of both K+ and Cl− in the extravesicular medium, being reduced 5 times when K+ was replaced by a nonphysiologic cation and 10 times when Cl− was replaced by a nonphysiologic anion. Sodium uptake was stimulated by gradients of either K+ or Cl− (relative to nongradient conditions) and could be driven against a sodium concentration gradient by a KCl gradient. No effect of membrane potentials on KCl-dependent sodium flux could be detected, indicating that this is an electroneutral process. A KCl-dependent component of sodium flux could also be demonstrated under equuilibrium exchange conditions, indicating a direct effect of K+ and Cl− on the sodium transport pathway. KCl-dependent sodium uptake exhibited a hyperbolic dependence on sodium concentration consistent with the existence of a single-transport system withK m =3.2mm at 80mm KCl and 23°C. Furosemide inhibited this transporter withK 0.5=2×10−4 m (23°C). When sodium uptake was measured as a function of potassium and chloride concentrations a hyperbolic dependence on [K] (Hill coefficient =1.31±0.07) were observed, consistent with a Na/K/Cl stoichiometry of 1∶1∶2. Taken together these data provide strong evidence for the electroneutral coupling of sodium and KCl movements in this preparation and strongly support the hypothesis that a Na+/K+/Cl− cotransport system thought to be associated with transepithelial chloride and water movements in many exocrine glands is present in the parotid acinar basolateral membrane.

Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine and p-chloromercuriphenyl sulfonate

Abstract: The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial,p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 μM) by NBMPR was biphasic −35% of the transport activity was inhibited with an IC50 value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 μM. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparentKm for uridine 50±18 and 163±28 μM, respectively). The two transport systems also differed in their sensitivity topCMBS. NBMPR-insensitive uridine transport was inhibited bypCMBS with an IC50 of ∼25μM, while 1 mMpCMBS had little effect on NBMPR-sensitive transport by intact cells.pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by β-mercaptoethanol, suggesting that thepCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [3H]NBMPR binding to the cell membrane (apparentKd46±25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25±6 molecules/site per sec at 22°C.pCMBS had no effect on [3H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [3H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent Mr on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR andpCMBS.

Mechanical study of the deformation and rupture of the plasma membranes of protoplasts during osmotic expansions

Abstract: The stress and strain (surface tension and fractional change in area) in the plasma membrane of protoplasts isolated from rye leaves (Secale cereale L. cv Puma) were measured during osmotic expansions from isotonic into a range of more dilute solutions. The membrane surface tension increases rapidly to a maximum and then decreases slowly with some protoplasts lysing in all phases of the expansion. The maximum surface tension is greater for rapid expansions, and protoplasts lyse earlier during rapid expansion. Over the range of expansion rates investigated, the area at which lysis occurs is not strongly dependent on expansion rate. The value of the maximum tension is determined by the expansion rate and the rate at which new material is incorporated into the membrane. During osmotic expansion, protoplasts isolated from cold-acclimated plants incorporate material faster than do those from nonacclimated plants and thus incur lower membrane tensions.

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: I. Tight-seal whole-cell recordings.

Abstract: Electrical currents associated with sodium-coupled alanine transport in mouse pancreatic acinar cells were studied using the method of whole-cell recording with patch pipettes. Single cells or small clusters of (electrically coupled) cells were isolated by collagenase treatment. The composition of the intracellular solution could be controlled by internal perfusion of the patch pipette. In this way both inward and outward currents could be measured under “zero-trans” conditions, i.e., with finite concentrations of sodium andl-alanine on one side and zero concentrations on the other. Inward andoutward currents for equal but opposite concentration gradients were found to be of similar magnitude, meaning that the cotransporter is functionally nearly symmetric. The dependence of current on the concentrations of sodium andl-alanine exhibited a Michaelis-Menten behavior. From the sodium-concentration dependence of current as well as from the reversal potential of the current in the presence of an alanine-concentration, gradient, a sodium/alanine stoichiometric ratio of 1:1 can be inferred. The finding that N-methylated amino acids may substitute, forl-alanine, as well as the observed pH dependence of currents indicate that the pancreatic alanine transport system is similar to (or identical with) the “A-system” which is widespread in animal cells. The transport system is tightly coupled with respect to Na+; alanine-coupled inward flow of Na+ is at least 30 times higher than uncoupled Na+ flow mediated by the cotransporter. The current-voltage characteristic of the cotransporter could be (approximately) determined from the difference of transmembrane current in the presence and in the absence ofl-alanine. The sodium-concentration dependence of the current-voltage characteristic indicates that a Na+ ion approaching the binding site from the extracellular medium has to cross part of the transmembrane electric field.

Membrane potentials and intracellular Cl- activity of toad skin epithelium in relation to activation and deactivation of the transepithelial Cl- conductance.

Abstract: The potential dependence of unidirectional36Cl fluxes through toad skin revealed activation of a conductive pathway in the physiological region of transepithelial potentials. Activation of the conductance was dependent on the presence of Cl− or Br− in the external bathing solution, but was independent of whether the external bath was NaCl-Ringer's, NaCl-Ringer's with amiloride, KCl-Ringer's or choline Cl-Ringer's To partition the routes of the conductive Cl− ion flow, we measured in the isolated epithelium with double-barrelled microelectrodes apical membrane potentialV a , and intracellular Cl− activity,a Cl , of the principal cells indentified by differential interference contrast microscopy. Under short-circuit conditionsI sc=27.0±2.0 μA/cm2, with NaCl-Ringer's bathing both surfaces,V a was −67.9±3.8mV (mean ±se,n=24, six preparations) anda Cl was 18.0±0.9mM in skins from animals adapted to distilled water. BothV a anda Cl were found to be positively correlated withI sc (r=0.66 andr=0.70, respectively). In eight epithelia from animals adapted to dry milieu/tap waterV a anda Cl were measured with KCl Ringer's on the outside during activation and deactivation of the transepithelial Cl− conductance (G Cl) by voltage clamping the transepithelial potential (V) at 40 mV (mucosa positive) and −100 mV. AtV=40 mV; i.e. whenG Cl was deactivated,V a was −70.1±5.0 mV (n=15, eight preparations) anda Cl was 40.0±3.8mm. The fractional apical membrane resistance (fR a) was 0.69±0.03. Clamping toV=−100 mV led to an instantaneous change ofV a to 31.3±5.6 mV (cell interior positive with respect to the mucosal bath), whereas neithera Cl norfR a changed significantly within a 2 to 5-min period during whichG Cl increased by 1.19±0.10 mS/cm2. WhenV was stepped back to 40 mV,V a instantaneously shifted to −67.8±3.9 mV whilea Cl andfR a remained constant during deactivation ofG Cl. Similar results were obtained in epithelia impaled from the serosal side. In 12 skins from animals adapted to either tap water or distilled water the density of mitochondria-rich (D MRC) cells was estimated and correlated with the Cl current (I Cl though the fully activated (V=−100mV) Cl− conductance). A highly significant correlation was revealed (r=−0.96) with a slope of −2.6 nA/m.r. (mitochondria-rich cell and an I-axis intercept not significantly different from zero. In summary, the voltage-dependent Cl currents were not reflected infR a anda Cl of the principal cells but showed a correlation with the m.r. cell density. We conclude that the pricipal cells do not contribute significantly to the voltage-dependent Cl conductance.

Chloride currents inChara—A patch-clamp study

Abstract: Ionic current steps were recorded with the patch-clamp technique from algal cells that had been prepared without enzyme treatment. Inward current steps with different conductance levels occurred, the lowest level being 7 pS. There were complex transitions between levels indicating either a lack of independence between single channels, or sublevels of a much larger conductance unit. The reversal potential was consistent with the permeant ion being Cl−. Furthermore, when a different concentration of Cl− was used in the patch electrode the reversal potential of the inward current shifted in a manner consistent with a Nernstian change in the Cl− reversal potential. The frequency of the current steps was voltage dependent and suggestive of the hyperpolarization-activated Cl− currents reported in voltage-clamp studies. Outward current steps, with conductances of 38 pS, were recorded when the membrane patch was depolarized by more than +120 mV. Their amplitude and frequency increased at more positive potentials. The current was probably carried by an efflux of cations through a different set of channels. The resting membrane potential, measured unambiguously without contamination from the tonoplast, was −190±5 mV.

Potassium transport in the rabbit renal proximal tubule: effects of barium, ouabain, valinomycin, and other ionophores.

Abstract: Potassium fluxes in a suspension of rabbit proximal tubules were monitored using a potassium-sensitive extracellular electrode. Ouabain (10−4 m) and barium (5mm) were used to selectively quantitate the potassium efflux pathway (105±5 nmol K+·mg protein−1·min−1) and the sodium pump-related potassium influx (108±7), respectively. These equal and opposite fluxes suggest that potassium accumulation in the cell occurs mainly through the sodium pump and that potassium efflux occurs mainly through barium-sensitive potassium channels. Thus the activity of the sodium pump (Na, K-ATPase) in the basolateral membrane of the proximal tubule is balanced by the efflux of potassium, presumably across the basolateral membrane, which has a high potassium permeability. In addition, the effect of valinomycin and other ionophores was examined on potassium fluxes and several metabolic parameters [oxygen consumption (QO2), ATP content]. The addition of valinomycin to the tubules produced a net efflux of potassium which was quantitatively equivalent to the efflux produced by the addition of ouabain. The valinomycin-induced efflux was mainly due to the activity of valinomycin as a mitochondrial uncoupler, which indirectly inhibited the sodium pump by allowing a rapid reduction of the intracellular ATP. Amphotericin, nystatin, and monensin all produced large net releases of intracellular potassium. The action of the ionophores could be localized to the plasma or mitochondrial membrane and classified into three groups, as follows: (a) those which demonstrated full mitochondrial uncoupler activity (FCCP, valinomycin), (b) those which had no uncoupler activity (amphotericin B, nystatin); and (c) those which displayed partial uncoupler activity (monensin, nigericin).

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models.

Abstract: In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the “simultaneous” mechanism the cotransporterC forms a ternary complexNCS with Na+ and the substrateS; coupled transport of Na+ andS involves a conformational transition between statesNC′S andNC″S with inward- and outward-facing binding sites. In the “consecutive” (or “ping-pong”) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the “simultaneous” model, but incompatible with the “consecutive” mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the “simultaneous” model can be estimated from the experimental results. The equilibrium dissociation constants of Na+ and alanine at the extracellular side are determined to beK N ″ <-64mm andK S ″ <-18mm. Furthermore, the ratioK N ″ /K N S″ of the dissociation constants of Na+ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na+ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na+ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.