Cotransport Mechanism Research Articles

Regulatory volume increase after hypertonicity- or vasoactive-intestinal-peptide-induced cell-volume decrease in small-intestinal crypts is dependent on Na(+)-K(+)-2Cl- cotransport.

The volume of intact crypts isolated from guinea-pig small intestine has been measured to assess the capacity of the cells to regulate their volume after hypertonic shock or vasoactive-intestinal-peptide (VIP)-induced shrinkage. Crypts exposed to anisotonic medium initially behave as perfect osmometers. Continued exposure to a hypertonic (400 mosmol/l) medium was followed by regulatory volume increase (RVI), which led to complete volume recovery in about 20 min. VIP produced a volume reduction, attributed to KCl loss through channels activated by the secretagogue, without any recovery during exposure to the polypeptide. Removal of VIP led to an increase of cellular volume towards control levels. This volume recovery after secretagogue-induced shrinkage is termed SVI. Both RVI and SVI were abolished by removal of Na+ or Cl- from the bathing solution, by addition of the loop diuretic bumetanide (1 microM), but not by addition of ethylisopropylamiloride (10 microM) or amiloride (1 mM). Cell shrinkage was also observed when tonicity was increased by addition of 100 mM NaCl or 200 mM D-mannitol, but RVI was seen only when NaCl was the added osmolyte. The ion dependence, pharmacological sensitivity and thermodynamic considerations of these effects are consistent with the operation of a Na(+)-K(+)-2Cl- cotransport mechanism activated by cell shrinkage and the secretagogue action of VIP.

Allosterism and Na(+)-D-glucose cotransport kinetics in rabbit jejunal vesicles: compatibility with mixed positive and negative cooperativities in a homo- dimeric or tetrameric structure and experimental evidence for only one transport protein involved.

We first present two simple dimeric models of cotransport that may account for all of the kinetics of Na(+)-D-glucose cotransport published so far in the small intestine. Both the sigmoidicity in the Na+ activation of transport (positive cooperativity) and the upward deviations from linearity in the Eadie-Hofstee plots relative to glucose concentrations (negative cooperativity) can be rationalized within the concept of allosteric kinetic mechanisms corresponding to either of two models involving sequential or mixed concerted and sequential conformational changes. Such models also allow for 2 Na+: 1 S and 1 Na+: 1 S stoichiometries of cotransport at low and high substrate concentrations, respectively, and for partial inhibition by inhibitors or substrate analogues. Moreover, it is shown that the dimeric models may present physiological advantages over the seemingly admitted hypothesis of two different cotransporters in that tissue. We next address the reevaluation of Na(+)-D-glucose cotransport kinetics in rabbit intestinal brush border membrane vesicles using stable membrane preparations, a dynamic approach with the Fast Sampling Rapid Filtration Apparatus (FSRFA), and both nonlinear regression and statistical analyses. Under different conditions of temperatures, Na+ concentrations, and membrane potentials clamped using two different techniques, we demonstrate that our data can be fully accounted for by the presence of only one carrier in rabbit jejunal brush border membranes since transport kinetics relative to glucose concentrations satisfy simple Michaelis-Menten kinetics. Although supporting a monomeric structure of the cotransporter, such a conclusion would conflict with previous kinetic data and more recent studies implying a polymeric structure of the carrier protein. We thus consider a number of alternatives trying to reconcile the observation of Michaelis-Menten kinetics with allosteric mechanisms of cotransport associated with both positive and negative cooperativities for Na+ and glucose binding, respectively. Such models, implying energy storage and release steps through conformational changes associated with ligand binding to an allosteric protein, provide a rational hypothesis to understand the long-time debated question of energy transduction from the Na+ electrochemical gradient to the transporter.

Bile acids as enhancers of steroid penetration through excised hairless mouse skin

Excised hairless mouse skin (EHMS) is used to evaluate the potential of sodium choleate (NaCOL), an ox bile extract containing the sodium salts of taurocholic, glycoeolic, desoxycholic and cholic acids, and of the free choleic acids (HCOL) to enhance the transcutaneous penetration of progesterone (PGT) and prednisolone (PDN). EHMS is pretreated with aqueous dispersions of the enhancers, then the steroids are allowed to permeate through the pretreated EHMS from normal saline under occluded conditions. NaCOL is ineffective whereas HCOL produces structural modifications of the stratum corneum, resulting in increased skin permeability of both steroids. The chloroform-soluble components of HCOL interact strongly with stratum corneum lipids, as demonstrated by differential scanning calorimetry, thus facilitating PGT penetration. The chloroform-insoluble components of HCOL interact with more polar structures of stratum corneum, thereby promoting PDN transport. The data also suggest the existence of a parallel drug co-transport mechanism by the more lipophilic HCOL components.

The effect of drug charge type and charge density on corneal transport

In order to understand the effect of charge type on corneal transport of peptides, it is useful to first investigate the mechanism of transport of amino acids across corneal tissue. The transport characteristics of l-[ 14C]lysine and l-[ 14C]glutamic acid were based on in vitro flux measurements. Net flux of l-lysine occurs via a sodium-dependent cotransport mechanism (with a coupling ratio of 1:1). The transport of l-lysine involves the Na +-K +-ATPase pump and requires a stereospecific carrier-mediated transport system. The permeability coefficient ( P) of l-lysine was decreased from 10.1 ± 0.80 to 2.34 ± 0.75 × 10 −6 (cm/s by blocking the active pump and carrier-mediated system. However, l-glutamic acid was apparently absorbed by a passive aqueous transport mechanism which was oubain-insensitive, [Na +]-independent, temperature-insensitive and non-stereospecific. Overall, the cornea was 2–3-fold more permeable to a cation ( l-lysine) than an anion ( l-glutamic acid). In addition, the negatively charged drug, salicylic acid, was 2.3 times less permeable than the cationic drug, benzylamine.

Human placental brush-border membrane Na(+)-pantothenate cotransport

Membrane transport pathways for transplacental transfer of the water-soluble vitamin pantothenate were investigated by assessing the possible presence of a Na(+)-pantothenate cotransport mechanism in the maternal facing membrane of human placental epithelial cells. The presence of Na(+)-pantothenate cotransport was determined from radiolabeled tracer flux measurements of pantothenate uptake using preparations of purified brush-border membrane vesicles. Compared with other cations the imposition of an inward Na+ gradient stimulated vesicle uptake of pantothenate to levels approximately 40-fold greater than those observed at equilibrium. The observed stimulation of pantothenate uptake was not the result of indirect electrostatic coupling to an inside positive Na+ diffusion potential. In the absence of Na+ and pantothenate concentration gradients an inside negative voltage difference induced a Na(+)-dependent net influx of pantothenate, suggesting the presence of an electrogenic Na(+)-pantothenate cotransport mechanism. The effect of biotin on the kinetics of Na(+)-dependent pantothenate uptake and the effect of pantothenate on the kinetics of Na(+)-dependent biotin uptake suggested that placental absorption of biotin and pantothenate from the maternal circulation occurs by a common Na+ cotransport mechanism in apical brush-border membrane.

Influence of Haemoglobin Conformation, Nitrite and Eicosanoids on K+ Transport Across the Carp Red Blood Cell Membrane

ABSTRACT The regulation of K+ transport across the red blood cell (RBC) membrane by haemoglobin (Hb) conformation was studied in carp, and the K+ transport mechanisms were identified. When a large proportion of Hb in the R quaternary structure was secured by oxygenation of blood at pH 8.14, a net RBC K+ efflux was induced, which was accompanied by RBC shrinkage. This K+ efflux was resistant to ouabain and inhibited by furosemide and DIDS and by substitution of NO3− for Cl−, showing it to result from a K+/C1− cotransport mechanism. Deoxygenation of the RBCs (Hb in T structure) eliminated the Cl−-dependent K+ efflux and resulted in a net K+ uptake via the Na+/K+ pump. These changes were fully reversible. Nitrite-induced methaemoglobin formation in deoxygenated blood, which converts a large fraction of the T structure Hb into an R-like conformation, shifted the K+ uptake to a Cl−-dependent K+ efflux similar to that seen in oxygenated cells. When the allosteric equilibrium between the R and T structures of Hb was gradually shifted towards the T state by decreases in pH, the Cl−-dependent K+ efflux from oxygenated cells decreased. At pH 7.52, where the Root effect caused a potent stabilisation of the T state, the K+ efflux was reversed to a net K+ uptake. A similar change was induced in methaemoglobin-containing deoxygenated blood, since low pH also favours a T-like conformation of metHb. The variable K+ fluxes could not be related to changes in membrane potential or pH but were always directly related to the experimental modulation of the relative proportions of R-and T-structure Hb. It is proposed that Hb conformation governs K+ movements via a different binding of T and R structures to integral membrane proteins, and that a large fraction of R-structure Hb triggers the Cl−-dependent K+ efflux mechanism. Application of inhibitors and a substrate of prostaglandin and leukotriene synthesis did not influence the K+ efflux from oxygenated erythrocytes. However, a fraction of the K+ efflux from nitrite-treated deoxygenated cells was inhibited by nordihydroguaiaretic acid, suggesting that a slightly larger K+ efflux from these RBCs than from oxygenated RBCs was related to leukotriene production caused by nitrite entry. A much larger influx of nitrite to deoxygenated than to oxygenated RBCs was positively correlated with the distribution ratio of H+ and the membrane potential, supporting the view that nitrite primarily enters the cells via conductive transport. The physiological implications of the results are discussed.

Human placental brush-border membrane Na(+)-biotin cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin biotin were investigated by assessing the possible presence of a Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells. The presence of Na(+)-biotin cotransport was determined from radiolabeled tracer flux measurements of biotin uptake using preparations of purified brush-border membrane vesicles. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of biotin to levels approximately 25-fold greater than those observed at equilibrium. The voltage sensitivity of Na+ gradient-driven biotin uptake suggested Na(+)-biotin cotransport is electrogenic occurring with net transfer of positive charge. A kinetic analysis of the activation of biotin uptake by increasing Na+ was most consistent with an interaction of Na+ at 2 sites in the transport protein. Static head determinations used to identify the magnitude of opposing driving forces bringing flux through the cotransport mechanism to equilibrium indicated a coupling ratio of 2 Na+ per biotin. Substrate specificity studies using chemical analogues of biotin suggested both the terminal carboxylic acid of the valeric acid side chain and a second nucleus of anionic charge were important determinants for substrate interaction with the cotransport protein. Initial rate determinations of biotin uptake indicate biotin interacts with a single saturable site (Km = 21 microM) with a maximal transport rate of 4.5 nmol/mg/min. The results of this study provide evidence for an electrogenic Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells.

Basolateral Cl −/HCO −3 exchange in rat jejunum: Evidence from H 14CO −3 uptake in membrane vesicles

Bicarbonate transport across basolateral membrane vesicles from rat jejunal enterocyte was studied at 28°C and pH 8.2. These experimental conditions make possible the determination of [ 14C]bicarbonate uptake. Inward gradients of Na +, K +, and Li + did not stimulate HCO − 3 uptake, suggesting that a cotransport mechanism with these cations does not occur. On the contrary a countertransport of bicarbonate driven by a Cl − gradient was evidenced. The ability of other inorganic anions to exchange with HCO − 3 was examined and results indicate that Cl − can be substituted by NO − 3, Br − and SCN −. The Cl −-dependent HCO − 3 uptake was strongly inhibited by SITS and DIDS, whereas acetazolamide was ineffective: thus transfer of labelled CO 2 is eliminated as a possible mode of HCO − 3 permeation. HCO − 3 uptake was also affected by the presence of superimposed membrane potentials, suggesting that a HCO − 3 conductive pathway is present in the jejunal basolateral membrane. These results show that there are no fundamental differences between data obtained performing H 14CO − 3 and 36Cl − (previously reported) uptake experiments.

Luminal influences on potassium secretion: chloride, sodium, and thiazide diuretics.

In the presence of Cl-, K+ secretion by the distal tubule saturates with increasing luminal Na+ concentration. Apparent maximal K+ secretion is attained with luminal Na+ concentrations of 40 mM. The results of the present study show that lowering the Cl- concentration of luminal fluid can increase the level of Na(+)-stimulated K+ secretion beyond the maximal level attained in the presence of Cl-. The effect of lowering luminal Cl- concentration to less than 10 mM on K+ secretion is greater with higher Na+ concentration. Under these conditions, chlorothiazide decreases K+ secretion. When chlorothiazide is present, changing the Na+ concentration does not affect K+ secretion. Because in rats a thiazide effect is attributed primarily to the distal convoluted tubule (DCT), we postulate that it is primarily DCT cells that increase K+ secretion when Na+ concentration is raised in the presence of low luminal Cl- concentration. We propose that the rat DCT cells have both an absorptive Na(+)-Cl- cotransport mechanism and a secretory K(+)-Cl- cotransport mechanism in the luminal membrane that can mediate the apparent exchange of Na+ for K+.

Mechanism of Cl- transport in eel intestinal brush-border membrane vesicles.

Cl- transport was studied in a preparation of brush-border membrane vesicles (BBMV) from seawater eel intestine. 36Cl- uptake appeared to be stimulated by a positive inside membrane diffusion potential generated (a) by a concentration gradient of salts, the cations of which are more permeable than the anions, (b) by a K+ diffusion potential obtained by imposing a K+ concentration gradient (Cout greater than Cin) in the presence of valinomycin, (c) an inwardly directed H+ ion concentration gradient. The membrane-potential-driven Cl- transport was inhibited by 1 mM 5-nitro-2-(4-phenylpropyl-amino)-benzoic acid. Arachidonic acid also inhibited Cl- uptake in eel intestinal BBMV, but the effect appeared to be unspecific, as the unsaturated fatty acid also affected the Na+ dependent D-glucose uptake. The effect of arachidonic acid was reversed in the presence of bovine serum albumin. Cl- influx was the same in the presence of inwardly directed gradients of Li+, Na+ or K+, arguing against the presence of Na(+)-Cl-, as well as K(+)-Cl- cotransport. The absence of a significant contribution of the Na(+)-K(+)-2Cl- cotransport mechanism to the Cl- uptake in seawater eel intestinal BBMV was indicated from the observations that Cl- uptake was not stimulated by the simultaneous presence of inwardly directed Na+ and K+ gradients, and that it was nearly insensitive to 1 mM bumetanide in the presence of extravesicular Na+ and K+. Furthermore, no evidence for the dependence of Cl- uptake on the Na+ gradient was obtained under a short-circuited membrane diffusion potential, i.e. in the presence of equilibrated K+ and valinomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular NH4+/K+ transport pathways in mouse medullary thick limb of Henle. Regulation by intracellular pH.

Fluorescence and electrophysiological methods were used to determine the effects of intracellular pH (pHi) on cellular NH4+/K+ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) from CD1 mice. Studies were performed in suspensions of MTAL tubules (S-MTAL) and in isolated, perfused MTAL segments (IP-MTAL). Steady-state pHi measured using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) averaged 7.42 +/- 0.02 (mean +/- SE) in S-MTAL and 7.26 +/- 0.04 in IP-MTAL. The intrinsic cellular buffering power of MTAL cells was 29.7 +/- 2.4 mM/pHi unit at pHi values between 7.0 and 7.6, but below a pHi of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pHi unit at pHi 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba(2+)-sensitive pathway and furosemide-sensitive Na+:K+(NH4+):2Cl- cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 mM/min, respectively. The apical Ba(2+)-sensitive cell conductance in IP-MTAL (Gc), which reflects the apical K+ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K0.5 at pHi approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba(2+)-sensitive NH4+ transport pathway was sensitive to reduction in cytosolic pH whether pHi was changed by acidifying the basolateral medium or by inhibition of the apical Na+:H+ exchanger with amiloride at a constant pHo of 7.4. The pHi sensitivities of Gc and apical, Ba(2+)-sensitive NH4+ influx in IP-MTAL were virtually identical. The pHi sensitivity of the Ba(2+)-sensitive NH4+ influx in S-MTAL when exposed to (apical+basolateral) NH4Cl was greater than that observed in IP-MTAL where NH4Cl was added only to apical membranes, suggesting an additional effect of intracellular NH4+/NH3 on NH4+ influx. NH4+ entry via apical Na+:K+ (NH4+):2Cl- cotransport in IP-MTAL was somewhat more sensitive to reductions in pHi than the Ba(2+)-sensitive NH4+ influx pathway; NH4+ entry decreased by 52.9 +/- 13.4% on reducing pHi from 7.31 +/- 0.17 to 6.82 +/- 0.14. These results suggest that pHi may provide a negative feedback signal for regulating the rate of apical NH4+ entry, and hence transcellular NH4+ transport, in the MTAL. A model incorporating these results is proposed which illustrates the role of both pHi and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.

Zinc uptake by proximal cells isolated from rabbit kidney: Effects of cysteine and histidine

The aim of this study was to characterize the mechanisms of zinc transport in proximal cells isolated from rabbit kidney cortex. Uptakes of 65Zn were assessed under initial rate conditions, after 0.5 min of incubation. The kinetic parameters obtained at 20 degrees C were a Km of 15.0 +/- 1.5 microM, a Jmax of 208.0 +/- 8.4 pmol min-1 (mg protein)-1, and an unsaturable constant of 0.259 +/- 0.104 (n = 8). Cadmium competitively inhibited the zinc uptake, with a Ki value of 13.0 +/- 2.8 microM, while zinc competitively inhibited 109Cd uptake by isolated cells. Cysteine and histidine stimulated zinc transport at an amino acid:zinc molar ratio ranging from 1:1 to 8:1. This stimulation was not observed in the absence of a sodium gradient. At a molar ratio greater than 16:1 (i.e. 400 microM cysteine or histidine and 25 microM Zn), there was evidence of inhibition. These data suggest that zinc enters renal proximal cells (a) as a free ion via a saturable carrier-mediated process or an unsaturable pathway and (b) complexed with cysteine or histidine, by means of a sodium/amino acid cotransport mechanism.

Transcellular sodium transport in cultured cystic fibrosis human nasal epithelium

Cystic fibrosis (CF) airway epithelia exhibit raised transepithelial Na+ transport rates, as determined by open-circuit isotope fluxes and estimates of the amiloride-sensitive equivalent short-circuit current (Ieq). To study the contribution of apical and basolateral membrane paths to raised Na+ transport in CF, CF nasal epithelial cultures were studied with double-barreled Na(+)-selective microelectrodes and the Ussing chamber technique. Intracellular Na+ activity (acNa) was 24.1 +/- 1.5 mM (n = 36), a value similar to acNa of normal nasal epithelial cells. Reduction of luminal [Na+] to 3 mM abolished Ieq and reduced acNa. Amiloride (10(-4) M) abolished Ieq but increased acNa from 20 +/- 2 to 36 +/- 7 mM (n = 10). Amiloride-induced increase in acNa was not affected by serosal [Na+] reduction but was blocked by preexposure to reduced luminal [Na+]. Amphotericin B increased Ieq during amiloride exposure, indicating that amiloride did not inhibit NA(+)-K(+)-ATPase. Ouabain abolished Ieq and slowly raised acNa. Reduction of serosal [Na+] led to a decrease in acNa that was blocked by bumetanide. It is concluded that 1) CF airway epithelia exhibit an increased apical membrane Na+ permeability, 2) acNa is regulated to a normal level in CF cells despite increased transcellular Na+ fluxes, 3) the abnormal increase in acNa in response to amiloride is dependent on luminal Na+, 4) Na+ is transported across the basolateral membrane by a bumetanide-sensitive cotransport mechanism, and 5) ouabain inhibits the basolateral Na(+)-K(+)-ATPase, causing slow dissipation of the chemical and electrical gradients across the cell membranes.

Dissecting the molecular mechanism of ion-solute cotransport: Substrate specificity mutations in theputP gene affect the kinetics of proline transport

Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of the putP gene, suggesting that they may replace amino acids at the active site of the enzyme. If putP substrate specificity mutations directly after the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of three putP substrate specificity mutants. One class of mutation increases the Km over 120 fold but only decreases the Vmax fourfold. Such Km mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases the Vmax 80-fold without changing the Km. Vmax mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both the Km and Vmax of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na+/proline symport.

Na+-coupled bicarbonate transport in renal proximal tubular epithelia

The renal proximal tubule cell is the functional unit of an epithelium that serves to maintain a normal acid–base balance by reclaiming HCO3− filtered at the glomerulus and returning it to the circulation. The proximal tubule cell performs transcellular HCO3− reabsorption by a process of H+ secretion at the luminal membrane and export of intracellular HCO3− at the basolateral membrane. The present review describes the identification and functional characterization of the major transport pathway mediating efflux of intracellular HCO3− across the basolateral membrane of the proximal tubule cell. Compelling experimental evidence obtained from investigations using isolated membrane vesicles and the perfused proximal tubule preparation is discussed that indicate this transport pathway is an electrogenic cotransport mechanism coupling transfer of Na+, HCO3−, and CO32− across the basolateral membrane. Key words: acid–base, bicarbonate reabsorption, renal epithelia.

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules

We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (Vbl) in rabbit proximal convoluted tubule (PCT, S1 segment). The present study is aimed at understanding the origin of the membrane repolarization following the initial effect of addition of luminal cotransported solutes. Luminal addition of 10-15 mM L-alanine produced a rapid and highly significant depolarization of Vbl (20.3 +/- 1.1 mV, n = 15) which was transient and associated with an increase in the fractional K+ conductance of the basolateral membrane (tK) from 8 to 29% (P less than 0.01, n = 6). Despite the significant increase in tK, the repolarization was only slightly reduced by the presence of basolateral Ba2+ (2 mM, n = 6) or quinine (0.5 mM, n = 5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4'isothiocyamostilbene-2,2'-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pHi) determined with the fluorescent dye 2',7'-bis-2-carboxyethyl-5(and -6)-carboxyfluorescein (BCECF), averaged 7.39 +/- 0.02 in control solution (n = 9) and increased to 7.50 +/- 0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16 +/- 0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K+ conductance but also through a cellular acidification reducing the basolateral HCO3- exit through the electrogenic Na-3(HCO3) cotransport mechanism.

Extra- and Intracellular pH and Membrane Potential Changes Induced by K+, Cl−, H2PO4−, and NO3− Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl(-) and H(2)PO(4) (-) uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K(+) uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K(+) absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl(-) FC induced intracellular acidification. Since H(+) fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K(+)/A(-) symport or HCO(3) (-)/A(-) antiport for anion uptake. Though not suitable to reveal the actual H(+)/A(-) stoichiometry, the results are consistent with an H(+)/A(-) cotransport mechanism.

Potassium-dependent volume regulation in retinal pigment epithelium is mediated by Na,K,Cl cotransport.

Changes in retinal pigment epithelial (RPE) cell volume were measured by monitoring changes in intracellular tetramethylammonium (TMA) using double-barreled K-resin microelectrodes. Hyperosmotic addition of 25 or 50 mM mannitol to the Ringer of the apical bath resulted in a rapid (approximately 30 s) osmometric cell shrinkage. The initial cell shrinkage was followed by a much slower (minutes) secondary shrinkage that is probably due to loss of cell solute. When apical [K+] was elevated from 2 to 5 mM during or before a hyperosmotic pulse, the RPE cell regulated its volume by reswelling towards control within 3-10 min. This change in apical [K+] is very similar to the increase in subretinal [K+]o that occurs after a transition from light to dark in the intact vertebrate eye. The K-dependent regulatory volume increase (RVI) was inhibited by apical Na removal, Cl reduction, or the presence of bumetanide. These results strongly suggest that a Na(K),Cl cotransport mechanism at the apical membrane mediates RVI in the bullfrog RPE. A unique aspect of this cotransporter is that it also functions at a lower rate under steady-state conditions. The transport requirements for Na, K, and Cl, the inhibition of RVI by bumetanide, and thermodynamic calculations indicate that this mechanism transports Na, K, and Cl in the ratio of 1:1:2.

Atrial peptide and cGMP effects on NaCl transport in inner medullary collecting duct

We examined the action of atrial natriuretic factor (ANF) on Na+ and Cl- transport in in vitro microperfused inner medullary collecting ducts (IMCD) isolated from rat kidneys. First we studied the isotopic fluxes at low perfusion rates (7 nl/min). The results showed that ANF added to bath decreased lumen-to-bath flux (Jl----b) of Na+ and increased Na+ bath-to-lumen flux (Jb----l). This was substantiated by a direct demonstration that ANF reduces net Na+ and Cl- absorption. The effect of ANF on Jl----b and Jb----l of Na+ was also observed at high perfusion rates (25 nl/min). The inhibitory effect of ANF was observed even when Na+ Jl----b was stimulated by vasopressin (VP). ANF (6 x 10(-11) M) added to bath increased Cl- Jb----l and generated a negative lumen potential difference (PD). These two effects were inhibited by furosemide and by the replacement of Na+ by choline and Cl- by SO4(2-) in the bath fluid. These observations are compatible with the existence of a Na(+)-Cl(-)-K+ cotransport mechanism stimulated by ANF. Moreover, the effects of guanosine 3',5'-cyclic monophosphate (cGMP) added to the bath on PD, Jl----b, and Jb----l of Na+ were similar to those observed with ANF. Thus, physiological concentrations of ANF inhibit directly Na+ and Cl- absorption in IMCD by two mechanisms, 1) by increasing cotransport Na(+)-Cl(-)-K+ secretion and 2) by inhibiting NaCl absorption both in the absence and in the presence of VP. These effects on NaCl transport appear to be mediated by cGMP.

Presteady-state kinetics and carrier-mediated transport: A theoretical analysis

Kinetic studies of cotransport mechanisms have so far been limited to the conventional steady-state approach which does not allow in general to resolve either isomerization or rate-limiting steps and to determine the values of the individual rate constants for the elementary reactions involved along a given transport pathway. Such questions can only be answered using presteady-state or relaxation experiments which, for technical reasons, have not yet been introduced into the field of cotransport kinetics. However, since two recent reports seem compatible with the observation of such transient kinetics, it would appear that theoretical studies are needed to evaluate the validity of such claims and to critically evaluate the expectations from a presteady-state approach. We thus report such a study which was performed on a simple four-state mechanism of carrier-mediated transport. The time-dependent equation for zero-trans substrate uptake was thus derived and then extended to models with p intermediary steps. It is concluded that (p-1) exponential terms will describe the approach to the steady state but that such equations have low analytical value since the parameters of the flux equation cannot be expressed in terms of the individual rate constants of the elementary reactions for models with p greater than 5. We thus propose realistic simplifications based on the time-scale separation hypothesis which allows replacement of the rate constants of the rapid steps by their equilibrium constants, thereby reducing the complexity of the kinetic system. Assuming that only one relaxation can be observed, this treatment generates approximate models for which analytical expressions can easily be derived and simulated through computer modeling. When performed on the four-state mechanism of carrier-mediated transport, the simulations demonstrate the validity of the approximate solutions derived according to this hypothesis. Moreover, our approach clearly shows that presteady-state kinetics, should they become applicable to (co)transport kinetics, could be invaluable in determining more precise transport mechanisms.