Electrogenic Transport Processes Research Articles

Comparison of electrogenic glucose transport processes and permeability between proximal and distal jejunum of laying hens

ABSTRACT1. The current objective was to assess (1) differences in mucosal transepithelial short-circuit current (Isc) and tissue conductance (GT), (2) the effect of a glucose stimulus and (3) epithelial paracellular permeability in the proximal and distal jejunum of laying hens.2. Proximal and distal jejunal segments used in the Ussing chambers were collected at 9 ± 0.5 and 73 ± 3.4% (SEM) of jejunal length, respectively. The proximal jejunal mucosa showed a small negative Isc (−1.3 µA/cm2), whereas the distal jejunum had a higher Isc (32.9 µA/cm2). Similarly, GT was 2.5-fold greater in the distal compared to the proximal jejunum.3. Increased paracellular permeability in the distal jejunum was displayed as demonstrated by a 5-fold higher mucosal to serosal flux of fluorescein isothiocyanate and horseradish peroxidase, representing molecules of low and high molecular weight, respectively.4. Addition of glucose to the mucosal side (5 mmol/l, final concentration in the chamber) to stimulate an absorptive effect caused 3-fold greater GT in the distal compared to the proximal jejunum.5. In conclusion, the present results supported site-specific electrogenic transport processes for the jejunal mucosa of laying hens. Therefore, precise description of the jejunal site may contribute to an improved comparability of electrophysiological data.

PH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones.

Gated ion transport across biological membranes is an intrinsic process regulated by protein channels. Synthetic anion carriers (anionophores) have potential applications in biological research; however, previously reported examples are mostly nonspecific, capable of mediating both electrogenic and electroneutral (nonelectrogenic) transport processes. Here we show the transmembrane Cl(-) transport studies of synthetic phenylthiosemicarbazones mimicking the function of acid-sensing (proton-gated) ion channels. These anionophores have remarkable pH-switchable transport properties with up to 640-fold increase in transport efficacy on going from pH 7.2 to 4.0. This "gated" process is triggered by protonation of the imino nitrogen and concomitant conformational change of the anion-binding thiourea moiety from anti to syn. By using a combination of two cationophore-coupled transport assays, with either monensin or valinomycin, we have elucidated the fundamental transport mechanism of phenylthiosemicarbazones which is shown to be nonelectrogenic, inseparable H(+)/Cl(-) cotransport. This study demonstrates the first examples of pH-switchable nonelectrogenic anion transporters.

A novel delta current method for transport stoichiometry estimation.

BackgroundThe ion transport stoichiometry (q) of electrogenic transporters is an important determinant of their function. q can be determined by the reversal potential (Erev) if the transporter under study is the only electrogenic transport mechanism or a specific inhibitor is available. An alternative approach is to calculate delta reversal potential (ΔErev) by altering the concentrations of the transported substrates. This approach is based on the hypothesis that the contributions of other channels and transporters on the membrane to Erev are additive. However, Erev is a complicated function of the sum of different conductances rather than being additive.ResultsWe propose a new delta current (ΔI) method based on a simplified model for electrogenic secondary active transport by Heinz (Electrical Potentials in Biological Membrane Transport, 1981). ΔI is the difference between two currents obtained from altering the external concentration of a transported substrate thereby eliminating other currents without the need for a specific inhibitor. q is determined by the ratio of ΔI at two different membrane voltages (V1 and V2) where q = 2RT/(F(V2 –V1))ln(ΔI2/ΔI1) + 1. We tested this ΔI methodology in HEK-293 cells expressing the elctrogenic SLC4 sodium bicarbonate cotransporters NBCe2-C and NBCe1-A, the results were consistent with those obtained with the Erev inhibitor method. Furthermore, using computational simulations, we compared the estimates of q with the ΔErev and ΔI methods. The results showed that the ΔErev method introduces significant error when other channels or electrogenic transporters are present on the membrane and that the ΔI equation accurately calculates the stoichiometric ratio.ConclusionsWe developed a ΔI method for estimating transport stoichiometry of electrogenic transporters based on the Heinz model. This model reduces to the conventional reversal potential method when the transporter under study is the only electrogenic transport process in the membrane. When there are other electrogenic transport pathways, ΔI method eliminates their contribution in estimating q. Computational simulations demonstrated that the ΔErev method introduces significant error when other channels or electrogenic transporters are present and that the ΔI equation accurately calculates the stoichiometric ratio. This new ΔI method can be readily extended to the analysis of other electrogenic transporters in other tissues.

Modulation of electrogenic transport processes in the porcine proximal colon by enteric neurotransmitters

The aim of our study was to evaluate the involvement of essential pro- and antisecretory neurotransmitters in regulation of secretion in porcine proximal colon. Choline acetyltransferase (ChAT), nitric oxide synthase (NOS), vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM) and neuropeptide Y (NPY) were located immunohistochemically in the epithelium and subepithelial layer. Modulation of epithelial secretion was studied in Ussing chambers. Application of carbachol (CA), sodium nitroprussid (SNP), VIP and SP but not of NPY or SOM resulted in a chloride dependent increase in short circuit current (I(sc) ). I(sc) increase induced by CA, VIP or SNP was not altered by preincubation with tetrodotoxin or indomethacin. In contrast, SP-induced I(sc) increase was diminished by preincubation with tetrodotoxin, indomethacin, L-nitro-arginin-methyl-ester, and atropine but not hexamethonium. Simultaneous application of CA and VIP, or CA and SNP increased the I(sc) stronger as expected. Applying SP/CA led to a smaller increase in I(sc) as calculated. It is concluded that mainly prosecretory neurotransmitters are involved in regulation of colonic secretion. Cross-potentiations of acetylcholine and nitric oxide and acetylcholine and VIP suggest activation of different intracellular cascades. Similar intracellular pathways may be stimulated by acetylcholine and SP, thus preventing an additive effect of the transmitters.

Extracellular signal-regulated kinase and GEF-H1 mediate depolarization-induced Rho activation and paracellular permeability increase

Plasma membrane depolarization activates the Rho/Rho kinase (ROK) pathway and thereby enhances myosin light chain (MLC) phosphorylation, which in turn is thought to be a key regulator of paracellular permeability. However, the upstream mechanisms that couple depolarization to Rho activation and permeability changes are unknown. Here we show that three different depolarizing stimuli (high extracellular K(+) concentration, the lipophilic cation tetraphenylphosphonium, or l-alanine, which is taken up by electrogenic Na(+) cotransport) all provoke robust phosphorylation of ERK in LLC-PK1 and Madin-Darby canine kidney (MDCK) cells. Importantly, inhibition of ERK prevented the depolarization-induced activation of Rho. Searching for the underlying mechanism, we have identified the GTP/GDP exchange factor GEF-H1 as the ERK-regulated critical exchange factor responsible for the depolarization-induced Rho activation. This conclusion is based on our findings that 1) depolarization activated GEF-H1 but not p115RhoGEF, 2) short interfering RNA-mediated GEF-H1 silencing eliminated the activation of the Rho pathway, and 3) ERK inhibition prevented the activation of GEF-H1. Moreover, we found that the Na(+)-K(+) pump inhibitor ouabain also caused ERK, GEF-H1, and Rho activation, partially due to its depolarizing effect. Regarding the functional consequences of this newly identified pathway, we found that depolarization increased paracellular permeability in LLC-PK1 and MDCK cells and that this effect was mitigated by inhibiting myosin using blebbistatin or a dominant negative (phosphorylation incompetent) MLC. Taken together, we propose that the ERK/GEF-H1/Rho/ROK/pMLC pathway could be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and regulate paracellular transport in the tubular epithelium.

Structural and Functional Analyses of Liver Cysts from the BALB/c-cpk Mouse Model of Polycystic Kidney Disease

Liver cysts arising from hepatic bile ducts are a common extra-renal pathology associated with both autosomal dominant and recessive polycystic kidney disease in humans. To elucidate the functional and structural changes inherent in cyst formation and growth, hepatic bile duct epithelia were isolated from the BALB/ c-cpk mouse model of polycystic kidney disease. Light and transmission electron microscopy revealed substantial fibrosis in the basal lamina surrounding hepatic bile duct cysts isolated from heterozygous (BALB/c-cpk/+) and homozygous (BALB/c-cpk/cpk) animals. Scanning electron microscopy and length analysis of normal, precystic and cystic bile ducts provided the unique observation that primary cilia in cholangiocytes isolated from bile ducts and cysts of animals expressing the mutated cpk gene had lengths outside the minimal and maximal ranges of those in cells lining bile ducts of wild-type animals. Based on the hypothesis that PKD is one of several diseases characterized as ciliopathies, this abnormal variability in the length of the primary cilia may have functional implications. Electrophysiological analyses of freshly isolated cysts indicate that the amiloride-sensitive epithelial Na(+) channel (ENaC) is inactive/absent and cAMP-mediated anion secretion is the electrogenic transport process contributing to cyst fluid accumulation. Anion secretion can be stimulated by the luminal stimulation of adenylyl cyclase.

Physiology and Pathophysiology of Potassium Channels in Gastrointestinal Epithelia

Epithelial cells of the gastrointestinal tract are an important barrier between the "milieu interne" and the luminal content of the gut. They perform transport of nutrients, salts, and water, which is essential for the maintenance of body homeostasis. In these epithelia, a variety of K(+) channels are expressed, allowing adaptation to different needs. This review provides an overview of the current literature that has led to a better understanding of the multifaceted function of gastrointestinal K(+) channels, thereby shedding light on pathophysiological implications of impaired channel function. For instance, in gastric mucosa, K(+) channel function is a prerequisite for acid secretion of parietal cells. In epithelial cells of small intestine, K(+) channels provide the driving force for electrogenic transport processes across the plasma membrane, and they are involved in cell volume regulation. Fine tuning of salt and water transport and of K(+) homeostasis occurs in colonic epithelia cells, where K(+) channels are involved in secretory and reabsorptive processes. Furthermore, there is growing evidence for changes in epithelial K(+) channel expression during cell proliferation, differentiation, apoptosis, and, under pathological conditions, carcinogenesis. In the future, integrative approaches using functional and postgenomic/proteomic techniques will help us to gain comprehensive insights into the role of K(+) channels of the gastrointestinal tract.

Plasma membrane depolarization: a possible coupling factor between epithelial transcellular and paracellular transport

The coordinated transport through transcellular and paracellular pathways ensures efficient transepithelial flux of water and solutes. Despite the fact that the coupling between these processes is of vital importance, their link is not well understood. To gain insight into the mechanism we asked whether 1) depolarization, induced by apical electrogenic transport processes, can serve as a signal coupling transcellular and paracellular transport; 2) increased cell contractility might play a role in this process. Paracellular permeability across confluent monolayer of proximal tubular (LLC-PK1) cells was followed as apical-to-basolateral transport of 4 kDa FITC-dextran. We show that depolarization, elicited either by high extracellular [K+], or by glucose or alanine, substrates of apical electrogenic transporters, substantially increased paracellular permeability. The effect of glucose required Na+-dependent cotransport, since 2-deoxy-glucose, which is not a substrate of the Na+/glucose cotransporter, failed to increase paracellular permeability. Importantly, depolarization was accompanied with Rho activation and Rho kinase (ROK)-dependent MLC phosphorylation. Inhibitors of ROK and myosin ATPase (Y27632, and blebbistatin) partially prevented the depolarization-induced rise in paracellular permeability, suggesting that the Rho-ROK-MLC pathway and the consequent increase in contractility contribute to the effect. Thus, plasma membrane depolarization is a good candidate as a coupling signal between apical and paracellular transport. Supported by Canadian Institute of Health Research.

Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells

Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular K(+), the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca(2+) signal, and removal of external Ca(2+) promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca(2+) did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na(+)-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na(+)-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but Ca(2+) signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport.

High-affinity glutamate transporters in the rat retina: a major role of the glial glutamate transporter GLAST-1 in transmitter clearance.

Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. The reverse transcriptase-polymerase chain reaction (RT-PCR) has revealed the presence of three different high-affinity glutamate transporters in the rat retina, viz. GLAST-1, GLT-1 and EAAC-1. No message has been found in the retina for EAAT-4, a transporter recently cloned from human brain. By using membrane vesicle preparations of total rat retina, we show that glutamate uptake in the retina is a high-affinity electrogenic sodium-dependent transport process driven by the transmembrane sodium ion gradient. Autoradiography of intact and dissociated rat retinae indicates that glutamate uptake by Müller glial cells dominates total retinal glutamate transport and that this uptake is strongly influenced by the activity of glutamine synthetase. RT-PCR, immunoblotting and immunohistochemistry have revealed that Müller cells express only GLAST-1. The Km for glutamate of GLAST-1 is 2.1+/-0.4 microM. This study suggests a major role for the Müller cell glutamate transporter GLAST-1 in retinal transmitter clearance. By regulating the extracellular glutamate concentration, the action of GLAST-1 in Müller cells may extend beyond the protection of neurons from excitotoxicity; we suggest a mechanism by which Müller cell glutamate transport might play an active role in shaping the time course of excitatory transmission in the retina.

Glucocorticoid stimulation of ileal Na+ absorptive cell brush border Na+/H+ exchange and association with an increase in message for NHE-3, an epithelial Na+/H+ exchanger isoform.

Methylprednisolone stimulates rabbit ileal neutral NaCl absorption; and aminoglutethimide, which decreases glucocorticoid levels, decreases NaCl absorption. Studies were carried out to determine the mechanism of these effects and to determine which members of the gene family of mammalian Na+/H+ exchangers were involved. Rabbits were treated subcutaneously with methylprednisolone (40 mg daily for 24 or 72 h), aminoglutethimide (100 mg twice daily for 72 h), or saline as a control. Ileal brush border membranes were prepared by magnesium precipitation, and brush border Na+/H+ exchange was determined by 22Na+ uptake over 3-8 s. The 22Na+ uptake experiments were performed in the presence of a voltage clamp using either valinomycin/potassium or tetramethylammonium/nitrate to eliminate potential contributions by other electrogenic transport processes. Methylprednisolone treatment approximately doubled ileal brush border Na+/H+ exchange, whereas aminoglutethimide led to a 50% decrease in Na+/H+ exchange. These effects were specifically on Na+ uptake with an acid inside pH gradient, whereas diffusive Na+ uptake (no pH gradient), glucose-dependent Na+ uptake, and glucose and Na+ equilibrium volumes were not affected. To determine if the increase in Na+/H+ exchange was associated with an increase in message expression, mRNA levels were measured by ribonuclease protection assay. Methylprednisolone stimulated the NHE-3 mRNA level by 4-6-fold at 24 h, which remained increased at 72 h. In contrast, messages for NHE-1 and NHE-2 were not affected by methylprednisolone. In summary, 1) methylprednisolone stimulation of rabbit ileal Na+ absorption is due to stimulation of ileal villus cell brush border Na+/H+ exchange; 2) basal ileal brush border Na+/H+ exchange is dependent on glucocorticoid levels; and 3) an increase in NHE-3 message, but not in NHE-1 or NHE-2 message, correlates with the stimulation of ileal brush border Na+/H+ exchange. It is likely that NHE-3 is an Na+/H+ exchanger that is involved in ileal Na+ absorption.

L-tryptophan uptake by segment-specific membrane vesicles from the proximal tubule of rabbit kidney.

1. The mechanism of the renal transport of L-tryptophan by basolateral and luminal membrane vesicles prepared from either the pars convoluta or the pars recta of the rabbit proximal tubule was studied. The uptake of L-tryptophan by basolateral membrane vesicles from the pars convoluta was found to be an Na(+)-dependent transport event. The Na(+)-conditional influx of the amino acid was stimulated in the presence of an inwardly directed H+ gradient. Lowering the pH without an H+ gradient had no effect, indicating that L-tryptophan is co-transported with H+. 3. On the other hand, no transient accumulation of L-tryptophan was observed in the presence or absence of Na+ in basolateral membrane vesicles from the pars recta. 4. In luminal membrane vesicles from the pars recta, the transient Na(+)-dependent accumulation of L-tryptophan occurred via a dual transport system. In addition, an inwardly directed H+ gradient could drive the uphill transport of L-tryptophan into these vesicles in both the presence and the absence of an Na+ gradient. 5. By contrast, the uptake of L-tryptophan by luminal membrane vesicles from the pars convoluta was a strictly Na(+)-dependent and electrogenic transport process, mediated by a single transport component. 6. Investigation of the coupling ratio in luminal membrane vesicles suggested that 1 Na+:1 L-tryptophan are co-transported in the pars convoluta. In the pars recta, examination of the stoichiometry indicated that approx. 1 H+ and 2 Na+ (high affinity) or 1 Na+ (low affinity) are involved in the uptake of L-tryptophan.

Ionic activities of the inner ear fluid and ionic permeabilities of the cochlear duct in endolymphatic hydrops of the guinea pig

Ionic activities (K +, Na +, and Cl −) of the perilymph and endolymph of the basal turn were measured using ion-selective microelectrodes in experimentally induced endolymphatic hydrops of the guinea pig. Three months following the obstruction of the endolymphatic duct and sac, the endocochlear potential (EP) of hydroptic ears was measured at 59.7 ± 9.6 mV (N = 12) which was significantly lower than the EP of the contralateral control ears (84.4 ± 2.8 mV, N = 12). A paired t-test ( P>0.05) showed no significant differences of ion concentrations of the inner ear fluid between the hydroptic and contralateral ears. Ion permeabilities of the cochlear duct following anoxia were calculated according to the Nernst-Planck equation. Comparing hydroptic and normal ears following anoxia, a statistically significant decrease was observed in the permeability coefficients for K +. Similarly, K + conductance was significantly lower in the hydroptic ears than in the normal ears. Total conductance of the cochlear duct, defined as the sum of each ion conductance, was 0.560 Siemens in the normal ears and 0.217 Siemens in the hydroptic ears. On the basis of the Goldman-Hodgkin-Katz equation, preexisting negative EP in the normal state was calculated to be −24.5 mV in normal ears and −21.4 mV in hydroptic ears. Therefore, the positive component of the EP was 108.9 mV in normal ears and 81.1 mV in hydroptic ears. These findings suggest that the pathophysiology of hydrops involves changes in K + permeability and the inhibition of the electrogenic transport processes.

ΔpH-Dependent Amino Acid Transport into Plasma Membrane Vesicles Isolated from Sugar Beet Leaves

Amino acid transport into plasma membrane vesicles isolated from mature sugar beet (Beta vulgaris L. cv Great Western) leaves was investigated. The transport of alanine, leucine, glutamine, glutamate, isoleucine, and arginine was driven by a trans-membrane proton concentration difference. DeltapH-Dependent alanine, leucine, glutamine, and glutamate transport exhibited simple Michaelis-Menten kinetics, and double-reciprocal plots of the data were linear with apparent K(m) values of 272, 346, 258, and 1981 micromolar, respectively. These results are consistent with carrier mediated transport. DeltapH-Dependent isoleucine and arginine transport exhibited biphasic kinetics, suggesting these amino acids may be transported by at least two transport systems. Symport mediated alanine transport was electrogenic as demonstrated by the effect of membrane potential (DeltaPsi) on DeltapH-dependent flux. In the absence of significant charge compensation, a low rate of alanine transport was observed. When DeltaPsi was held at 0 millivolt with symmetric potassium concentrations and valinomycin, the rate of flux was stimulated fourfold. In the presence of a negative DeltaPsi, alanine transport increased sixfold. These results are consistent with an electrogenic transport process which results in a net flux of positive charge into the vesicles. The effect of changing DeltaPsi on the kinetics of alanine transport altered V(max) with no apparent change in K(m). Amino acid transport was inhibited by the protein modifier diethyl pyrocarbonate, but was insensitive to N-ethylmaleimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, p-chloromercuribenzenesulfonic acid, phenylglyoxal, and N,N'-dicyclohexylcarbodiimide. Four amino acid symport systems, two neutral, one acidic, and one basic, were resolved based on inter-amino acid competition experiments. One neutral system appears to be active for all neutral amino acids while the second exhibited a low affinity for isoleucine, threonine, valine, and proline. Although each symport was relatively specific for a given group of amino acids, each system exhibited some crossover specificity for amino acids in other groups.

Modulation of the ionic permeability of renal cortical brush-border membranes by cAMP

The effects of adenosine 3',5'-cyclic monophosphate (cAMP) on potassium chloride permeability, relative ionic permeabilities, and Na+-dependent glucose transport were examined in rat renal cortical membrane vesicles. Brush-border membrane vesicles were prepared from paired control and cAMP- or forskolin-exposed homogenates by use of a magnesium aggregation technique. These studies demonstrate that exposure to exogenous cAMP or increases in endogenous cAMP significantly increase KCl permeability and ionic chloride permeability relative to that of potassium (PCl/PK) as determined with the fluorescent potential-sensitive probe 3,3'-dipropylthiadicarbocyanine iodide [diS-C3-(5)] and 36Cl uptake. Because PNa/PK did not change, PCl/PNa was also significantly increased by cAMP. These changes in ionic permeabilities were associated with a significant stimulation of Na+-dependent glucose transport that was unassociated with either an alteration in the kinetics of glucose transport or delayed dissipation of the inwardly directed Na+ gradient. These findings indicate that the cAMP-induced stimulation of glucose transport resulted from hyperpolarization of the vesicles secondary to the increase in PCl/PNa. These studies suggest that in vivo variations in intracellular cAMP concentration may modulate electrogenic transport processes by altering relative ionic permeabilities and membrane potential in renal proximal tubule cells.

Effects of Nitrogen Mustard-N-Oxide on Ionic Activities of Inner Ear Fluid and Ionic Permeabilities of the Cochlear Partition in the Guinea Pig

The effect of nitrogen mustard-N-oxide (NMO) on the endocochlear potential (EP) was investigated from the aspect of the ion concentrations and permeabilities in the cochlea. Compared with the untreated animals, in NMO-treated animals 20 to 30 hours after administration, the EP was decreased (30.8 +/- 3.5 mV in NMO versus 82.4 +/- 1.6 mV in control), the K+ concentration in perilymph of the scala tympani was increased (8.2 +/- 1.0 mM versus 5.3 +/- 0.7 mM), the K+ concentration in endolymph was decreased (128.5 +/- 10.6 mM versus 157.9 +/- 7.9 mM), and the Na+ concentration in endolymph was increased (9.6 +/- 3.6 mM versus 2.5 +/- 0.4 mM). The permeability coefficient for Na+ of the cochlear partition in the NMO-treated animals significantly decreased, while that for Cl- significantly increased. The negative EP, which presumably exists in the normal state, diminished further (-2.7 mV versus -27.8 mV), and the calculated electrogenic potential of the EP was depressed remarkably (33.5 mV versus 110.2 mV). The results suggest that the effects of NMO involved changes in ion permeabilities of the partition and the inhibition of electrogenic transport processes in the cochlea.

Simultaneous measurement of changes in current and tracer flux in voltage-clamped squid giant axon

A method is described for the simultaneous measurement of changes in membrane current and unidirectional radiotracer flux in internally dialyzed voltage-clamped squid giant axons. The small currents that are produced by electrogenic transport processes or steady-state ionic currents can be resolved using this method. Because the use of grounded guard electrodes in the end pools is not, by itself, an adequate means of eliminating end-effects, two ancillary end pool clamp circuits are described to eliminate extraneous current flow from the ends of the axon. The end pool voltage-clamp circuits serve to minimize net current flow between the end pools and center pool, and employ stable, low-impedance calomel electrodes to monitor the potentials of the end and center pools. The adequacy of the method is demonstrated by experiments in which unidirectional 22Na efflux and current, flowing through tetrodotoxin (TTX)-sensitive Na channels into Na-free seawater, under K-free conditions, are shown to be equal. The equality of unidirectional TTX-sensitive flux and current is maintained over the entire range of membrane potentials examined (-60 to +20 mV). The method has been applied to a series of experiments in which the voltage dependence and stoichiometry of the Na/K pump have been measured (Rakowski et al., 1989), and can be applied in general to the simultaneous measurement of changes in current and flux of other electrogenic transport processes, and of currents through ionic channels that open under steady-state conditions.

Characteristics of uptake of short chain fatty acids by luminal membrane vesicles from rabbit kidney

The mechanisms of renal transport of short chain fatty acids by luminal membrane vesicles prepared from pars convoluta or pars recta of rabbit proximal tubule were studied by a Millipore filtration technique and by a spectrophotometric method using a potential-sensitive carbocyanine dye. Both luminal membrane vesicle preparations take up propionate and butyrate by strictly Na +-dependent transport systems, although with different characteristics. The uptake of short chain fatty acids by membrane vesicles from the pars convuluta was insensitive to changes in membrane potential, which is indicative of electroneutral transport of these compounds. Furthermore, kinetic studies showed that the Na +-dependent, but electrically silent transport of propionate is saturable ( K m = 10.9 ± 1.1 mM and V max = 3.6 ± 0.2 nmol/mg protein per 20 s) and is unaffected by the presence of l- and d-lactate, indicating that these monocarboxylic acids did not share the same common transport system. In the luminal membrane vesicles from the pars recta, the uptake of propionate and butyrate was mediated by an Na +-dependent electrogenic transport process, since addition of the organic compounds to these vesicle/dye suspensions depolarized the membrane vesicles and the renal uptake of propionate and butyrate was enhanced by K + diffusion potential induced by valinomycin. Competition experiments revealed that in contrast to the transport of propionate by vesicles from the pars convoluta, the Na +-dependent electrogenic transport of short chain fatty acids in vesicles from the pars recta occurred via the same transport system that is responsible for the reabsorption of l- and d-lactate in this region of rabbit kidney proximal tubule.

Identification and characterization of ATP-dependent proton transport by rat liver multivesicular bodies.

Multivesicular bodies (MVB), prelysosomal organelles in the endocytic pathway, were prepared from estrogen-treated rat livers and examined for the presence of ATP-dependent proton transport. Vesicle acidification, assessed by acridine orange fluorescence quenching, was ATP dependent (ATP much greater than GTP, UTP), was enriched 25-fold over homogenate, was abolished by pretreatment with protonophores or a nonionic detergent, exhibited a pH optimum of 7.5, was inhibited by N-ethylmaleimide (NEM) (IC50 approximately 5 microM) and N,N'-dicyclohexylcarbodiimide (IC50 approximately 5 microM), and was resistant to inhibition by vanadate, ouabain, and oligomycin. Acidification exhibited no specific cation requirement; however, maximal rates of acidification depended upon the presence of Cl- (Km approximately 20 mM). Other anions were less effective in supporting acidification (Cl- greater than Br- greater than much greater than gluconate, NO-3, SO2-4, and mannitol), and indeed NO-3 inhibited acidification even in the presence of 150 mM Cl-. The proton transport mechanism appeared to be electrogenic based on: (a) enhancement of acidification by valinomycin in the presence of K gluconate, and (b) ATP-dependent fluorescence quenching of bis(3-phenyl-5-oxoisoxasol-4-yl)pentamethine oxonol, a membrane potential-sensitive anionic dye. Furthermore, the magnitude of the pH and electrical gradients generated by the proton transport mechanism appeared to vary inversely in the presence and absence of Cl-. Finally, MVB exhibited ATPase activity that was resistant to ouabain and oligomycin, but was inhibited 32.3% by 1 mM NEM, 33.7% by 200 microM dicyclohexylcarbodiimide, and 18.7% by KNO3. In isolated MVB, therefore, the NEM-sensitive ATPase activity may represent the enzymatic equivalent of a proton pump. These studies identify and characterize an ATP-dependent electrogenic proton transport process in rat liver MVB which shares many of the properties of the proton pump described in clathrin-coated vesicles, endosomes, lysosomes, Golgi, and endoplasmic reticulum from liver and other tissues. Acidification of MVB differed somewhat from that of rat liver clathrin-coated vesicles in response to Br- and NO-3, suggesting that membrane properties of these two organelles might differ.

Electrical properties of Madin-Darby canine kidney cells. Effects of extracellular potassium and bicarbonate.

To gain some insight into electrogenic transport processes across the plasma membrane of Madin-Darby canine kidney (MDCK)-cells, continuous measurements of the potential difference across the plasma measurements of the potential difference across the plasma membrane (PD) were made during step changes of extracellular ion composition as well as application of barium or valinomycin. During control conditions mimicking in vivo extracellular fluid, PD approaches -51.5 +/- 0.8 mV (n = 62). Step increase of extracellular potassium concentration from 5.4 to 10, to 20 or to 35 mmol/l, depolarizes PD by +5.5 +/- 0.8 mV (n = 7), by +15.8 +/- 0.5 mV (n = 64) and by +23.8 +/- 1.2 mV (n = 12), respectively. 1 mmol/l barium depolarizes PD by +19.8 +/- 0.6 mV (n = 38) and abolishes the effect of increasing extracellular potassium from 5.4 to 10 mmol/l but not to 35 mmol/l. Ten mumol/l valinomycin hyperpolarizes PD to -69.3 +/- 2.9 mV (n = 7). In the presence of valinomycin, increase of extracellular potassium from 5.4 to 20 mmol/l depolarizes PD by +31.0 +/- 1.0 mV (n = 7). Ouabain depolarizes PD and reduces the sensitivity of PD to extracellular potassium concentration. Omission of extracellular bicarbonate and carbon dioxide as well as increase of extracellular bicarbonate at constant carbon dioxide lead to a hyperpolarization and enhanced sensitivity of PD to extracellular potassium. In the presence of barium, the effects of omitted bicarbonate and carbon dioxide of MDCK-cells is highly conductive to potassium.(ABSTRACT TRUNCATED AT 250 WORDS)