Lysine and arginine transport in the photosynthetic bacterium Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum can take up both arginine and lysine in the light and, to a lesser extent, in the dark. Competitive inhibition experiments suggest the likely presence of two transport systems in this bacterium: One capable of transporting either lysine or arginine and a second capable of transporting arginine but not lysine. Uptake of both amino acids is electrogenic and appears to involve the cotransport of neither protons nor sodium ions. It is suggested that the transport occurs via an electrogenic uniport.     

Ion dependence of cystine and lysine uptake by rat renal brush-border membrane vesicles.

The shared transport system for uptake of L-cystine and L-lysine was examined in isolated rat renal brush-border membrane vesicles for the ionic requirements for activation of the system. No requirement for sodium was seen for either cystine or lysine influx. However, the efflux of lysine from the vesicle was stimulated by Na+. Therefore, the transport system appears to be asymmetric in its requirement for sodium. Two different divalent cations were used in the membrane isolations which resulted in different responses of cystine uptake to the electrogenic movement of K+ out of the vesicle. Membranes prepared by Mg-aggregation showed no stimulation of cystine influx by the imposition of a transient interior negative potential while vesicles prepared by Ca-aggregation did respond to electrogenic stimulation by an outwardly directed K-diffusion potential in the presence of valinomycin. Lysine influx was stimulated by electrogenic potassium efflux in both Mg-prepared and Ca-prepared membranes. No difference in sodium requirement for cystine influx was seen between the vesicles isolated by different cation-aggregation methods.     

Electrogenic and nonelectrogenic ion fluxes across lipid and mitochondrial membranes mediated by monensin and monensin ethyl ester

Monensin is a carrier of cations through lipid membranes capable of exchanging sodium (potassium) cations for protons by an electroneutral mechanism, whereas its ethyl ester derivative ethyl-monensin is supposed to transport sodium (potassium) cations in an electrogenic manner. To elucidate mechanistic details of the ionophoric activity, ion fluxes mediated by monensin and ethyl-monensin were measured on planar bilayer lipid membranes, liposomes, and mitochondria. In particular, generation of membrane potential on liposomes was studied via the measurements of rhodamine 6G uptake by fluorescence correlation spectroscopy. In mitochondria, swelling experiments were expounded by the additional measurements of respiration, membrane potential, and matrix pH. It can be concluded that both monensin and ethyl-monensin can perform nonelectrogenic exchange of potassium (sodium) ions for protons and serve as electrogenic potassium ion carriers similar to valinomycin. The results obtained are in line with the predictions based on the crystal structures of the monensin complexes with sodium ions and protons (Huczyński et al., Biochim. Biophys. Acta, 1818 (2012) pp. 2108–2119). The functional activity observed for artificial membranes and mitochondria can be applied to explain the activity of ionophores in living systems. It can also be important for studying the antitumor activity of monensin.     

Cyclooxygenase blockade and exogenous glutamine enhance sodium absorption in infected bovine ileum

We have previously shown that prostanoids inhibit electroneutral sodium absorption in Cryptosporidium parvum-infected porcine ileum, whereas glutamine stimulates electroneutral sodium absorption. We postulated that glutamine would stimulate sodium absorption via a cyclooxygenase (COX)-dependent pathway. We tested this hypothesis in C. parvum-infected calves, which are the natural hosts of cryptosporidiosis. Tissues from healthy and infected calves were studied in Ussing chambers and analyzed via immunohistochemistry and Western blots. Treatment of infected tissue with selective COX inhibitors revealed that COX-1 and -2 must be blocked to restore electroneutral sodium absorption, although the transporter involved did not appear to be the expected Na(+)/H(+) exchanger 3 isoform. Glutamine addition also stimulated sodium absorption in calf tissue, but although this transport was electroneutral in healthy tissue, sodium absorption was electrogenic in infected tissue and was additive to sodium transport uncovered by COX inhibition. Blockade of both COX isoforms is necessary to release the prostaglandin-mediated inhibition of electroneutral sodium uptake in C. parvum-infected calf ileal tissue, whereas glutamine increases sodium uptake by an electrogenic mechanism in this same tissue.     

Biology of the 2Na+/1H+ antiporter in invertebrates

The functional expression of membrane transport proteins that are responsible for exchanging sodium and protons is a ubiquitous phenomenon. Among vertebrates the Na+/H+ antiporter occurs in plasma membranes of polarized epithelial cells and non-polarized cells such as red blood cells, muscle cells, and neurons, and in each cell type the transporter exchanges one sodium for one hydrogen ion, is inhibited by amiloride, and regulates intracellular pH and sodium concentration within tight limitations. In polarized epithelial cells this transporter occurs in two isoforms, each of which is restricted to either the brush border or basolateral cell membrane, and perform somewhat different tasks in the two locations. In prokaryotic cells, sodium/proton exchange occurs by an electrogenic 1Na+/2H+ antiporter that is coupled to a primary active proton pump and together these two proteins are capable of tightly regulating the intracellular concentrations of these cations in cells that may occur in environments of 4 M NaCl or pH 10-12. Invertebrate epithelial cells from the gills, gut, and kidney also exhibit electrogenic sodium/proton exchange, but in this instance the transport stoichiometry is 2Na+/1H+. As with vertebrate electroneutral Na+/H+ exchange, the invertebrate transporter is inhibited by amiloride, but because of the occurrence of two external monovalent cation binding sites, divalent cations are able to replace external sodium and also be transported by this system. As a result, both calcium and divalent heavy metals, such as zinc and cadmium, are transported across epithelial brush border membranes in these animals and subsequently undergo a variety of biological activities once accumulated within these cells. Absorbed epithelial calcium in the crustacean hepatopancreas may participate in organismic calcium balance during the molt cycle and accumulated heavy metals may undergo complexation reactions with intracellular anions as a detoxification mechanism. Therefore, while the basic process of sodium/proton exchange may occur in invertebrate cells, the presence of the electrogenic 2Na+/1H+ antiporter in these cells allows them to perform a wide array of functions without the need to develop and express additional specialized transport proteins. J. Exp. Zool. 289:232-244, 2001.     

Molecular mechanisms of ANP inhibition of renal sodium transport.

ANP, a hormone secreted by the atria of mammalian hearts in response to volume expansion, increases urinary sodium excretion in part by inhibiting sodium reabsorption across the inner medullary collecting duct. A number of nephron segments may contribute to the ANP-induced natriuresis; however, this review will focus on the cellular mechanisms of ANP inhibition of electrogenic sodium reabsorption by the inner medullary collecting duct. Patch-clamp studies conducted on rat inner medullary collecting duct cells in primary culture revealed that ANP, via its second messenger cGMP, inhibits electrogenic sodium reabsorption by reducing the open probability of a cation channel located in the apical membrane. Cyclic GMP inhibits the cation channel and thereby sodium reabsorption by two mechanisms. First, cGMP inhibits the channel by a phosphorylation-independent mechanism, by binding either to an allosteric modifier site on the channel or to a regulatory subunit. Second, cGMP inhibits the channel by activating cGMP-dependent protein kinase, which by a sequential pathway involving the GTP-binding protein, Gi, inhibits the channel. These cGMP-dependent mechanisms inhibiting sodium reabsorption across the inner medullary collecting duct account for a substantial component of the natriuresis following a rise in ANP levels.     

Electrogenic plasma membrane H<Superscript>+</Superscript>-ATPase activity using voltage sensitive dyes

Fast responding voltage sensitive dyes, RH421 and di-4-ASPBS, were used to study the electrogenic properties of plant plasma membrane proton pumps on sealed plasma membrane vesicles extracted by two-phase partitioning from Beta vulgaris and Avena sativa cv Swan root material. Fluorescence spectroscopy in the presence of the dye RH421 (10.8 nM) was sufficiently sensitive to detect electrogenic activity of the extracted plant vesicles. The dye detection system could detect inhibition of electrogenic activity of vesicles by vanadate (75 μM) and stimulation by nigericin (0.5 μM). The newly developed dye di-4-ASPBS was less sensitive to detecting the electrogenic proton pump activity. This study represents an important innovation in plant biophysics as this class of fast responding voltage sensitive dyes have never to our knowledge been used to study electrogenic proton pump activity derived from plant membranes and represents a novel approach for carrying out such studies.     

Inhibition of the acetylcholine-induced relaxation of canine isolated basilar artery by potassium-conductance blockers

Canine basilar artery rings precontracted with 5-hydroxytryptamine (0.1-0.5 microM) relaxed in the presence of acetylcholine (25-100 microM), sodium nitroprusside (0.1 microM), or stimulation of the electrogenic sodium pump by restoration of extracellular K+ (4.5 mM) after K(+)-deprivation. Acetylcholine-induced relaxation is believed to be caused by the release of endothelium-derived relaxing factor (EDRF) and is prevented by mechanical removal of the endothelium, while relaxations induced by sodium nitroprusside or restarting of the sodium pump are endothelium-independent. Acetylcholine-induced relaxation was selectively blocked by pretreatment of the tissue with the nonselective K+ conductance inhibitors, 4-aminopyridine (4-AP, 3 mM), Ba2+ (1 mM), and tetraethylammonium (20 mM), 4-AP also blocked ACh-mediated relaxation in muscles contracted with elevated external K+. Relaxation of 5-hydroxytryptamine-induced contraction by sodium nitroprusside, or by addition of K+ to K(+)-deprived muscle, was not affected by 4-AP. Relaxation of basilar artery with acidified sodium nitrite solution (containing nitric oxide) was reduced by 4-AP. These results suggest that 4-AP and possibly Ba2+ inhibit acetylcholine-induced endothelium-dependent relaxation by inhibition of the action of EDRF on the smooth muscle rather than through inhibition of release of EDRF. The increase in K+ conductance involved in acetylcholine-induced relaxation is not due to ATP-inhibited K+ channels, as it is not blocked by glyburide (10(-6) M). Endothelium-derived relaxant factor(s) may relax smooth muscle by mode(s) of action different from that of sodium nitroprusside or by hyperpolarization due to the electrogenic sodium pumping.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kidney epithelial cells of monkey origin (BSC-1) express a sodium bicarbonate cotransport. Characterization by 22Na+ flux measurements

Na movement across the plasma membranes of confluent monolayers of monkey kidney epithelial cells (BSC-1) was studied using 22Na+ uptake and efflux techniques in the presence of 10(-4) M ouabain. In the presence of 28 mM bicarbonate, uptake was inhibited by both 10(-3) M amiloride and 10(-3) M 4,4'diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In DIDS-pretreated cells, 10(-3) M amiloride led to a further reduction of 22Na+ uptake, while 10(-5) furosemide was ineffective. DIDS also inhibited sodium efflux, indicating that the DIDS-sensitive pathway mediates both influx and efflux of 22Na+. DIDS-sensitive 22Na+ uptake, as studied in the presence of both 10(-4) M ouabain and 10(-3) M amiloride, was abolished by the absence of bicarbonate, which could not be substituted by other plasma membrane-permeable buffers. In 28 mM HCO3-, DIDS-sensitive uptake of 28 mM Na+ was cis-inhibited by 124 mM Na+, but no significant inhibition by K+ or Li+ was found. DIDS-sensitive 22Na+ uptake was a saturable function of both Na+ concentration (apparent Km between 20 and 40 mM at 28 mM HCO3-) and HCO3- concentration (apparent Km between 7 and 14 mM at 151 mM Na+). Intracellular microelectrode measurements showed that net Na+ transport in the presence of HCO3- is electrogenic, i.e. that there is anion cotransport with Na+. This effect is abolished by 1 mM DIDS. It is concluded that monkey kidney epithelial cells possess a stilbene-sensitive, electrogenic sodium bicarbonate symport, which may play an important role in bicarbonate reabsorption in the mammalian kidney.     

Inhibition of adenylate cyclase by arsenite and cadmium: evidence for a vicinal dithiol requirement.

The effect of sodium arsenite and cadmium chloride on adenylate cyclase activity was examined in turkey erythrocyte membranes. Sodium arsenite was a weak inhibitor of adenylate cyclase -7mM produced only 60% inhibition. Its effect, however, was greatly potentiated by equimolar 2,3 dimercaprol- wherein 0.7 mM sodium arsenite inhibited 100% with an apparent Ki of 0.1 mM. Equimolar mercaptoethanol was less effective in potentiating sodium arsenite inhibition. Thus 0.7mM sodium arsenite in the presence of equimolar mercaptoethanol inhibited adenylate cyclase 56%. Excess 2,3 dimercaprol reversed inhibition by sodium arsenite or cadmium chloride. Sodium arsenite or cadmium chloride inhibited all forms of adenylate cyclase activity tested, including nonhormonal stimulation. Equimolar sodium arsenite and dimercaprol, at concentrations that caused 100% inhibition of adenylate cyclase activity, reduced the binding of the beta-receptor specific ligand iodohydroxybenzylpindolol by less than 15%. These results suggest that turkey erythrocyte membranes contain closely juxtaposed thiol groups and that interaction of such groups with arsenate interferes with the catalytic function of adenulate cyclase.     

Analyzing an electrogenic cotransporter

A model for the sodium-dependent accumulation of glutamate by synaptosomes has been presented which fits the data of Wheeler and his coworkers and supports their hypothesis of an electrogenic cotransporter. Since their hypothesis was based on experimental data on the operation of the cotransporter on the outer membrane, the model was expanded to predict events when the cotransporter was operating on both sides of the membrane. The model predicts that the accumulation of glutamate is sensitive to the synaptosomal sodium and emphasizes the importance of the sodium/potassium pump to maintain this value. A model which uses only an electrogenic form of the cotransporter on the external membrane and a neutral form on the inside of the membrane predicts too much or too little accumulation of glutamate at different membrane potentials. A model which uses an electrogenic cotransporter on the external membrane and a concentration-dependent sodium glutamate leak would require a significant increase in the permeability of sodium glutamate when the membrane depolarizes. Only the operation of all four mentioned mechanisms will fit experimental data at two different external sodium concentrations and over the range of membrane potentials measured experimentally.     

The relationship between transepithelial sodium movement and potential difference in the larva of Camptochironomus tentans (Fabr.) and some observations on the accumulation of other ions.

In fourth instar larvae of Camptochironomus tentans, net sodium uptake from 2 mM-NaCl has an electrogenic component. During net uptake the transepithelial potential (TEP) alters from a value of approximately - 40 mV (sign refers to haemolymph), in depleted animals, to approximately o mV. The TEP in depleted larvae is dependent upon external sodium concentration above about I mM-Na+, becoming increasingly electropositive (haemolymph relative to medium) at high sodium concentrations. This effect is exaggerated in Na2SO4 compared with NaCl. At an external concentration of 2mM-NaCl, chloride is carried by an electroneutral mechanism, probably a closely coupled Cl-/anion exchange. However, it is possible that chloride transport could become somewhat electrogenic at higher concentrations. Lithium competes with sodium for the electrogenic pump. Observed TEPs differ greatly from those required to maintain passively the haemolymph concentrations of sodium and chloride.     

Sodium-dependent taurocholate uptake by isolated rat hepatocytes occurs through an electrogenic mechanism

The uptake mechanism for the bile salt, taurocholate, by the liver cell is coupled to sodium but the stoichiometry is controversial. A one-to-one coupling ratio would result in electroneutral transport, whereas cotransport of more than one sodium ion with each taurocholate molecule cause an electrogenic response. To better define the uptake of this bile salt, we measured the effect of taurocholate on the membrane potential and resistance of isolated rat hepatocytes using conventional microelectrode electrophysiology. The addition of 20 microM taurocholate caused transient but significant depolarization accompanied by a significant decrease in membrane resistance. The electrical effect induced by taurocholate mimicked that induced by L-alanine (10 mM), the uptake of which is known to occur through an electrogenic, sodium-coupled mechanism. The sodium dependence of taurocholate-induced depolarization was further confirmed by: (1) replacing Na+ with choline +, and (2) preincubating cells with ouabain (2 mM) or with the Na+-ionophore, gramicidin (25 micrograms/ml); both suppressed the electrogenic response. Further, cholic acid, which inhibits sodium-coupled taurocholate uptake in hepatocytes, inhibited taurocholate evoked depolarization. These results support the hypothesis that sodium-coupled taurocholate uptake by isolated hepatocytes occurs through an electrogenic process which transports more than one Na+ with each taurocholate molecule.     

Glutamine transport by rat basolateral membrane vesicles

Glutamine, a neutral amino acid, is unlike most amino acids, has two amine moieties which underlies its importance as a nitrogen transporter and a carrier of ammonia from the periphery to visceral organs. The gastrointestinal tract utilizes glutamine as a respiratory substrate. The intestinal tract receives glutamine from the luminal side and from the arterial side through the basolateral membranes of the enterocyte. This study characterizes the transport of glutamine by basolateral membrane vesicles of the rat. Basolateral membranes were prepared by a well validated technique of separation on a percoll density gradient. Membrane preparations were enriched with Na+/K+-ATPase and showed no 'overshoot' phenomena with glucose under sodium-gradient conditions. Glutamine uptake represented transport into the intravesicular space as evident by an osmolality study. Glutamine uptake was temperature sensitive and driven by an inwardly directed sodium gradient as evident by transient accumulation of glutamine above the equilibrium values. Kinetics of glutamine uptake under both sodium and potassium gradients at glutamine concentrations between 0.01 and 0.6 mM showed saturable processes with Vmax of 0.39 +/- 0.008 and 0.34 +/- 0.05 nmol/mg protein per 15 s for both sodium-dependent and sodium-independent processes, respectively. Km values were 0.2 +/- 0.01 and 0.55 +/- 0.01 mM, respectively. pH optimum for glutamine uptake was 7.5. Imposition of negative membrane potential by valinomycin and anion substitution studies enhanced the sodium-dependent uptake of glutamine suggesting an electrogenic process, whereas the sodium-independent uptake was not enhanced suggesting an electroneutral process. Other neutral amino acids inhibited the initial uptake of glutamine under both sodium-dependent and sodium-independent conditions. We conclude that glutamine uptake by basolateral membranes occurs by carrier-mediated sodium-dependent and sodium-independent processes. Both processes exhibit saturation kinetics and are inhibited by neutral amino acids. The sodium-dependent pathway is electrogenic whereas the sodium-independent pathway is electroneutral.     

Lactate rescues neuronal sodium homeostasis during impaired energy metabolism

Recently, we established that recurrent activity evokes network sodium oscillations in neurons and astrocytes in hippocampal tissue slices. Interestingly, metabolic integrity of astrocytes was essential for the neurons' capacity to maintain low sodium and to recover from sodium loads, indicating an intimate metabolic coupling between the 2 cell types. Here, we studied if lactate can support neuronal sodium homeostasis during impaired energy metabolism by analyzing whether glucose removal, pharmacological inhibition of glycolysis and/or addition of lactate affect cellular sodium regulation. Furthermore, we studied the effect of lactate on sodium regulation during recurrent network activity and upon inhibition of the glial Krebs cycle by sodium-fluoroacetate. Our results indicate that lactate is preferentially used by neurons. They demonstrate that lactate supports neuronal sodium homeostasis and rescues the effects of glial poisoning by sodium-fluoroacetate. Altogether, they are in line with the proposed transfer of lactate from astrocytes to neurons, the so-called astrocyte-neuron-lactate shuttle.     

Lactate rescues neuronal sodium homeostasis during impaired energy metabolism

Recently, we established that recurrent activity evokes network sodium oscillations in neurons and astrocytes in hippocampal tissue slices. Interestingly, metabolic integrity of astrocytes was essential for the neurons'' capacity to maintain low sodium and to recover from sodium loads, indicating an intimate metabolic coupling between the 2 cell types. Here, we studied if lactate can support neuronal sodium homeostasis during impaired energy metabolism by analyzing whether glucose removal, pharmacological inhibition of glycolysis and/or addition of lactate affect cellular sodium regulation. Furthermore, we studied the effect of lactate on sodium regulation during recurrent network activity and upon inhibition of the glial Krebs cycle by sodium-fluoroacetate. Our results indicate that lactate is preferentially used by neurons. They demonstrate that lactate supports neuronal sodium homeostasis and rescues the effects of glial poisoning by sodium-fluoroacetate. Altogether, they are in line with the proposed transfer of lactate from astrocytes to neurons, the so-called astrocyte-neuron-lactate shuttle.     

Molecular basis of an inherited epilepsy

Epilepsy is a common neurological condition that reflects neuronal hyperexcitability arising from largely unknown cellular and molecular mechanisms. In generalized epilepsy with febrile seizures plus, an autosomal dominant epilepsy syndrome, mutations in three genes coding for voltage-gated sodium channel alpha or beta1 subunits (SCN1A, SCN2A, SCN1B) and one GABA receptor subunit gene (GABRG2) have been identified. Here, we characterize the functional effects of three mutations in the human neuronal sodium channel alpha subunit SCN1A by heterologous expression with its known accessory subunits, beta1 and beta2, in cultured mammalian cells. SCN1A mutations alter channel inactivation, resulting in persistent inward sodium current. This gain-of-function abnormality will likely enhance excitability of neuronal membranes by causing prolonged membrane depolarization, a plausible underlying biophysical mechanism responsible for this inherited human epilepsy.     

The electrogenic sodium pump activity in Aplysia neurons is not potential dependent

We have investigated the potential dependence of the electrogenic sodium pump in Aplysia neurons by recording the potential and current induced by sudden change of the artificial sea water from one containing K+ at various concentrations to K+ -free sea water in the presence or absence of ouabain. Both K+ free sea water and ouabain block sodium transport and result in a significant depolarization due to removal of a maintained outward current that is a result of transport of more Na+ out of the cell than K+ into the cell during pump operation. In the presence of ouabain there is, however, an inward current induced by changing external K+ concentration from zero to some value between 1 and 20 mM, and this current is greater with a greater K+ concentration gradient. The current induced by change from zero to 1 mM K+ does not show any potential dependence, although those currents induced by higher K+ concentrations are potential dependent. We conclude that the activity of the electrogenic sodium pump is not potential dependent, but that the potential independence is obscured if higher concentrations of K+ are used to activate the electrogenic sodium pump.     

Interaction of sodium with the sarcolemmal calcium system.

Sarcolemma isolated from guinea pig heart binds calcium in an ATP-dependent manner. Sodium ions decrease the total amount of calcium bound by the membranes. ATP-dependent calcium binding is more sensitive to sodium than the non-ATP-dependent calcium binding. The ATPase active during calcium binding is affected by sodium ions to the same extent as the ATP-dependent calcium binding process. The inhibition of the calcium binding process and of ATPase activity by sodium was more pronounced when the membranes were preincubated with sodium. The effect of sodium on calcium binding is dependent on both the time of contact between sodium and the membranes and the concentration of sodium. It is suggested that the effect of sodium on the calcium binding system in the sarcolemma may be a link between the inhibition of Na+K+-ATPase (EC 3.6.1.3) by cardiac glycosides and the subsequent increase in intracellular calcium.