Cl Gradient Research Articles

Flow Characteristics around Rotating Circular Cylinder with Arc Grooves

The drag and lift coefficient characteristics of rotating cylinder with arc grooves is investigated. The circular cylinders with grooves have thirty-two arc grooves of different depth on the cylinder surface. Pressure of the cylinder surface is measured with the Reynolds number Re= (0.4-1.8) ×105, and rotation of 0-4500rpm.The drag and lift coefficients are calculated from pressure distribution. At Re=0.4×105, the drag coefficient CD of smooth cylinder is constant until the spin rate ratio α (α=rotating speed/uniform flow) =0.6, and CD decreases afterwards at 0.6<α<1.0. As Reynolds number increase, this decreasing part shift to low a side. In the case of cylinder with grooves, the decreasing part of CD exists only in Re= (0.4-0.8) ×105 (subcritical, critical region of stationary condition), and CD has monotonic increase at Re>1.0×105.As groove depth becomes deep, CD becomes constant value as α increase. The lift coefficient CL of smooth cylinder increases as a increase.However, CL decreases to the minimum value as α further increase and CL increases again with the increase of α, CL indicated negative lift in Re>1.0×105.CL of cylinder with grooves become monotonous increases tendency in Re>1.0×105.As groove depth becomes deep, the increasing gradient of CL becomes small.

Vasopressin‐stimulated CFTR Cl− currents are increased in the renal collecting duct cells of a mouse model of Liddle's syndrome

Liddle's syndrome is a genetic form of hypertension linked to Na(+) retention caused by activating mutations in the COOH terminus of the beta or gamma subunit of the epithelial sodium channel (ENaC). In this study, we used the short-circuit current (I(sc)) method to investigate the effects of deamino-8-d-arginine vasopressin (dDAVP) on Na(+) and Cl(-) fluxes in primary cultures of cortical collecting ducts (CCDs) microdissected from the kidneys of mice with Liddle's syndrome carrying a stop codon mutation, corresponding to the beta-ENaC R(566) stop mutation (L) found in the original pedigree. Compared to wild-type (+/+) CCD cells, untreated L/+ and L/L CCD cells exhibited 2.7- and 4.2-fold increases, respectively, in amiloride-sensitive (Ams) I(sc), reflecting ENaC-dependent Na(+) absorption. Short-term incubation with dDAVP caused a rapid and significant increase (approximately 2-fold) in Ams I(sc) in +/+, but not in L/+ or L/L CCD cells. In sharp contrast, dDAVP induced a greater increase in 5-nitro-2-(3-phenylpropamino)benzoate (NPPB)-inhibited apical Cl(-) currents in amiloride-treated L/L and L/+ cells than in their +/+ counterparts. I(sc) recordings performed under apical ion substituted conditions revealed that the dDAVP-stimulated apical secretion of Cl(-), which was absent in cultured CCDs lacking CFTR, was 1.8-fold greater in L/+ and 3.7-fold greater in L/L CCD cells than in their +/+ CCD counterparts. After the basal membrane had been permeabilized with nystatin and a basal-to-apical Cl(-) gradient had been imposed, dDAVP also stimulated larger Cl(-) currents across L/L and L/+ CCD layers than +/+ CCD layers. These findings demonstrate that vasopressin stimulates greater apical CFTR Cl(-) conductance in the renal CCD cells of mice with Liddle's syndrome than in wild-type mice. This effect could contribute to the enhanced NaCl reabsorption observed in the distal nephron of patients with Liddle's syndrome.

Bicarbonate secretion plays a role in chloride and water absorption of the European flounder intestine

Experiments performed on isolated intestinal segments from the marine teleost fish, the European flounder (Platichthys flesus), revealed that the intestinal epithelium is capable of secondary active HCO3(-) secretion in the order of 0.2-0.3 micromol x cm(-2) x h(-1) against apparent electrochemical gradient. The HCO3(-) secretion occurs via anion exchange, is dependent on mucosal Cl(-), results in very high mucosal HCO3(-) concentrations, and contributes significantly to Cl(-) and fluid absorption. This present study was conducted under in vivo-like conditions, with mucosal saline resembling intestinal fluids in vivo. These conditions result in a transepithelial potential of -16.2 mV (serosal side negative), which is very different from the -2.2 mV observed under symmetrical conditions. Under these conditions, we found a significant part of the HCO3(-) secretion is fueled by endogenous epithelial CO2 hydration mediated by carbonic anhydrase because acetazolamide (10(-4) M) was found to inhibit HCO3(-) secretion and removal of serosal CO(2) was found not to influence HCO3(-) secretion. Reversal of the epithelial electrochemical gradient for Cl(-) (removal of serosal Cl(-)) and elevation of serosal HCO3(-) resulted in enhanced HCO3(-) secretion and enhanced Cl(-) and fluid absorption. Cl(-) absorption via an anion exchange system appears to partly drive fluid absorption across the intestine in the absence of net Na(+) absorption.

A Creatine Transporter Is Operative at the Brush Border Level of the Rat Jejunal Enterocyte

Although ergogenic effects and health benefits have been reported for creatine used as nutritional supplement, to date little is known about the mechanism of creatine absorption in the small intestine. Thus the current study was undertaken to elucidate the mechanism of creatine intake in rat jejunum with the use of well-purified brush border membrane vesicles, isolated from jejunal enterocyte. Creatine uptake was found markedly stimulated by inwardly directed Na(+) and Cl(- )gradients, potential-sensitive, strongly reduced by the substitution of Na(+) and Cl(-) with various cations and anions and positively affected by intravesicular K(+). Moreover, creatine uptake is: 1) significantly inhibited by creatine structural analogs, 2) abolished by low concentrations of 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), 3) saturable as a function of creatine concentration with an apparent Michaelis-Menten constant of 24.08 +/- 0.80 muM and a maximal velocity of 391.30 +/- 6.19 pmoles mg protein(-1) 30 s(-1). The transport is electrogenic since at least two Na(+) and one Cl(-) are required to transport one creatine molecule. Western blot analysis showed the same amount of creatine transport protein in the jejunal apical membrane when compared to ileum. Thus, these data demonstrate the existence of a Na(+)- and Cl(-)-dependent, membrane potential-sensitive, electrogenic carrier-mediated mechanism for creatine absorption in rat jejunal apical membrane vesicles, which is biochemically and pharmacologically similar to those observed in other tissues. However, in other cell types the stimulatory effect of intravesicular K(+) was never detected.

Chloride transport in the renal proximal tubule.

The renal proximal tubule is responsible for most of the renal sodium, chloride, and bicarbonate reabsorption. Micropuncture studies and electrophysiological techniques have furnished the bulk of our knowledge about the physiology of this tubular segment. As a consequence of the leakiness of this epithelium, paracellular ionic transport--in particular that of Cl(-)--is of considerable importance in this first part of the nephron. It was long accepted that proximal Cl(-) reabsorption proceeds solely paracellularly, but it is now known that transcellular Cl(-) transport also exists. Cl(-) channels and Cl(-)-coupled transporters are involved in transcellular Cl(-) transport. In the apical membrane, Cl(-)/anion (formate, oxalate and bicarbonate) exchangers represent the first step in transcellular Cl(-) reabsorption. A basolateral Cl(-)/HCO(3)(-) exchanger, involved in HCO(3)(-) reclamation, participates in the rise of intracellular Cl(-) activity above its equilibrium value, and thus also contributes to the creation of an outwardly directed electrochemical Cl(-) gradient across the cell membranes. This driving force favours Cl(-) diffusion from the cell to the lumen and to the interstitium. In the basolateral membrane, the main mechanism for transcellular Cl(-) reabsorption is a Cl(-) conductance, but a Na(+)-driven Cl(-)/HCO(3)(-) exchanger may also participate in Cl(-) reabsorption.

Modulation of spontaneous Ca2+-activated Cl- currents in the rabbit corpus cavernosum by the nitric oxide-cGMP pathway.

The whole-cell perforated patch clamp technique was used to study membrane currents in isolated rabbit corpus cavernosum smooth muscle cells. Depolarization from -80 mV to the range -40 to -10 mV evoked a nifedipine-sensitive Ca(2+) current that was followed by a slower inward current that activated over several hundred milliseconds. The slow current reversed near the Cl(-) equilibrium potential (E(Cl)) and was reduced by anthracene-9-carboxylic acid (A9C; 1 mm) and niflumic acid (100 microm), suggesting that it was a Ca(2+)-activated Cl(-) current. When held constantly at -60 mV, over 70% of cells fired spontaneous transient inward currents (STICs), the amplitudes of which were reduced by A9C and niflumic acid. STICs reversed near E(Cl) in a symmetrical Cl(-) gradient and when [Cl(-)](o) was substituted with glutamate or I(-), the reversal potential shifted to more positive or more negative values, respectively, confirming that STICs were mediated by Cl(-) channels. STICS were also blocked by cyclopiazonic acid, 2-aminoethoxydiphenyl borate (2-APB) and 2-nitro-4-carboxyl-N,N-diphenylcarbamate (NCDC), suggesting that they depended on IP(3)-mediated Ca(2+)-release from the sarcoplasmic reticulum. Modulation by the NO-cGMP pathway was investigated by applying nitrosocysteine, 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1), and 8-bromo cGMP, all three of which abolished STIC activity. YC-1 also reduced noradrenaline-evoked inward currents, but had no effect on similar currents evoked by caffeine, suggesting that cGMP selectively inhibited IP(3)-mediated Ca(2+) release. We propose that Ca(2+)-activated Cl(-) currents underlie detumescent tone in the corpus cavernosum, and that modulation of this mechanism by the NO-cGMP pathway is important during penile erection.

円弧型溝付回転円柱まわりの流動特性(G05-1 物体周りの流れ,G05 流体工学部門一般講演)

The drag and lift coefficient characteristics of rotating cylinder with arc grooves is investigated. The circular cylinders with grooves have thirty-two arc grooves of different depth on the cylinder surface. Pressure of the cylinder surface is measured with the Reynolds number Re= (0.4-1.8) ×105, and rotation of 0-4500rpm.The drag and lift coefficients are calculated from pressure distribution. At Re=0.4×105, the drag coefficient CD of smooth cylinder is constant until the spin rate ratio α (α=rotating speed/uniform flow) =0.6, and CD decreases afterwards at 0.6 1.0×105.As groove depth becomes deep, CD becomes constant value as α increase. The lift coefficient CL of smooth cylinder increases as a increase.However, CL decreases to the minimum value as α further increase and CL increases again with the increase of α, CL indicated negative lift in Re>1.0×105.CL of cylinder with grooves become monotonous increases tendency in Re>1.0×105.As groove depth becomes deep, the increasing gradient of CL becomes small.

Expression and Function of Chloride Transporters during Development of Inhibitory Neurotransmission in the Auditory Brainstem

Glycine and GABA, the dominant inhibitory neurotransmitters in the CNS, assume a depolarizing role in early development, leading to increased cytoplasmic Ca2+ levels and action potentials. The effect is thought to be of some significance for maturation. The depolarization is caused by Cl- efflux, and chloride transporters contribute to the phenomenon by raising the intracellular Cl- concentration ([Cl-]i) above equilibrium, thereby generating an outward-directed electrochemical gradient for Cl-. In mature neurons, the [Cl-]i is reduced below equilibrium, thus rendering glycine activity hyperpolarizing. Here, we investigated the temporal expression of the K-Cl cotransporter KCC2 and the Na-K-Cl cotransporter NKCC1 in the lateral superior olive (LSO) of rats and mice. The two cation cotransporters normally extrude and accumulate Cl-, respectively. As evidenced by several methods, KCC2 mRNA was present in LSO neurons during both the depolarizing and hyperpolarizing periods. Western blots confirmed a constant level of KCC2 in the brainstem, and immunohistochemistry showed that the protein is diffusely distributed within neonatal LSO neurons, becoming integrated into the plasma membrane only with increasing age. The glycine reversal potential in KCC2 knock-out mice differed significantly from that determined in wild-type controls at postnatal day 12 (P12) but not at P3, demonstrating that KCC2 is not active in neonates, despite its early presence. NKCC1 mRNA was not detected during the depolarizing phase in the LSO, implying that this transporter does not contribute to the high [Cl-]i. Our results reveal major differences in the development of [Cl-]i regulation mechanisms seen in brainstem versus forebrain regions.

Facilitated diffusion of urate in avian brush-border membrane vesicles.

Membrane transport pathways mediating transcellular secretion of urate across the proximal tubule were investigated in brush-border membrane vesicles (BBMV) isolated from avian kidney. An inside-positive K diffusion potential induced a conductive uptake of urate to levels exceeding equilibrium. Protonophore-induced dissipation of membrane potential significantly reduced voltage-driven urate uptake. Conductive uptake of urate was inhibitor sensitive, substrate specific, and a saturable function of urate concentration. Urate uptake was trans-stimulated by urate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate. Urate uptake was unaffected by an outward alpha-ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in the presence and absence of an imposed inside-alkaline pH gradient or an outward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux across the brush border membrane of urate-secreting proximal tubule cells.

Amygdala kindling induces upregulation of mRNA for NKCC1, a Na(+), K(+)-2Cl(-) cotransporter, in the rat piriform cortex.

GABA, the main inhibitory neurotransmitter in the brain, elicits a hyperpolarizing response by activation of the GABA(A)-receptor/chloride-channel complex under conditions of normal Cl(-) homeostasis. Thus the pathogenesis of epilepsy could involve an impairment of GABA(A)-receptor-mediated inhibition due to a collapse of the Cl(-) gradient. We examined the expression patterns of Cl(-) transporters and a Cl(-) channel in a rat amygdala-kindling model. Activity-dependent increases were observed in the mRNA for NKCC1, an inwardly-directed Cl(-) transporter, in the piriform cortex. This suggests that an increase in [Cl(-)](i) and a resultant reduction in GABAergic inhibition may occur in the kindled piriform cortex.

Platelet-activating factor regulates chloride transport in colonic epithelial cell monolayers.

Platelet-activating factor (PAF) has been implicated in the pathogenesis of gastrointestinal diseases such as necrotizing enterocolitis, Crohn's disease, and ulcerative colitis. However, neither the physiologic role of PAF in the intestine, nor the mechanisms by which PAF participates in the pathogenesis of disease are well understood. The aim of the present study was to determine the direct effect of PAF on intestinal epithelial cell ion transport, and to delineate the mechanisms of regulation. Ion transport was evaluated by measuring short circuit current (I(sc)) in HT29-CL19A cell monolayers using Ussing chambers. PAF receptor polarity was assessed using domain-selective biotinylation followed by immunoprecipitation and streptavidin blotting of intact epithelial monolayers. PAF (1-200 microM) stimulated I(sc) that followed the direction of a Cl(-) gradient and was specifically inhibited by the Cl(-) channel blockers glybenclamide, 2,2' iminodibenzoic acid and 4,4' diisothiocyanostilbene-2, 2' disulfonic acid, but was unaffected by the inhibition of prostaglandin synthesis with indomethacin. Stimulated I(sc) was only detected after apical addition of PAF, correlating with the results of biotinylation experiments indicating an exclusive apical polarity of the PAF receptor. PAF receptor antagonists CV6209 and octylonium bromide abolished PAF-stimulated I(sc). Thus, mucosal acting PAF directly and specifically stimulates ion transport via activation of an apical Cl(-) channel in intestinal epithelial cell monolayers independent of prostaglandin biosynthesis.

Ion channel formation from a calix[4]arene amide that binds HCl.

The ion transport activity of calix[4]arene tetrabutylamide 1,3-alt 2 was studied in liposomes, planar lipid bilayers, and HEK-293 cells. These experiments, when considered together with (1)H NMR and X-ray crystallography data, indicate that calix[4]arene tetrabutylamide 2 (1) forms ion channels in bilayer membranes, (2) mediates ion transport across cell membranes at positive holding potential, (3) alters the pH inside liposomes experiencing a Cl(-) gradient, and (4) shows a significant Cl(-)/SO(4)(2)(-) transport selectivity. An analogue, calix[4]arene tetramethylamide 1, self-assembles in the presence of HCl to generate solid-state structures with chloride-filled and water-filled channels. Structureminus signactivity studies indicate that the hydrophobicity, amide substitution, and macrocyclic framework of the calixarene are essential for HCl binding and transport. Calix[4]arene tetrabutylamide 2 is a rare example of an anion-dependent, synthetic ion channel.

Partial correction of defective Cl(-) secretion in cystic fibrosis epithelial cells by an analog of squalamine.

Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl(-) transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl(-) transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl(-) gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ~30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl(-) currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl(-) solution, indicating restoration of Cl(-) secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl(-) secretion.

Mixed excitatory and inhibitory GABA-mediated transmission in chick cochlear nucleus.

1. Neurons of the chick nucleus magnocellularis (NM) receive depolarizing GABAergic input from the superior olivary nucleus (SON). We examined the response to exogenous GABA or to stimulation of GABAergic fibres in order to identify the ionic basis of GABAergic synaptic transmission and its physiological implications. 2. Reversal potentials of GABA responses (E(GABA)) were determined exclusively by the Cl(-) gradient, measured using whole-cell recording. With gramicidin-perforated patch recording, E(GABA) was -25 +/- 5 mV (mean +/- S.D.), and was stable between embryonic day 17 and post-hatch day 10. With normal intracellular Cl(-), GABA depolarized neurons by 12 mV. 3. In current clamp, repetitive activation of the GABAergic axons reduced the probability of spiking in response to simultaneous stimulation of excitatory axons. However, IPSPs could themselves elicit action potentials, and facilitation of IPSPs by repetitive activation could lead to a characteristic pattern of spiking. 4. These data indicate that IPSPs with reversal potentials positive to spike threshold may have dual functions, depending on the context of their activation.

Properties of the Na+-H+ exchanger in renal microvillus membrane vesicles. 1980.

Am. J. Physiol. 238 (Renal Fluid Electrolyte Physiol. 7): F461-F469, 1980.-Transport of Na + and H + was evaluated in brush border membrane vesicles isolated from the rabbit renal cortex. Na + transport was assayed by a rapid filtration technique; H + transport was monitored with 5,5-dimethoxazolidine-2,4-dione by flow dialysis. Uphill Na + uptake was induced by imposition of an in > out H + gradient, and uphill H + efflux by imposition of an out > in Na + gradient, consistent with the action of a Na + -H + exchanger. The uptake of Na + was electroneutral either in the presence or absence of a H + gradient, indicating a fixed 1:1 stoichiometry for the exchange process. Na + transport was saturable and inhibited by Li + and NH 4 + but not by K + , Rb + , Cs + , or choline. Uphill H + efflux was induced by imposition of an out > in Li + gradient. Neither the uptake of Na + nor H + efflux were influenced by out > in gradients of Cl - compared to gradients of SCN - or SO 4 2- . If transport systems mediating Na + -Cl - co-transport and/or Cl - -OH - exchange are present in the microvillus membrane, their respective rates must be slow compared to the rate of Na + -H + exchange. Transport of Na + was inhibited by harmaline and amiloride, but not by acetazolamide, furosemide, or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate. We conclude that isolated renal microvillus membranes contain a tightly coupled Na + -H + exchanger that may play an important role in proximal tubular acidification.

Immunolocalization of the Na+–K+–2Cl− cotransporter in peripheral nervous tissue of vertebrates

Efflux of Cl(-) through GABA(A)-gated anion channels depolarizes the cell bodies and intraspinal terminals of sensory neurons, and contributes to the generation of presynaptic inhibition in the spinal cord. Active accumulation of Cl(-) inside sensory neurons occurs through an Na(+)-K(+)-2Cl(-) cotransport system that generates and maintains the electrochemical gradient for this outward Cl(-) current. We studied the immunolocalization of the Na(+)-K(+)-2Cl(-) cotransporter protein using a monoclonal antibody (T4) against a conserved epitope in the C-terminus of the molecule. Western blots of frog, rat and cat dorsal root ganglion membranes revealed a single band of cotransporter immunoreactivity at approximately 160kDa, consistent with the molecular mass of the glycosylated protein. Deglycosylation with N-glycosidase F reduced the molecular mass to approximately 135kDa, in agreement with the size of the core polypeptide. Indirect immunofluorescence revealed strong cotransporter immunoreactivity in all types of dorsal root ganglion cell bodies in frog, rat and cat. The subcellular distribution of cotransporter immunoreactivity was different amongst species. Membrane labeling was more apparent in frog and rat dorsal root ganglion cell bodies than in cat. In contrast, cytoplasmic labeling was intense in cat and weak in frog, being intermediate in the rat. Cotransporter immunoreactivity also occurred in satellite cells, particularly in rat and cat dorsal root ganglia. The membrane region and axoplasm of sensory fibers were heavily labeled in cat and rat and less in frog. Three-dimensional reconstruction of confocal optical sections and dual immunolocalization with S-100 protein showed that the cotransporter immunoreactivity was prominently expressed in the nodal and paranodal regions of the Schwann cells. Ultrastructural immunolocalization confirmed the presence of immunoreactivity on the membranes of the axon and the Schwann cell in both the nodal region and the paranode. Treatment with sodium dodecylsulfate and beta-mercaptoethanol also uncovered intense cotransporter immunoreactivity in Schmidt-Lanterman incisures at the light microscopic level. The localization of the Na(+)-K(+)-2Cl(-) cotransporter protein is consistent with its function as a Cl(-)-accumulating mechanism in sensory neurons. Its distinctive presence in Schwann cells suggests that it could also be involved in K(+) uptake from the extracellular space, particularly in the paranodal region of myelinated axons, thereby regulating the extracellular ionic environment and the excitability of axons.

Gamma-Aminobutyric acid(A) neurotransmission and cerebral ischemia.

In this review, we present evidence for the role of gamma-aminobutyric acid (GABA) neurotransmission in cerebral ischemia-induced neuronal death. While glutamate neurotransmission has received widespread attention in this area of study, relatively few investigators have focused on the ischemia-induced alterations in inhibitory neurotransmission. We present a review of the effects of cerebral ischemia on pre and postsynaptic targets within the GABAergic synapse. Both in vitro and in vivo models of ischemia have been used to measure changes in GABA synthesis, release, reuptake, GABA(A) receptor expression and activity. Cellular events generated by ischemia that have been shown to alter GABA neurotransmission include changes in the Cl(-) gradient, reduction in ATP, increase in intracellular Ca(2+), generation of reactive oxygen species, and accumulation of arachidonic acid and eicosanoids. Neuroprotective strategies to increase GABA neurotransmission target both sides of the synapse as well, by preventing GABA reuptake and metabolism and increasing GABA(A) receptor activity with agonists and allosteric modulators. Some of these strategies are quite efficacious in animal models of cerebral ischemia, with sedation as the only unwanted side-effect. Based on promising animal data, clinical trials with GABAergic drugs are in progress for specific types of stroke. This review attempts to provide an understanding of the mechanisms by which GABA neurotransmission is sensitive to cerebral ischemia. Furthermore, we discuss how dysfunction of GABA neurotransmission may contribute to neuronal death and how neuronal death can be prevented by GABAergic drugs.

Na+- and Cl--coupled active transport of carnitine by the amino acid transporter ATB(0,+) from mouse colon expressed in HRPE cells and Xenopus oocytes.

1. ATB(0,+) is an amino acid transporter energized by transmembrane gradients of Na+ and Cl(-) and membrane potential. We cloned this transporter from mouse colon and expressed the clone functionally in mammalian (human retinal pigment epithelial, HRPE) cells and Xenopus laevis oocytes to investigate the interaction of carnitine and its acyl esters with the transporter. 2. When expressed in mammalian cells, the cloned ATB(0,+) was able to transport carnitine, propionylcarnitine and acetylcarnitine. The transport process was Na(+) and Cl(-) dependent and inhibitable by the amino acid substrates of the transporter. The Michaelis constant for carnitine was 0.83 +/- 0.08 mM and the Hill coefficient for Na(+) activation was 1.6 +/- 0.1. 3. When expressed in Xenopus laevis oocytes, the cloned ATB(0,+) was able to induce inward currents in the presence of carnitine and propionylcarnitine under voltage-clamped conditions. There was no detectable current in the presence of acetylcarnitine. Carnitine-induced currents were obligatorily dependent on the presence of Na(+) and Cl(-). The currents were saturable with carnitine and the Michaelis constant was 1.8 +/- 0.4 mM. The analysis of Na(+)- and Cl(-)-activation kinetics revealed that 2 Na(+) and 1 Cl(-) were involved in the transport of carnitine via the transporter. 4. These studies describe the identification of a novel function for the amino acid transporter ATB(0,+). Since this transporter is expressed in the intestinal tract, lung and mammary gland, it is likely to play a significant role in the handling of carnitine in these tissues. 5. A Na(+)-dependent transport system for carnitine has already been described. This transporter, known as OCTN2 (novel organic cation transporter 2), is expressed in most tissues and transports carnitine with high affinity. It is energized, however, only by a Na(+) gradient and membrane potential. In contrast, ATB(0,+) is a low-affinity transporter for carnitine, but exhibits much higher concentrative capacity than OCTN2 because of its energization by transmembrane gradients of Na(+) and Cl(-) as well as by membrane potential.

Chloride channel function is linked to epithelium-dependent airway relaxation.

We previously reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of mouse tracheal smooth muscle. Since both SP and ATP are known to evoke transepithelial Cl- secretion across epithelial monolayers, we tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on Cl- channel function. In perfused mouse tracheas, the responses to SP and ATP were both inhibited by the Cl- channel inhibitors diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was unaffected by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and relaxation to SP was unaffected by either DIDS or DNDS. Replacing Cl- in the buffer solutions with the impermeable anion gluconate on both sides of the trachea inhibited relaxation to SP or ATP. In contrast, increasing the gradient for Cl- secretion using Cl- free medium only in the tracheal lumen enhanced the relaxation to SP or ATP. We conclude that Cl- channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS suggested the involvement of a DIDS-insensitive Cl- channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. To test this hypothesis, we evaluated tracheas from CFTR-deficient mice and found that the peak relaxation to SP or ATP was not significantly different from those responses in wild-type littermates. This suggests that a DIDS-insensitive Cl- channel other than CFTR is active in the SP response. This work introduces a possible role for Cl- pathways in the modulation of airway smooth muscle function and may have implications for fundamental studies of airway function as well as therapeutic approaches to pulmonary disease.

A patch-clamp study of ion channels in protoplasts prepared from the marine alga Valonia utricularis.

The giant marine alga Valonia utricularis is a classical model system for studying the electrophysiology and water relations of plant cells by using microelectrode and pressure probe techniques. The recent finding that protoplasts can be prepared from the giant "mother cells" (Wang, J., Sukhorukov, V.L., Djuzenova, C.S., Zimmermann, U., Müller, T., Fuhr, G., 1997, Protoplasma 196:123-134) allowed the use of the patch-clamp technique to examine ion channel activity in the plasmalemma of this species. Outside-out and cell-attached experiments displayed three different types of voltage-gated Cl(-) channels (VAC1, VAC2, VAC3, Valonia Anion Channel 1,2,3), one voltage-gated K(+) channel (VKC1, Valonia K(+) Channel 1) as well as stretch-activated channels. In symmetrical 150 mm Cl(-) media, VAC1 was most frequently observed and had a single channel conductance of 36 +/- 7 pS (n = 4) in the outside-out and 33 +/- 5 pS (n = 10) in the cell-attached configuration. The reversal potential of the corresponding current-voltage curves was within 0 +/- 4 mV (n = 4, outside-out) and 9 +/- 7 mV (n = 10, cell-attached) close to the Nernst potential of Cl(-) and shifted towards more negative values when cell-attached experiments were performed in asymmetrical 50:150 mm Cl(-) media (bath/pipette; E(Cl(-)) -20 +/- 7 mV (n = 4); Nernst potential -28 mV). Consistent with a selectivity for Cl(-), VAC1 was inhibited by 100 micronM DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid). VAC1 was activated by a hyperpolarization of the patch. Boltzmann fits of the channel activity under symmetrical 150 mm Cl(-) conditions yielded a midpoint potential of -12 +/- 5 mV (n = 4, outside-out) and -3 +/- 6 mV (n = 9, cell-attached) and corresponding apparent minimum gating charges of 15 +/- 3 (n = 4) and 18 +/- 5 (n = 9). The midpoint potential shifted to more negative values in the presence of a Cl(-) gradient. VAC2 was activated by voltages more negative than E(Cl(-)) and was always observed together with VAC1, but less frequently. It showed a "flickering" gating. The single channel conductance was 99 +/- 10 pS (n = 6). VAC3 was activated by membrane depolarization and frequently exhibited several subconductance states. The single channel conductance of the main conductance state was 36 +/- 5 pS (n = 5). VKC1 was also activated by positive clamped voltages. Up to three conductance states occurred whereby the main conductance state had a single channel conductance of 124 +/- 27 pS (n = 6). In the light of the above results it seems to be likely that VAC1 contributes mainly to the Cl(-) conductance of the plasmalemma of the turgescent "mother cells" and that this channel (as well as VAC2) can operate in the physiological membrane potential range. The physiological significance of VAC3 and VKC1 is unknown, but may be related (as the stretch-activated channels) to processes involved in turgor regulation.