Chloride channel blockers promote relaxation of TEA-induced contraction in airway smooth muscle.

Enhanced airway smooth muscle (ASM) contraction is an important component in the pathophysiology of asthma. We have shown that ligand gated chloride channels modulate ASM contractile tone during the maintenance phase of an induced contraction, however the role of chloride flux in depolarization-induced contraction remains incompletely understood. To better understand the role of chloride flux under these conditions, muscle force (human ASM, guinea pig ASM), peripheral small airway luminal area (rat ASM) and airway smooth muscle plasma membrane electrical potentials (human cultured ASM) were measured. We found ex vivo guinea pig airway rings, human ASM strips and small peripheral airways in rat lungs slices relaxed in response to niflumic acid following depolarization-induced contraction induced by K(+) channel blockade with tetraethylammonium chloride (TEA). In isolated human airway smooth muscle cells TEA induce depolarization as measured by a fluorescent indicator or whole cell patch clamp and this depolarization was reversed by niflumic acid. These findings demonstrate that ASM depolarization induced contraction is dependent on chloride channel activity. Targeting of chloride channels may be a novel approach to relax hypercontractile airway smooth muscle in bronchoconstrictive disorders.

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule.

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.

Distinct transport activity of tetraethylammonium from L-carnitine in rat renal brush-border membranes.

We investigated the contribution of the Na(+)/L-carnitine cotransporter in the transport of tetraethylammonium (TEA) by rat renal brush-border membrane vesicles. The transient uphill transport of L-carnitine was observed in the presence of a Na(+) gradient. The uptake of L-carnitine was of high affinity (K(m)=21 microM) and pH dependent. Various compounds such as TEA, cephaloridine, and p-chloromercuribenzene sulfonate (PCMBS) had potent inhibitory effects for L-carnitine uptake. Therefore, we confirmed the Na(+)/L-carnitine cotransport activity in rat renal brush-border membranes. Levofloxacin and PCMBS showed different inhibitory effects for TEA and L-carnitine uptake. The presence of an outward H(+) gradient induced a marked stimulation of TEA uptake, whereas it induced no stimulation of L-carnitine uptake. Furthermore, unlabeled TEA preloaded in the vesicles markedly enhanced [14C]TEA uptake, but unlabeled L-carnitine did not stimulate [14C]TEA uptake. These results suggest that transport of TEA across brush-border membranes is independent of the Na(+)/L-carnitine cotransport activity, and organic cation secretion across brush-border membranes is predominantly mediated by the H(+)/organic cation antiporter.

Tetraethylammonium and amantadine identify distinct organic cation transporters in rat renal cortical proximal and distal tubules.

Tetraethylammonium (TEA) and amantadine are two organic cations that are secreted by the kidney. It appears that each cation may characterize distinct renal tubule organic cation transport pathways. To test this hypothesis, we investigated the renal proximal and distal tubule energy-dependent transport properties of TEA and amantadine. Isolated tubules were incubated at 25 degrees C in bicarbonate buffer (Krebs-Henseleit solution) and nonbicarbonate buffer (Cross-Taggart) with varying concentrations of [14C]TEA or [3H]amantadine to determine initial rates of energy-dependent uptake of TEA and amantadine, respectively. The uptake of TEA could best be described by two transport sites, a high-affinity site and a lower affinity site. TEA uptake was not influenced by the presence of bicarbonate. Consistent with our previously reported data, amantadine uptake could also be described by two transport sites, a high-affinity-capacity site that is bicarbonate-dependent and a lower-affinity-capacity transport site that is bicarbonate-independent. The renal tubule uptake of amantadine into proximal and distal tubules, in Krebs-Henseleit solution or Cross-Taggart buffers, was not inhibited by 10 to 1000 microM of TEA. However, tubule accumulation of TEA could be inhibited (>90%) by amantadine in proximal and distal tubules in Krebs-Henseleit solution and Cross-Taggart buffers. In proximal tubules, N1-methylnicotinamide was not able to inhibit amantadine uptake but it reduced TEA uptake by 60 to 70% at similar concentrations. These data support the existence of multiple renal tubule organic cation transporters that have different substrate affinity and controlling mechanisms. It is also apparent that amantadine characterizes organic cation transporters that are distinct from those characterized by TEA.

Cloning and functional expression of a mouse liver organic cation transporter.

Hepatic uptake of organic cations is essential for the metabolism and secretion of numerous endobiotics and drugs. Several hepatic organic cation transporters have been kinetically defined, yet have not been isolated or cloned. We have isolated a complementary DNA (cDNA) from both murine liver and kidney cDNA libraries (mOct1/Slc22a1), and have functionally expressed it in Xenopus laevis oocytes. Although mOct1/Slc22a1 is homologous to previously cloned rat and human organic cation transporters, organic cation transport kinetics differed markedly. mOct1/Slc22a1-RNA injection of oocytes resulted in the saturable, time- and temperature-dependent uptake of the quaternary organic cation [14C]-tetraethylammonium ([14C]-TEA), with a Km of 38 micromol/L. TEA uptake was inhibited by several other organic cation drugs, but was not inhibited by the organic cation n-methyl-nicotinamide (NMN), being instead stimulated by it (fourfold). [14C]-TEA uptake was also stimulated by an inside-outside proton gradient. mOct1/Slc22a1-injected oocytes transported the organic cations [3H]-1-methyl-4-phenylpyridium and [3H]-choline chloride, but did not transport other classes of organic compounds. mOct1/Slc22a1 encodes for a hepatic and renal organic cation transporter which may be important for the uptake and secretion of cationic drugs and endobiotics.

A nucleoside-sensitive organic cation transporter in opossum kidney cells.

Renal secretion of organic cations and anions are pleiotropic, active processes in mammals. Some nucleosides such as deoxyadenosine (dAdo), 2-chlorodeoxyadenosine, and azidothymidine are secreted by human and rodent kidneys. Previous work (J. A. Nelson, J. F. Kuttesch, Jr., and B. H. Herbert. Biochemical Pharmacology 32: 2323-2327, 1983) indicated a role for the classic organic cation transporter (OCT) in the secretion of the dAdo analog, 2'-deoxytubercidin, by mouse kidney. Using [14C]tetraethylammonium bromide ([14C]TEA) as a substrate, we tested several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a cimetidine-sensitive and metabolic-dependent ability to efflux TEA. Other classic OCT inhibitors and several nucleosides also inhibit TEA efflux by these cells in a manner reflecting structural specificity for the carrier. Inhibition of OCT by nucleosides is not a universal feature of OCTs, since TEA transport mediated by cloned rat kidney OCT2 in the Xenopus laevis oocyte system was not inhibited by the same nucleosides. In conclusion, OK cells appear to possess an OCT that may also transport some nucleosides by a novel carrier.