Inhibition Of Cation Transport Research Articles

Involvement of the Pyrilamine Transporter, a Putative Organic Cation Transporter, in Blood-Brain Barrier Transport of Oxycodone

The purpose of this study was to characterize blood-brain barrier (BBB) transport of oxycodone, a cationic opioid agonist, via the pyrilamine transporter, a putative organic cation transporter, using conditionally immortalized rat brain capillary endothelial cells (TR-BBB13). Oxycodone and [3H]pyrilamine were both transported into TR-BBB13 cells in a temperature- and concentration-dependent manner with Km values of 89 and 28 microM, respectively. The initial uptake of oxycodone was significantly enhanced by preloading with pyrilamine and vice versa. Furthermore, mutual uptake inhibition by oxycodone and pyrilamine suggests that a common mechanism is involved in their transport. Transport of both substrates was inhibited by type II cations (quinidine, verapamil, and amantadine), but not by classic organic cation transporter (OCT) substrates and/or inhibitors (tetraethylammonium, 1-methyl-4-phenylpyridinium, and corticosterone), substrates of OCTN1 (ergothioneine) and OCTN2 (L-carnitine), or organic anions. The transport was inhibited by metabolic inhibitors (rotenone and sodium azide) but was insensitive to extracellular sodium and membrane potential for both substrates. Furthermore, the transport of both substrates was increased at alkaline extracellular pH and decreased in the presence of a protonophore (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone). Intracellular acidification induced with ammonium chloride enhanced the uptakes, suggesting that the transport is driven by an oppositely directed proton gradient. The brain uptake of oxycodone measured by in situ rat brain perfusion was increased in alkaline perfusate and was significantly inhibited by pyrilamine. These results suggest that blood-brain barrier transport of oxycodone is at least partly mediated by a common transporter with pyrilamine, and this transporter is an energy-dependent, proton-coupled antiporter.

Renal accumulation of [111In]DOTATOC in rats: influence of inhibitors of the organic ion transport and diuretics

Radiation exposure to the kidney limits therapy with radiometal labelled DOTATOC. This study evaluates the organic anion and cation transport (inhibitors: probenecid and cimetidine/dexamethasone) as well as diuresis (furosemide and mannitol) regarding renal uptake of [(111)In]DOTATOC. One hundred eight male Fisher rats were injected with [(111)In]DOTATOC via the tail vein. Prior to activity injection a total of 84 rats underwent injection with probenecid vs. sodium chloride 0.9% (48 rats), cimetidine vs. dexamethasone vs. sodium chloride 0.9% (18 rats), and furosemide vs. mannitol vs. sodium chloride 0.9% (18 rats). Rats were sacrificed at predetermined time points up to 48 h after activity injection. Kidneys, adrenal glands, pancreas, spleen, blood, liver, and muscle were harvested and injected activity per gram tissue was determined. Autoradiographic images of the kidneys were acquired in a total of 24 rats. Probenecid led to a reduction in renal uptake by up to 30% while not significantly changing the activity accumulation in the other organs investigated. This reduction was attributable to the renal cortex (ratio cortex/medulla 1.72 vs. 1.99; p = 0.006). Cimetidine and dexamethasone had no effect in any of the organs. Furosemide led to a 44% increase in renal activity accumulation attributable to enhanced renal medullary uptake (ratio cortex/medulla 1.44 versus 1.69; p = 0.006). Mannitol had no effect on renal activity uptake. Inhibition of the organic anion transport by probenecid may help reduce renal uptake regarding therapy with radiometal labelled DOTATOC. The enhancing effect of furosemide may be unfavourable for therapy. The results must be confirmed by human studies.

Inhibition of carrier‐mediated uptake of epirubicin reduces cytotoxicity in primary culture of rat hepatocytes

Epirubicin, an antineoplastic drug, is considered to be taken up by tumor cells via a common carrier by facilitated diffusion and is then pumped out in an energy-dependent manner because epirubicin is a substrate for P-glycoprotein (P-gp). However, this study investigated the details of the influx mechanism of epirubicin and demonstrated that epirubicin uptake was mediated by active carrier systems in addition to facilitated diffusion in the primary culture of rat hepatocytes. The uptake of epirubicin gradually increased in a saturated manner when the concentrations were between 1 x 10(-7) M and 1 x 10(-6) M. In contrast, the uptake increased progressively in a linear manner when the concentration was high (greater than 1 x 10(-6) M). The uptake of epirubicin at a clinical concentration (7.5 x 10(-7) M) was significantly reduced at 4 degrees C and significantly inhibited when pretreated with metabolic inhibitors (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone and sodium azide) by nearly 25%. Furthermore, an organic anion transporter inhibitor, namely, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS); organic anion transport substrates, namely, para-aminohippurate (PAH), taurocholic acid and estradiol 17-beta-D-glucuronide; and organic cation transporter inhibitors, namely, verapamil and tetraethylammonium significantly reduced the uptake of epirubicin. Furthermore, pretreatment with verapamil and PAH significantly prevented epirubicin-induced reduction of proliferative activity in rat hepatocytes. These results indicated that the uptake of epirubicin was induced, at least in part, by the active transport protein in rat hepatocytes; the inhibition of the probable transport protein protected the intact normal cells from the injury induced by the cytotoxicity of epirubicin.

Osmotic swelling-provoked release of organic osmolytes in human intestinal epithelial cells.

Human Intestine 407 cells respond to osmotic cell swelling by the activation of Cl(-)- and K(+)-selective ionic channels, as well as by stimulating an organic osmolyte release pathway readily permeable to taurine and phosphocholine. Unlike the activation of volume-regulated anion channels (VRAC), activation of the organic osmolyte release pathway shows a lag time of approximately 30-60 s, and its activity persists for at least 8-12 min. In contrast to VRAC activation, stimulation of organic osmolyte release did not require protein tyrosine phosphorylation, active p21(rho), or phosphatidylinositol 3-kinase activity and was insensitive to Cl(-) channel blockers. Treatment of the cells with putative organic anion transporter inhibitors reduced the release of taurine only partially or was found to be ineffective. The efflux was blocked by a subclass of organic cation transporter (OCT) inhibitors (cyanine-863 and decynium-22) but not by other OCT inhibitors (cimetidine, quinine, and verapamil). Brief treatment of the cells with phorbol esters potentiated the cell swelling-induced taurine efflux, whereas addition of the protein kinase C (PKC) inhibitor GF109203X largely inhibited the response, suggesting that PKC is involved. Increasing the level of intracellular Ca(2+) by using A-23187- or Ca(2+)-mobilizing hormones, however, did not affect the magnitude of the response. Taken together, the results indicate that the hypotonicity-induced efflux of organic osmolytes is independent of VRAC and involves a PKC-dependent step.

Effects of organic anion, organic cation, and dipeptide transport inhibitors on cefdinir in the isolated perfused rat kidney.

Cefdinir (Omnicef; Abbott Laboratories) is a cephalosporin antibiotic primarily eliminated by the kidney. Nonlinear renal elimination of cefdinir has been previously reported. Cefdinir renal transport mechanisms were studied in the erythrocyte-free isolated perfused rat kidney. Studies were performed with drug-free perfusate and perfusate containing cefdinir alone to establish the baseline physiology and investigate cefdinir renal elimination characteristics. To investigate cefdinir renal transport mechanisms, inhibition studies were conducted by coperfusing cefdinir with inhibitors of the renal organic anion (probenecid), organic cation (tetraethylammonium), or dipeptide (glycylsarcosine) transport system. Cefdinir concentrations in biological samples were determined using reversed-phase high-performance liquid chromatography. Differences between treatments and controls were evaluated using analysis of variance and Dunnett's test. The excretion ratio (ER; the renal clearance corrected for the fraction unbound and glomerular filtration rate) for cefdinir was 5.94, a value indicating net renal tubular secretion. Anionic, cationic, and dipeptide transport inhibitors all significantly affected the cefdinir ER. With probenecid, the ER was reduced to 0.59, clearly demonstrating a significant reabsorptive component to cefdinir renal disposition. This finding was confirmed by glycylsarcosine studies, in which the ER was elevated to 7.95, indicating that reabsorption was mediated, at least in part, by the dipeptide transporter system. The effects of the organic cation tetraethylammonium, in which the ER was elevated to 7.53, were likely secondary in nature. The anionic secretory pathway was found to be the predominant mechanism for cefdinir renal excretion.

Effects of anion and cation inhibitors and carbonic anhydrase inhibitors upon the activity of the gypsy moth (Lepidoptera: Lymantriidae) nucleo-polyhedrovirus.

Twenty chemicals that were reported to act as anion transport inhibitors, cation transport inhibitors, and inhibitors of carbonic anhydrase activity were tested at a 1% concentration (wt:wt) for their effects upon the biological activity of the gypsy moth, Lymantria dispar (L.), nucleopolyhedrovirus (LdMNPV). Among the five anion transport inhibitors tested, flufenamic acid acted as a viral enhancer. None of the seven inhibitors of K+ enhanced viral activity and three (4-aminopyridine, diacetyl, and procaine) significantly reduced the activity of LdMNPV. All four Na+ transport inhibitors (abietic acid, amiloride, dicyclohexylcarbodiimide, triamterene) acted as viral enhancers. Triamterene was the most active enhancer, as the LC50 was reduced by approximately 1,750-fold. Five carbonic anhydrase inhibitors were tested and four (acetazolamide, hydrochlorothiazide, methazolamide, sulfanilamide) enhanced the activity of LdMNPV. Acetazolamide (a carbonic anhydrase inhibitor), amiloride (a Na+ transport inhibitor), and flufenamic acid (an anion transport inhibitor) were tested singly and in different combinations. Every combination tested (acetazolamide/amiloride, acetazolamide/flufenamic acid, amiloride/flufenamic acid, acetazolamide/amiloride/flufenamic acid) significantly decreased the LC50 from 7.79 PIB/mm2 to a value as low as 0.008 PIB/mm2 (amiloride/flufenamic acid).

A transfected cell model for the renal toxin transporter, rOCT2.

A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.

Effects of ion transport inhibitors on D 2O induced action potential in Characeae

Substitution of the solvent H 2O, with D 2O, induced excitation in the Chara cell. The phenomenon, `D 2O-induced action potential' (DAP), was characterised by abrupt membrane depolarisation with incomplete repolarisation. Electrophysiological studies on the subcellular level (perfused plasmalemma and reconstituted channels in BLM) revealed a mechanism of channel activation similar to the electrically evoked action potential. In the present study the origin of DAP was analysed pharmacologically on intact internodal cells and thus the previous findings obtained on subcellular systems could be checked. The first pre-depolarisation DAP phase was significantly prolonged by depletion of external Ca 2+ with EGTA. The DAP spike was diminished with Cl − channel inhibitors, 9-anthracenecarboxilic acid and ethacrynic acid. The repolarisation phase was prolonged with tetraethylammonium chloride and Cs + (K + channel inhibitors) or could be abolished by less specific cation transport inhibitors, La 3+ and diethylstilbestrol. Thus, it was suggested, that the Cl − current was the main ion flux underlying the DAP spike, and that it was activated by a Ca 2+ inward current (i.e. rise in [Ca 2+] i), which was the first membrane response to D 2O. In fact, D 2O-induced rise in [Ca 2+] i was observed by fura-2 fluorescence measurements. The K + channels could give the main contribution to the repolarisation phase, but they were apparently partially blocked by D 2O.

Competitive and non-competitive inhibition of l-3,4-dihydroxyphenylalanine uptake in Opossum kidney cells

The present study aimed to determine the kinetics of l-3,4-dihydroxyphenylalanine ( l-DOPA) uptake in Opossum kidney (OK) cells and to define the type of inhibition produced by l-5-hydroxytryptophan ( l-5-HTP), cyanine 863 and 3,3″-diethyloxacarbocyanine (3,3″-DOC). Non-linear analysis of the saturation curves revealed for l-DOPA a K m (in μM) of 129 (114, 145) and a V max (in nmol mg protein −1 6 min −1) of 30.0±0.4. IC 50 values for l-5-HTP (1454 μM) obtained in the presence of a nearly saturating (250 μM) concentration of l-DOPA were almost 4-fold those obtained when non-saturating (25 μM) concentrations of l-DOPA were used (330). IC 50 values for cyanine 863 and 3,3″-DOC (638 and 353 μM) obtained in the presence of a nearly saturating (250 μM) concentration of l-DOPA were similar to those obtained when non-saturating (25 μM) concentrations of l-DOPA were used (654 and 339 μM). V max values (in nmol mg protein −1 6 min −1) for l-DOPA uptake were identical in the absence (36.4±0.7) and the presence of l-5-HTP (39.2±1.3), but K m values ( μM) were significantly greater ( P<0.05) when l-DOPA uptake was studied in the presence of l-5-HTP (121 (100, 142) versus 318 (237, 399)). In contrast, the effect of cyanine 863 and 3,3″-DOC was to cause a significant reduction in V max values without significant changes in K m values. It is concluded that l-5-HTP exerts a competitive type of inhibition of l-DOPA uptake in cultured OK cells, whereas both cyanine 863, an organic cation transport inhibitor and 3,3″-DOC behave as non-competitive inhibitors.

Rhodamine 123 accumulates extensively in the isolated perfused rat kidney and is secreted by the organic cation system

Rhodamine 123 has been shown to be a substrate for P-glycoprotein in multidrug resistant cells. In the present investigation the disposition of rhodamine 123 was studied in the isolated perfused rat kidney. After exposing the kidneys to perfusate concentrations ranging from 10 to 1000 ng/ml, the renal clearance was 4–1 times the clearance by glomerular filtration, respectively, indicating active and saturable secretion of rhodamine 123. The rate-limiting step in secretion was found to be membrane passage from cell to tubular lumen. Suprisingly, renal clearance was not influenced by the P-glycoprotein inhibitors cyclosporin A or digoxin. However, pretreatment of the kidneys with verapamil and quinidine (inhibitors of both P-glycoprotein and organic cation transport) or cimetidine (organic cation transport inhibitor) resulted in a significantly reduced rhodamine 123 clearance, indicating that the renal organic cation carrier may be involved in active secretion. Rhodamine 123 accumulated extensively in the isolated perfused rat kidney; tissue concentrations of 270–360 times the perfusate concentration were determined. Similar accumulation ratios at different perfusate concentrations were found, suggesting that the compound enters the tubular cells by (facilitated) diffusion. In conclusion, rhodamine 123 accumulated extensively in the isolated perfused rat kidney and active renal secretion appears to be preferentially mediated by the organic cation carrier and not by P-glycoprotein.

The role of calcium in UVB-induced damage in irradiated ocular lenses.

The purpose of this study was to evaluate the role of altered calcium homeostasis in the development of irreversible membrane damage in the UVB-irradiated ocular lens. In particular, experiments were designed to determine whether restricting calcium influx could prevent membrane damage that typically leads to ion imbalances and lens opacification following short-term exposure to ultraviolet light (UVB). The influx of calcium was reduced by culturing lenses in a low-calcium culture medium containing 0.3 mM Ca2+ rather than physiological concentrations of 1.6 mM. This low-calcium protocol retarded calcium accumulation in UVB-irradiated lenses for 2 days of culture, and opacification was delayed by 24 h. Loss of transparency did occur during the second day of culture, but more slowly than in irradiated lenses cultured in normal-calcium medium. Membrane damage was assessed by evaluating loss in cation transport activity, assessed by measuring 86Rb uptake into cultured lenses. Uptake was markedly inhibited in UVB-irradiated lenses and low-calcium culture did not prevent this inhibition of cation transport, a finding that explains why low-calcium protocol did not help maintain sodium homeostasis in irradiated lenses. Inhibition of cation transport and sodium accumulation eventually caused lens hydration and light scattering during extended culture in the absence of significant calcium elevation. Additional experiments were done to establish whether initial damage sustained by membranes could be repaired through the biosynthesis of new membrane proteins. Incorporation of 14C-histidine in membranes of the UVB-exposed lens was measured to assess membrane synthesis essential for repairing membrane damage. The rate of membrane protein synthesis, assessed by measuring incorporation of labeled amino acids, declined in UVB cataract, despite the prevention of calcium accumulation. These results suggest that one explanation for irreversible gain in sodium and calcium content accompanying opacification is the inability of lenses to replenish damaged membrane proteins comprising ion channels or transporters.

Interaction of dipyridamole, a nucleoside transport inhibitor, with the renal transport of organic cations by LLCPK&lt;SUB&gt;1&lt;/SUB&gt; cells

Dipyridamole is a well-known inhibitor of nucleoside transport by various cell membranes and is frequently used in in vitro studies that characterize nucleoside transport properties. Because interactions between the renal transport of organic cations and nucleosides have previously been suggested, we studied the effect of dipyridamole on the renal transport of the typical organic cations cimetidine and N1-methylnicotinamide by LLCPK1 monolayer cells grown on a permeable support. [14C]Mannitol was used to correct for extracellular flux. Basolateral to apical transcellular flux (transepithelial flux-extracellular flux) of [3H]cimetidine was significantly reduced by the monolayer cells (90%) in the presence of 50 microM dipyridamole. In addition, the effect of dipyridamole on cimetidine renal transport was dose dependent (IC50 = 7.7 microM). The dipyridamole inhibitory effect was nearly comparable with the effect of 1 mM quinine (a typical organic cation transport inhibitor), which led to 95% inhibition of cimetidine renal transport over time. The dipyridamole effect on N1-methylnicotinamide renal transport was less potent. The effect of 1 mM of typical probes of the nucleoside transporters (i.e., thymidine, adenosine, uridine) and the effect of 100 nM of another nucleoside transport inhibitor, dilazep, were also studied on cimetidine transport by LLCPK1 monolayer cells. These compounds did not exert any significant effect. These results suggest that dipyridamole, a widely used nucleoside transport inhibitor, is also an inhibitor of organic cation renal transport and they alert us to possible interactions between the renal transport of nucleosides and organic cations. This finding also has relevance to the interpretation of in vitro studies using this agent as a nucleoside membrane transport inhibitor.

Studies on the pharmacology of the inward transport of L-DOPA in rat renal tubules.

1. The accumulation of L-DOPA in suspensions of renal tubules obtained from male Wistar rats occurred through non-saturable and saturable mechanisms. The kinetics of the saturable component of L-DOPA uptake in renal tubules was as follows: Vmax = 46.3 +/- 2.8 nmol mg-1 protein h-1 and Km = 114.4 (95% confidence limits; 83.8, 156.2) microM (n = 5). The diffusion constant (in nmol-1) of the non-saturable component for the accumulation of L-DOPA was 1.3 (1.1, 1.6; n = 8). 2. The effect of 2,4-dinitrophenol was a marked reduction in the tubular uptake of L-DOPA, with an IC50 value of 12.1 (4.0, 36.9) microM. Cocaine produced a slight (P = 0.08) decrease (22% reduction at 50 microM) in the tubular uptake of L-DOPA. Corticosterone produced a considerable inhibitory effect on the uptake of L-DOPA with an IC50 value of 11.0 (3.6, 33.5) microM. The maximal inhibitory effect of probenecid was a 24% decrease in the uptake of L-DOPA; however, the more selective organic anion transport inhibitor 4,4'-diisothiocyantostilbene-2,2'-disulphonic acid (DIDS) was, found not to affect the tubular uptake of L-DOPA. The organic cation transport inhibitor, cyanine 863, was found to produce a marked decrease in the uptake of L-DOPA (IC50 = 2.02 [1.07, 3.79]). The cyanine derivatives 1,1'-diethyl-2,2'-cyanine (decynium 22) and 1,1'-diethyl-2,4'-cyanine (decynium 24) also potently inhibited L-DOPA uptake with IC50 values (in microM) of 0.63 (0.39, 1.01) and 0.10 (0.08, 0.13), respectively, both compounds were found to be more potent than cyanine 863. 3. The inhibitory effect of decynium 24 (0.2 microM) on the tubular uptake of L-DOPA was dependent on the pH of the incubation medium; at pH = 6.5 the accumulation of L-DOPA was reduced up to 37 +/- 2% of control values, whereas at pH = 7.4 and pH = 8.2 the accumulation of L-DOPA was reduced by 56 +/- 1% and 60 +/- 6%, respectively. Cyanine 863 (2 microM), decynium 22 (1 microM) and decynium 24 (0.2 microM) were found to decrease the Vmax values for the saturable component of L-DOPA uptake without changes in Km values. 4. It is concluded that the tubular uptake of L-DOPA might be promoted through a mechanism which is dependent on the activity of the organic cation transport system.

Anti-idiotypic antibodies to delineate epitope specificity of anti-amiloride antibodies.

Amiloride and related compounds have found widespread use as cation transport inhibitors. We have previously raised a series of polyclonal anti-amiloride antibodies using different amiloride-protein conjugates as immunogens, where amiloride was coupled to protein either through its guanidino moiety or through its 5-aminopyrazinyl moiety. The anti-amiloride antibodies recognized distinct sites on amiloride, and the site of attachment of amiloride to carrier protein was a critical factor in determining which part of the amiloride molecule was recognized by the anti-amiloride antibody. The specificity of binding of amiloride analogues to these polyclonal anti-amiloride antibodies mimicked the specificity of binding of amiloride analogues to selected isoforms of the epithelial Na+ channel or the Na+/H+ exchanger, suggesting that antigen binding site of these antibodies might be similar in structure to amiloride binding sites on selected Na+ transport proteins. We previously generated monoclonal anti-idiotypic antibodies RA2.4 and RA6.3 by an auto-anti-idiotypic approach, using amiloride coupled to albumin through the guanidinium moiety (amiloride-A1). We have now raised a series of monoclonal anti-idiotypic antibodies, T6, T26, T40, and T181, using amiloride coupled to keyhole limpet hemocyanin through the 5-aminopyrazinyl moiety (amiloride-A5) as an immunogen with the same auto-anti-idiotypic approach. These monoclonal anti-idiotypic antibodies recognized both polyclonal anti-amiloride-A1 and anti-amiloride-A5 antibodies, suggesting that idiotype-anti-idiotype interaction was not epitope restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute renal toxicity of thiabendazole (TBZ) in ICR mice

The acute toxic effects of thiabendazole [2-(4′-thiazolyl)benzimidazole; TBZ] on the kidneys of ICR mice were investigated. The mice were given 0, 250, 500 or 1000 mg TBZ/kg body weight by gavage (using olive oil as a vehicle), and the kidneys were subjected to pathological examination at 1, 3, 5 or 24 hr after dosing. Gross findings were slight enlargement and the presence of whitish areas (white maculae) in kidneys of treated mice at 3, 5 or 24 hr after dosing. Histological findings were desquamation of degenerated cells in proximal tubules of treated mice at 1 hr. Dilation of proximal, distal and collecting tubules was apparent in treated mice at 3, 5 and 24 hr. TBZ-induced renal injury was reduced by pretreatment with inducers of the microsomal monooxygenase system (sodium phenobarbital, β-naphthoflavone and 3-methylcholanthrene) and were enhanced by pretreatment with inhibitors of that system (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride and piperonyl butoxide). The concentration of TBZ in blood at 1 or 5 hr after dosing was lower in mice pretreated with microsomal monooxygenase system inducers and was higher in those pretreated with the inhibitors, than in those given TBZ alone. These results suggest that TBZ-induced renal injury may be attributable to the parent compound rather than its metabolites. Measurement of organic ion uptake into renal slices revealed significant depression of [1- 14C]tetraethylammonium bromide (TEA) uptake in treated mice at 1 or 5 hr, whereas uptake of p-[glycyl-2- 3H]aminohippurate (PAH) was not depressed at 1 or 5 hr after dosing. The reduction in uptake of TEA is interpreted as the result of competitive suppression of the tubular transport of TEA by TBZ. TBZ-induced renal injury was reduced by organic cation transport inhibitors [N′-methylnicotinamide (NMN) or thiamine] but not by organic anion transport inhibitor [ p-(dipropylsulphamyl)benzoic acid probenecid], suggesting that the reduction of TBZ-induced renal injury is the result of competitive suppression of the tubular transport of TBZ by NMN or thiamine.

Pathways of NH3/NH4+ permeation across Xenopus laevis oocyte cell membrane.

While acid loading with extracellular NH4Cl solutions usually first alkalinizes the cells through NH3 influx, and acidifies only when NH4Cl is removed, Xenopus oocytes became immediately acidic upon NH4Cl addition and the cells did not acidify further upon its removal. Since NH4Cl solutions also collapsed the membrane potential (Vm) and resistance (Rm), we conclude that primarily NH4+ entered the cells where it liberated H+, with NH3 being trapped in intracellular lipid stores. To identify the NH4+ permeation pathway we have used K+ channel blockers (Ba2+, Cs+, tetraethylammonium, quinidine), various cation transport inhibitors (ouabain, bumetanide, amiloride) and other inhibitors, some of which block non-selective cation channels (La3+, diphenylamine-2-carboxylate, and p-chloromercuribenzoate). However, only the latter substances partially prevented the collapse of Vm and Rm. This suggests, that NH4+ passes through non-selective cation channels. In accordance with the voltage dependence and/or stretch activation of such channels NH4+ fluxes appeared to be asymmetric. NH4+ influx, which depolarized and swelled the cells, was large and acidified rapidly, while the efflux, which repolarized and shrank the cells, was slow and alkalinized only slowly.

Interactions of magnesium and chloride ions on tone and contractility of vascular muscle

Replacement of extracellular chloride ions ([Cl −] o) by other anions, on contractility and the effects of extracellular magnesium ions ([Mg 2+] o]) on spontaneous mechanical activity, as well as on agonist-induced responses of rat aorta ana portal vein, were studied. Replacement of [Cl −] o With acetate (Ac −) or isethionate (Ise −) ions resulted in an increase and decrease, respectively, of the spontaneous mechanical activity frequency in portal vein; the amplitudes of the spontaneous mechanical activity were attenuated by Ac − and Ise − substitution. Withdrawal of [Mg 2+] o in Cl −-containing media resulted in elevation of tension development in rat aortas, whereas a similar maneuver in media with Ac − or Ise −, substituted for [Cl −] o, resulted in abrogation of this tension development. Use of disulfonic stilbene anion-channel blockers, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate, 400–600 μM) and SITS (4,4'-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid, 400–600 μM), failed to influence either spontaneous mechanical activity or basal tone of rat portal portal vein or aortas. Incubation of DIDS or SITS in Mg 2+-free media also failed to influence mechanical responses to withdrawal of [Mg 2+] o. Use of the Cl − cation transport inhibitor bumetanide (30–80 μM) also failed to alter spontaneous mechanical activity or basal tone in either the presence or absence of [Mg 2+] o. Ac − and Ise − substitution attenuated norepinephrine- and K +-induced contractile responses in portal vein and aorta, Caffeine-induced contractions of aortas were potentiated by withdrawal of [Mg 2+] o, in Cl −-containing media but inhibited in Ac −- or Ise − substituted solutions. In the presence of [Mg 2+] o, substitution of foreign anions resulted in alterations in the agonist contractile dose-response curves; EC 50s were increased whereas maximum tensions were depressed. Withdrawal of [Mg 2+] o amplified these effects. Substitution of Ac − or Ise −for [Cl −] o, in the presence or absence of [Mg 2+] o depressed contractions induced by Ca acetate in aortas and portal vein. These results suggest that: (1) Cl − plays an important role in regulating spontaneous mechanical activity, basal tone, and contractility in rat aorta and portal vein; and (2) Cl − probably physiologically mediates some of the effects and actions of [Mg 2+] o. on intracellular release of and influx of Ca 2+ in these smooth muscles.

Mechanism of organic cation transport in rabbit renal basolateral membrane vesicles

The transports of [3H]tetraethylammonium (TEA), [3H]procainamide (PCA), and N1-[3H]methylnicotinamide (NMN) were studied in rabbit renal basolateral membrane vesicles (BLMVs) by use of a rapid filtration assay. All three compounds exhibited a similar uptake profile into the BLMVs and reached equilibrium by 1 h. In the presence of valinomycin, a K+ ionophore and K+ gradients (in to out), an inside-negative potential difference (PD) was generated that stimulated the uptake of TEA, PCA, and NMN by 1.9-, 1.9-, and 2.1-fold, respectively. The effect of PD could be blocked by the organic cation transport inhibitor mepiperphenidol. An inside-negative PD was also generated by a pH gradient (inside acidic). An overshoot of TEA uptake was produced, which was blocked by a valinomycin voltage clamp. Counterflow studies revealed that 1 mM TEA was capable of trans-stimulating 50 microM [3H]TEA uptake and producing a peak overshoot of nearly three times the equilibrium value, which was not abolished in the presence of a valinomycin voltage clamp or a gramicidin pH clamp. When an inside-negative PD was imposed on 1 mM TEA-loaded BLMVs, the uptake of [3H]TEA was 33% less. In contrast, neither NMN nor PCA produced a trans-stimulation of [3H]NMN or [3H]PCA transport, respectively. In addition, the effect of several organic cations on the TEA-TEA exchange mechanism was studied. Mepiperphenidol, PCA, choline, cimetidine, and NMN all demonstrated cis inhibition (82, 81, 58, 51, and 20%, respectively). Arginine, a basic amino acid, and probenecid, an organic anion transport inhibitor, had no effect. Choline was capable of trans-stimulating TEA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells.

In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.

Different cation transport inhibitor in malignant renal hypertension and in uraemia.

1. We investigated whether the previously reported circulating frusemide-like factor in rats with malignant renal hypertension was specific to this syndrome, or was also present in rats with chronic uraemia. 86Rb uptake (K+ transport) of monolayers of vascular smooth muscle cells was measured in the plasma of rats with malignant one-kidney, one-clip hypertension and of uraemic rats (60-70% nephrectomy). 2. Compared with control plasma, ouabain-insensitive 86Rb uptake of cells was reduced and ouabain-sensitive 86Rb uptake was unchanged in the plasma of rats with malignant hypertension. The reverse was found in the plasma of uraemic rats. 3. Inhibition of ouabain-sensitive 86Rb uptake of cells occurred in the plasma of rats with malignant hypertension when an angiotensin II antagonist was added to the reaction mixture. 4. The findings confirm the presence of a frusemide-like plasma factor in malignant hypertension and a ouabain-like plasma factor in uraemic. The presence of a ouabain-like plasma factor in malignant hypertension is camouflaged by an elevated circulating angiotensin II level, which stimulates K+ transport.