piperidines and mepiperphenidol

Recently, an organic cation:H+ antiport was selectively identified on the sinusoidal domain of rat liver with the use of the endogenous organic cation N1-methylnicotinamide (NMN). Absence of NMN+:H+ exchange on canalicular membrane suggested that this transport process was primarily involved in organic cation uptake, leaving the mechanism(s) for organic cation secretion into bile unknown. To further define hepatic organic cation transport, we examined the characteristics of tetraethylammonium (TEA) transport in basolateral (blLPM) and canalicular (cLPM) rat liver plasma membrane vesicles. In cLPM vesicles, under voltage-clamped conditions, an outwardly directed H+ gradient stimulated [14C]TEA uptake compared with [14C]TEA uptake under pH-equilibrated conditions, consistent with electroneutral TEA:H+ exchange. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone had no effect on [14C]TEA uptake, demonstrating that pH-dependent [14C]TEA uptake was not the result of a H+ diffusion potential. In the absence of a pH gradient, the intravesicular presence of TEA trans-stimulated uptake of [14C]TEA. Procainamide ethobromide (PAEB), vecuronium, and tributylmethylammonium (TBuMA), organic cations selectively excreted in bile, cis-inhibited pH-dependent TEA uptake. In contrast, in blLPM vesicles, no pH gradient-dependent [14C]TEA uptake was demonstrated. Instead, basolateral [14C]TEA uptake was significantly stimulated by a valinomycin-induced intravesicular-negative K+ diffusion potential. Basolateral [14C]TEA uptake was also cis-inhibited by PAEB, vecuronium, and TBuMA, but not by NMN. Conversely, PAEB, vecuronium, and TBuMA had no effect on basolateral pH-dependent [3H]NMN uptake. These findings suggest that organic cation transport, with TEA as a model quaternary amine, across the canalicular membrane is driven by an electroneutral organic cation:H+ exchange and that the transport of certain organic cations across the basolateral membrane is via a carrier-mediated system stimulated by an inside-negative membrane potential.

Topics: Animals; Biological Transport; Carrier Proteins; Cations; Cell Membrane; Electrophysiology; Hydrogen-Ion Concentration; Ion Exchange; Kinetics; Liver; Piperidines; Rats; Tetraethylammonium; Tetraethylammonium Compounds

The effect of vancomycin, a putatively nephrotoxic amine glycopeptide antibiotic, on the transport of organic cations was examined in rabbit renal basolateral and brush border membrane vesicles. The studies were conducted using a rapid filtration technique and the prototypic organic cation tetraethylammonium. In basolateral membrane vesicles, vancomycin cis-inhibited the electrogenic transport of tetraethylammonium with an IC50 value of 260 microM. In contrast, gentamicin, an aminoglycoside, was without effect. Inhibition by mepiperphenidol, a classical organic cation transport inhibitor, was observed with an IC50 value of 24 microM. Countertransport, that is, trans-stimulation experiments, were initiated in order to determine whether or not vancomycin was capable of traversing the plasma membrane. Vancomycin caused trans-stimulation of tetraethylammonium uptake. The specificity of inhibition was assessed by determining the effect of vancomycin on the transport of p-aminohippurate, an organic anion. Vancomycin did not inhibit transport, whereas probenecid, a classical organic anion inhibitor, did. In the brush border membrane, vancomycin had no effect on the transport of tetraethylammonium. These data are consistent with mediated transport for vancomycin across the basolateral membrane, but not across the brush border membrane. This implies that the nephrotoxicity of vancomycin may be due to entry through the basolateral membrane and the absence of mediated egress at the brush border membrane.

Topics: Animals; Basement Membrane; Biological Transport; Cations; Ganglionic Blockers; Gentamicins; Kidney; Kidney Tubules, Proximal; Microvilli; Piperidines; Rabbits; Vancomycin

The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.

Topics: Animals; Azides; Biological Transport, Active; Cations; Cell Line; Cell Membrane; Cell-Free System; Epithelium; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney Tubules, Proximal; Niacinamide; Opossums; Piperidines; Temperature; Tetraethylammonium Compounds

The mechanisms involved in the transport of the organic cation N1-methylnicotinamide (NMN) were investigated in the isolated perfused rabbit S2 proximal tubule. NMN underwent a small reabsorptive transport which appeared to be passive. NMN secretory transport was saturable, inhibited by mepiperphenidol (10(-4) M) and presented a relatively low apparent affinity (apparent Km of 852 microM) for the organic cation transport system, with transport against a concentration gradient being observed only at low flow rates. Acidification of the perfusate, by either buffering it at pH 6.8 with MES, or by bubbling the bath with a mixture of 80% O2-20% CO2, resulted in a decrease, rather than an increase, of NMN secretion. Carbonic anhydrase inhibition with ethoxyzolamide (10(-4) M in the bath) did not modify NMN secretion. Addition of 20 to 40 microM NMN in the perfusate also did not change NMN secretion. Proton or organic cation counterexchange seemed therefore not to play a major role in NMN apical step of secretion, the basolateral step appearing to be the general organic cation system of transport for which NMN shows a low affinity.

Topics: Absorption; Animals; Biological Transport; Cell Membrane Permeability; Female; Ganglionic Blockers; Kidney Tubules, Proximal; Male; Niacinamide; Piperidines; Rabbits

Transport of two organic cations, N1-methylnicotinamide (NMN) and tetraethylammonium (TEA), was investigated in LLC-PK1 cells grown on Transwell collagen coated Nuclepore filters. Two min NMN or TEA unidirectional transepithelial flux and simultaneous cellular uptake were measured. Transport of NMN and TEA from basolateral to apical side was temperature-dependent, saturable and competitively inhibited by each other or by mepiperphenidol. NMN and TEA transport from the apical to the basolateral side was very slow, only slightly faster than that of mannitol. Apparent kinetic parameters of basolateral to apical transcellular flux were measured. For NMN apparent Km = 133.6 +/- 35.4 microM, Vmax = 48.3 +/- 5.6 pmol/cm2.2 min. For TEA apparent Km = 11.4 +/- 2.2 microM, Vmax = 42.1 +/- 1.8 pmol/cm2.2 min. Kinetic parameters of cellular uptake were also estimated. For NMN apparent Km = 436.9 +/- 139.8 microM, Vmax = 143.7 +/- 30.5 pmol/micrograms DNA. For TEA apparent Km = 50.3 +/- 7.2 microM, Vmax = 26.5 +/- 2.5 pmol/micrograms DNA. It is concluded that LLC-PK1 cells transport NMN and TEA from the basolateral to the apical side; this flux corresponds to the secretory transport of the renal proximal tubule. NMN and TEA share the same transport system, but NMN has a lower affinity.

Topics: Biological Transport; Cell Line; Epithelium; Kidney; Niacinamide; Piperidines; Tetraethylammonium Compounds

Mechanisms of tetraethylammonium (TEA) transport were investigated in basolateral membrane (BLM) vesicles from rabbit renal cortex. Preloading vesicles with 10 mM TEA or 1 mM mepiperphenidol stimulated the 15-s uptake of [14C]-TEA compared with control vesicles (258 and 200%, respectively) and produced an overshoot of the equilibrium value (3 and 1.6 times, respectively). In the presence of a K+ gradient, net TEA uptake was also increased (and showed an overshoot of 2-fold) when the membrane potential of vesicles was made interior negative by adding valinomycin. Both TEA-TEA exchange and the voltage-driven net TEA transport were cis-inhibited by other organic cations, and a similar affinity order was found for both transport mechanisms (quinine greater than amiloride greater than morphine greater than mepiperphenidol greater than choline = N1-methylnicotinamide). This data suggests that the same transport protein might be responsible for both phenomena. Other experiments determined that the BLM vesicles had no TEA-H+ exchange, and that contamination of the vesicle population by brush-border membranes was negligible in terms of their contribution to TEA transport. These results demonstrate the presence of an exchange reaction for TEA in the rabbit renal BLM and thus imply carrier-mediated transport of TEA by these membranes.

Topics: Acridine Orange; Amiloride; Animals; Biological Transport; Cell Membrane; Choline; Female; Ganglionic Blockers; Kidney Cortex; Kinetics; Male; Monensin; Morphine; Piperidines; Rabbits; Sodium; Sodium-Potassium-Exchanging ATPase; Spectrometry, Fluorescence; Tetraethylammonium; Tetraethylammonium Compounds

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were tested as a model system for assessing the role of calcium in gentamicin-induced nephrotoxicity. Cell viability was evaluated by a vital dye exclusion procedure, and intracellular free calcium [Ca2+]i was measured employing Fura-2 fluorescence. Exposing cell suspensions (10(6)/ml) to concentrations of the drug, which had no apparent effect on viability, produced a rapid and prolonged increase in intracellular [Ca2+]. The perturbation of calcium homeostasis could be blocked by the addition of mepiperphenidol, an inhibitor of the organic cation transport system. We propose that LLC-PK1 cells are an appropriate model to study drug-induced nephrotoxicity. Gentamicin disrupts calcium homeostasis and causes plasma membrane alterations. Since mepiperphenidol blocked the gentamicin-induced Ca2+ increases, the data suggest that aminoglycosides enter the cell via the organic cation transporter.

Topics: Animals; Calcium; Cell Line; Cytosol; Dose-Response Relationship, Drug; Gentamicins; Homeostasis; Kidney; Piperidines; Swine

The mechanisms involved in the transport of tetraethylammonium (TEA) in the rabbit renal brush border were investigated by using membrane vesicles. Transport of [14C]TEA (0.2 mM) was measured by a rapid filtration method. We have reported previously that an imposed pH gradient (pHi = 6, pHo = 7.4) stimulates the uptake of TEA markedly, yielding a transient overshoot of 200% above the equilibrium value. Here we demonstrate that an overshoot of similar magnitude can also be obtained in the presence of indirectly induced proton gradients, via the naturally occurring Na+/H+ exchanger or via the artificial K+/H+ exchanger nigericin. The TEA exchange mechanism is shown to be electroneutral, temperature-dependent and saturable [Km, 0.328 (CL for P less than .05: 0.250-0.425) mM; Vmax, 2.13 (CL for P less than .05: 1.98-2.32) nmole/mg of protein X 15 sec]. Other organic cations interact with this exchange mechanism: mepiperphenidol and morphine both cis-inhibited and trans-stimulated TEA uptake. Quinine, which was the most potent inhibitor of TEA uptake (57% inhibition at 10(-5) M), did not trans-stimulate. The mechanism appears to be specific for organic cations inasmuch as the organic anion p-aminohippurate did not cis-inhibit or trans-stimulate TEA uptake. These results demonstrate the presence of a H+/TEA exchange mechanism in the rabbit renal brush border membrane showing many similarities with that shown to be present in the rat.

Topics: Animals; Carrier Proteins; Dose-Response Relationship, Drug; Female; Hydrogen-Ion Concentration; Kidney; Kinetics; Male; Membrane Potentials; Microvilli; Morphine; Nigericin; Piperidines; Potassium-Hydrogen Antiporters; Quinine; Rabbits; Temperature; Tetraethylammonium Compounds

N1-methylnicotinamide (NMN) transport was studied in isolated perfused snake (Thamnophis spp.) proximal renal tubules. Unidirectional lumen-to-bath (Jl----bNMN) and bath-to-lumen (Jb----lNMN) fluxes saturated. Although Jl----bNMN and Jb----lNMN were similar, mean Jl----bNMN tended to exceed mean Jb----lNMN at all concentrations studied. Direct measurements confirmed a net reabsorptive flux equal to the difference between the unidirectional fluxes. This transport, opposite in direction to tetraethylammonium (TEA) transport, was not inhibited by TEA. Transport into the cells across both the luminal and peritubular membranes during flux measurements was apparently down an electrochemical gradient by a mediated process that was sodium dependent. Inhibition with NMN analogues suggested that transport into the cells across the luminal membrane during Jl----bNMN was more specific than transport into the cells across the peritubular membrane during Jb----lNMN. Transport out of the cells across both the luminal and peritubular membranes during flux measurements was apparently against an electrochemical gradient.

Topics: Animals; Biological Transport; Body Water; Epithelium; In Vitro Techniques; Kidney Tubules, Proximal; Kinetics; Niacinamide; Osmolar Concentration; Perfusion; Piperidines; Snakes; Sodium; Tetraethylammonium; Tetraethylammonium Compounds

Cisplatin, an effective antineoplastic agent, is toxic to the kidney. Since the kidney's vulnerability to cisplatin may originate in its ability to accumulate and retain platinum to a greater degree than other organs, we studied the characteristics of the renal accumulation of platinum and investigated the nature of intracellular platinum. Cisplatin and ethylenediamminedichloroplatinum, nephrotoxic and antineoplastic liganded platinum compounds, were concentrated in rat renal cortical slices fivefold above medium concentration. Platinum uptake was energy- and temperature-dependent and could be inhibited by drugs which inhibit base transport. The organic anions para-aminohippurate and pyrazinoate did not reduce renal slice platinum uptake. Unbound platinum in the blood and urine was predominantly cisplatin but unbound platinum in kidney cytosol was not. This latter compound, in contrast to cisplatin, was not active as a mutagen. These studies suggest that the kidney accumulates platinum in part by transport or specific binding to the base transport system in the kidney and biotransforms it intracellularly. Unbound platinum in the cell is not cisplatin and may no longer be toxic.

Topics: Animals; Binding Sites; Biological Transport; Biotransformation; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Cisplatin; Kidney; Kidney Cortex; Male; Mutagenicity Tests; Organoplatinum Compounds; Piperidines; Platinum; Probenecid; Rats; Rats, Inbred Strains; Thiamine; Tolazoline

The secretory transport of tetraethylammonium (TEA) was investigated in perfused and nonperfused isolated S1, S2, and S3 segments of proximal tubules from rabbit kidneys. In the perfused tubules the transepithelial net secretory flux and in nonperfused tubules the TEA cellular uptake were saturable (Km = 67 microM, Vmax = 2,480 fmol X min-1 X mm-1 in perfused S2 segments), energy dependent, and inhibited by mepiperphenidol. The net secretory flux of TEA (J b leads to j TEA) at a bath TEA concentration of 40 microM differed for the three segments and decreased in the order S1 greater than S2 greater than S3. The concentration of TEA in the perfusate leaving the tubule was approximately twice as great and the intracellular TEA concentration approximately 40 times as great as that in the bath. In nonperfused segments (40 microM TEA in the incubation medium) the TEA tissue water-to-medium ratio reached 100. In the three segments the ability to accumulate TEA across the peritubular membrane, thus, was similar, but the transepithelial secretory flux differed significantly. The differences in secretory rate between the three segments presumably result from differences in the luminal membrane permeability.

Topics: Animals; Biological Transport; Ganglionic Blockers; In Vitro Techniques; Kidney Tubules, Proximal; Kinetics; Perfusion; Piperidines; Rabbits; Tetraethylammonium; Tetraethylammonium Compounds

The accumulation of organic cations and anions by the isolated rabbit choroid plexus was measured and the results were considered in comparison to in vivo measurements of transport using the ventriculocisternal perfusion technique also in rabbits. The accumulation in vitro of 3H-p-aminohippurate (PAH) and 14C-N1-methylnicotinamide (NMN) was shown to be organic anion and cation specific and dependent upon oxidative metabolism since tissue/medium ratios were reduced by competitive inhibitors including dibenamine and mepiperphenidol for cation transport and probenecid for anion transport and were also reduced by cyanide and dinitrophenol. Rabbits pretreated with chlorpromazine and anesthetized with pentobarbital were perfused lateral ventricle to cisterna magna with artificial cerebrospinal fluid containing inulin, 3H-PAH and 14C-NMN. Ratios of concentrations of these substances in the perfusate to those infused were calculated. Significant reductions in the ratios for PAH and NMN compared to unulin were obtained when low rates of perfusion, 40mul/min, were employed with low concentrations of PAH (2 mug/ml) and NMN (0.2 mug/ml). Probenecid (10(-3)M) specifically increased the ratio for PAH without affecting the NMN ratios. Mepiperhenidol (10(-o) M) blocked NMN transport but no PAH. The data and calculated clearances indicate that these prototype anions and cations may be actively transported out of cerebrospinal fluid, but for cations only at a low rate.

Topics: Aminohippuric Acids; Animals; Biological Transport; Choroid Plexus; Cyanides; Dibenzylchlorethamine; Dinitrophenols; In Vitro Techniques; Niacinamide; Perfusion; Piperidines; Probenecid; Quaternary Ammonium Compounds; Rabbits; Time Factors

The renal excretion of N'1-methylnicotinamide (NMN) was studied in the rat. Renal clearance experiments clearly demonstrated that: 1) NMN is secreted; 2)a tubularmaximum (Tm), 7 mumol/min per kg, could be reached; and 3)NMN secretion is inhibitedby a competitive inhibitor, mepiperphenidol. In free-flow micropuncture experiments, animals were infused with plasma concentrations of NMN ABOVE Tm; the TF/P NMNto TF/P inblin ratio for proximal and distal samples was 2.34 and 2.28, respectively, indicating that NMN is secreted in the proximal tubules and is not secreted orreabsorbed in the distal tubules. This finding was further confirmed by intratubularmicroinjections of ['14C]NMN into rats. In diuretic animals approxiamately 10%of the NMN injected into early proximal tubules was reabsorbed, but no reabsorption could be detected after distal injections. The nondiuretic animals showed no significant reabsorption of NMN. It was concluded that NMN transport is a carrier-mediated process and that reabsorption, if it occurs, plays only a minor role.

Topics: Absorption; Animals; Binding, Competitive; Diuresis; Ganglionic Blockers; Kidney; Kidney Tubules, Distal; Kidney Tubules, Proximal; Male; Microinjections; Niacinamide; Piperidines; Punctures; Rats

Topics: Dogs; Kidney; Mecamylamine; Metabolism; Parasympatholytics; Piperidines; Research

Topics: Atropine; Central Nervous System; Humans; Parasympatholytics; Peripheral Nerves; Piperidines

Topics: Bromides; Cholinergic Antagonists; Parasympatholytics; Piperidines