Competitive inhibition of the uptake of demethylphalloin by cholic acid in isolated hepatocytes

Summary Cholic acid inhibits the uptake of demethylphalloin (DMP), in a competitive manner. The bile acid increases the Michaelis constant but not V _max of the inward transport. The inhibition constant K _i for cholate was found to be 8 M. Cholate competes for the transport system but not for intracellular binding of phallotoxins. Various experimental data presented in this paper exclude an accumulation of phallotoxins in hepatocytes by intracellular binding only.Preincubation of hepatocytes with small concentrations of either (³H)-demethylphalloin or (¹⁴C)-cholate and subsequent treatment with high concentrations of the nonlabelled compounds reduces the intracellular concentration of both radioactive substrates. In accordance with earlier findings the above results suggest a common component needed for the transport of both phallotoxins and cholic acid.This work was supported by the Deutsche Forschungsgemeinschaft     

Active transport of polypeptides in rabbit nasal mucosa: Possible role in the sampling of potential antigens

Transepithelial pathways of macromolecule transport have been studied in vitro in rabbit nasal respiratory mucosa, maintained at 27° C. Transepithelial electrical potential difference, short-circuit current and resistance were 3.4±0.5mV (submucosa positive), 65.0±6.7 A cm^–2 and 52.1±5.6 cm^–2 respectively (n=15). These electrical characteristics are those of a leaky epithelium allowing macromolecules to permeate paracellularly. A detailed permeation study of a polypeptide (elcatonin, M _w=3362) was also undertaken. Elcatonin mucosa-submucosa (J _ms) and submucosa-mucosa (J _sm) fluxes were measured by radioimmunoassay. With 10 g/ml elcatonin, J _ms was significantly larger than J _sm for the whole 120-min period of observation; net flux showed a maximum in the first 30 min (J _ms=13.6±1.0 ng cm^–2 h^–1, J _sm=1.4±0.1 ng cm^–2 h^–1, n=10). J _ms fell towards the value of J _sm if the temperature was reduced to 4°C or if the mucosa was simultaneously treated with 0.1 mM dinitrophenol and 3 mM monoiodoacetate. J _ms and J _net followed saturation kinetics with increasing elcatonin concentrations. Adrenocorticotropic hormone (M _r=4500) produced a similar pattern to elcatonin. However, J _ms and J _sm were not significantly different from each other at any time either for [³H]sucrose (M _w=342) or for [¹⁴C]polyethyleneglycol-4000 (M _w=4000) when present in the bathing medium at 500 M concentration. The results show active transport of polypeptides in parallel with passive permeation (possibly through leaky intercellular junctions). Active transport does not appear to be related to nonspecific pinocytosis but to receptor-mediated endocytosis. The latter may be important for the sampling of potential antigens from the nasal lumen.     

Oxalate transport in cultured porcine renal epithelial cells

Summary Oxalate-containing kidney stones are the most common type (75%) of renal stones. In order to control oxalate excretion in the urine, a basic understanding of the cellular transport of oxalate is imperative. We have utilized the technique of continuous cell culture to establish and characterize a model system to study renal epithelial cell (LLCPK1) oxalate transport. Our data demonstrate that oxalate uptake in these cells is dependent on time, concentration and energy. TheK _m for oxalate uptake was 200 m. Oxalate uptake was decreased at lower temperatures and elevated in an acidic extracellular environment. Both anion exchange inhibitors DIDS and SITS inhibited oxalate oxalate uptake. Sulfate, chloride, and bicarbonate decreased oxalate uptake, as did the diuretics bumetanide and furosemide. There was no evidence for the co-transport of oxalate with sodium. Our data show that monolayers of cultured kidney epithelial cells are a valuable model system for study of the basic cellular mechanisms of oxalate transport.     

Potassium channels in crustacean glial cells.

Unitary currents through single ion channels in the glial cells, which ensheath the abdominal stretch receptor neurons of the crayfish, were characterized with respect to their basic kinetic properties. In cell-attached and excised patches two types of Ca(++)-independent K+ channels were observed with slope conductances of 57 pS and 96 pS in symmetrical K+ solution. The 57 pS K+ channel was weakly voltage-dependent with a slope of the Po vs. membrane potential relationship of +95 mV for an e-fold change in Po. In addition to the main conductance level, the channel displayed conductance levels of 80 and 109 pS. In excised patches, channel activity of this "subconductance" K+ channel showed "rundown" that could be prevented with 2 mM ATP-Mg on the cytoplasmic side of the membrane. The 96 pS K+ channel was strongly voltage-dependent with a slope of +12 mV for an e-fold change in Po. Averaged single-channel currents elicited by voltage jumps proved the channel to be of the delayed rectifying type. Channel activity persisted in excised patches with minimal salt solution and in virtually Ca(++)-free saline. Because of its dependence on intracellular ATP-Mg, the subconductance K+ channel is discussed as a target of modulation by transmitters or peptides via phosphorylation of the channel.     

Isoprenaline increases brain concentrations of administered l-dopa and l-tryptophan in the rat

A small dose of isoprenaline or saline was administered intraperitoneally to rats 20 min before the administration of one of the amino acids l-dopa or l-tryptophan. Isoprenaline caused a marked increase in the brain concentration of the administered amino acid. Isoprenaline has previously been shown to cause a decrease in at least some of those plasma amino acids which compete with l-dopa and tryptophan for carrier-mediated transport into the brain. The effect of isoprenaline on the concentrations of dopa and tryptophan in the brain is suggested to be at least partly caused by a change in the relationship between endogeneous and administered amino acids. It is also possible that a direct effect of isoprenaline on the blood-brain barrier transport system contributes to the effect.The reported finding might be of clinical interest in view of the therapeutic importance of aromatic amino acids with a central site of action.     

The effect of naringenin on the intestinal and renal transport of organic solutes

Summary Naringenin (4,5,7-trihydroxy-flavanone) inhibits the accumulation of glycine, -methyl-glucoside, p-amino-hippurate and N¹-methyl-nicotinamide in dog renal cortex slices. It also inhibits oxygen consumption by this tissue. Since the sensitivity of amino-acid uptake to the drug is less than the sensitivity of oxygen consumption, the inhibition of this transport might be secondary to an effect on intermediary metabolism. The inhibition of PAH uptake occurs at a lower concentration of the drug, and so naringenin may affect this process at the membrane level.Naringenin inhibits the transport of sugars and amino-acids by dog, guinea-pig and rat small intestine. Both steady-state accumulation and initial rates of entry are affected. Amino-acid uptake is depressed both in the presence or absence of sodium ions. The inhibition is reversible provided short contact times are employed. According to kinetic analysis, naringenin appeared to be a fully non-competitive inhibitor of phenylalanine influx. Examination of the unidirectional transmural fluxes of phenylalanine across guinea-pig intestine revealed that only the mucosal-serosal flux was affected, and then only if the flavanone was added to the solution bathing the mucosal face of the tissue. Naringenin does not inhibit mucosal Na⁺-K⁺-ATPase, but it does alter the intracellular ion concentrations.Although some of the results can be explained in terms of an effect of naringenin on metabolism, others can not. It is argued that naringenin has a direct action on cell membranes.Some of these results have been presented to the Physiological Society and a short report has appeared in their proceedings (Robinson, 1979)     

Responses of fetal rat bone cells and bone organ cultures to the ionophore,A23187

The ionophore A23187 produced a rapid transient increase in the rate of calcium uptake by isolated fetal rat bone cells. There was no effect on calcium efflux or total cellular calcium. The magnitude of the effect on influx was amplified when the cell were incubated at 4°C. Cellular metabolic functions and resorption of cultured fetal rat bones (release of⁴⁵Ca from pre-labeled long bone) were affected by A23187 in a biphasic manner: cell cyclic AMP (cAMP) was increased by 0.1 and 0.3 g/ml of the ionophore, whereas 10 g/ml was either ineffective or lowered the cAMP levels. The high A23187 concentration abolished the stimulatory effects of parathyroid hormone and methylisobutylxanthine. Concentrations of 0.1 and 0.3 g/ml A23187 stimulated bone resorption. The effect was abolished by calcitonin. Ionophore concentrations above 1 g/ml produced less bone resorption. These higher concentrations antagonized the bone-resorbing effect of parathyroid hormone and 1,25-dihydroxyvitamin D₃. A23187 at 5 and 10 g/ml decreased bone cell lactate and ATP. Thus at low concentrations, A23187 produced effects on bone similar to those of parathyroid hormone, suggesting that calcium is the primary initiator of PTH-induced bone resorption. At the higher concentrations A23187 may have a general inhibitory effect on cell metabolism.