Activity and Stoichiometry of Na+:HCO− 3 Cotransport in Immortalized Renal Proximal Tubule Cells

The proximal tubule Na⁺-HCO⁻ ₃ cotransporter is located in the basolateral plasma membrane and moves Na⁺, HCO⁻ ₃, and net negative charge together out of the cell. The presence of charge transport implies that at least two HCO⁻ ₃ anions are transported for each Na⁺ cation. The actual ratio is of physiological interest because it determines direction of net transport at a given membrane potential. To determine this ratio, a thermodynamic approach was employed that depends on measuring charge flux through the cotransporter under defined ion and electrical gradients across the basolateral plasma membrane. Cells from an immortalized rat proximal tubule line were grown as confluent monolayer on porous substrate and their luminal plasma membrane was permeabilized with amphotericin B. The electrical properties of these monolayers were measured in a Ussing chamber, and ion flux through the cotransporter was achieved by applying Na⁺ or HCO⁻ ₃ concentration gradients across the basolateral plasma membrane. Charge flux through the cotransporter was identified as difference current due to the reversible inhibitor dinitro-stilbene disulfonate. The cotransporter activity was Cl⁻ independent; its conductance ranged between 0.12 and 0.23 mS/cm² and was voltage independent between −60 and +40 mV. Reversal potentials obtained from current-voltage relations in the presence of Na⁺ gradients were fitted to the thermodynamic equivalent of the Nernst equation for coupled ion transport. The fit yielded a cotransport ratio of 3HCO⁻ ₃:1Na⁺. Received: 19 January 1996/Revised: 24 April 1996     

Hydrolysis of fucosyl-GM1 ganglioside by purified pellet-associated human brain and human liver alpha-L-fucosidases without activator proteins or detergents.

Pellet-associated human brain alpha-L-fucosidase was solubilized with 0.5% (w/v) Triton X-100 and purified by affinity chromatography on agarose-6-aminohexanoyl-fucosamine resin. The procedure resulted in a 290,000-fold purification, a 58% yield and a final specific activity of 11,500 nmol/min per mg of protein. Isoelectric focusing indicated that all six major isoforms (with pI values between 4.1 and 5.3) present in crude brain pellet preparations were purified by the affinity technique. SDS/PAGE indicated the presence of one subunit (54 kDa) and a minor protein band at 67 kDa, which presumably is a contaminant since it was not immunoreactive on Western blotting. The pH optimum of the brain enzyme and its apparent Km for the synthetic substrate 4-methylumbelliferyl alpha-L-fucopyranoside were 5.5 and 0.07 mM respectively. Pellet-associated human brain and liver alpha-L-fucosidases were both capable of hydrolysing fucosyl-GM1 ganglioside without activator proteins or detergents. Linear hydrolysis rates were found only for short incubation times (1-5 min). Optimal enzymic activity at 37 degrees C was found at pH 3.4 for both alpha-L-fucosidases, with no activity at pH values above 4.0.     

Dual modulation of chloride conductance by nucleotides in pancreatic and parotid zymogen granules.

The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.     

Active alanine transport in isolated brush border membranes.

Uptake of L-alanine against a concentration gradient has been shown to occur with isolated brush border membranes from rat small intestine. An alanine transport system, displaying the following characteristics, was shown: (a) L-alanine was taken up and released faster than D-alanine; (b) Na+ as well as Li+ stimulated the uptake of both stereoisomers; (c) the uptake of L- and D-alanine showed saturation kinetics; (d) countertransport of L-alanine was shown; (e) other neutral amino acids inhibited L-alanine but not D-alanine entry when an electrochemical Na+ gradient across the membrane was present initially during incubation. No inhibition occurred in the absence of a Na+ gradient. The electrogenicity of L-alanine transport was established by three types of experiments: (a) Gradients of Na+ salts across the vesicle membrane (medium concentration greater than intravesicular concentration) supported a transient uptake of L-alanine above equilibrium level, and the lipophilic anion SCN- was the most effective counterion. (b) A gradient of K= across the membrane (vesicle greater than medium) likewise supported active transport of L-alanine into the vesicles provided the K= conductance of the membrane was increased with valinomycin. (c) Similarly, a proton gradient (vesicle greater than medium) in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, an agent known to increase the proton conductance of membranes, produced an overshooting L-alanine uptake. A consideration of the possible forces, existing under the experimental conditions, suggests that the gradients of SCN-, K+ in the presence of valinomycin, and H+ in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone contribute to the driving force for L-alanine transport by creating a diffusion potential. Since the presence of Na+ was required in all experiments with active L-alanine transport these results support the existence of a transport system in the brush border membrane which catalyzes the co-transport of Na+ and L-alanine across this membrane.     

Die Verteilung der Gc-Phänotypen und Gc-Allele bei einigen Krankheiten (Diabetes mellitus,Leberparenchymschaden, Psoriasis vulgaris)

Zusammenfassung Bei 239 Probanden mit Diabetes mellitus, 213 Leberkranken—bes. Patienten mit akuter und chronischer Hepatitis sowie Lebercirrhosen—und 203 Kranken mit Psoriasis vulgaris wurden die Gc-Phänotypen bestimmt und mit der Gc-Typenverteilung bei 1733 gesunden Kontrollpersonen verglichen. Die Frequenz für das Allel Gc¹ beim Diabetes mellitus liegt mit 0,7490 sowie bei den Leberkrankheiten mit 0,7371 höher, bei der Psoriasis vulgaris mit 0,6749 niedriger als bei der gesunden Kontrollgruppe mit Gc¹ 0,7185. Der Unterschied im Vergleich der Diabetiker sowie der Leberkranken mit den Gesunden ist jedoch nicht signifikant. In der Gruppe mit Psoriasis vulgaris dagegen kommt der Phänotyp Gc2-1 signifikant häufiger vor als der Typ 1–1, allerdings ist diese Stichprobe in sich möglicherweise nicht hinreichend homogen.

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The frequency of the three Gc-phenotypes has been determined in 239 propositi with diabetes mellitus,213 patients with liver diseases—especially acute and chronic hepatitis as well as liver cirrhosis—and 203 persons with psoriasis vulgaris. The distribution in these diseases is compared with 1733 serum samples of healthy inhabitants of South-Niedersachsen with a Gc¹ frequency of 0,7185. The frequency of the gene Gc¹ in diabetes mellitus is 0,7490, in liver diseases 0,7371 and in psoriasis vulgaris 0,6749. These differences are statistically not significant in diabetes and in liver diseases. A significant difference has been found in psoriasis vulgaris between the both phenotypes Gc 1–1 and Gc 2-1 (X ²=10,7164, p(m=1)0,001). It may, however, be discussed wether this sample has a sufficient homogeneity.

     

Liposomal amphotericin B is toxic to fungal cells but not to mammalian cells

Amphotericin B is an efficacious but extremely toxic anti fungal drug. Recently it has been shown that the incorporation of Amphotericin B in multilamellar liposomes results in a marked reduction in drug toxicity in mice with no loss of anti fungal potency. Until now, the mechanistic basis of the enhanced therapeutic index of liposomal Amphotericin B has been unclear. In this report, however, we show that the in vivo effects can be mimicked in vitro where free but not liposomal Amphotericin B causes lysis of erythrocytes while both free and liposomal drug kill fungal cells. These results suggest that the markedly improved therapeutic index of liposomal Amphotericin B is largely due to a fundamental alteration in the ability of the drug to interact with mammalian cell membranes rather than to alterations in pharmacokinetics or drug distribution.     

Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.