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F Lang

Publications and source records attributed to F Lang.

At least 271 records · Page 15Linked to original sources
PubMed1995

Effects of membrane-permeant and -impermeant thiol reagents on Ca2+ and K+ channel currents of mouse pancreatic B cells.

The membrane permeant thiol reagent diazene dicarboxylic acid bis-(N'-methylpiperazide) (DIP) has been shown to inhibit insulin secretion and Ca2+ uptake in pancreatic B cells in the presence of a stimulating glucose concentration (20 mM), whereas the nonpenetrating analog of DIP (bis-N'-methyliodide; DIP + 2) stimulates insulin release and Ca2+ uptake at a low glucose concentration (3 mM). The effects of DIP and DIP + 2 were tested on currents through ATP-sensitive K+ (K+ATP) channels and voltage-dependent Ca2+ channels (with Ba2+ as the charge carrier) in mouse pancreatic B cells in the whole-cell mode of the patch-clamp technique. DIP (0.1 mM) almost completely inhibited both the K+ATP and Ca2+ channel currents. In contrast, DIP + 2 (0.1 mM) did not affect the Ca2+ channel current but reduced the whole-cell K+ATP current by about 40%. The data strongly suggest that the suppression of insulin secretion previously observed with DIP is due to a reduction of the current through voltage-dependent Ca2+ channels, whereas the stimulation of hormone release induced by DIP + 2 is caused by the partial inhibition of K+ATP channel current.

Animals↗
PubMed1995

Properties of electrogenic Pi transport by a human renal brush border Na+/Pi transporter.

Inorganic phosphate (Pi) induced an inward current (IP) in Xenopus oocytes expressing the human renal Na+/Pi cotransporter NaPi-3. At 100mM Na+, Pi-transport was independent of the holding potential and resulted in an apparent Km of 0.08 mM; lowering the Na+ concentration to 50 mM resulted in an increase of the apparent Km to 0.22 mM at -50 mV and to 0.31 mM at -90 mV. In contrast, the apparent Km for Na+ was not significantly influenced by the holding potential. A decrease of the pH from 7.8 to 6.8 resulted in a decrease of IP at 50 mM Na+, but not at 150 mM Na+. Arsenate induced inward currents through NaPi-3 and decreased the apparent Km in measurements of IP. Phosphonoformic acid itself induced no currents, but inhibited Pi-induced currents with an apparent Ki of 3.6 mM. In summary, NaPi-3 displays characteristic Na+/Pi cotransporter properties with relevant interactions with arsenate (transport substrate) and phosphonoformic acid (inhibitor). Monovalent and divalent Pi both appear to be transported by NaPi-3.

Animals↗
PubMed1995

New antiinflammatory compounds that inhibit tumor necrosis factor production: probable interaction with protein kinase C activation.

We have previously described a family of benzamide derivatives that showed antiinflammatory activity in vivo on carragenin-induced paw edema and experimental cerebral edema. Those compounds inhibited eicosanoids production from activated macrophages (M phi) without inhibiting cyclooxygenase. To further investigate their antiinflammatory activity and compare it to that of classical cyclooxygenase inhibitors, we analyzed their effect on the production of a major proinflammatory cytokine, tumor necrosis factor (TNF-alpha), by in vitro-activated peritoneal macrophages. We show that, in marked contrast with ibuprofen, flurbiprofen and indomethacin which all significantly enhanced TNF production, the two benzamide derivatives tested, JM34 and JM42, significantly inhibited TNF-alpha production by zymosan or lipopolysaccharide-activated M phi. Those compounds did not interfere with the calcium-dependent pathway because they did not affect TNF production of either mice peritoneal M phi or human T cell clones induced by the calcium ionophore A23187 alone. More likely, these benzamide derivatives acted mainly at the level of the protein kinase C (PKC) pathway because: 1) After treatment of M phi with PKC inhibitors which significantly inhibited TNF production, our compounds showed no additional inhibition. 2) Our compounds significantly inhibited TNF production of M phi stimulated with the phorbol ester phorbol di-butyrate alone or in combination with A23187. 3) After depletion of PKC by prolonged phorbol di-butyrate treatment of M phi, inhibition of TNF production by our compounds was markedly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Effects of calcium channel blockers on NIH 3T3 fibroblasts expressing the Ha-ras oncogene.

NIH 3T3 fibroblasts expressing the ras oncogene (+ras cells) respond to bradykinin, bombesin or serum with sustained oscillations of cell membrane potential reflecting oscillations of intracellular calcium activity and subsequent activation of calcium-sensitive K+ channels. In contrast, identical cells not expressing the oncogene (-ras cells) respond to bradykinin with a single, transient hyperpolarization of the cell membrane. Furthermore, +ras cells are characterized by a serum-independent proliferation, an increase in cell volume and a marked reorganization of the cytoskeleton. It has been shown previously that the calcium channel blocker nifedipine, but not verapamil and diltiazem, inhibits oscillations of cell membrane potential as well as proliferation. In this study, we have examined the effect of several calcium channel blockers (bepridil, nifedipine, verapamil, diltiazem) on the proliferation, volume and cytoskeletal reorganization of +ras cells. Bepridil (10 mumol/l), which is also shown here to inhibit oscillations of cell membrane potential, and nifedipine (10 mumol/l) caused a decrease in cell number, whereas verapamil and diltiazem (10 mumol/l each) resulted in growth rates which did not differ from untreated +ras cells. The increase in cell volume as observed in untreated +ras cells was also observed for cells treated with verapamil and diltiazem, whereas cell volumes of +ras cells treated with bepridil and nifedipine were markedly reduced and similar to the values obtained for -ras cells. In addition, bepridil and nifedipine markedly inhibited cytoskeletal rearrangement, i.e depolymerization of actin-containing stress fibers. This inhibitory effect was not observed for verapamil and diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1995

Renal hemodynamic response to intravenous and oral amino acids in animals.

Oral or parenteral application of amino acids leads to marked hyperfiltration and increased of renal plasma flow. Amino acids stimulate the release of glucagon, which increases hepatic production and release of cyclic adenosine monophosphate (cAMP). In the kidney, the combined effect of cAMP and glucagon increases glomerular filtration rate (GFR), possibly by reducing NaCl concentration at the macula densa and depression of the tubuloglomerular feedback. Vasopressin-dependent urea recycling and delivery to the thick ascending limb could similarly reduce NaCl concentration at the macula densa. Beyond that, amino acids may trigger a hepatorenal reflex or directly interfere with renal function. Mechanisms invoked include dopamine from renal nerves, prostaglandins, nitric oxide (NO), and angiotensin II. At this point, it is not clear to which extent the described mechanisms participate in, permit, or fully account for the hyperfiltrative effect of amino acids.

Administration, Oral↗
PubMed1994

Effects of osmotic changes in extracellular solution on electrical activity of mouse pancreatic B-cells.

The influence of changes in the osmolarity of the extracellular solution on electrical activity of mouse pancreatic B-cells was studied with intracellular microelectrodes. In the presence of 15 mmol/l glucose the membrane potential of B-cells oscillates. A reduction of the osmolarity by 40 mosmol/l caused a small temporary hyperpolarization in six out of eight cells. After 2 to 3 min electrical activity was increased in all cells. However, this effect was also transient. 5 to 8 min after onset of exposure to hypotonic solution the cells repolarized again and electrical activity decreased. Increasing the osmolarity of the extracellular solution by 40 mosmol/l led to a sustained and reversible depolarization of the membrane potential. However, the electrical activity was transiently suppressed. The changes in electrical activity observed in hypotonic solution might explain the previously described transient rise in insulin secretion provoked by osmotic cell swelling.

Animals↗
PubMed1994

Rat interleukin-2 immunoglobulin M fusion proteins are cytotoxic in vitro for cells expressing the IL-2 receptor and can abolish cell-mediated immunity in vivo.

A hybrid cDNA coding for a fusion protein between rat interleukin 2 (IL-2) and a truncated heavy chain from rat immunoglobulin M (IgM) was constructed. The rat IL-2 and rat IgM CH2-3-4 hybrid gene was subcloned into a vector (PKCR6) for expression of the fusion molecule in Chinese hamster ovary (CHO) cells. Cells transfected with the hybrid cDNA secrete multimeric forms of the fusion protein (IL-2-Mu). Size analysis of the construct revealed that the majority (95%) of the secreted proteins have a high mw (> 500 kDa). The IL-2-Mu construct bind specifically to cells bearing the IL-2 receptors (IL-2R) with a binding affinity around 5 nM. The specific binding to IL-2R leads to T cell proliferation or, if rabbit complement is added, to T cell lysis. Multimeric forms (> 500 kDa) of the fusion protein mediate complement-dependent lysis but trigger only weak proliferation when compared with the low-mw forms (< 500 kDa). In contrast, the latter only efficiently mediate T cell proliferation without inducing complement-dependent lysis. After intravenous administration of CHO supernatant containing IL-2-Mu, or purified IL-2-Mu proteins into rats, the fusion proteins disappeared from the circulation with a t1/2 of 1 hr. The circulating IL-2-Mu constructs in the rat serum retained their capacity to induce complement-dependent lysis of IL-2R-bearing T cells in vitro. Furthermore, the IL-2-Mu construct was able to suppress the delayed-type hypersensitivity (DTH) reaction (an IL-2R, T helper cell-dependent event) in mice. A weak immune response (antirat IL-2-Mu antibodies) was observed when rats received multiple daily injections of the construct.

Animals↗
PubMed1994

Opposite directed currents induced by the transport of dibasic and neutral amino acids in Xenopus oocytes expressing the protein rBAT.

Voltage- and current-clamp studies have been performed on a renal and intestinal protein (rBAT) which induces transport for neutral and dibasic amino acids when expressed in Xenopus oocytes. In current-clamp mode, superfusion with L-leucine caused a hyperpolarization while superfusion with L-arginine depolarized the oocyte. Accordingly, in voltage-clamp experiments dibasic amino acids and neutral amino acids induced inward and outward currents, respectively. The relationship between currents and substrate concentrations could be fitted by simple Michaelis-Menten kinetics. Currents induced by L-arginine and L-leucine were also voltage-dependent. pH changes from 6.25 to 8.75 did not affect the currents induced by saturating concentrations of L-arginine and L-leucine, but reversed the direction of L-histidine-induced currents from inward to outward. The reversal potentials as well as the apparent Km for L-histidine-induced currents were altered by the ambient pH. Currents induced by individual amino acids decreased during extended superfusion periods. However, extended superfusion with neutral amino acids increased dibasic amino acid induced currents, while prior superfusion with dibasic amino acid resulted in an increase of currents induced by neutral amino acids. The reversal potentials for L-leucine- and L-arginine-induced currents were depending on their intra- (after preloading) and extracellular concentrations. In conclusion, rBAT-mediated transport of neutral and dibasic amino acids is associated with net outward or inward currents, respectively, which may be caused by an exchange of neutral with dibasic amino acids.

Amino Acid Transport Systems, Basic↗
PubMed1994

Blockade of human IsK channels expressed in Xenopus oocytes by the novel class III antiarrhythmic NE-10064.

cRNA encoding the human IsK protein was injected into Xenopus oocytes and the expressed channels were investigated using the two-microelectrode voltage-clamp method. The novel class III antiarrhythmic NE-10064 (1-[[[5-(4-chlorophenyl)-2-furanyl]methylene]-amino]-3- [4-(4-methyl-1-piperazinyl)-butyl]-2,4-imidazolidinedione dihydrochloride) was tested for its ability to block these channels. The compound displayed potent inhibitory effects with an EC50 of 5.4 microM. The block caused by NE-10064 was use-dependent, i.e. channels had to be activated for the inhibition to occur. Further, the reversal of the inhibition during the wash-out period was use-dependent. Finally, the blockade of human IsK channels by NE-10064 appeared to be voltage-dependent, being more pronounced at depolarized potentials. We conclude that this novel class III antiarrhythmic is a potent inhibitor of human IsK channels and suggest that such effects could be involved in its antiarrhythmic action.

Animals↗
PubMed1994

Effects of aniso-osmolarity and hydroperoxides on intracellular pH in isolated rat hepatocytes as assessed by (2',7')-bis(carboxyethyl)-5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextran fluorescence.

Freshly isolated rat hepatocytes were plated for 4-6 h and either loaded with (2',7)-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran in order to study the effects of aniso-osmotic exposure and oxidative stress on cytosolic (pHcyt) and apparent vesicular pH (pHves) by single-cell fluorescence recordings. In the presence of normo-osmotic (305 mosmol/l) medium pHcyt was 7.23 +/- 0.03 (n = 108), whereas an apparent pH of 6.07 +/- 0.02 (n = 156) was found in the vesicular compartment accessible to endocytosed FITC-dextran. Substitution of 60 mM NaCl against 120 mM raffinose had no effect on pHcyt or apparent pHves, whereas addition of NH4Cl increased both pHcyt and apparent pHves. Hypo-osmotic cell swelling lowered pHcyt, whereas simultaneously apparent pHves increased. These effects were rapidly reversible upon re-institution of normo-osmotic media. Similarly, an increase of apparent pHves was observed when cell swelling was induced by Ba2+, glutamine or histidine. Conversely, hyperosmotic cell shrinkage due to addition of NaCl or raffinose led to a cytosolic alkalinization and a vesicular acidification. Both, H2O2 (0.2 mmol/l) and t-butyl-hydroperoxide (0.2 mmol/l) were without effect on pHcyt, but lowered apparent pHves by about 0.2 pH units. Ba2+ (1 mmol/l) diminished the acidifying effect of the hydroperoxides by about 50%. Pretreatment of the cells with colchicine, but not with lumicolchicine, largely abolished the effects of aniso-osmolarity and hydroperoxides on pHves. The data suggest that hepatocellular hydration affects the proton gradients built up across the membranes of endocytotic FITC-dextran-accessible compartments in a microtubule-dependent way. They further suggest that hydroperoxides induce vesicular acidification in a colchicine- and Ba(2+)-sensitive way. Because hydroperoxides induce Ba(2+)-sensitive cell shrinkage [Hallbrucker, Ritter, Lang, Gerok and Häussinger (1992) Eur. J. Biochem. 211, 449-458], the results are compatible with the view that hydroperoxide-induced cell shrinkage mediates vesicular acidification. It is concluded that modulation of vesicular pH by the hepatocellular hydration state may play a role in triggering some metabolic changes in response to cell swelling/shrinkage.

Ammonium Chloride↗
PubMed1994

Role of N-linked glycosylation in rat renal Na/Pi-cotransport.

Our laboratory recently identified a sodium-dependent transport system for phosphate from rat kidney cortex (NaPi-2; Magagnin, S., Werner, A., Markovich, D., Sorribas, V., Stange, G., Biber, J., and Murer, H. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 5979-5983). In the present study we have investigated whether or not this cotransporter is glycosylated and the role of N-glycosylation in determining its function. Glycosidase digestion of the NaPi-2 protein from rat brush border membranes, in vitro translation studies, or oocyte expression of the NaPi-2 cRNA indicate that the mature protein is glycosylated. Glycosidase treatment reduces the size of the protein from approximately 70-110 kDa to approximately 60-65 kDa. We therefore used site-directed mutagenesis to identify which of the putative consensus sites for N-linked glycosylation are utilized in the mature NaPi-2 protein. Altering the nucleotide sequences encoding both of the Asn-298 and Asn-328 residues to Gln produced mutants that are completely devoid of glycosylation, whereas mutants in which each of these sites were mutated separately are glycosylated when expressed in oocytes. These results suggest that both of these sites are modified by N-linked glycosylation in the mature protein. Surface expression of glycosylated and unglycosylated NaPi-2-related proteins was documented by biotinylation experiments. In contrast to the wild-type (fully glycosylated) transporter, immunocytochemistry provides evidence for a partial intracellular localization of mutant unglycosylated cotransporters. Na/Pi cotransport was studied in oocytes expressing wild-type or mutagenized NaPi-2 proteins using tracer or electrophysiological techniques. Although the transport rates are lower (by a factor of 2-3) after expression of the unglycosylated NaPi-2 protein, the Pi transport characteristics (pH dependence, apparent affinity for Pi or Na+) are similar in oocytes expressing either wild-type or glycosylation-deficient proteins.

Animals↗
PubMed1994

Involvement of microtubules in the link between cell volume and pH of acidic cellular compartments in rat and human hepatocytes.

Cell swelling is shown to induce an increase in acridine orange fluorescence intensity, an effect pointing to the alkalinization of acidic vesicles. Since autophagic hepatic proteolysis is accomplished by pH-sensitive proteinases within acidic lysosomes, this effect may contribute to the well-known inhibitory effect of cell swelling on proteolysis. In the present study, the role of microtubules in volume-dependent alterations of pH in acidic vesicles of rat and human hepatocytes was studied. Colcemid and colchicine were used to depolymerize microtubules and vesicular pH was monitored using two different fluorescent dyes, fluorescein isothiocyanate conjugated-dextran and acridine orange. Colcemid and colchicine, but not the inactive stereoisomer gamma-lumicolchicine, blunted the increase of pH during osmotic cell swelling. The alkalinization of acidic vesicles by NH4Cl was not significantly modified by colcemid or colchicine, indicating that the vesicles were still sensitive to alkalinizing procedures other than cell swelling. Further, colchicine, but not gamma-lumicolchicine, inhibited the antiproteolytic action of osmotic cell swelling. The present observations point to an involvement of the microtubule network in the link of cell volume, lysosomal pH, and proteolysis.

Animals↗
PubMed1994

Electrophysiological analysis of Na+/Pi cotransport mediated by a transporter cloned from rat kidney and expressed in Xenopus oocytes.

Phosphate (Pi) reabsorption in renal proximal tubules involves Na+/Pi cotransport across the brush border membrane; its transport rate is influenced by the Na(+)-coupled transport of other solutes as well as by pH. In the present study, we have expressed a cloned rat renal brush border membrane Na+/Pi cotransporter (NaPi-2) in Xenopus laevis oocytes and have analyzed its electrophysiologic properties in voltage- and current-clamp studies. Addition of Pi to Na(+)-containing superfusates resulted in a depolarization of the membrane potential and, in voltage-clamped oocytes, in an inward current (IP). An analysis of the Na+ and/or Pi concentration dependence of IP suggested a Na+/Pi stoichiometry of 3:1. IP was increased by increasing the pH of the superfusate; this phenomenon seems to be mainly related to a lowering of the affinity for Na+ interaction by increasing H+ concentration. The present data suggest that known properties of Pi handling at the tubular/membrane level are "directly" related to specific characteristics of the transport molecule (NaPi-2) involved.

Animals↗
PubMed1994

Inhibition of human IsK channels expressed in Xenopus oocytes by calmodulin antagonists.

The calmodulin antagonists, trifluoperazine, chlorpromazine and W7 (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluomethyl)-10H-phen othiazine , 2-chloro-10-(dimethylaminopropyl)-phenothiazine and N-(6-aminohexyl)-5-chloro-1-naphtalen-sulfonamide, respectively), were tested for their effects on human IsK channels expressed in Xenopus oocytes and their interference with the previously described [Ca2+]i-mediated regulation of IsK. An increase in [Ca2+]i accelerated IsK activation and increased the current amplitude, as has been previously observed. Chlorpromazine, trifluoperazine and W7 inhibited depolarization-activated IsK channels with an EC50 between 70 and 100 microM. None of the calmodulin antagonists abolished the regulation of IsK by A23187 (calcimycin) or hypotonic extracellular fluid, although the inhibitory effects of these compounds were also obvious after enhancement of [Ca2+]i. In conclusion, the calmodulin antagonists inhibit IsK at both physiological and enhanced [Ca2+]i.

Animals↗
PubMed1994

Effects of SH-group reagents on Ca2+ and K+ channel currents of pancreatic B-cells.

The whole-cell patch-clamp technique has been applied to elucidate the effects of H2O2 and the SH-group reagent 2,2'-dithio-bis(5-nitropyridine) (DTBNP) on Ca2+ currents of mouse pancreatic B-cells. We also tested the effect of DTBNP on the whole-cell K+ ATP current. DTBNP (20 and 100 mumol/l) blocked both the current through Ca2+ channels (with Ba2+ as the charge carrier) and the K+ATP current in a concentration-dependent manner. The results suggest that the channels themselves or regulating proteins possess free SH-groups which may be of functional relevance. However, H2O2 did not influence the Ca2+ channel currents under standard whole-cell conditions as well as when recorded with the perforated patch technique. These results indicate that the two agents act differently on ion channel currents in B-cells although both are known to oxidize SH-groups.

Adenosine Triphosphate↗
PubMed1994

Electrogenic cotransport of Na+ and sulfate in Xenopus oocytes expressing the cloned Na+SO4(2-) transport protein NaSi-1.

The Na+/sulfate cotransporter cloned from rat kidney cortex (NaSi-1) has been expressed in oocytes of Xenopus laevis and subjected to electrophysiological analysis by current and voltage clamp methods. In current-clamped oocytes, superfusion with 1 mM sulfate resulted in a 12-mV depolarization of the cell membrane. Accordingly, in voltage-clamped oocytes sulfate induced an inward current IS, which was dependent on both the concentration of Na+ and sulfate in the superfusate. Half-maximal IS was observed at about 0.1 mM sulfate and 70 mM Na+. The Hill coefficients were 1 and 2.8 for sulfate and Na+, respectively. Thiosulfate and selenate created similar currents as sulfate with a similar Km. At saturating concentrations of thiosulfate and selenate, addition of sulfate could not induce an additive current. Phosphate (1 mM) did not inhibit sulfate-induced currents. Finally, IS was dependent on the holding potential being larger at more negative potentials. The results of this study strongly suggest an electrogenic cotransport of sulfate and Na+ with a stoichiometry of 1:3.

Animals↗