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A E Busch

Publications and source records attributed to A E Busch.

At least 73 records · Page 4Linked to original sources
PubMed1996

The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells.

We have shown previously that secretagogues acting via the second messenger adenosine 3',5'-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl- conductance, a K+ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K+ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 micromol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K+ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency (fc) of 308 +/- 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K+ selective. 293B reduced the power density of the noise (So) to 46 +/- 8.7% of its control value and shifted fc from 291 +/- 26 to 468 +/- 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5'-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba2+ and was completely absent when K+ in the bath was replaced by Na+. These data suggest that forskolin activates a K+ channel of very small conductance which can be inhibited directly and reversibly by 293B.

Animals↗
PubMed1996

cAMP stimulation of CFTR-expressing Xenopus oocytes activates a chromanol-inhibitable K+ conductance.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel in a large variety of cells expressing this protein. Recently evidence has accumulated that it also regulates other ion channels. A coordinated increase in Cl- and K+ conductances is necessary in many Cl--secreting epithelia. This has, for example, recently been demonstrated for the colonic crypt, for which a new type of K+ channel and a specific inhibitor of this channel, the chromanol 293B, have been described. In the present study we have examined whether the cAMP-evoked activation of CFTR, overexpressed in Xenopus oocytes, in addition to its known activation of a Cl- conductance, also upregulates endogenous K+ channels. It is shown that CFTR-cRNA-injected but not water-injected oocytes possess a cAMP-activated Cl- conductance. Of the cAMP-induced whole-cell current increase, 15-25% was due to a 293B-, Ba2+and TEA+-inhibitable K+ conductance. The cRNA of the mutated CFTR (DeltaF508 CFTR) had no such effect. We conclude that cAMP activated CFTR and an endogenous IsK-type and 293B-sensitive K+ conductance. Similar events, occurring, for example, in the colonic crypt possessing CFTR and 293B-sensitive K+ channels, might explain the coordinated cAMP-mediated increase in Cl- and K+ conductances.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Blockade of epithelial Na+ channels by triamterenes - underlying mechanisms and molecular basis.

The three subunits (alpha, beta, gamma) encoding for the rat epithelial Na+ channel (rENaC) were expressed in Xenopus oocytes, and the induced Na+ conductance was tested for its sensitivity to various triamterene derivatives. Triamterene blocked rENaC in a voltage-dependent manner, and was 100-fold less potent than amiloride at pH 7.5. At -90 mV and -40 mV, the IC50 values were 5 microM and 10 microM, respectively. The blockage by triamterene, which is a weak base with a pKa of 6.2, was dependent on the extracellular pH. The IC50 was 1 microM at pH 6.5 and only 17 microM at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analysis, the apparent inhibition constants at -90 mV were 0.74 microM for the charged and 100.6 microM for the uncharged triamterene. The main metabolite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibited rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substituted by acidic or basic residues, inhibited rENaC with IC50 values in the range of 0.1-20 microM. Acidic and basic triamterenes produced a rENaC blockade with a similar voltage and pH dependence as the parent compound, suggesting that the pteridinemoiety of triamterene is responsible for that characteristic. Expression of the rENaC alpha-subunit-deletion mutant, Delta278-283, which lacks a putative amiloride-binding site, induced a Na+ channel with a greatly reduced affinity for both triamterene and amiloride. In summary, rENaC is a molecular target for triamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage- and pH-dependent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms.

Amiloride↗
PubMed1996

Inhibition of IKs in guinea pig cardiac myocytes and guinea pig IsK channels by the chromanol 293B.

The chromanol derivative 293B was previously shown to inhibit a cAMP regulated K+ conductance in rat colon crypts. Subsequent studies on cloned K+ channels from the rat demonstrated that 293B blocks specifically IsK channels expressed in Xenopus oocytes, but does not affect the delayed and inward rectifier Kv1.1 and Kir2.1, respectively. In the present study, the specificity of 293B for the cardiac K+ conductances IKs and IKr, and for the cloned guinea pig IsK channel and the human HERG channel, which underly IKs and IKr, respectively, was analyzed. 293B inhibited both the slowly activating K+ conductance IKs in cardiac myocytes and guinea pig IsK channels expressed in Xenopus oocytes with a similar IC50 (2-6 micromol/l). In contrast, high concentrations of 293B had only a negligible effect on the more rapid activating IKr. Similarly, 293B exerted no effect on HERG channels expressed in Xenopus oocytes. In summary, 293B appears to be a rather specific inhibitor of IKs and the underlying IsK channels.

Animals↗
PubMed1996

Coexpression and stimulation of parathyroid hormone receptor positively regulates slowly activating IsK channels expressed in Xenopus oocytes.

Expression of the IsK protein in Xenopus oocytes induced the characteristically slow, voltage-dependent outward currents. Superfusion with the parathyroid hormone (PTH) peptide 1-34 had no effect on IsK when expressed alone, but increased IsK when IsK was coexpressed with the PTH-receptor. PTH receptor stimulation caused a shift of IsK conductance-voltage relationship to more negative potentials, and a decrease of both the rate of IsK activation and deactivation. IsK regulation by PTH was independent of extracellular Ca2+, and was also present IsK protein mutants lacking the protein kinase C consensus site. However, regulation of IsK by PTH was mimicked by activators of protein kinase A (PKA) and greatly reduced in the presence of the kinase inhibitors staurosporine and H89. These results suggest that PTH regulates IsK by a mechanism involving phosphorylation independent of protein kinase C (PKC). Such regulation may play a role in proximal tubule cells of the kidney, where both PTH receptor and the IsK protein are expressed.

Animals↗
PubMed1996

Heavy metals inhibit Pi-induced currents through human brush-border NaPi-3 cotransporter in Xenopus oocytes.

Heavy metal intoxication with Hg2+, Pb2+ and Cd2+ commonly leads to phosphaturia. In this study, we examined the effects of these heavy metals on Pi-induced currents (Ip) through NaPi-3, the human renal cotransporter for Na+ and Pi. Hg2+ inhibited Ip in a dose- and time-dependent fashion. Hg2+ decreased the extrapolated maximal current but did not alter the apparent affinity for Pi. This inhibition was also observed with the membrane-permeable oxidizing agent 2,2'-dithio-bis(5-nitropyridine) (DTNP) but not with the membrane-impermeable 5,5'-dithiobis(2-nitrobenzoic acid). Hg(2+)- and DTNP-mediated inhibition of Ip was reversible only in the presence of the reducing agent 2,3-dihydroxybutane-1,4-dithiol. Cd2+ and Pb2+ also inhibited Ip. However, while CD2+ did not significantly alter the apparent affinity for Pi, the apparent concentration needed for half-maximal current (Km) for Pi was increased by Pb2+. In contrast to Hg2+, the inhibition of Ip by Cd2+ and Pb2+ was rapidly reversible upon washout. In the presence of the Na(+)-K(+)-adenosinetriphosphatase inhibitor ouabain, Ip was not reduced, and the effects of the heavy metals were maintained. In summary, the three heavy metals Hg2+, Cd2+, and Pb2+ inhibit Ip through the Na+/Pi cotransporter NaPi-3 by distinct mechanisms. Heavy metal-mediated inhibition of NaPi-3 may be responsible for the phosphaturia observed after intoxication with these compounds.

2,2'-Dipyridyl↗
PubMed1996

Function and dysfunction of renal transport molecules: lessons from electrophysiology.

The availability of cloned transport molecules achieved by efforts in expression cloning has allowed their electrophysiological analysis in the Xenopus oocyte expression system. We describe the electrogenic uptake of various substrates by their corresponding transport molecules originally expressed in brush border membranes of proximal tubules. The functional properties of the following transport molecules are discussed: the sodium-coupled glucose transporter, the sodium-coupled phosphate transporter, the sodium-coupled sulfate transporter and the sodium-independent transporter of neutral and dibasic amino acids. Additionally, functional consequences of naturally occurring disease-causing mutations in some of these transport molecules are described.

Animals↗
PubMed1995

Subunit-specific inhibition of inward-rectifier K+ channels by quinidine.

Distinct inward-rectifier K+ channel subunits were expressed in Xenopus oocytes and tested for their sensitivity to the channel blocker quinidine. The 'strong' inward-rectifier K+ channel IRK1 was inhibited by quinidine with an EC50 of 0.7 mM, while the 'weak' rectifier channel ROMK1 was only moderately inhibited. ROMK1(N171D)-IRK1C-term chimeric channels, which carry both sites for strong rectification of IRK1 channels (the negatively charged D171 in the second transmembrane domain and the IRK1-C-terminus including E224), displayed strong rectification like IRK1, but showed weak sensitivity to quinidine-like ROMK1, suggesting independence of quinidine binding and rectification mechanisms. Moreover, BIR10 and BIR11, two strong rectifier subunits originally cloned from rat brain, exerted subunit-specific sensitivity to quinidine, being much higher for BIR11. Quinidine blockade of IRK1 was not voltage-dependent, but strongly dependent on the pH in the superfusate. These results strongly suggest a subunit-specific interaction of inward-rectifier K+ channels with neutral quinidine within membrane lipid bilayers.

Animals↗
PubMed1995

Effect of channel modulation and pH on IsK inhibition by the novel class III antiarrhythmic azimilide (NE-10064).

Inhibition of human IsK channels expressed in Xenopus oocytes by the novel class III antiarrhythmic azimilide was studied under distinct treatments known to increase IsK (hypotonic solution, A23187 and isoproterenol) Azimilide inhibited IsK under all conditions with similar potency. Reduction of ionic strength or pH changes from pH 6.5 to 8.5 did not alter IsK amplitude. However, inhibition of IsK by azimilide was decreased by reduced pH, but not by reduced ionic strength. Further, the apparent affinity of azimilide was increased more than tenfold by increasing pH from 6.5 to 8.5. The data suggest that the neutral form of azimilide, a weak base, inhibits IsK via a lipophilic protein-drug interaction. pH-dependence of azimilide may significantly alter its effects on IsK under distinct pathophysiological conditions (acidosis vs. alkalosis) and in distinct locations (heart vs. kidney).

Animals↗
PubMed1995

Molecular basis of IsK protein regulation by oxidation or chelation.

Slowly activating IsK channels were expressed in Xenopus oocytes and exposed to oxidative agents. Oxidative treatment reduced the resulting current IsK, while no inhibition was observed for IsK protein mutants carrying a Ser mutation instead of a highly conserved Cys residue in the intracellular domain. In contrast, Hg2+, which may not only oxidize thiol groups but also form chelates with dibasic amino acids, caused a use-dependent, positive regulation of IsK. This effect was reversed in an IsK protein mutant with a deletion in the extracellular domain. These data suggest opposite effects of peroxides and Hg2+ on IsK, a peroxide-mediated IsK inhibition by intracellular oxidation and a Hg(2+)-mediated IsK increase, caused by extracellular Hg2+ chelation of the IsK protein.

Animals↗
PubMed1995

The nitroso-donor S-nitroso-cysteine regulates IsK expressed in Xenopus oocytes via a c-GMP independent mechanism.

In Xenopus oocytes expressing slowly activating IsK channels superfusion with the nitroso-donor S-Nitroso-Cysteine (SNOC) resulted in an increase of IsK, which was greatly enhanced when the amino acid-exchanger rBAT was coexpressed. The effects of SNOC on IsK could not be prevented by the guanylate cyclase inhibitor LY-83,583 and the cGMP kinase inhibitor H8, but was abolished in the presence of staurosporine. SNOC also increased the currents induced by the expression of protein mutants lacking intracellular sites, previously described to be involved in IsK regulation by oxidation and phosphorylation. These data suggest that the NO-donor SNOC regulates IsK indirectly via a cGMP independent, but staurosporine sensitive, pathway.

Alkaloids↗
PubMed1995

Protein kinase C consensus sites and the regulation of renal Na/Pi-cotransport (NaPi-2) expressed in XENOPUS laevis oocytes.

Renal brush border membrane sodium/phosphate (Na/Pi)-cotransport activity is inhibited by hormonal mechanisms involving activation of protein kinases A and C. The recently cloned rat renal Na/Pi-cotransporter (NaPi-2) contains several protein kinase C but no protein kinase A consensus sites [17, 20]. In the present study we have expressed wild type and polymutant (protein kinase C consensus sites removed) NaPi-2-transporters in Xenopus laevis oocytes. The expression of transport function as well as the basic transport properties were unaffected by the removal of the consensus sites. Pharmacological activation of protein kinase C with phorbol 12,13-didecanoate (PDD) led to a time-dependent inhibition of expressed wild type Na/Pi-cotransport function; simultaneous exposure to staurosporine (0.3) prevented the PDD induced (50 nM) inhibition. The kinase-C-mediated inhibition was not prevented by the removal of the protein kinase C consensus sites. Pharmacological activation of protein kinase A (dibutyryl adenosine 3':5':cyclic monophosphate (cAMP)/forskolin) had no effect on wild type NaPi-2-induced oocyte Na/Pi-cotransport. It is concluded that the protein-kinase-C-mediated regulation of expressed Na/Pi-cotransport does not involve the predicted consensus sites. The involvement of "cryptic" phosphorylation sites and/or of a phosphorylated "regulatory" protein is discussed.

Amino Acid Sequence↗
PubMed1995

Transport characteristics of a murine renal Na/Pi-cotransporter.

A complementary deoxyribonucleic acid (cDNA) corresponding to a murine renal cortical Na/phosphate-(Na/Pi-) cotransporter was isolated and its transport properties characterized by electrophysiological techniques after expression in Xenopus laevis oocytes. A Na-dependent inward movement of positive charges ("short-circuit current") was observed upon superfusion with Pi (and with arsenate). Increasing the Na concentration led to a sigmoidal elevation in Pi-induced short-circuit current; the apparent Michaelis constant, Km, (around 40 mM Na) was increased by lowering the pH of the superfusate but was not influenced by altering the Pi concentration. Increasing the Pi (and arsenate) concentration led to a hyperbolic elevation in Na-dependent short-circuit current (apparent Km for Pi at 100 mM Na was around 0.1 mM; apparent Km for arsenate was around 1 mM); lowering the Na concentration decreased the apparent affinity for Pi. The Pi-induced short-circuit current was lower at more acidic pH values (at pH 6.3 it was about 50% of the value at pH 7.8); this pH dependence was similar if the Pi concentration was calculated in total, or if distinction was made between its mono- and divalent forms. Thus, the pH dependence of Na-dependent Pi transport (total Pi) may not be related primarily to a pH-dependent alteration in the availability of divalent Pi, but includes also a competitive interaction of Na with protons. The effect of Pi and Na concentration on the apparent Km values for Na or Pi, respectively, provides evidence for an ordered interaction of "cosubstrate" (Na first) and "substrate" (Pi or arsenate second).

Amino Acid Sequence↗
PubMed1995

Heavy metal mediated inhibition of rBAT-induced amino acid transport.

rBAT, a protein which is located in the brush border membranes of intestine and renal proximal tubule cells, was recently shown to induce electrogenic countertransport of neutral and dibasic amino acids after its expression in Xenopus oocytes. Here, we studied the effects of heavy metals on rBAT induced amino acid transport in Xenopus oocytes to clarify a possible involvement of rBAT in heavy metal-induced aminoaciduria. The heavy metals Hg2+ and Pb2+ inhibited rBAT-induced amino acid transport with a different profile of action. The Pb2+ mediated inhibition occurred rapidly upon superfusion and was readily reversible upon washout. The maximal inhibition caused by Pb2+ was about 50% of the amino acid-induced currents at an apparent affinity (Km) of about 10 microM. In contrast, the Hg(2+)-mediated inhibition occurred rather slowly, depending on its concentration, and was not reversible during washout with control solution. However, the Hg(2+)-mediated amino acid transport inhibition could be reversed with Hg2+ chelating agents and reducing compounds. Other oxidative agents, such as the membrane permeable 2,2'-Dithio-bis(5-Nitropyridine) (DTNP), but not the membrane impermeable 5,5'-Dithio-bis (2-Nitrobenzoic acid) (DTNB), mimicked the effect of Hg2+, and their effect could similarly be reversed with 2,3-Dihydroxybutane-1,4-dithiol (DTE). In conclusion, Pb2+ and Hg2+ inhibit rBAT-induced amino acid transport in a noncompetitive, allosteric fashion. Blockade of rBAT-induced amino acid transport may be involved in aminoaciduria following mercury or lead intoxication.

Amino Acid Transport Systems, Basic↗
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↗
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↗