Biomedical subjects

R Greger

Publications and source records attributed to R Greger.

At least 19 recordsLinked to original sources

PubMed2002

The oxidant thimerosal modulates gating behavior of KCNQ1 by interaction with the channel outer shell.

Thimerosal (o-Ethylmercurithio)benzoic acid, TMS), a membrane-impermeable, sulfhydryl-oxidizing agent, has been described to increase the K+ current IKs in KCNE1-injected Xenopus laevis oocytes. Since there are no cysteine residues in the extracellular domain of KCNE1, it has been proposed that TMS interacts with its partner protein KCNQ1. The aim of this study was therefore to investigate the interaction of TMS with KCNQ1 and the respective K+current IK. In CHO cells stably transfected with KCNQ1/KCNE1, TMS increased IKs, whereas in CHO cells expressing KCNQ1 alone, TMS initially decreased IK. TMS also affected the cytosolic pH (pHi) and the cytosolic Ca2+ activity ([Ca2+]i) in these cells. TMS slowly decreased pHi. With a short delay, TMS increased [Ca2+]i by store depletion and capacitative influx. The time course of the effects of TMS on pHi and [Ca2+]i did not correlate with the effect of TMS on IK. We therefore anticipated a different mode of action by TMS and investigated the influence of TMS on cysteine residues of KCNQ1. For this purpose, KCNQ1wt and two mutants lacking a cysteine residue in the S6 or the S3 segment (KCNQ1C331A and KCNQ1C214A, respectively) were expressed in Xenopus laevis oocytes. A sustained current decrease was observed in KCNQ1wt and KCNQ1C331A, but not in KCNQ1C214A-injected oocytes. The analysis of tail currents, I/V curves and activation kinetics revealed a complex effect of TMS on the gating of KCNQ1wt and KCNQ1C331A. In another series we investigated the effect of TMS on IKs. TMS increased IKs of KCNQ1C214A/KCNE1-injected oocytes significantly less than IKs in KCNQ1wt/KCNE1- or KCNQ1C331A/KCNE1-injected cells. These results suggest that thimerosal interacts with the cysteine residue C214 in the S3 segment of KCNQ1, leading to a change of its gating properties. Our results support the idea that not only the inner shell, but also the outer shell of the channel is important for the gating behavior of voltage dependent K+ channels.

Animals↗

PubMed2002

Membrane potential dependent modulations of calcium oscillations in insulin-secreting INS-1 cells.

This study was undertaken to examine the role of K(+) channels on cytosolic Ca(2+) ([Ca(2+)](i)) in insulin secreting cells. [Ca(2+)](i) was measured in single glucose-responsive INS-1 cells using the fluorescent Ca(2+) indicator Fura-2. Glucose, tolbutamide and forskolin elevated [Ca(2+)](i) and induced [Ca(2+)] oscillations. Whereas the glucose effect was delayed and observed in 60% and 93% of the cells, in a poorly and a highly glucose-responsive INS-1 cell clone, respectively, tolbutamide and forskolin increased [Ca(2+)](i) in all cells tested. In the latter clone, glucose induced [Ca(2+)](i) oscillations in 77% of the cells. In 16% of the cells a sustained rise of [Ca(2+)](i) was observed. The increase in [Ca(2+)](i) was reversed by verapamil, an L-type Ca(2+) channel inhibitor. Adrenaline decreased [Ca(2+)](i) in oscillating cells in the presence of low glucose and in cells stimulated by glucose alone or in combination with tolbutamide and forskolin. Adrenaline did not lower [Ca(2+)](i) in the presence of 30mM extracellular K(+), indicating that adrenaline does not exert a direct effect on Ca(2+) channels but increases K(+) channel activity. As for primary b-cells, [Ca(2+)](i) oscillations persisted in the presence of closed K(ATP) channels; these also persisted in the presence of thapsigargin, which blocks Ca(2+) uptake into Ca(2+) stores. In contrast, in voltage-clamped cells and in the presence of diazoxide (50mM), which hyperpolarizes the cells by opening K(ATP) channels, [Ca(2+)](i) oscillations were abolished. These results support the hypothesis that [Ca(2+)](i) oscillations depend on functional voltage-dependent Ca(2+) and K(+) channels and are interrupted by a hyperpolarization in insulin-secreting cells.

Adrenergic alpha-Agonists↗

PubMed2001

Synthesis and activity of novel and selective I(Ks)-channel blockers.

Since the discovery of the I(Ks)-potassium channel as the slowly activating component of the delayed rectifier current (I(k)) in cardiac tissue, the search for blockers of this current has been intense. During the screening of K(ATP)-channel openers of the chromanol type we found that chromanol 293B was able to block I(Ks). Chromanol 293B is a sulfonamide analogue of the K(ATP)-channel openers but had no activity on this target. Experiments were initiated to improve the activity and properties based on this lead compound. As a screening model we used Xenopus oocytes injected with human minK (KCNE1). Variations of the aromatic substituent and the sulfonamide group were prepared, and their activity was evaluated. We found that the greatest influence on activity was found in the aromatic substituents. The most active compounds were alkoxy substituted. We chose HMR1556 ((3R, 4S)-(+)-N-[-3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methyl-ethanesulfonamide) 10a for development as an antiarrhythmic drug. The absolute configuration, resulting from an X-ray single-crystal structure analysis, was determined.

Animals↗

PubMed2001

Regulation of slowly activating potassium current (I(Ks)) by secretin in rat pancreatic acinar cells.

1. The secretagogue-activated K(+) conductance is indispensable for the electrogenic Cl(-) secretion in exocrine tissue. In this study, we investigated the effect of secretin and other cAMP-mediated secretagogues on the slowly activating voltage-dependent K(+) current (I(Ks)) of rat pancreatic acinar cells (RPAs) with the whole-cell patch clamp technique. 2. Upon depolarization, RPAs showed I(Ks) superimposed upon the instantaneous background outward current. Secretin (5 nM), vasoactive intestinal peptide (5 nM), forskolin (5 microM), isoprenaline (10 microM) or 3-isobutyl-1-methylxanthine (IBMX, 0.1 mM) increased the amplitude of I(Ks) two- to fourfold. 3. The physiological concentration of secretin (50 pM) had a relatively weak effect on I(Ks) (160 % increase), which was significantly enhanced by transient co-stimulation with carbachol (CCh) (10 microM). However, the secretin-induced production of cAMP, which was measured by enzyme-linked immunosorbent assay, was not augmented by co-stimulation with CCh. 4. This study is the first to demonstrate the regulation of K(+) channels in RPAs by cAMP-mediated agonists. The I(Ks) channel is a common target for both Ca(2+) and cAMP agonists. The vagal stimulation under the physiological concentration of secretin facilitates I(Ks), which provides an additional driving force for Cl(-) secretion.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Cystic fibrosis and CFTR.

Cystic fibrosis (CF) is a complex disease affecting epithelial ion transport. There are not many diseases like CF that have triggered such intense research activities. The complexity of the disease is due to mutations in the CFTR protein, now known to be a Cl(-) channel and a regulator of other transport proteins. The various interactions and the large number of disease-causing CFTR mutations is the reason for a variable genotype-phenotype correlation and sometimes unpredictable clinical manifestation. Nevertheless, the research of the past 10 years has resulted in a tremendous increase in knowledge, not only in regard to CFTR but also in regard to molecular interactions and completely new means of ion channel and gene therapy.

Cystic Fibrosis↗

PubMed2001

Cloning and function of the rat colonic epithelial K+ channel KVLQT1.

KVLQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of KVLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to KVLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of KVLQT1 in crypt cells and surface epithelium. Expression of rKVLQT1 in Xenopus oocytes induced a typical delayed activated K+ current, that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that KVLQT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias.

KCNQ1 (KVLQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study the properties and regulation of the cloned sKVLQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (<3 pS) K+ channels, in parallel with other K+ channels. sKCNQ1 generated similar small-conductance K+ channels upon expression in CHO cells and Xenopus oocytes. The results suggest the presence of low-conductance KCNQ1 K+ channels in RGT, which are probably regulated by changes in intracellular cAMP, Ca2+ and pH.

Animals↗

PubMed2001

Induction of the epithelial Na+ channel via glucocorticoids in mineralocorticoid receptor knockout mice.

Epithelial Na+ channel (ENaC) activity in kidney and colon is stimulated by aldosterone acting on the mineralocorticoid receptor (MR). MR and the glucocorticoid receptor (GR) show high homology in their DNA-binding domain and have similar affinities to mineralo- and glucocorticoids. We therefore asked whether the glucocorticoid-mediated activation of ENaC is restricted to the presence of MR and used the MR knockout mouse model to address this question. Due to their MR deficiency and the consecutive reduction of ENaC activity these mice die as neonates, and even after appropriate substitution therapy adult MR knockout mice suffer from high Na+ loss and hyperkalemia. In the present study, glucocorticoid treatment restored plasma K+ and almost normalized the fractional excretions of Na+ (FENa+) and K+ (FEK+) in adult salt-substituted MR knockout mice, while the effect of amiloride on FENa+ and FEK+ was augmented in these animals. In order to estimate ENaC activity, measurements of transepithelial equivalent short-circuit current (Isc) were performed. Glucocorticoids induced an amiloride-sensitive Na+ absorption in renal cortical collecting duct and distal colon of MR-/- of about 25% and 50% of the currents observed in glucocorticoid-treated wild-type mice, respectively. In the colon glucocorticoid treatment increased the mRNA abundance of all three ENaC subunits, in the kidney only alpha-ENaC was increased. The regulation of ENaC expression was the same in both genotypes and thus irrespective of the presence of MR. These data show that MR is no prerequisite for the activation of ENaC transcription and activity, and that the respective mechanisms can be stimulated via GR.

Amiloride↗

PubMed2001

The distal convoluted tubule of rabbit kidney does not express a functional sodium channel.

We sought to assess whether the distal convoluted tubule (DCT) segment of the rabbit nephron expresses a functional epithelial sodium channel. First, the transepithelial voltage (V(te), lumen vs. bath) was measured in isolated perfused DCT segments (assessed separately in the upstream half and the downstream half of the DCT). V(te) was zero and not affected by amiloride or barium in the upstream DCT. V(te) was sometimes negative in the downstream DCT and depolarized by amiloride and hyperpolarized by barium, suggesting inclusion of connecting tubule (CNT) cells. To determine expression of epithelial sodium channel (ENaC) mRNA subunits by the upstream DCT, rabbit alpha-, beta-, and gamma-ENaC cDNA fragments were cloned and primers were selected for single-nephron RT-PCR analysis. Although alpha-ENaC was expressed by the DCT, beta- and gamma-ENaC were not detected in the DCT. In contrast, the CNT, CCD, and outer medullary collecting duct (OMCD) expressed all three subunits. Nedd4 was also not detected in the DCT but was expressed by the CNT, CCD, and OMCD. When upstream DCT fragments were grown to confluent monolayers in primary culture, the epithelia exhibited negative voltages and high transepithelial resistances and expressed mRNA for all three ENaC subunits as well as for Nedd4. The absence of a negative voltage and failure to detect transcript for beta- and gamma-ENaC and Nedd4 in the native rabbit DCT suggest that the sodium channel is not a significant pathway for sodium absorption by this segment. The phenotype conversion observed when DCT cells are grown in culture does not rule out the possibility that there may be conditions in which the DCT in the intact kidney expresses sodium channel activity. The results are consistent with the notion that DCT sodium transport is predominantly, if not exclusively, electroneutral.

Animals↗

PubMed2000

Aquaporin 3 cloned from Xenopus laevis is regulated by the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is essential for epithelial electrolyte transport and has been shown to be a regulator of epithelial Na(+), K(+), and Cl(-) channels. CFTR also enhances osmotic water permeability when activated by cAMP. This was detected initially in Xenopus oocytes and is also present in human airway epithelial cells, however, the mechanisms remain obscure. Here, we show that CFTR activates aquaporin 3 expressed endogenously and exogenously in oocytes of Xenopus laevis. The interaction requires stimulation of wild type CFTR by cAMP and an intact first nucleotide binding domain as demonstrated for other CFTR-protein interactions.

Amino Acid Sequence↗

PubMed2000

A constitutively open potassium channel formed by KCNQ1 and KCNE3.

Mutations in all four known KCNQ potassium channel alpha-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the beta-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current I(Ks) that is affected in some forms of cardiac arrhythmia. Here we show that the novel beta-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.

Amino Acid Sequence↗

PubMed2000

pH regulation in isolated in vitro perfused rat colonic crypts.

We investigated disorders and regulation of cytosolic pH (pHi) in isolated perfused crypts from rat distal colon using the pH-sensitive dye BCECF. This preparation allows distinct examination of either luminal or basolateral transport. The effects of luminal weak organic acids and bases on pHi were examined. The physiological concentrations of both luminal CO2/HCO3- and acetic acid/acetate acidified pHi significantly, but less than when applied from the basolateral side. Corresponding changes (luminal versus basolateral) in pHi were -0.17+/-0.04 versus -0.39+/-0.04, (n=8) and -0.15+/-0.02 versus -0.41+/-0.04, (n=8), respectively. Basolateral versus luminal application of NH3/NH4+ led to a more marked change in pHi, namely 0.35+/-0.03 versus 0.008+/-0.007 pH units, (n=19). The luminal perfusion of NH3/NH4+ was controlled by applying fura-2 acid to the luminal side and at the same time recording fura-2-specific fluorescence. Hence, the influence of luminal acid/base on colonic pHi homeostasis was limited. To examine pHi regulation, we investigated the recovery from an intracellular acid load using the NH3/NH4+ pulse method. Recovery was completely dependent on basolateral Na+, indicating that luminal acid/base transport does not play a major role in pHi homeostasis. The basolateral transporters involved in pHi recovery are probably the EIPA- and HOE694-inhibitable (IC50=0.2 and 2 micromol/l, respectively) Na+/H+ exchanger NHE1 and the DIDS-inhibitable Na+-dependent HCO3- importer.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Inwardly rectifying K+ channels in the basolateral membrane of rat pancreatic acini.

Previous studies of the whole-cell K+ conductance suggest the presence of inwardly rectifying K+ channels (Kir) in rat pancreatic acini (RPAs). Here we investigate the properties of Kir of RPAs using patch-clamp techniques. The whole-cell current-to-voltage relationship of freshly isolated RPAs was steeper for inward currents than for outward currents when the extracellular K+ concentration ([K+]o) was raised. With a high [K+]o (145 mM), external application of Ba2+ and Cs+ blocked the inward K+ current in a voltage-dependent manner. The apparent IC50 of Ba2+ was 8.5+/-1.9 microM and 1.1+/-0.2 microM at -70 mV and -130 mV, respectively (n=5). The IC50 of Cs+ was 3.5+1.1 mM and 0.2+0.1 mM at -60 mV and -120 mV, respectively (n=4). Application of Ba2+ (0.1 mM) to the extracellular solution reversibly depolarized RPAs from -43+1.1 mV to -37+/-1.2 mV (n=20). In the cell-attached configuration with 145 mM KC1 in the pipette solution, we observed inwardly rectifying channels with a high open probability (PO) of 0.85+/-0.02 (n=6) and a slope conductance (Gs) of 30+/-2.8 pS (n=13). The same type of channel was observed in the outside-out patch. We could also observe a very small conductance K+ channel which was resistant to 0.1 mM Ba2+ and did not show inward rectification (n=11). RT-PCR analysis of RPA confirmed the presence of transcripts for Kir2.1, Kir2.3 and Kir7.1 subfamilies as molecular candidates for the observed channels. The above results demonstrate the presence of Kir channels in the basolateral membrane of the RPA, which may be important for the K+ recycling process during electrolyte secretion as well as for maintaining a hyperpolarized membrane.

Animals↗

PubMed2000

KCNE1 reverses the response of the human K+ channel KCNQ1 to cytosolic pH changes and alters its pharmacology and sensitivity to temperature.

Previous studies have shown that heteromultimeric KCNQ1/KCNE1 (KvLQT1/minK) channels and homomultimeric KCNQ1 (KvLQT1) channels exhibit different current properties, e.g. distinct kinetics and different sensitivities to drugs. In this study we report on the divergent responses to internal pH changes and further characterize some of the current properties of the human isoforms of KCNQ1 and KCNE1 expressed in Chinese hamster ovary (CHO) cells or Xenopus laevis oocytes. Decreasing the bath temperature from 37 degrees C to 20 degrees C increased the half-activation time by a factor of 5 for KCNQ1/KCNE1 currents (IKs) but by only twofold (not significant) for KCNQ1 currents (IK) in CHO cells. Acidification of cytosolic pH (pHi) increased IKs but decreased 1K whereas intracellular alkalinization decreased I(Ks) but increased IK. pHi-induced changes in intracellular Ca2+ activity ([Ca2+]i) did not correlate with the current responses. At 20 degrees C mefenamic acid (0.1 mM) significantly augmented IKs but slightly decreased IK. It changed the slow activation kinetics of I(Ks) to an instantaneous onset. The form of the current/voltage (I/V) curve changed from sigmoidal to almost linear. In contrast, at 37 degrees C, mefenamic acid also increased I(Ks) but slowed the activation kinetics and shifted the voltage activation to more hyperpolarized values without markedly affecting the sigmoidal shape of the I/V curve. The potassium channel blockers clotrimazole and tetrapentylammonium (TPeA) inhibited I(Ks) with a lower potency than I(K). These results show that coexpression of KCNE1 reversed pH regulation of KCNQ1 from inhibition to activation by acidic pHi. In addition, KCNE1 altered the pharmacological properties and sensitivity to temperature of KCNQ1. The pH-dependence of I(Ks) might be of clinical and pathophysiological relevance in the pathogenesis of ischaemic cardiac arrhythmias.

Animals↗

PubMed2000

Regulation of the Na+2Cl-K+ cotransporter in isolated rat colon crypts.

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.

Algorithms↗

PubMed2000

Deoxycholic acid (DOC) affects the transport properties of distal colon.

Secondary bile acids can induce diarrhea. In the present study we examined the effects of deoxycholic acid (DOC) on equivalent short-circuit current (Isc) in rabbit colon and the cellular mechanisms involved in DOC action (rabbit and rat). Luminal DOC inhibited amiloride-sensitive Na+ absorption. In the presence of amiloride luminal DOC had a concentration dependent effect on Isc. Low concentrations (1-10 micromol/l) induced a lumen-positive current (51+/-3 microA/cm2, 10 micromol/l, n=7) which was inhibited by luminal Ba2+ suggesting the activation of a luminal K+ conductance. Higher luminal concentrations induced a lumen-negative current (-76+/-9 microA/cm2, 100 micromol/l, n=11). Basolateral application of DOC, also in the presence of amiloride, only induced lumen-negative Isc, (-58+/-10 microA/cm2, 100 micromol/l, n=6, EC50= 3 micromol/l). This current could be abolished completely by the K+ channel blocker 293B, a selective inhibitor of cAMP-dependent Cl- secretion. This action of DOC on Isc was additive to the effect of carbachol (CCH) but not additive to that of cAMP. In intact rat colon mucosa pre-treated with DOC a significant increase in cAMP production was observed. Fura-2 measurements of cytosolic Ca2+ activity ([Ca2+]i) in isolated colonic crypts (rabbit and rat) showed that 100 micromol/l DOC induced a weak [Ca2+]i increase. Whole-cell measurements of membrane voltage in isolated rat colonic crypts revealed a hyperpolarization by DOC (4.9+/-0.8 mV, 100 micromol/l, n=8) but a depolarization by prostaglandin E2 (PGE2, via cAMP) (24+/-7 mV, n=8). The present data show that DOC acts at more than one target in the colon: in the intact mucosa it activates luminal K+ channels and Cl- secretion and this is paralleled by an increase in cAMP production. In isolated crypts DOC probably activates a Ca(2+)-regulated K+ conductance but has no effect on cAMP. Hence DOC probably activates ion channels or channel-regulating factors in colonocytes and acts on non-epithelial cells to activate Cl- secretion indirectly.

Amiloride↗

PubMed2000

Effect of genistein on native epithelial tissue from normal individuals and CF patients and on ion channels expressed in Xenopus oocytes.

The flavonoid genistein has been shown to activate a Cl(-) conductance in various cell types expressing CFTR. We examined if similar effects can be observed when genistein is applied to native ex vivo tissues from human respiratory tract and rectum. We further compared the effects when genistein was applied to oocytes of Xenopus laevis expressing CFTR. In oocytes, both wtCFTR and DeltaF508-CFTR were activated by genistein while both cyclic AMP (K(v)LQT1) and Ca(2+) (SK4) activated K(+) channels were inhibited at high concentrations of genistein. Biopsies from nasal polyps and rectal mucosa were obtained from normal individuals (non-CF) and CF patients and in the presence of amiloride (10 micromol l(-1); mucosal side) the effects of genistein were assessed using a perfused Ussing chamber. In non-CF airway epithelia, genistein (50 micromol l(-1); mucosal side) increased lumen negative I(sc) but had no additional effects on tissues pre-stimulated with IBMX and forskolin (100 micromol l(-1) and 1 micromol l(-1); both sides). In non-CF rectal biopsies, in the presence of amiloride (10 micromol l(-1); mucosal side) and indomethacin (10 micromol l(-1); basolateral side), genistein increased lumen negative I(sc) and enabled cholinergic (carbachol; CCH, 100 micromol l(-1); basolateral side) stimulation of Cl(-) secretion indicating activation of luminal CFTR Cl(-) channels. However, after stimulation with IBMX/forskolin, genistein induced opposite effects and significantly inhibited CCH activated I(sc). In CF airway and intestinal tissues genistein failed to induce Cl(-) secretion. Thus, genistein is able to activate luminal CFTR Cl(-) conductance in non-CF tissues and mutant CFTR in oocytes. However, additional inhibitory effects on basolateral K(+) conductance and missing effects in native CF tissues do not support the use for pharmacological intervention in CF.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus.

UNLABELLED: Angiotensin II increases the intracellular calcium activity in podocytes of the intact glomerulus. BACKGROUND: Knowledge about biological functions of podocytes in the glomerulus is limited because of its unique anatomical location. Here we introduce a new method for measuring the intracellular calcium activity ([Ca2+]i) in the podocyte in the intact glomerulus. METHODS: With the help of fluorescence high-resolution digital imaging and a recently developed ultraviolet laser-scanning microscope, [Ca2+]i was measured in fura-2-loaded glomeruli and single podocytes of intact microdissected rat glomeruli. RESULTS: Angiotensin II (Ang II) increased [Ca2+]i reversibly in a biphasic and concentration-dependent manner. In contrast to Ang II, bradykinin, thrombin, arginine vasopressin, and serotonin did not change [Ca2+]i in the glomerulus. At reduced extracellular Ca2+ activity, Ang II released [Ca2+]i from intracellular stores, but the second phase, corresponding to a Ca2+ influx from the extracellular space, was absent. The L-type Ca2+ channel blocker nicardipine did not influence the Ang II-mediated [Ca2+]i increase, and an increase of the extracellular K+ concentration did not change [Ca2+]i in the glomerulus. The angrotensin II type I (AT1) receptor antagonist losartan inhibited the Ang II-mediated [Ca2+]i increase. Confocal [Ca2+]i measurements using fura-2 or fluo-3 or fluo-4 on the single cell level show that some of the Ang II-mediated [Ca2+]i response originated from podocytes. Costaining with calcein allowed the identification of podocytes because of the characteristic morphology and location in relationship to the capillary network. CONCLUSIONS: These data suggest that podocytes in the intact glomerulus respond to Ang II with an increase of [Ca2+]i via an AT1 receptor.

Angiotensin II↗