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R Greger

Publications and source records attributed to R Greger.

At least 127 records · Page 7Linked to original sources
PubMed1996

Extracellular ATP regulates glomerular endothelial cell function.

1. Glomerular endothelial cells form the inner part of the filtration barrier and are involved in pathophysiological processes in the glomerulum. New techniques for culturing glomerular endothelial cells have been established recently. The effect of extracellular ATP on membrane voltage and intracellular calcium activity was examined in bovine glomerular endothelial cells (GEC) in culture. 2. Membrane voltage was measured with the patch clamp technique in the fast whole cell configuration. GEC possess a stable membrane voltage of -88 mV. ATP induced a small transient hyperpolarization, which was followed by a depolarization. The ATP-induced depolarization was significantly inhibited by flufenamate, a blocker of non-selective ion channels. 3. The intracellular calcium activity [Ca2+]i was measured in single cells with the fura-2 technique. ATP stimulated an increase of [Ca2+]i. The increase of [Ca2+]i was biphasic with an initial peak followed by a sustained plateau. The [Ca2+]i peak was still present in an extracellular Ca(2+)-free solution, whereas the plateau was inhibited. 4. The order of potency of different purine nucleotides in stimulating [Ca2+]i and inositol formation was UTP = ATP > ATP-gamma-S > 2-methylthio ATP > [alpha,beta-CH2]ATP. 5. The data indicate that ATP regulates membrane voltage and [Ca2+]i in glomerular endothelial cells by a P2y2 receptor.

Adenosine Triphosphate↗
PubMed1996

Regulation of the intracellular calcium concentration in epithelial cells.

Epithelia, like other vertebrate cells, are equipped with an extensive network of signaling pathways, involved in the regulation of a large variety of distinct cellular functions. Amongst many the intracellular Ca2+ [Ca2+]i signal is the most common one. On the one hand, [Ca2+]i is involved in regulation functions of general cell physiology, such as the control of cell volume, cell growth or differentiation. On the other hand, [Ca2+]i controls epithelia-specific functions like the regulation of transepithelial solute transport. This short overview addresses the various aspects of Ca2+ signaling in epithelia. It is emphasized that [Ca2+]i is a spatially restricted signal, permitting the distinct regulation of one cellular response. Evidence will be discussed indicating that the functional epithelial polarity is paralleled by polarized membrane receptors and polarized [Ca2+]i signals, regulating the apical and basolateral membranes differentially.

Animals↗
PubMed1996

The mechanisms of renal tubule electrolyte and water absorption, 100 years after Carl Ludwig.

Some 154 years after Carl Ludwig's Habilitationsschrift "Contributions to the theory of the mechanism of urine secretion" renal physiology has come a long way. The mechanisms of urine formation are now understood as the result of glomerular filtration and tubule absorption of most of the filtrate. The detailed understanding of tubule transport processes has become possible with the invention of several refined techniques such as the micropuncture techniques; the microchemical analysis of nanolitre tubule fluid samples; the in vitro perfusion of isolated tubule segments of defined origin; electrophysiological analysis of electrolyte transport including micropuncture and patch-clamp techniques; transport studies in membrane vesicle preparations; recordings of intracellular electrolyte concentrations and cloning techniques of the individual membrane transport proteins. With this wealth of information we are now starting to build an integrative understanding of the function of the individual nephron segments, the regulatory processes, the integrated function of the nephron and hence the formation of the final urine. Like anatomists of previous centuries we still state that the kidney is an "organum mirable" and we recognize that basic research in this area has fertilized the analysis of the function of a large number of other organs and cells.

Absorption↗
PubMed1995

Mutations in the putative pore-forming domain of CFTR do not change anion selectivity of the cAMP activated Cl- conductance.

Cystic fibrosis transmembrane conductance regulator (CFTR) apparently forms Cl- channels in apical membranes of secretory epithelial cells. A detailed model describes molecular structure and biophysical properties of CFTR and the impact of various mutations as they occur in cystic fibrosis. In the present report mutations were introduced into the putative 6th alpha-helical transmembrane pore forming domain of CFTR. The mutants were subsequently expressed in Xenopus oocytes by injection of the respective cRNAs. Whole cell (wc) conductances could be reversibly activated by IBMX (1 nmol/l) only in oocytes injected with wild-type (wt) or mutant CFTR but not in oocytes injected with water or antisense CFTR. The activated conductance was partially inhibited by (each 100 mumol/l) DIDS (27%) and glibenclamide (77%), but not by 10 mumol/l NPPB. The following mutations were examined: K335E, R347E, R334E, K335H, R347H, R334H. They did not measurably change the wt-CFTR anion permeability (P) and we conductance (G) sequence of: PCl- > PBr- > P1- and GCl- > GBr- > G1-, respectively. Moreover, anomalous mole fraction behavior for the cAMP activated current could not be detected: neither in wt-CFTR nor in R347E-CFTR. Various mutants for which positively charged amino acids were replaced by histidines (K335H, R347H, R334H) did not show pH sensitivity of the IBMX activated wc conductance. We, therefore, cannot confirm previous results. CFTR might have a different molecular structure than previously suggested or it might act as a regulator of ion conductances.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Actin-dependent activation of ion conductances in bronchial epithelial cells.

Activation of Cl- and K+ channels is necessary to drive ion secretion in epithelia. There is substantial evidence from previous reports that vesicular transport and exocytosis are involved in the regulation of ion channels. In the present study we examined the role of cytoskeletal elements and components of intracellular vesicle transport on ion channel activation in bronchial epithelial cells. To this end, cells were incubated with a number of different compounds which interact with either microtubules or actin microfilaments, or which interfere with vesicle transport in the Golgi apparatus. The effectiveness of these agents was verified by fluorescence staining of cellular microtubules and actin. The function was examined in 36Cl- efflux studies as well as in whole-cell (WC) patch-clamp and cell-attached studies. The cells were studied under control conditions and after exposure to (in mmol/l) ATP (0.1), forskolin (0.01), histamine (0.01) and hypotonic bath solution (HBS, NaCl 72.5). In untreated control cells, ATP primarily activated a K+ conductance whilst histamine and forskolin induced mainly a Cl- conductance. HBS activated both K+ and Cl- conductances. Incubation of the cells with brefeldin A (up to 100 mumol/l) did not inhibit WC current activation and 36Cl- efflux. Nocodazole (up to 170 mumol/l) reduced the ATP-induced WC current, and mevastatin (up to 100 mumol/l) the cell-swelling-induced WC current. Neither had any effect on the WC current induced by forskolin and histamine. Also 36Cl- efflux induced by HBS, ATP, forskolin and histamine was unaltered by these compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1995

Effect of NH4+/NH3 on cytosolic pH and the K+ channels of freshly isolated cells from the thick ascending limb of Henle's loop.

The conductance properties of the luminal membrane of cells from the thick ascending limb of Henle's loop of rat kidney (TAL) are dominated by K+. In excised membrane patches the luminal K+ channel is regulated by pH changes on the cytosolic side. To examine this pH regulation in intact cells of freshly isolated TAL segments we measured the membrane voltage (Vm) in slow-whole-cell (SWC) recordings and the open probability (Po) of K+ channels in the cell-attached nystatin (CAN) configuration, where channel activity and part of Vm can be recorded. The pipette solution contained K+ 125 mmol/l and Cl- 32 mmol/l. Intracellular pH was determined by 2',7'bis(2-carboxyethyl)-5,(6)-carboxyfluorescein (BCECF) fluorescence. pH changes were induced by the addition of 10 mmol/l NH4+/NH3 to the bath. In the presence of NH4+/NH3 intracellular pH acidified by 0.53 +/- 0.11 units (n = 7). Inhibition of the Na+2Cl-K+ cotransporter by furosemide (0.1 mmol/l) reversed this effect and led to a transient alkalinisation by 0.62 +/- 0.14 units (n = 7). In SWC experiments Vm of TAL cells was -72 +/- 1 mV (n = 70). NH4+/NH3 depolarised Vm by 22 +/- 2 mV (n = 25). In 11 SWC experiments furosemide (0.1 mmol/l) attenuated the depolarising effect of NH4+ from 24 +/- 3 mV to 7 +/- 3 mV. Under control conditions the single-channel conductance of TAL K+ channels in CAN experiments was 66 +/- 5 pS and the reversal voltage for K+ currents was 70 +/- 2 mV (n = 35). The Po of K+ channels in CAN patches was reduced by NH4+/NH3 from 0.45 +/- 0.15 to 0.09 +/- 0.07 (n = 7). NH4+/NH3 exposure depolarised the zero current voltage of the permeabilised patches by -9.7 +/- 3.6 mV (n = 5). The results show that TAL K+ channels are regulated by cytosolic pH in the intact cell. The cytosolic pH is acidified by NH4+/NH3 exposure at concentrations which are physiologically relevant because Na+2Cl-K+(NH4+) cotransporter-mediated import of NH4+ exceeds the rate of NH3 diffusion into the TAL. K+ channels are inhibited by this acidification and the cells depolarise. In the presence of furosemide TAL cells alkalinise proving that NH4+ uptake occurs by the Na+2Cl-K+ cotransporter. The findings that, in the presence of NH4+/NH3 and furosemide, Vm is not completely repolarised and that K+ channels are not activated suggest that the respective K+ channels may in addition to their pH regulation be inhibited directly by NH4+/NH3.

Ammonia↗
PubMed1995

Na+ and Cl- conductances in airway epithelial cells: increased Na+ conductance in cystic fibrosis.

Na+ and Cl- conductances in the apical membrane of respiratory epithelial cells are essential for electrolyte and water transport in the airways. Apart from the well described defect in adenosine 3' : 5' cyclic monophosphate-(cAMP-) dependent activation of Cl- conductances in cystic fibrosis (CF), an increased Na+ conductance has also been reported from transepithelial measurements. In the present experiments we tried to identify these conductances in nasal epithelial cells using patch-clamp and microelectrode techniques. With these methods we found identical and relatively low membrane voltages of about -36 mV in both freshly isolated and primary cultured normal and CF nasal epithelial cells. A Cl- conductance could be activated by cAMP in normal (deltaG = 0.3 +/- 0.8 nS, n = 10) but not in CF (deltaG = 0.3 +/- 0.1 nS, n = 11) cells, whereas Ca2+-dependent Cl- currents activated by adenosine 5'-triphosphate (ATP) and bradykinin were present in both types of cells. Cell-attached membrane patches from stimulated cells did not reveal discernible single-channel events when activated with any of the agonists. A Na+ conductance was also detected in freshly isolated ciliated respiratory cells in impalement studies, as evidenced by the hyperpolarization induced by 10 micromol/l amiloride (deltaV = -5.2 +/- 0.6 mV, n = 56) and when Na+ was replaced in the bath by N-methyl-D-glucamine (NMDG) (deltaV = -5.7 +/- 0.9 mV, n = 14). In whole-cell patch-clamp experiments, the amiloride-induced hyperpolarization was significantly larger in CF (deltaV = 9.7 +/- 2.4 mV, n = 22) when compared to normal (deltaV = -3.3 +/- 0.9 mV, n = 27) cells in short-term culture. Reverse transcriptase polymerase chain reaction analysis of normal respiratory cells identified messenger RNA of both the cystic fibrosis transmembrane conductance regulator (CFTR) as well as the human epithelial Na+ channel (hNaCh). The present experiments confirm the absence of a cAMP-dependent Cl- conductance in CF respiratory epithelial cells and support previous findings obtained in transepithelial and microelectrode studies which indicate an increased Na+ conductance in respiratory epithelial cells from CF patients.

Amiloride↗
PubMed1995

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in Ht29 colonic carcinoma cells.

Cl- secretion in HT29 cells is regulated by agonists such as carbachol, neurotensin and adenosine 5'-triphosphate (ATP). These agonists induce Ca2+ store release as well as Ca2+ influx from the extracellular space. The increase in cytosolic Ca2+ enhances the Cl- and K+ conductances of these cells. Removal of extracellular Ca2+ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca2+ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n=6) inhibited ATP (0.1 mmol.l-1) induced increases in whole-cell conductance (Gm). When Cl- and K+ currents were inhibited by the presence of Cs2SO4 in the patch pipette and gluconate in the bath, ATP (0.1 mmol.l-1) still induced a significant increase in Gm from 1.2 +/- 0.3 nS to 4.7 +/- 1 nS (n = 24). This suggests that ATP induces a cation influx with a conductance of approximately 3-4 nS. This cation influx was inhibited by flufenamate (0.1 mmol.l-1, n = 6) and Gd3+ (10 micromol.l-1, n = 9). When Ba2+ (5 mmol.l-1) and 4,4'-diisothiocyanato-stilbene-2-2'-disulphonic acid (DIDS, 0.1 mmol.l-1) were added to the KCl/K-gluconate pipette solution to inhibit K+ and Cl- currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol.l-1) reduced the membrane current (Im) significantly from 86 +/- 14 pA to 54 +/- 11 pA (n = 13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5-10 mmol.l-1 1,2-bis-(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The zero-current membrane voltage (Vm) and Im (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30-120 s after membrane rupture. Vm depolarised significantly from -33 +/- 2 mV to -12 +/- 1 mV, and Im fell significantly from 17 +/- 2 pA to 8.9 +/- 1.0 pA (n = 71). This negative current, representing a cation inward current, was activated when Ca2+ stores were emptied and was reduced significantly ( Im) when Ca2+ and/or Na+ were removed from the bathing solution: removal of Ca2+ in the absence of Na+ caused a Im of 5.0 +/- 1.2 pA (n = 12); removal of Na+ in the absence of Ca2+ caused a Im of 12.8 +/- 3.5 pA (n = 4). The cation inward current was also reduced significantly by La3+, Gd3+, and flufenamate. We conclude that store depletion induces a Ca2+/Na+ influx current in these cells. With 145 mmol.l-1 Na+ and 1 mmol.l-1 Ca2+, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.

Adenosine Triphosphate↗
PubMed1995

Polarized ion transport during migration of transformed Madin-Darby canine kidney cells.

Epithelial cells lose their usual polarization during carcinogenesis. Although most malignant tumours are of epithelial origin little is known about ion channels in carcinoma cells. Previously, we observed that migration of transformed Madin-Darby canine kidney (MDCK-F) cells depended on oscillating K+ channel activity. In the present study we examined whether periodic K+ channel activity may cause changes of cell volume, and whether K+ channel activity is distributed in a uniform way in MDCK-F cells. After determining the average volume of MDCK-F cells (2013+/-270 microm3; n=8) by means of atomic force microscopy we deduced volume changes by calculating the K+ efflux during bursts of K+ channel activity. Therefore, we measured the membrane conductance of MDCK-F cells which periodically rose by 22.3+/-2.5 nS from a resting level of 6.5+/-1.4 nS (n=12), and we measured the membrane potential which hyperpolarized in parallel from -35.4+/-1.2 mV to -71.6+/-1.8 mV (n=11). The distribution of K+ channel activity was assessed by locally superfusing the front or rear end of migrating MDCK-F cells with the K+ channel blocker charybdotoxin (CTX). Only exposure of the rear end to CTX inhibited migration providing evidence for "horizontal" polarization of K+ channel activity in transformed MDCK-F cells. This is in contrast to the "vertical" polarization in parent MDCK cells. We propose that the asymmetrical distribution of K+ channel activity is a prerequisite for migration of MDCK-F cells.

Animals↗
PubMed1995

The effect of secretagogues on ion conductances of in vitro perfused, isolated rabbit colonic crypts.

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca2+ activity and are known to increase Cl- secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (Vte), transepithelial resistance (Rte), membrane voltage across the basolateral membrane (Vbl), and fractional basolateral membrane resistance (FRbl), were estimated. Basolateral prostaglandin E2 (PGE2, > or = 0.1 mumol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of Vbl. In the case of adenosine, the initial depolarization of Vbl was by 31 +/- 2 mV (n = 47). Rte fell significantly from 16.4 +/- 3.6 to 14.2 +/- 3.7 omega.cm2 (n = 6), and FRbl increased significantly from 0.11 +/- 0.02 to 0.51 +/- 0.10 (n = 6). In the second phase the repolarisation of Vbl amounted 11 +/- 2 mV (n = 47) and a steady-state (Vbl) of -51 +/- 2 mV (n = 47) was reached. Rte fell further and significantly to a steady-state value of 12.4 +/- 3.8 omega.cm2 (n = 6) and FRbl fell significantly to 0.42 +/- 0.13 (n = 6). In 30% of the experiments, a transient hyperpolarisation of Vbl by 8 +/- 2 mV (n = 14) was seen during wash out of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1994

Effects of P-glycoprotein expression on cyclic AMP and volume-activated ion fluxes and conductances in HT-29 colon adenocarcinoma cells.

The tissue distribution of P-glycoprotein (Pgp) and the structurally related cystic fibrosis transmembrane conductance regulator (CFTR) is apparently mutually exclusive, particularly in epithelial; where one protein is expressed the other is not. To study the possible function(s) of Pgp and its potential effects on CFTR expression in epithelia, HT-29 colon adenocarcinoma cells, which constitutively express CFTR, were pharmacologically adapted to express the classical multidrug resistance (MDR) phenotype (Pgp+). Concomitant with the appearance of Pgp and MDR phenotype (drug resistance, reduced drug accumulation and increased drug efflux), CFTR levels and cAMP-stimulated Cl conductances were markedly decreased compared to wild-type HT-29 (Pgp-) cells (as shown using the whole cell patch clamp technique). Removal of drug pressure led to the gradual decrease in Pgp levels and MDR phenotype, as evidenced by increased rhodamine 123 accumulation (Pgp-Rev). Concomitantly, CFTR levels and cAMP-stimulated Cl- conductances increased. The cell responses of Pgp/Rev cells were heterogeneous with respect to both Pgp and CFTR functions. We also studied the possible contribution to Pgp to hypotonically activated (HCS) ion conductances. K+ and Cl- effluxes from Pgp- cells were markedly increased by HCS. This increase was twice as high as that induced by the cation ionophore gramicidin; it was blocked by the Cl- channel blocker DIDS (4,4'-disothiocyano-2,2'-disulfonic stilbene) and required extracellular Ca2+. In Pgp+ cells, the HCS-induced fluxes were not significantly different from those of Pgp- cells. Verapamil (10 microM), which caused 80% reversal of Pgp-associated drug extrusion, failed to inhibit the HCS-evoked Cl- efflux of Pgp+ cells. Similarly, HCS increased Cl- conductance to the same extent in Pgp-, Pgp+ and Pgp-Rev cells. Verapamil (100 microM), but not 1,9-dideoxyforskolin (50 and 100 microM), partially inhibited the HCS-evoked whole cell current (WCC) in all three lines. Since the inhibition by verapamil was not detected in the presence of the K+ channel blocker Ba2+ (3 mM), it is suggested that verapamil affects K+ and not Cl- conductance. We conclude that hypotonically activated Cl- and K+ conductances are similar in HT-29 cells irrespective of Pgp expression. Expression of high levels of Pgp in HT-29 cells confers no physiologically significant capacity for cell volume regulation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Flufenamate and Gd3+ inhibit stimulated Ca2+ influx in the epithelial cell line CFPAC-1.

The relevant influx pathway for stimulated Ca2+ entry into epithelial cells is largely unknown. Using flufenamate (Flu) and Gd3+, both known pharmacological blockers of non-selective cation currents in other epithelial preparations, we tested whether the stimulated Ca2+ entry in CFPAC-1 cells was inhibited by these agents. Transmembraneous Ca2+ influx into CFPAC-1 cells was stimulated by either ATP (10(-4) and 10(-5) mol/l), carbachol (CCH, 10(-4) mol/l) or thapsigargin (TG, 10(-8) mol/l). Three different experimental approaches were used. (1) Because the plateau phase of an agonist-induced [Ca2+]i transient reflects Ca2+ influx into these cells, we investigated the influence of Flu and Gd3+ on the level of the stimulated [Ca2+]i plateau. (2) The fura-2 Mn(2+)-quenching technique was used to visualise divalent cation entry and monitor its inhibition. (3) During the "refilling period" after agonist-induced discharge of the intracellular pools the putative influx inhibitors Flu and Gd3+ were given and subsequently the filling state of the agonist-sensitive intracellular stores tested. The results from the first experimental approach showed that both Flu and Gd3+ were potent inhibitors of the stimulated Ca2+ entry in CFPAC-1 cells. Flu reversibly decreased the ATP-induced [Ca2+]i plateau in a concentration dependent manner, with an IC50 value of 33 mumol/l (n = 6). Similar results were obtained for the CCH- (n = 5) and the TG-induced (n = 5) [Ca2+]i plateau. Gd3+ concentration dependently inhibited the stimulated Ca2+ plateau. A complete block of the ATP-induced [Ca2+]i plateau was seen at 0.5 mumol/l (ATP 10(-5) mol/l, n = 8).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

cAMP-dependent activation of ion conductances in bronchial epithelial cells.

The cAMP-dependent activation of Cl- channels was studied in a bronchial epithelial cell line (16HBE14o-) in fast and slow whole-cell, and cell-attached patch-clamp experiments. The cells are known to express high levels of cystic fibrosis transmembrane conductance regulator mRNA and protein. Isoproterenol, forskolin and histamine (all 10 mumol/l) reversibly and significantly depolarized the membrane voltage (Vm) and increased the whole-cell Cl- conductance significantly by 34.0 +/- 0.9 (n = 3), 18.1 +/- 2.7 (n = 50), and 25 +/- 4.5 (n = 37) nS respectively. The effect of histamine was blocked by cimetidine (10 mumol, n = 5) but not by diphenhydramine (10 mumol/l, n = 4), which suggests binding of histamine to H2 receptors. The forskolin-induced current was not inhibited significantly by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (0.5 mmol/l, n = 9) nor glibenclamide (10 mumol/l, n = 3) and had an anion-permeability sequence of Cl = Br- > I- (n = 9). In cell-attached recordings forskolin (10 mumol/l) increased the conductance of the patched membrane from 65.5 +/- 13.6 pS to 150.8 +/- 33.2 pS (n = 30). Although the conductance was increased significantly, clear ion channel events occurring in parallel with the current activation were not detected in the cell-attached membrane. In 4 out of 30 cell-attached recordings single-channel currents were observed. These channels, with a single-channel conductance of about 6 pS, were already active before forskolin was added. No effect of forskolin on the channel amplitude, open probability or kinetics of these channels was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchi↗
PubMed1994

Ca(2+)- and swelling-induced activation of ion conductances in bronchial epithelial cells.

The present study was performed to examine Ca(2+)-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (Vte) and resistance (Rte) were measured in confluent monolayers. Resting cells had a membrane voltage (Vm) of -36 +/- 1.1 mV (n = 137) which was mainly caused by K+ and Cl- conductances and to a lesser extent by a Na+ conductance. Vte was apical-side-negative after stimulation. Equivalent short-circuit (Isc = Vte/Rte) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1 - 10 mumol/l). and ATP (0.1 - mumol/l). The histamine-induced Is was blocked by either basolateral diphenhydramine (0.1 mmol/l, n = 4) or apical cimetidine (0.1 mmol/l, n = 4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K+ conductance and hyperpolarized Vm. This effect was mimicked by the Ca2+ ionophore ionomycin (1 mumol/l, n = 11). Inhibition of the bradykinin-induced Isc by the blocker HOE140 (1 mumol/l, n = 3) suggested the presence of a BK2 receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP approximately ATP > ITP > GTP approximately CTP approximately [beta, gamma-methylene] ATP approximately 2-methylthio-ATP = 0 and was obtained in Isc measurements and patch-clamp recordings. This suggests the presence of a P2u receptor. Hypotonic cell swelling activated both Cl- and K+ conductances.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP, Ca2+ and hypotonic cell swelling.

HT29 cells were preincubated with forskolin (10(-5) mol/l, FORHT) or phorbol 12-myristate 13-acetate (PMA) (10(-7) mol/l, PMAHT) for 20 h, which has been shown previously and is also shown here, to upregulate and downregulate, respectively, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFPAC-1 cells underwent the same protocols. HT29 cells were examined by slow (SWC) and fast (FWC) whole-cell patch-clamp techniques. The results of SWC and FWC were indistinguishable and were pooled. CFPAC-1 cells were examined with FWC. The membrane voltage (V) of FORHT was -41.8 +/- 1.4 mV (n = 77) and that of PMAHT was -43.6 +/- 2.4 mV (n = 76). The conductance (G) of FORHT (9.4 +/- 0.9 nS, n = 77) was significantly larger than that of PMAHT (3.7 +/- 0.4 nS, n = 76). Acute application of forskolin (10(-5) mol/l, FOR) plus 0.5 mmol/l 8-(4-chlorophenylthio)-cAMP (cAMP) depolarized V by 12 (FORHT) and 8 (PMAHT) mV, respectively. The acute increase of G by FOR plus cAMP was by 7.6 +/- 1.9 nS for FORHT (n = 22) and only 2.2 +/- 1 nS for PMAHT (n = 13). ATP (10(-4) mol/l) depolarized V in both types of cells. It enhanced G by 16.7 +/- 4.1 nS in FORHT (n = 14) and significantly less (by 5.5 +/- 1.2 nS, n = 14) in PMAHT. Also the G increase lasted longer in FORHT. Neurotensin (NT, 10(-8) mol/l) also had a stronger and longer lasting effect in FORHT.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Analysing ion channels with hidden Markov models.

Ion channel current amplitudes (mu) and open probabilities (Po) have been analysed so far by defining a 50% threshold to distinguish between open and closed states of the channels. With this standard method (SM) it is very difficult or even impossible to analyse channels of different size in one membrane patch correctly. A stochastical model, named the hidden Markov model (HMM), separates between observation noise and the stochastic process of opening and closing of ion channels. The HMM allows the independent analysis of mu, Po, and mean dwell times (tau) of different channels in one membrane patch, without defining threshold levels. Using this method errors in the analysis are not summarized like in the SM because all different analysing procedures (e.g. filtering, setting of threshold, fitting processes) are done in one step. Two different K+ channels in excised basolateral membranes of the cortical collecting duct of rat (CCD) were analysed by the SM and the HMM. The mu value of the intermediate-conductance K+ channel (i-K+) was 3.9 +/- 0.1 pA (SM) and 3.8 +/- 0.2 pA (HMM) for 11 observations. The Po value of this channel was 10.2 +/- 4.2% (SM) and 10.1 +/- 4.0% (HMM). The mean tau values were 5.4 +/- 0.6 ms for the open state and 9.6 +/- 2.2 ms and 145 +/- 21 ms for the closed states (SM) and 7.8 +/- 1.1 ms, 7.7 +/- 0.9 ms and 148 +/- 24 ms (HMM), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1994

Effects of dinitrophenol on active-transport processes and cell membranes in the Malpighian tubule of Formica.

In Formica Malpighian tubules KCl secretion is driven by a V-type H+ ATPase in the luminal membrane in parallel with a H+/K+ antiporter. The effect of the protonophore dinitrophenol (DNP) was investigated on the isolated, symmetrically perfused tubule. DNP was applied in two different concentrations: 0.2 mmol/l and 1 mmol/l. The effects were fast and rapidly reversible. The equivalent short-circuit current (Isc) was reduced significantly to respectively 25 +/- 3% Cn = 4) and -3 +/- 7% (n = 11) of the control value when 0.2 mmol/l or 1 mmol/l was added to the bath. When 1 mmol/l DNP was applied the transepithelial resistance (Rte) decreased significantly to 74 +/- 11% of the control value (n = 11), and the luminal over basolateral voltage divider ratio (VDR), providing an estimate of luminal over basolateral membrane resistance, decreased to 37 +/- 12% of the control (n = 6). A concentration of 1 mmol/l DNP was also applied from the lumen. The decrease in Isc was significant, but much less pronounced (74 +/- 5% of control; n = 6) and no significant changes in Rte and VDR were observed. It is argued that, when the concentration in the bath is high enough, DNP may cross the cell and have a protonophoric effect not only on the mitochondria but also across the luminal cell membrane explaining the drop in transepithelial and in relative luminal membrane resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗