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Biomedical subjects

R Greger

Publications and source records attributed to R Greger.

At least 37 records · Page 2Linked to original sources
PubMed1991

Simultaneous recording of the cell membrane potential and properties of the cell attached membrane of HT29 colon carcinoma and CF-PAC cells.

Reconciliation of the properties of excised single channels and whole cell conductances is one of the major problems in the interpretation of patch clamp data. To combine cell attached and whole cell recordings we have modified the nystatin technique. Low concentrations of nystatin (less than or equal to 3 * 10(-5) mol/l) were added to the filling solution of the patch pipettes. This permeabilized the cell attached membrane partially and made it possible to measure the potential difference (PD) of the cell in current clamp mode. The input resistance (Rl) of the cell attached patch was only slightly decreased by nystatin and stayed in the GO range, allowing for the simultaneous recording of single channel activity and the input conductance of the cell attached membrane. This technique was examined in HT29 colon carcinoma and CF-PAC cells. In both cells it was shown that this method provides reliable PD measurements. The method was used then to test which type of Cl- channel is activated by carbachol. The PD of HT29 cells was depolarized by carbachol. The depolarization was mainly due to an increase in the Cl- conductance of the cell membrane and was followed by a slight and transient hyperpolarization. No detectable Cl- channels (conductance greater than 4-8 pS, 300 Hz) were activated in the cell attached membrane, but the input conductance (Go) increased concomitantly with cell depolarization. These results suggest that carbachol induces the opening of very small conductance or very rapidly opening and closing Cl- channels in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbachol↗
PubMed1991

Potassium conductance of smooth muscle cells from rabbit aorta in primary culture.

Vascular smooth muscle cells were obtained from rabbit aorta and were studied in primary culture on days 1-7 after seeding with electrophysiological techniques. In impalement experiments a mean membrane potential difference (PD) of -50 +/- 0.3 mV (n = 387) was obtained with Ringer-type solution in the bath. PD was depolarized by 6 +/- 0.3 mV (n = 45) and 16 +/- 2 mV (n = 5) when the bath K+ concentration was increased from the control value of 3.6 mmol/l to 13.6 and 23.6 mmol/l, respectively. Ba2+ (0.1-1 mmol/l) depolarized PD. Tetraethylammonium (TEA, 10 mmol/l) depolarized PD only slightly but significantly. Verapamil (0.1 mmol/l) and charybdotoxin (10 nmol/l) had no effect on PD. The conductance properties of these cells were further examined with the patch-clamp technique. K+ channels were spontaneously present in cell-attached patches. When the pipette was filled with 145 mmol/l KCl, a mean conductance (gK) of 209.6 +/- 4.6 mV (n = 17) was read from the current/voltage curves at a clamp voltage (Vc) of 0 mV. After excision K+ channels were found in 129 patches with inside-out and in 50 with outside-out configuration. With KCl on one and NaCl on the other side the mean gK at a Vc of 0 mV was 134.6 +/- 3.9 pS (n = 179). The mean permeability was 0.89 +/- 0.03 x 10(-12) cm3/s. With symmetrical KCl solution the mean gK was 227 +/- 6 pS (n = 17). The conductance sequence was gK much greater than gRb = gCs = gNa = 0. TEA blocked dose-dependently only from the outside (1-10 mmol/l). Lidocaine (5 mmol/l) quinidine (0.01-1 mmol/l) and quinine (0.01-1 mmol/l) blocked from both sides. Charybdotoxin (0.5-5 nmol/l) blocked only from the extracellular side. Ba2+ blocked from the cytosolic side and the inhibition was increased by depolarization and reduced by hyperpolarization. At a Vc of 0 mV a half-maximal inhibition (IC50) of 2 mumol/l was obtained. Verapamil and diltiazem blocked from both sides, verapamil with an IC50 of 2 mumol/l and diltiazem with an IC50 of 10 mumol/l. The open probability of this channel was increased by CA2+ on the cytosolic side at activities greater than 0.1 mumol/l. Half-maximal activation occurred at Ca2+ activities exceeding 1 mumol/l. The present data indicate that the vascular smooth muscle cells of rabbit aorta in primary culture possess a K+ conductance. In excised patches only a maxi K+ channel was detected. This channel has properties different from the macroscopic K+ conductance.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells.

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37 degrees C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was -50 +/- 0.4 mV (n = 53). Cromakalim (BRL 34915), 1 mumol/l and 10 mumol/l, hyperpolarized the membrane potential by 9 +/- 1 mV (n = 11) and 15 +/- 1 mV (n = 53) respectively. Glibenclamide (10 mumol/l) abolished the hyperpolarizing effect of chromakalim (n = 6). Simultaneous addition of cromakalim and glibenclamide (both 10 mumol/l, n = 11) and glibenclamide itself (10 mumol/l, n = 7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca(2+)-sensitive K+ channels, cromakalim increased the open probability (Po) only slightly and only with a cytosolic Ca2+ activity of 1 mumol/l. In all other series cromakalim had no effect on the Po of these channels. Forskolin (10 mumol/l) hyperpolarized PD by 6 +/- 1 mV (n = 13). The nucleotides UTP, ATP and ITP (10 mumol/l) depolarized PD by 12 +/- 1 mV (n = 7), 8 +/- 1 mV (n = 65) and 5 +/- 1 mV (n = 6) respectively. GTP, [alpha, beta-methylene]ATP and adenosine had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Effect of bicarbonate on potassium conductance of isolated perfused rat pancreatic ducts.

The aim of this study was to investigate the role of the K+ conductance in unstimulated and stimulated pancreatic ducts and to see how it is affected by provision of exogenous HCO3-/CO2. For this purpose we have applied electrophysiological techniques to perfused pancreatic ducts, which were dissected from rat pancreas. The basolateral membrane potential PDbl of unstimulated duct cells was between -60 mV and -70 mV, and the cells had a relatively large K+ conductance in the basolateral membrane as demonstrated by (a) 20-22 mV depolarization of PDbl in response to increase in bath K+ concentration from 5 mmol/l to 20 mmol/l and (b) the effect of a K+ channel blocker, Ba2+ (5 mmol/l), which depolarized PDbl by 30-40 mV. These effects on unstimulated ducts were relatively independent of bath HCO3-/CO2. The luminal membrane seemed to have no significant K+ conductance. Upon stimulation with secretin or dibutyryl cyclic AMP, PDbl depolarized to about -35 mV in the presence of HCO3-/CO2. Notably, the K+ conductance in the stimulated ducts was now only apparent in the presence of exogenous HCO3-/CO2 in the bath solutions. Upon addition of Ba2+, PDbl depolarized by 13 +/- 1 mV (n = 7), the fractional resistance of the basolateral membrane, FRbl increased from 0.66 to 0.78 (n = 6), the specific transepithelial resistance, Rte, increased from 52 +/- 13 omega cm2 to 59 +/- 15 omega cm2 (n = 11), and the whole-cell input resistance, Rc, measured with double-barrelled electrodes, increased from 20 M omega to 26 M omega (n = 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Inhibition of epithelial chloride channels by cytosol.

Chloride channels that have an intermediate conductance and are outwardly rectifying were studied by the patch-clamp technique in cell-excised membrane patches from respiratory epithelial cells in primary culture (REC) of normal and cystic fibrosis tissue, HT29 and T84 human colon carcinoma cells and placenta trophoblast cells (PTC). Chloride channels were immediately activated by the exposure of the cytosolic side of the patch to a Ringer-type solution, which lacked cytosolic components normally inhibiting chloride channels in the "on" cell configuration. Tentatively, we labelled the cytosolic component (or components) responsible for this inhibition cytosolic inhibitor (CI). The presence of CI in cytosol derived from HT29 cells was shown by assaying crude cytosol extracts from these cells on Cl- channels from HT29 cells (n = 2) and REC from normal subjects and cystic fibrosis patients (n = 4). In order to examine CI further, PTC were used as a source of cytosol. The cytosol of PTC inhibited HT29 Cl- channels in a dose-dependent manner with a half-maximal inhibition observed at a 1:6 dilution (n = 11) of the native cytosol. CI from PTC was heat-stable (10 min at 100 degrees C, n = 8). When cytosol extract was partitioned into a chloroform phase, Cl- channel inhibition was shown for the lipophilic extract (n = 12) as well as for the aqueous phase (n = 10). The inhibitory potency of the lipid extract was slightly larger than that of the aqueous phase. Several separation procedures were used to determine the molecular size of CI. When CI was filtered through 30-kDa filters at 6000 rpm for 45 min, inhibitory potency was observed in the filtrate and the retained fraction (n = 3). The same was observed with 10-kDa filters (n = 6). When CI was dialysed through a 12-kDa membrane, inhibitory capacity was recovered from the dialysate. Similarly, gel filtration indicated that CI was less than 5 kDa (n = 13) and probably less than 1.5 kDa (n = 11), but greater than 700 kDa (n = 9). CI was exposed to bead-coupled hydrolysing enzymes (trypsin, non-specific protease, lipase, alpha-amylase, nucleotidase), but none of the enzymes used destroyed the inhibitory potency of CI. These data indicate that CI is present in HT29 as well as in PTC. It inhibits reversibly intermediate-conductance outwardly rectifying Cl- channels in REC, HT29, and PTC.(ABSTRACT TRUNCATED AT 400 WORDS)

Chloride Channels↗
PubMed1991

Macromolecular conjugates of transport inhibitors: new tools for probing topography of anion transport proteins.

Macromolecular-conjugated, water-soluble, membrane-impermeant compounds were designed and assessed as topological probes for chloride-transporting agencies. The novel compounds were derivatives of either disulfonic stilbene (DS) and benzylaminoethylsulfonate (BS), "classical" inhibitors of erythrocyte chloride-bicarbonate exchange, or of phenylanthranilates (PA), high-affinity blockers of epithelial chloride channels. Covalent reactive derivatives of various DS, BS, and PA were synthesized and coupled either directly to polyethylene glycol or via spacer arms of different lengths to dextrans. The macromolecular conjugates were demonstrably inhibitory to red blood cell anion exchange when the ligands were appropriately coupled: inhibitory efficacy strongly depended on the chemical structure of the coupled ligand and the spacer length between the inhibitory moiety and the macromolecule. Mechanistic studies indicated that impermeant DS and PA derivatives acted exofacially on sites, which although different in their affinity for chloride, shared geographical proximity. BS derivatives were unique in that they affected transport from either surface. The results suggest asymmetric aqueous access routes leading to the functional domain of the anion transporter from either membrane surface.

Anion Transport Proteins↗
PubMed1991

How do loop diuretics act?

In the thick ascending limb of the loop of Henle, NaCl reabsorption is mediated by a Na+/2Cl-/K+ cotransport system, present in the luminal membrane of this nephron segment. Loop diuretics such as furosemide (frusemide), piretanide, bumetanide and torasemide bind reversibly to this carrier protein, thus reducing or abolishing NaCl reabsorption. This leads to a decrease in interstitial hypertonicity and thus to a reduced water reabsorption. In nephron segments other than the thick ascending limb, loop diuretics have no quantitative importance with respect to their saluretic and diuretic activities. Loop diuretics also reduce Ca++ and Mg++ reabsorption in the thick ascending limb in a way which is still not clear. Furthermore, these drugs increase the urinary K+ excretion by enhancing distal tubular K+ secretion and reducing K+ reabsorption in the loop of Henle. Finally, by reduction of active NaCl transport, loop diuretics drastically reduce the substrate requirement and oxygen dependence of the thick ascending limb cells. This renders these cells, which are characterised by high transport rates and only limited substrate reserves, less vulnerable in acute renal failure.

Animals↗
PubMed1991

Macula densa cell function.

Studies concerning the sensing step in the tubuloglomerular feedback (TGF) mechanism have been conflicting. To study this step, we measured macula densa (MD) cell volume and membrane potentials in the isolated perfused ascending limb of the loop of Henle with attached glomerulus with MD segments (cTAL-MD). Addition of furosemide reduced cell volume rapidly and the effect could be reversed on removal of the drug. From the time course of cell volume changes hydraulic conductivity could be measured both in the basolateral and apical cell membrane. It was found that the apical cell membrane constituted the main barrier for water flow with a low hydraulic conductance, while the basolateral hydraulic conductance was quite high. Measurements of the basolateral electrical potential in the MD cells have shown a mean electrical potential of -56 mV. This potential was hyperpolarized by the addition of furosemide, the Cl channel blocker NPPB, or during a reduction of luminal NaCl from 150 to 30 mM, and depolarized when bath Cl concentration was reduced from 150 to 30 mM. These results are consistent with the following model for electrolytes transported and similar to the one described in the cTAL [15]. In the luminal cell membrane there is an Na-K-2Cl cotransporter that takes these ions into the MD cells and there is a potassium recycling through a K channel. On the basolateral membrane side there is an Na-K pump and a Cl channel through which chloride is transported out of the MD cell. The Na-K pump activity seems to be only 1/40 of that in the cTAL cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Effects of parathyroid hormone and calcitonin on Na+, Cl-, K+, Mg2+ and Ca2+ transport in cortical and medullary thick ascending limbs of mouse kidney.

The effect of parathyroid hormone (PTH) on transepithelial Na+, Cl-, K+, Ca2+ and Mg2+ transport was investigated in isolated perfused cortical thick ascending limbs (cTAL) and that of human calcitonin (hCT) was tested in both cortical and medullary thick ascending limbs (mTAL) of the mouse nephron. The transepithelial ion net fluxes (Jx) were determined by electron probe analysis of the perfused and collected fluids. Simultaneously, the transepithelial voltage (PDte) and resistance (Rte) were recorded. In cTAL segments, PTH and hCT significantly stimulated the reabsorption of Na+, Cl-, Ca2+ and Mg2+, hCT generated a net K+ secretion towards the lumen and PTH tended to exert the same effect. Neither PDte nor Rte were significantly altered by either PTH or hCT. However, in the post-experimental period a significant decrease in PDte was noted. Time control experiments carried out under similar conditions revealed a significant decrease in PDte with time, which could have masked the hormonal response. In mTAL segments, Mg2+ and Ca2+ transport was close to zero, hCT did not exert any detectable effect on either PDte or Jcl-, JNa+, JK+, JMg2+ and JCa2+ in these segments. In conclusion, our data demonstrate that PTH and hCT stimulate NaCl reabsorption as well as Mg2+ and Ca2+ reabsorption in the cTAL segment of the mouse. These data are in agreement with and extend data obtained in vivo in the rat.

Animals↗
PubMed1990

Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl- transport.

The rat cortical collecting duct (CCD) exhibits high rates of NaCl reabsorption when stimulated by mineralocorticoid and antidiuretic hormone (ADH). The present study was undertaken to determine if there is significant transcellular Cl- movement across the principal cells of the rat CCD. CCDs were dissected from kidneys of rats that had been injected with deoxycorticosterone (5 mg, i.m.) 2-9 days prior to the experiment. The ducts were perfused in vitro with identical perfusing and bathing solutions, except that 200 pmol.l-1 ADH was added to the bathing solutions. The basolateral membrane voltage (PDbl) of principal cells was -77 +/- 1 mV and the luminal membrane voltage (PD1) was -68 +/- 1 mV (mean +/- SEM, n = 124). Separate impalements with single-barrelled Cl(-)-selective microelectrodes gave an apparent intracellular Cl- activity of principal cells of 17 +/- 2 mmol.l-1. Transepithelial PD and PDbl were unaffected by luminal furosemide, hydrochlorothiazide (HCT), 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid, (SITS), or the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB); bath addition of SITS or the Cl- channel blocker diphenylamino-2-carboxylic acid; or replacement of bath HCO3- by Cl-. The intracellular Cl- activity (a(cell)Cl) also remained unchanged with the addition of HCT, SITS or the Cl- channel blockers to either the perfusing or bathing solutions, or with replacement of the bathing solution HCO3-.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1990

The luminal K+ channel of the thick ascending limb of Henle's loop.

In vitro perfused rat thick ascending limbs of Henle's loop (TAL) were used (n = 260) to analyse the conductance properties of the luminal membrane applying the patch-clamp technique. Medullary (mTAL) and cortical (cTAL) tubule segments were dissected and perfused in vitro. The free end of the tubule was held and immobilized at one edge by a holding pipette kept under continuous suction. A micropositioner was used to insert a patch pipette into the lumen, and a gigaohm seal with the luminal membrane was achieved in 455 instances out of considerably more trials. In approximately 20% of all gigaohm seals recordings of single ionic channels were obtained. We have identified only one single type of K+ channel in these cell-attached and cell-excised recordings. In the cell-attached configuration with KCl or NaCl in the pipette, the channel had a conductance of 60 +/- 6 pS (n = 24) and 31 +/- 7 pS (n = 4) respectively. In cell-free patches with KCl either in the patch pipette or in the bath and with a Ringer-type solution (NaCl) on the opposite side the conductance was 72 +/- 4 pS (n = 37) at a clamp voltage of 0 mV. The permeability was 0.33 +/- 0.02 . 10(-12) cm3/s. The selectivity sequence of this channel was: K+ = Rb+ = NH4+ = Cs+ greater than Li+ much greater than Na+ = 0; the conductance sequence was K+ much greater than Li+ much greater than Rb+ = Cs+ = NH4+ = Na+ = 0. In excised patches Rb+, Cs+ and NH4+ when present in the bath at 145 mmol/l all inhibited K+ currents out of the pipette. The channel kinetics were described by one open (9.5 +/- 1.5 ms, n = 18) and by two closed (1.4 +/- 0.1 and 14 +/- 2 ms) time constants. The open probability of this channel was increased by depolarization. The channel open probability was reduced voltage dependently by Ba2+ (half maximal inhibition at 0 mV: 0.07 mmol/l) from the cytosolic side. Verapamil, diltiazem, quinine and quinidine inhibited at approximately 1 mumol/l -0.1 mmol/l from either side. Similarly, the amino cations lidocaine, tetraethylammonium and choline inhibited at 10-100 mmol/l. The channel was downregulated in its open probability by cytosolic Ca2+ activities greater than 10(-7) mol/l and by adenosine triphosphate greater than or equal to 10(-4) mol/l. The open probability was downregulated by decreasing cytosolic pH (2-fold by a decrease in pH by less than or equal to 0.2 units).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells.

The present study examines the properties of Cl- channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl- channels were larger at positive as compared to negative clamp voltages (Vc): 74 +/- 2.6 (Vc greater than 0) and 47 +/- 2.0 pS (Vc less than 0) for CF (n = 57) and 69 +/- 3.6 (Vc greater than 0) and 45 +/- 2.3 pS (Vc less than 0) for N (n = 35). The open probability (Po) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of Po contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl- = Br- = I- greater than NO-3 much greater than gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10(-7) mol/l to 10(-5) mol/l. While Cl- channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl- channels was 76 +/- 3.2 pS for positive clamp voltages (Vc) and 46 +/- 3.9 pS for negative Vc (n = 8). When the membrane patches were excised from CF cells Cl- currents appeared spontaneously (n = 19). The immediate appearance (within 1 s) of Cl- channels after excision was observed at positive (n = 6) as well as at negative clamp voltage (n = 13). "Excision activation" of CF Cl- channels was observed at low (less than 10(-9) mol/l) or high (10(-3) mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca+ activity (less than 10(-9)-10(-3) mol/l) or pH (6.5-8.5) on the cytosolic side exerted no effects on these Cl- channels. These results suggest that Cl- channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic "inhibitor" leads to an activation of Cl- channels. The Cl- channels in excised patches of N and CF cells have identical properties.

Calcium↗
PubMed1989

Effects of glucagon on Na+, Cl-, K+, Mg2+ and Ca2+ transports in cortical and medullary thick ascending limbs of mouse kidney.

The effects of glucagon on transepithelial Na+, Cl-, K+, Ca2+ and Mg2+ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (JNa+, JCl-, JK+, JCa2+, JMg2+) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PDte) and the transepithelial resistance (Rte) were recorded. In cTAL-segments (n = 8), glucagon (1.2 x 10(-8) mol.1-1) stimulated significantly the reabsorption of Na+, Cl-, Ca2+ and Mg2+: JNa+ increased from 204 +/- 20 to 228 +/- 23 pmol.min-1.mm-1, JCl- from 203 +/- 18 to 234 +/- 21 pmol.min-1.mm-1, JCa2+ from 0.52 +/- 0.13 to 1.34 +/- 0.30 pmol.min-1.mm-1 and JMg2+ from 0.51 +/- 0.08 to 0.84 +/- 0.08 pmol.min-1.mm-1.JK+ remained unchanged: 3.2 +/- 1.3 versus 4.0 +/- 1.9 pmol.min-1.mm-1. Neither PDte (16.3 +/- 1.5 versus 15.9 +/- 1.4 mV) nor Rte (22.5 +/- 3.0 versus 20.3 +/- 2.6 omega cm2) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PDte and increase in Rte were noted. In mTAL-segments (n = 9), Mg2+ and Ca2+ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na+ and Cl-, however, were strongly stimulated: JNa+ increased from 153 +/- 17 to 226 +/- 30 pmol.min-1.mm-1 and JCl- from 151 +/- 23 to 243 +/- 30 pmol.min-1.mm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Versatile supplement device with remote control for the control of patch clamp experiments.

A versatile device for a patch-clamp amplifier is described. This device contains: (i) an acoustic indicator to monitor the input resistance of the patch pipette, which is used in search-mode to indicate the formation of seals; (ii) two pulse generators; and (iii) a staircase generator to produce various pulse and voltage step programs; (iv) a low-pass filter which is used to filter the output of the patch clamp amplifier; and (v) a remote control which is used to control the entire patch clamp experiment. This remote control is used to switch between search-, current clamp-, and voltage clamp-mode, to activate the respective stimulus potential programs, and to control the tape recorder. This electronic device can be easily connected to patch clamp amplifiers.

Animals↗