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Biomedical subjects

R Greger

Publications and source records attributed to R Greger.

At least 199 records · Page 11Linked to original sources
PubMed1988

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane.

In order to study the mechanism of pancreatic HCO3- transport, a perfused preparation of isolated intra- and interlobular ducts (i.d. 20-40 microns) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PDte) in ducts bathed in HCO3(-)-free and HCO3(-)-containing solution was -0.8 and -2.6 mV, respectively. The equivalent short circuit current (Isc) under similar conditions was 26 and 50 microA . cm-2. The specific transepithelial resistance (Rte) was 88 omega cm2. In control solutions the PD across the basolateral membrane (PDbl) was -63 +/- 1 mV (n = 314). Ouabain (3 mmol/l) depolarized PDbl by 4.8 +/- 1.1 mV (n = 6) within less than 10 s. When the bath K+ concentration was increased from 5 to 20 mmol/l, PDbl depolarized by 15.9 +/- 0.9 mV (n = 50). The same K+ concentration step had no effect on PDbl if the ducts were exposed to Ba2+, a K+ channel blocker. Application of Ba2+ (1 mmol/l) alone depolarized PDbl by 26.4 +/- 1.4 mV (n = 19), while another K+ channel blocker TEA+ (50 mmol/l) depolarized PDbl only by 7.7 +/- 2.0 mV (n = 9). Addition of amiloride (1 mmol/l) to the bath caused 3-4 mV depolarization of PDbl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PDbl. An increase in the bath HCO3- concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PDbl by 8.5 +/- 1.0 mV (n = 149). It was investigated whether the effect of HCO3- was due to a Na+-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K+ conductance caused by lowered intracellular pH. Experiments showed that the HCO3- effect was present even when the bath Na+ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO3- effect remained unchanged after Ba2+ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na+ + K+)-ATPase, a Ba2+ sensitive K+ conductance and an amiloride sensitive Na+/H+ antiport. The HCO3- effect on PDbl is most likely due to rheogenic anion exit across the luminal membrane.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport.

The aim of the present study was to investigate by what transport mechanism does HCO-3 cross the luminal membrane of pancreatic duct cells, and how do the cells respond to stimulation with dibutyryl cyclic AMP (db-cAMP). For this purpose a newly developed preparation of isolated and perfused intra- and interlobular ducts of rat pancreas was used. Responses of the epithelium to inhibitors and agonists were monitored by electrophysiological techniques. Addition of HCO-3/CO2 to the bath side of nonstimulated ducts depolarized the PD across the basolateral membrane (PDbl) by about 9 mV, as also observed in a previous study [21]. This HCO-3 effect was abolished by Cl- channel blockers or SITS infused into the lumen of the duct: i.e. 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 10(-5) M) hyperpolarized PDbl by 8.2 +/- 1.6 mV (n = 13); 3',5-dichlorodiphenylamine-2-carboxylic acid (DCl-DPC, 10(-5) M) hyperpolarized PDbl by 10.3 +/- 1.7 mV (n = 10); and SITS hyperpolarized PDbl by 7.8 +/- 0.9 mV (n = 4). Stimulation of the ducts with db-cAMP in the presence of bath HCO-3/CO2 resulted in depolarization of PDbl, the ductal lumen became more negative and the fractional resistance of the luminal membrane decreased. Together with forskolin (10(-6) M), db-cAMP (10(-4) M) caused a fast depolarization of PDbl by 33.8 +/- 2.5 mV (n = 6). When db-cAMP (5 x 10(-4) M) was given alone in the presence of bath HCO-3/CO2, PDbl depolarized by 25.3 +/- 4.2 mV (n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1988

Differential effects of ADH on sodium, chloride, potassium, calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron.

The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na+, Cl-, K+, Ca2+ and Mg2+ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (JNa+, JCl-, JK+, JCa2+, JMg2+) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PDte) and transepithelial resistance (Rte) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO3- containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO3- free Ringer solutions. In cTAL segments, AVP (10(-10) mol.l-1) significantly increased JMg2+ and JCa2+ from 0.39 +/- 0.08 to 0.58 +/- 0.10 and from 0.86 +/- 0.13 to 1.19 +/- 0.15 pmol.min-1 mm-1 respectively. Neither JNa+ nor JCl-, (JNa+: 213 +/- 30 versus 221 +/- 28 pmol.min-1 mm-1, JCl-: 206 +/- 30 versus 220 +/- 23 pmol.min-1 mm-1) nor PDte (13.4 +/- 1.3 mV versus 14.1 +/- 1.9 mV) or Rte (24.6 +/- 6.5 omega cm2 versus 22.6 +/- 6.4 omega cm2) were significantly changed by AVP. No significant effect of AVP on net K+ transport was observed. In mTAL segments, Mg2+ and Ca2+ net transports were close to zero and AVP (10(-10) mol.l-1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone, JNa+ increased from 107 +/- 33 to 148 +/- 30 and JCl- from 121 +/- 33 to 165 +/- 32 pmol.min-1 mm-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Stimulation of NaCl secretion in the rectal gland of the dogfish Squalus acanthias.

1. The rectal gland of the dogfish (Squalus acanthias) secretes NaCl when stimulated by a hormone related to vasointestinal peptide. 2. Patch clamp and microelectrode techniques are used to examine the changes in membrane conductances occurring with hormonal stimulation. 3. The conductance of the "resting" cell is dominated by basolateral K+ channels. 4. Hormonal stimulation "opens" apical Cl- channels. 5. This opening of apical chloride channels appears to be mediated by cAMP-dependent phosphorylation of pre-existing closed channels.

Animals↗
PubMed1988

Inhibition of active NaCl reabsorption in the thick ascending limb of the loop of Henle by torasemide.

The new diuretic torasemide (1-isopropyl-3- ([4-(3-methyl-phenylamino)pyridine]-3-sulfonyl)urea) was tested in isolated in vitro perfused cortical thick ascending limbs of Henle loops of rabbit kidney. The equivalent short circuit current (Isc) corresponding in this nephron segment to the rate of secondarily active Cl-reabsorption was measured continuously. Torasemide led to a rapid and reversible inhibition of Isc when added to the luminal perfusate. The IC50 for this effect was 3 x 10(-7) mol/l. A similar effect was also exerted by torasemide addition to the peritubular bath. However, 100 times larger doses were needed. The data are compatible with the view that torasemide interacts with both, the Na+2Cl-K+ carrier, localized in the luminal membrane, and, at higher concentrations, with the chloride channels present in the basolateral membrane of this nephron segment. Under normal conditions, and in the intact kidney, the former effect, i.e. interference with the Na+2Cl-K+ carrier will be the dominant action of this drug.

Absorption↗
PubMed1987

Potassium conductance in straight proximal tubule cells of the mouse. Effect of barium, verapamil and quinidine.

The present study has been performed to test for the influence of verapamil and quinidine on the potential difference across the basolateral cell membrane (PDbl) and on the basolateral potassium conductance of isolated perfused segments of the mouse proximal tubule. PDbl was recorded continuously with conventional microelectrodes during rapid alterations of bath or luminal perfusate composition. The contribution of the basolateral potassium conductance to the conductance of both cell membranes (tk) was estimated from the effects of altered bath potassium concentration on PDbl. Under control conditions tk approaches 0.8, i.e. the basolateral cell membrane is mainly conductive to potassium. Neither quinidine nor verapamil affect PDbl at concentrations below 10 mumol/l. At higher concentrations both substances depolarize the basolateral cell membrane mimicking the effect of 1 mmol/l barium. In the presence of 0.1 mmol/l verapamil tk is virtually abolished at 5 to 10 mmol/l bath potassium concentration but is almost unaffected at bath potassium concentrations between 20 and 40 mmol/l. 1 mumol/l ionophore A-23187 does not change the depolarizing effect of 0.1 mmol/l verapamil on cell membrane potential. In the presence of 0.1 mmol/l quinidine, tk is reduced to some 50%, irrespective of the bath potassium concentration. It is concluded that the potassium conductance in straight proximal tubules is inhibited not only by barium but as well by high concentrations of verapamil and quinidine. The effect is probably direct and not related to alterations in the intracellular calcium activity.

Animals↗
PubMed1987

Properties of single K+ channels in the basolateral membrane of rabbit proximal straight tubules.

The basolateral membrane of rabbit straight proximal tubules, which were cannulated and perfused on one side, was investigated with the patch clamp technique. Properties of inward and outward directed single K+ channel currents were studied in cell-attached and inside-out oriented cell-excised membrane patches. In cell-attached patches with NaCl Ringer solution both in pipette and bath, outward K+ currents could be detected after depolarization of the membrane patch by about 20-30 mV. The current-voltage (i/V) relationship could be fitted by the Goldman-Hodgkin-Katz (GHK) current equation, with the assumption that these channels were mainly permeable for K+ ions. A permeability coefficient PK of (0.17 +/- 0.04).10(-12) cm3/s was obtained, the single channel slope conductance at infinite positive potential g(V infinity) was 50 +/- 12 pS and the single channel conductance at the membrane resting potential g(Vbl) was 12 +/- 3 pS (n = 4). In cell-excised patches, with NaCl in the pipette and KCl in the bath, the data could also be fitted to the GHK equation and yielded PK = (0.1 +/- 0.01).10(-12) cm3/s, g(V infinity) = 40 +/- 4 pS and g(Vbl) = 7 +/- 1 pS (n = 8). In cell-attached patches with KCl in the pipette and NaCl in the bath, inward K+ channels occurred at clamp potentials less than or equal to 60 mV, whereas outward K+ channel current was detected at more positive voltages. The current-voltage curves showed slight inward rectification.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

The diuretic effect of muzolimine.

Muzolimine produces a diuresis in the loop of Henle. Unlike other diuretics of this kind, the effect of muzolimine is slow and its action is long lasting. The present study was designed to examine the mechanism of action of muzolimine in isolated in vitro perfused rabbit cortical thick ascending limb segments (cTAL). In a first series it was confirmed that muzolimine itself in a concentration of up to 10(-4) mol/l had no effect on the equivalent short circuit current (Isc), corresponding to the rate of active NaCl reabsorption, in cTAL segments neither from the lumen nor from the bath side. In a second series of experiments, muzolimine was administered intravenously (70 mumol/kg) to male Wistar rats, and the clearances of inulin, Na+ and K+ were calculated. Muzolimine, after a lag phase of 5 to 20 min caused a marked diuresis and natriuresis, but had only a minor effect on glomerular filtration rate and on K+ excretion. The urines of these animals were diluted with isotonic saline, and were examined in rabbit cTAL segments for their ability to block Isc. It was found that the urines of the antidiuretic period were devoid of effects, whereas the diuretic urines inhibited Isc strongly down to a dilution of 300 times when they were perfused in the lumen. The same diluted urines had no effect from the bath side. In a third series, probenecid (100 mumol/kg) was administered i.v. to rats undergoing a muzolimine induced saluresis. It was found that probenecid inhibited the diuresis and saluresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Analogues of torasemide--structure function relationships--experiments in the thick ascending limb of the loop of Henle of rabbit nephron.

The aim of the present study was to examine compounds related to torasemide with respect to their ability to block the equivalent short circuit current, corresponding to the rate of chloride reabsorption, in isolated in vitro perfused cortical thick ascending limbs of Henle of the rabbit. The torasemide molecule was modified with respect to the anionic sulfonylurea group, and the secondary amine linked to the pyridine ring. Our results indicate that only few of the tested 48 torasemide-related compounds were able to inhibit from both epithelial sides like torasemide. Only few of the tested compounds were equally effective as torasemide from the lumen side. Some analogues were acting only from the luminal side and some only from the peritubular side. The correlations between structure and potency of inhibition from the luminal side allow the following conclusions: The secondary amine moiety linked to the pyridine ring (toluidine in case of torasemide) can be replaced by a cycloalkylamine or, with some loss of inhibitory potency, by alkylamines. The inhibitory potency is increased with the number of C-atoms in the cycloalkylamine substituted compounds (optimum C7 to C8) and is also depending on the length of the alkylamines (optimum C4). The secondary amine seems to be required since nitrogen cannot be replaced by -S- or -SO2-. The sulfonylurea group cannot be substituted by other anionic groups such as -SO-3 or -COO-. If the pyridine ring is replaced by a NO2-substituted phenyl ring, the inhibitory potency from the luminal side is lost. However, these compounds act still (with some loss of potency) from the peritubular side.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Potassium channels in the basolateral membrane of the rectal gland of the dogfish (Squalus acanthias).

Previous studies in isolated, in vitro perfused rectal gland tubules (RGT) have revealed that the basolateral membrane possesses a K+ conductive pathway. In the present study, we have utilized the patch clamp technique in RGT segments to characterize this pathway. The basolateral membrane was approached with patch pipettes at the open end of in vitro perfused segments. Recordings were obtained in cell-attached as well as in excised inside-out patches. In cell-attached patches with the pipette filled with a KCl solution (274 mmol/l) and the bath containing NaCl shark Ringer (275 mmol/l), inward K+ currents (from pipette into cell) with a mean slope conductance of 123 +/- 26 pS (n = 3) were observed. We were unable to generate outward K+ currents at high depolarizing (cell more positive) clamp voltages. This indicates inward rectification of this channel. To examine the rectification properties further, excised (inside out) patches were exposed to K+ concentration gradients, directed out of, as well as into the pipette. With NaCl in the pipette and KCl in the bath, K+ outward currents were observed. The current-voltage (IV) relation revealed Goldman-type rectification, with a mean single channel conductance of 185 +/- 28 pS (n = 7) at high positive voltages (linear range of the IV curve). The single-channel permeability coefficient for K+ was 0.26 +/- 0.04 X 10(-12) cm3/s (n = 7). In the reversed experiment (pipette KCl, bath NaCl), inward currents of similar kinetics and amplitude were obtained. The single channel conductance was 146 +/- 21 pS (n = 7) at high negative voltages (linear range of the IV curve).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Potassium channels in the basolateral membrane of the rectal gland of Squalus acanthias. Regulation and inhibitors.

The present study examines the influences of pH and Ca2+ and several putative inhibitors on the basolateral K+ channel of the rectal gland of Squalus acanthias. Excised membrane patches were examined using the patch clamp technique. It is shown that reduction of the calcium activity on the cytosolic side to less than 10(-9) mol/l has no detectable inhibitory effect on this channel. Conversely, increase in calcium activity to some 10(-3) mol/l reduced the activity of this channel. Variations in cytosolic pH had only a moderate effect on the current amplitude: alkalosis by one pH unit increased and acidosis reduced the single current amplitude by some 15%. Several inhibitors were tested in excised patches when added to the cytosolic side. Ba2+ (approximately 5 X 10(-3) mol/l), quinine (approximately 10(-3) mol/l), quinidine (approximately 10(-4) mol/l), lidocaine (approximately equal to 1 mmol/l), tetraethylammonium (approximately 10 mmol/l), Cs+ (approximately 10 mmol/l), and Rb+ (approximately 20 mmol/l) all blocked this K+ channel reversibly. We conclude that the basolateral K+ channel of the rectal gland is distinct from other epithelial K+ channels inasmuch as it is not stimulated by Ca2+ directly, but that it is qualitatively similar to many other known K+ channels with respect to its sensitivity towards blockers.

Animals↗
PubMed1987

Chloride channels in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias). Properties of the "larger" conductance channel.

The rectal gland of the dogfish (Squalus acanthias) secretes chloride via a chloride channel present in the apical cell membrane. Using the patch clamp technique in isolated perfused rectal gland tubules, two types of chloride channels are demonstrable in the apical membrane of cyclic AMP treated tubule segments. A small channel of about 11 pS and another channel of 40-50 pS are present. The small channel is described in the succeeding report. With NaCl on both sides (excised patches) the current amplitude of the larger channel is an almost linear function of the voltage (+/- 50 mV). However, the open probability of this channel is grossly reduced at negative clamp potentials (corresponding to cell hyperpolarization). Therefore, the macroscopic Cl- current through this channel is reduced with hyperpolarization on the cytosolic side. An analysis of time constants of this channel reveals that at depolarized voltages two open and two closed time constants of about 1 ms and of about 10 ms, respectively, are demonstrable. With hyperpolarized voltages the larger open state time constant is reduced significantly. This type of chloride channel is blocked reversibly by diphenylamine-2-carboxylate (10(-4) mol/l) and by 5-nitro-2-(3-phenylpropylamino)-benzoate (10(-5) mol/l). The channel is selective for Cl- over Na+ and K+ as well as over Br-. It is, however, permeable for NO3-. Since this channel is very rare or absent in nonstimulated rectal gland tubules, it is very likely that this type of channel is responsible for hormone and cAMP dependent chloride secretion in this organ.

Animals↗
PubMed1987

The "small" conductance chloride channel in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias).

Besides the "larger" Cl- channel, with a single channel conductance of about 45 pS, a "small" channel was observed in the luminal membrane of the dogfish rectal gland. In cell excised (inside out) patches with NaCl solution on both sides, the latter channel had a single channel conductance of 11 +/- 1 pS (n = 21), and its current-voltage relationship was linear in the voltage range +90 to -90 mV. The open state probability increased moderately with negative clamp potentials. Ionic replacement studies revealed a high selectivity of Cl- over gluconate, sulfate, and iodide, whereas bromide was permeable to some extent. Also the channel is impermeable for Na+. The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate did not affect this "small" conductance Cl- channel. It can be concluded that the luminal membrane of stimulated rectal gland cells possesses two types of Cl- channels, which differ markedly in their characteristics.

Animals↗
PubMed1987

Characterization of human sweat duct chloride conductance by chloride channel blockers.

To characterize the chloride conductance of human sweat duct the effect of various analogues of diphenylamine-2-carboxylate was investigated on the transepithelial potential difference (PDT) and resistance (RT) of isolated microperfused sweat ducts. Although the most powerful analogues which block Cl- channels in various secretory and absorptive epithelia were ineffective, a number of analogues (in particular Cl substituted ones) were found which at high concentrations significantly and reversibly increased PDT and RT. The data suggest that the main chloride conductance pathway of sweat duct epithelium resides in the cell membranes rather than in the tight junctions. In addition the different blocking spectra of the chloride conductances of sweat duct and tracheal epithelium (Welsh MJ, Science 232:1648, 1986) suggest that the combined impairment of both conductances in cystic fibrosis does not result from a molecular defect in the Cl- channels.

Chlorides↗
PubMed1987

Characteristics of apical chloride channels in human colon cells (HT29).

Recent studies have demonstrated that active chloride secretion in mammalian colon and other epithelia, is dependent on the induction of an increase of apical chloride conductance. Since the physical characteristics of apical chloride channels in man have not been elucidated, patch clamp analysis of human colon cells (HT29), in culture, was performed, after stimulation with db-cAMP 10(-4) mol/l. In excised inside out patches of apical membranes two types of channels were found. The smaller and less frequent channel had a mean conductance of 15 +/- 1 pS (n = 9). This type of channel showed identical I/V curves in NaCl and KCl solutions. It was inhibited by a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The more frequently observed larger conductance channel was selective for anions and was impermeable to Na+ and K+. Regarding anion selectivity, the channel was similarly permeable to Cl-, Br-, I-, and NO3-, but was impermeable to gluconate. The channel was completely inhibited by the potent Cl- channel blocker NPPB (10(-6) mol/l). This channel exhibited rectification: The conductance was 50 +/- 4 pS at positive clamp potentials (sign referred to bath with respect to pipette interior) and 32 +/- 3 (n = 33) pS at negative voltages. Moreover, the open state probability was doubled when the clamp potential was increased from -20 to +20 mV. These results demonstrate the existence of chloride channels in the apical membrane of db-cAMP treated colonic carcinoma cells.

Chlorides↗
PubMed1987

Sidedness of action of loop diuretics and ouabain on nonsensory cells of utricle: a micro-Ussing chamber for inner ear tissues.

It is known that nonsensory tissues of the utricle produce a lumen-positive transepithelial electrical potential difference (VT). This potential has been shown previously to be inhibited by ouabain and bumetanide applied to the bathing medium in vitro. In order to more fully characterize the origin of this potential we mounted the utricle as a flat sheet in a newly designed Ussing chamber and measured the VT and transepithelial resistance (RT) while perfusing the endolymphatic and perilymphatic surfaces independently with identical solutions. The aperture of the chamber was 1.5 X 10(-4) cm2. VT averaged 5.6 +/- 0.46 mV and RT was 24.0 +/- 2.43 omega X cm2 (n = 45). Ouabain and loop diuretics of the furosemide family were found to inhibit the VT only from the serosal side. The KI for ouabain was 7.63 X 10(-5) M. The loop diuretics tested inhibited the VT in the same order as in other tissues known to contain a Na/2 Cl/K cotransporter (KI: 2-benzylamino-4-cyclohexylsulfonyl-5-sulfamoylbenzolsulfonate++ + (BCSB), 1.72 X 10(-7) M; bumetanide, 1.10 X 10(-6) M; piretanide, 5.67 X 10(-6) M; furosemide, 4.14 X 10(-5) M). It is concluded that this tissue produces a lumen-positive VT (i) in the absence of a transepithelial chemical gradient; the generation of which is dependent upon the activity of (ii) Na,K-ATPase and (iii) a Na/2 Cl/K cotransporter; (iv) in the basolateral membranes of the nonsensory cells; (v) which is not depressed by luminal application of inhibitors of these transporters.

Animals↗