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R Greger

Publications and source records attributed to R Greger.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1987

Atrial natriuretic peptide stimulates salt secretion by shark rectal gland by releasing VIP.

Salt secretion by the isolated perfused rectal gland of the spiny dogfish shark, Squalus acanthias, is stimulated by synthetic rat atrial natriuretic peptide (ANP II) as well as extracts of shark heart, but not by 8-bromo-cyclic guanosine 5'-monophosphate. Cardiac peptides have no effect on isolated rectal gland cells or perfused tubules, suggesting that stimulation requires an intact gland. The stimulation of secretion by ANP II is eliminated by maneuvers that block neurotransmitter release. These include: perfusion with procaine (10(-2) M), perfusion with high Mg2+ (9.5 mM) and low Ca2+ (0.5 mM) concentrations, and addition to the perfusate of the calcium channel blockers nifedipine (10(-6)M), diltiazem (5 X 10(-5)M), or verapamil (10(-4)M). Cardiac peptides stimulate the release of vasoactive intestinal peptide (VIP), known to be present in rectal gland nerves, into the venous effluent or perfused glands in parallel with their stimulation of salt secretion, but the release of VIP induced by ANP II is prevented by perfusion with procaine. Cardiac peptides thus appear to regulate rectal gland secretion by releasing VIP from neural stores within the gland. It is possible that other physiological effects of these hormones might be explained by an action to enhance local release of neurotransmitters.

Adenosine↗
PubMed1987

Loop diuretics.

Loop diuretics such as furosemide, piretanide, and bumetanide bind reversibly to the Na+2Cl-K+ carrier. This transporter is responsible for the uptake of Cl- into the thick ascending limb segment. As a consequence, these compounds reduce or abolish NaCl reabsorption in this nephron segment and lead to a decreased interstitial hypertonicity and thus to a reduced water absorption. Apart from these profound effects, loop diuretics also inhibit Ca2+ and Mg2+ reabsorption in the thick ascending limb in a manner which is still not clear. They also increase urinary K+ excretion by an increase in distal tubule NaCl and water delivery and probably also by the reduction of K+ reabsorption which goes on under in vivo conditions in the thick ascending limb segment. Finally, by the reduction of the transport work in the thick ascending limb, these compounds reduce dramatically the substrate and oxygen dependence of this nephron segment.

Animals↗
PubMed1987

[Pathophysiology of renal ischemia].

Renal ischaemia results, early in its course, in cell swelling of those tubule segments which are specifically susceptible to damage. These segments are the straight proximal tubules in the outer stripe of the outer medulla and the medullary thick ascending limb of the loop of Henlé in the outer and inner stripe of the outer medulla. One causal predisposing factor for both nephron segments is the sharp decline in the partial pressure of oxygen in the outer medulla. Another important cause for the vulnerability of the thick ascending limb is its strict dependence on a continuous supply of metabolic substrates. The cell swelling caused by ischaemia can be explained, for all nephron segments, as an unchanged uptake of Na+ across the luminal membrane in the face of a reduced pump rate of the basolateral (Na+ + K+)-ATPase. The swelling can be prevented if, for example, in the thick ascending limb the Na+ uptake is inhibited by furosemide. In addition to cell damage by cell swelling, other pathomechanisms are operative in ischaemia. One such mechanism is the damage caused by oxygen radicals. The concentration of these radicals is increased during ischaemia, especially during the initiation of reperfusion. Additionally, the concentration of radical scavengers such as glutathion, is reduced during ischaemia. Future work is needed to define more closely the damage produced by oxygen radicals in terms of lipid peroxidation, ATP and enzyme degradation.

Animals↗
PubMed1986

Torasemide inhibits NaCl reabsorption in the thick ascending limb of the loop of Henle.

Torasemide (1-isopropyl-(4-(3-methylphenylamino)pyrid-3-yl)urea) is a new diuretic. The present study examines the effects of this substance in the isolated perfused thick ascending limb (TAL) of mouse and rabbit kidney. In cortical TAL segments of the rabbit, torasemide added to the lumen perfusate led to a fall in equivalent short circuit current ( = transepithelial voltage divided by transepithelial resistance, which corresponds to the rate of chloride reabsorption) with a half maximal inhibition concentration of 3 X 10(-7) mol/l. This effect was accompanied by a hyperpolarization of the luminal and basolateral membrane from -78 to -81 mV and from -72 to -81 mV, respectively. A similar hyperpolarization of both membrane voltages was also observed in medullary TAL segments of the mouse. Torasemide, added to the basolateral perfusate of cortical TAL segments of the rabbit, also inhibited the equivalent short circuit current. However, 3 X 10(-5) mol/l were necessary for a half maximal inhibition. The fall in the equivalent short circuit current was accompanied by a significant increase in transepithelial resistance from 34 to 38 omega cm2, by an increase in the fractional resistance of the basolateral membrane, and by a hyperpolarization mainly of the basolateral membrane. Again, similar results were obtained in the medullary TAL segment of the mouse. The strong inhibitory effect of torasemide from the lumen side can be explained by an interference with the Na+ 2Cl-K+ carrier in the luminal membrane. In fact, torasemide apparently is structurally related to furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Cl(-)-channel blockers in the thick ascending limb of the loop of Henle. Structure activity relationship.

On the basis of our findings with diphenylamine-2-carboxylate we have searched for compounds which possess an even higher affinity for the Cl(-)-channels in the basolateral membrane of the thick ascending limb of the loop of Henle. To quantity the inhibitory potency, we performed measurements of the equivalent short circuit current, corresponding to the secondary active transport of Cl- and measurements of the voltage across the basolateral membrane. A survey of 219 compounds reveals that relatively simple modifications in the structure of diphenylamine-2-carboxylate led to very potent blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate which inhibits the short circuit current half maximally (IC50) at 8 X 10(-8) mol/l. A comparison of the structural formula and the respective IC50 values leads to several empirical conclusions: The potent compounds are lipophilic due to the apolar residue (e.g. phenyl- or cycloalkyl group). Replacing this part of the molecule by an aliphatic chain (up to 4 C-atoms) leads to inactive compounds. Most of the inhibitors are secondary amines. Linking other than with -NH- between the phenyl ring and the benzoic acid results in inactive compounds. Tertiary amines, such as in case of 2-(N,N-diphenylamine)benzoic acid or N-methylphenylamine-benzoic acid are poorly active. The carboxylate group of the benzoate moiety must be in ortho position to the amino group. Introduction of substituents into the benzoate moiety e.g. -NO2 (in meta position to the carboxylate group), or by -Cl (in para position to the carboxylate group) results in an increase of inhibitory potency. A -CH2-, -C2H4-, -C3H6- spacer between the amino bridge and the phenyl ring increases the affinity for the Cl(-)-channel by several orders of magnitude. The above described structure activity relationship renders it likely that these chloride channel blockers possess several sites of interaction: The negatively charged carboxylate group, the secondary amine group which probably carries a positive partial charge, and for the very potent agents (nos. 130, 143, 144, and 145) an additional negative partial charge at the respective -Cl or -NO2 substituent. Finally, also an apolar interaction with an cycloalkyl or cycloaryl residue seems to be required, and this site of interaction has a defined spacing from the secondary amino nitrogen.

Animals↗
PubMed1986

A voltage-dependent ionic channel in the basolateral membrane of late proximal tubules of the rabbit kidney.

The patch-clamp method was applied to the lateral membrane of late proximal tubules of the rabbit kidney. Tubule segments were cannulated on one side by a perfusion system. At the noncannulated end of the tubules, the lateral membrane was accessible to a patch pipette. In cell-attached, as well as cell-excised (presumably inside-out oriented) membrane patches, a voltage sensitive channel was observed. The open-state probability of this channel increased with depolarizing potentials. In cell-excised patches bathed with NaCl-Ringer on both sides, the single channel conductance g was 28.0 +/- 1.2 pS (n = 10). With KCl-Ringer in the pipette and NaCl-Ringer in the bath g was 24.7 +/- 1.3 pS (n = 7) and the current-voltage curve crossed the axis at 0 mV. Therefore, the channel does not discriminate between K+ and Na+ ions. Replacing half of NaCl by mannitol on the bath side yielded a permeability for cations about twice as high as for Cl-. The channel could be reversibly blocked by diphenylamine-2-carboxylate (DPC), whereas its inhibition by SITS was only partially reversible. In cell-attached patches, the channel was nearly inactivated at zero clamp potential, but became active when the membrane patch was depolarized. The significance of this nonselective channel for proximal tubule cell function is still unclear. It could be involved in the contraluminal exit mechanism of various anions. However, it could also play a role in cell volume regulation processes.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1986

Effects of ouabain and temperature on cell membrane potentials in isolated perfused straight proximal tubules of the mouse kidney.

In isolated perfused segments of the mouse proximal tubule, the potential difference across the basolateral cell membrane (PDbl) was determined with conventional microelectrodes. Under control conditions with symmetrical solutions it amounted to -62 +/- 1 mV (n = 118). The potential difference across the epithelium (PDte) was -1.7 +/- 0.1 mV (n = 45). Transepithelial resistance amounted to 1.82 +/- 0.09 k omega cm (n = 28), corresponding to 11.4 +/- 0.6 omega cm2. Increasing bath potassium concentration from 5 to 20 mmol/l depolarized PDbl by +24 +/- 1 mV (n = 103), and PDte by +1.6 +/- 0.1 mV (n = 19). Thus, the basolateral cell membrane is preferably conductive to potassium. Rapid cooling of the bath perfusate from 38 degrees C to 10 degrees C led to a transient hyperpolarization of PDbl from -60 +/- 1 to -65 +/- 1 mV (n = 21) within 40 s followed by gradual depolarization by +18 +/- 1% (n = 14) within 5 min. The transepithelial resistance increased significantly from 1.78 +/- 0.11 k omega cm to 2.20 +/- 0.21 k omega cm (n = 15). Rapid rewarming of the bath to 38 degrees C caused a depolarization from -61 +/- 2 mV (n = 17) to -43 +/- 2 mV (n = 16) within 15 s followed by a repolarization to -59 +/- 2 mV (n = 10) within 40 s. Ouabain invariably depolarized PDbl. During both, sustained cooling or application of ouabain, the sensitivity of PDbl to bath potassium concentration decreased in parallel to PDbl pointing to a gradual decrease of potassium conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Na+ selective channels in the apical membrane of rabbit late proximal tubules (pars recta).

Using the patch-clamp technique, Na+ selective channels were observed in the luminal membrane of rabbit straight proximal tubule segments. In the cell-attached configuration (NaCl-Ringers in pipette and bath) influx of Na+ ions from the pipette into the cell through fluctuating channels was observed. The current-voltage curve of these Na+ channels yielded a zero-current potential of 84.3 +/- 30.9 mV (n = 10), reflecting the electrochemical driving force for Na+ influx under resting conditions. The single channel conductance was 12.0 +/- 2.1 pS (n = 13). In inside-out oriented cell-excised patches the single channel conductance was not significantly different with NaCl-Ringers on both sides. At clamp potentials ranging from +50 mV to -50 mV the single channel current was ohmic and channel kinetics were independent of the voltage. With KCl-Ringers on the bath side (corresponding to cell interior), the zero current potential was 62 +/- 19 mV (n = 4), indicating a high selectivity of Na+ over K+ ions. Addition of 10(-5) mol/l amiloride to the bathing solution decreased the mean channel open time slightly. This effect was more pronounced with 10(-4) mol/l amiloride, whereas the single channel conductance was unaffected by the diuretic. 10(-3) mol/l amiloride caused a complete block of the channel. It is concluded that amiloride sensitive Na+ channels, with similar properties to those observed in tight epithelia, contribute to Na+ reabsorbtion in the straight portion of proximal tubules.

Amiloride↗
PubMed1985

Diphenylamine-2-carboxylate, a blocker of the Cl(-)-conductive pathway in Cl(-)-transporting epithelia.

The present study examines the effects of diphenylamine-2-carboxylate (DPC) in Cl(-)-transporting epithelia. This substance blocks reversibly the Cl(-)-conductance present under normal circumstances in the basolateral membrane of the thick ascending limb of the loop of Henle (TAL) and in the apical membrane of shark rectal gland tubules (RGT). This leads to a reduction in active NaCl reabsorption (TAL) and NaCl secretion (RGT) respectively, as measured by the equivalent short circuit current. The cells hyperpolarize as the membrane voltage drifts from the control value (some compromise between the chemical potential of Cl- and K+) towards the chemical potential of K+. The resistance of the basolateral (TAL) or apical membrane (RGT) increases and this leads to a moderate increase in transepithelial resistance. In addition, the Cl(-)-concentration step induced membrane voltage changes, which can be produced under control conditions, disappear in the presence of the blocker. Finally, experiments in excised membrane patches indicate that this substance inhibits the single current events of individual Cl(-)-channels.

Animals↗
PubMed1985

Cl- -channels in the apical cell membrane of the rectal gland "induced" by cAMP.

Isolated rectal gland tubules (n approximately equal to 1000) of dogfish (Squalus Acanthias) were perfused in vitro. Individual channels in the apical and basolateral cell membrane were recorded with the patch clamp method. K+-channels were present in excised membrane patches of the basolateral membrane in stimulated (dbcAMP + forskolin + adenosine) and in nonstimulated state. Cl- -channels were found only in patches of the apical cell membrane when the tubule was stimulated. Cell attached recordings and simultaneous transepithelial PD measurements were obtained while the segment was stimulated. It is shown that concomitant with the increase in lumen negative PD "silent" membrane patches of the apical cell membrane suddenly develop Cl- -channel activity. It is concluded that stimulation of rectal gland tubules "activates" Cl- -channels in the apical cell membrane.

Animals↗
PubMed1985

cAMP increases the basolateral Cl- -conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse.

The effect of cAMP on transepithelial and transmembrane potential differences and resistances was examined in isolated in vitro perfused mouse medullary thick ascending limbs of Henle's loop (mTAL). The effects of furosemide and barium were tested. Stimulation of NaCl transport by ADH 10(-9) + dbcAMP 4 X 10(-4) + forskolin 10(-6) mol X l-1 (paired experiments) resulted in: a) an increase in transepithelial potential difference, referenced to the grounded bath, from +6.7 +/- 0.3 mV to +12.0 +/- 0.4 mV (n = 47); b) a decrease in transepithelial resistance from 25 +/- 1 omega cm2 to 20 +/- 1 omega cm2 (n = 47); c) a depolarization of the basolateral membrane by 12 mV and of the apical membrane by 7 mV (n = 36); d) a decrease in the fractional resistance of the basolateral membrane from 0.27 +/- 0.05 to 0.15 +/- 0.06 (n = 12). Furosemide (10(-4) mol X l-1) abolished the active transepithelial transport potential and hyperpolarized the basolateral membrane potential to values which were similar in both control and cAMP treated mTAL segments. Barium increased the transepithelial resistance and depolarized PDb1 to similar values in both functional states. An increase in the fractional conductance of the basolateral membrane was also seen, if, prior to the cAMP treatment, the luminal Na+2Cl-K+ cotransport was inhibited by furosemide. Thus, we propose that stimulation of active NaCl reabsorption in the mTAL segment of the mouse by ADH, mediated via cAMP, increases primarily the basolateral chloride conductance.

Animals↗