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

A membrane fraction, rich in basal-lateral plasma membranes, was prepared from the rectal gland of the spiny dogfish, Squalus acanthias, and the uptake ofNa into the plasma membrane vesicles was investigated by a rapid filtration technique. Sodium uptake was greatest in the presence of a chloride gradient directed into the vesicles; it was strikingly reduced when chloride was replaced with nitrate and was even slower with gluconate. If the membrane vesicles were pre-equilibrated with potassium chloride or potassium nitrate plus valinomycin, to minimize any electrical driving forces on sodium movement, the uptake of sodium was still greatest in the presence of chloride and remarkably decreased in the presence of nitrate. Furosemide, 10 and 10 M, decreased sodium uptake into the vesicles in a dose dependent manner only in the presence of chloride. Furthermore, saturation of sodium uptake by increasing sodium chloride concentrations was observed. The above results provide direct evidence for a coupling of sodium and chloride fluxes across the plasma membrane of the rectal gland via a cotransport system sensitive to furosemide. They support the hypothesis that chloride secretion of the rectal gland is a secondary active transport and is driven by the sodium gradient across the basal-lateral membranes of the cell. [ABSTRACT FROM AUTHOR]

The present study examines the influences of pH and Ca 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 mol/l has no detectable inhibitory effect on this channel. Conversely, increase in calcium activity to some 10 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. Ba (≈5·10 mol/l), quinine (≈10 mol/l), quinidine (≈10 mol/l), lidocaine (≈1 mmol/l), tetraethylammonium (≈10 mmol/l), Cs (≈10 mmol/l), and Rb (≈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 Ca directly, but that it is qualitatively similar to many other known K channels with respect to its sensitivity towards blockers. [ABSTRACT FROM AUTHOR]

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 [7], 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 mol/l) and by 5-nitro-2-(3-phenylpropylamino)-benzoate (10 mol/l). The channel is selective for Cl over Na and K as well as over Br. It is, however, permeable for NO. 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. [ABSTRACT FROM AUTHOR]

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 [9]. 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. [ABSTRACT FROM AUTHOR]

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 [5]. 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 ·10 cm/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). The single channel permeability coefficient for K was 0.21±0.03·10 cm/s ( n=7). We were not able to reverse the currents in any of these experiments, indicating that this channel is highly selective for K over Na. In all three series of experiments, the kinetic appearance of the channels was similar. Bursts of activity were followed by interburst pauses. The open state was described by a single time constant of 3.0±0.2 ms, whereas the closed state was described by two time constants of 0.7±0.2 ms and 2.8±0.5 ms ( n=8). It can be concluded that these channels permit K inward and outward currents. They are probably the equivalent of the basolateral K conductance as observed in a previous study [12]. Under physiological conditions a single channel conductance of some 20 pS is predicted from the present data. In cell-attached patches, with a high K concentration in the pipette, the channel behaves as an inward rectifier. [ABSTRACT FROM AUTHOR]

Rectal gland tubule (RGT) segements of the spiny dogfish ( Squalus acanthias) were perfused in vitro. The effects of inhibitors of known mode of action on transepithelial PD (PD resistance (R), the PD across the basolateral membrane (PD), the fractional resistance of this membrane (FR), and intracellular activities of Na, Cl, K ( a) were examined. Furosemide (5·10 mol·l) reduced PD from −12±0.7 to −2.3±0.2 mV ( n=63), hyperpolarized PD from −71±1.3 to −79±0.9 mV ( n=59), FR decreased from 0.2±0.03 to 0.13±0.01 ( n=21), a fell from 38±4 to 11±2 mmol·l ( n=14), a fell from 37±4 to 17±2 mmol·l ( n=12) and a was constant [113±14 vs. 117±15 mmol·l ( n=6)]. Furosemide exerted its effects within some 20-40 s. Its action was completely reversible. Analysis of the time courses revealed that the furosemide induced initial fall in a was approximately twice as rapid when compared to that of a. Ba 0.5 mmol·l (bath) reduced PD from −7.4±1.2 to −4.1±0.6 mV ( n=24), increased R from 18±2 to 22±2.5, Ωcm ( n=14). PD depolarized from −75±2 to −48±2 mV ( n=42), FR increased from 0.2±0.02 to 0.34±0.04 ( n=14) and a increased from 143±28 to 188±40 mmol·l ( n=4). Ouabain (50·10 mol·l, bath) reduced PD from −12±2 to −3±0.5 mV ( n=9), R increased from 18±3 to 21±3 Ωcm ( n=5), PD depolarized from −67±4 to −26±3 mV ( n=14), FR increased from 0.23±0.04 to 0.45±0.05 ( n=6), a fell only slightly from 135±15 to 112±30 mmol·l ( n=4), but a increased from 35±12 to 111±37 mmol·l ( n=3). These effects of ouabain were slow when compared to those exerted by furosemide or Ba. The ouabain effects on PD and PD were completely prevented if furosemide was applied first. Amiloride (≤10 mol·l, both sides), and anthracene-9-carboxylate (≤10 mol·l, lumen) were devoid of effects on PD and PD. The stilbene disulfonate derivate SITS (≤10 mol·l, both sides) led to a 36±9% inhibition of PD. The present data, apart from supporting the cell model proposed in a preceding report, allow the following conclusions: The transient analysis of the furosemide effects is suggestive for a 2 Cl∶1 Na stoichiometry of the carrier. The furosemide induced reduction in FR suggests that the fall in aleads to a marked reduction in the apical Cl-conductance. Furosemide apparently drives a Cl/cell to passive distribution and PD to the K equilibrium potential. a then approaches a minimal value which is close to that present in nonstimulated RGT segments. Ba leads to an increase in a because it blocks the conductive recycling pathway for this ion. Ouabain does not only inhibit the (Na+K)-ATPase, it also reduces the K-conductance of the basolateral membrane. This explains why a stays high. The cell PD then is dominated by a. The Na 2ClK-carrier increases a further and the cell continues to depolarize. Thus, the depolarization of the cell is not a sign of K loss but indicative of Cl gain. In the presence of furosemide ouabain is devoid of any effect on PD, suggesting that the Na2 ClK-carrier is the only quantitatively important source of Na entry into the RGT cell. [ABSTRACT FROM AUTHOR]

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD), resistance (R), the PD across the basolateral membrane (PD) and intracellular chloride and potassium activities ( a, a) were measured. In a first series, 67 RGT segments were perfused with symmetric shark 'Ringers' solution. The bath perfusate contained in addition db-cAMP 10, forskolin 10, and adenosine 10 mol · l. PD was −11±1 ( n=67) mV lumen negative, R 27±2 ( n=47) Ω cm. PD −75±0.4 ( n=260) mV. a and a were 109±22 ( n=4) and 38±4 ( n=36) mmol · l respectively. These data indicate that Cl secretion across the RGT must be an uphill transport process, whereas secretion of Na could be driven by the lumen negative PD. Intracellular K is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K concentration led to a rapid hyperpolarization of PD with a mean slope of 19 mV per decade of K concentration change. Addition of 0.5 mmol · l Ba to the bath solution lead to a marked depolarization and abolished the response to K concentration steps. In the lumen a Cl concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I=PD/R) was determined as a function of symmetrical changes in Na concentration, with Cl held at 276 mmol · l, and as a function of symmetrical changes in Cl concentration, with Na held at 278 mmol · l I was a saturable function of Na concentration (Hill coefficient 0.9±0.1, K 4.4 mmol · l, n=7) and also a saturable function of Cl concentration (Hill coefficient 2.0±0.1, K 75 mmol · l, n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na per 2 Cl stoichiometry. In series 4, the effect of a K concentration downward step on PD and a transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10 to 10 mol · l) in an attempt to examine whether K reduction on the bath side inhibits NaCl uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na+K)-ATPase, probably the Na 2 ClK carrier, and a K conductance. In the apical membrane a Cl conductance; and a Na conductive paracellular pathway. [ABSTRACT FROM AUTHOR]

Previously we have shown that stimulation of in vitro perfused rectal gland tubules (RGT) of the dogfish Squalus acanthias by adenosine 3',5'-cyclic monophosphate (cAMP), (as a cocktail comprising 0.1 mmol/l dibutyryl-cAMP, 10 µmol/l forskolin and 0.1 mmol/l adenosine, hereafter termed STIM) leads to an increase in cytosolic Ca2+ ([Ca2+]i) and that this assists Cl– secretion by enhancing basolateral K+ conductance. In the present study we examined the mechanism of the cAMP-induced increase in [Ca2+]i. [Ca2+]i was measured using the fura-2 technique in isolated in vitro perfused RGT. As before, STIM enhanced [Ca2+]i. This elevation of [Ca2+]i was prevented completely when STIM was added in the presence of the Na+2Cl–K+ cotransport inhibitor furosemide (0.5 mmol/l). This suggests that the increase in [Ca2+]i induced by STIM is caused by a concomitant increase in cytosolic Na+ ([Na+]i) and not by the activation of second messenger cascades. Furosemide prevents this increase in [Na+]i and hence the elevation of [Ca2+]i. Moreover, the plateau phase of the [Ca2+]i transient produced by carbachol (CCH, 0.1 mmol/l) was augmented strongly when bath Na+ was reduced to 5 mmol/l. These data suggest that the level of [Ca2+]i is determined by Na+-dependent Ca2+ export, most likely via a Na+/Ca2+ exchanger. The increase in [Na+]i accompanying stimulation of Cl– secretion reduces the rate of Ca2+ export leading to an elevation of [Ca2+]i, as does a reduction in bath Na+ which augments the [Ca2+]i plateau produced by CCH. [ABSTRACT FROM AUTHOR]

An abstract of the article "Activation of Shark Na+,K+,2Cl- Cotransporter s-NKCCl by the Serine/Threonine Kinase h-sgk" is presented. The study aims to test the influence of h-sgk, a serine/threonine glucocorticoid kinase, on the shark's sodium, potassium, and chlorine cotransporter shark rectal gland (s-NKCCl). As a result, h-sgk activates the sNKCCl by stimulating its insertion into the cell membrane.

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. [ABSTRACT FROM AUTHOR]