Biomedical subjects

R Warth

Publications and source records attributed to R Warth.

At least 37 records · Page 2Linked to original sources

PubMed1999

Molecular and functional characterization of the small Ca(2+)-regulated K+ channel (rSK4) of colonic crypts.

Colonic crypt cells possess basolateral Ca(2+)-regulated K+ channels which support Cl- secretion by providing the necessary driving force. The pharmacological characteristics of these channels were examined in Ussing chamber experiments of rat and rabbit colon mucosa by the use of blockers. The chromanol 293B, a blocker of KVLQT1 channels, and clotrimazole (CTZ), a blocker of small Ca(2+)-activated K+ channels, blocked stimulated Cl- secretion completely. Small-conductance Ca(2+)-activated K+ channels (SK) in excised basolateral patches of rat colonic crypts were inhibited concentration dependently by the imidazoles CTZ, NS004 and NS1619 and activated by 1-EBIO. These properties are similar to those of the known human SK channel (hSK4). hSK4-expressing Xenopus laevis oocytes showed ionomycin-activated and CTZ-inhibited K+ currents. When P2Y2 receptors were coexpressed these currents were also activated by ATP. The concentration/response curve was identical to that of rat SK channels. In human colonocytes (T84) exposed to hSK4 antisense probes, but not to sense probes, carbachol-induced K+ currents were attenuated. With RT-PCR an hSK4 could be demonstrated in human colon and in T84 colonocytes. By homology cloning the SK of the rat colon (rSK4) was identified. This protein has a high homology to hSK4 and mouse IK1. These data indicate that the Ca(2+)-activated and imidazole-inhibited basolateral K+ current in the colon is caused by SK4 channels.

Animals↗

PubMed1999

Evidence for Na+/Ca2+ exchange in the rectal gland of Squalus acanthias.

Previously we have shown that stimulation of in vitro perfused rectal gland tubules (RGT) of the dog-fish Squalus acanthias by adenosine 3',5'-cyclic monophosphate (cAMP), (as a cocktail comprising 0.1 mmol/l dibutyryl-cAMP, 10 micromol/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.

Animals↗

PubMed1998

Mineralocorticoid receptor knockout mice: pathophysiology of Na+ metabolism.

Mineralocorticoid receptor (MR)-deficient mice were generated by gene targeting. These animals had a normal prenatal development. During the first week of life, MR-deficient (-/-) mice developed symptoms of pseudohypoaldosteronism. They finally lost weight and eventually died at around day 10 after birth from dehydration by renal sodium and water loss. At day 8, -/- mice showed hyperkalemia, hyponatremia, and a strong increase in renin, angiotensin II, and aldosterone plasma concentrations. Methods were established to measure renal clearance and colonic transepithelial Na+ reabsorption in 8-day-old mice in vivo. The fractional renal Na+ excretion was elevated >8-fold. The glomerular filtration rate in -/- mice was not different from controls. The effect of amiloride on renal Na+ excretion and colonic transepithelial voltage reflects the function of amiloide-sensitive epithelial Na+ channels (ENaC). In -/- mice, it was reduced to 24% in the kidney and to 16% in the colon. There was, however, still significant residual ENaC-mediated Na+ reabsorption in both epithelia. RNase protection analysis of the subunits of ENaC and (Na++ K+)-ATPase did not reveal a decrease in -/- mice. The present data indicate that MR-deficient neonates die because they are not able to compensate renal Na+ loss. Regulation of Na+ reabsorption via MR is not achieved by transcriptional control of ENaC and (Na+ + K+)-ATPase in RNA abundance but by transcriptional control of other as yet unidentified genes. MR knockout mice will be a suitable tool for the search of these genes.

Amiloride↗

PubMed1998

The basolateral Ca2+-dependent K+ channel in rat colonic crypt cells.

Previous studies have indicated that a 16-pS K+ channel (KCca) in the basolateral membrane is responsible for the acetylcholine-induced whole-cell K+ conductance in these cells. In the present study we have examined this channel in excised inside-out patches of the basolateral membrane. Over a wide voltage range this channel showed inward rectification. The Ca2+ sensitivity was very marked, with a Hill coefficient of three and with half-maximal activation at 330 nmol/l. After several minutes most channels showed a slow run-down. Channel activity could be refreshed by addition of ATP (1 mmol/l) to the bath solution. The non-metabolizable derivative 5'-adenylylimidodiphosphate (AMP-PNP) had no such effect. In contrast, it inhibited channel activity by some 50%. ATP and its derivatives had no effect on the Ca2+ sensitivity. Channels activated by ATP were subsequently studied in the presence of alkaline (10 kU/l) or acidic (1 kU/l) phosphatase. Both phosphatases reduced channel activity significantly. These data suggest that the 16-pS K+ channel is directly controlled by cytosolic Ca2+. This regulatory step is probably distal to an activation produced by protein-kinase-C-dependent phosphorylation. As is the case for several other K+ channels, high concentrations of non-metabolizable ATP analogues inhibit this channel.

Acid Phosphatase↗

PubMed1998

The role of cytosolic Ca2+ in the secretion of NaCl in isolated in vitro perfused rectal gland tubules of Squalus acanthias.

In many exocrine glands cytosolic Ca2+ ([Ca2+]i) plays a pivotal role in stimulation-secretion coupling. In the rectal gland of the dogfish Squalus acanthias this appears not to be the case and it is believed that secretion is mainly controlled by the Cl- conductance of the luminal membrane. We have examined this question in a study of isolated in vitro perfused rectal gland tubules (RGT). Three types of measurements were performed: (1) measurements of [Ca2+]i by the fura-2 technique; (2) measurements of transepithelial electrical parameters, i.e. transepithelial voltage (Vte), transepithelial resistance (Rte), the equivalent short-circuit current (Isc) and the voltage across the basolateral membrane (Vbl), and (3) whole-cell patch-clamp measurements of cellular voltage (Vm), conductance (Gm) and membrane capacitance (Cm). The data indicates that carbachol (CCH) increases [Ca2+]i by increasing store release and Ca2+ influx. Other agonists, producing cytosolic cAMP, also increased [Ca2+] by enhancing Ca2+ influx. CCH hyperpolarized these cells and enhanced Gm significantly. The effect of CCH on Vte and Isc was most marked under control conditions and disappeared in RGT otherwise stimulated by agonists that lead to cAMP production. It is concluded that [Ca2+]i plays a major role in the stimulation of NaCl secretion in RGT by enhancing the basolateral K+ conductance. cAMP-producing agonists enhance [Ca2+]i by increased Ca2+ influx. CCH releases Ca2+ from respective stores. CCH, unlike the cAMP-producing agonists, only increases basolateral K+ conductance. It modulates secretion especially under conditions in which the cAMP pathway is not fully activated.

Adenosine Triphosphate↗

PubMed1998

Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts.

It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca2+ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from -78+/-2.0 to -66+/-3.8 mV (n=22) and from -78+/-1. 3 to -56+/-2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20+/-8.0 to 14+/-6.6 nS (n=7) and from 20+/-3.0 to 9.8+/-1.6 nS (n=19), respectively. In cell-attached patches K+ channels with a single-channel conductance of approximately 16 pS were found in most recordings. The activity of these channels (NxPo, N=number, Po=open channel probability) was reduced from 2.08+/-0.37 to 0.98+/-0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75+/-0.26 to 0.77+/-0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K+ channel is controlled by cytosolic Ca2+ ([Ca2+]i). Therefore we measured [Ca2+]i by the fura-2 method and found that hypertonic solution reduced [Ca2+]i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca2+]i falls in hypertonic solution leading to a reduction in the value of K+ channel NxPo, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell.

Animals↗

PubMed1998

pH-regulatory mechanisms in in vitro perfused rectal gland tubules of Squalus acanthias.

Isolated in vitro perfused rectal gland tubules (RGT) were preincubated with the pH-sensitive dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and pH-regulatory mechanisms were studied. A reduction of bath Cl- concentration from 269 to 6 mmol/l increased the fluorescence ratio 488/436 [corresponding to cytosolic pH (pHi)] slightly but significantly (n=10). Depolarization by Ba2+ (1 mmol/l) or a bath solution containing 30 mmol/l K+ (n=4-6) increased the fluorescence ratio (pHi). These data suggest that HCO3- uptake and/or H+ extrusion is dependent on Cl- and/or voltage. A reduction of bath Na+ from 278 to 5 mmol/l reduced the ratio significantly (n=3). Addition of trimethylamine (Trima+, 20 mmol/l) alkalinized cytosolic pH (n=7). Similarly, addition of NH4+ (20 mmol/l) led to an initial alkalinization and a strong acidification when NH4+ was removed (n=59). The initial pHi-recovery rates after NH4+ removal were quantified and the responsible H+ extrusion and/or HCO3- import systems were examined. The recovery was almost completely abolished when the extracellular Na+ concentration was reduced to 5 mmol/l. In the presence of normal Na+, recovery was slower in the absence as compared to the presence of HCO3- (n=5). It was inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (0.5 mmol/l, n=11) in the presence of HCO3- and in the absence of HCO3- by the Na+/H+-exchange blocker HOE694 (0.5 mmol/l, n=6). These data suggest that acid extrusion probably occurs by basolateral Na+-2HCO3-/Cl- exchange in the presence of HCO3- and by basolateral Na+/H+ exchange in the absence of HCO3-. Luminal perfusion with a solution containing a low Cl- concentration (6 mmol/l) increased the fluorescence ratio (pHi) (n=5). The ratio (pHi) was further increased and pH recovery further delayed by basolateral addition of Trima+ (20 mmol/l, n=3). These data suggest that the HCO3-/Cl- exchanger is present in the luminal membrane. Luminal HCO3-/Cl- exchange and basolateral Na+-2HCO3-/Cl- exchange may work in tandem to secrete HCO3- and exchange it for luminal Cl-.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

Regulation of the Na+2Cl–K+ cotransporter in in vitro perfused rectal gland tubules of Squalus acanthias.

Previously it has been shown that the Na+2Cl–K+ cotransporter accepts NH4 + at its K+ binding site. This property can be used to estimate its transport rates by adding NH4 + to the bath and measuring the initial furosemide-dependent rates of change in BCECF fluorescence. We have utilized this technique to determine the regulation of the furosemide-inhibitable Na+2Cl–K+ cotransporter in in vitroperfused rectal gland tubules (RGT) of Squalus acanthias. Addition of NH4 + to the bath (20 mmol/l) led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4 + uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (Δfluorescence/time). This acidification could be completely inhibited by furosemide. In the absence of any secretagogue preincubation of RGT in a low Cl– solution (6 mmol/l, low Cl–) for 10 min enhanced the uptake rate significantly from 4.04±0.51 to 12.7±1.30 (n=5). The addition of urea (200 mmol/l) was without effect, but the addition of 300 mmol/l mannitol (+300 mannitol) enhanced the rate significantly from 7.24±1.33 to 14.7±4.6 (n=6). Stimulation of NaCl secretion by a solution maximizing the cytosolic cAMP concentration (Stim) led to a significant increase in NH4 + uptake rate from 5.00±1.33 to 13.3±1.54 (n=6). Similar results were obtained in the additional presence of Ba2+ (1 mmol/l): the uptake rate was increased significantly from 4.23±0.34 to 15.1±1.86 (n=16). In the presence of Stim low Cl– had no additional effect on the uptake rate: 15.1±3.1 versus 15.2±2.8 in high Cl– (n=6). The uptake rate in Stim containing additional +300 mannitol (22.3±4.0, n=5) was not significantly different from that obtained with Stim or +300 mannitol alone. By whatever mechanism the NH4 + uptake rate was increased furosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced furosemide-inhibitable uptake rates of the Na+2Cl–K+ cotransporter probably independently: (1) lowering of cytosolic Cl– concentration; (2) cell shrinkage; and (3) activation by cAMP.

Animals↗

PubMed1998

Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance?

NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl– channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl– channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl– channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 µmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G m). The membrane capacitance (C m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C m correlated with the increase in G m with a slope of 51 fF/nS. Next the effect of furosemide (500 µmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na+2Cl–K+ uptake and to reduce cell volume but not membrane trafficking of Cl– channels. Furosemide reduced G m slightly (due to the fall in cytosolic Cl– concentration) and C m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC m/ΔG m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 µmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G m or C m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C m changes observed with Stim reflect a minor cell volume increase and an ”unfolding” of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl– channels in these cells.

Adenosine↗

PubMed1998

Osmotically induced conductance and capacitance changes in in vitro perfused rectal gland tubules of Squalus acanthias.

The rectal gland of Squalus acanthias is critically involved in the homeostasis of NaCl and water metabolism and hence in overall osmoregulation. In the present study, we have examined the acute responses of rectal gland slices and in vitro perfused rectal gland tubule (RGT) cells to the exposure to dilute and hypertonic peritubule solutions. Five series were performed. (i) With changes in osmolality, Western blots to monitor tyrosine, threonine and serine phosphorylation in rectal gland slices did not reveal clear-cut changes in phosphorylation patterns. All other series were performed in in vitro perfused RGT. (ii) Relative cell volume was estimated by fura-2 fluorescence using the emission at the isosbestic excitation wavelength of 360 nm. Hypotonic solution (-100 mmol/l NaCl) reduced fura-2 fluorescence by 16% and hypertonic solution (+100 mmol/l NaCl) had the opposite effect (+12%). (iii) Transepithelial resistance was increased markedly by hypotonic solution, probably by cell swelling, and the opposite was seen with hypertonic solutions. (iv) Whole-cell patch clamp experiments indicated that hypotonic solution hyperpolarized the cells, and increased membrane conductance and membrane capacitance. The latter two changes correlated significantly with each other. Hypertonic solution had the opposite effect. (v) Measurements of the fura-2 fluorescence ratio (340/380 nm) revealed that hypotonic solution (-NaCl) increased cytosolic Ca2+ activtiy ([Ca2+]i). Hypertonic solution had no detectable effect on [Ca2+]i. These data indicate that RGT cells are swollen by removal of NaCl from the bath solution. This causes an increase in [Ca2+]i and a predominant increase in K+ conductance and hyperpolarization. Urea apparently permeates these cells quite well and its addition (+U) or its removal (-U) had only moderate osmotic effects. The removal of urea and replacement by mannitol produced effects similar to those seen with hypertonic NaCl solution.

Animals↗

PubMed1997

New types of K+ channels in the colon.

The mechanism of epithelial Cl- secretion requires K+ recycling via basolateral ion channels. Two types of K+ channels were identified. A Ca(2+)-activated, and a very small cAMP-regulated K+ channel. Increase of cAMP inhibits the Ca(2+)-activated K+ channel and thus makes small K+ channel the limiting step for cAMP-dependent Cl- secretion.

Animals↗

PubMed1997

The role of K+ channels in colonic Cl- secretion.

Cl- secretion in the rat colonic crypt base cell (bc) requires the coordinated (a) opening of Cl- channels in the luminal membrane; (b) activation of the Na+2Cl-K+ cotransporter; (c) enhanced conductive K+ exit from the cell; and (d) increased pumping by the (Na+ + K+)-ATPase. In this study we focus on the importance of conductive K+ exit. After stimulation with the cholinergic agonist carbachol (CCH, 0.1-10 mumol/l) bc respond with a marked increase in whole cell (wc) conductance and a hyperpolarization of the membrane voltage (Vm). This is paralleled by a marked increase in the (Cl- secretory) short-circuit current (Isc) in Ussing chamber studies of the intact distal colon. Current evidence favors the view that CCH, via IP3, enhances cytosolic Ca2+ activity, and that Ca2+ increases the open probability of Cl- channels indirectly and that of K+ channels directly. After stimulation with PGE2 bc also enhance the wc conductance, but this is paralleled by a marked depolarization of Vm. Again these effects correspond to a marked increase in (Cl- secretory) Isc. The depolarization and enhanced wc conductance is partly due to the activation of Cl- channels. However, current evidence suggests that these effects on Cl- channels are paralleled by an activation of K+ channels. The chromanol 293B, by inhibiting these K+ channels specifically, abolishes PGE2-induced Cl- secretion completely, but has no effect on basal K+ conductance or on CCH-induced Cl- secretion. CCH apparently activates a Ca(2+)-dependent K+ channel with a conductance of 10-20 pS, whilst PGE2 (or cAMP) activate a much smaller K+ channel. Only the latter K+ channel can be inhibited by 293B in excised patches. Noise analysis suggests that this K+ channel has a conductance of < 3 pS and fast kinetics. The complete 293B induced inhibition of Cl- secretion caused by PGE2 can be explained by the fact that PGE2 induces a marked depolarization and that this depolarization reduces the basal K+ conductance. Current evidence suggests that this inhibition of the basal K+ conductance is caused by a depolarization induced inhibition of Ca2+ entry.

Animals↗

PubMed1997

Functional analysis of the yeast 40 kDa cyclophilin Cyp40 and its role for viability and steroid receptor regulation.

We have identified and characterized a homolog of the 40 kDa cyclophilins in the budding yeast Saccharomyces cerevisiae. At the amino acid level, this novel yeast cyclophilin, termed Cyp40, is 47% identical to human cyclophilin-40. Recombinant Cyp40 produced in bacteria has a peptidyl-prolyl cis-trans isomerase activity with a catalytic efficiency (k[cat]/K[m]) of 0.5 x 10(6)M(-1)s(-1), which can be inhibited by cyclosporin A with an IC50 value of 60nM. Using a polyclonal antibody against Cyp40 we have found that Cyp40 is predominantly cytoplasmic, and that its expression is induced 3-4-fold by heat shock. Moreover, Cyp40 can be coprecipitated from yeast extracts with the cytosolic molecular chaperone Hsp90. Surprisingly, a Cyp40-deficient yeast strain is fully viable at normal and elevated temperatures. Cyp40 is also dispensable for normal regulation of vertebrate steroid receptors in yeast. While other immunophilins could conceivably compensate a Cyp40 defect, our results are compatible with the notion that immunophilins may be fortuitous partners in the biochemically established steroid receptor-Hsp90 complex.

Amino Acid Isomerases↗

PubMed1996

Two eukaryote-specific regions of Hsp82 are dispensable for its viability and signal transduction functions in yeast.

The 90-kDa heat shock protein (Hsp90) is a molecular chaperone that is very abundant even at normal temperature. It is highly conserved and essential for viability in yeast. To delineate functional domains of Hsp90, we have performed a deletion analysis of one of the two Hsp90 isoforms from budding yeast, Hsp82. The Hsp82 derivatives were tested for complementation of a Hsp90-deficient yeast strain and for their ability to function in two signal transduction pathways that depend on Hsp90. Surprisingly, we found that two salient features of Hsp90 sequences from eukaryotes, the N-terminal charged domain and the extremely conserved C-terminal pentapeptide MEEVD, are dispensable for viability as well as for proper regulation of vertebrate steroid receptors and for pheromone signaling. Moreover, we describe, to our knowledge, the first dominant negative mutant of Hsp90; A Hsp82 derivative that lacks amino acids 538-552 fails to complement but has a dominant negative effect on viability of wild-type strains at moderately elevated temperatures. This mutant may become a valuable tool to study Hsp90 functions both in yeast and in mammalian cells.

Amino Acid Sequence↗

PubMed1996

Regulation of epithelial ion channels by the cystic fibrosis transmembrane conductance regulator.

In most epithelia ion transport is tightly regulated. One major primary target of such regulation is the modulation of ion channels. The present brief review focuses on one specific example of ion channel regulation by the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a cAMP-regulated Cl- channel. Its defect leads to the variable clinical pictures of cystic fibrosis (CF), which today is understood as a primary defect of epithelial Cl- channels in a variety of tissues such as the respiratory tract, intestine, pancreas, skin, epididymis, fallopian tube, and others. Most recent findings suggest that CFTR also acts as a channel regulator. Three examples are discussed by which CFTR regulates other Cl- channels, K+ channels, and epithelial Na+ channels. From this perspective it is evident that CFTR may play a major role in the integration of cellular function.

Animals↗

PubMed1996

The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells.

We have shown previously that secretagogues acting via the second messenger adenosine 3',5'-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl- conductance, a K+ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K+ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 micromol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K+ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency (fc) of 308 +/- 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K+ selective. 293B reduced the power density of the noise (So) to 46 +/- 8.7% of its control value and shifted fc from 291 +/- 26 to 468 +/- 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5'-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba2+ and was completely absent when K+ in the bath was replaced by Na+. These data suggest that forskolin activates a K+ channel of very small conductance which can be inhibited directly and reversibly by 293B.

Animals↗

PubMed1996

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells.

We have previously shown that a new type of K+ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K+ channel of 16 +/- 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0. 1 mmol/l, n = 26). Reduction of extracellular Ca2+ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SKCa). It was also inactivated by forskolin (5 micromol/l, n = 38), whilst the K+ channel noise caused by the very small K+ channel increased. Activity of non-selective cation channels (NScat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NScat, with a mean conductance of 49 +/- 1.0 pS (n = 96), was Ca2+ activated and required >10 micromol/l Ca2+ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3',5-dichloro-diphenylamine-2-carboxylate (10-100 micromol/l, n = 5). SKCa was also Ca2+ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 micromol/l (cs) and then fell sharply to almost zero at 0.1 micromol/l Ca2+ (cs, n = 12). SKCa was inhibited by Ba2+ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca2+ activity ([Ca2+]i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K+ concentration to 30 mmol/l, reduced [Ca2+]i markedly (n = 8-10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca2+-sensitive K+ channel. This channel is activated promptly by very small increments in [Ca2+]i and is inactivated by a fall in [Ca2+]i induced by forskolin.

Adenosine Triphosphate↗

PubMed1995

A new class of inhibitors of cAMP-mediated Cl- secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance.

Previously we have shown that arylaminobenzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (Isc) induced by prostaglandin E2 (n = 7), vasoactive intestinal polypeptide (VIP, n = 5), adenosine (n = 3), cholera toxin (n = 4) and cAMP (n = 6), but not by ionomycin (n = 5) in distal rabbit colon half maximally (IC50) at 2 mumol/l from the mucosal and at 0.7 mumol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116 +/- 16 omega.cm2 to 136 +/- 21 omega.cm2 (n = 5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound within this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mumol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mumol/l 293 B had no effect on the membrane voltage across the basolateral membrane (Vbl) in non-stimulated crypt cells: -69 +/- 3 mV versus -67 +/- 3 mV (n = 10), whilst in the same cells 1 mmol/l Ba2+ depolarised Vbl significantly. However, 293 B depolarised Vbl significantly in the presence of 1 mumol/l forskolin: -45 +/- 4 mV versus -39 +/- 5 mV (n = 7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised Vbl from -40 +/- 5 mV to -30 +/- 4 mV (n = 19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mumol/l 293 B: -75 +/- 6 mV versus -75 +/- 6 mV (n = 6). Also 293 B had no effect on basal K+ conductance (n = 4). Hence, we conclude that 293 B inhibits the K+ conductance induced by cAMP. This conductance is apparently relevant for Cl- secretion and the basal K+ conductance is insufficient to support secretion.

Amiloride↗