Search PubMed⌕ Search

Biomedical subjects

R Greger

Publications and source records attributed to R Greger.

At least 37 records · Page 2Linked to original sources
PubMed2000

Mineralocorticoid receptor knockout mice: lessons on Na+ metabolism.

The mineralocorticoid receptor (MR) binds aldosterone and glucocorticoids with equal affinity. In aldosterone target tissues, like the epithelial cells of the distal colon and the principal cells of the collecting ducts in the kidney, the MR is protected from glucocorticoids by the action of the enzyme 11beta-hydroxysteroid-dehydrogenase type 2 (11betaOHSD2), allowing aldosterone to specifically activate the receptor. However, in MR-expressing cells, which lack 11betaOHSD2, like the neurons of the limbic system in the brain, MR is mainly activated by glucocorticoids. MR knockout mice die in the second week after birth, showing at day 8 symptoms of pseudohypoaldosteronism with hyponatremia, hyperkalemia, high renal salt wasting, and a strongly activated renin-angiotensin-aldosterone system (RAAS). The activity of the amiloride-sensitive epithelial Na+ channel (ENaC) is strongly reduced in colon and kidney, but there is no down-regulation of the mRNA abundance of the three ENaC subunits. Daily subcutaneous injections of isotonic NaCl solution until weaning and continued oral NaCl supply lead to survival of the MR knockout mice. The NaCl-rescued MR knockout mice display a strongly enhanced fractional renal excretion of Na+, hyperkalemia, and a persistently strongly activated RAAS. There is almost no renal ENaC activity. The renal mRNA abundance of alphaENaC is reduced by 30%, whereas betaENaC and gammaENaC are not altered.

Animals↗
PubMed2000

Physiology of renal sodium transport.

A wealth of studies performed with a spectrum of methods spanning simple clearance studies to the molecular identification of ion transporters has increased our understanding of how approximately 1.7 kg of NaCl and 180 L of H2O are absorbed by renal tubules in man and how the urinary excretion is fine-tuned to meet homeostatic requirements. This review will summarize our current understanding. In the proximal nephron, approximately 60 to 70% of the filtered Na+ and H2O is absorbed together with approximately 90% of the filtered HCO3-. The exact quantities are determined by many regulatory factors, such as glomerulotubular balance, angiotensin II, endothelin, sympathetic innervation, parathyroid hormone, dopamine, acid base status and others. The essential components of absorption are luminal membrane Na+/H+ exchange and the basolateral (Na+ + K+)-ATPase. In the thick ascending limb of the loop of Henle, 20 to 30% of the filtered NaCl is absorbed via Na+2Cl-K+ cotransport driven by the basolateral (Na+ + K+)-ATPase. No H2O is absorbed at this nephron site. The transport rate is determined by the Na+ load and by several hormones and neurotransmitters, including prostaglandins, parathyroid hormone, glucagon, calcitonin, arginine vasopressin (AVP), and adrenaline. In the distal tubule, some 5 to 10% of the filtered load is absorbed via Na+Cl- cotransport in the luminal membrane driven by the basolateral (Na+ + K+)-ATPase. The rate of transport is again determined by the delivered load and by several hormones and neurotransmitters. One of the tasks of the collecting duct is to control the absorption of approximately 10 to 15% of the filtered H2O, regulated by AVP, and just a few percent of the filtered Na+, controlled by aldosterone and natriuretic hormone. The water absorption proceeds through the luminal membrane via aquaporin 2 and through the basolateral membrane via aquaporin 3 channels and is driven by the osmotic gradient built up by the counter current concentrating system. The Na+ absorption occurs via Na+ channels present in the luminal membrane driven by the basolateral (Na+ + K+)-ATPase. With no pharmacological interference, urinary excretion of Na+ can vary between less than 0.1% and no more than 3% of the filtered load, and that of H2O can vary between 0.3 and 15%.

Absorption↗
PubMed2000

Role of CFTR in the colon.

In contrast to the airways, the defects in colonic function in cystic fibrosis (CF) patients are closely related to the defect in CFTR. The gastrointestinal phenotype of CF transgenic mice closely resembles the phenotype in CF patients, which clearly indicates the crucial role of CFTR in colonic Cl- secretion and the absence of an effective compensation. In the colon, stimulation of CFTR Cl- channels involves cAMP- or cGMP-dependent phosphorylation. Exocytosis is not involved. Activation of CFTR leads to coactivation of basolateral KVLQT1-type K+ channels and inhibition of luminal Na+ channels (ENaC). In contrast to cultured cells, Ca2+ does not activate luminal Cl- channels in intact enterocytes. It activates basolateral SK4-type K+ channels and luminal K+ channels, which provide additional driving force for Cl- exit. The magnitude of Cl- secretion, however, completely depends on the presence of at least a residual CFTR function in the luminal membrane. These findings have been clearly demonstrated by Ussing chamber experiments in colon epithelium biopsies of CF and normal individuals: Colonic Cl- secretion in CF patients is variable and reflects the genotype; a complete defect of CFTR is paralleled by the absence of Cl- secretion and unmasks Ca(2+)-regulated K+ channels in the luminal membrane; overabsorption of Na+ in CF reflects the absence of ENaC inhibition by CFTR; and the functional status of CF colon can be mimicked by the complete suppression of cAMP stimulation in enterocytes of healthy individuals.

Animals↗
PubMed2000

Defective cholinergic Cl(-) secretion and detection of K(+) secretion in rectal biopsies from cystic fibrosis patients.

Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl(-) secretion and an inverse response of the short-circuit current (I(sc)) toward stimulation with carbachol (CCh). Alternative Cl(-) channels are found in airway epithelia and have been attributed to residual Cl(-) secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed I(sc) upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca(2+)-dependent Cl(-) conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and I(sc) were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I(sc) in CF rectal biopsies but caused a negative I(sc) in non-CF subjects. The CCh-induced negative I(sc) in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive I(sc) was significantly enhanced in CF and was caused by activation of a luminal K(+) conductance, as shown by the use of the K(+) channel blockers Ba(2+) and tetraethylammonium. Moreover, a cAMP-dependent luminal K(+) conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl(-) channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl(-) conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca(2+)- and cAMP-dependent K(+) secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.

Adolescent↗
PubMed2000

A Bartter's syndrome mutation of ROMK1 exerts dominant negative effects on K(+) conductance.

Mutations in the gene encoding the renal epithelial K(+) channel ROMK1 (Kir 1.1) is one of the causes for Bartter's syndrome, an autosomal recessive disease. It results in defective renal tubular transport in the thick ascending limb of the loop of Henle that leads to hypokalemic metabolic alkalosis and loss of salt. Two novel ROMK1 mutations, L220F/A156V, have been described recently in a compound heterozygote patient demonstrating typical manifestations of Bartter's syndrome. Functional properties of these ROMK1 mutants were studied by coexpressing in Xenopus oocytes and by means of double electrode voltage clamp experiments. When both ROMK1 mutants were coexpressed no K(+) conductance could be detected. The same was found in oocytes expressing A156V-ROMK1 only or coexpressing wild type (wt) ROMK1 together with A156V-ROMK1. In contrast, K(+) conductances were indistinguishable from that of wt-ROMK1 when L220F-ROMK1 was expressed alone. Activation of protein kinase C signaling inhibited the conductance in both L220F-ROMK1 and wt-ROMK1 expressing oocytes. These effects were not seen in A156V-ROMK1 expressing oocytes. Because no further abnormalities in the properties or regulation of L220F-ROMK1 were detected, we conclude that A156V-ROMK1 has a dominant negative effect on L220F-ROMK1 thereby causing Bartter's syndrome type two in this patient.

Bartter Syndrome↗
PubMed2000

Role of K(V)LQT1 in cyclic adenosine monophosphate-mediated Cl(-) secretion in human airway epithelia.

Ion transport defects underlying cystic fibrosis (CF) lung disease are characterized by impaired cyclic adenosine monophosphate (cAMP)-dependent Cl(-) conductance. Activation of Cl(-) secretion in airways depends on simultaneous activation of luminal Cl(-) channels and basolateral K(+) channels. We determined the role of basolateral K(+) conductance in cAMP- dependent Cl(-) secretion in native human airway epithelium obtained from non-CF and CF patients. CF tissues showed typical alterations of short-circuit currents with enhanced amiloride-sensitive Na(+) conductance and defective cAMP-mediated Cl(-) conductance. In non-CF tissues, Cl(-) secretion was significantly inhibited by the chromanol 293B (10 micromol/liter), a specific inhibitor of K(V)LQT1 K(+) channels. Inhibition was increased after cAMP-dependent stimulation. Similar effects were obtained with Ba(2+) (5 mmol/liter). In patch-clamp experiments with a human bronchial epithelial cell line, stimulation with forskolin (10 micromol/liter) simultaneously activated Cl(-) and K(+) conductance. The K(+) conductance was reversibly inhibited by Ba(2+) and 293B. Analysis of reverse-transcribed messenger RNA from non-CF and CF airways showed expression of human K(V)LQT1. We conclude that the K(+) channel K(V)LQT1 is important in maintaining cAMP-dependent Cl(-) secretion in human airways. Activation of K(V)LQT1 in CF airways in parallel with stimulation of residual CF transmembrane conductance regulator Cl(-) channel activity or alternative Cl(-) channels could help to circumvent the secretory defect.

1-Methyl-3-isobutylxanthine↗
PubMed1999

Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.

1-Methyl-3-isobutylxanthine↗
PubMed1999

The first-nucleotide binding domain of the cystic-fibrosis transmembrane conductance regulator is important for inhibition of the epithelial Na+ channel.

The cystic-fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-regulated Cl- channel and as a regulator of other membrane conductances. cAMP-dependent activation of CFTR inhibits epithelial Na+ channels (ENaC). The specificity of interaction between CFTR and ENaC was examined by coexpression of ENaC and ATP-binding cassette (ABC) proteins other than CFTR. In addition, we identified domains within CFTR that are of particular importance for the inhibition of ENaC. To that end, two-electrode voltage-clamp experiments were performed on Xenopus oocytes coexpressing ENaC together with CFTR, the multidrug resistance protein MDR1, the sulfonyl urea receptor SUR1, or the cadmium permease YCF1. Except for CFTR, none of the other ABC proteins were able to inhibit ENaC. Several truncated versions of CFTR were examined for their inhibitory effects on ENaC. In fact, it is shown that C-terminal truncated CFTR is able to inhibit ENaC on activation by intracellular cAMP. Moreover, the data also show that an intact first-nucleotide binding domain (NBF-1) is important for inhibition of ENaC. We conclude that NBF-1 of CFTR contains a CFTR-specific regulatory site that down-regulates ENaC. It is speculated that this regulatory site also is needed for CFTR-mediated interactions with other membrane proteins and that it is not present in NBF-1 of other ABC proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells.

Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibrosis transmembrane conductance regulator (CFTR) protein. Subsequent studies have shown that CFTR activates an endogenous water permeability in oocytes, but that CFTR itself is not the water channel. Here, we show CFTR-dependent activation of endogenous water permeability in normal but not in cystic fibrosis human airway epithelial cells. Cell volume was measured by novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells. Regulatory interaction was confirmed by coexpression of CFTR and AQP3 cloned from human airways in Xenopus oocytes and of CFTR and rat AQP3 in Chinese hamster ovary cells. These findings indicate that CFTR is a regulator of AQP3 in airway epithelial cells.

Animals↗
PubMed1999

Lack of correlation between CFTR expression, CFTR Cl- currents, amiloride-sensitive Na+ conductance, and cystic fibrosis phenotype.

Cystic fibrosis (CF) is characterized by defective Cl- and enhanced Na+ conductance, both due to malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in airway epithelial cells. In the present study we examined whether expression of CFTR mRNA (CFTR messenger ribonucleic acid) is different in airway epithelia derived from either CF patients or healthy volunteers. Moreover, we tried to correlate differences in epithelial Cl- and Na+ conductance with the level of CFTR mRNA expression and studied whether these properties correlate to the clinical phenotype of CF patients. To that end, CFTR mRNA was determined by means of quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and cyclic adenosine monophosphate (cAMP)-activated Cl- and epithelial Na+ conductances were examined in airway epithelial cells using microelectrode techniques. Complementary in vitro data were obtained from cultured CF and non-CF airway epithelial cell lines. Genotype and Shwachman score were assessed for each patient. We found variable levels of CFTR mRNA expression in airway cells of both CF patients and healthy volunteers. As expected, epithelial Na+ conductance was enhanced and CFTR Cl- conductance was absent in airway cells from CF patients. However, CFTR mRNA expression did not correlate with either electrophysiological properties or Shwachman scores obtained from CF patients. In addition, CFTR mRNA expression did not correlate to Cl- conductance in cultured CF and non-CF airway epithelial cells. These results indicate a lack of correlation between levels of CFTR mRNA and CFTR function, and that only small amounts of CFTR are required for expression of the CFTR Cl- conductance.

Amiloride↗
PubMed1999

Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of Squalus acanthias.

Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.

Animals↗
PubMed1999

The cellular mechanisms of Cl- secretion induced by C-type natriuretic peptide (CNP). Experiments on isolated in vitro perfused rectal gland tubules of Squalus acanthias.

We have examined the mechanism whereby C-type natriuretic peptide (CNP), an agonist acting through the second messenger cGMP, enhances NaCl secretion in the rectal gland of Squalus acanthias. Single rectal gland tubules (RGT) were dissected manually, perfused in vitro and equivalent short-circuit current [Isc=transepithelial voltage/transepithelial resistance (Rte)] as well as basolateral membrane voltage (Vbl) were measured. CNP was added to luminal and basolateral perfusates at concentrations between 1 and 1000 nmol/l and its effects on the above parameters were compared to those of a "stimulation cocktail" (Stim, containing dibutyryl cAMP, adenosine and forskolin) that maximally enhances cytosolic cAMP, and other agonists and hormones such as guanylin, vasoactive intestinal peptide (VIP), and adenosine. CNP had no effect from the luminal side (n=6). Its effects from the basolateral side consisted of a substantial increase in Isc (-31.6+/-7.7 to -316+/-82.2 microA/cm2, n=15). CNP significantly depolarized the luminal membrane from -87. 4+/-1.0 to -82.3+/-2.6 mV (n=12). Vbl was not changed (n=12) but the fractional conductance for K+ was increased (n=3). These effects were qualitatively and even quantitatively comparable to those of other agonists acting via cytosolic cAMP, but were less marked than those caused by Stim (n=64). The effects of VIP and CNP on Isc were not additive (n=5). The cytosolic Ca2+ concentration ([Ca2+]i) was monitored using the fura-2 fluorescence ratio (FFR 340/380 nm) and it was found that CNP, like agonists acting via cAMP, enhances FFR significantly from 1.02+/-0.05 to 1.32+/-0.05 (n=8) with a time constant in the 1-2 min in range. Our data suggest that CNP, acting via the second messenger cGMP, induces a marked increase in Isc in the rectal gland. The concomitant fall in Rte corresponds to increases in the luminal membrane Cl- conductance and in the basolateral membrane K+ conductance. The latter effect is probably due to an increase in [Ca2+]i.

Animals↗
PubMed1999

The Na+2Cl-K+ cotransporter in the rectal gland of Squalus acanthias is activated by cell shrinkage.

Effects of cAMP on Cl- secretion, intracellular Cl- activity and cell volume were studied in isolated perfused rectal gland tubules (RGT) of Squalus acanthias with electrophysiological and fluorescence methods. Recording of equivalent short-circuit current (Isc) showed that cAMP stimulates Na+Cl- secretion in a biphasic manner. The first and rapid phase corresponds to Cl- exit via the respective protein-kinase-A- (PKA-) phosphorylated Cl- conductance. The inhibitory effect of the loop diuretic furosemide (0.5 mmol/l, n=12) indicates that second phase reflects the delayed (1-2 min) activation of the Na+2Cl-K+ cotransporter. During the first phase cytosolic Cl- activity, as monitored by 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) fluorescence, fell to 78% (n=23) of the control value. Concomitantly, a transient fall in cell volume was recorded by calcein fluorescence to 92% (n=5) of the control value. Preincubation of the RGT with phalloidin (0.1 mmol/l, n=6) or cytochalasin D (0.1 mmol/l, n=4) almost completely prevented the development of the second phase of Isc activation. When cytosolic Cl- activity was increased by exposing the RGT to a high K+ concentration (25 mmol/l), in the presence of mannitol to prevent volume increases, stimulation was unaffected and biphasic. In contrast, when cell volume was clamped to an increased value (115%, n=8) by removing extracellular NaCl, the second phase was abolished completely (n=11). These data suggest that the primary and key process for triggering the Na+2Cl-K+ cotransport is transient cell shrinkage.

Animals↗
PubMed1999

Rescue of the mineralocorticoid receptor knock-out mouse.

The mineralocorticoid receptor knock-out mouse (MR-/-), resembling inborn pseudohypoaldosteronism, dies 8-12 days after birth in circulatory failure with all the signs of terminal volume contraction. The present study aimed to examine the functional defects in the kidney and colon in detail and to attempt to rescue these mice. In neonatal (nn) MR-/- the amiloride-sensitive short-circuit current in the colon was reduced to approximately one-third compared to controls (MR+/+ and MR+/-). In isolated in vitro perfused collecting ducts the amiloride-induced hyperpolarization of the basolateral membrane (Vbl) of nn MR-/- was similar to that of controls, but urinary Na+ excretion was markedly increased to 4.3 micromol/day.g (BW). Based on this measured urinary Na+ loss we tried to rescue nn MR-/- mice by injecting NaCl twice daily (3.85 micromol/g BW), corresponding to 22 microliter of isotonic saline/g BW subcutaneously. This regimen was continued until the animals had reached a body mass of 8.5 g. Thereafter, in addition to normal chow and tap water, NaCl drinking water (333 mmol/l) and pellets soaked in 333 mmol/l NaCl were offered. Unlike the untreated nn MR-/- most of these mice survived. The adult animals were examined between days 27 and 41, some were used for breeding. When compared to age-matched controls the growth of MR-/- was delayed until day 20. Then their growth curve increased in slope and reached that of controls. MR-/- retained their Na+-losing defect. Amiloride's effect on urinary Na+ excretion was not significant in MR-/- mice and the effect on Vbl in isolated cortical collecting ducts was attenuated. The renin-producing cells were hypertrophic and hyperplastic. Plasma renin and aldosterone concentrations were significantly elevated in MR-/- mice. These data indicate that MR-/- can be rescued by timely and matched NaCl substitutions. This enables the animals to develop through a critical phase of life, after which they adapt their oral salt and water intake to match the elevated excretion rate; however, the renal salt-losing defect persists.

Absorption↗
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

Carbachol activates a K+ channel of very small conductance in the basolateral membrane of rat pancreatic acinar cells.

Secretion of Cl- requires the presence of a K+ conductance to hyperpolarize the cell, and to provide the driving force for Cl- exit via luminal Cl- channels. In the exocrine pancreas Cl- secretion is mediated by an increase in cytosolic Ca2+ ([Ca2+]i). Two types of Ca2+-activated K+ channels could be shown in pancreatic acinar cells of different species. However, there are no data on Ca2+-activated K+ channels in rat pancreatic acini. Here we examine the basolateral K+ conductance of freshly isolated rat pancreatic acinar cells in cell-attached and cell-excised patch-clamp experiments. Addition of carbachol (CCH, 1 micromol/l) to the bath led to the activation of very small conductance K+ channels in cell-attached patches (n=27), producing a noisy macroscopic outward current. The respective outward conductance increased significantly by a factor of 2.1+/-0.1 (n=27). Noise analysis revealed a Lorentzian noise component with a corner frequency (f(c)) of 30.3+/-3.5 Hz (n=19), consistent with channel activity in these patches. The estimated single-channel conductance was 1.5+/-0.4 pS (n=19). In cell-excised patches (inside out) from cells previously stimulated with CCH, channel activity was only observed in the presence of K+ in the bath solution. Under these conditions f(c) was 47.6+/-11.9 Hz (estimated single-channel conductance 1.1+/-0.2 pS, n=20). The current/voltage relationship of the noise showed weak inward rectification and the reversal potential shifted towards E(K+) when Na+ in the bath was replaced by K+. Channel activity in cell-excised patches was slightly reduced by 10 mmol/l Ba2+ (23.6+/-2.1% of the total outward current) and was completely absent when K+ in the bath was replaced by Na+. Reduction of the [Ca2+]i from 1 mmol/l to 1 micromol/l in cell-excised experiments decreased the current by 52.3+/-12.3% (n=5). Expression of K(v)LQT1, one of the possible candidates for a small-conductance K+ channel in rat pancreatic acinar cells, was shown by reverse transcriptase polymerase chain reaction (RT-PCR). In fact, a K(V)LQT-blocker (chromanol 293B) reduced channel activity in seven excised patches. These data suggest that CCH activates very small conductance K+ channels in rat pancreatic acinar cells, most likely via an increase in [Ca2+]i.

Animals↗
PubMed1999

Voltage-dependent, slowly activating K+ current (I(Ks)) and its augmentation by carbachol in rat pancreatic acini.

Acetylcholine-stimulated exocrine secretion of Cl- and water requires the concomitant activation of K+ channels. However, there has not been much investigation of the carbachol- (CCH-) activated K+ channel of rodent pancreatic acini. Here, in a study of rat pancreatic acini, we characterize a voltage-dependent, slowly activating outward current (I(Ks)) that is augmented by CCH. Intact acini were obtained by enzymatic digestion and fast-whole-cell patch-clamp was applied. With symmetrical [Cl-] (32 mmol/l) in the pipette and bath solution, acinar cells had resting membrane voltages of -45+/-0.8 mV (n=97) under current-clamp conditions. CCH (10 micromol/l), which is known to activate Cl- channels via a Ca2+-mediated pathway, sharply depolarized the membrane to -4+/-0.5 mV, which was more negative than E(Cl) (0 mV), and reversed it to -41+/-0.9 mV (n=83) by washout. A clamp voltage of 0 mV activated I(Ks) under control conditions (91+/-8.6 pA, n=83). During CCH application an increase of outward current was observed at 0 mV, and at -50 mV a marked increase of inward Cl current occurred. In the presence of CCH the slow activation of I(Ks) was rarely distinguishable because of interference by the huge Cl- conductance. During CCH washout and decrease of inward current, a persistent augmentation of I(Ks) was revealed (486+/-36.3 pA, n=83). I(Ks) and its augmentation were abolished by substituting K+ in the pipette solution with Cs+. Augmentation of I(Ks) was mimicked by applying ionomycin (0.1 micromol/l), a Ca2+ ionophore. Pharmacological blockers were tested. The chromanol 293B and clotrimazole blocked I(Ks) at micromolar concentrations (IC50=3 micromol/l and 9 micromol/l, respectively) and Ba2+ was a poor blocker (IC50=3 mmol/l). In the presence of CCH (0.2 micromol/l), the membrane was depolarized to around -20 mV and the addition of 293B (10 micromol/l) further depolarized the membrane by 11+/-3 mV (n=5). These data suggest the presence of I(Ks) channels in rat pancreatic acini and their muscarinic activation.

Animals↗