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• W2013222540 abstract "The estrogen sex steroid 17β-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl– secretion in the female rat distal colonic crypt by the inhibition of basolateral K+ channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K+ current was shown to be attenuated by pretreatment with rottlerin, a PKCδ-specific inhibitor. In whole cell patch-clamp analysis, 17β-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKCδ in comparison to male tissue. PKCδ and PKA were activated at 5 min in response to 17β-estradiol in female distal colonic crypts only. Both PKCδ- and PKA-associated with the KCNQ1 channel in response to 17β-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKCδ as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKCδ- and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels. The estrogen sex steroid 17β-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl– secretion in the female rat distal colonic crypt by the inhibition of basolateral K+ channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K+ current was shown to be attenuated by pretreatment with rottlerin, a PKCδ-specific inhibitor. In whole cell patch-clamp analysis, 17β-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKCδ in comparison to male tissue. PKCδ and PKA were activated at 5 min in response to 17β-estradiol in female distal colonic crypts only. Both PKCδ- and PKA-associated with the KCNQ1 channel in response to 17β-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKCδ as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKCδ- and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels. Fluid and electrolyte secretion is an important function in the distal colon as it regulates whole body fluid homeostasis and also maintains mucosal hydration (1Barrett K.E. Keely S.J. Annu. Rev. Physiol. 2000; 62: 535-572Crossref PubMed Scopus (379) Google Scholar). Chloride secretion in the distal colon occurs as a two-step transport process. Cl– is transported into the cell basolaterally with Na+ and K+ via the Na+/K+/2Cl– isoform 1 (NKCC1) co-transporter. Cl– is secreted across the apical membrane into the lumen via a chloride ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). 3The abbreviations used are: AbbreviationsCFTRcystic fibrosis transmembrane conductance regulatorPKCprotein kinase CPKAcAMP-dependent protein kinase A 3The abbreviations used are: AbbreviationsCFTRcystic fibrosis transmembrane conductance regulatorPKCprotein kinase CPKAcAMP-dependent protein kinase A Secretion of Cl– ions is driven by the activity of Na+-K+-ATPase located in the basolateral membrane. Basolateral K+ ion channels carry out the K+ recycling required to establish the favorable membrane electrical potential for Cl– secretion. The activity of ion transporters involved in Cl– secretion may be modulated by secretagogues acting through cAMP activity or intracellular Ca2+ concentration. Disorders resulting in increased Cl– secretion, for example in bacterial or viral infections, are a primary cause of secretory diarrhea (2Field M. Semrad C.E. Annu. Rev. Physiol. 1993; 55: 631-655Crossref PubMed Scopus (79) Google Scholar). cystic fibrosis transmembrane conductance regulator protein kinase C cAMP-dependent protein kinase A cystic fibrosis transmembrane conductance regulator protein kinase C cAMP-dependent protein kinase A The biologically active estrogen 17β-estradiol (E2) plays an important role in the normal development and maturation of the female. In addition to this classical role, E2 has also been implicated in the regulation of whole body fluid and electrolyte balance (3Crocker A.D. J. Physiol. 1971; 214: 257-264Crossref PubMed Scopus (14) Google Scholar, 4Harvey B.J. Condliffe S. Doolan C.M. News Physiol. Sci. 2001; 16: 174-177PubMed Google Scholar). The distal colon has recently been recognized as a target of E2. The colonic crypts express both isoforms of the nuclear estrogen receptor, ERα and ERβ, similar to classical E2-responsive tissues such as uterus and breast tissue (5Thomas M.L. Xu X. Norfleet A.M. Watson C.S. Endocrinology. 1993; 132: 426-430Crossref PubMed Scopus (102) Google Scholar). We have previously demonstrated that E2 inhibits secretagogue stimulated Cl– secretion in the rat distal colonic crypt (6Condliffe S.B. Doolan C.M. Harvey B.J. J. Physiol. 2001; 530: 47-54Crossref PubMed Scopus (72) Google Scholar). The anti-secretory response to E2 was rapid, occurring within 10 min and was dependent on protein kinase C (PKC). In human distal colonic tissue, blocking basolateral K+ channel activity decreases forskolin-stimulated Cl– secretion (7McNamara B. Winter D.C. Cuffe J.E. O'Sullivan G.C. Harvey B.J. J. Physiol. 1999; 519: 251-260Crossref PubMed Scopus (60) Google Scholar). In rat distal colon the basolateral K+ conductance is formed by at least two different types of K+ channels, activated by Ca2+ or cAMP-dependent agonists (8Liao T. Wang L. Halm S.T. Lu L. Fyffe R.E. Halm D.R. Am. J. Physiol. Cell Physiol. 2005; 289: C564-C575Crossref PubMed Scopus (30) Google Scholar). Electrophysiological studies in these cells have revealed the candidate K+ channel involved in Cl– secretion to be KCNQ1, a low conductance (1–3 pS) K+ channel, which is activated during cAMP-stimulated Cl– secretion (9Kunzelmann K. Hubner M. Schreiber R. Levy-Holzman R. Garty H. Bleich M. Warth R. Slavik M. von Hahn T. Greger R. J. Membr. Biol. 2001; 179: 155-164Crossref PubMed Scopus (77) Google Scholar). Other types of K+ channels have also been implicated in chloride secretion, including the Ca2+-dependent KCNN4 channel (10Halm S.T. Liao T. Halm D.R. Am. J. Physiol. Cell Physiol. 2006; 291: C636-C648Crossref PubMed Scopus (28) Google Scholar). The rat KCNQ1 channel was cloned from colonic tissue and expression was demonstrated in both the crypt and surface cells (9Kunzelmann K. Hubner M. Schreiber R. Levy-Holzman R. Garty H. Bleich M. Warth R. Slavik M. von Hahn T. Greger R. J. Membr. Biol. 2001; 179: 155-164Crossref PubMed Scopus (77) Google Scholar). KCNQ1 is a voltage-gated channel, which plays a crucial role in controlling salt and water homeostasis in a number of epithelia (11Jespersen T. Grunnet M. Olesen S.P. Physiology (Bethesda). 2005; 20: 408-416Crossref PubMed Scopus (213) Google Scholar). KCNQ1 is located in the basolateral membrane of the rat distal colonic crypt (8Liao T. Wang L. Halm S.T. Lu L. Fyffe R.E. Halm D.R. Am. J. Physiol. Cell Physiol. 2005; 289: C564-C575Crossref PubMed Scopus (30) Google Scholar) and is regulated by forming a complex with the β-subunit KCNE3 (MiRP2) to form the basolateral K+ conductance that is required for transepithelial cAMP-stimulated chloride secretion (12Warth R. Bleich M. Rev. Physiol. Biochem. Pharmacol. 2000; 140: 1-62Crossref PubMed Google Scholar). We propose KCNQ1 as a candidate membrane target for E2 in the inhibition of Cl– secretion in the rat distal colonic crypt. A previous study in cardiac tissue demonstrated a concentration-dependent inhibition of the KCNQ1/KCNE1-mediated K+ current by E2 (13Moller C. Netzer R. Eur. J. Pharmacol. 2006; 532: 44-49Crossref PubMed Scopus (32) Google Scholar). PKCδ is a serine/threonine kinase belonging to the novel PKC subgroup and plays a key role in cell cycle progression, transcriptional regulation and tissue remodeling (14Steinberg S.F. Biochem. J. 2004; 384: 449-459Crossref PubMed Scopus (325) Google Scholar). In the gastrointestinal tract PKCδ has been implicated in the maintenance of the epithelial barrier integrity (15Di Mari J.F. Mifflin R.C. Powell D.W. Gastroenterology. 2005; 128: 2131-2146Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and is an important regulator of Cl– secretion (16Song J.C. Hanson C.M. Tsai V. Farokhzad O.C. Lotz M. Matthews J.B. Am. J. Physiol. Cell Physiol. 2001; 281: C649-C661Crossref PubMed Google Scholar). We have previously demonstrated that physiological concentrations of E2 (0.01–100 nm) produced a rapid (15 min) stimulation of PKC and PKA activities in female rat distal colonic crypts (17Doolan C.M. Harvey B.J. J. Biol. Chem. 1996; 271: 8763-8767Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The phosphorylation of ion channels is a major mechanism for their regulation. Indeed it has been demonstrated recently that KCNQ1 can be phosphorylated at Ser27 in the N terminus of the channel by PKA (18Kurokawa J. Motoike H.K. Rao J. Kass R.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16374-16378Crossref PubMed Scopus (102) Google Scholar). In the present study, we examined the female gender-specific regulation of the KCNQ1 channel by E2 and the role of PKA and PKCδ as mediators of the rapid anti-secretory response to estrogen in rat distal colonic crypts. Materials—Phospho-PKCδ (Ser643) antibody was obtained from Cell Signaling Technologies. Phosphoserine (Clone IC8) antibody was obtained from Calbiochem. Total PKCδ, total PKA regulatory subunit isoform I (PKARI), and total PKA catalytic subunit isoform I (PKACI) antibodies from BD Transduction. Anti-rabbit anti-goat horseradish peroxidase-linked secondary antibody from Santa Cruz Biotechnology. Anti-rabbit and anti-mouse horseradish peroxidase-linked secondary antibodies from Sigma-Aldrich. Total KCNQ1 antibody was obtained from Sigma-Aldrich and Santa Cruz Biotechnology. Protein-G Sepharose beads and the ECL plus detection system were from Amersham Biosciences and Bradford reagent from Bio-Rad. Chromanol 293B was obtained from Tocris and rottlerin from Calbiochem. All other reagents were obtained from Sigma-Aldrich. Animals—Male (∼350 g) and female (∼300 g) Sprague-Dawley rats at three-months-old were used for all experiments. Animals were kept on a 12-h light, 12-h dark cycle, and were given ad libitum access to food and water. Following anesthesia rats were killed by cervical dislocation. Cervical smears were obtained from female rats, and the stage of the estrous cycle was determined histologically as previously described (19Hubscher C.H. Brooks D.L. Johnson J.R. Biotech. Histochem. 2005; 80: 79-87Crossref PubMed Scopus (163) Google Scholar). All female rats were used at the estrus stage of the cycle where estradiol is present at a circulating level of ∼75 pg/ml and progesterone at ∼32.5 pg/ml (20Nequin L.G. Alvarez J. Schwartz N.B. Biol. Reprod. 1979; 20: 659-670Crossref PubMed Scopus (230) Google Scholar). The distal colon was removed to below the pelvic rim. The faecal contents were rinsed and distal colonic crypts were isolated as previously described (17Doolan C.M. Harvey B.J. J. Biol. Chem. 1996; 271: 8763-8767Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Isolations and treatments were carried out at room temperature to avoid colonic crypt disintegration (17Doolan C.M. Harvey B.J. J. Biol. Chem. 1996; 271: 8763-8767Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21Schultheiss G. Lan Kocks S. Diener M. Biol. Proced. Online. 2002; 3: 70-78Crossref PubMed Scopus (20) Google Scholar). Sheets of colonic mucosa were obtained by blunt dissection for transepithelial transport measurements. All procedures were approved by the RCSI Ethics Committee. Transepithelial Transport Studies—Colonic epithelia were mounted in Ussing chambers (Physiologic Instruments) on inserts exposing an area of 0.5 cm2. Transepithelial potential difference was clamped to 0 mV using an EVC-4000 voltage-clamp apparatus (World Precision Instruments). The transepithelial short circuit current (ISC) was recorded using Ag-AgCl electrodes in 3 m KCl agar bridges. Apical and basolateral baths were filled with Krebs bicarbonate buffer (in mm: 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, 10 glucose), pH 7.4 maintained at 37 °C by heated water jackets and oxygenated with a 95% O2/5% CO2 mixture. All preparations were allowed to equilibrate for 30–45 min before the experiments were performed. The Isc was defined as positive for anion flow from the basolateral to apical chamber and for cation flow in the opposite direction. To investigate the activity of basolateral K+ channels in Ussing chamber experiments, the apical membrane was permeabilized by addition of 10 μm of the K+ ionophore amphotericin B in the presence of a mucosal to serosal K+ gradient established by the following bath solutions: apical (in mm), 145 K-gluconate, 3 KH2PO4, 0.8 K2HPO4, 1.2 Mg(gluconate)2, 4 Ca(gluconate)2, 10 glucose, and 10 HEPES; basolateral, 145 Nagluconate, 3.3 NaH2PO4, 0.8 Na2HPO4, 1.2 Mg(gluconate)2, 4 Ca(gluconate)2, 10 glucose, and 10 HEPES. Ouabain (1 mm) was added to the serosal bath to inhibit Na+-K+-ATPase. The resulting Isc was generated by the movement of K+ through channels in the basolateral membrane (IK) as the current collapses to zero in equimolar K+ solutions bathing both sides of the epithelium. For measurement of IK current-voltage relationships, currents were elicited in asymmetrical K+ gluconate solutions by imposition of 1-s test potentials between −100 and +100 mV in 20-mV increments. Patch-clamp—A small aliquot (100 μl) of freshly isolated colonic crypts was transferred into 1 ml of superfusion chamber mounted on the stage of an inverted microscope (TE 2000-S, Nikon Ltd). Pipettes were prepared from capillary glass (GC150F-10, Harvard Apparatus Ltd, Edenbridge, UK). Patch pipettes were pulled and fire-polished using a programmable horizontal puller (DMZ-Universal, Zeitz-Instruments GmbH) and had an electrical resistance of 2–5 mΩ when filled with K+ solutions. The patch-clamp apparatus consisted of a CV-203BU head stage (Axon Instruments Incorporated) connected to an Axopatch 200B series amplifier (Axon Instruments). Patch-clamp experiments were performed at 37 °C in the standard whole cell recording configuration (22Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pflugers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15118) Google Scholar) and recorded membrane currents were filtered at 1 kHz through an 8 pole, low-pass Bessel filter, and digitized at 5 kHz. The voltage clamp protocol consisted of a series of voltage steps from −100 mV to +100 mV in 20-mV increments from an initial holding potential of −50 mV. Colonic crypts were superfused at a rate of 1 ml/min in a standard bath solution containing (in mm): NaCl 140, KCl 5.4, MgCl2 1, CaCl2 1.25, HEPES 10, glucose 12.2, buffered at pH 7.4 with NaOH. The patch pipette solution contained (mm): K-gluconate 95, KCl 30, Na2ATP 4.8, KH2 PO4 1.2, EGTA 1, Ca-gluconate 0.73, MgCl2 1, ATP 3, d-glucose 5 (pH 7.2). Once whole cell access to the inside of the cell was obtained, cells were allowed to stabilize for up to 10 min before the experiment began. The protocols for patch-clamp and data analysis were established with routines using pClamp 9.2 software (Axon Instruments), and data were stored for subsequent analysis. Co-immunoprecipitation and Western Blotting—Isolated distal colonic crypts resuspended in Krebs solution were treated with drugs for the indicated time points. The samples were then immediately centrifuged for 30 s at 4 °C at 4,000 rpm, and the supernatant removed. Cells were lysed by hypotonic shock on ice for 45 min (lysis buffer: 20 mm Tris, pH 7.4, 0.5% Nonident P-40, 250 mm NaCl, 3 mm EDTA, 3 mm EGTA, leupeptin 1 μg/μl, 500 mm dithiothreitol, 5 mm phenylmethylsulfonyl fluoride, complete mini EDTA-free protease inhibitor mixture tablets (1 tablet/7 ml of lysis buffer; Roche Applied Science) and phosphatase inhibitors). Following incubation the samples were clarified at 12,000 rpm for 10 min. The cleared supernatant was collected and the protein content was quantified by the Bradford method (23Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214351) Google Scholar). For activation assays 50 μg of sample were combined with 2× Laemmli buffer, boiled at 95 °C for 5 min, and spun at 12,000 rpm for 2 min. All KCNQ1 immunoprecipitations were carried out using lysis buffers as described previously (24Kurokawa J. Chen L. Kass R.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2122-2127Crossref PubMed Scopus (107) Google Scholar). 500 μg of sample was removed for co-immunoprecipitation of KCNQ1 and associated kinases. KCNQ1 was immunoprecipitated as follows: 1 μg of total KCNQ1 antibody was added to 500 μg of sample and incubated on a rotor for one hour at 4 °C. 40 μl of washed protein G-Sepharose beads were combined with the immunocomplex. The samples were then incubated overnight on a rotor at 4 °C. Complexes were spun at 12,000 rpm for 10 min and washed three times with ice-cold sterile 1× phosphate-buffered saline. The supernatant was removed, and 35 μl of Laemmli buffer were added and samples were boiled at 95 °C for 5 min and spun at 14,000 rpm for 5 min. Western blot analysis was carried out as standard. Protein was transferred to polyvinylidene difluoride membranes, blocked in 1× TBS with Tween (0.3%) (1× TBST) and 5% nonfat dry milk for 1 h. Membranes were incubated with the appropriate primary antibody overnight at 4 °C and incubated for 1 h at room temperature with the appropriate secondary antibody. Membranes were washed in 1× TBST 0.3% three times for 15 min. Bands were detected using autoradiographic film and chemiluminescence. PKA Activation Assay—PKA activation was detected using PepTag Assay (Promega) for non-radioactive detection of cAMP-dependent protein kinase according to the manufacturer's instructions with minor modifications. 2.5 μl of the F-Kemptide PepTag and 2.5 μl of the cAMP activator solution were added instead of 5 μl. RNA Preparation and Reverse Transcriptase (RT) PCR—Total RNA was extracted from male and female rat distal colonic crypt preparations using Qiagen RNeasy kit (Qiagen). Single-strand cDNA was synthesized using the Improm II reverse transcriptase kit (Promega). cDNA was quantified and corrected for loading into RT-PCR reaction mixes. PKCδ was amplified using the following primers: forward; 5-caccatcttccagaaagaacg-3′ and reverse; 5′-cttgccataggtcccgttgttg-3′. β-Actin was amplified using the following primers: forward; 5′-cagtaatctccttctgcatcc-3′ and reverse; 5′-actacctcatgaagatcctga-3′. GoTaq® polymerase mix from Promega was used in the amplification. Touch-down PCR was used to amplify PKCδ cDNA for 25 cycles over an annealing temperature range of 65–55 °C. β-Actin was amplified for 25 cycles at an annealing temperature of 52 °C. The RT-PCR product was analyzed on a 1.5% 1× Tris acetate-EDTA (TAE) agarose gel and imaged using a UV light source. Statistical and Densitometric Analysis—Data are presented as mean ± S.E. for a series of the indicated number of experiments. Statistical analysis of the data was obtained by analysis using paired Student's t test for analysis between two groups. One-way analysis of variance and Tukeys post-hoc test was used for multiple analysis of more than two groups. Patch-clamp data analysis was performed using Clampfit software of the p-clamp suite version 9.2 and Origin 7.5 (OriginLab Corp.). Densitometric analysis of Western blots, PKA and RT-PCR images were performed using GeneTools software (SYNGENE). 17β-Estradiol Inhibits Cl– Secretion Evoked by Forskolin in Rat Colonic Epithelia—We first set out to examine the effect of 17β-estradiol (E2) on intestinal secretion elicited by the cAMP agonist forskolin. Basolateral addition of 20 μm forskolin induced an almost instantaneous and sustained increase in ISC (Fig. 1A). E2 (10 nm) reduced the forskolin-effect on ISC by 60 ± 6% (n = 5, p < 0.01). The estrogen inhibition of forskolin-induced secretion was observed to be gender dependent. E2 inhibition of forskolin-induced chloride secretion was only observed in tissues from female rats with no significant response to E2 in male tissues (female 60 ± 6% ISC decrease versus male 6 ± 3%, p < 0.01, n = 5) (Fig. 1A). All Ussing chamber values are maximal inhibition achieved at E2 treatment between 15 and 20 min. Previous data from our group have demonstrated direct and rapid activation of PKCδ in response to E2 (25Doolan C.M. Condliffe S.B. Harvey B.J. Br. J. Pharmacol. 2000; 129: 1375-1386Crossref PubMed Scopus (82) Google Scholar). We therefore investigated a potential role for this kinase in mediating the anti-secretory effects of E2. We used rottlerin as an inhibitor of PKCδ as it has been reported to inhibit this kinase more potently than other kinases. A previous publication on the inhibitor reported that it inhibits the PKC isoforms at different IC50 concentrations; PKCδ:3–6 μm, PKCα, PKCβ and PKCγ: 30–42 μm, PKCϵ, PKCζ and PKCθ: 80–100 μm (26Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (759) Google Scholar). Pretreatment of colonic mucosa with rottlerin (10 μm) completely abolished the anti-secretory action of E2 (E2: 59 ± 6%, E2 + rottlerin, 5 ± 3%, n = 5, p < 0.01) (Fig. 1B). These results indicate that PKCδ activity is required for the E2-mediated inhibition of forskolin-induced Cl– secretion. The Effect of 17β-Estradiol on Basolateral Membrane K+ Currents in Rat Colonic Epithelia—Basolaterally directed K+ currents were generated in female rat colonic epithelia permeabilized apically by pretreatment with amphotericin B. Fig. 2A shows the current/voltage (IK/V) relationship for apically permeabilized epithelia before and after the addition of the cAMP-dependent agonist forskolin (10 μm). Treatment with forskolin-activated outward currents (apical to basolateral) that displayed slight outward rectification at positive transepithelial voltages and shifted apparent reversal potentials to more negative values consistent with activation of basolateral membrane K+ current IK. Treatment with chromanol 293B (10 μm), a specific KCNQ1 channel blocker, inhibited both the basal and forskolin-activated basolateral membrane K+ current suggesting the main component of this current is due to KCNQ1 channel activity (Fig. 2A). Treatment with E2 (10 nm) produced a marked inhibition of forskolin-stimulated IK (at Vp +100, forskolin 185 ± 19 μA/cm2; forskolin + E2 44 ± 6 μA/cm2, n = 3, p < 0.01) and subsequent addition of chromanol 293B had little effect on the remaining K+ current (Fig. 2B). Pretreatment of colonic epithelia with rottlerin (10 μm) for 15 min before addition of forskolin did not modify the increase in IK induced by forskolin but prevented the inhibition by E2 (at Vp +100, forskolin + rottlerin 182 ± 17 μA/cm2; forskolin + rottlerin + E2 158 ± 21 μA/cm2, n = 3, p < 0.01) (Fig. 2C). These results suggest that the anti-secretory effect of 17β-estradiol is mediated by the inhibition of basolateral KCNQ1 channels via PKCδ. The Effect of Chromanol 293B on Whole Cell KCNQ1 Channel Current in Female Rat Colonic Crypts—Membrane currents were recorded in the whole cell mode of the patch-clamp technique from colonic crypt cells at 37 °C. The mean maximal whole cell current under control conditions was 817 ± 84 pA (at Vp = 100 mV, n = 4, p < 0.01) and subsequent addition of chromanol 293B (100 μm) produced a reduction of currents by 91% (corresponding to a reduction to 69 ± 50 pA, at Vp =+100 mV, n = 4, p < 0.01). Consistent with the decrease in whole cell current, chromanol 293B also reduced the whole cell conductance (Gc) at +100 mV (γ+100) from 397 ± 33 pS to 25 ± 4 pS (n = 4, p < 0.01 n = 4) (figure shown as supplemental data). The KCNQ1 channel activity is temperature-sensitive and increasing the temperature to 37 °C is known to activate the channel (27Unsold B. Kerst G. Brousos H. Hubner M. Schreiber R. Nitschke R. Greger R. Bleich M. Pflugers Arch. 2000; 441: 368-378Crossref PubMed Scopus (47) Google Scholar). Supplementary data show the mean maximal whole cell KCNQ1 currents recorded at room temperature (22 °C), Vp +100 mV and at 37 °C prior to the addition of chromanol 293B (100 μm). Chromanol 293B inhibited the mean maximal whole cell KCNQ1 currents and reduced the K+ current toward the level measured at room temperature (22 °C). The mean maximal temperature-stimulated increase in current was 822 ± 52 pA (n = 4, p < 0.02) and chromanol 293B addition produced a reduction of current by 69% (corresponding to 252 ± 45 pA), (n = 4, p < 0.05). Taken together, these results demonstrate the functional activity of KCNQ1 channels in female rat distal colonic crypts. E2 Effect on KCNQ1 Whole Cell Currents in Female Rat Colonic Crypts—Following membrane breakthrough to the whole cell patch-clamp configuration, the patched cells were allowed to stabilize and dialyze for 5 min. Currents were recorded over this time period before the addition of E2 to ensure stability of the current recording and to check for channel rundown. The experiments involved exposing the isolated colonic crypts to E2 (100 nm) and measuring the whole cell currents at 30-s intervals. Fig. 3A shows the effects of E2 (100 nm) treatment over time on the whole cell current-voltage relationships. E2 application caused a decrease in whole cell current and its outward rectification over 9 min with a concomitant fall in whole cell membrane conductance at +100 mV from control 278 ± 22 pS to: 160 ± 13 pS at 1 min; 158 ± 11 pS at 3 min; 107 ± 9 pS at 5 min; 82 ± 16 pS at 7 min and 81 ± 25 pS at 9 min (n = 4, p < 0.01). The effect of E2 treatment on whole cell current over time is shown in Fig. 3B. The mean maximal whole cell currents recorded over 5 min prior to E2 addition was 526 ± 50 pA (at Vp =+100 mV). Addition of E2 (100 nm) inhibited the mean maximal current after 5 min to 248 ± 38 pA and to 73 ± 10 pA after 15 min (n = 4, p < 0.05). Taken together, these results support the conclusion that KCNQ1 channel activity generates the main basolateral membrane K+ current, in female colonic crypts, consistent with the Ussing chamber results, and the channel is a target for E2 causing an anti-secretory response. Gender Comparisons of PKA, PKCδ, and KCNQ1 Basal Expression Levels in Rat Distal Colonic Crypts—Total untreated cellular lysates of isolated rat distal colonic crypts were prepared, subjected to Western blot analysis, and probed using specific antibodies to endogenous levels of PKARI, PKACI, PKCδ, and KCNQ1. In all cases expression differences were normalized for loading by probing for total β-actin levels. Differences are expressed as fold values of female expression levels compared with male. PKA isoform I is a cytoplasmic PKA, unlike isoform II which is membrane bound (28Tasken K. Aandahl E.M. Physiol. Rev. 2004; 84: 137-167Crossref PubMed Scopus (620) Google Scholar). Previous studies in rat colonic crypts demonstrated no significant activation of PKA in the membrane fractions of crypts and a significant activation of PKA in cytosolic fractions (25Doolan C.M. Condliffe S.B. Harvey B.J. Br. J. Pharmacol. 2000; 129: 1375-1386Crossref PubMed Scopus (82) Google Scholar). Isoform I, the soluble cytosolic isoform, was therefore investigated in this study. Basal expression amounts of PKARI and PKACI were similar between male and female rat distal colonic crypts (PKACI: 1.1 ± 0.2, p > 0.05, n = 3; PKARI: 1 ± 0.2, n = 3, p > 0.05) (Fig. 4, A and B). A comparison of KCNQ1 basal expression levels between male and female distal colonic crypts was also investigated. No significant difference was found between male and female crypts for total basal expression levels of KCNQ1 (1.1 ± 0.03, n = 3, p > 0.05) (Fig. 4E). We have found gender differences to exist in the basal expression for novel PKCδ. In comparison to male, distal colonic crypts from female rats displayed a 3.2 ± 0.42-fold higher expression of the PKCδ protein (n = 6, p < 0.01) (Fig. 4C). To further verify the gender difference in expression of PKCδ we also amplified the messenger transcript levels of the kinase by RT-PCR. Female distal colonic crypts had a significantly higher level of the PKCδ transcript present compared with male distal colonic crypts (2.1 ± 0.1-fold higher, n = 3, p < 0.01) (Fig. 4D). We conclude from these expression studies that a significant gender difference exists for PKCδ, with a higher expression level in female rat distal colonic crypts. No gender differences were detected for PKACI, PKARI, or" @default.
• W2013222540 created "2016-06-24" @default.
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• W2013222540 creator A5023649116 @default.
• W2013222540 creator A5024709364 @default.
• W2013222540 creator A5036569831 @default.
• W2013222540 creator A5041989030 @default.
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• W2013222540 date "2007-08-01" @default.
• W2013222540 modified "2023-10-15" @default.
• W2013222540 title "Female Gender-specific Inhibition of KCNQ1 Channels and Chloride Secretion by 17β-Estradiol in Rat Distal Colonic Crypts" @default.
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