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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Prostaglandin E₂ Enhances Acetylcholine-Induced, Ca²⁺-Dependent Ionic Currents in Swine Tracheal Mucous Gland Cells

Department of Pharmacology & Toxicology, University of Mississippi Medical Center, Jackson, Mississippi

Received for publication January 19, 2007
Accepted May 3, 2007.

	Abstract

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Airway submucosal gland cell (SMGC) secretions are under the control of various neurotransmitters and hormones. Interactions between different pathways, such as those mediated by cAMP and Ca²⁺, in controlling mucus or electrolyte secretions are not well understood. Prostaglandin E₂ (PGE₂) or forskolin has been shown to enhance acetylcholine (ACh)-induced short circuit current (I_SC) in SMGC mucous cell monolayers. We show that PGE₂, by activating cAMP-dependent protein kinase A (PKA), enhanced ACh-induced, Ca²⁺-mediated current and changes in [Ca²⁺]_i in mucous cells. PGE₂ pretreatment sensitized ACh-induced I_SC (I_SC) by activating endoprostanoid (EP₂) receptors. PKA inhibitors 14-22 amide PKI (PKI) and Rp-diastereomer (Rp) of cAMPs prevented the effect of PGE₂. Removing external Ca²⁺ or pretreatment with the Ca²⁺ entry blocker, SKF96365 [1-[-(3-(4-methoxyphenyl) propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl] imidazole], shifted the concentration-response relationships for ACh to the right but did not abolish PGE₂-induced sensitization of the ACh response. An inositol 1,4,5-trisphosphate (IP₃) receptor antagonist and Ca²⁺ entry blocker, 2-aminoethoxydiphenyl borate, abolished the ACh-induced response. Charybdotoxin, but not iberiotoxin (IbTX), inhibited the ACh-induced I_SC. Clotrimazole, but not IbTX, inhibited the ACh-induced serosal K⁺ current. Under whole-cell patch clamp, ACh-induced K⁺ and Cl^– currents were coincident with increases in [Ca²⁺]_i in single mucous cells. PGE₂ or forskolin pretreatment did not induce current or [Ca²⁺]_i changes but enhanced ACh-induced currents, membrane hyperpolarization, and [Ca²⁺]_i changes. Intra-cellular dialysis with the PKA-catalytic subunit enhanced ACh-induced whole-cell current as well. These findings demonstrate that PGE₂, via EP₂ receptors and the cAMP/PKA pathway, activates Ca²⁺ entry-independent mechanisms, possibly by increasing IP₃-mediated Ca²⁺ release, resulting in the sensitization of ACh-induced currents.

The activation of K⁺ and Cl^– channels are crucial to SMGC secretion, permitting net ion movements followed by fluid secretion (Petersen, 1992). Serous cells express abundant CFTR Cl^– channels (Engelhardt et al., 1992) and are responsible for most of the cAMP-activated fluid secretion in the airway (Wine and Joo, 2004). Agents such as forskolin induce serous cell secretion by increasing [cAMP]_i and activating CFTR (Wine and Joo, 2004) and possibly cAMP-activated K⁺ channels (Cowley and Linsdell, 2002). The activation of CFTR allows apical anion (Cl^–/HCO^–₃) exit, driving water transport through AQP4 and AQP5 water channels (Song and Verkman, 2001), whereas the activation of serosal K⁺ channels hyperpolarizes the membrane and facilitates and sustains anion flux by maintaining a favorable electrochemical driving force for such flux (Welsh, 1983). Ca²⁺-elevating agents, such as muscarinic agonists, activate K_Ca that hyperpolarizes the membrane and drive Cl^– or HCO^–₃ exit through apical Cl^– channels (Devor et al., 1999). In humans, CFTR is the most important Cl^– channel for airway serous fluid secretion, as demonstrated in cystic fibrosis patients whose CFTR is genetically defective, leading to reduced fluid secretion. Much less is known about the ionic currents of mucous cells, a cell whose major function is to release mucin. In isolated cystic fibrosis SMGC, forskolin-induced mucus secretion was totally lost, whereas carbachol-induced secretion was partially retained, raising the possibilities that mucous cells secrete fluid via CFTR-independent mechanisms, possibly via Ca²⁺-activated Cl^– channels (CaCC) (Wine and Joo, 2004). Mucous cells express little CFTR (Engelhardt et al., 1992) but do have CaCC that are activated by muscarinic receptors (Griffin et al., 1996). Mucous and serous cells are differentiated by their whole-cell current response to ACh; mucous cells produce dominant K⁺ current, whereas in serous cells, Cl^– current dominates (Iwase et al., 2002).

Mucus secretion from SMGC is controlled by secretagogues and neurotransmitters, such as ACh, ATP, vasoactive intestinal peptide (VIP), and prostanoids (Ballard and Inglis, 2004). ACh, a Ca²⁺-elevating agonist, is the most important neurotransmitter released by the respiratory parasympathetic nervous system (Coulson and Fryer, 2003), whereas ATP is released locally by epithelial cells (Donaldson et al., 2000). PGE₂ is a known cAMP-elevating agonist released by alveolar macrophages (Liu et al., 2005). VIP is another important cAMP-elevating agonist found in the airway, coexisting in cholinergic nerve terminals (Fischer et al., 1996). Ca²⁺ and cAMP are the most important second messengers responsible for the secretion of mucin and electrolyte in SMGC. Intracellular elevation of either Ca²⁺ or cAMP can induce mucus secretion by submucosal glands (Wine and Joo, 2004). Furthermore, these agents interact in controlling mucus secretion. For example, VIP or forskolin suppresses mucus secretion controlled by cholinergic nerve endings by activating large conductance calcium-activated K⁺ channels (BK), located in parasympathetic nerve endings, inhibiting ACh release (Liu et al., 1999). At the cellular level, Shimura et al. (1994) showed that isoproterenol, a cAMP-elevating agonist, enhances ATP-induced whole-cell current and [Ca²⁺]_i changes (possibly via Ca²⁺ entry) and increases ATP-induced mucus glycoprotein secretion in SMGC. The interactions of Ca²⁺- and cAMP-mediated pathways during SMGC secretion deserve further investigation because it is very possible that such interactions play important roles in regulating airway fluid and mucin secretion in pulmonary diseases, such as asthma, bronchitis, and chronic obstructive pulmonary disease (Rogers, 2002).

In this study, we focused on such interactions in controlling ionic current and changes in [Ca²⁺]_i in mucous cells. So far, little is known about the ion and fluid secretion in mucous cells and their coordinated control by multiple secretagogues and hormones. In our previous study, PGE₂, released by activated alveolar macrophage, enhanced ACh-induced I_SC by increasing the apparent sensitivities of the ACh-induced response (up to 3-fold decrease in EC₅₀ for the ACh response) in monolayers of primary cultured SMGC that were predominantly mucous cells (Liu et al., 2005). Prostanoids such as PGE₂ are released during airway inflammation and may be involved in mucus hypersecretion. Therefore, the mechanisms of PGE₂-mediated sensitization of ACh-induced ionic current in mucous cells were examined by measuring I_SC, whole-cell K⁺ and Cl^– currents, membrane potential changes, and [Ca²⁺]_i using Ussing chamber studies and whole-cell patch clamp with concurrent measurement of [Ca²⁺]_i.

	Materials and Methods

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Isolation and Culture of SMGC. Isolation of SMGC was performed according to methods from this laboratory (Yang et al., 1991). Male swine from local vendors (25–30 kg) were sacrificed by exsanguination after being anesthetized with 5% isoflurane (Rhodia Organique Fine Ltd., Bristol, UK). Fresh tracheas were also obtained from a local abattoir (Wilson Meat Packing, Crystal Springs, MS). Each trachea was quickly removed and transported to the laboratory in ice-cold physiological saline containing 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml kanamycin. The epithelium was stripped off as a single layer, minced, and digested with 1 mg/ml collagenase IV (Worthington CLS-4; Worthington Biochemical Corporation, Freehold, NJ), 0.5 mg/ml dithiothreitol, and 0.5 mg/ml DNase I (DN-25l; Sigma-Aldrich, St. Louis, MO) in Dulbecco's modified Eagle's culture media at 37°C for 4 to 6 h. Isolated cells were centrifuged through a discontinuous 10, 20, 30, 40, and 60% Percoll gradient at 500g for 10 min. SMGC concentrated at the interface between the 40 and 60% layers and were removed, washed, and resuspended in PC-1 medium containing 2 mM GlutaMAX (Invitrogen, Carlsbad, CA), serum substitutes (Cambrex Bio Science Walkersville, Inc., Walkersville, MD), and antibiotics. Cells were then plated on inserts (described below) for Ussing chamber studies. Some cells were plated on glass coverslips (VWR, West Chester, PA) coated with poly-L-lysine (P-4707; Sigma-Aldrich) and collagen type IV for patch-clamp studies. SMGC were attached to the coverslip within 2 h after plating. Cells were then used immediately for patch-clamp studies.

SMGC Culture at an Air-Liquid Interface Using Culture Inserts. Millicell-CM inserts (0.45 µm pore size, 0.6 cm² area; Millipore, Bedford, MA) were each coated with 75 µl of human placental collagen (0.5 mg/ml in 0.2% acetic acid and further diluted in 70% ethanol 1:3 v/v). The collagen coating was air-dried and then crosslinked by exposure to ammonium hydroxide vapors (3%) for 10 min followed by immersion in glutaraldehyde (2.5%) for 10 min. The coated inserts were washed once in distilled water, once in 70% ethanol, three times in distilled water, and finally incubated in culture media for 30 min before plating the cells (Takacs-Jarrett et al., 1998). Approximately 10⁶ cells were plated on each insert. One day after plating, the medium inside the inserts was removed, allowing the cells to grow at an air interface to maintain phenotype (Finkbeiner et al., 1994). Medium under the insert was changed every 2 days, and any solution inside the inserts was removed. Cells were maintained in culture for 3 to 5 days before measurement of I_SC.

I_SC Measurement. Confluent mucous cell monolayers grown on Millicell-CM inserts were mounted in Ussing chambers (Corning Life Sciences, Acton, MA), each modified to accept the Millicell inserts. The chambers were maintained at 37°C and continuously bubbled with gas mixture (95% O₂ + 5% CO₂). The bubbling also served to drive the circulation that quickly mixed drugs after the addition to the serosal or apical chamber, each having a maximal volume of 10 ml. The transepithelial I_SC was measured using VCC600 voltage-clamp amplifiers (Physiologic Instruments, San Diego, CA) connected to the chambers via salt bridges and silver/silver chloride pellet electrodes. The current across the confluent monolayers of SMGC was measured under voltage clamp, with the potential across the membrane set at 0 mV. Compounds (ACh, forskolin, 2-APB, and so on) were added to the serosal or apical solution from concentrated stock solutions (at least 1000 times concentrated). At the beginning of each experiment, amiloride (10 µM) was applied apically to block the epithelial Na⁺ channel activity, which can be found in the gland duct epithelia of submucosal gland acini and in the primary cultured SMGC (Yamaya et al., 1991). For cumulative ACh treatments, ACh was added serosally in the order of 0.003, 0.01, 0.03, 0.1, 0.3, 1, and 3 µM final concentrations. Only a small amiloride-sensitive current was recorded as shown in a previous study (Liu et al., 2005), suggesting a minimal contamination of surface or duct epithelial cells. The increases in I_SC in response to ACh, PGE₂, or forskolin were measured as the peak currents obtained after the addition of agonist at each concentration subtracted from the baseline current measured before the addition of initial concentration of the agonist. The currents were normalized to the area of the insert (0.6 cm²). Ussing chamber solution contained 128 mM NaCl, 4.6 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄, 11.2 mM glucose, pH adjusted to 7.4 before adding 25 mM NaHCO₃ (Moon et al., 1997). In Ca²⁺-free solution, CaCl₂ was replaced with 2.5 mM MgCl₂ and 0.5 mM EGTA.

Serosal Membrane K⁺ Current Measurement. Methanesulfonic acid (MeSO₄)-based Ussing chamber solutions were used to study the serosal K⁺ current. The serosal solution contained 145 mM NaMeSO₄ and 5 mM KMeSO₄; the apical solution contained 150 mM KMeSO₄; and the remaining components of both serosal and apical solution were 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄, 11.2 mM glucose, and 10 mM HEPES. Osmolarity was adjusted to 320 mOsm, with mannitol and the pH adjusted to 7.4 at 37°C with NaOH. These solutions were bubbled with 100% O₂. Nystatin (100 µM) was used to permeabilize the apical membrane to monovalent ions, such as Cl^–, Na⁺, and K⁺ (Liu et al., 2005). When these solutions were used, a 30:1 apical to serosal K⁺ gradient was established after Nystatin permeabilization.

Patch-Clamp Experiments. Whole-cell current and membrane potential recording were performed according to a previous report (Hamill et al., 1981). Patch pipettes were pulled from borosilicate glass (FMG 15; Dagan Corp., Minneapolis, MN) using a DMZ Universal Electrode Puller (Zeitz, Augsburg, Germany) and then fire-polished immediately before filling with pipette solution. The pipettes had resistances of approximately 4 M when filled with internal solution. Gigaohm seals were readily formed between the mucous cell membrane and pipette by applying gentle suction to the pipette. The seal resistances obtained were greater than 2 G. The whole-cell configuration was formed by rupture of the cell membrane by further suction. For whole-cell current recording, the membrane potential was held at–40 mV, and the potential was ramped over 200 ms from –100 to +40 mV every 2 s. This permitted measurement of both K⁺ current and Cl^– current at 0 and –80 mV, the equilibrium potentials for Cl^– and K⁺, when external and internal solutions as described below were used (as demonstrated in Fig. 6). The K⁺ and Cl^– current amplitudes at these potentials were measured by subtracting baseline currents from the currents obtained after agonist stimulation. Whole-cell membrane potentials were recorded using the current-clamp mode. Current and voltage data were acquired at 2 kHz and filtered at 1 KHz using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The data acquisition software used was Axon pClamp version 9. Patch-clamp data were analyzed using Clampfit 9.0 (Molecular Devices) and Origin 7.0 (Origin Lab, Northampton, MA). External solution for whole-cell measurements consisted of 140 mM NaCl, 6 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, with pH adjusted to 7.4 with NaOH. The internal solution consisted of 140 mM KCl, 8 mM NaCl, 1 mM MgCl₂, 0.05 mM EGTA, 5 mM MgATP, 1 mM Na₃GTP, and 10 mM HEPES, with pH adjusted to 7.2 with NaOH. External solutions were superfused over cells by gravity feed. All recordings were performed at room temperature.

View larger version (32K): [in this window] [in a new window]   Fig. 6. ACh-induced SMGC whole-cell current and Ca2+ fluorescence signal. A, stacked current sweeps (top) generated by the voltage-ramp protocol (bottom) and whole-cell current recording during the application of ACh are shown. The membrane potential was held at –40 mV, and the potential was ramped over 200 from –100 to +40 mV every 2 s. K+ and Cl– currents were recorded at 0- and –80-mV membrane potentials (equilibrium potentials for Cl– and K+, respectively). B, raw current traces (top) and voltage ramps (bottom) during the same experiment are shown. C, the top panel shows the development of K+ and Cl– currents induced by 10 µM ACh (marked at arrow) extracted from current traces in A and B. The bottom panel shows the development of Ca2+ signal measured concurrently using fluo-4 dye. D, I-V relationships derived from individual current traces during the voltage ramp at times marked by letters a, b, and c in C (a, before ACh treatment; b, time point at peak ACh current response; c, recovery). E1 shows a bright-field image of patched mucous cell; E2 through E4 show the fluorescence images at times a, b, and c shown in C (representative of six cells from three animals).

Reagents. AH6809 (6-isopropoxy-9-oxoxanthene-2-carboxylic acid) and SC19220 [8-chloro-dibenzo[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide] were purchased from Cayman Chemical (Ann Arbor, MI). PKI and Rp-cAMPs were purchased from BIOMOL International, L. P. (Plymouth Meeting, PA); and SKF96365 was purchased from Fisher Scientific Co. (Pittsburgh, PA). Other chemicals, such as 2-APB, ChTX, clotrimazole, forskolin, IbTX, and PGE₂, were purchased from Sigma-Aldrich, unless specifically noted in the text.

Statistics. Data are presented as mean ± S.E.M., and n is the number of inserts or animals used as described in the text. EC₅₀ values for the ACh-induced I_SC responses are presented with 95% confidence interval (95% CI). Because there were considerable differences among the ACh-induced maximal I_SC in primary cultures from different animals, for I_SC measurements, matched inserts with identical culture and experimental conditions from the same animal were subject to different treatments. Cells from at least three animals per experimental protocol were performed. The statistical method used to differentiate differences between treatments was one-way repeated measures analysis of variance (or on ranks whenever appropriate) plus post hoc tests. ACh-induced I_SC was normalized to the maximal I_SC from each insert before calculating average concentration responses (average maximal I_SC from each treatment is also provided). For whole-cell current, membrane potential, and Ca²⁺ measurements, the same treatment data from a single animal were averaged before calculating mean ± S.E.M. One-way ANOVA (or on ranks) was used whenever appropriate to test statistical difference between multiple treatments. *, p < 0.05 indicates significance of difference.

	Results

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Effect of PGE₂ or Forskolin Pretreatment on the ACh-Induced I_SC. The resting transepithelial potentials, currents, and resistances for confluent SMGC monolayers were reported in a previous study (Liu et al., 2005); in the same study we also reported that PGE₂ pretreatments (10 nM to 1 µM) sensitized ACh-induced I_SC. Similar Ussing chamber studies on ACh-induced I_SC across mucous gland cell monolayers grown on CM inserts were performed. ACh alone induced average maximal I_SC of 36.2 ± 7.3 µA/cm², with an EC₅₀ of 151 nM (95% CI 113–191 nM; n = 4) in Control. PGE₂ or forskolin pretreatment induced persistent increases in I_SC (14.3 ± 4.9 and 12.8 ± 3.4 µA/cm², respectively, n = 4 each). EC₅₀ values for the ACh-induced I_SC after PGE₂ or forskolin pretreatment were 47 nM (95% CI 38–58 nM; n = 4) and 43 nM (95% CI 34–52 nM; n = 4), respectively, both significantly smaller than EC₅₀ in Control (p < 0.01). The maximal I_SC induced by ACh after PGE₂ and forskolin pretreatments were 41.7 ± 6.0 and 39.9 ± 7.1 µA/cm², respectively, not significantly different from Control (p > 0.05).

EP₂ Receptors and PKA Were Involved in the PGE₂-Induced Sensitization of Mucous Cells to ACh-Induced Increase in I_SC. The EP_{1 + 2} receptor antagonist AH6809 (30 µM) partially reversed the effect of PGE₂ in sensitizing ACh-induced I_SC (Fig. 1, A and B). EC₅₀ values for the ACh-induced I_SC in Control (no PGE₂ pretreatment), PGE₂ pretreatment group (0.1 µM), and AH6809 + PGE₂ group were 147 nM (95% CI 85–223 nM), 59 nM (95% CI 27–91 nM; p < 0.05 versus Control), and 87 nM (95% CI 47–128 nM; p < 0.05 versus Control or PGE₂ group), respectively (n = 4 each). Pretreating mucous cell monolayers with AH6809 before PGE₂ and ACh applications shifted the ACh concentration-response relationships to the right compared with those in PGE₂ group (Fig. 1B). The EP₁ receptor antagonist SC19220 did not reverse the PGE₂-induced sensitization of mucous cells in response to ACh to increase I_SC (EC₅₀ = 35 nM, 95% CI 31–38 nM; n = 4; p > 0.05 versus PGE₂ group).

View larger version (35K): [in this window] [in a new window]   Fig. 1. EP2 receptors and PKA were involved in the PGE2-induced sensitization of mucous cells in response to ACh to increase ISC. A, trace 1 is a control recording that shows the ISC induced by ACh alone (cumulative application marked at arrows). As shown in trace 2, PGE2 (0.1 µM, serosal) was applied before cumulative ACh applications. In trace 3, AH6809 (30 µM, serosal) was applied 5 min before PGE2 pretreatment followed by cumulative ACh applications. Similar experiments were performed using SC19220 (30 µM). B, concentration-response relationships are shown for the ACh-induced ISC, demonstrating that AH6809 (upward triangles), but not SC19220 (downward triangles), modulates the PGE2-induced sensitization of mucous cell monolayers to ACh in increasing ISC (normalized to maximal ISC from each insert), indicative of the involvement of EP2 receptors in the sensitization. C, the PKA inhibitor 14-22 amide myristoylated (2 µM, serosal, trace 3) was applied 30 min before the PGE2 pretreatment (0.1 µM, serosal, not shown), followed by cumulative ACh application. D, concentration-response relationships for the ACh-induced ISC show the effect of PKI and PGE2 on the apparent sensitivities of the ACh-induced ISC (normalized to maximal ISC from each insert, four inserts each from three animals). PKI decreased the PGE2-induced sensitization of mucous cells to ACh in increasing ISC.

PGE₂-Sensitized ACh-Induced I_SC in the Absence of Extracellular Ca²⁺. We tested whether extracellular Ca²⁺ is required for the effect of PGE₂ on the ACh-induced I_SC. As shown in Fig. 2A, in 0 mM [Ca²⁺]_o cumulative application of ACh (serosal) induced increases in I_SC similar to those found in Fig. 1 but with a more rapidly declining tail. In Control, without PGE₂ pretreatment, the average EC₅₀ for the ACh-induced I_SC (Fig. 2, A, trace 1, and B, gray squares) was 217 nM (95% CI 151–289 nM; n = 4). Compared with control, PGE₂ pretreatments (0.1 and 1 µM in Fig. 2, A, traces 2 and 3, and B, gray circles and triangles, respectively) significantly decreased EC₅₀ values for the ACh response (115 nM, 95% CI 96–134 nM and 80 nM, 95% CI 71–89 nM for 0.1 and 1 µM PGE₂ groups, respectively; n = 4 each; p < 0.05 versus Control).

View larger version (22K): [in this window] [in a new window]   Fig. 2. PGE2 treatment sensitized mucous cells in response to ACh in inducing ISC under 0 mM [Ca2+]o. A, CaCl2 in the Ussing chamber solution was replaced with MgCl2 and EGTA, and PGE2 (0.1 and 1 µM, serosal, in traces 2 and 3, respectively) was applied to –10 min before ACh application. Trace 1 is a control recording without PGE2 pretreatment. B, the concentration-response relationships are shown for the ACh-induced ISC in the absence of [Ca2+]o (gray symbols) or in the presence of normal [Ca2+]o (2.5 mM, black symbols). Under both normal and 0 mM [Ca2+]o conditions, PGE2 pretreatment (0.1 or 1 µM) sensitized mucous cell monolayers in response to ACh, as shown by the left shift of the ACh concentration-response relationships. Note that 0 mM [Ca2+]o reduced the sensitivity to ACh but did not prevent the left shift of the ACh concentration response caused by PGE2 pretreatment (four inserts in each group; data from three animals).

Note that in Fig. 2B, under both 0 mM [Ca²⁺]_o (gray symbols) and 2.5 mM [Ca²⁺]_o (black symbols) conditions, PGE₂ pretreatments caused left shifts of the ACh-induced concentration-response relationships to a similar extent; whereas 0 mM [Ca²⁺]_o treatments caused right shifts of the ACh concentration-response relationships relative to those in 2.5 mM [Ca²⁺]_o (gray squares and circles versus black squares and circles, respectively).

PGE₂ pretreatments did not significantly change the ACh-induced maximal I_SC under conditions of both 0 and 2.5 mM [Ca²⁺]_o. In 0 mM [Ca²⁺]_o, average maximal I_SC responses were 36.2 ± 10.5, 41.7 ± 7.6, and 39.9 ± 9.8 µA/cm² for 0 (Control), 0.1, and 1 µM PGE₂-pretreatment groups, respectively. In 2.5 mM [Ca²⁺]_o, average maximal I_SC responses were 118.7 ± 23.1 and 108.5 ± 12.4 µA/cm² in 0 (Control) and 0.1 µM PGE₂ group, respectively. However, the latter two maximal I responses in 2.5 mM [Ca²⁺_SC]_o were significantly larger than those in 0 mM [Ca²⁺]_o (p < 0.05).

Also shown in Fig. 2A, PGE₂ induced increases in I_SC to a peak followed by a stable plateau. The average peak I_SC was 29.3 ± 7.1 µA/cm², and plateau I_SC was 14.4 ± 2.4 µA/cm² (0.1 µM PGE₂ in 0 mM[Ca²⁺]_o; n = 4). In the presence of 2.5 mM [Ca²⁺]_o, average peak and plateau I_SC responses induced by 0.1 µM PGE₂ were 58.6 ± 5.7 and 36.9 ± 3.3 µA/cm² (n = 4), significantly larger than those in 0 mM [Ca²⁺]_o (p < 0.05).

Effect of SKF96365 on the ACh-Induced I_SC and PGE₂-Induced Sensitization of ACh Response. SKF96365, a Ca²⁺ entry blocker (Merritt, 1990), was used to examine the role of Ca²⁺ entry in the ACh-induced I_SC and PGE₂-induced sensitization of the ACh response. As shown in Fig. 3A, trace 1 (Control), ACh (0.1 µM, a submaximal concentration) induced transient I_SC with average peak I_SC of 33.8 ± 6.7 µA/cm² (Fig. 3B, top graph). Pretreatment with 1 µM PGE₂ (Fig. 3A, trace 2) significantly increased ACh-induced I_SC compared with Control, with average maximal I_SC being 66.5 ± 7.0 µA/cm² (n = 3, p < 0.05). SKF96365 (5, 25, and 50 µM in Fig. 3A, traces 3, 4, and 5, respectively) applied before PGE₂ and ACh applications significantly decreased ACh-induced peak I_SC response compared with that in PGE₂ group (p < 0.05), with average I_SC responses being 45.4 ± 5.4, 35.4 ± 6.1, and 23.9 ± 2.6 µA/cm², respectively.

View larger version (32K): [in this window] [in a new window]   Fig. 3. Effects of SKF96365 and PGE2 on the ACh-induced ISC. A, a single submaximal concentration of ACh (0.1 µM, serosal, trace 1, Control) induced a transient increase in ISC. PGE2 (1 µM, serosal, trace 2), applied 5 min before ACh application, induced a relatively persistent increase in ISC, after which ACh induced a larger increase in ISC that declined with a similar rate to Control. SKF96365 (5, 25, and 50 µM, serosal: traces 3, 4, and 5, respectively) applied 5 to 10 min before PGE2 and ACh applications, caused the ACh-induced ISC to decline more rapidly. B, summary of analysis of data, as in A, are shown. Top panel shows the effect of PGE2 and SKF96365 on the ACh-induced peak ISC, and the middle and bottom panels show the duration for the peak ISC to decline to 50% maximal ISC (T1/2, in seconds) and to 25% maximal ISC (T3/4, in seconds), respectively. Note that SKF96365 significantly decreased the peak current induced by ACh (* p < 0.05 compared with PGE2 group, bar no.2) and at 25 and 50 µM enhanced the rate of ISC decline (*, p < 0.05 compared with control). Total of three inserts from three animals for each experiment. C, trace 1, ACh was cumulatively applied in the presence of PGE2 (1 µM) to induce increases in ISC similar to those shown in Fig. 1. SKF96365 (trace 2, 25 µM, and trace 3, 50 µM, serosal) was applied before PGE2 and ACh treatments. D, the concentration-response relationships for the ACh-induced ISC are shown (normalized to maximal ISC from each insert) in the presence of SKF96365 (0, 5, 25, and 50 µM) with PGE2 pretreatment (1 µM, filled black labels, top graph) or without PGE2 pretreatment (empty labels, bottom graph); n is the number of inserts used, three to four animals were used for each group. Note that SKF96365 caused right shifts in concentration-response relationships of the ACh-induced response in both graphs. PGE2 treatment in top graph caused similar magnitude of left shifts in the concentration-response relationships relative to groups treated with identical concentrations of SKF96365 in the bottom graph.

SKF96365 (25 and 50 µM) significantly inhibited 1 µM PGE₂-induced I_SC (Fig. 3A). Average plateau I_SC responses were 17.8 ± 5.9 µA/cm² for PGE₂ alone and 9.8 ± 2.5 and 8.9 ± 3.6 µA/cm² for PGE₂ in the presence of 25 or 50 µM SKF96365 (p < 0.05 versus PGE₂ alone; n = 3 each).

The effect of SKF96365 on concentration-response relationships for ACh-induced I_SC was also examined. SKF96365 (25 and 50 µM; Fig. 3C, traces 2 and 3, respectively), applied before PGE₂ pretreatment (1 µM), decreased the ACh-induced I_SC at lower ACh concentrations (0.01–0.3 µM), causing rapid decline of I_SC after the peak response, relative to the ACh-induced increase after pre-exposure to PGE₂ (PGE₂ group, trace 1). EC₅₀ values for the ACh-induced responses after 1 µM PGE₂ pretreatment only (Fig. 3D, top graph, black squares), PGE₂ + 25 µM SKF96365 (Fig. 3D, black upward triangles), and PGE₂ + 50 µM SKF96365 (Fig. 3D, black downward triangles) were 104 nM (95% CI 64–126 nM), 211 nM (95% CI 114–266 nM), and 227 nM (95% CI 166–269 nM), respectively. The latter two were significantly larger than EC₅₀ in PGE₂ group (p < 0.05). Furthermore, SKF96365 at 50 µM, but not at 25 µM, significantly decreased the ACh-induced average maximal I_SC (17.2 ± 5.0 and 23.1 ± 4.2 µA/cm², respectively) compared with that in PGE₂ group (28.6 ± 2.6 µA/cm²)(P < 0.05). SKF96365 at 5 µM did not change either the maximal I_SC (27.8 ± 6.0 µA/cm²) or EC₅₀ (104 nM, 95% CI 58–134 nM; Fig. 3D, black circles) for the ACh response (P > 0.05 versus PGE₂ group).

In the absence of PGE₂ pretreatment, SKF96365 at 5 and 25 µM did not change either maximal I_SC or EC₅₀ for the ACh response. ACh-induced maximal I_SC were 24.2 ± 2.2 µA/cm² (Control: ACh alone), 25.3 ± 6.1 µA/cm² (5 µM SKF96365), and 22.1 ± 4.0 µA/cm² (25 µM SKF96365) (p > 0.05; n = 3). EC₅₀s for the ACh-induced I_SC were 279 nM (95% CI 136–432 nM) in Control, 242 nM (95% CI 122–374 nM) in 5 µM SKF96365, and 369 nM (95% CI 139–690 nM) in 25 µM SKF96365 (p > 0.05 versus Control). SKF96365 at 50 µM significantly inhibited ACh-induced maximal I_SC (14.8 ± 2.9 µA/cm²) and increased EC₅₀ for the ACh response (482 nM, 95% CI 324–668 nM) (P < 0.05 versus Control, n = 3). As shown in Fig. 3D, SKF96365 treatments (25 and 50 µM) shifted the ACh concentration-response relationships to the right, both in the presence (top graph, black symbols) or absence of PGE₂ (bottom graph, open symbols), whereas PGE₂ induced left shifts of ACh concentration-response relationships in the presence of equal concentrations of SKF96365 to a similar extent (Fig. 3D), suggesting that PGE₂ induced the sensitization of ACh response by a Ca²⁺ entry-independent mechanism. However, as shown for the effects of Ca²⁺ removal, Ca²⁺ entry is important in the response to ACh.

Effect of 2-APB on the ACh-Induced I_SC and PGE₂-Induced Sensitization of ACh Response. 2-APB, a known IP₃-receptor antagonist and Ca²⁺ entry blocker (Bootman et al., 2002), was used to examine the role of Ca²⁺-release (IP₃-induced) and Ca²⁺ entry in the ACh-induced I_SC and PGE₂-induced sensitization of the ACh response. Inserts used to test the effect of 2-APB on the ACh response (0.1 µM) and time course were from the same animals used in SKF96365 experiments (Control and PGE₂-treatment groups are the same as those in Fig. 3, A and B). Serosal application of 2-APB (5, 25, or 100 µM in Fig. 4A, traces 3, 4, and 5, respectively) before PGE₂ and ACh application significantly decreased ACh-induced peak I_SC compared with PGE₂ group (P < 0.05). The average peak I_SC responses were 48.8 ± 11.7, 26.9 ± 4.3, and 1.4 ± 0.4 µA/cm², respectively (5, 25, or 100 µM 2-APB; Fig. 4B, top panel). There were significant differences in ACh-induced peak I_SC between 5 and 25 µM groups (P < 0.05) and between 25 and 100 µM groups (P < 0.01). Furthermore, each 2-APB treatment significantly accelerated the decline of ACh-induced I_SC compared with PGE₂ group (Fig. 4B, middle, T_1/2 data; and bottom, T_3/4 data). As shown in Fig. 4A, PGE₂-induced plateau I_SC responses were significantly reduced by all 2-APB pretreatments at 5, 25, and 100 µM(P < 0.05). PGE₂-induced average plateau I_SC responses were 21.1 ± 3.2 (PGE₂ alone), 14.5 ± 2.3, 13.2 ± 4.6, and 3.1 ± 0.6 µA/cm² (5, 25, and 100 µM 2-APB, respectively).

View larger version (27K): [in this window] [in a new window]   Fig. 4. Effects of 2-APB and PGE2 on ACh-induced ISC. A, using protocol of Fig. 3A, 2-APB (5, 25, and 100 µM in traces 3, 4, and 5, respectively) was added serosally before PGE2 and ACh applications. Both PGE2 and ACh-induced peak ISC were decreased, and the decline of ACh-induced ISC was more rapid than control in the presence of 2-APB. B, the effects of 2-APB and PGE2 on the ACh-induced peak ISC (top) and also the time to decay to 50 (middle) and 25% (lower) of peak are shown in bar graphs (three inserts from three animals for each treatment; *, p < 0.05 compared with the respective values in bar no 2: PGE2-only). C, 2-APB (100 µM, trace 2) was added serosally 5 to 10 min before PGE2 (1 µM) and cumulative ACh application. ACh-induced ISC in the presence of PGE2 were greatly reduced by 100 µM 2-APB (typical of three inserts from three animals).

The effect of 2-APB on concentration-response relationships for the ACh-induced I_SC was also tested. Serosal application of 100 µM 2-APB before PGE₂ and ACh treatments greatly reduced ACh-induced peak I (4.0 ± 1.1 µA/cm²_SC; Fig. 4C, trace 2, n = 5) compared with Control (50.7 ± 6.4 µA/cm²; Fig. 4C, trace 1, n = 5). Not shown in the figure, serosal application of 25 µM 2-APB before PGE₂ treatment (1 µM) significantly decreased ACh-induced peak I_SC and increased EC₅₀ for the ACh response. Peak I_SC responses were 26.9 ± 5.6 and 13.1 ± 3.4 µA/cm² (n = 7, p < 0.05), and the average EC₅₀ values were 99 (95% CI 58–133 nM) and 155 nM (95% CI 78–214 nM) for Control and 2-APB group (25 µM), respectively. At 5 µM, 2-APB did not change the peak I_SC or EC₅₀ for the ACh response (data not shown).

Intermediate Conductance Ca²⁺-Activated K⁺ Channel (IK), but Not BK, Were Involved in the ACh-Induced I_SC. Previously, we showed that ChTX, a BK and IK channel blocker, inhibited ACh-induced I_SC (Liu et al., 2005). Here we used IbTX, a specific blocker for BK in addition to ChTX, to determine the type of K⁺ channels involved in the ACh -induced I_SC. ChTX, but not IbTX, significantly inhibited ACh-induced I_SC in the presence of 1 µM PGE₂ (Fig. 5, A and B). ACh-induced maximal I_SC responses were 52.9 ± 8.2, 49.3 ± 7.3, and 11.5 ± 0.8 µA/cm² in Control (PGE₂ pretreatment only), IbTX group (PGE₂ + 100 nM IbTX), and ChTX group (PGE₂ + 100 nM ChTX), respectively (n = 3 each, p < 0.05). Neither IbTX nor ChTX significantly changed EC₅₀ for the ACh-induced I_SC response, with EC₅₀ values at 33 (95% CI 27–38 nM), 35 (95% CI 19–51 nM), and 40 nM (95% CI 16–72 nM) in the Control, IbTX group, and ChTX group, respectively (P > 0.05).

View larger version (33K): [in this window] [in a new window]   Fig. 5. IK, but not BK, were involved in the ACh-induced ISC. A, ChTX (100 nM, trace 2) or IbTX (100 nM, trace 3) was added serosally after PGE2 (1 µM) but before ACh application. In Control (trace 1), no K+ blockers were applied. Note that the baselines for the traces in A (also in C) were offset for clarity. B, summary of concentration-response relationships for the ACh-induced ISC (normalized to percentage of the ACh-induced maximal ISC in Control, n = 3 each) are shown. ChTX, but not IbTX, decreases the magnitude of ACh-induced ISC compared with that in Control. C, the apical membranes of monolayers were permeabilized with 100 µM nystatin (at the first arrow) to monovalent ions, and a 30:1 apical to serosal K+ gradient was established as described under Materials and Methods. Trace 1 shows currents induced by cumulative ACh in a control recording without forskolin treatment. In trace 2, Forskolin (10 µM, serosal), added 5 to 10 min before cumulative ACh treatment, enhanced ACh-induced K+ current. In trace 3, IbTX (40 nM) was added serosally before forskolin and ACh additions. D, clotrimazole (50 µM, serosal, trace 2) was added before the forskolin and ACh treatments. Trace 1 shows a forskolin pretreatment recording without K+ channel blocker treatment (similar to trace 2 in A). E, the effects of forskolin and K+ channel blockers on concentration-response relationships for the ACh-induced K+ current from experiments presented in C and D are shown (n is the number of inserts used, two- to three animals were used in each group). Forskolin treatment sensitizes the ACh-induced serosal K+ current.

PGE₂ pretreatment sensitized the ACh-induced serosal K⁺ current (Liu et al., 2005). Here we used clotrimazole, an IK or KCNN4 blocker (Devor et al., 1999), to examine the types of Ca²⁺-activated K⁺ channels (K_Ca) involved in ACh-induced I_SC and cAMP-mediated sensitization of the ACh response. As shown in Fig. 5C, trace 1 (Control), after nystatin permeabilization of the apical membrane, ACh induced increases in serosal K⁺ current, with an average maximal current of 187.3 ± 40.5 µA/cm² and EC₅₀ of 161 nM (95% CI 121–206 nM; n = 3). Forskolin pretreatment (10 µM, serosal; Fig. 5C, trace 2) sensitized the ACh-induced K⁺ current (EC₅₀ = 67 nM, 95% CI 56–78 nM; n = 3, p < 0.05 versus Control) but did not change the ACh-induced maximal K⁺ current (148.2 ± 37.3 µA/cm²; p > 0.05 versus Control). In the presence of 40 nM IbTX and forskolin (Fig. 5C, trace 3), the average ACh-induced maximal K⁺ current was 181.0 ± 48.5 µA/cm², and EC₅₀ for the ACh response was 73 nM (95% CI 48–102 nM; n = 3), and each was not significantly different from that in forskolin group (P > 0.05).

In separate experiments, after forskolin pretreatment (10 µM, serosal; Fig. 5D, trace 1), ACh induced an average maximal K⁺ current of 167.0 ± 27.8 µA/cm², with an EC₅₀ of 61 nM (95% CI 40–81 nM; n = 5), similar to those found in Fig. 5A. Clotrimazole (50 µM, serosal) was applied before forsko-lin pretreatment (Fig. 5D, trace 2); afterward, the ACh-induced maximal K⁺ current was 58.7 ± 15.5 µA/cm², and EC₅₀ for the ACh response was 174 nM (95% CI 112–241 nM; n = 5). Clotrimazole significantly decreased ACh-induced maximal K⁺ current and increased EC₅₀ for ACh response (P < 0.05 versus forskolin group). Figure 5E summarizes the effect of forskolin, IbTX, and clotrimazole on the concentration-response relationships for ACh-induced K⁺ current.

As shown in the Fig. 5C, forskolin (10 µM) alone induced a peak average K⁺ current of 155.5 ± 14.6 µA/cm², whereas in the presence of 40 nM IbTX, forskolin induced an average K⁺ current of 177.1 ± 33.8 µA/cm² (n = 3, p > 0.05 versus forskolin alone, paired t test). As shown in Fig. 5B, forskolin (10 µM) alone induced an average K⁺ current of 153.8 ± 16.8 µA/cm² (n = 5), whereas in the presence of 50 µM clotrimazole, forskolin-induced average K⁺ current was 76.4 ± 19.6 µA/cm² (n = 5, p < 0.05 versus forskolin alone). Therefore clotrimazole but not IbTX inhibited forskolin and ACh-induced serosal K⁺ current.

PGE₂ and Forskolin Enhanced ACh-Induced Mucous Cell Whole-Cell K⁺ and Cl^– Current and [Ca²⁺]_i. Upon whole-cell formation, mucous cells were dialyzed with internal solution with a Cl^– concentration equal to that of external solution. The average mucous cell capacitance was 6.48 ± 0.15 pF, and cell capacitances among cells from different animals were not significantly different (P < 0.05, 28 cells from five animals). Whole-cell membrane resistance and access resistance were 2.8 ± 0.3 G and 23 ± 3 M (total of 30 cells from six animals), respectively, at a 0-mV holding potential.

Voltage-ramp protocols were used to detect changes in ACh-induced whole-cell K⁺ and Cl^– channel current as described under Materials and Methods (Fig. 6, A and B). Cells enriched by discontinuous Percoll gradient were large with abundant large nonhomogeneous granules (Fig. 6E₁) and previously identified using periodic acid Schiff-Alcian Blue staining methods as mucous cells (Yang et al., 1991). As shown in Fig. 6C, top, for a mucous cell, superfusion with 10 µM ACh induced a K⁺ current (triangles) and a smaller Cl^– current (circles). I-V relationships for ACh-induced whole-cell current are shown in Fig. 6D. The reversal potential of ACh-induced current trace is close to–80 mV, the equilibrium potential for K⁺ using these solutions. Both K⁺ and Cl^– currents developed transiently to peaks that were followed by declining tails in the continuous presence of ACh. In some experiments, fluo-4 potassium salt (10 µM) was included in the patch pipette to measure Ca²⁺ concurrently (Fig. 6, E₂–E₄). Ca²⁺ signals were measured as fluo-4 fluorescence changes and expressed as (F–F₀)/F₀. ACh-induced Ca²⁺ fluorescence signals were coincident with ACh-induced changes in whole-cell currents (Fig. 6C, bottom).

View larger version (30K): [in this window] [in a new window]   Fig. 7. Measurement of Ca2+ signal during PGE2, forskolin, and ACh-induced whole-cell K+ and Cl– current. A, the protocol in Fig. 6 was used, and ACh was applied at 0.3 or 10 µM marked by gray bars. The development of whole-cell current at –80 (Cl– current) and 0 mV (K+ current) is shown in bottom panel. The top panel shows the fluo-4 fluorescence signal reflecting intracellular [Ca2+]i in the same cell. ACh (0.3 µM) induced little or no increase in current or [Ca2+]i. B, current (bottom) and [Ca2+]i changes (top) induced by PGE2 (1 µM, open bar) and ACh (0.3 µM, gray bar) are shown. Note that ACh (0.3 µM) induced a robust response in the presence of PGE2. C, current (bottom) and changes in [Ca2+]i (top) induced by forskolin (10 µM, open bar) and ACh (0.3 µM, gray bar). Similar to PGE2, forskolin did not induce current or [Ca2+]i changes but enhanced the response to 0.3 µM ACh. D, the inclusion of 250 U/ml PKA-catalytic subunit in the internal solution of the patch pipette mimicked the effect of 1 µM PGE2 and 10 µM forskolin in enhancing 0.3 µM ACh-induced whole-cell currents. Note that the response to 0.3 µM ACh is similar to the 10 µM ACh response. E, summary of ACh, PGE2, and forskolin-induced cytosolic peak Ca2+ changes as measured using fluo-4 florescence signals are shown (**, p < 0.01 compared with 0.3 µM ACh, the numbers of animal used are shown in the figure). F, summary of the effects of PGE2, forskolin, and PKA treatment on the ACh-induced K+ and Cl– currents are shown. Positive bars indicate changes in K+ current at 0 mV, and negative bars indicate changes in Cl– current at –80 mV. The numbers in parentheses are the number of animals used in each treatment group, and two to three cells were used in each treatment per animal (*, p < 0.05 from 0.3 µM ACh control). PGE2 and forskolin enhance both K+ and Cl– currents as well as the associated rise in [Ca2+]I, suggesting that sensitization of the Ca2+-release process occurred via activation of PKA.

When 250 U/ml PKA catalytic subunit was included in the patch pipette, 0.3 µM ACh-induced K⁺ and Cl^– currents were 163 ± 13 and –40 ± 4 pA/pF, respectively, and 10 µM ACh induced 170 ± 17 and –50 ± 10 pA/pF currents (Figs. 7D and 8E, five cells from three animals). The current induced by 0.3 µM ACh in the presence of PKA catalytic sub-units was significantly larger than that caused by 0.3 µM ACh alone, but not different from that induced by 10 µM ACh alone, or by 0.3 µM ACh in the presence of forskolin (10 µM) or PGE₂ (1 µM) (Fig. 7E).

View larger version (20K): [in this window] [in a new window]   Fig. 8. ACh- and PGE2-induced changes in membrane potential in single mucous cells. A, membrane potential changes induced by ACh at 0.3 (six cells, five animals) or 10 µM (five cells, five animals) are shown. ACh at 0.3 µM caused a small hyperpolarization, whereas 10 µM ACh-hyperpolarized cell near EK. Also note the transient spontaneous hyperpolarizations that occur due to opening of single K+ channels. B, a similar experiment to A except that the cell is exposed to 1 µM PGE2 (open bar) before 0.3 µM ACh (gray bar) (seven cells, five animals). ACh (0.3 µM) now induces hyperpolarization near EK. PGE2 before ACh application depolarizes the cell. C, average data for ACh and PGE2-induced membrane potential changes. PGE2 significantly enhanced the hyperpolarization induced by 0.3 µM ACh. **, statistically significant difference from 0.3 µM ACh group (p < 0.01).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present study demonstrated that PGE₂ increased the apparent sensitivities of ACh-induced I_SC in mucous cells mediated by EP₂ receptors via PKA that was independent of Ca²⁺ entry. Ca²⁺ entry was important to maintain the basal sensitivity of the ACh response. IK but not BK channels were also shown to be involved. In addition, PGE₂ or forskolin enhanced ACh-induced whole-cell K_Ca and CaCC currents and membrane hyperpolarization by enhancing Ca²⁺ release from internal stores.

Role of Endoprostanoid Receptors and PKA. ACh-induced I_SC was sensitized by PGE₂ or forskolin pretreatment in mucous cells grown on CM inserts as shown by left shifts of concentration-response relationships for ACh-induced I_SC (Fig. 1), results similar to our previous findings (Liu et al., 2005). All four endoprostanoid receptors were present in SMGC but the sensitization effect of PGE₂ was dependent on the EP₂ receptor; The PGE₂ receptor antagonist AH6809 (EP_{1 + 2}), but not SC19220 (EP₁), partially inhibited PGE₂-induced I_SC (Liu et al., 2005) and reversed the PGE₂-induced sensitization of the ACh response (Fig. 1). Furthermore PKA-specific blockers PKI 14-22, a pseudosubstrate for PKA (Glass et al., 1989), and Rp-cAMP_S, an antagonist of cAMP in binding to PKA (Schaap et al., 1993), reduced PGE₂-induced sensitization (Fig. 1). PKA-catalytic subunit enhanced ACh-induced whole-cell currents (Fig. 7). These data implicate the EP₂ receptor in sensitization because it is known to be coupled to the activation of PKA (Bos et al., 2004). Besides activating PKA via EP₂ receptors, PGE₂ can also activate EP₄ receptors coupled to G_s-protein capable of transactivating epidermal growth factor (EGF) receptors and receptor tyrosine kinases (Pai et al., 2002). EGF receptor activation-prolonged ACh-induced Ca²⁺ mobilization in SMGC has been shown to be dependent on tyrosine kinase activity and extracellular Ca²⁺ (Iwase et al., 2002). However, neither the tyrosine kinase inhibitor genistein nor the EGF receptor tyrosine kinase inhibitor AG1478 altered sensitization by PGE₂. Thus, PKA-dependent processes are primarily responsible for sensitization.

Role of Ca²⁺ Influx and Ca²⁺ Release. A known effect of cAMP/PKA is to modulate [Ca²⁺]_i in SMGC by enhancing ATP-induced Ca²⁺ entry (Shimura et al., 1994). Removing external Ca²⁺ (Fig. 2) or treatment with SKF96365 (Fig. 3), the Ca²⁺ entry blocker, caused similar right shifts in the ACh concentration-response relationships in both control and after PGE₂ pretreatment, demonstrating that Ca²⁺ influx is necessary to maintain "normal" sensitivities of the ACh response. ACh-induced I_SC declined to baseline more quickly under 0 mM [Ca²⁺]_o or SKF96365 treatments compared to control conditions, indicating that Ca²⁺ influx is essential for sustaining the ACh-induced response. Furthermore, maximal I_SC responses were decreased by these treatments, indicating that Ca²⁺ influx is involved in the ACh-induced "transient" peaks in I_SC responses, as is Ca²⁺ release from internal stores.

The sensitization effect of PGE₂ was not reversed by 0 mM [Ca²⁺]_o or SKF96365 treatments. Under 0 mM [Ca²⁺]_o, PGE₂ at 0.1 and 1 µM still caused 1.9- and 2.7-fold increases in the apparent sensitivity to ACh, as measured by ratios of EC₅₀s (without/with PGE₂ treatments) for ACh-induced I_SC (Fig. 2). Under normal [Ca²⁺]_o, 0.1 µM PGE₂ caused 2.8-(Fig. 2) and 2.5-fold (Fig. 1) and 1 µM PGE₂ caused 2.7-fold (Fig. 3) increase in the apparent sensitivity to ACh. Likewise, after SKF96365 treatment, the -fold shifts in EC₅₀s for the ACh response were 2.7-, 2.3-, 1.7-, and 2.1-fold in the presence of 0, 5, 25, and 50 µM SKF96365, respectively (Fig. 3D).

2-APB decreased ACh-induced maximal I_SC and shortened the response. At 100 µM, 2-APB greatly reduced both peak and sustained I_SC induced by ACh in the presence of PGE₂ (Fig. 4, A and C), effects that were not achieved by 0 mM [Ca²⁺]_o or SKF96365 treatments (Fig. 2, A and B, I_SC and T_1/2 data). These data demonstrated that 2-APB, in addition to inhibiting ACh-induced Ca²⁺ entry, inhibited ACh-induced/IP₃ receptor-mediated Ca²⁺ release from internal stores, which was essential for the sensitization effect of PGE₂/PKA on the ACh response.

Whole-cell current and Ca²⁺ measurements in single mucous cells demonstrated that ACh induced K_Ca and that CaCC currents were coincident with changes in [Ca²⁺]_i.Enhanced currents were a result of enhanced Ca²⁺ mobilization (Figs. 6 and 7). The existence of K_Ca and CaCC in SMGC are consistent with previous reports (Griffin et al., 1996). PGE₂ or forskolin enhanced ACh-induced peak currents and changes in peak [Ca²⁺]_i, indicating that ACh-induced Ca²⁺ release from internal stores was enhanced. PGE₂ also prolonged ACh-induced ionic current [Ca²⁺]_i and membrane hyperpolarization (Figs. 7 and 8), suggesting that prolonged Ca²⁺ entry also occurred.

Role of K⁺ Channels Involved. Direct sensitization of ion channels (for example K_Ca)to Ca²⁺ by PKA is another possible mechanism responsible for PGE₂-induced sensitization. A known effect of PKA is to enhance BK activities through phosphorylation (Tian et al., 2001). Therefore, we examined the ability of forskolin to sensitize ACh-induced serosal K⁺ current. ChTX, the blocker for both IK and BK, abolished ACh-induced I_SC (Fig. 5) (Liu et al., 2005). In contrast, IbTX, the specific blocker for BK, was ineffective in inhibiting ACh-induced I_SC (Fig. 5). Therefore, IK but not BK channels are involved in the ACh-induced I_SC. Forskolin sensitized ACh-induced serosal K⁺ current also (Fig. 5) with a 2.4-fold increase in the apparent sensitivity of ACh. IbTX did not alter the maximal response or EC₅₀ for the ACh-induced serosal K⁺ current. Clotrimazole, a blocker for IK (KCNN4), inhibited ACh-induced serosal K⁺ current. Clotrimazole also reversed the effect of forskolin in increasing the apparent sensitivity of the ACh response and reduced the maximal ACh-induced current (Fig. 5). Forskolin-induced increases in K⁺ current were also blocked by clotrimazole but not by IbTX. Thus, IK instead of BK channels are the primary K⁺ channels activated during ACh-induced I_SC. Direct effects of PKA in enhancing IK (KCNN4) activities are controversial (von Hahn et al., 2001).

PGE₂ or forskolin alone did not induce increases in [Ca²⁺]_i and whole-cell K⁺ or Cl^– current (Fig. 7), consistent with the report that cAMP-elevating agonists alone did not induce significant increases in [Ca²⁺]_i and whole-cell currents in mucous cells (Shimura et al., 1994). PGE₂ induced membrane depolarization instead of hyperpolarization under current clamp (Fig. 8), possibly because PGE₂ altered electrogenic ion transport or slightly enhanced Cl^– or Na^– channel activities. These data indicated that cAMP or PKA does not greatly activate or enhance K⁺ channel activities in single mucous cells. The PGE₂ responses in Ussing chamber and patch-clamp studies differ due to the fact that the former study used a mixed cell population and that patch clamp was performed using only mucus-containing cells. The serous cells in the primary culture expressed the cAMP-responsive ion channels CFTR and cAMP-activated K⁺ channels. However, blocking both channels did not change the sensitization effect of PGE₂ (Liu et al., 2005). PGE₂-induced I_SC responses were also significantly reduced by 0 mM [Ca²⁺]_o, SKF96365, and 2-APB treatments (Figs. 3, 4, and 5), suggesting that normal Ca²⁺ influx is also important in maintaining ion channel activities (such as KCa) that are associated with I_SC response induced by intracellular cAMP elevation in serous cells.

Role of Ion Channels and [Ca²⁺]_i Changes. Ca²⁺ is important for mucin condensation and release (Verdugo, 1991). CaCC are reportedly correlated with mucin production (Toda et al., 2002) in addition to mediating anion secretion. However, K⁺ current dominates the response to ACh (Iwase et al., 2002), because mucous cells hyperpolarized in response to ACh close to the equilibrium potential for K⁺ (–80 mV; Fig. 8). Membrane hyperpolarization facilitates CaCC-dependent Cl^– secretion and also facilitates Ca²⁺ entry. Nonexcitable cells often lack voltage-dependent Ca²⁺ channels, and Ca²⁺ entry relies on a favorable electrochemical gradient (Penner and Fleig, 2004). K⁺ channel-mediated membrane hyperpolarization increases and sustains a favorable driving force for Ca²⁺, enhancing Ca²⁺ entry. Ca²⁺ entry is responsible for the prolonged muscarinic actions and is more efficient than Ca²⁺-release from internal stores in causing glycoconjugate secretion (Ishihara et al., 1990).

Target of PKA Actions. How does PKA enhance ACh-induced Ca²⁺ mobilization? In SMGC, ACh activates M₃ receptors and phospholipase C-catalyzed IP₃ release, resulting in elevation in [Ca²⁺]_i (Hall, 1992), activation of CaCC and K_Ca (Griffin et al., 1996), increases in I_SC (Liu and Farley, 2005), and glycoprotein secretion (Yang et al., 1991). PKA is known to phosphorylate IP₃ receptors, enhancing ACh-induced Ca²⁺ release from internal stores (Straub et al., 2004). Furthermore, enhanced Ca²⁺ entry may occur due to more efficient Ca²⁺ store depletion.

In summary, the elevated Ca²⁺ levels induced at lower ACh concentrations after PGE₂ pretreatment resulted in an increase in apparent sensitivity to ACh. Such mechanisms may be involved in the enhanced airway fluid and mucus secretion during inflammation. Mediators released during airway inflammation, such as PGE₂, play important roles as regulators of ACh-mediated airway fluid secretion and possibly mucin release. Ion channels such as IK and those responsible for Ca²⁺ entry are potential targets for the treatment of mucus hypersecretion.

	Acknowledgements

 
We thank Wen-Shou Chung for assistance during the experiments and Joe Ed Smith for building the micromanipulator mounting stage, chambers for the patch-clamp setup, and modifications to the Ussing chambers.

	Footnotes

 
This study was supported in part by a grant from the American Heart Association (to J.M.F.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.120154.

ABBREVIATIONS: SMGC, submucosal gland cells; 2-APB, 2-aminoethoxydiphenyl borate; ACh, acetylcholine; PGE₂, prostaglandin E₂; AH6809, 6-isopropoxy-9-oxoxanthene-2-carboxylic acid; ANOVA, analysis of variance; BK, large conductance, calcium-activated K⁺ channels; CaCC, Ca²⁺-activated Cl^– channels; CFTR, cystic fibrosis transmembrane conductance regulator; ChTX, charybdotoxin; DMSO, dimethyl sulfoxide; EP, endoprostanoid; IbTX, iberiotoxin; IK, intermediate conductance calcium-activated K⁺ channel; I_SC, short circuit current; PG, prostaglandin; PKA, cAMP-dependent protein kinase A; PKI, PKA inhibitor, 14-22 amide; SC19220, 8-chloro-dibenzo[b,f][1,4]oxazepine-10(11H)-carboxylic acid, 2-acetylhydrazide; IP₃, inositol 1,4,5-trisphosphate; SKF96365, 1-[-(3-(4-methoxyphenyl) propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl] imidazole; VIP, vasoactive intestinal peptide; Rp, Rp-diastereomer; EGF, epidermal growth factor; AG1478, 4-(3-chloroanillino)-6,7-dimethoxyquinazoline.

Address correspondence to: Dr. Jerry M. Farley Sr., Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4624. E-mail: jfarley{at}pharmacology.umsmed.edu

	References

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