Under conditions of high dietary salt intake, prostaglandin E2 (PGE2) production is increased in the collecting duct and promotes urinary sodium chloride (NaCl) excretion; however, the molecular mechanisms by which PGE2 increases NaCl excretion in this context have not been clearly defined. We used the mouse inner medullary collecting duct (mIMCD)-K2 cell line to characterize mechanisms underlying PGE2-regulated NaCl transport. When epithelial Na+ channels were inhibited, PGE2 exclusively stimulated basolateral EP4 receptors to increase short-circuit current (IscPGE2). We found that IscPGE2 was sensitive to inhibition by H-89 and CFTR-172, indicating that EP4 receptors signal through protein kinase A to induce Cl− secretion via cystic fibrosis transmembrane conductance regulator (CFTR). Unexpectedly, we also found that IscPGE2 was sensitive to inhibition by BAPTA-AM (Ca2+ chelator), 2-aminoethoxydiphenyl borate (2-APB) (inositol triphosphate receptor blocker), and flufenamic acid (FFA) [Ca2+-activated Cl− channel (CACC) inhibitor], suggesting that EP4 receptors also signal through Ca2+ to induce Cl− secretion via CACC. Additionally, we observed that PGE2 stimulated an increase in Isc through crosstalk between cAMP and Ca2+ signaling; BAPTA-AM or 2-APB inhibited a component of IscPGE2 that was sensitive to CFTR-172 inhibition; H-89 inhibited a component of IscPGE2 that was sensitive to FFA inhibition. Together, our findings indicate that PGE2 activates basolateral EP4 receptors and signals through both cAMP and Ca2+ to stimulate Cl− secretion in IMCD-K2 cells. We propose that these signaling pathways, and the crosstalk between them, may provide a concerted mechanism for enhancing urinary NaCl excretion under conditions of high dietary NaCl intake.
- prostaglandin E2
- EP4 receptor
- collecting duct
- cystic fibrosis transmembrane conductance regulator
- Ca2+-activated Cl− channel
the collecting duct of the kidney maintains total body sodium chloride (NaCl) balance by fine-tuning urinary NaCl excretion in response to changes in dietary salt intake. Local changes in the hormonal milieu of the collecting duct play a critical role in this response mechanism. For example, high salt feeding of mice induces renal medullary production of prostaglandin E2 (PGE2) (12, 14, 23), which could operate as a paracrine hormone to promote urinary NaCl excretion. Targeted deletion of microsomal prostaglandin E synthase (mPGES-1) in mice disrupts renal PGE2 production and induces salt-sensitive hypertension, indicating that PGE2 signaling contributes to NaCl balance and blood pressure regulation (13, 29).
The molecular mechanisms through which PGE2 increases urinary NaCl excretion, in the context of high dietary salt intake, have not been completely defined. PGE2 activates four subtypes of prostanoid receptors: EP1, EP2, EP3, and EP4 (9). EP2 and EP4 receptors are classic Gαs-coupled receptors, which increase intracellular cyclic AMP (cAMP) concentration, whereas EP1 receptors are Gq-coupled receptors, which increase intracellular calcium (Ca2+) concentration through an inositol triphosphate (IP3)-dependent pathway (9, 28). EP3 receptors have multiple splice variants, which, depending on the interacting G protein, activate either Gαs, Gi, or Gq proteins (9).
PGE2 can stimulate various prostanoid receptors to increase Cl− secretion in the collecting duct (12, 16, 57, 68). The physiological significance of Cl− secretion in the collecting duct may be most important under salt-loading conditions when extracellular fluid volume expansion leads to low levels of plasma aldosterone concentration and, as a consequence, diminished Na+ reabsorption through the epithelial sodium channel (ENaC) (32, 40, 41, 43, 56). Under these circumstances, paracrine hormones, such as ATP, adenosine, and possibly PGE2, are synthesized and released by kidney epithelial cells and stimulate respective signaling pathways that promote Cl− secretion (12, 50, 60). Notably, the initial segment of the inner medullary collecting duct (IMCD) is the only nephron segment that has been shown to secrete Cl− under salt-loading conditions (22, 62, 69).
We used the mIMCD-K2 cell line as a model system to study the direct effects of PGE2 on NaCl transport across IMCD epithelia under conditions of diminished ENaC activity. The mIMCD-K2 cell line was derived from the initial segment of the IMCD and retains characteristic signaling and ion transport pathways of the IMCD (6, 7, 33, 66). We demonstrate here that PGE2 activates basolateral EP4 receptors to increase Cl− secretion through cystic fibrosis transmembrane conductance regulator (CFTR). Although EP4 receptors are linked to Gαs and cAMP signaling, we found that EP4 receptor activation also leads to stimulation of Cl− secretion through calcium-activated Cl− channels (CACC). Moreover, we found that PGE2-stimulated Cl− secretion involves crosstalk between cAMP and Ca2+-signaling pathways, in which CFTR-mediated short-circuit current (Isc) is sensitive to inhibition by BAPTA-AM (Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB) (IP3 receptor blocker) and CACC-mediated Isc is sensitive to inhibition by H-89 [protein kinase A (PKA) inhibitor]. These findings indicate that PGE2 acts through a cAMP and Ca2+-signaling network to stimulate Cl− secretion in IMCD cells, which may serve as an adaptive response to enhance urinary NaCl excretion under conditions of high dietary NaCl intake.
MATERIALS AND METHODS
The mIMCD-K2 cell line was kindly provided by Dr. Bruce Stanton (Dartmouth Medical School, Hanover, NH). Cells of passages 38–46 were expanded and plated on polycarbonate Snapwell inserts (surface area of 1.12 cm2, Corning Costar, Corning, NY), as described previously (55). Transepithelial voltage (Vte) and resistance (Rte) were measured with an epithelial voltohmmeter “chopstick” voltmeter (World Precision Instruments, Sarasota, FL). Cells were grown in defined medium until Rte reached values greater than 800 Ω/cm2.
Ussing chamber measurements.
Cell sheets were mounted between the Lucite half chambers of the Ussing chamber apparatus (Physiologic Instruments, San Diego, CA) for electrophysiological studies, as described previously (52, 55). Cell sheets were bathed in Krebs-Henseleit solution (in mM: 140 NaCl, 25 NaHCO3, 5 KCl, 5 glucose, 2 CaCl2, and 1 MgCl2) and gassed with a mixture of 95% O2 and 5% CO2. In ion-substitution experiments, Cl− in the Krebs-Henseleit was replaced with glutamate and sulfate, and HCO3− was replaced with HEPES to identify the ions responsible for PGE2-stimulated Isc (IscPGE2). Vte across cell sheets was clamped to 0 mV, and a set voltage pulse of 1 mV was applied across cell sheets for 200 ms every 20 s. The Isc and Rte across cell sheets were continuously recorded using Acquire and Analyze Software (Physiologic Instruments). Electrophysiological responses were characterized in cells from at least three different passages to account for interpassage variability.
Once Isc and Rte across cell sheets stabilized in the Ussing chamber, typically within 2–4 min of mounting, a series of pharmacological agents was added to cell sheets. In these experiments, no washout step was included in between addition of each agent. The apical side of all cell sheets was treated with the ENaC inhibitor amiloride (10−5 M; Sigma, St. Louis, MO). PGE2 (7.7 × 10−8 M) was then added sequentially to the apical and basal sides of cell sheets or vice versa. In some experiments, the EP1/EP3 receptor agonist sulprostone (10−6 M; Tocris Bioscience, Bristol, UK), the EP2 receptor agonist butaprost (10−5 M, Sigma), or the EP4 agonist TCS2510 (10−6 M, Tocris Bioscience) was added to the apical or basal side of cell sheets. In other experiments, the EP1/EP2 receptor antagonist AH-6809 (10−6 M; Cayman Chemicals, Ann Arbor, MI), the EP1/EP3 receptor antagonist ONO-8713 (10−6 M, Cayman Chemicals), or the EP4 receptor antagonist L-161,982 (10−6 M, Tocris Bioscience) was added to cell sheets before PGE2 stimulation. Bromocresol green (3 × 10−5 M, Sigma) was used as a prostaglandin transport inhibitor to confirm transport of PGE2 across cell sheets.
To identify the classes of ion channels or transporters that respond to EP4 receptor activation, several small molecule inhibitors were used. CFTR inhibitor-172 (CFTR-172, 10−5 M, Tocris Bioscience) or flufenamic acid (FFA, 2 × 10−4 M, Sigma) was added to the apical side of cell sheets to block CFTR (39) or CACC (19, 71), respectively. Bumetanide (2 × 10−4 M, Sigma), diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (10−4 M, Sigma), or acetazolamide (2 × 10−4 M, Sigma) was added to the basal side of cell sheets to inhibit Na+/K+/2 Cl− cotransporters or carbonic anhydrase, respectively.
To determine whether PGE2-stimulated Isc (IscPGE2) was dependent on PKA, phospholipase C, PI3-kinase, or protein kinase C, the following respective compounds were added to cell sheets: H-89 (10−5 M, Sigma), U73122 (10−6 M, Tocris Bioscience), LY294002 (2.5 × 10−5 M, Sigma), or Go6983 (2 × 10−5 M, Tocris Bioscience). To determine whether IscPGE2 was dependent on intracellular Ca2+ or IP3 receptor activation on IscPGE2, the cell-permeable Ca2+ chelator BAPTA-AM (5 × 10−5 M, Tocris Bioscience) or the IP3 receptor blocker 2-APB (10−4 M, Tocris Bioscience) was added to both sides of cell sheets, respectively.
To characterize involvement of cAMP in stimulating FFA-sensitive Isc, forskolin (10−5 M, Tocris Bioscience) was added to the apical side of cell sheets before the addition of either CFTR-172 or FFA. In other experiments, BAPTA-AM was added to forskolin-stimulated cell sheets to probe the role of intracellular Ca2+ in regulating CFTR-mediated Isc.
mIMCD-K2 cells were grown to resistance on permeable supports, and total RNA was harvested using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer instructions. Total RNA from mouse kidney was obtained from Zyagen (San Diego, CA) to serve as positive control. Reverse transcriptase (RT) reactions were performed according to manufacturer instructions (New England BioLaboratories, Ipswich, MA). Thermal cycling parameters were the following: incubation at 98°C for 30 s followed by 35 cycles at 98°C for 10 s, 58°C for 10 s, 72°C for 50 s, and then a final extension at 72°C for 5 min. PCR primers were designed and used for detecting gene amplification of EP1, EP2, EP3, and EP4 receptors. The sequences were as follows: mouse EP1 Forward: 5′-AGCCTGCTTGCCATCGACCTA-3′, mouse EP1 Reverse: 5′-GCCAGGGTACTGTAACTCGTAGCGAC-3′; mouse EP2 Forward: 5′-AGGACTTCGATGGCAGAGGAGACGGACCACCTCATTCTCC-3′, mouse EP2 Reverse: 5′-CAGCCCCTTACACTTCTCCAATGAGGCCATTTAAAGACTT-3′; mouse EP3 Forward: 5′-CCAGCCACATGAAGACTCGCG-3′, mouse EP3 Reverse: 5′-TCATTATCAATAGCGGCGACCAA-3′; mouse EP4 Forward: 5′-CCATCGTAGTATTGTGCAAGTCGCG-3′, mouse EP4 Reverse: 5′-AAGGAGCTGAAGCCGGCGTAC-3′. Specificity of each set of primers was confirmed by BLAST search against GenBank and by direct sequencing of PCR products. PCR products were resolved using a 1% agarose gel dissolved Tris-Acetate EDTA buffer and visualized with ethidium bromide.
Statistical analyses for comparisons between different treatment groups of mIMCD-K2 cells were performed using paired or unpaired two-tailed Student's t-tests. Differences were considered significant at P < 0.05.
PGE2 activates basolateral EP4 receptors to induce Cl− secretion.
Amiloride was first added to the apical side of mIMCD-K2 cells to study the effects of PGE2 on Cl− transport in the absence of ENaC activity. The addition of amiloride induced no significant changes to Isc; we next added PGE2 (7.7 × 10−8 M PGE2) to either side of mIMCD-K2 cells and examined the Isc response. We chose a concentration of 7.7 × 10−8 M for PGE2 because this concentration has previously been used in IMCD cells to stimulate cAMP signaling (16, 38). Addition of PGE2 to either side of cell sheets induced an initial transient followed by a sustained increase in Isc (Fig. 1A).
We next generated a cumulative dose-response curve of the Isc response to basal addition of PGE2. We found that 10−8 M was the smallest dose of PGE2 that increased Isc. Sequential addition of 10−7 M and 10−6 M of PGE2 progressively increased Isc; addition of higher concentrations of PGE2, however, did not further increase Isc (Fig. 1B). We measured the sustained increase in IscPGE2 and plotted these values against the logarithm of PGE2 concentration (Fig. 1C). The EC50 of the PGE2 Isc response was 3.1 ± 0.2 × 10−8 M.
We observed that PGE2 induced a different Isc response depending on which side PGE2 is added. Addition of PGE2 to the basal side elicited a maximal IscPGE2 response, so addition of PGE2 to the apical side did not further stimulate Isc (Fig. 2A, dashed line; Fig. 2B). On the other hand, addition of PGE2 to the apical side elicited a partial IscPGE2 response, which could be further enhanced with addition of PGE2 to the basal side (Fig. 2A, solid line; Fig. 2C). We also found that addition of PGE2 to either side of cells increased transepithelial conductance, a response that closely mirrored the increases in Isc (Fig. 2, D and E). These findings suggest that a class of prostanoid receptors is preferentially localized to the basal side of mIMCD-K2 cells to initiate the IscPGE2 response.
To identify which class of prostanoid receptors is responsible for IscPGE2, we stimulated mIMCD-K2 cells with a series of EP receptor agonists and examined the Isc response. Apical addition of the EP4 agonist TCS-2510 (10−6 M) induced no change in Isc (Fig. 3, A and B), whereas basal addition of TCS-2510 (10−6 M) induced an increase in Isc (Fig. 3, A and C), which closely mirrored and accounted for IscPGE2. Addition of EP1/EP3 receptor agonist sulprostone (10−6 M) and the EP2 receptor agonist butaprost (10−5 M) to either side of cell sheets induced no change in Isc (Fig. 3, B and C).
As a complementary approach for identifying the EP receptors responsible for IscPGE2, we also pretreated mIMCD-K2 cells with a panel of EP receptor antagonists and then induced with PGE2. Addition of maximal doses of the nonselective EP1/EP2 receptor antagonist AH-6809 (10−6 M) or the EP1/EP3 receptor antagonist ONO-8713 (10−6 M) to both sides of cells induced no attenuation of IscPGE2 (data not shown). On the other hand, pretreatment of cells with the specific EP4 receptor antagonist L-161,982 (10−7 M, added to both sides of cell sheets) completely blocked IscPGE2 (Fig. 4), as well as TCS-2510-stimulated Isc (data not shown), indicating that EP4 receptor activation is responsible for IscPGE2 in mIMCD-K2 cells.
We observed that L-161,982 produced a different inhibitory response depending on which side L-161,982 was added. When L-161,982 (10−7 M) was added to the apical bath before basal addition of PGE2, the Isc response was partly inhibited (Fig. 4C, solid line); in contrast, addition of L-161,982 to the basal bath before addition of PGE2 almost completely inhibited the Isc response (Fig. 4C, dashed line). When L-161,982 was used at a lower concentration and added after PGE2 treatment, the sidedness of the inhibitory response became more apparent. Apical addition of L-161,982 (2.0 × 10−8 M) to PGE2-stimulated cells did not decrease Isc at 1 min (Fig. 5A, dashed line; Fig. 5B, open bars); basal addition of L-161,982 at the same concentration inhibited Isc (Fig. 5A, solid line; Fig. 5B, solid bars). Moreover, the latency of Isc inhibition was different depending on which side L-161,982 was added. When L-161,982 was added to the apical side of cells, the lag time for inhibition of IscPGE2 was 75 ± 5 s, whereas it was 26.0 ± 2.0 s when L-161,982 was added to the basal side. These findings again suggest that EP4 receptors are localized strictly to the basal membrane.
Although we observed a sidedness to the latency period for inhibition of IscPGE2 by L-161,982, we observed no significant difference in sidedness to the latency period for PGE2-stimulated Isc. The lag time for Isc stimulation was 33 ± 4 s and 28 ± 2 s when PGE2 was added to the apical and basal side of cells, respectively. We hypothesized that addition of PGE2 to the apical side increases Isc because it is rapidly transported across mIMCD-K2 cell sheets to stimulate EP4 receptors present in the basolateral membrane. To test this, we incubated mIMCD-K2 cells with bromocresol green (3.0 × 10−5 M), an inhibitor of the prostaglandin transporter (5, 25, 31, 51), and then added PGE2 to either side of cell sheets. We found that pretreatment of mIMCD-K2 cells with bromocresol green significantly inhibited IscPGE2 when PGE2 was added to the apical [Fig. 6A, dashed line, PGE2(a); Fig. 6B], but not basal, side of cell sheets [Fig. 6A, dashed line, PGE2(b); Fig. 6C]. These findings suggest the following: 1) PGE2 activates EP4 receptors localized to the basolateral membrane; 2) apical addition of PGE2 elicits an Isc response because a prostaglandin transporter transports PGE2 to the basolateral membrane; and 3) unlike PGE2, the EP4 receptor agonist TCS2510 does not appear to be a substrate for the prostaglandin transporter.
We next used RT-PCR to evaluate EP4 receptor mRNA expression in mIMCD-K2 cells. Mouse kidney cDNA were run in parallel as a positive control. We detected all four classes of EP receptors in mouse kidney homogenates. We also found that EP4 receptors, as well as EP1 and EP2 receptors, are expressed in mIMCD-K2 cells (Fig. 7).
PGE2 stimulates Cl− secretion through CFTR and CACC.
Because ENaC activity was blocked with addition of amiloride to the apical side of cell sheets in all experiments, we concluded that IscPGE2 is likely caused by stimulation of electrogenic anion secretion. To verify that PGE2 stimulates Cl− secretion in mIMCD-K2 cells, we performed similar experiments in a Cl−-free, HEPES-buffered Krebs-Henseleit solution. As expected, addition of PGE2 to mIMCD-K2 cells in this bath solution induced no increase in Isc (Fig. 8). We and others have identified CFTR and CACC as being candidate Cl− channels that mediate apical Cl− secretion in mIMCD-K2 cells (6, 53–55, 66, 67). To identify the apical ion channels mediating Cl− secretion in the IscPGE2 response, we treated PGE2-stimulated cells with CFTR inhibitor-172 (CFTR-172) and FFA. Apical addition of CFTR-172 (10−5 M) attenuated IscPGE2 by 60.1 ± 2.7% (Fig. 9, A and B); surprisingly, apical addition of FFA (2 × 10−4 M), following CFTR-172 addition, also reduced IscPGE2 by 25.7 ± 1.7% (Fig. 9, A and B). Addition of CFTR-172 and FFA also similarly inhibited transepithelial conductance (Fig. 9C). Moreover, the inhibitory effects of CFTR-172 and FFA on IscPGE2 were similar regardless of the order in which they were added to cell sheets (Fig. 9D), suggesting that the inhibitory effects of these compounds on IscPGE2 do not overlap. Because PGE2 activates EP4 receptors, which are linked to the Gαs/adenylate cyclase pathway, the possible involvement of CACC in the IscPGE2 response was unexpected.
We next evaluated the transport pathways responsible for basolateral Cl− uptake in the IscPGE2 response. We first treated PGE2-stimulated cells with bumetanide (Na+/K+/2 Cl− cotransporter inhibitor) and DIDS (a nonselective anion transport inhibitor). Basal addition of bumetanide decreased IscPGE2 by 41.8 ± 3.7%; basal addition of DIDS further decreased IscPGE2 by 49.1 ± 3.2% (Fig. 10). To further characterize the transport pathway inhibited by DIDS, we next added acetazolamide (a carbonic anhydrase inhibitor) after bumetanide to PGE2-stimulated cells. In these experiments, basal addition of bumetanide decreased IscPGE2 by 39.1 ± 2.5% (Fig. 11, A and C); basal addition of acetazolamide (2.0 × 10−4 M) further decreased IscPGE2 by 32.4 ± 3.6% (Fig. 11, B and C). These findings suggest that both Na+/K+/2 Cl− cotransporters and Cl−/HCO3− exchangers mediate basolateral Cl− uptake during the IscPGE2 response, similar to mechanisms underlying basolateral Cl− uptake in primary human IMCD cells (68).
PGE2 stimulates PKA and Ca2+-signaling pathways.
Because EP4 receptors classically couple to the Gαs subunit to stimulate adenylate cyclase and PKA activity, we next evaluated whether PKA activity is required for IscPGE2. We incubated mIMCD-K2 cells with the PKA inhibitor H-89 (10−5 M) before addition of PGE2. Pretreatment of cells with H-89 inhibited the transient phase of IscPGE2 by 69.2 ± 5.8%: vehicle-treated cells demonstrated a transient IscPGE2 response of 18.0 ± 2.4 μA/cm2, whereas H-89 treated cells demonstrate a much smaller transient IscPGE2 response of 5.0 ± 1 μA/cm2. Similarly, H-89 attenuated the sustained phase of IscPGE2 by 62.7 ± 4.4%: vehicle-treated cells showed a sustained IscPGE2 response of 8.2 ± 0.9 μA/cm2, whereas H-89-treated cells showed a sustained IscPGE2 response of only 3.4 ± 0.5 μA/cm2 (Fig. 12B). In contrast, addition of the PI3-kinase inhibitor LY-294002 (5 × 10−5 M), the phospholipase C inhibitor U73122 (10−6 M), or the protein kinase C inhibitor Go6983 (2 × 10−5 M) before PGE2 stimulation induced no change in IscPGE2 (Fig. 13).
Because FFA, an inhibitor of CACC, could attenuate IscPGE2, we next tested whether a Ca2+-signaling pathway regulates a component of IscPGE2. We used the cell-permeable Ca2+ chelator BAPTA-AM to decrease intracellular Ca2+ concentration in mIMCD-K2 cells. Addition of BAPTA-AM (5 × 10−5 M) diminished IscPGE2 from 8.86 ± 0.72 μA/cm2 to 4.01 ± 0.56 μA/cm2, a decrease of 60.6 ± 5.6% (Fig. 14, A and B). Addition of FFA to cells had no effect on Isc PGE2 following BAPTA-AM treatment (Fig. 14D).
Crosstalk exists between cAMP and Ca2+-signaling pathways.
In the course of our experiments, we observed that a PKA-dependent pathway controls a component of IscPGE2 that is sensitive to inhibition by FFA. In paired studies, addition of FFA to PGE2-stimulated cells led to a decrease in Isc of 1.8 ± 0.2 μA/cm2 (Fig. 12C); in comparison, addition of FFA to PGE2-stimulated cells, pretreated with H-89, led to a decrease in Isc of only 0.75 ± 0.2 μA/cm2 (Fig. 12C). These findings suggest that inhibiting PKA inhibits, not only CFTR-mediated IscPGE2, but also CACC-mediated IscPGE2 and indicate that Ca2+ signaling may be downstream of PKA in mIMCD-K2 cells.
We also observed that a Ca2+-dependent pathway controls a component of IscPGE2 that is sensitive to inhibition by CFTR-172. Addition of CFTR-172 to PGE2-stimulated cells led to a decrease in Isc of 6.67 ± 0.53 μA/cm2, which represented 64.2 ± 3.7% of IscPGE2 (Fig. 14, C and D); in comparison, addition of CFTR-172 to PGE2-stimulated cells, pretreated with BAPTA-AM, led to a decrease in Isc of only 2.39 ± 0.41 μA/cm2, which represented 26.9 ± 3.5% of IscPGE2 (Fig. 14, C and D). From these experiments, we conclude that an increase in intracellular Ca2+ is required for maximal PGE2-stimulated CFTR activity.
Several studies have demonstrated that PKA can induce release of Ca2+ from intracellular stores by phosphorylating IP3 receptors (18, 63). We therefore tested whether mobilization of Ca2+ from intracellular stores contributes to IscPGE2 by using 2-APB to inhibit IP3 receptor activation in mIMCD-K2 cells. Addition of 2-APB (10−4 M) significantly diminished IscPGE2 from 8.8 ± 0.7 μA/cm2 to 2.6 ± 0.7 μA/cm2, a decrease of 78.5 ± 4.4% (Fig. 15B); addition of FFA to 2-APB-treated cells induced no further inhibition of IscPGE2 (Fig. 15, C and D). Because the FFA-sensitive component of IscPGE2 is completely blocked by 2-APB, we conclude that PGE2 signals through IP3 receptors to activate CACC. Similar to the effect of BAPTA-AM on the CFTR-172-sensitive component of IscPGE2, addition of CFTR-172 to PGE2-stimulated cells, pretreated with 2-APB, led to a decrease in Isc of 1.05 ± 0.14 μA/cm2, which represented only 13.84 ± 1.2% of IscPGE2 (Fig. 15, C and D). This finding also confirms that intracellular Ca2+ is required for maximal PGE2-stimulated CFTR activity.
We also observed that the magnitude of inhibition of IscPGE2 after BAPTA-AM or 2-APB is much larger than the magnitude of inhibition of IscPGE2 by FFA treatment. Addition of BAPTA-AM or 2-APB to PGE2-stimulated mIMCD-K2 cells inhibited IscPGE2 by 60.6 ± 5.6% (Fig. 14B) or 78.5 ± 4.5% (Fig. 15B), respectively. In contrast, addition of FFA to paired PGE2-stimulated cells treated with vehicle decreased IscPGE2 by only 34.0 ± 2.9% (Figs. 14 and 15). The discrepancy between the magnitude of inhibition of IscPGE2 with BAPTA-AM or 2-APB with that after FFA treatment further suggests that BAPTA-AM or 2-APB may inhibit, not only CACC, but also CFTR activity in PGE2-stimulated mIMCD-K2 cells.
To evaluate whether direct stimulation of adenylate cyclase activity would also stimulate Ca2+ signaling in mIMCD-K2 cells, we treated mIMCD-K2 cells with forskolin and examined the Isc response. Addition of forskolin (10−5 M) to the apical side of cell sheets induced a large increase in Isc, which was inhibited by FFA (Fig. 16A, solid line; Fig. 16B) or BAPTA-AM (Fig. 16A, dashed line; Fig. 16B), indicating that an increase in cAMP signaling, independent of PGE2 action, can also stimulate CACC activity. Addition of BAPTA-AM to cells inhibited forskolin-stimulated Isc to a greater extent than the addition of FFA (Fig. 16B), suggesting again that intracellular Ca2+ stimulates, not only CACC, but also a component of CFTR activity.
To rule out the possibility that CFTR activity itself might regulate CACC, we incubated mIMCD-K2 cells with CFTR-172 before addition of PGE2. We found that PGE2 could still induce an increase in Isc, which was sensitive only to FFA inhibition (Fig. 17A, solid line). When cells were incubated sequentially with CFTR-172, PGE2, and FFA, we observed no further inhibition of Isc with CFTR-172 treatment. Conversely, if cells were first incubated with FFA before addition of PGE2, PGE2 could still induce an increase in Isc (Fig. 17A, dashed line), which was almost completely inhibited by the subsequent addition of CFTR-172. These findings indicate that, although there is significant crosstalk between cAMP and Ca2+ signaling in stimulating IscPGE2, CFTR-172-sensitive and FFA-sensitive Cl− channel activities are distinct and can be regulated independent of one another. To further test whether FFA and CFTR-172 inhibit distinct Cl− transport pathways, we used genistein, a tyrosine kinase inhibitor, to activate CFTR in a manner that is independent of Ca2+ or cAMP-PKA signaling (1, 20, 26). Addition of FFA induced no decrease in genistein-stimulated Isc, but addition of CFTR-172 completely inhibited genistein-stimulated Isc (Fig. 18). This finding provides additional support that FFA and CFTR-172 can be used to probe distinct Cl− transport pathways in mIMCD-K2 cells.
In this study, we used the mIMCD-K2 cell line to examine direct effects of PGE2 on NaCl transport across IMCD epithelium. Our data demonstrate that PGE2 exclusively activates basolateral EP4 receptors to increase Cl− secretion (Fig. 18). The following findings support this conclusion. First, addition of PGE2 to the basal side of mIMCD-K2 cells elicited a maximal IscPGE2 response; in contrast, addition of PGE2 to the apical side elicited a partial IscPGE2 response, which could be further enhanced with basal addition of PGE2 (Fig. 2). Second, basal, but not apical, addition of the EP4 receptor agonist TCS-2510 induced an increase in Isc, which closely mirrored the IscPGE2 response (Fig. 3). Third, addition of the EP4 receptor antagonist L-161,982 completely blocked IscPGE2 (Fig. 4), whereas addition of other classes of EP receptor antagonists had no effect on IscPGE2. The magnitude and speed of this inhibition differed depending on whether L-161,982 was added to the apical or basal side of cells. Apical addition of L-161,982 led to partial inhibition of IscPGE2, whereas basal addition of L-161,982 led to complete and almost instantaneous inhibition of IscPGE2 (Fig. 5), suggesting that EP4 receptors are expressed on the basolateral side of mIMCD-K2 cells. Fourth, addition of a prostaglandin transporter inhibitor significantly inhibited IscPGE2 but only when PGE2 was added to the apical side of mIMCD-K2 cells (Fig. 6).
If our findings in mIMCD-K2 cells can be extrapolated to the in vivo setting, we suggest that PGE2 could arrive at the basolateral membrane of the IMCD from either the medullary interstitium or the collecting duct, which is known to synthesize and release PGE2 into the urinary space (8, 10, 27, 61). Although it is apparent how PGE2 could directly access basolateral EP4 receptors from the medullary interstitium, PGE2 must take a more circuitous route to reach the basolateral membrane from the urinary space. The prostaglandin transporter (PGT) is expressed in the apical or subapical membrane of the collecting duct (2, 3, 30, 45) and mediates PGE2 uptake from the urinary space via an anion exchange mechanism, exchanging lactate for PGE2 (11). In a Madin-Darby canine kidney cell model system, PGT extracts PGE2 from the apical solution and transports it across the epithelium to the basolateral solution (17). Although the molecular details for how PGE2 traverses the kidney cell are not yet clear, PGE2 may reach the exterior of the basolateral membrane through the organic anion transporter 3, which is expressed on the basolateral surface of renal tubules (44, 58). We infer from these and our own experiments that PGT carries PGE2 from the apical to basal side of IMCD cells, where it activates EP4 receptors in an autocrine or paracrine fashion (Fig. 18). It is noteworthy that high dietary salt intake induces transcription of PGT gene in the mouse renal collecting duct, which may represent an adaptive mechanism for increasing delivery of PGE2 to the basolateral membrane of IMCD cells to stimulate EP4 receptors (14).
Studies with EP4 receptor knockout mice and EP4 receptor-selective agonists/antagonists have demonstrated that EP4 receptors play a critical role in numerous physiological functions, including promoting urinary salt and water excretion. EP4 receptors are expressed in rat IMCD cells (65) and isolated mouse IMCD tubules (35). Moreover, addition of EP4 receptor agonists increase intracellular cAMP concentration in freshly isolated IMCD tubules, indicating that EP4 receptor signaling is intact in isolated IMCD tubules (35). In a hyperprostaglandin E/antenatal Bartter syndrome model in which mice are treated chronically with furosemide, genetic deletion or pharmacological inhibition of EP4 receptors results in a blunted diuretic and natriuretic response, which is associated with an attenuation of the expected rise in plasma renin concentration. The blunted diuretic/natriuretic response in EP4 knockout mice is not accompanied by a change in glomerular filtration (46); these findings are consistent with the notion that PGE2 stimulates EP4 receptors in IMCD cells to increase tubular NaCl secretion.
In a similar study examining PGE2 effects on Cl− secretion, Sandrasagra and colleagues (57) found that, in the presence of amiloride, addition of PGE2 to either side of M1 cortical collecting duct cells stimulates diphenylamine-2-carboxylic acid-sensitive Cl− secretion. In our study with mIMCD-K2 cells, we conclude that PGE2, by activating basolateral EP4 receptors, increases Cl− secretion by stimulating basolateral Cl− entry through Cl−/HCO3− exchange and NKCC transport and apical Cl− exit through CACC and CFTR. Moreover, our experiments demonstrate that PGE2 stimulates Cl− secretion through crosstalk between cAMP and Ca2+ signaling pathways in which cAMP and intracellular Ca2+ also regulate CACC and CFTR activities, respectively (Fig. 18). To our knowledge, this is the first demonstration of crosstalk between these two signaling pathways in kidney epithelial cells.
Several lines of evidence support the existence of crosstalk between cAMP and Ca2+-signaling pathways. First, FFA did not inhibit IscPGE2 to the same extent as BAPTA-AM or 2-APB: the magnitude of inhibition of IscPGE2 after BAPTA-AM or 2-APB was much larger than the magnitude of inhibition of IscPGE2 after FFA treatment (Figs. 14 and 15). This discrepancy suggests that BAPTA-AM or 2-APB may inhibit, not only CACC, but also CFTR activity in PGE2-stimulated mIMCD-K2 cells. Second, maneuvers that decrease intracellular Ca2+ concentration (BAPTA-AM or 2-APB treatment) significantly decreased CFTR-172-sensitive IscPGE2 in mIMCD-K2 cells (Figs. 14 and 15). Because CFTR is classically linked to PKA signaling, the sensitivity of CFTR activity to chelation of intracellular Ca2+ suggests that a component of CFTR activity responds to Ca2+ signaling. This finding adds to growing evidence in other epithelia that Ca2+-mobilizing secretagogues can activate CFTR (4, 15, 42, 64). Third, the PKA inhibitor H-89 significantly blocked FFA-sensitive IscPGE2 (Fig. 12). The sensitivity of CACC activity to H-89 inhibition suggests that PKA, in addition to regulating CFTR, may also regulate CACC activity. Fourth, PGE2 could stimulate FFA-sensitive IscPGE2, even after maximal CFTR-172 inhibition (Fig. 17). Additionally, CFTR-172, but not FFA, decreased genistein-stimulated Isc. Genistein activates CFTR independent of Ca2+ or cAMP/PKA signaling; the last two findings indicate that FFA and CFTR-172 can be used to probe distinct Cl− transport pathways and that activity of CFTR or CACC does not directly regulate the activity of the other channel in mIMCD-K2 cells.
We used a wide array of small molecule inhibitors to characterize the activities of ion transporters and channels and their signaling intermediates in the IscPGE2 response. Our findings should be interpreted with some caution because small molecule inhibitors may not always specifically inhibit their intended targets. For example, we tried to confirm the inhibitory effects of H-89 with myristolated PKA inhibitor (mPKI), a more specific inhibitor of PKA, but we were unable to recapitulate the inhibitory effects of H-89 on IscPGE2 with low doses of mPKI (data not shown). In another example, the inhibitory effects of BAPTA-AM on IscPGE2 occurred over a period of 20 min, which we suggest is consistent with Ca2+ chelation and gradual inhibition of CACC activity, but we cannot rule out the possibility that BAPTA-AM inhibits other pathways regulating CACC activity. In a third example, FFA may inhibit other Cl− transport pathways in mIMCD-K2 cells. During the preparation of this manuscript, small molecule compounds that are potentially more specific for CACC became available through Tocris Bioscience. These new inhibitors may prove useful in characterizing IscPGE2 and confirming CACC activity in mIMCD-K2 cells. To address some of these limitations, we used agonists or antagonists targeting different components of signaling and transport pathways, and the aggregate findings provide a coherent set of mechanisms for the IscPGE2 response in mIMCD-K2 cells. Future studies using new biochemical and genetic approaches to modulate these signaling and transport pathways will ultimately be required to provide a more complete understanding of mechanisms underlying PGE2 action in these cells.
The role of cAMP/PKA signaling in regulating CACC is currently not clear. Several studies have shown that inhibiting components of the cAMP/PKA signaling pathway, including CFTR itself, can decrease CACC-mediated Cl− transport (47–49, 70). We observed that inhibition of PKA significantly decreased, not only the CFTR-172-sensitive component of IscPGE2, but also the FFA-sensitive component, suggesting that PKA activation potentiates Ca2+-activated Cl− secretion in mIMCD-K2 cells. In contrast, we observed no change in the FFA-sensitive component of IscPGE2 when mIMCD-K2 cells were pretreated with CFTR inhibitor-172, indicating that CFTR does not simply regulate the activity of CACC. The mechanisms by which cAMP/PKA signaling potentiates CACC remain to be characterized, but we suggest one mechanism in which PGE2 induces PKA-mediated phosphorylation of IP3 receptors, which may sensitize them to basal levels of IP3 (24, 63). This could lead to an augmented IP3 receptor response and provide a mechanism through which PGE2 activates PKA and subsequently contributes to Ca2+ signaling (Fig. 18).
The role of Ca2+ signaling in regulating CFTR is also not entirely clear. In bronchial serous acinar cells, vasoactive intestinal polypeptide stimulates CFTR activity in part by increasing intracellular Ca2+ concentration through release of Ca2+ from IP3-sensitive Ca2+ stores. Foskett and colleagues (34) have proposed that Ca2+-dependent CFTR-mediated Cl− secretion involves activation of basolateral K+ channels, which mediates K+ efflux, stimulates Cl− influx via NKCC1 at the basolateral membrane, and drives eventual Cl− secretion via CFTR at the apical membrane (34). Similar to serous acinar cells, mIMCD-K2 cells also express basolateral K+ channels that participate in basolateral Cl− uptake (53) and may also drive Ca2+-activated Cl− secretion.
Our findings suggest a molecular mechanism through which PGE2 enhances urinary NaCl excretion under high-salt conditions. High-salt feeding increases expression of enzymes that increase PGE2 production, including cyclooxygenase (COX)-1, COX-2, and mPGES1, in the renal medulla (27, 29, 59). As a consequence, high-salt feeding increases the PGE2 concentration in the renal medulla and urine (12, 21, 36, 37, 72). When PGE2 production is disrupted through deletion of COX-1 or mPGES-1 in mice, salt-sensitive hypertension ensues (29, 72).
We propose that PGE2 activates basolateral EP4 receptors in IMCD cells to stimulate Cl− secretion as an adaptive mechanism in which local increases in PGE2 production in the collecting duct serve to enhance urinary NaCl excretion under conditions of high dietary NaCl intake. The activation of these signaling pathways may be particularly important under states of extracellular fluid volume expansion, when levels of serum aldosterone are low and ENaC activity is suppressed. To date, the contribution of EP4 receptors to the control of NaCl balance and blood pressure in vivo is unclear and awaits phenotypic analysis of EP4 receptor knockout mice challenged with NaCl loading.
This work was supported by a grant from the National Institutes of Health (K08-DK-073487 to A. Pao) and (T32-335 DK07357-26A1 to P. Kathpalia and S. Thomas).
No conflicts of interest, financial or otherwise, are declared by the authors.
Author contributions: M.R. and A.C.P. conception and design of research; M.R., S.V.T., P.P.K., and Y.C. performed experiments; M.R., S.V.T., P.P.K., Y.C., and A.C.P. analyzed data; M.R., S.V.T., P.P.K., Y.C., and A.C.P. interpreted results of experiments; M.R., S.V.T., P.P.K., Y.C., and A.C.P. prepared figures; M.R. and A.C.P. drafted manuscript; M.R. and A.C.P. edited and revised manuscript; M.R. and A.C.P. approved final version of manuscript.
We are grateful to Dr. Bruce Stanton (Dartmouth Medical School) for providing mIMCD-K2 cells for the study. We thank Dr. Rajeev Rohatgi (Mount Sinai School of Medicine) and Dr. Glenn Chertow (Stanford University) for valuable discussions.