“Store-operated” cAMP signaling contributes to Ca²⁺-activated Cl⁻ secretion in T84 colonic cells

Abstract

Apical cAMP-dependent CFTR Cl⁻ channels are essential for efficient vectorial movement of ions and fluid into the lumen of the colon. It is well known that Ca²⁺-mobilizing agonists also stimulate colonic anion secretion. However, CFTR is apparently not activated directly by Ca²⁺, and the existence of apical Ca²⁺-dependent Cl⁻ channels in the native colonic epithelium is controversial, leaving the identity of the Ca²⁺-activated component unresolved. We recently showed that decreasing free Ca²⁺ concentration ([Ca²⁺]) within the endoplasmic reticulum (ER) lumen elicits a rise in intracellular cAMP. This process, which we termed “store-operated cAMP signaling” (SOcAMPS), requires the luminal ER Ca²⁺ sensor STIM1 and does not depend on changes in cytosolic Ca²⁺. Here we assessed the degree to which SOcAMPS participates in Ca²⁺-activated Cl⁻ transport as measured by transepithelial short-circuit current (I_sc) in polarized T84 monolayers in parallel with imaging of cAMP and PKA activity using fluorescence resonance energy transfer (FRET)-based reporters in single cells. In Ca²⁺-free conditions, the Ca²⁺-releasing agonist carbachol and Ca²⁺ ionophore increased I_sc, cAMP, and PKA activity. These responses persisted in cells loaded with the Ca²⁺ chelator BAPTA-AM. The effect on I_sc was enhanced in the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), inhibited by the CFTR inhibitor CFTR_inh-172 and the PKA inhibitor H-89, and unaffected by Ba²⁺ or flufenamic acid. We propose that a discrete component of the “Ca²⁺-dependent” secretory activity in the colon derives from cAMP generated through SOcAMPS. This alternative mode of cAMP production could contribute to the actions of diverse xenobiotic agents that disrupt ER Ca²⁺ homeostasis, leading to diarrhea.

apical cl⁻ channels are essential for maintaining hydration of the luminal contents of the intestine. Pathological imbalance of this process commonly results in intestinal disorders including obstruction and diarrhea. In the colon, the secretion of anions (Cl⁻ and, to a smaller extent, HCO₃⁻) occurs mainly at the level of the cells of the colonic crypt to create the osmotic gradient necessary to attract water into the lumen. This process is under the physiological control of various neurotransmitters, endocrine, paracrine, and immune mediators. These diverse agents typically converge on two fundamental intracellular signaling systems, cAMP and Ca²⁺, to orchestrate the cooperative activity of channels, pumps, and transporters needed for efficient electrolyte and fluid secretion (1).

The tremendous synergism observed when colonic cells are stimulated concurrently by both cAMP and Ca²⁺ has been known for years (18, 35), but details of this process are still not completely understood. There is extensive reciprocal modulation at the level of individual messenger pathways (cAMP-Ca²⁺ “cross talk”) in colon, as in all cells (25, 55, 56), and this profoundly affects the secretory response (6).

In colonic cells, the dominant Cl⁻ exit pathway is the apical cAMP-dependent cystic fibrosis transmembrane regulator (CFTR), although expression of other Cl⁻ pathways (e.g., ClC-2) has also been suggested (15). Intracellular uptake of Cl⁻ is mediated mainly by the basolateral Na⁺-K⁺-2Cl⁻ cotransporter. Ca²⁺- and cAMP-activated K⁺ channels (37) provide the driving force for the secretion of Cl⁻ and other anions by maintaining a hyperpolarized membrane voltage (7, 17, 22, 23, 30). Ca²⁺-activated Cl⁻ channels (CaCCs) such as TMEM16A are clearly involved in anion secretion in many types of epithelia but might not necessarily constitute a major pathway in colon (41). Therefore, it appears that some component of cAMP-dependent Cl⁻ secretion is likely needed to initiate efficient vectorial transport of ions in the colon.

Recently we described a new mode for eliciting elevations in intracellular cAMP that is triggered directly by a decrease in free Ca²⁺ concentration ([Ca²⁺]) within the endoplasmic reticulum (ER) Ca²⁺ store. We termed this process “store-operated cAMP signaling” (SOcAMPS) (33). SOcAMPS is completely independent of cytosolic [Ca²⁺], requires the luminal ER Ca²⁺ sensor STIM1 (stromal interaction molecule), and is particularly prominent in NCM460 cells, colon-derived cultured cells of epithelial origin (34, 40, 44). The same mechanism was also described in other Cl⁻ secretory cells, CFTR-expressing airway epithelia (47), and in cholangiocytes (49). The physiological importance of SOcAMPS in the native colon is unknown, but such a pathway would be expected to permit Ca²⁺-mobilizing agents that persistently deplete the ER Ca²⁺ store to generate cAMP, which in turn would stimulate cAMP-dependent ion transport processes (i.e., via CFTR), leading to fluid secretion that is independent of cytosolic [Ca²⁺].

In this work we investigated whether SOcAMPS has functional consequences in colonic cells. To this end, we took advantage of newer-generation fluorescence resonance energy transfer (FRET)-based sensors to measure cAMP and PKA activation dynamics in single T84 cells, a human colonic adenocarcinoma cell line, and correlated these measurements with Cl⁻ secretion evaluated with classical short-circuit current (I_sc) techniques. We found that depletion of intracellular Ca²⁺ stores by a variety of agents in the absence of extracellular Ca²⁺ initiates cAMP/PKA signaling, and thereby elicits a measurable functional secretory response. We propose that this store depletion-dependent PKA activation assists in driving the increase in Cl⁻ secretion observed upon mobilization of Ca²⁺ stores via the Ca²⁺/phosphoinositide pathway.

MATERIALS AND METHODS

Materials and solutions.

Reagents were purchased from Sigma-Aldrich (St. Louis, MO) with the following exceptions: 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid AM ester (BAPTA-AM), fura-2 AM, and N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) (Invitrogen, Grand Island, NY); ionomycin, KT5720, and H-89 hydrochloride (EMD Chemicals, Billerica, MA); and CFTR_inh-172 (Cayman Chemicals, Ann Arbor, MI). These reagents were stored as 1,000× stock solutions in either dimethyl sulfoxide or water.

Ca²⁺-free bicarbonate-buffered Ringer solution contained (in mM) 120 NaCl, 2.09 K₂HPO₄, 0.34 KH₂PO₄, 24 NaHCO₃, 1 MgSO₄, and 10 d-glucose. One millimolar CaCl₂ was added to Ca²⁺-containing Ringer solution. HEPES-buffered solution contained 125 NaCl, 10 HEPES, 2.4 K₂HPO₄, 0.39 KH₂PO_4, 1 MgSO₄, and 10 d-glucose, pH 7.43. In solutions containing Ba²⁺, MgCl₂ was substituted for MgSO₄.

Cell culture and transfection.

T84 cells (ATCC, Manassas, VA) were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells plated on glass coverslips were transiently transfected with FRET-based cAMP, Ca²⁺, or PKA activity sensors with FuGENE transfection reagent (Promega) 24–48 h prior to imaging. For I_sc measurements, cells were plated 14–21 days prior to experiments on 1.12-cm² Snapwell inserts with a pore size of 0.4 μm (Corning, NY) at a density of ∼1.5E5 cells/well. When FRET imaging was performed with Snapwells, cells were transfected with sensors prior to seeding and used when high-resistance monolayers formed, as for the I_sc experiments.

Experiments with rat colon.

Distal or proximal colon segments, harvested from adult male Sprague-Dawley rats euthanized by CO₂, were opened along the mesenteric line and rinsed with ice-cold saline. Colonic segments were dissected from submucosa and muscular layers and mounted in a six-pin circular aperture slider (8-mm diameter; Physiologic Instruments, San Diego, CA) for use in I_sc experiments. Experimental protocols were approved by the VA Boston Healthcare System Institutional Animal Care and Use Committee.

Short-circuit current measurements.

Ussing chambers (Physiologic Instruments) were mounted with Snapwell inserts containing either confluent T84 cells or segments of rat colon and bathed in 3.5 ml/well of bicarbonate-buffered solution gassed with 95% O₂-5% CO₂ at 37°C. I_sc was measured with a VCC MC6 multichannel I/V clamp (Physiologic Instruments), and data were recorded and analyzed with PowerLab 4/30 (AD Instruments, Castle Hill, Australia). All solutions were added symmetrically to both sides of the Ussing chamber, unless otherwise noted.

Live cell imaging protocols.

Glass coverslips were mounted in a perfusion chamber (Warner Instruments, Hamden, CT) that enabled laminar flow of HEPES-buffered Ringer solution. Ratio images were recorded on a TE2000-U microscope using a ×40 (1.3 NA) or ×60 (1.45 NA) oil-immersion objective lens. Images were taken every 5–10 s during the time-lapse capture. Excitation and emission filter wheels (Sutter Instruments, Novato, CA) were controlled with MetaFluor software (Molecular Devices, Sunnyvale, CA). This allowed excitation of the FRET-based sensors with 440-nm excitation light and measurement of a 480 nm-to-535 nm FRET emission ratio for the cAMP sensors (EpacH30 or EpacH90; a kind gift of Kees Jalink, Netherlands Cancer Institute) (43, 53) and a 535 nm-to-480 nm FRET emission ratio for A-Kinase Activity Reporter 4 (AKAR4) (kindly provided by Jin Zhang, Johns Hopkins University) (16) and the Ca²⁺ sensor D3cpv (42). For fura-2 imaging of Ca²⁺, cells were first incubated with 1.5–2.5 μM fura-2 AM in DMEM for 30 min at 37°C. Fura-2 fluorescence was measured in 340/385-nm excitation mode (510-nm emission). For experiments using BAPTA-AM and fura-2 AM, cells were coloaded with both compounds as above.

Data analysis and statistics.

Values are expressed as means ± SE for n individual experiments. The significance of the observations was evaluated by Student's t-test for paired or unpaired data as appropriate, and P < 0.05 denotes a statistical difference.

RESULTS

ER Ca²⁺ store depletion induces measurable anion secretion in T84 monolayers in the absence of external Ca²⁺.

Agents that elevate intracellular cAMP such as the adenylyl cyclase (AC) activator forskolin are well established to stimulate changes in anion secretion, also in the absence of extracellular Ca²⁺. As demonstrated in Fig. 1A, addition of forskolin to T84 monolayers bathed bilaterally in nominally Ca²⁺-free solutions produced changes in I_sc that were largely reversed by treatment with the CFTR inhibitor CFTR_inh-172 and further reversed by the Na⁺-K⁺-2Cl⁻ cotransporter inhibitor bumetanide.

Fig. 1.

Short-circuit current (I_sc) response to forskolin and ionomycin in the absence of external Ca²⁺ in T84 monolayers and colonic epithelium. A: response to forskolin in the absence of Ca²⁺ and reversal by the cystic fibrosis transmembrane regulator (CFTR) inhibitor CFTR_inh-172 and bumetanide. The deflections in the traces are due to transepithelial current pulses applied to measure transepithelial resistance (R_t). B: response to ionomycin compared in Ca²⁺-free solutions and after acute addition of Ca²⁺. C and D: carbachol (C) and ionomycin (D) stimulate a significant CFTR_inh-172-sensitive Cl⁻ secretion in the absence of external Ca²⁺. E: PKA inhibitor H-89 significantly accelerated the recovery of the response to ionomycin in Ca²⁺-free solutions. Bar graph summarizes results. *P = 0.033 compared with controls done in parallel on the same day (control: n = 11, H-89: n = 5). F, left: summary of results obtained with forskolin (FSK; n = 13, P < 0.0001), ionomycin (ion; n = 23, P = 0.00023), and carbachol (carb; n = 7, P = 0.0018). Right: I_sc responses were accompanied by significant changes in R_t (forskolin P = 0.016; ionomycin P < 0.0001; carbachol P = 0.027). G: changes in I_sc following ionomycin (top) and forskolin (bottom) treatment in Ca²⁺-free solutions in rat proximal colonic epithelium. Note that the subsequent change in I_sc following addition of forskolin in the presence of CFTR_inh-172 (top) was greatly reduced compared with the control response (bottom). Bar graph summarizes the responses to ionomycin (ΔI_sc) obtained in proximal (n = 11) and distal (n = 5) rat colon.

It is also well established that maneuvers that mobilize internal Ca²⁺ stores to elevate intracellular [Ca²⁺] are able to induce significant Cl⁻ secretion in colonic cells (3, 5, 14, 19, 21, 46, 50). Release of ER Ca²⁺ stores with agonists or ionophores typically causes a transient increase in intracellular Ca²⁺. The corresponding reduction in free intraluminal ER [Ca²⁺] then stimulates store-operated Ca²⁺ entry pathways in the plasma membrane (2, 10, 48). This leads to a second phase of sustained Ca²⁺ influx that is terminated only upon store refilling. As shown in Fig. 1B, ionomycin caused a small but significant increase in basal I_sc when added in the absence of extracellular Ca²⁺ (observed in 19/24 monolayers). Readdition of Ca²⁺ produced a large increase in I_sc that was partially sensitive to CFTR_inh-172 and fully terminated by bumetanide. Of note, acute addition of CFTR_inh-172 by itself had no effect on I_sc (not shown). These results confirm previous findings that Ca²⁺ entry strongly enhances I_sc across T84 monolayers. However, we were struck by the persistent nature of the small initial increase in I_sc in Ca²⁺-free Ringer solution, as it frequently did not mirror the expected time course of the transient Ca²⁺ release phase of the signal. As seen in Fig. 1B, the response in Ca²⁺-free solutions was fully reversed by CFTR_inh-172 and amounted to ∼40% of the I_sc change induced by 5 μM forskolin under the same conditions (see Fig. 1A for comparison and Fig. 1F for data summary). A small but significant increase in I_sc was also obtained upon addition of the cholinergic Ca²⁺ mobilizing agonist carbachol (Fig. 1C).

As expected, these increases in I_sc were accompanied by a proportional decrease in transepithelial resistance (R_t;, summarized in Fig. 1F, right), indicating an increase in the transepithelial conductance. Interestingly, acute addition of the PKA inhibitor H-89 resulted in a fast reversal of ionomycin-induced anion secretion (Fig. 1E), consistent with a cAMP/PKA-mediated action of the ionophore. Overall, the data of Fig. 1 suggest that there is a small but sustained I_sc elicited by maneuvers that empty ER Ca²⁺ stores in the absence of extracellular Ca²⁺. This effect was inhibited by H-89 and CFTR_inh-172, leaving open the possibility that the action of ionomycin is in part mediated via PKA-dependent stimulation of CFTR. The finding that a detectable anion flux can be elicited by store depletion in the absence of external Ca²⁺ seems to also hold true for the native tissue, since we observed similar results in both proximal and distal rat colon (Fig. 1G).

It is known that changes in I_sc elicited by ionomycin or other Ca²⁺ store-depleting agents in T84 cells can be amplified in the face of elevated cytosolic cAMP (35). We tested whether this is also the case in Ca²⁺-free conditions. Figure 2 shows the significant effect of ionomycin after pretreatment with the general phosphodiesterase (PDE) inhibitor IBMX (1 mM; Fig. 2A) or when measured after pretreatment with a submaximal dose of forskolin (Fig. 2B). In both cases the increase in I_sc induced by ionomycin was significantly higher than that evoked by ionomycin alone. Similar results were obtained with carbachol (Fig. 2C) or the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) pump inhibitor cyclopiazonic acid (CPA; Fig. 2D). As expected, a much smaller effect was observed when the ionophore was administered after internal Ca²⁺ stores had already been partially emptied by CPA (Fig. 2D).

Fig. 2.

I_sc change following store depletion in the absence of external Ca²⁺ was enhanced after cAMP elevation. A and B: I_sc response to ionomycin after IBMX (A; −19.31 ± 1.74 μA/cm², n = 10) and after forskolin (B; −31.85 ± 5.96 μA/cm², n = 5); both responses were higher than ionomycin alone (−11.89 ± 2.59); the response in the presence of forskolin was significantly higher (P < 0.0001). C: the change in I_sc due to carbachol in the presence of IBMX (−18.36 ± 0.11 μA/cm², n = 3) was significantly different from the control response (−5.5 ± 1.04 μA/cm², P < 0.0001). D: response to cyclopiazonic acid (CPA) in the presence of forskolin (−4.4 ± 1.4 μA/cm², n = 7).

Effect of ionomycin on I_sc persists after blockade of Ca²⁺-activated Cl⁻ channels and other parallel ion-conducting pathways.

Even though the experiments above were conducted in Ca²⁺-free solutions, we considered the possibility that the transient spike of Ca²⁺ elicited by ionomycin or other Ca²⁺-releasing agents might lead to opening of CaCCs, thus accounting for the increase in I_sc. However, the stilbene derivative 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), a known nonspecific CaCC inhibitor (used also in T84 cells) (3, 29, 38, 52), did not eliminate the response to ionomycin, here shown in the presence of IBMX (Fig. 3A). Similar results (not shown) were also obtained with the fenamate compound flufenamic acid, another nonspecific CaCC inhibitor (45, 54). While our data do not exclude non-CFTR anion pathways in T84 cells, we do show that the response to store depletion can be maintained even when several known Cl⁻-conducting pathways are eliminated.

Fig. 3.

4,4′-Diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) or Ba²⁺ did not inhibit the response to ionomycin. A: I_sc response to ionomycin in the presence of DIDS (−12.35 ± 3.47 μA/cm²; typical of n = 5) was not significantly different from that measured in control experiments (−19.31 ± 1.74 μA/cm², n = 28). B: response in the presence of Ba²⁺ after pretreatment of T84 monolayers with IBMX was also not significantly different from the control response (−34.56 ± 8.62 μA/cm²; typical of n = 7 experiments). Similar results were observed after preincubation with forskolin.

Optimal efficiency of anion flux across the colonic epithelium is obtained when there is parallel activation of K⁺-conducting channels, leading to membrane hyperpolarization. For example, it is known that K⁺ channel openers (e.g., 1-EBIO compound, which targets Ca²⁺-activated K⁺ channels) augment Cl⁻ secretion in T84 cells (17). The initial Ca²⁺ spike after ionomycin might therefore activate K⁺ channels and indirectly enhance I_sc, but the presence of the nonspecific K⁺ channel blocker Ba²⁺ in the serosal bath did not affect the change in I_sc induced by ionomycin (Fig. 3B). Of note, in a limited series of experiments, we also applied K⁺ channel blockers to both sides of the monolayer (2 experiments with Ba²⁺ and 1 with flufenamic acid), with a similar lack of effect. This suggests that Ca²⁺-stimulated opening of basolateral K⁺ channels (and possibly apical channels as well) cannot account entirely for the action of ionomycin, although it is undisputed that these channels are permissive for the secretory response.

Evidence for store-operated cAMP signaling as assessed with FRET sensors in single T84 cells.

Our data indicated that Ca²⁺ mobilization activates an electrogenic anion conductive pathway having many features in common with CFTR. However, CFTR is generally held to require cAMP/PKA-dependent phosphorylation for its activation. One possible explanation for this residual H-89- and CFTR_inh-172-sensitive I_sc is that T84 cells, like several other colonic epithelial cell lines, manifest the SOcAMPS pathway such that lowering of intraluminal ER Ca²⁺ leads to cAMP production. This would in turn activate CFTR. We therefore assessed the effect of store depletion on cAMP levels in single T84 colonocytes, using ratiometric FRET-based cAMP sensors (Fig. 4).

Fig. 4.

Evidence for store-operated cAMP signaling (SOcAMPS) in T84 cells. A: real-time measurements of intracellular cAMP, expressed as fluorescence resonance energy transfer (FRET) emission ratio (480 nm/535 nm), in single T84 cells transiently transfected with the cAMP sensor EpacH30 in Ca²⁺-free solutions. TPEN and ionomycin increased cAMP. Supramaximal concentrations of forskolin and IBMX were given at the end of the experiments to saturate the sensor. B: removal of Ca²⁺ from extracellular solution enhanced cAMP production. C: carbachol and ionomycin increased cytosolic cAMP. Other cAMP FRET sensors (EpacH74 and EpacH90) were also used with similar results. D: bar graph summarizes results represented as % of the maximal (FSK+IBMX) response: forskolin (4 exp/7 cells), TPEN (9 exp/9 cells), carbachol (23 exp/32 cells), ionomycin in 0 Ca²⁺ (11 exp/12 cells) and in 1 mM Ca²⁺ (n = 6 exp/8 cells). The response recorded in 1 mM Ca²⁺ was significantly lower than that observed in the absence of Ca²⁺ (column labeled “Ion”). **P = 0.0028.

Cells grown on glass coverslips were transfected with a genetically encoded cAMP reporter (either EpacH30 or EpacH90) (43, 53). To specifically manipulate intraluminal ER [Ca²⁺], we exposed cells to a low-affinity membrane-permeant Ca²⁺ buffer, TPEN. We have previously shown in other cell lines that 1 mM TPEN rapidly, reversibly, and dose-dependently lowers free [Ca²⁺] in the ER without affecting resting cytosolic calcium levels, also in the absence of external Ca²⁺ (11, 26, 33). As shown in Fig. 4A, TPEN added in Ca²⁺-free Ringer solution caused the 480 nm-to-535 nm FRET emission ratio of EpacH30 to gradually increase, indicating elevated cAMP concentration in the cytosol. Rinsing off TPEN fully reversed the response. Under these conditions, we expect that ER [Ca²⁺] should be restored to its original levels upon removal of TPEN. This would occur in the absence of Ca²⁺ entry across the plasma membrane because there is no Ca²⁺ in the bathing solution (26, 33). TPEN binds avidly to heavy metals (4, 33), but low doses (10 μM) of the chelator, expected to scavenge these other metal species, had no effect on the FRET ratio (not shown). The significant response to the subsequent addition of ionomycin is also depicted in Fig. 4A. At the end of every cAMP imaging experiment we applied a cocktail of IBMX and a supramaximal concentration of forskolin (50 μM) to establish maximal ratio response.

As illustrated in Fig. 4B, Ca²⁺ influx that takes place in the presence of extracellular Ca²⁺ attenuates SOcAMPS in T84 cells, as it does in several other cell types (33, 47, 49). Treatment with ionomycin in the presence of external Ca²⁺ yielded only a minor increase in the FRET ratio, but switching to a Ca²⁺-free solution enhanced cAMP production by ∼22%. Figure 4C also shows that the cholinergic agonist carbachol was able to induce an increase in FRET ratio, indicative of increased cAMP levels. Taken together, our data are consistent with the presence of a mechanism in which lowering of free [Ca²⁺] within internal Ca²⁺ stores can drive the production of cAMP, as shown previously in other cell types (33, 47, 49). Figure 4D summarizes the changes in cAMP (as measured by changes in the FRET ratio) obtained with forskolin alone, TPEN, ionomycin, and carbachol expressed as percentage of the maximal response to forskolin + IBMX.

Activation of PKA in response to store depletion.

We also evaluated the effect of store depletion on signaling downstream of cAMP, using a complementary FRET-based probe for PKA activity known as AKAR4. In Fig. 5A it is shown that depleting Ca²⁺ stores with ionomycin resulted in an increase in the 535 nm-to-480 nm FRET emission ratio of AKAR4 in T84 cells, consistent with an increase in PKA phosphorylation. The process was inhibited when extracellular Ca²⁺ was readmitted to the bath.

Fig. 5.

Activation of PKA as measured by the FRET sensor A-Kinase Activity Reporter 4 (AKAR4). A: ionomycin induced PKA activity. Addition of Ca²⁺ inhibited this process (typical of n = 5 exp/5 cells). B: effect of the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor CPA illustrated in 2 different cells (4 exp/4 cells). C: response to carbachol (13 exp/16 cells) was reversed by the PKA inhibitor H-89. PKA activity expressed as % of maximal (forskolin+IBMX) response: ionomycin, 72.15 ± 7.05% (5 exp/5 cells); CPA, 31.43 ± 5.32% (4 exp/4 cells); carbachol 34.08 ± 6.34% (16 exp/16 cells).

As depicted in Fig. 5, B and C, similar results were obtained when alternative strategies were used to release Ca²⁺ stores. In fact, both CPA (Fig. 5B) and carbachol (Fig. 5C) were able to activate PKA, as measured by AKAR4. The response could be fully reversed by the PKA inhibitor H-89 (Fig. 5C).

A component of I_sc response in T84 monolayers is independent of cytosolic Ca²⁺: effect of BAPTA-AM.

Our data indicated that lowering free [Ca²⁺] in the ER with diverse tools led to change in I_sc, cAMP production, and PKA activation. However, with the exception of the TPEN experiments, the maneuvers used to lower ER [Ca²⁺] imply an obligate release of Ca²⁺ into the cytosol. It is well established that changes in cytosolic [Ca²⁺] can either stimulate or inhibit cAMP production through the various Ca²⁺-sensitive isoforms of ACs or PDEs. Unfortunately, we could not use TPEN on the monolayers because even low concentrations caused a loss of R_t. We therefore performed experiments in which the monolayers were exposed to the high-affinity cell-permeant Ca²⁺ chelator BAPTA-AM, thereby clamping excursions of cytosolic free [Ca²⁺] following store release.

First we needed to directly assess the ability of BAPTA-AM to effectively buffer intracellular Ca²⁺ changes in high-resistance T84 monolayers. The diffuse background of the monolayers grown on semipermeable supports interfered with our initial attempts to monitor Ca²⁺ changes with fura-2. We therefore performed these control experiments with the newer-generation genetically encoded cameleon Ca²⁺ reporter D3cpv (42). Cells were first transfected with D3cpv, seeded onto Snapwell filters, and used when a high-resistance monolayer was formed. With this approach it was possible to locate individual cells within the monolayers that expressed the Ca²⁺ reporter. Figure 6A compares typical experiments in which intracellular Ca²⁺ changes were recorded in controls and in BAPTA-AM-preloaded monolayers. These experiments demonstrate that BAPTA-AM was highly effective in preventing cytosolic Ca²⁺ spikes following stimulation with carbachol or ionomycin. Parallel I_sc experiments showed that the increase in I_sc observed after stimulation with carbachol was independent of changes in cytosolic Ca²⁺ levels, as the response in cells pretreated with the intracellular Ca²⁺ buffer was not different from the control in Ca²⁺-free solutions (Fig. 6B). Imaging experiments to measure cytosolic cAMP changes and PKA activation, respectively, confirmed that BAPTA-AM preincubation did not influence the response to store depletion with carbachol (Fig. 6C). Therefore, we propose that lowering of free [Ca²⁺] per se within the lumen of internal stores results in cAMP production in T84 cells. This cAMP signal contributes in a small but not insignificant manner to the I_sc response of the monolayer, likely through activation of PKA-dependent CFTR.

Fig. 6.

Effect of the intracellular Ca²⁺ buffer BAPTA-AM. A: intracellular Ca²⁺ concentration ([Ca²⁺]) changes, measured with cameleon D3cpv in T84 cell monolayers, in controls (top) and in cells preloaded (45 min) with 20 μM BAPTA-AM (bottom). Bar graph: the response to carbachol in BAPTA-AM-pretreated cells (12 exp/35 cells) was significantly different (P = 0.00017) from control (7 exp/35 cells). B: changes in I_sc after addition of carbachol and ionomycin in control (top) and BAPTA-AM-pretreated (bottom) monolayers. Bar graph: response to carbachol was not different in control (n = 5) vs. BAPTA-AM-pretreated (n = 4) monolayers. C: FRET experiments with EpacH30 (top) and with AKAR4 (bottom) showed that cAMP and PKA activity did not change after preincubation with BAPTA-AM. EpacH30: controls 23 exp/46 cells; BAPTA-AM 7 exp/18 cells. AKAR4: controls 20 exp/25 cells; BAPTA-AM 6 exp/10 cells.

DISCUSSION

cAMP-dependent gating of CFTR is a point of convergence for the Cl⁻ and fluid secretory activity of many epithelia. In the conventional view, cytosolic Ca²⁺ can potentially increase epithelial Cl⁻ secretion by two general mechanisms: 1) by activating conductances (e.g., basolateral cation pathways and cotransporters) that are permissive for vectorial anion transport via CFTR and 2) by acting on CaCCs that appear to be located in parallel with CFTR in many cell types. However, in the case of the colon, while the first mechanism seems to be universally accepted the physiological relevance of the second scenario is controversial, and difficult to reconcile with the profound deficits in colonic secretion observed in CFTR-null models (3, 5, 7, 14, 24, 30, 35, 38, 46).

Although the powerful synergism imposed by Ca²⁺ and cAMP on epithelial secretory activity has been known for many years, nuances on this theme continue to emerge. Billet and Hanrahan (9) recently commented on the “secret life” of CFTR as a Ca²⁺-activated anion conductance. Thus while not directly gated by Ca²⁺, there are numerous indirect modes by which Ca²⁺ signaling impacts this important pathway (9, 31). For example, Ca²⁺ can potentially stimulate CFTR gating via tyrosine kinases and inhibit protein phosphatase type 2A to alter the termination of channel activity (9, 20). Ca²⁺-dependent AC has also been proposed to generate apical cAMP microdomains that positively regulate CFTR in colon (9).

Conversely, cAMP also affects Ca²⁺ signaling in colon in many ways. These include recently described PKA-dependent phosphorylation of IRBIT, a protein that dissociates from the inositol 1,4,5-trisphosphate receptor to modify both ER Ca²⁺ release and ion transporters that participate in the secretory response (1, 57). CFTR inserted into the ER membrane as it traffics along to the plasma membrane may potentially affect ER Ca²⁺ handling and signaling upon its activation with cAMP (27, 36). In addition, stimulation of the cAMP effector Epac is known to influence PLCε activity to elicit Ca²⁺ signaling, also in T84 cells (28).

The fact that such a multiplicity of mechanisms, both Ca²⁺ and cAMP dependent, converge to activate CFTR highlights its essential role in colonic physiology. Here we provide evidence that a discrete component of Ca²⁺-activated Cl⁻ secretion observed in T84 colonic cells may be partially explained by the previously described “store-operated” cAMP signaling mechanism that links lowering of free Ca²⁺ within stores to activation of AC (33, 34, 44) to gate cAMP-dependent CFTR. Key FRET imaging results shown in Figs. 4–6 indicate that lowering Ca²⁺ in ER stores with different strategies resulted in increased cAMP/PKA activity. In agreement with our previous findings in another colonic cell line (33, 34), the effects of store depletion on cAMP/PKA were actually attenuated by Ca²⁺ entering from the extracellular space in T84 cells. While depletion of ER Ca²⁺ usually implies an obligatory elevation of cytosolic Ca²⁺, our data obtained in BAPTA-AM- and TPEN-treated cells indicate that this effect is independent of excursions in cytosolic [Ca²⁺]. These data would also argue that Ca²⁺-activated ACs (or PDEs inactivated by Ca²⁺) are not responsible for the effects of store depletion on cAMP/PKA activity. This latter interpretation is also consistent with the finding that Ca²⁺ entry from the extracellular space attenuated rather than activated cAMP production.

As assessed by I_sc experiments, the increase in cAMP/PKA activity in the absence of external Ca²⁺ resulted in stimulation of H-89- and CFTR_inh-172-sensitive anion secretion, and this response was not affected by BAPTA-AM treatment (Fig. 6). Under these conditions, we expect any potential contribution of CaCCs to be minimized (14, 46), although we cannot exclude the existence of cytosolic “nanodomains” of Ca²⁺ that might not be perfectly clamped by the chelator. However, experiments with two nonspecific but effective CaCC inhibitors (flufenamic acid and DIDS; Fig. 3) would argue against this point. In light of these findings we propose that, by analogy with what we observed earlier in NCM460 cells (33), SOcAMPS has the potential to contribute to the functional secretory response in T84 cells, and possibly also in the native colon (Fig. 1G).

We do not dispute that cytosolic Ca²⁺ has a powerful activating effect on the overall secretory response of the monolayer (Fig. 1B), nor do our data specifically preclude the existence of CaCCs in T84 cells. However, our findings do show that the actions of Ca²⁺-mobilizing hormones on anion flux involve a component of cAMP production. We would suggest that this second messenger production permits secretion to occur in the complete absence of hormones that directly generate cAMP. We propose that SOcAMPS would be most pronounced after prolonged depletion of ER stores, when store-operated Ca²⁺ entry becomes downregulated, e.g., through Ca²⁺ release-activated Ca²⁺ (CRAC) channel inactivation (10). Interestingly, as we have already observed in another colonic cell line (33, 34), in T84 cells store-operated cAMP production is not entirely eliminated in the presence of external Ca²⁺ (see summary bar graph, Fig. 4D). This might be sufficient to provide a minimum level of “trigger” cAMP able to sustain the secretory response following store depletion. Another consideration is that in vivo, where extracellular volumes and diffusion are quite limited compared with the effectively infinite bath of our perfusion chamber, it is possible to generate localized depletion of external Ca²⁺ at the site of Ca²⁺ entry. We measured this directly in intact gastric epithelium (12, 13) and found that store-operated Ca²⁺ entry can cause [Ca²⁺] in the extracellular basolateral spaces to fall to ∼0.5 mM, while Ca²⁺ export at the apical side of the tissue experiences a corresponding increase in extracellular [Ca²⁺]. Thus basolateral [Ca²⁺] can theoretically approach the concentration at which Ca²⁺ entry channels cease to effectively take up Ca²⁺ (∼0.5 mM).

CFTR is emerging as a central organizing platform for numerous proteins in the apical membrane (31). Redundant regulation of CFTR by the store-operated cAMP pathway ensures that anion fluxes can be activated under a wide range of pathophysiological conditions. The intestine is particularly susceptible to exogenous agents (e.g., viral and bacterial toxins, bile acids) that alter second messenger levels to produce unregulated secretion (7). Many of the diverse agents (intestinal pathogens, chemotherapy drugs, bioactive dietary constituents, bile acids, inflammatory mediators, and environmental toxins) that cause diarrhea also cause the release of Ca²⁺ from the ER (8, 14, 24, 30, 32, 38, 39, 51). This can profoundly affect Cl⁻ and water transport across the colonic epithelium. Activation of SOcAMPS in the colon may even be protective in the face of such pathological store depletion; by providing a certain level of “trigger” cAMP, the epithelium can still mount an effective defense to flush away such noxious agents through synergistic Ca²⁺/cAMP-dependent activation of the fluid secretory machinery.

GRANTS

We gratefully acknowledge support from the Harvard Digestive Diseases Center (5P30DK034854-24), the National Institute of Diabetes and Digestive and Kidney Diseases (1R21DK088197-01), and the Medical Research Service of the Department of Veterans Affairs (VA-ORD 1 I01 BX000968-01), all to A. M. Hofer.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: J.M.N., I.M., J.A.-J., S.C., and A.M.H. performed experiments; J.M.N., I.M., J.A.-J., S.C., and A.M.H. analyzed data; J.M.N., I.M., J.A.-J., S.C., and A.M.H. interpreted results of experiments; J.M.N., J.A.-J., S.C., and A.M.H. drafted manuscript; J.M.N., I.M., J.A.-J., S.C., and A.M.H. edited and revised manuscript; J.M.N., I.M., J.A.-J., S.C., and A.M.H. approved final version of manuscript; I.M., S.C., and A.M.H. conception and design of research; S.C. prepared figures.