Modulation of K+ channels by arachidonic acid in T84 cells. I. Inhibition of the Ca2+-dependent K+ channel

Daniel C. Devor and Raymond A. Frizzell

Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The Cl- secretory response of colonic cells to Ca2+-mediated agonists is transient despite a sustained elevation of intracellular Ca2+. We evaluated the effects of second messengers proposed to limit Ca2+-mediated Cl- secretion on the basolateral membrane, Ca2+-dependent K+ channel (KCa) in colonic secretory cells, T84. Neither protein kinase C (PKC) nor inositol tetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affected KCa in excised inside-out patches. In contrast, arachidonic acid (AA; 3 µM) potently inhibited KCa, reducing NPo, the product of number of channels and channel open probability, by 95%. The apparent inhibition constant for this AA effect was 425 nM. AA inhibited KCa in the presence of both indomethacin and nordihydroguaiaretic acid, blockers of the cyclooxygenase and lipoxygenase pathways. In the presence of albumin, the effect of AA on KCa was reversed. A similar effect of AA was observed on KCa during outside-out recording. We determined also the effect of the cis-unsaturated fatty acid linoleate, the trans-unsaturated fatty acid elaidate, and the saturated fatty acid myristate. At 3 µM, all of these fatty acids inhibited KCa, reducing NPo by 72-86%. Finally, the effect of the cytosolic phospholipase A2 inhibitor arachidonyltrifluoromethyl ketone (AACOCF3) on the carbachol-induced short-circuit current (Isc) response was determined. In the presence of AACOCF3, the peak carbachol-induced Isc response was increased ~2.5-fold. Our results suggest that AA generation induced by Ca2+-mediated agonists may contribute to the dissociation observed between the rise in intracellular Ca2+ evoked by these agonists and the associated Cl- secretory response.

protein kinase C; inositol tetrakisphosphate; potassium channels; chloride secretion; intestine

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

CALCIUM-MEDIATED INTESTINAL Cl- secretion was proposed by Dharmsathaphorn and Pandol (22) to be dependent on the opening of a basolateral membrane K+ conductance (GK) in the absence of any change in apical membrane Cl- conductance (GCl). Thus activation of GK would hyperpolarize the apical and basolateral membrane potentials, increasing the electrochemical driving force for Cl- exit from the cell through apical membrane Cl- channels that were constitutively active (22). An increase in intracellular Ca2+ alone is sufficient to induce a Cl- secretory response in the T84 cell line (27). However, there is a dissociation between both the time course and magnitude of the cellular Ca2+ rise and the associated Cl- secretory response, measured as the agonist-induced short-circuit current (Isc; Refs. 21, 51, 52). These results have led to speculation that second messengers other than Ca2+ may produce a secondary inhibition of Isc that causes the secretory response to Ca2+-mediated agonists to be transient. Numerous inhibitory second messengers have been postulated, with the most prominent being protein kinase C (PKC; Refs. 10, 31, 37, 45, 46), inositol tetrakisphosphate (InsP4; Refs. 26, 47), and arachidonic acid (AA; Refs. 5, 6, 31).

This model for Ca2+-mediated Cl- secretion was based initially on isotopic flux assays, but more recent electrophysiological data have supported this concept. Measurements of membrane potential in T84 cells (12, 16, 48) indicated that the resting potential of these cells is between -30 and -40 mV. These values are very near the predicted Cl- equilibrium potential, suggesting that the GCl of these cells is dominant under resting conditions. In the absence of secretory agonists, a GCl was detected in ion replacement studies using both whole cell current-clamp (16, 48) and intracellular microelectrode measurements (48). Stimulation by muscarinic agonists resulted in hyperpolarization of the membrane potential (7, 12, 16, 48, 50), due to an increase in GK (7, 12, 16, 50). In contrast, muscarinic agonists failed to increase Cl- current (ICl) during voltage-clamp recordings in T84 cells (16) or dissociated crypts (7).

In contrast to these findings, muscarinic agonists were shown to increase GCl in T84 cells using the perforated whole cell patch-clamp technique (4, 12), and GCl was stimulated by Ca2+ ionophores during whole cell recordings (9). This Ca2+-dependent Cl- conductance was confirmed at the single-channel level in the HT-29 colonic cell line (34). These results appear contradictory to the original model for Ca2+-dependent Cl- secretion, but it is now apparent that expression of the Ca2+-dependent GCl is differentiation dependent. In polarized epithelial monolayers, increasing intracellular Ca2+ fails to increase apical GCl. Several lines of evidence support this conclusion. 1) The Ca2+ ionophore A-23187 failed to increase apical Cl- uptake into T84 cells (32). 2) In nystatin-permeabilized T84 monolayers, Ca2+-dependent agonists failed to increase apical GCl (3, 18, 37, 53). 3) Using single-channel recording techniques, Morris et al. (34) demonstrated a decrease in Ca2+-dependent Cl- channels after polarization of HT-29 cells. 4) In cystic fibrosis (CF), Ca2+-dependent Cl- secretion is lacking in the intestine (49), yet this disease impairs only the adenosine 3',5'-cyclic monophosphate (cAMP)-activated GCl. In the CF knockout mouse, disease severity is inversely correlated with the magnitude of Ca2+-dependent GCl in various tissues (8). The mice die of intestinal obstruction because their intestinal cells lack an apical Ca2+-dependent GCl; this can be corrected by expression of the CF transmembrane conductance regulator (CFTR) Cl- conductance in the intestinal epithelium (54). The CF mouse does not suffer from airway disease due to the presence of a significant apical Ca2+-dependent GCl (8). These results support the concept that the basolateral membrane Ca2+-dependent K+ channel (KCa) is the site at which Ca2+-dependent agonists regulate intestinal Cl- secretion.

We previously characterized the basolateral membrane K+ channel activated by Ca2+-dependent agonists (KCa) in the T84 cell line using single-channel patch-clamp techniques (14). Thus we can determine directly whether the second messengers proposed to limit the Ca2+-dependent secretory response act on KCa. Our results demonstrate that neither PKC nor InsP4 has any direct effect on KCa. In contrast, AA in particular, and fatty acids in general, are potent inhibitors of KCa. Indeed, inhibition of cytosolic phospholipase A2 (cPLA2) results in a potentiated carbachol-dependent Cl- secretory response. These results suggest that an increase in fatty acid liberation is responsible for the dissociation between an agonist-induced secretory response and the rise in intracellular Ca2+.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. T84 cells were grown in Dulbecco's modified Eagle's medium and Ham's F-12 (1:1) supplemented with 15 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 14 mM NaHCO3, and 10% fetal bovine serum. The cells were incubated in a humidified atmosphere containing 5% CO2 at 37°C. For measurements of Isc, T84 cells were seeded onto Costar Transwell cell culture inserts (0.33 cm2), and the culture media were changed every 48 h. Isc measurements were performed on filters after 14-21 days in culture. Patch-clamp experiments were performed on single cells plated onto glass coverslips 18-48 h before use.

Solutions. For measurements of Isc, the bath solution contained (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. The pH of this solution was 7.4 when gassed with a mixture of 95% O2-5% CO2 at 37°C. The effects of 4beta -phorbol 12-myristate 13-acetate (PMA) and 1-ethyl-2-benzimidazolinone (1-EBIO) on apical membrane ICl were assessed after permeabilization of the serosal membrane with nystatin (360 µg/ml) and the establishment of a mucosa-to-serosa Cl- concentration gradient. Serosal NaCl was replaced by equimolar sodium gluconate, and CaCl2 was increased to 4 mM to compensate for the Ca2+ buffering capacity of the gluconate anion. Nystatin was added to the serosal membrane 15-30 min before the addition of drugs (17). Successful permeabilization of the basolateral membrane was based on the recording of a negative ICl as described (17).

The effects of PMA, 1-EBIO, and thapsigargin on basolateral membrane K+ currents (IK) were assessed after permeabilization of the apical membrane with nystatin (180 µg/ml) for 15-30 min and establishment of a mucosa-to-serosa K+ concentration gradient. For measurements of IK, mucosal NaCl was replaced by equimolar potassium gluconate, whereas serosal NaCl was substituted with equimolar sodium gluconate. CaCl2 was increased to 4 mM to compensate for the Ca2+ buffering capacity of the gluconate. Cl- was removed from these solutions to prevent cell swelling that may be associated with the limited Cl- permeability of the nystatin pore as previously reported (53).

During inside-out patch-clamp recordings, the bath contained (in mM) 145 potassium gluconate, 5 KCl, 1 MgCl2, 1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 0.71 CaCl2 (calculated free Ca2+ = 400 nM), and 10 HEPES (adjusted to pH 7.2 with KOH). A free Ca2+ concentration of 400 nM was chosen because this provides a level of channel activity from which either an increase or decrease in NPo, the product of the number of channels and the channel open probability, can be readily detected as previously described (14, 19). The pipette solution contained (in mM) 140 potassium gluconate, 5 KCl, 1 CaCl2, 1 MgCl2, and 10 HEPES (adjusted to pH 7.2 with KOH). For outside-out recordings, the bath contained 1 mM CaCl2 in the absence of added EGTA while the pipette solution Ca2+ was buffered to 200 nM with EGTA (1 mM).

Isc measurements. Costar Transwell cell culture inserts were mounted in an Ussing chamber (Jim's Instruments, Iowa City, IA), and the monolayers were continuously short circuited (model EC-825; Warner Instrument). Transepithelial resistance was measured by applying a 5-mV pulse at 60-s intervals, and the resistance was calculated using Ohm's law. The T84 monolayers had resistances of 500-2,000 Omega  · cm2. 1-EBIO, forskolin, thapsigargin, PMA, glibenclamide, arachidonyltrifluoromethyl ketone (AACOCF3), and luffariellolide were added to both sides of the monolayers at the indicated concentrations. Bumetanide and charybdotoxin (CTX) were added only to the serosal bathing solution. Changes in Isc are calculated as a difference current between the sustained phase of the response and their respective baseline values.

Single-channel recordings. Single-channel currents were recorded using a List EPC-7 amplifier (Medical Systems) and recorded on videotape for later analysis as described previously (14). All recordings were performed during continuous perfusion of the patch-clamp chamber. Pipettes were fabricated from KG-12 glass (Willmad Glass). All recordings were done at a holding voltage of -100 mV. The voltage is referenced to the extracellular compartment, as is standard for membrane potentials. Inward currents are defined as the movement of positive charge from the extracellular compartment to the intracellular compartment and are presented as downward deflections from baseline in all recording configurations.

Single-channel analysis was performed on records sampled after low-pass filtering at 400 Hz. Because of the poor reversibility of the effects of fatty acids on KCa, only a single concentration of fatty acid was employed per experimental paradigm. The average length of record analyzed to determine NPo was 94 ± 2 s (n = 236). The NPo was determined using Biopatch software (version 3.11; Molecular Kinetics). NPo was calculated from the mean total current (I) divided by the single-channel current amplitude (i), i.e., NPo = I/i, where i was determined from the amplitude histogram of the current record.

Chemicals. Nystatin was a generous gift from Dr. S. Lucania (Bristol Meyers-Squibb). 1-EBIO was obtained from Aldrich. Glibenclamide, bumetanide, PMA, and the fatty acids were obtained from Sigma Chemical. Inositol 1,4,5-trisphosphate (InsP3), inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4], and sn-1,2-dioctanoyl-glycerol (DiC8) were obtained from Calbiochem. The inositol polyphosphates were made as 3 mM stock solutions in water buffered with HEPES to pH 7.2. CTX was obtained from Accurate Chemical and Scientific Corporation and made as a 10 µM stock solution in standard bath solution. Thapsigargin was obtained from Research Biochemicals. AACOCF3, luffariellolide, indomethacin, and nordihydroguaiaretic acid (NDGA) were obtained from Biomol. 1-EBIO, thapsigargin, DiC8, PMA, glibenclamide, AACOCF3, and luffariellolide were made as >= 1,000-fold stock solutions in dimethyl sulfoxide (DMSO). Nystatin was made as a 180 mg/ml stock solution in DMSO and sonicated for 30 s just before use. Bumetanide was made as a 1,000-fold stock solution in ethanol. All fatty acids were made as >= 1,000-fold stock solutions in DMSO and stored under N2 at -80°C. The fatty acids were dissolved to the final working concentration just before use, and all solutions were continuously bubbled with N2 during perfusion through the patch-clamp chamber. Cell culture medium was obtained from GIBCO.

Data analysis. All data are presented as means ± SE, where n indicates the number of experiments. Statistical analysis was performed using the Student's t-test. A value of P < 0.05 was considered statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of PKC activation on Cl- secretory responses that involve KCa. Initially, we wished to determine whether activation of PKC would affect Cl- secretory responses in which the current flow across the basolateral membrane is due to activation of KCa. PKC has been proposed to be an inhibitory modulator of Ca2+-mediated Cl- secretion (27, 37, 46, 53). This experiment is complicated by the fact that Ca2+-dependent agonists such as carbachol induce only a transient Cl- secretory response, and nonreceptor agonists such as thapsigargin or Ca2+ ionophores only weakly stimulate Cl- secretion in T84 cells. However, we recently identified a novel pharmacological opener of KCa in both intestinal and airway epithelia, 1-EBIO, which stimulates a sustained transepithelial Cl- secretory response (17, 19). We took advantage of this sustained response to determine whether PKC activation would inhibit KCa and hence the transepithelial Cl- secretory response. The results of one experiment are shown in Fig. 1A. 1-EBIO (300 µM) stimulated a sustained Isc response as previously described (17, 19). Rather than inhibiting Isc, addition of PMA (100 nM) stimulated an additional increase in Isc that was sensitive to block by bumetanide. PMA alone had no effect on basal Isc (data not shown), as described by others (28). In eight experiments, 1-EBIO increased Isc an average of 49 ± 9 µA/cm2 from a baseline of 2.6 ± 0.7 µA/cm2. In five of these experiments, the subsequent addition of PMA further increased Isc by 65 ± 8 µA/cm2. In three additional experiments, the inactive phorbol ester 4alpha -PMA failed to stimulate an increase in Isc subsequent to 1-EBIO (data not shown).


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Fig. 1.   A: effect of 1-ethyl-2-benzimidazolinone (1-EBIO; 300 µM, serosal and mucosal) and phorbol 12-myristate 13-acetate (PMA; 100 nM, serosal and mucosal) on short-circuit current (Isc) in a T84 monolayer. 1-EBIO induced a sustained current that was further increased by PMA. This current was inhibited by bumetanide (20 µM, serosal). B: effect of PMA on an 1-EBIO-induced Cl- current (ICl) after permeabilization of serosal membrane with nystatin and establishment of a mucosa-to-serosa Cl- gradient. 1-EBIO- and PMA-induced ICl was inhibited by glibenclamide (gliben; 300 µM, serosal and mucosal). C and D: effect of PMA on an 1-EBIO-induced (C) or thapsigargin-induced (D) transepithelial K+ current (IK) after permeabilization of mucosal membrane with nystatin and establishment of a mucosa-to-serosa K+ gradient. 1-EBIO- and thapsigargin-induced currents were both susceptible to block by charybdotoxin (CTX; 100 nM, serosal). Monolayer illustrations indicate direction of ion gradient and membrane that was permeabilized with nystatin (dashed line). BL, basolateral.

Effect of PKC activation on transepithelial IK and ICl. The above results suggest that activation of PKC increases either a basolateral membrane GK or apical membrane GCl subsequent to addition of 1-EBIO. To resolve the conductance pathways activated by PMA, the pore-forming antibiotic nystatin was used to permeabilize either the apical or basolateral membrane, and the appropriate transepithelial ion gradients were established to measure IK or ICl, respectively (see METHODS). The effect of PMA on IK after 1-EBIO stimulation is shown in Fig. 1C. After nystatin permeabilization, 1-EBIO (300 µM) induced a sustained increase in IK as previously described (17, 19). The subsequent addition of PMA (100 nM) had no effect on the 1-EBIO-induced IK; CTX (100 nM) inhibited this current, confirming activation of KCa by 1-EBIO (17, 19). Similar results were obtained in six experiments. 1-EBIO increased IK an average of 205 ± 59 µA/cm2, and this was not affected by PMA. The subsequent addition of CTX inhibited IK an average of 60 ± 6%.

It is possible that an inhibitory effect of PMA on KCa requires a Ca2+-dependent PKC isoform, and 1-EBIO does not reproduce this aspect of the response to a Ca2+-mediated agonist. Therefore, we determined the effect of PMA on a thapsigargin-induced IK (Fig. 1D). In contrast to thapsigargin's effect on Isc, it induces a sustained increase in IK (Fig. 1D). Similar to what we observed with 1-EBIO, PMA had no effect on IK after activation of KCa by thapsigargin. Again, CTX inhibited the thapsigargin-induced current, confirming activation of KCa. Similar results were obtained in four experiments. Thapsigargin (1 µM) increased IK by 229 ± 34 µA/cm2, and this was not affected by PMA. The subsequent addition of CTX reduced IK an average of 87 ± 3%. These results further indicate that activation of PKC does not modulate the activity of KCa in T84 cells.

Because PMA did not affect IK, these results suggest that PMA is increasing Isc subsequent to 1-EBIO by increasing an apical GCl. Therefore, we determined the effect of PMA on ICl after stimulation by 1-EBIO (Fig. 1B). After nystatin permeabilization, 1-EBIO increased ICl as described previously (17). The addition of PMA (100 nM) stimulated an additional transient increase in ICl that was sensitive to block by glibenclamide (300 µM); this is consistent with activation of CFTR (13, 39, 41). In five experiments, 1-EBIO increased ICl by 51 ± 5 µA/cm2, and PMA transiently increased ICl an additional 17 ± 7 µA/cm2.

Effect of PKC on KCa in excised patches. We next determined whether PKC activation would modulate the activity of KCa in excised patch-clamp recordings. Initially, we attempted to use the lipid analog of 1,2-diacylglycerol (DAG), DiC8, as an activator of PKC in our patch-clamp experiments. However, as illustrated in Fig. 2, DiC8 (3 µM) alone potently inhibited KCa during inside-out recording; the bath contained no ATP. In six patches, DiC8 reduced NPo by 88 ± 4%, from 1.48 ± 0.64 to 0.14 ± 0.04. 


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Fig. 2.   Effect of sn-1,2-dioctanoyl-glycerol (DiC8; 3 µM) on Ca2+-dependent K+ channel (KCa) activity in an excised, inside-out patch. Pipette and bath contained symmetric potassium gluconate, and patch was voltage clamped to -100 mV (inside negative). Bath Ca2+ was 400 nM. Arrows indicate closed state of channel.

In a second series of experiments, we used PMA as an activator of PKC while relying on the patch membrane to supply the phosphatidylserine required for activation. In these experiments, the patch was initially exposed to ATP (1 mM) plus PMA (100 nM), with PKC being added directly to the cytoplasmic side of the excised patch subsequently. The addition of ATP plus PMA resulted in a significant increase in NPo from 1.20 ± 0.43 to 2.12 ± 0.45 (P < 0.05). ATP alone has previously been shown to increase the activity of KCa in T84 cells (43; unpublished observations). However, the subsequent addition of PKC directly to the cytoplasmic side of the excised patch had no significant effect on NPo (1.85 ± 0.51). These results are consistent with our transepithelial measurements and further indicate that PKC does not directly interact with the basolateral membrane KCa of T84 cells.

Effect of InsP3 and InsP4 on KCa. Traynor-Kaplan and colleagues (26, 47) have shown that stimulation by carbachol induces a prolonged rise in InsP4. They speculated that the generation of InsP4 may be an inhibitory modulator of KCa (26). We directly assessed the effect of two isoforms of InsP4 (1,3,4,5 and 3,4,5,6), as well as InsP3, on the NPo of KCa in excised inside-out patches. Neither Ins(1,3,4,5)P4 (control, 1.07 ± 0.28; InsP4, 0.91 ± 0.24; n = 3) nor Ins(3,4,5,6)P4 (control, 0.79 ± 0.39; InsP4, 0.74 ± 0.39; n = 5) affected KCa NPo when added directly to the bathing solution at concentrations previously shown to be produced by carbachol in T84 cells (i.e., 6-12 µM; Ref. 47). Similarly, InsP3 (6-12 µM) had no effect on the NPo of KCa (control, 0.92 ± 0.69; InsP3, 0.76 ± 0.53; n = 4). These results demonstrate that inositol polyphosphates do not directly modulate the activity of KCa in the T84 cell line.

Effect of AA on KCa in excised, inside-out patches. Ca2+-dependent agonists are known to increase AA subsequent to the cellular Ca2+ rise in a wide variety of tissues (33, 36). Therefore, we determined the effect of AA on KCa in excised, inside-out patches. The results of one experiment are shown in Fig. 3A. AA (3 µM) dramatically inhibited the activity of KCa at a holding potential of -100 mV. In 22 patches, AA (3 µM) inhibited NPo by 95 ± 1%, from 1.36 ± 0.21 to 0.07 ± 0.02. An average concentration-response curve is shown in Fig. 3B. AA inhibited KCa with a predicted inhibition constant (Ki) of 425 nM. Although we did not routinely voltage clamp to positive holding potentials, a similar inhibition was observed at +100 mV (data not shown), indicating this inhibition is voltage independent.


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Fig. 3.   A: effect of arachidonic acid (3 µM) on KCa in an excised, inside-out patch. Arachidonic acid was added at asterisk. Data shown are from a continuous recording. Pipette and bath contained symmetric potassium gluconate, and patch was voltage clamped to -100 mV (inside negative). Bath Ca2+ was clamped to 400 nM. Arrows indicate closed state of channel. B: average concentration-inhibition curve for arachidonic acid on KCa in excised, inside-out patches. Data are plotted as a relative inhibition (NPo-control - NPo-AA/NPo-control), where N is number of channels and Po is channel open probability. Data were fitted to a Michaelis-Menten inhibition function with a predicted inhibition constant (Ki) of 0.42 µM (shown by arrow). Numbers above symbols indicate number of times experiment was performed at each concentration.

One possible mechanism to explain the inhibitory effect of AA is the generation of oxidative metabolites via either the cyclooxygenase or lipoxygenase pathways. Therefore, we determined the effect of AA on KCa in the presence of phenidone (250 µM), an inhibitor of both cyclooxygenase and lipoxygenase activities. In the presence of phenidone, AA reduced NPo by 94 ± 1%, from 1.76 ± 0.51 to 0.09 ± 0.02 (n = 5), which is not different from AA alone. Similar results were obtained with the more specific cyclooxygenase (indomethacin; 1 µM) and lipoxygenase (NDGA; 2 µM) inhibitors; AA (3 µM) reduced NPo from 0.90 ± 0.30 to 0.09 ± 0.03 (n = 5). These results indicate that generation of leukotrienes and/or prostaglandins is not responsible for the observed inhibition.

Effect of elevated Ca2+ on the AA-induced inhibition of KCa. We determined whether the inhibition of KCa by AA could be overcome by increasing Ca2+ at the cytoplasmic face of the channel. For these experiments, the channel was first inhibited by AA (3 µM) in the presence of 400 nM free Ca2+. Then, in the continued presence of AA, free Ca2+ was increased to 10 µM. The results of one experiment are illustrated in Fig. 4. Increasing the free Ca2+ concentration after inhibition by AA failed to induce recovery of channel activity. In six experiments, AA reduced NPo from 1.22 ± 0.27 to 0.09 ± 0.04, and the subsequent increase in Ca2+ failed to induce a recovery of NPo (0.07 ± 0.03). These results suggest that AA is not simply displacing Ca2+ from its binding site to cause the reduction in NPo.


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Fig. 4.   Effect of increasing free Ca2+ from 400 nM to 10 µM on KCa activity after inhibition by arachidonic acid (AA; 3 µM). Pipette and bath contained symmetric potassium gluconate, and patch was voltage clamped to -100 mV (inside negative). Arrows indicate closed state of channel.

The unbound form of AA is required for inhibition of KCa. AA is normally transported in the bloodstream bound to albumin. Therefore, we determined the effect of AA in the presence of albumin. In the presence of 2.5 g/l albumin, AA (3 µM) reduced the NPo of KCa from 1.09 ± 0.27 to 0.62 ± 0.09 (n = 5; P < 0.001). This 37% inhibition of NPo is significantly less than the 95% inhibition seen in the absence of albumin (P < 0.001). The subsequent removal of albumin in the continued presence of AA (3 µM) resulted in a further reduction of NPo to 0.03 ± 0.01 (n = 5). This suggests that AA must be free in solution to inhibit KCa.

After inhibition of KCa by AA, removal of AA results in only a modest recovery of channel activity, suggesting that AA remains bound to its inhibitory site. Based on the above results, we determined whether albumin would induce a further recovery of channel activity. The results of one experiment are shown in Fig. 5. AA (3 µM) nearly abolished channel activity, as shown above. After removal of AA from the bathing solution, a small increase in channel activity is apparent. However, the subsequent addition of albumin in the continued absence of AA results in a significant increase in channel activity. In five experiments, AA reduced NPo from 1.30 ± 0.56 to 0.02 ± 0.01, and NPo increased to 0.15 ± 0.08 upon removal of AA. The subsequent addition of albumin resulted in NPo, increasing to 65% of control (0.98 ± 0.56). This result suggests that albumin competes with membrane-associated AA, thus removing the block of KCa.


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Fig. 5.   Effect of albumin (2.5 g/l) on recovery of KCa activity after inhibition by arachidonic acid (3 µM). Arachidonic acid nearly completely inhibited KCa activity (2nd trace). Removal of arachidonic acid from bath solution resulted in only a partial recovery of channel activity (3rd trace), while subsequent addition of albumin resulted in a further increase in activity of KCa (4th trace); see text for NPo data. Pipette and bath contained symmetric potassium gluconate, and patch was voltage clamped to -100 mV (inside negative). Bath Ca2+ was clamped to 400 nM. Arrows indicate closed state of channel.

Effect of AA from the external side of KCa. We determined whether AA would inhibit KCa from the extracellular side of the channel using the excised, outside-out recording technique. The results of one experiment are shown in Fig. 6. AA (3 µM) inhibited KCa with an apparently similar affinity to that seen in inside-out recordings. Similar results were observed in four additional patches.


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Fig. 6.   Effect of arachidonic acid (3 µM) on KCa in an excised, outside-out patch. Arachidonic acid was added at asterisk. Data shown are from a continuous recording. Pipette and bath contained symmetric potassium gluconate, and patch was voltage clamped to -100 mV (inside negative). Pipette contained 200 nM free Ca2+. Arrows indicate closed state of channel.

Specificity of the AA effect on KCa. We next determined whether the observed inhibitory effect of AA on KCa was specific for AA or whether additional fatty acids would also modulate KCa activity. For these experiments, we used an additional cis-unsaturated fatty acid, linoleic acid (C18; cis,cis-Delta 9,Delta 12), the trans-unsaturated fatty acid elaidic acid (C18; trans-Delta 9), and a saturated fatty acid, myristic acid (C14). The results of these experiments are shown in Fig. 7. At 3 µM, all of these fatty acids significantly inhibited KCa. Both linoleic acid (n = 11) and elaidic acid (n = 6) reduced NPo by 86 ± 2%. This inhibition is significantly less than that observed in the presence of AA (P < 0.01). Myristic acid reduced NPo by 72 ± 2% (n = 11). This effect is less than that caused by any of the unsaturated fatty acids (P < 0.001). These results demonstrate that fatty acids in general are potent inhibitors of the basolateral membrane KCa in T84 cells, although some structural specificity is apparent.


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Fig. 7.   Effect of various fatty acids on KCa activity in excised, inside-out patches. Data are plotted as a percent inhibition of control NPo. Number of repetitions are indicated in parentheses. All fatty acids were used at 3 µM. Lin, linoleic acid; Ela, elaidic acid; Myr, myristic acid. * Inhibition significantly less than AA (P < 0.01).

Effect of PLA2 inhibition on the carbachol-induced Isc response. Our results demonstrate that AA is a potent inhibitor of KCa in T84 cells. Therefore, we wished to determine whether inhibition of PLA2 would induce a potentiation of the carbachol-induced Cl- secretory response. Initially, we evaluated the effect of the cPLA2 inhibitor AACOCF3. In 14 control filters, carbachol (100 µM) increased Isc from a baseline of 1.1 ± 0.2 µA/cm2 to a peak of 32.6 ± 2.7 µA/cm2 (Fig. 8). Although AACOCF3 (100 µM) increased baseline Isc by only 1.0 ± 0.2 µA/cm2 (n = 10), the subsequent response to carbachol was dramatically potentiated (82.4 ± 7.6 µA/cm2; n = 10; P < 0.001; Fig. 8). In contrast, the secreted PLA2 inhibitor luffariellolide (2 µM) failed to potentiate the carbachol-mediated Cl- secretory response (37.1 ± 4.7 µA/cm2; n = 6; Fig. 8).


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Fig. 8.   Effect of phospholipase A2 (PLA2) inhibition on carbachol (CCh)-induced increase in Isc (Delta Isc). cPLA2 inhibitor arachidonylktrifluoromethyl ketone (AACOCF3) (100 µM, serosal and mucosal) potentiated peak carbachol (100 µM; serosal)-induced increase in Isc. In contrast, secreted PLA2 inhibitor luffariellolide (Luffar; 2 µM, serosal and mucosal) had no effect on carbachol-induced Isc response. AACOCF3 and luffariellolide were added 10 min before addition of carbachol. Number of experiments is indicated in parentheses. * P < 0.001.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

A clear dissociation exists between the agonist-induced rise in intracellular Ca2+ and the concomitant Cl- secretory response in the colonic cell line T84 (21, 51, 52). It has been proposed that additional second messengers modulate the effects of Ca2+ on secretory mechanisms to produce the characteristic transient response to Ca2+-dependent agonists. The candidate messengers that have received the greatest attention in this regard are PKC, InsP4, and AA. In the present study, we evaluated the effects of PKC, InsP4, and AA on KCa in both excised patches and Ussing chamber experiments.

PKC does not acutely regulate KCa. Ca2+-mediated agonists, such as carbachol, histamine, and taurodeoxycholic acid, are known to activate phospholipase C, resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate and subsequent increases in cell Ca2+ and PKC activity in the T84 cell line (10, 15, 23, 52, 53). Although the InsP3-mediated release of Ca2+ from intracellular stores has been convincingly linked to the Cl- secretory current evoked by these agonists (15, 16, 21, 22, 53), it has been speculated that PKC likely contributes to the secondary downregulation of the secretory response that follows this initial stimulation, despite a continuing elevation of cell Ca2+ (10, 27, 31, 37, 46). As summarized in the introduction, any negative regulation of Cl- secretion by PKC likely occurs at the level of KCa. Because Ca2+-mediated Isc responses in T84 cells are virtually complete within 5-10 min, we focused our experiments on determining the acute effect of PKC activation on KCa activity.

Employing several approaches, we have failed to find an acute inhibitory effect of PKC on KCa. We recently demonstrated that the benzimidazolone 1-EBIO directly activates KCa in excised patches (19) and stimulates a sustained Cl- secretory current in colonic and airway epithelia (17, 19). Rather than inhibiting the 1-EBIO-induced Isc, PKC activation by PMA increased Cl- secretion. Using nystatin-permeabilized monolayers, we demonstrated that this effect was not due to upregulation of KCa but was due to an increase in apical GCl, which is likely CFTR. Consistent with this interpretation, Hanrahan and co-workers (44) demonstrated that PKC directly activates CFTR in excised patches. Acute exposure to phorbol esters has been shown to potentiate the effects of Ca2+-mediated agonists (28, 31), although, in one of these studies (28), this potentiation could not be blocked by PKC inhibitors. Our results suggest that this potentiation is caused by the activation of CFTR rather than an effect on KCa. Finally, inhibitors of PKC fail to modulate carbachol-dependent Cl- secretion (31), suggesting that PKC is not affecting the ion-conductive pathways regulated by carbachol.

The results from our patch-clamp studies were consistent with the Ussing chamber experiments. PKC directly applied to an excised patch had no effect on KCa activity. However, the addition of ATP before PKC increased the NPo of KCa significantly. This ATP-dependent activation of KCa has previously been reported by Tabcharani et al. (43). In contrast to our results with PKC, Tabcharani et al. (43) found that PKC inhibited KCa activity in excised, inside-out patches from T84 cells. However, these conflicting results are due to differences in the protocols employed in these studies. Tabcharani et al. (43) simultaneously added DiC8 (5 µM) and PKC to their excised patches (Fig. 5C of Ref. 43) in the presence of Ca2+ and ATP. Here, we show that DiC8 (3 µM) alone inhibits KCa (Fig. 2); this is consistent with the other lipid inhibitory effects we observed. Thus DiC8 is likely responsible for the inhibitory effect reported by Tabcharani et al. (43). We conclude that an acute increase in PKC, which might occur during a Ca2+-mediated response, is not responsible for the inhibition of KCa that limits the Cl- secretory response to Ca2+.

Although our results clearly demonstrate that PKC does not acutely regulate KCa, other investigators have found that long-term treatment with PMA (30 min to 12 h) inhibits the activity of a basolateral membrane GK (28, 37, 46). The reason for this inhibition is unclear, although this long-term treatment is known to inhibit the carbachol-induced Ca2+ release process (28) as well as decrease CFTR mRNA levels (40) in T84 cells. Thus these or other effects not directly related to KCa gating may be responsible for the decreased activity of GK after the long-term activation of PKC.

Inositol polyphosphates do not acutely regulate KCa. When carbachol stimulates Cl- secretion in T84 cells, it inhibits the subsequent secretory responses to other Ca2+-dependent agonists (26). This inhibitory effect correlated with a sustained, carbachol-induced elevation of InsP4 that was not observed with histamine (26). The use of cell-permeant analogs of InsP4 suggested that Ins(3,4,5,6)P4 was the isoform most likely involved in inhibiting Isc (47). It was postulated that InsP4 may attenuate Cl- secretion by inhibiting KCa (26). Based on these observations, we determined the effect of inositol polyphosphates (InsP3, InsP4) on KCa in excised, inside-out patches. Our results demonstrate that neither InsP4 (1,3,4,5 or 3,4,5,6) nor InsP3 modulates the activity of KCa in excised patches from T84 cells, demonstrating that this is not a site for inhibitory modulation by InsP4. Consistent with this notion is the recent report demonstrating inhibition of a Ca2+-activated Cl- channel from bovine trachea by InsP4 (25). These results suggest an apical GCl may be the inhibitory site of action for InsP4. However, as outlined in the introduction, the role of a Ca2+-activated Cl- channel in mediating intestinal Cl- secretion in an intact epithelium remains obscure and awaits direct electrophysiological confirmation.

Inhibition of KCa by fatty acids. Ca2+-dependent agonists are known to increase AA levels in a wide range of tissues. This can occur in several ways (1): 1) Ca2+ can directly activate PLA2, 2) either DAG itself or PKC can activate PLA2, or 3) DAG lipase can directly generate AA from DAG. The generation of AA by Ca2+-mediated agonists lags behind the rise in intracellular Ca2+. Thus an effect of AA on the transporters associated with Cl- secretion would be temporally appropriate to explain the dissociation between the Ca2+ and Isc. Numerous agonists, both Ca2+ dependent and cAMP dependent, including the kinins (30), bile acids (11), adenosine (6), vasoactive intestinal polypeptide (5), and Ca2+ ionophores (23), have been shown to increase the levels of AA in intestinal tissues. We previoulsy demonstrated that both taurodeoxycholic acid and Ca2+ ionophores activate KCa in the colonic cell line T84 (14). In the present communication, we demonstrate that an additional second messenger known to be generated by these agonists, AA, potently inhibits KCa. Thus AA may serve as an important second messenger in the Cl- secretory response to these agonists.

Fatty acids are known to affect a wide variety of ion channels, including those for K+, Na+, Ca2+, and Cl- (33, 36). Although the effects of AA on voltage-dependent channels in neuronal and muscle tissues have received a great deal of attention, the effects of AA on channels involved in transepithelial transport have been little studied (33, 36). Although AA has been shown to inhibit Cl- channels in airway (2, 24) and intestinal epithelium (29), the role of AA in modulating intestinal K+ channels has heretofore not been characterized.

In the present communication, we demonstrate that AA potently (Ki 425 nM) inhibits the basolateral membrane K+ channel that is activated by Ca2+-dependent agonists (14, 38, 43). Our results suggest this inhibition is not dependent on the generation of either cyclooxygenase- or lipoxygenase-dependent oxidative metabolites of AA. Indeed, native colonic epithelia have a limited ability for the oxidative conversion of AA by the cyclooxygenase pathway (30). The concentration of AA found to inhibit KCa is well within the physiological levels produced after agonist stimulation. In in vitro assays, the Michaelis constant for either the cyclooxygenase- and lipoxygenase-dependent oxidation of AA is 3-5 µM (35). We attempted to rule out the possibility that a cytochrome P-450-dependent metabolite was responsible for the AA-induced inhibition of KCa using the cytochrome P-450 inhibitor clotrimazole. However, clotrimazole, as well as several additional cytochrome P-450 inhibitors, inhibited KCa directly in excised, inside-out patches (20). However, because cytochrome P-450 requires NADP and ATP for the oxidative metabolism of AA, it is unlikely that cytochrome P-450 is involved in the effects of AA on excised membrane patches.

The inhibitory effects of AA on KCa can occur from either the intracellular or extracellular side of the channel. A similar lack of sidedness of AA effects has previously been reported for other K+ channels (33, 36). An extracellular effect could be important for two reasons: 1) because fatty acids are highly lipophilic, they may cross from the cytoplasm to the extracellular space where they could act as autocrine factors modulating the Cl- secretory response of adjacent cells; and 2) during an inflammatory response, AA released from migrating neutrophils may be important in modulating the transport properties of the epithelium.

The effect of AA on KCa is not specific, and other fatty acids were capable of inhibiting KCa in excised patches. The specificity of fatty acid effects on K+ channels is well known to differ widely in various tissues (33, 36). Nevertheless, the effects we observe allow us to draw several conclusions. 1) These effects are unlikely to be caused by general membrane fluidity changes, because cis-unsaturated fats and saturated fats (myristic acid) should have opposite effects on membrane fluidity. However, it is possible that the membrane lipid composition maintains an optimal fluidity set point for channel activity such that deviations in either direction will influence channel gating. 2) Neither elaidic acid nor myristic acid is a substrate for oxidation by cyclooxygenase or lipoxygenase (36). This is consistent with the lack of effect of inhibitors of these enzymes employed in our studies. 3) AA is an activator of PKC, but neither myristic acid nor elaidic acid, which inhibits KCa, is capable of activating PKC (42). This is consistent with the absence of a direct effect of PKC on KCa activity (see above).

Effect of cPLA2 inhibition. Our results demonstrate that inhibition of cPLA2 potentiates the subsequent response to the Ca2+-dependent agonist carbachol. This is consistent with the notion that the generation of AA by carbachol attenuates the Cl- secretory response. As we have demonstrated that AA is a potent negative modulator of KCa, and KCa is known to be activated by carbachol (14), the most parsimonious explanation of these results is that the activation of cPLA2 by carbachol results in the liberation of AA, which subsequently inhibits KCa, resulting in an attenuation of the Cl- secretory response. Others have previously reported that inhibition of AA generation by blocking DAG lipase activity results in a potentiation of both carbachol-induced (31) and histamine-induced (5) Cl- secretory responses in T84 cells. These results are consistent with the hypothesis that AA acts as a negative modulator of Ca2+-dependent Cl- secretion. Also, the kinins (30) and bile acids (11), both of which are Ca2+-dependent agonists, have previously been shown to increase intestinal AA levels. We previously demonstrated that the bile acid taurodeoxycholic acid similarly activates KCa in T84 cells (14). However, direct measurements of AA generation and cPLA2 activation during agonist stimulation in T84 cells are required to confirm this proposal.

In conclusion, we have characterized the effects of several potential second messengers on the activity of the basolateral membrane K+ channel activated during a Ca2+-dependent Cl- secretory response in the colonic cell line T84. Two previously proposed candidate second messengers, PKC and InsP4, had no effect on KCa. These results suggest that if PKC and InsP4 affect Isc during a Ca2+-dependent Cl- secretory response, it is at a site distinct from KCa. Because Ca2+-dependent agonists do not increase apical GCl in intestinal tissue (3, 18, 37, 53), this is unlikely to be the site of action. However, additional possibilities that need to be explored include the Na+-K+-2Cl- cotransporter as well as the Ca2+ entry step at the basolateral membrane. AA potently inhibited KCa in single-channel recordings. Also, inhibition of cPLA2 potentiated the subsequent carbachol-induced Cl- secretory response. These results suggest that AA is a pivotal second messenger in modulating Ca2+-dependent responses in intestinal epithelia.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the excellent technical assistance of Cheng Zhang Shi in both tissue culture and Ussing chamber experiments.

    FOOTNOTES

This work was supported by Cystic Fibrosis Foundation Grant DEVOR96PO (to D. C. Devor) and National Institute of Diabetes and Digestive and Kidney Diseases Research Grant DK-31091 (to R. A. Frizzell).

Address for reprint requests: D. C. Devor, Dept. of Cell Biology and Physiology, S312 BST, 3500 Terrace St., University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261 (E-mail: dd2+{at}pitt.edu).

Received 7 February 1997; accepted in final form 20 September 1997.

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