INTRODUCTION

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FLUID SECRETION ACROSS epithelial cells involves the activation of several apical Cl channels, including those dependent on intracellular Ca²⁺ (1). The properties of Ca²⁺-activated currents from different cell types are similar, conceivably suggesting the existence of a family of Ca²⁺-dependent Cl channels. One of the best-studied secretory cell model systems is the human colonic cell line designated T84. We have investigated Ca²⁺-activated Cl channels in rat parotid acinar cells (3-5) and found that these channels share many properties with those in the T84 cell line (7, 8, 25). The similarities of these Ca²⁺-activated Cl channels include 1) activation that is both voltage and time dependent, 2) currents that exhibit an outwardly rectifying current-voltage relation, and 3) channel activation that is inhibited by intracellular acidification.

The Ca²⁺-dependent channels present in the T84 cell line are regulated by a multifunctional Ca²⁺- and calmodulin-dependent protein kinase (CaM kinase) II mechanism. This conclusion results from several studies of the sensitivity of these channels to activated CaM kinase II as well as CaM kinase II inhibitors (7, 8, 25, 26). Ca²⁺-activated channels in several other epithelial cells appear to be regulated by a similar mechanism (9, 12, 18, 24). Because the mechanism of activation of the channels in rat parotid acinar cells has not previously been investigated, we examined the possibility that these apparently similar channels are also activated by a CaM kinase II mechanism. We compared the actions of several CaM kinase II inhibitors on the Ca²⁺-activated Cl channels in both T84 and in rat parotid acinar cells. All these agents markedly reduced Ca²⁺-activated Cl currents in T84 cells but, under identical conditions, were without effect on the channels in parotid cells. These results indicate that it is unlikely that CaM kinase II is involved in the activation of the Cl channels in parotid acinar cells.

	METHODS

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Cell culture. T84 cells (CCL-248, American Type Culture Collection, Rockville, MD) were grown at 37°C in a humidified chamber gassed with 95% air and 5% CO₂. Cells cultured in 50% Ham's F-12 medium-50% Dulbecco's modified Eagle's medium containing 5% fetal bovine serum were dispersed by incubation for 20 min with Ca²⁺- and Mg²⁺-free phosphate-buffered solution. Dispersed cells were plated on glass coverslips (Rochester Scientific, Rochester, NY) and incubated for an additional 48 h before use.

Single cell dissociation. Single acinar cells were dissociated from parotid glands of male (150-250 g) Wistar strain rats (Charles River, Kingston, NY) as previously described (2). Glands were minced in Ca²⁺-free minimum essential medium (MEM; GIBCO BRL, Gaithersburg, MD) containing 1% bovine serum albumin (BSA; fraction V, Sigma Chemical, St. Louis, MO). Tissue was treated for 20 min at 37°C with a 0.02% trypsin solution (Ca²⁺-free MEM + 1 mM EDTA + 2 mM glutamine + 1% BSA). After the reaction had been quenched with 2 mg/ml soybean trypsin inhibitor (Sigma), the tissue was further dispersed by two sequential treatments with collagenase (100 U/ml, type CLSPA, Worthington Biochemical, Freehold, NJ) in Ca²⁺-free MEM + 2 mM glutamine + 1% BSA. The dispersed tissue was then centrifuged and washed with basal medium Eagle (GIBCO BRL) supplemented with 2 mM glutamine, and the resuspended cells were plated onto poly-L-lysine-coated coverslips.

Whole cell patch clamp. The whole cell patch-clamp technique (13) was used to record Cl currents from single parotid acinar and T84 cells (3). The actions of CaM kinase II inhibitors were assayed at least 8 min after achieving whole-cell recording mode to allow for equilibration of the pipette solution with the cell interior. Cells were bathed in a solution containing (in mM) 135 tetraethylammonium (TEA) chloride, 0.5 CaCl₂, 20 N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), and 60 D-mannitol, pH 7.3. In most experiments, Cl currents were activated by application of the Ca²⁺ ionophore ionomycin (4 µM). The Ca²⁺-sensitive current was obtained by subtracting the nonstimulated basal current from the current in the presence of ionomycin. These cells were dialyzed with (in mM) 135 TEACl, 5 TEAF, 1 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-TEA, and 20 TES, pH 7.3. Possible differences in ionomycin-induced intracellular Ca²⁺ levels were eliminated by directly activating the channels with 250 nM free Ca²⁺ in a buffered pipette solution containing (in mM) 36.2 EGTA-TEA, 26.2 CaCl₂, 2.5 TEAF, and 50 TES, pH 7.3. The membrane potential was changed by delivering square pulses of 500 or 2,500 ms every 5 or 10 s, respectively, from a holding potential of 50 mV. Current-voltage relations were determined from the currents at the end of the test pulse. Under the conditions used in this study, these currents are close to steady-state levels. An Ag-AgCl pellet was used to ground the bath through a 1 M CsCl agar bridge. Membrane potentials were corrected for liquid junction potentials. Data were collected using custom-designed software and hardware.

Reverse transcription-polymerase chain reaction amplification. Total RNA was isolated from rat parotid glands using TRIzol reagent (GIBCO BRL), and cDNA was synthesized according to previously described methods (5). The cDNA was amplified using polymerase chain reaction (PCR) primer sets that recognized the calmodulin-binding region and the association domain of rat CaM kinase II genes (23). The 50-µl PCR reaction mixture contained 2 µl of the cDNA-containing solution, 0.2 µM upper (U) and lower (L) primers, 3 µl of 25 mM MgCl₂, 4 µl of 2.5 mM dNTPs, 5 µl of 10× PCR buffer [500 mM KCl and 100 mM tris(hydroxymethyl)aminomethane (Tris) · HCl, pH 8.3] and 0.2 µl (1 U) of AmpliTaq DNA polymerase (Perkin-Elmer, Norwalk, CT). PCR amplification was carried out for 30 cycles in a DNA thermal cycler (MJ Research, Watertown, MA), and each cycle was set to 94°C for 30 s, 69°C for 30 s, and 72°C for 1 min. A 2-µl aliquot of the first-round PCR reaction mixture was reamplified using the same PCR conditions. PCR amplification products were separated on 1% Tris base-EDTA-boric acid-agarose gels and further analyzed by DNA sequencing.

View larger version (93K): [in this window] [in a new window]   Fig. 1.   Reverse transcription-polymerase chain reaction (RT-PCR) of CaM kinase II- transcripts in rat parotid gland. Amplified products from parotid gland (P), molecular weight markers (M; 1-kilobase ladder, GIBCO BRL), and a negative control () with no template added are shown. Sequence analysis of ~220-base pair RT-PCR product verified that CaM kinase II- transcripts are present in rat parotid gland.

	RESULTS

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Ca²⁺-activated Cl currents. Figure 2 illustrates the general similarity of Ca²⁺-activated Cl currents in parotid acinar (Fig. 2, A and C) and T84 cells (Fig. 2, B and D). Figure 2, A and B, shows currents recorded in the absence of the Ca²⁺ ionophore ionomycin (unstimulated). Illustrated are currents recorded during voltage-clamp steps to 80 and +80 from a holding potential of 50 mV. The currents from both cell types are quite small (<0.2 nA) in the absence of ionomycin and increase to high levels (1-2 nA) in the presence of ionomycin. The magnitude and time course of the currents in the two types of cells are generally similar and consistent with the high levels of intracellular Ca²⁺ induced by this concentration of ionomycin (4). Although we did not do an in-depth pharmacological analysis of the Ca²⁺-activated Cl currents in rat parotid acinar cells, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) produced a voltage-dependent block similar to that previously reported for T84 cells (3). In five acinar cells treated with 500 µM DIDS, the currents were inhibited by 49 ± 4 at +60 mV and 17 ± 9% at 60 mV, whereas in T84 cells the currents were inhibited by 52 ± 12 at +50 mV and 20 ± 14% at 50 mV with 200 µM DIDS (3).

Effects of CaM kinase inhibition on Ca²⁺-activated Cl currents. Although Fig. 2 shows certain similarities between the Ca²⁺-activated Cl channels in the two cell types, Fig. 3 shows that they differed in their response to the presence in the patch pipette of the CaM kinase inhibitor KN-62. KN-62 selectively inhibits CaM kinases by competing with calmodulin for the calmodulin-binding site on the kinase with an inhibition constant (K_i) of ~1 µM for CaM kinase II (14). Even at 10 times this concentration, KN-62 does not inhibit protein kinase A or protein kinase C (14). As in Fig. 2, currents to potentials of 80 and +80 mV are shown in Fig. 3. Figure 2, A and C, shows that ionomycin was still able to substantially activate current in parotid cells in the presence of KN-62. In contrast, this compound inhibited the ability of ionomycin to activate the channels in T84 cells (Figure 2, B and D).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Ca2+-activated Cl currents from a parotid acinar cell and from a T84 cell. Raw currents from a parotid acinar cell (A and C) and from a T84 cell (B and D) before (A and B) and during (C and D) application of 4 µM ionomycin. Displayed currents were obtained during voltage-clamp steps to +80 and 80 mV from a 50 mV holding potential. Dashed lines, 0 current.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Actions of KN-62 on Ca2+-activated Cl currents from a parotid acinar cell and from a T84 cell. Raw currents from a parotid acinar cell (A and C) and from a T84 cell (B and D) dialyzed with 10 µM KN-62 before (A and B) and during (C and D) application of 4 µM ionomycin. Displayed currents were obtained during voltage-clamp steps to +80 and 80 mV from a 50 mV holding potential. Dashed lines, 0 current.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Effects of intracellular dialysis with the multifunctional Ca2+- and calmodulin-dependent protein kinase (CaM kinase) II inhibitor KN-62 on current-voltage relations for currents recorded from parotid acinar cells (A) and from T84 cells (B). Ionomycin-induced (4 µM) Cl currents were recorded from control cells (; n = 5 for parotid cells and n = 5 for T84 cells) and from cells dialyzed with 10 µM KN-62 (; n = 4 for parotid cells and n = 5 for T84 cells). Currents for parotid cells in presence of KN-62 were not significantly different from controls (P > 0.05). Em, membrane potential.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of pretreatment with the CaM kinase II inhibitor KN-62 on current-voltage relations recorded from parotid acinar cells (A) and from T84 cells (B). Current-voltage relations were constructed for currents activated by 250 nM free Ca2+ from untreated parotid acinar and T84 cells (; n = 12 for parotid cells and n = 6 for T84 cells) or cells pretreated for at least 30 min with 10 µM KN-62 (; n = 6 for parotid cells and n = 7 for T84 cells).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Actions of the CaM kinase II inhibitor peptide 281-302 on activation of Ca2+-dependent Cl current in rat parotid acinar cells. Current-voltage relations were constructed for ionomycin-activated Cl currents recorded from cells dialyzed without (; n = 7) and with (; n = 4) 40 µM peptide 281-302.

Effects of a specific calmodulin inhibitor on Ca²⁺-activated Cl currents. Unlike peptide 281-302, which binds at the catalytic site on CaM kinase II, the calmodulin-binding domain (CBD) peptide 290-309 binds to calmodulin (with a K_i of 52 nM) and so inhibits CaM kinase II (6, 15, 20) as well as other calmodulin-dependent processes. Figure 7 summarizes the current-voltage relations obtained from parotid cells (A) and from T84 cells (B) dialyzed with and without 0.5 µM CBD peptide. The magnitude of ionomycin-induced Cl currents in T84 cells was inhibited ~70% by 0.5 µM peptide (n = 5). In contrast, the currents in rat parotid cells were not changed by the CBD peptide.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effects of the calmodulin antagonist peptide 290-309 [calmodulin-binding domain (CBD) peptide] on activation of Ca2+-dependent Cl current in parotid acinar cells (A) and T84 cells (B). Current-voltage relations were constructed for ionomycin-activated Cl currents recorded from cells dialyzed without (; n = 4 for parotid cells and n = 5 for T84 cells) and with (; n = 4 for parotid cells and n = 5 for T84 cells) 0.5 µM peptide 290-309.

	DISCUSSION

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Our results demonstrated an inhibition of Ca²⁺-dependent Cl channel current in T84 cells by KN-62 and the CBD peptide 290-309 and are thus consistent with the requirement for CaM kinase II activation of the channels in these cells (7, 8, 25, 26). Conversely, under identical conditions, we found that selective inhibitors of calmodulin and CaM kinase II were without effect on the Ca²⁺-activated Cl channel in rat parotid acinar cells. Thus CaM kinase II activity is not required for activation of Ca²⁺-dependent Cl channels in these exocrine cells. This indicates that the Ca²⁺-activated Cl channels in parotid acinar cells are rather different from the channels in T84 cells (7, 8, 25, 26) and in the other cell types (9, 12, 18, 19, 21, 24) in which CaM kinase II involvement has been established.

Because peptide 290-309 mimics the calmodulin-binding site on CaM kinase II, it acts by binding to calmodulin and so inhibits all calmodulin-dependent processes. Consequently, our results indicate that Cl channel activation in parotid acinar cells is not only independent of CaM kinase II but of calmodulin-dependent activity as well.

The Ca²⁺-sensitive Cl channels in cell-free patches from human airway epithelia (11), guinea pig hepatocytes (16), and submandibular gland cells (17) are activated by application of Ca²⁺ to the intracellular surface, suggesting that Ca²⁺ may directly activate these channels. Our results showing the failure of CaM kinase antagonists to inhibit channel activation in parotid acinar cells are consistent with a direct activation of the Cl channel by Ca²⁺. We have shown previously that Ca²⁺ activation of the channels in rat parotid acinar cells is voltage dependent (4). This result indicates that the Ca²⁺-binding site is located within the electrical field of the plasma membrane. One interpretation of these results is that intracellular Ca²⁺ directly bind to Cl channels to open the pore.

In conclusion, there appear to be two distinct Ca²⁺-dependent Cl channels in secretory epithelia: channels in exocrine gland cells directly activated by Ca²⁺ and channels like those found in other secretory cells, including airway and colonic epithelia (7-9, 12, 18, 24-26), whose activation requires CaM kinase II. A calmodulin-dependent Cl channel has recently been cloned from bovine tracheal epithelium (10). Functional expression of this channel suggests that it shares many of the properties of the Ca²⁺-dependent Cl channels present in T84 cells and so may represent the prototypical CaM kinase II-dependent Cl pathway. Further studies will be necessary to establish the molecular identity of Ca²⁺-activated Cl channels that are not dependent on calmodulin-sensitive processes.