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Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role

To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free [Ca] ([Ca]i) and the potassium conductance (gK) of the basolateral membrane in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value ([Ca]'i) normalized to control. To assess changes in basolateral gK, we measured the short circuit current (Isc) in the presence of luminal amphotericin and a transepithelial mucosa-to-serosa K+ gradient (Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson. 1986. J. Gen. Physiol. 88:237-251). Treatment of the monolayers with carbachol resulted in a parallel increase and then decrease in [Ca]'i and gK. The carbachol-induced changes in gK appeared to be dependent on the increase in [Ca]i because stimulation of gK was significantly diminished when the hormone-induced increase in [Ca]'i was blunted, either by loading the cells with BAPTA or by reducing the extracellular [Ca]. The carbachol-stimulated increase in gK appeared to be the direct result of the increase in steady-state [Ca]'i. The changes in gK and [Ca]'i after stimulation with carbachol were correlated and ionomycin also increased gK and [Ca]'i in a parallel manner. The carbachol- induced delta gK per delta[Ca]'i, however, was greater than that after ionomycin. Because ionomycin and carbachol appear to open the same channel, a conclusion based on inhibitor and selectivity experiments, carbachol may have a second action that amplifies the effect of calcium on gK.

Earlier studies (McRoberts et al., 1985;Dharmsathaphorn and Pandol, 1986) suggested that one of the sites of calcium's action is a potassium channel in the basolateral membrane. Both carbachol and the calcium ionophore ionomycin stimulated a quinidine-sensitive rubidium flux across the basolateral membrane.
The mechanism by which carbachol, acting via calcium, might increase the basolateral gx in T84 cells could be similar to that demonstrated in exocrine cells (for reviews, see Petersen andMaruyama, 1984 andPetersen, 1986). In numerous tissues carbachol increases cell [Ca], which in turn opens large conductance calciumactivated channels in the basolateral membrane (Maruyama et al., 1983;Trautmann and Marty, 1984;Petersen and Findlay, 1987;Richards et al., 1989). These channels are sensitive to [Ca] in a range well within that attained during stimulation with carbachol.
Recent experiments suggest that the basolateral potassium channels in T84 cells are not likely to be of the large conductance variety and probably are of the small conductance class found in trachea (Welsh and McCann, 1985) and MDCK cells (Kolb et al., 1987). The carbachol-stimulated gx in T84 cells is insensitive to both barium and TEA (Devor et al., 1990), quite unlike the large conductance channels (Latorre and Miller, 1983). Carbachol might nevertheless act via an increase in cell [Ca] because the carbachol and ionomycin-stimulated gx have similar characteristics, indicating that gx is calcium activated (Devor et al., 1990).
If carbachol increases gx in T84 cells via a change in steady-state [Ca]i, as it does in exocrine cells, the range of [Ca] in which calcium-activated channels open should be similar to the range of cell [Ca] achieved after stimulation with carbachol. This comparison cannot be made in T84 cells because the calcium sensitivity of gx is presently unknown. However, single channel calcium activation studies of the small conductance channel have been performed in other chloride-secreting epithelial tissues. In the trachea the channel remains in a closed state when [Ca] is 100 nM and opens submaximally when [Ca] is increased to 1 #M (Welsh and McCann, 1985), whereas in MDCK cells the channel is more sensitive to calcium, opening at 100 nM and reaching maximal conductance at 1 #M (Kolb et al., 1987). Given the fact that carbachol rarely increases [Ca]i to levels significantly above 300 nM in T84 cells (Dharmsathaphom et al., 1989;Wong et al., 1989), it would appear that the small conductance channel might remain unopened during carbachol's action.
Carhachol thus might not activate gx via changes in cell [Ca] in T84 cells if the small conductance calcium-activated channel does not open at levels of [Ca] i achieved during stimulation with carbachol. There are other explanations accounting for this discrepancy, however. First, the conditions under which calcium-activation curves and cell [Ca] are measured are quite different. Patch clamp studies are performed by exposing naked membrane to EGTA-calcium buffered solutions, whereas measurements of cell calcium with calcium-sensitive dyes are performed in intact cells. Thus, if the cytoplasm contained factors that increased the affinity of the channel for calcium, the affinity measured in an intact cell might be lower than that measured in a cell-detached patch of membrane. Second, carbachol might have another action that raises the affinity of the channel for calcium, such as phosphorylation (Ewald et al., 1985). Third, the action of calcium on the basolateral potassium conductance might be an indirect mechanism. The rise in cell [Ca] could trigger a second event which in turn opens the channel. If this were the case, gr might not be a function of the steady-state [Ca]i.
There are understandable difficulties in using a comparison of calcium activation curves with measurements of cell [Ca] to reach a conclusion regarding calcium's role in regulating gK in T84 cells. We sought a more direct approach to this problem and devised a method to make simultaneous measurements of intracellular [Ca] and basolateral gx (in the same tissue). Our results suggest that the action of carbachol on the basolateral gx is mediated by changes in the steady-state [Ca] i. Carbachol increases gx and [Ca] i in a parallel manner throughout the effect of the hormone, the hormone-induced changes in gx are correlated to the carbachol-induced changes in [Ca] i, and the increase in gx is dependent on the carbachol-induced increase in [Ca] i. However, carbachol appears to be more effective than ionomycin at stimulating gK, per change in [Ca]i, suggesting that carbachol might have a second action that enhances the effect of calcium on gx.

Cell Culture
T84 cells, derived from human colonic cancer (Murakami and Masui, 1980), were cultured as described previously (Dharmsathaphorn et al., 1984;Wong et al., 1989). To measure [Ca]i and short circuit current (I~), cells were plated on collagen-coated filters (Transwell filters; Costar, Cambridge, MA) at a density of 2.4 • 106 cells/4.7 cm ~. It took ~2 wk for the cells to reach confluence and develop a transepithelial resistance high enough to allow for measurements of I~ (>600 f~-cm~).

Measurement of [Ca]i
[Ca]i was measured with fura-2 and fluorescence microscopy using methods described previously (Chase and Wong, 1988;Wong and Chase, 1988;Wong et al., 1989). In brief, cells grown on filters were removed from the incubator and bathed in Hanks buffer containing (in mmol/liter): 137 NaCI, 4.2 NaHCO s, 0.3 Na~HPO 4, 5.4 KC1, 0.4 KI-I2PO 4, 1.3 CaCI2, 0.5 MgCI~, 0.8 MgSO 4, and 20 HEPES, brought to a pH of 7.5. The cells were loaded by incubation with a Hanks solution containing 10 tzM fura-2 acetoxymethylester and 0.1% albumin on the serosal side. After loading was completed the cells were washed free of the ester by rinsing numerous times with Hanks buffer. Approximately 30 min later the cells were placed into the Ussing chamber on the stage of the microscope for fluorescence readings. When solutions were changed during an experiment, the lower or upper chamber was emptied using light suction and the solution was replaced with buffer. All experiments were performed at room temperature.
Epifluorescence was measured using an upright microscope (Carl Zeiss, Inc., Thomwood, NY) equipped with a 75-W xenon light source, a water immersion 40 x lens, and a silicon intensifier target camera (SIT camera, series 66; Dage-MTI, Inc., Michigan City, IN). The sample was excited at 380 and 350 nm using band pass filters that were changed automatically. The image from the camera was recorded by a video recorder and displayed on a video monitor. The signal output from the video recorder first passed through a video analyzer (Instrumentation for Physiology & Medicine; San Diego, CA) before going to the monitor. The fluorescence of the sample was measured using the video analyzer, which transformed the light intensity of the image on the video screen to a voltage yielding a "real time" strip-chart record of the changes in light intensity. THE JOUt~AL oF GENERAL PHYSIOLOGY 9 VOLUME 96 -1990

Calculation of [Ca]i
One can derive a value for [Ca] i by calculating the fractional saturation of the dye. We used 380/350 nm ratios (r), rather than fluorescence at a single wavelength, to make this calculation (Grynkiewicz et al., 1985). Thus, where rm~ and r,u n are the maximum and minimum fluorescence 380/350 ratios and s is the fluorescence at 350 nm when the dye is either free of calcium (sf) or saturated with calcium (sb). By arrangement To calculate [Ca]~ the maximum and minimum signals must be known to define the boundaries of dye fluorescence. With these ratios [Ca]i is calculated by inserting the value for the K~ (160 nM, based on the published K d of 200 nM [Grynkiewicz et al., 1985] and the value of sf/s b of 0.8, experimentally determined at 350 nm). To obtain the minimal fluorescence ratio, we ended each experiment by adding 5 ~M ionomycin, a calcium ionophore, and excess calcium (~30 rain). To obtain the maximal fluorescence we added excess EGTA to a calcium-free buffer. Using this protocol we determined in a previous publication (Wong et al., 1989) that the average resting [Ca] i was 25 • 5 nM (n ----4).

Normalization of [Ca]i
In many experiments ionomycin was added to increase [Ca]~ to very high levels. Once [Ca]~ rose to these levels, however, it was often impossible to lower [Ca]i with EGTA to obtain the maximal fluorescence ratio. Without a maximal value of fluorescence, a precise calculation of [Ca]i could not be made. To present the data in a consistent manner we derived a normalized value of [Ca]i ([Ca]~) (Wong et al., 1989). This was accomplished by assigning a value for the missing maximum fluorescence so that at the start of each experiment the derived value of [Ca]i was 25 nM, the average control [Ca]~ (see above).

Measurement of Potassium Conductance of the Basolateral Membrane
The basolateral potassium conductance was measured using the technique developed by Dawson and co-workers (Kirk and Dawson, 1983;Germann et al., 1986). A potassium gradient (m----s) is first established across the monolayer by substituting KCI for NaO in the Hanks buffer bathing the mucosal side. Amphotericin is then added to the mucosal side to eliminate the mucosal barrier as rate limiting to transepithelial potassium movement. Under the conditions of the experiment, in which the monolayer is short circuited (i.e., voltage-clamped at zero potential) and the transepithelial potassium gradient is constant, the amphotericindependent I~ becomes a measure of the rate of the transepithelial potassium flux. Because the flux of potassium across the cell is limited by the basolateral potassium conductance, a change in I x represents, in turn, a change in gK.
I~ was measured using calomel electrodes, 3 M KCl-agar bridges, and a DVC-1000 voltage clamp (World Precision Instruments, Inc., New Haven, CT). Resistance was measured by pulsing the tissue with enough current to increase the transepithelial potential 5 or 10 inV.

Use of Gluconate Buffers
In a number of experiments gluconate was substituted for chloride. Because of the significant chelation of calcium by gluconate, the total [Ca] of the gluconate-Hanks buffers had to be increased to 5-7 mM so that the free [Ca] would be the same (1 mM) as the chloridecontaining buffers. Free [Ca] was measured using a calcium electrode (Radiometer America, Inc., Westlake, OH).

Statistics
Results are expressed as the mean • SEM. We used the Student's t test to compare different groups in individual experiments or the percent increase over control. A result was considered significant if P < 0.05. For each group of experiments n refers to the number of experiments performed.

Effects of Amphotericin and Carbachol on [Ca]i and Potassium Conductance of the Basolateral Membrane
A typical experiment, in which the transepithelial potassium flux (I~ and [Ca]i were recorded simultaneously, is depicted in Fig. 1 Fig. 1 also demonstrates that the addition of amphotericin unmasks a very large gK. Previous work established that this large increase in gK is due to cell swelling, observed only when chloride is present in the buffers (Germann et al., 1986). To eliminate any effect that cell swelling might have on the cell's response to carbachol, we examined the effect of carbachol on I~ and [Ca]~ in chloride-free buffers. As shown in Fig. 2, the addition of amphotericin to the mucosal bathing solution had no effect on I~ when chloride was absent from the buffers. When carbachol was then added to the serosal solution the same parallel increase in I~ and [Ca]~ occurred (Fig.  2 A). I,r and [Ca]~ first increased and decreased quickly and then declined further over the next 5 min. The responses of I~= and [Ca]~ to carbachol were reversed by washing the hormone from the basolateral compartment. Not only were the carbachol-induced changes in I~c and [Ca]~ parallel, they were also correlated ( Fig.  2 B). In a plot of Isc as a function of [Ca]~, the control, carbachol peak, 2-and 5-rain points, and wash all fall on the same line.

Dependence of the Carbachol-induced Changes in gx on the Increase in [Ca]i
To determine if the carbachol-induced increase in gK was dependent on the rise in steady-state   (Tsien, 1980). As demonstrated in Fig. 3 A, the effect of carbachol on I~ and [Ca]~ in BAPTA-loaded cells was delayed, and the peak increases in Itr and [Ca]~ were significantly blunted (compare with the control response depicted in Fig. 1). The peak in I~ and [Ca]~ did not occur for almost 2 min and the magnitude of the peak was well below the response in the control monolayers. The blunted response of the BAPTA-loaded cells is best demonstrated in a plot of I= as a function of [Ca]~ (Fig. 3 B), showing that the increases in I= and [Ca]~ were clearly not of the same magnitude as in the control cells.
We used a second procedure, lowering extracellular [Ca], to reduce the magnitude of the carbachol-induced increase in [Ca]~ (Penner et al., 1988). Fig. 4 (Fig.  4 B, left). A Hill analysis (Fig. 4 B, Fig. 5 B, where it is shown that the slope of the relationship (&/~./A[Ca]~) is steeper after carbachol than after ionomycin. A similar result was obtained if we reversed the order of the experiment: first adding carbachol, washing, and then adding ionomycin (Fig. 6). The quantitative dissimilarity between the effects of carbachol and ionomycin suggested that carbachol might open a second channel in addition to the calciumactivated channel also opened by ionomycin. To explore this issue we examined the inhibitor profile and ion selectivity of the carbachol and ionomycin-stimulated gK. As shown in Table I, the hormone-and ionomycin-induced gK had the same inhibitor sensitivity and ion selectivity, clearly different from the gg observed after the addition of amphotericin alone: it was blocked by quinidine, was resistant to both barium and TEA, and permitted rubidium to pass through as easily as potassium. Carbachol and ionomycin are thus likely to activate the same basolateral potassium channel.

Role of Calcium in the Action of Carbachol on the BasolaWral Potassium Conductance
The carbachol-activated changes in basolateral gK in T84 cells are probably due to the changes in the steady-state [Call. This conclusion follows from the observations that the hormone-induced changes in gK and [Ca] i are parallel throughout the entire effect of the hormone (Fig. 2 B), that the increase in KK is correlated to the rise in [Ca]i (Figs. 3 and 4  All experiments were performed in Hanks buffer with chloride. In each group at least four experiments were performed. 5 mM barium and 10 mM TEA were added to the serma while 250 ~tM q 'umidine was added to the mucosa.

B), and that increasing cell [Ca] with ionomycin increases [Ca]i
*Though TEA actually inhibited the response to carbachol by 42 + 8%, the response was probably due to the atropineqike action of TEA (Sutliff et al., 1989). Using a K~ for TEA of 1.4 mM and a Kd for carbachol of 170 #M, 10 mM TEA would be expected to inhibit the action of 100 ~M carbachoi by 52%. Thus, the effect of TEA on carbachol's stimulation ofg~ was negligible. :Not significantly different from 1.0. tll,.b value was determined by dividing the ionomycin-induced &/sc using rubidium (179 • 19/~A) by A/sc using potassium (133 • 27). There is no significant difference between these two means. and gK in a parallel manner (Fig. 5 A) by opening the same channel as that activated by carbachol. Calcium thus appears to serve as carbachol's second messenger in T84 cells, as it does in exocrine tissues (Maruyama et al., 1983;Trautmann and Marty, 1984;Petersen and Findlay, 1987;Richards et al., 1989). This is an interesting observation considering that the basolateral channel activated in T84 cells is more likely to be of the small conductance variety, identified in both epithelial and nonepithelial tissues (Welsh and McCann, 1985;Sauve et al., 1986Sauve et al., , 1987Kolb et al., 1987;Furuya et al., 1989;Gallin, 1989), than of the large conductance type found in exocrine cells (Petersen, 1986). This conclusion is based on the observations that the calciumactivated gK in T84 cells (our studies and Devor et al., 1990) is barium and TEA insensitive, and, like the barium-and TEA-insensitive basolateral channel in turtle colon (Chang and Dawson, 1988), conductive to rubidium. This particular inhibitor and selectivity profile is uncharacteristic of the large conductance channel (Latorre and Miller, 1983) and more representative of the small conductance channel which is resistant to barium (Gallin, 1989) and TEA (Furuya et al., 1989). Demonstration that the basolateral calcium-activated potassium channel in T84 cells is of the small conductance variety will require single channel studies.
The mechanism by which calcium increases gK remains unknown. The action could be a direct one in which calcium interacts with the channel to open it, or an indirect one in which calcium associates with an intermediary to open the channel. For example, calmodulin could serve as middleman as it does in Paramecium (Hinrichsen et al., 1986). Future studies should be designed to elucidate the precise mechanism by which calcium increases gK. If carbachol stimulates a second process to enhance calcium's effect on basolateral gK it could do so in one of a variety of ways, including activation of a calciumdependent kinase such as protein kinase C (Audigier et al., 1988) or the calciumcalmodulin dependent kinase (Connelly et al., 1987), stimulation of exocytosis and insertion of calcium-activated potassium channels into the basolateral membrane (van Adelsberg and AI-Awqati, 1986), or increasing cell pH (Melvin et al., 1988) making gK more sensitive to changes in calcium (Christensen and Zeuthen, 1987).

Does Car~chol Act Exclusively via Changes in
Before concluding that carbachol has a second action, however, it must be demonstrated that the apparent dissimilarity of the carbachol-and ionomycininduced A/~/A[Ca] i is genuine. It is possible that the carbachol-stimulated increase in [Ca]i is predominantly in the vicinity of the basolateral membrane, while the increase in [Ca]i after ionomycin is more evenly distributed throughout the cell. A similar conclusion was reached in studies of parotid acinar cells in which it was shown that though the carbachol-stimulated gK Was dependent on a rise in [Ca] i, activation of gK could be observed before any detectible change in [Ca]i (Foskett et al., 1989). It seemed as if the true [Ca]i in the vicinity of the basolateral channel was much higher than that recorded from whole cell fluorescence measurements.
Using our present methodology, in which fluorescence readings represent an average value throughout the whole cell, we are unable to ascertain if the carbachol-In previous studies of T84 cells and sweat glands (Sato and Sato, 1988;Dharmsathaphorn et al., 1989;Wong et al., 1989) carbachol was found to be more effective than ionomycin in stimulating secretion. However, because secretion is a multi-step process, carbachol's superior action could be explained by a caicium-activated action at one step and a calcium-independent action at another. Our present results suggest, however, that there may be two carbachol-stimulated actions converging on a final common pathway. induced ~k/tc//A[Ca]i is the same or greater than that after ionomycin. Studies using confocal fluorescence microscopy will be required to determine directly if there are differences in the spatial localization of changes in [Call after ionomycin and carbachol. If A/~JA [Ca] i in the vicinity of the potassium channel after the two agents are indeed the same, then it will not be necessary to invoke a second carbacholactivated process.
One other explanation of the dissimilar response of the cells to ionomycin and carbachol is that ionomycin has a second, inhibitory action which reduces the sensitivity of gK for calcium. This is a less likely reason because carbachol increased gK in ionomycin-treated cells to a degree comparable to that observed in the non-ionomycin-treated control cells (Fig. 5).

Role of the Calcium-activated gx in Transepithelial Chloride Secretion
Previous studies have shown the carbachol-induced increase in chloride secretion in T84 cells is insensitive to both barium and TEA and inhibited by quinidine (Dharmsathaphorn and Pandol, 1986). The fact that the inhibitor profile is identical to that of the carbachol-stimulated glt suggests that hormone activation of gK is essential in the hormone-activated increase in transepithelial chloride secretion.
We thank Dr. Myles Akabas for his critical review and helpful suggestions and Heinz Rosskothen for making our Ussing chambers.