INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

K CHANNELS IN THE BASOLATERAL membrane of the medullary thick ascending limb (mTAL) play an important role in the regulation of epithelial transport in the loop of Henle. The basolateral K channels in the mTAL are responsible for K recycling across the basolateral membrane and participate in generating the cell membrane potential (Fig. 1), which partially determines the Cl diffusion potential across the basolateral membrane (26). Cl ions enter the cell across the apical membrane via Na-K-2Cl cotransporters and leave the cell across the basolateral membrane through KCl cotransporters or Cl channels along its electrochemical gradient (10, 11, 16). The driving force for the Cl exit across the basolateral membrane is the Cl electrochemical gradient, which is the difference between the chemical gradient of Cl and the cell membrane potential. An increase in basolateral K channel activity hyperpolarizes the basolateral membrane potential and augments the Cl diffusion potential, whereas a decrease in K channel activity reduces the driving force for Cl exit. Therefore, it is conceivable that changes in basolateral K channel activity are expected to affect the Cl diffusion rate across the basolateral membrane in the mTAL (11). Because the intracellular Cl concentration has been demonstrated to be a factor regulating the Na-K-2Cl cotransporter (22), it is possible that changes in basolateral K channel activity can indirectly affect the turnover rate of the cotransporter in the mTAL.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   The thick ascending limb (TAL) cell showing the major ion channels and transporters that are involved in mediating NaCl transport in the medullary TAL (mTAL) is shown. PD, membrane potential difference; ECl, electrochemical gradient for Cl.

Eicosanoids have been demonstrated to regulate the transepithelial NaCl transport in the mTAL (1, 2, 13, 15, 20). Moreover, a large body of evidence indicates that cytochrome P-450-dependent metabolites of arachidonic acid (AA) play an important role in regulating a variety of ion transporters in the kidney (8, 23, 27, 29). It has been shown that cytochrome P-450-dependent metabolites of AA inhibit the activity of the Na-K-2Cl cotransporter (8), Na-K-ATPase in the proximal tubule (23), the Ca²⁺-activated K channel of renal vascular smooth muscle (29), and the apical 70-pS K channels in the mTAL (27). Because an integrated mechanism of NaCl transport in the mTAL requires the involvement of basolateral K channels, it is conceivable that basolateral K channel activity must work in concert with other ion transporters, such as Na-K-2Cl cotransporters, to achieve the transport function of the tubule. Therefore, it is possible that AA may also regulate the basolateral K channels in the mTAL. This possibility is examined in the present investigation.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparation of the TAL. Pathogen-free Sprague-Dawley rats of either sex (90-100 g) were purchased from Taconic Farms (Germantown, NY). The animals were kept on a normal chow for 1 wk before use. Rats were killed by cervical dislocation, and the kidneys were removed immediately. Thin coronal sections were cut with a razor blade, and several small bundles of tubules separated from slices of the kidneys were incubated in a buffer solution containing collagenase type 1A (1 mg/ml; Sigma, St. Louis, MO) at 37°C for 45-60 min. After the collagenase treatment, the mTALs were isolated under a dissecting microscope. The dissection buffer solution contained (in mM) 140 NaCl, 5 KCl, 1.5 MgCl_2, 1.8 CaCl_2, and 10 HEPES (pH 7.4 with NaOH) at 22°C. The isolated tubule was transferred onto a 5 × 5-mm cover glass coated with Cell-Tak (Collaborative Research, Bedford, MA) to immobilize the tubule. The cover glass was placed in a chamber mounted on an inverted microscope (Nikon), and the tubules were superfused with HEPES-buffered NaCl solution composed of (in mM) 140 NaCl, 5 KCl, 1.8 CaCl₂, 1.8 MgCl₂, and 10 HEPES (pH = 7.4). The temperature of the chamber (1,000 µl) was maintained at 37 ± 1°C by circulating warm water around the chamber.

Patch-clamp technique. We used an Axon 200A patch-clamp amplifier to record channel current. The current was low-pass filtered at 1 kHz with an eight-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA) and digitized by an Axon interface (Digidata 1200). Data were collected by an IBM-compatible Pentium computer at a rate of 4 kHz and analyzed by using pClamp software system 6.04 (Axon Instruments, Burlingame, CA). Opening and closing transitions were detected with 50% of the single-channel amplitude as the threshold. Channel activity was defined as NP_o, which was obtained by using the following equation
in which t_i is the fractional open time spent at each of the observed current levels. NP_o was calculated from data samples of 60-s duration, which were always at the end of each experimental maneuver and in the steady state. Specifically, we used an all-point histogram (pClamp 6.04) to obtain the channel activity after determining the channel-closed level, which was easily detected because fewer than three channel levels were observed in >95% patches. No efforts were made to determine whether the change was induced by an alteration in channel number or P_o. To determine the mean channel open and closed times and P_o, we selected patches in which only one level of channel current was visible for at least 10 min. The slope conductance of the channel was determined by measurement of the K current at different holding potentials.

Chemicals and experimental solution. The pipette solution contained (in mM) 140 KCl, 1.8 mM MgCl₂, and 10 HEPES (pH = 7.4). The bath solution was composed of (in mM) 140 NaCl, 5 KCl, 1.8 CaCl₂, 1.8 MgCl₂, and 10 HEPES (pH = 7.4). AA and cis-11,14,17-eicosatrienoic acid were purchased from Nu-Chek Prep (model 56028, Elysian) and dissolved in 100% ethanol. Indomethacin was obtained from Sigma, and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) was synthesized at Dr. J. R. Falck's laboratory, University of Texas Southwestern Medical Center at Dallas.

Statistics. Data are shown as means ± SE. We used paired Student's t-tests to determine the significance of the difference between the control and experimental periods. Statistical significance was taken as P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We used the method described by Guinamard et al. (14) to patch the basolateral membrane of the mTAL. We observed channel activity in at least 245 patches out of a total of 817 high-resistance seals. Figure 2A is a typical channel trace recorded from a cell-attached patch with 140 mM KCl in the pipette and Ringer solution (5 mM KCl) in the bath. From an inspection of the channel recording, it is apparent that channel activity decreased when cell membrane potential hyperpolarized. Figure 2B shows the relationship between P_o and changes in the cell membrane potential; P_o was 0.51 ± 0.05 at the spontaneous membrane potential and decreased by 50% to 0.25 ± 0.03 by 30-mV hyperpolarization. Therefore, depolarization increases, whereas hyperpolarization decreases, channel activity. Channel activity was completely blocked by 1 mM Ba²⁺ (Fig. 2C), indicating that it is a K channel. The conductance of the K channel was inwardly rectifying, and the current-voltage curve yielded an inward slope conductance of 50 ± 2 pS between 20 and 20 mV (Fig. 2D). Figure 2E illustrates channel open and closed time histograms. The K channel has one open state with a mean open time of 5.8 ± 0.5 ms and two closed states (₁ = 1.9 ± 0.2 ms, ₂ = 14.9 ± 1 ms).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   A: typical recording showing the activity of the basolateral K channel in a cell-attached patch. Holding potential is indicated on the top of each trace. C, channel-closed level. B: relationship between open probability (Po) and changes in the cell membrane potential illustrates that depolarization activates the channel activity. C: channel recording showing the effect of 1 mM Ba2+ on channel activity in an inside-out patch. Ba2+ was added to the bath solution. D: current (I) and voltage (V) curves that were obtained from cell-attached patches () and from inside-out patches (). E: channel closed (a) and open (b) time histograms showing that two exponentials are required to fit the channel closed time (1 = 1.9 and 2 = 14.9 ms) and that the channel open time can be fitted with one exponential ( = 5.8 ms).

After confirming the presence of a 50-pS K channel in the basolateral membrane of the mTAL, we investigated the effect of AA on the activity of the 50-pS K channel. The reason for studying the effect of AA is because AA has been demonstrated to be an important player in the regulation of the apical K channels and Na-K-2Cl cotransporters in the mTAL (8, 27). Therefore, it is highly possible that AA may also be involved in the regulation of basolateral K channel activity. Figure 3A is a representative recording showing that AA inhibits the basolateral 50-pS K channel in a cell-attached patch. Addition of 5 µM AA decreased channel activity by 86 ± 8%, and NP_o fell from 0.58 ± 0.05 to 0.08 ± 0.01 (n = 8). The AA-induced channel inhibition was expected to depolarize the cell membrane potential, which was evidenced by a decrease in the channel current amplitude after AA application. However, addition of AA did not transiently increase NP_o, suggesting that the depolarization-induced increase in channel activity can be observed only when the K channel is in the open state. The effect of AA can be observed in inside-out patches, and AA decreased channel activity from 0.5 ± 0.05 to 0.03 ± 0.02 (n = 4; Fig. 3B). Also, the effect of AA is reversible. Figure 3C is a dose-response curve between AA concentrations and channel activity in cell-attached patches, and it shows that K_i, a concentration required to decrease channel activity by 50%, is ~3 µM.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   A: effect of arachidonic acid (AA) on the K channel activity in the basolateral membrane of the mTAL. The experiment was performed in a cell-attached patch with 140 mM K in the pipette and 5 mM K/140 mM Na in the bath. The top trace shows the time course of the experiment. Two parts of the trace, indicated by numbers, are extended to show the fast time resolution. Holding potential was 0 mV. The decrease in the current amplitude shown in trace 2 was presumably the result of depolarization of cell membrane potential induced by AA. B: effect of AA on channel activity in an inside-out patch in the presence of 0.5 mM of reduced NADP; AA (5 µM) was added to the bath solution. C: dose-response curve of the AA effect on the basolateral K channels in cell-attached patches.

To test the specificity of the AA effect, we also examined the effect of cis-11,14,17-eicosatrienoic acid, a 20-carbon fatty acid with three double bonds, on the 50-pS K channel in a cell-attached patch. Figure 4 is a channel recording demonstrating that addition of 10 µM cis-11,14,17-eicosatrienoic acid did not inhibit the activity of the 50-pS K channel and NP_o was 0.64 ± 0.05 (n = 7), which is not different from the control value (0.68 ± 0.05). This suggests that the effect of AA is not mediated by changing lipid fluidity or other nonspecific fatty acid-induced modulation of membrane proteins.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Effect of 11,14,17-eicosatrienoic acid on the basolateral K channel in the mTAL. Holding potential was 0 mV. The experiment was performed in a cell-attached patch. Traces with 5 and 10 µM AA were obtained 10 and 20 min after addition of AA.

AA can be metabolized by three pathways: cyclooxygenase (COX), lipoxygenase, and cytochrome P-450 monooxygenase (2, 19). It has been shown that both COX and cytochrome P-450 monooxygenase are expressed in the mTAL (6, 9, 20, 24). Therefore, we explored the possibility that the effect of AA was mediated by either COX-dependent or cytochrome P-450-dependent metabolites of AA. Figure 5 is a representative channel recording demonstrating the effect of AA in the presence of indomethacin, an inhibitor of COX. Addition of indomethacin (5 µM) significantly increased channel activity from 0.54 ± 0.05 to 0.90 ± 0.1 (n = 9). This indicates that the channel activity was suppressed by endogenous COX-dependent metabolites of AA. However, it is apparent that addition of 5 µM AA can still inhibit the K channel, and NP_o drops from 0.9 ± 0.1 to 0.2 ± 0.03 (n = 9). This suggests that it is unlikely that the AA-induced acute inhibition of the 50-pS K channel was mediated by a COX-dependent metabolite of AA.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of AA on the basolateral K channel activity in the presence of indomethacin. Arrows, application of indomethacin and AA. The top trace shows the experimental course. Three parts of the trace, indicated by numbers, are extended to show the fast time resolution. Holding potential was 0 mV.

We next examined the effect of AA on K channel activity in the presence of DDMS (25), an inhibitor of cytochrome P-450-dependent -oxidation of AA. Figure 6 is a channel recording illustrating the effect of AA on channel activity in a cell-attached patch in the presence of 5 µM DDMS. Inhibition of the cytochrome P-450-dependent -oxidation of AA not only significantly increased channel activity from 0.49 ± 0.04 to 0.83 ± 0.1 but also completely abolished the inhibitory effect of AA, because 5 µM AA did not decrease NP_o (0.89 ± 0.1, n = 7). The notion that the effect of AA is mediated by cytochrome P-450-dependent metabolites of AA is further supported by observations that inhibiting the cytochrome P-450 -oxidation of AA can completely reverse the AA-induced channel blockade (Fig. 7). Application of AA reduced NP_o from 0.50 ± 0.06 to 0.11 ± 0.02, and addition of 5 µM DDMS increased NP_o to 0.8 ± 0.1 (n = 7). In contrast, inhibition of COX did not restore the AA-induced decrease in channel activity (Fig. 8). From the inspection of Fig. 8, it is clear that addition of 5 µM AA decreased channel activity from 0.51 ± 0.05 to 0.12 ± 0.02 (n = 7) and that application of indomethacin did not abolish the AA-induced decrease in channel activity.

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Effect of AA on the basolateral K channel activity in a cell-attached patch in the presence of N-methylsulfonyl-12,12-dibromodec-11-enamide (DDMS). The top trace shows the experimental course. Three parts of the trace, indicated by numbers, are extended to show the fast time resolution. Holding potential was 0 mV.

View larger version (45K): [in this window] [in a new window]   Fig. 7.   Depicted is a recording showing that addition of DDMS reversed the AA-induced decrease in channel activity. The experiment was carried out in a cell-attached patch, and the holding potential was 0 mV. The top trace shows the time course of the experiment. Three parts of the trace, indicated by numbers, are extended to demonstrate the fast time resolution.

View larger version (34K): [in this window] [in a new window]   Fig. 8.   Depicted is a recording showing that addition of indomethacin did not reverse the AA-induced decrease in channel activity. The experiment was carried out in a cell-attached patch, and the holding potential was 0 mV. The top trace shows the time course of the experiment. Three parts of the trace, indicated by numbers, are extended to demonstrate the fast time resolution.

After establishing that the AA-induced acute decrease in channel activity was mediated by cytochrome P-450-dependent metabolites of AA, we examined the effect on the 50-pS K channel of 20-hydroxyeicosatetraenoic acid (HETE), a main product of cytochrome P-450 metabolism of AA in the mTAL (6). Figure 9 is a typical channel recording showing that application of 100 nM 20-HETE reversibly inhibited the activity of the 50-pS K channel in an inside-out patch and that NP_o fell from 0.52 ± 0.05 to 0.10 ± 0.02 (n = 5). The effect of 20-HETE on channel activity was specific, because addition of 100 nM 19-HETE, another metabolite of the cytochrome P-450-dependent pathway (3, 4), had no significant effect on channel activity in inside-out patches (data not shown). Moreover, the effect of 20-HETE could also be observed in cell-attached patches in the presence of DDMS. Figure 10 is a representative recording showing that 20-HETE decreased NP_o from 0.7 ± 0.1 to 0.05 ± 0.02 in the presence of DDMS (n = 5). This indicates that 20-HETE is the most likely candidate to mediate the effect of AA on the basolateral 50-pS K channel.

View larger version (37K): [in this window] [in a new window]   Fig. 9.   Depicted is a recording showing the effect of 100 nM 20-hydroxyeicosatetraenoic acid (20-HETE) on the basolateral K channel activity in an inside-out patch. The top trace shows the time course of the experiment. Three parts of the data, indicated by numbers, are extended to show the detail of the channel activity. Holding potential was 0 mV. The bath solution contains 140 mM KCl and 100 nM of free Ca2+.

View larger version (27K): [in this window] [in a new window]   Fig. 10.   Depicted is a recording demonstrating the effect of 100 nM 20-HETE on the basolateral K channel in a cell-attached patch in the presence of DDMS. The top trace shows the time course of the experiment. Two parts of the trace, indicated by numbers, are extended to show a fast time resolution of channel activity. Holding potential was 0 mV.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The mTAL is responsible for the reabsorption of 25% of filtered Na load and plays a key role in mediating urinary concentrating ability (10, 11, 16). Na and Cl enter the cell across the apical membrane through the Na-K-2Cl cotransporters, and Na is then actively transported across the basolateral membrane by Na-K-ATPase, whereas Cl leaves the cell via KCl cotransporters or Cl channels (10, 11). The basolateral K channels serve two cell functions: 1) they are responsible for K recycling across the basolateral membrane; and 2) they are involved in generating the cell membrane potential. Although changes in the cell membrane potential are not expected to directly affect the turnover rate of the Na-K-2Cl cotransporters, it is possible that an alteration in the cell membrane potential can indirectly influence the function of the cotransporter. For instance, changes in the cell membrane potential can alter the electrochemical gradient of K across the apical membrane, which can affect K recycling. Because K recycling is essential for maintaining the activity of the Na-K-2Cl cotransporters, changes in cell membrane potential can affect the function of the Na-K-2Cl cotransporters. Moreover, Cl exit across the basolateral membrane via Cl channels is expected to depolarize the basolateral membrane and, accordingly, to diminish the driving force for Cl exit. A decrease in Cl driving force leads to an increase in intracellular Cl concentrations, which have been shown to inhibit the activity of the Na-K-2Cl cotransporters (22). Therefore, activation of the basolateral K channels can hyperpolarize the basolateral membrane and maintain the driving force for Cl diffusion.

Although the importance of the basolateral K channel in the regulation of the transport function in the mTAL is well established, the information regarding the structure and biophysical properties of basolateral K channels is very limited. This is largely because the basolateral membrane is not accessible for patch-clamp studies without removal of the basement membrane. Hurst et al. (17) carried out a patch-clamp study in the collagenase-digested TALs from rabbit kidneys and identified a 41- to 43-pS K channel in cell-attached patches. Recently, Paulais et al. (21) successfully characterized the biophysical properties of the basolateral K channels by using collagenase-treated cTALs from mouse kidneys (21). They have identified an inwardly rectifying K channel with an inward conductance of 50 pS and an outward conductance of 11 pS. This K channel was inhibited by Mg²⁺ and spermine and was sensitive to cell pH (21).

In the present investigation, we confirmed that there is an inwardly rectifying 50-pS K channel in the basolateral membrane of the mTAL from rat kidneys and that the activity of the 50-pS K channel increased by depolarization (22). The depolarization-induced increase in channel activity has physiological significance in maintaining a constant driving force for Cl diffusion, because the K channel activity was expected to increase in response to Cl diffusion across the basolateral membrane. Although it is possible that K channels other than the 50-pS K channel are also present in the basolateral membrane of the mTAL, the 50-pS K channel may be one of the major K channels responsible for the basolateral K conductance in the mTAL. This speculation is supported by the observation that we detected the 50-pS K channel in ~30% of cell-attached patches, and the channel P_o was relatively high (0.5) at the spontaneous cell membrane potential. Thus factors that regulate the 50-pS K channel should have an effect on the basolateral K conductance and cell membrane potential.

In the present study, we have demonstrated that eicosanoids play an important role in regulating the basolateral K channels as follows: 1) inhibiting COX increased the activity of the 50-pS K channel; and 2) blocking the cytochrome P-450 -oxidation of AA could also augment the channel activity. This suggests that COX-dependent metabolites and cytochrome P-450-dependent metabolites of AA are involved in the regulation of the basolateral K channels. The COX-dependent AA metabolites such as PGE₂ have been shown to attenuate the stimulatory effect of vasopressin on Cl reabsorption (7). We have previously demonstrated that PGE₂ at low concentrations (<1 µM) abolished the vasopressin-induced increase in the activity of the apical 70-pS K channel, whereas PGE₂ at high concentrations decreased the activity of the 70-pS K channel by a PKC-dependent mechanism (18). In addition to PGE₂, the cytochrome P-450-dependent metabolites of AA, such as 20-HETE, have been indicated as potent inhibitors of the Na-K-2Cl cotransporters (9) and the apical 70-pS K channel in the mTAL (27).

Although both COX-dependent and cytochrome P-450-dependent AA metabolites are involved in the regulation of basolateral K channel activity, it is unlikely that the inhibitory effect of AA on channel activity is induced by a COX-dependent metabolite, because indomethacin did not abolish the AA-induced inhibition of channel activity. Our results strongly suggest that the inhibitory effect of AA is mediated by a cytochrome P-450-dependent metabolite of AA. First, inhibition of the cytochrome P-450-dependent -oxidation abolished the effect of AA on channel activity. Second, the AA-induced decrease in channel activity was completely reversed by DDMS. In contrast, indomethacin was not able to reverse the AA-induced inhibition. Third, addition of 20-HETE could inhibit the 50-pS K channel even in the presence of DDMS, suggesting that 20-HETE is a mediator for the effect of AA. Therefore, it is most likely that the effect of AA on channel activity under the present experimental conditions is mediated by a cytochrome P-450-dependent metabolite of AA, such as 20-HETE, rather than a COX-dependent metabolite of AA.

Under our experimental conditions, there are two possibilities that can be employed to explain why blocking cytochrome P-450 monooxygenase rather than inhibiting COX can abolish the inhibitory effect of AA on channel activity. One possibility is that AA is preferably metabolized by the cytochrome P-450-dependent pathway compared with the COX-dependent metabolism, although both COX and cytochrome P-450 monooxygenase are present in the mTAL. Therefore, inhibition of the cytochrome P-450-dependent metabolism of AA abolishes the inhibitory effect of AA. Alternatively, AA cannot be effectively converted to a COX-dependent metabolite that can inhibit channel activity. Thus the inhibitory effect of AA cannot be blocked by indomethacin. The second possibility is that AA is first metabolized by the cytochrome P-450-dependent pathway and that the cytochrome P-450-dependent metabolites of AA are further converted to prostaglandins by COX. Therefore, only DDMS can abolish the inhibitory effect of AA on the channel activity. In this regard, it has been shown that the cytochrome P-450-dependent metabolites of AA can be further converted to prostaglandins by COX (5). We need additional experiments to determine the metabolites of AA in the mTALs after addition of exogenous AA to examine why only inhibition of cytochrome P-450 metabolism of AA can block the AA-induced inhibition.

The physiological event in which 20-HETE serves as a mediator to regulate the basolateral K channels is not known. Stimulation of the Ca²⁺-sensing receptor has been shown to increase 20-HETE production (28). Because the Ca²⁺-sensing receptor is located in the basolateral membrane of the mTAL, it is conceivable that the stimulation of the Ca²⁺-sensing receptor may also inhibit the basolateral 50-pS K channel. Moreover, we have previously demonstrated that 20-HETE production increased in the mTAL obtained from rats on a K-deficient diet (12). Therefore, it is possible that basolateral K channel activity differs between mTALs from animals on a normal-K diet and from those on a low-K diet. Further experiments are required to test this possibility.

We conclude that an inwardly rectifying 50-pS K channel is expressed in the basolateral membrane of the mTAL and is inhibited by AA and that the effect of AA is mediated by cytochrome P-450-dependent metabolites of AA.