Angiotensin II stimulates basolateral K channels in ratcortical collecting duct - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:25 |只看该作者 |倒序浏览 |打印

作者：YuanWei and WenhuiWang作者单位：Department of Pharmacology, New York Medical College, Valhalla, NewYork 10595 6 g9 L" [2 _2 E5 g) M  d, C
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      【摘要】
   We used the patch-clamp techniqueto study the effects of angiotensin II (ANG II) on basolateral Kchannels in cortical collecting ducts (CCDs). Application of ANG II(100 pM-100 nM) increased the activity of basolateral 18-pS Kchannels. This effect of ANG II was completely abolished by losartan,which is an antagonist of type 1 angiotensin (AT 1 )receptors. In contrast, inhibition of type 2 angiotensin(AT 2 ) receptors did not block the stimulatory effect of ANGII. Also, application of ANG II significantly increased intracellularCa 2  concentrations, which were measured with fura 2 dye.To explore the role of Ca 2  -dependent pathways in theregulation of basolateral K channels, the effects of ANG II on channelactivity were examined in the presence of arachidonyltrifluoromethylketone to inhibit phospholipase A 2 (PLA 2 ),GF-109203X [a protein kinase C (PKC) inhibitor], and N G -nitro- L -arginine methyl ester( L -NAME) to inhibit nitric oxide synthase. Inhibition ofeither PLA 2 or PKC did not block the effect of ANG II onbasolateral K-channel activity. However, the stimulatory effect of ANGII was absent in the CCDs treated with L -NAME. Moreover, addition of the membrane-permeant 8-bromo-guanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) not only increased channel activity butalso abolished the stimulatory effect of ANG II on channel activity. Weconclude that ANG II increases basolateral K-channel activity via thestimulation of AT 1 receptors, and the stimulatory effect ofANG II is mediated by a nitric oxide-dependent cGMP pathway.
      【关键词】 Angiotensin stimulates basolateral channels ratcortical collecting
   guanosine 3',5'-cyclic monophosphate; nitric oxide; angiotensintype 1 receptor; potassium secretion

INTRODUCTION8 Y9 W9 F; Z! C7 V5 ]

BASOLATERAL K CHANNELS PLAY an important role in generating cell-membrane potential inthe cortical collecting ducts (CCDs; Refs. 7, 33 ). Because Na reabsorption and K secretion in the CCDsare electrogenic processes ( 24, 30 ), a change in thecell-membrane potential is expected to have effects on Na reabsorptionand K secretion ( 17, 25, 26, 29 ). Moreover, basolateral Kchannels can serve as potential routes for K to enter the cell when thecell-membrane potential exceeds the K equilibrium potential( 28 ). There are at least three types of K channels, whichare measured here according to inside-out and cell-attached patches,respectively: small conductance, 18 and 27 pS; intermediateconductance, 28 and 67 pS; and large conductance, 85 and 148 pS.Although the regulatory mechanisms of these types of K channels aredifferent, all three are activated by guanosine 3',5'-cyclicmonophosphate (cGMP)-dependent protein kinase ( 10, 18, 32 ).
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The production of cGMP can be stimulated by nitric oxide (NO), which isa potent activator of soluble quanylate cyclase (GC; Ref. 14 ). We previously demonstrated that NO linksapical Na transport to basolateral K-channel activity: an increase inapical Na entry leads to augmentation of the intracellularCa 2  concentration [Ca 2  ] i, whichin turn stimulates the activity of Ca 2  -dependent NOsynthase (NOS). As a consequence, NO increases cGMP generation, whichfurther activates basolateral K channels ( 20 ). Thissuggests that the Ca 2  -NO-cGMP pathway plays an importantrole in the regulation of basolateral K-channel activity. However, itis not clear which hormone regulates basolateral K channels via acGMP-dependent pathway. Because the stimulation of type 1 angiotensin(AT 1 ) receptors has been shown to increase[Ca 2  ] i in renal tubules ( 4 ) andAT 1 receptors are also present in CCDs ( 9 ), itis possible that angiotensin (ANG) II may have an effect on basolateralK channels via Ca 2  -NO-cGMP pathways. Therefore, in thepresent study, we explored the role of ANG II in the regulation ofbasolateral K channels.

METHODS

Preparation of CCDs. Male pathogen-free Sprague-Dawley rats (age 5-6 wk) were used inthe experiments. The rats were purchased from Taconic Farms (Germantown, NY) and were fed a normal rat chow. We also used rats thatwere fed a high-K diet (10%, wt/wt) in some experiments. Because theresponses of basolateral K channels to ANG II were the same for rats onnormal rat chow, data were pooled. The body weight of the rats used forthe experiments was 100-120 g. Rats were killed by cervicaldislocation, and the kidneys were removed immediately. Several thin( placed in ice-cold Ringersolution until dissection. The dissection was carried out at roomtemperature, and two watchmakers' forceps were used to isolate thesingle CCD. To immobilize the tubules, they were placed on a 5 × 5-mm cover-glass that was coated with D -polylysin (BectonDickinson, Bedford, MA) and were then transferred to a 1-ml chamberthat was mounted on an inverted Nikon microscope. The CCDs weresuperfused with HEPES-buffered NaCl-Ringer solution, and thetemperature of the chamber was maintained at 37°C by circulation ofwarm water around the chamber. We followed previously described methodsto prepare the basolateral membrane for patch-clamp experiments. Briefly, after the CCD was split open, the intercalated cell was removed to gain access to the lateral membrane principal cell. In addition, we also patched the CCD that was treated with 1% collagenase. Because the effects of ANG II on the basolateral Kchannels from the collagenase-treated and untreated CCDs were identical, we pooled the data.
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Patch-clamp technique. Single-channel current was recorded by an Axon 200A patch-clampamplifier and was low-pass filtered at 1 kHz through an eight-pole Bessel filter (model 902LPF; Frequency Devices, Haverhill, MA). Thedata were digitized by an Axon interface (Digidata 1200) and stored inan IBM-compatible Pentium II computer. We used pClamp software system6.04 (Axon Instruments, Burlingame, CA) to generate an all-pointhistogram, which was then fitted to calculate the channel activity.Channel activity was defined as NP o, a product of channel number ( N ) and open probability( P o ) that was calculated from data samples of60-s duration in the steady state as o =&Sgr;  ( t 1 + 2 t 2 +... it i

where t i is the fractional open timespent at each of the observed current levels. No efforts were made todetermine whether an increase in channel activity results from a changein N or P o.. m" |! v! P. V% a
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Measurement of[Ca 2 ] i. [Ca 2  ] i was measured with fura2-acetoxymethyl ester (Molecular Probes, Eugene, OR). Fluorescence wasimaged digitally with an intensified video-imaging system that includeda SIT 68 camera, controller, and HR 1000 video monitor. The exiting andemitted light passed through a ×40 fluorite objective (numericalaperture, 1.30; Nikon, Melville, NY). We followed methods publishedpreviously ( 20 ) to measure and calculate the[Ca 2  ] i.' V1 ^2 a  \& z8 l3 [
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Experimental solution and statistics. The pipette solution contained (in mM) 140 KCl, 1.8 MgCl 2,and 10 HEPES (pH 7.4). The bath solution was composed of (in mM) 140 NaCl, 5 KCl, 1.8 CaCl 2, 1.8 MgCl 2, 5 glucose,and 10 HEPES (pH 7.4). Herbimycin A, GF-10239X, andarachidonyltrifluoromethyl ketone (AACOCF 3 ) were purchasedfrom Biomol (Plymouth Meeting, PA) and were dissolved in DMSO solution.The final concentration of DMSO was ANG II, 8-bromo-cGMP, losartan, PD-123319,and N G -nitro- L -arginine methyl ester( L -NAME) were purchased from Sigma Chemical (St. Louis,MO). Data are shown as means ± SE, and paired Student's t -test was used to determine the significance between thetwo groups. Statistical significance was taken as P

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In our previous study, we patched the lateral membranes ofprincipal cells using an approach that removed the intercalated cellnext to the principal cell ( 35 ) and identified a 27-pS Kchannel in cell-attached patches in the CCD. When the bath and pipettesolutions were switched to a symmetrical 140-mM KCl solution, thechannel conductance in inside-out patches was 18 pS. This 18-pS Kchannel can be found in 35% of patches in the CCDs obtained from ratsthat were fed normal rat chow and 52% from rats on a high-K diet.Because the P o was 0.5-0.8, it was assumedthat the 18-pS K channel is one of the major K channels thatcontributes to basolateral K conductance in CCDs. This conclusion isalso supported by experiments performed on collagenase-treated CCDs. Weobserved the 18-pS K channels in 11 of 31 cell-attached patches. Moreover, the P o was 0.4-0.7 and was notsignificantly different from that observed for channels fromcollagenase-untreated CCDs. Also, it has been found that theresponse of the 18-pS K channel to ANG II in both tubule preparationswas the same. Therefore, we pooled the data obtained from bothcollagenase-treated and untreated CCDs.
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Several investigations have demonstrated that ANG II receptors areexpressed in CCDs ( 4, 9 ); however, it has not been explored whether ANG II is involved in the regulation of the transport function in CCDs. Therefore, we examined the effect of ANG II on theactivity of basolateral 18-pS K channels. Figure 1 A is a representativerecording that shows the effect of 100 nM ANG II on the basolateral Kchannel from a cell-attached patch. It is apparent that ANG IIsignificantly increased NP o from 0.76 ± 0.2 under control conditions to 1.74 ± 0.25 ( n = 7). Figure 1 B is a dose-response curve of the ANG II effect,which demonstrates that ANG II stimulates the 18-pS K channel at aconcentration of 100 pM and that the stimulatory effect of ANG IIreaches its plateau at 100 nM. To determine which type of ANG IIreceptor was responsible for mediating the effect of ANG II on thebasolateral K channels, we examined the effects of 100 nM ANG II onbasolateral 18-pS K channels in the presence of losartan, an antagonistof AT 1 receptors (1 µM) and PD-123319 (1 µM), aselective antagonist of AT 2 receptors. Addition of eitherlosartan or PD-123319 had no significant effect on channel activity(Fig. 2 ). However, inhibition ofAT 2 receptors did not abolish the stimulatory effect of ANG II on basolateral K channels. Figure 3 isa representative recording that shows the effect of ANG II on 18-pS Kchannels in the presence of an AT 2 -receptor inhibitor.Clearly, PD-123319 did not block the effect of ANG II on channelactivity, because addition of ANG II increased NP o from 0.75 ± 0.2 to 1.75 ± 0.22 ( n = 9). In contrast, inhibition of AT 1 receptors completely abolished the effect of ANG II on 18-pS K channels(see Fig. 2 ), because application of 100 nM ANG II failed to increasechannel activity (control, 0.70 ± 0.2; ANG II, 0.8 ± 0.2; n = 5). This suggests that the effects of ANG II onchannel activity are mediated by stimulation of AT 1 receptors.

Fig. 1. A : effect of angiotensin II (ANG II) onbasolateral K-channel activity in a cell-attached patch. Time course ofthe experiment is shown ( top ); two parts of the traceindicated by numbers are expanded ( middle and bottom ) to illustrate the fast resolution. Time when 100 nMANG II was added to the bath is indicated (arrow). Pipette holdingpotential was 30 mV (hyperpolarization of cell membrane potential by 30 mV), and the channel closed level is indicated (C). B :dose-response curve of effect of ANG II on basolateral K-channelactivity. Experimental number for each group was 5-10. Data werenormalized using the equation [ NP o (experimental)/ NP o (control)], where N is channel number and P o is openprobability. *Data are significantly different from the control value(without ANG II). AII, angiotensin II.
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Fig. 2. Effects of 100 nM ANG II on basolateral 18-pS K channelsin the presence of PD-123319 [an inhibitor of type 2 angiotensin(AT 2 ) receptors] and losartan [an inhibitor of type 1 angiotensin (AT 1 ) receptors], respectively. *Result issignificantly different from the control value.+ K9 I9 ^. ~6 [8 ?4 b8 o1 `9 Z
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Fig. 3. Effects of 100 nM ANG II on basolateral K channels in acell-attached patch in the presence of PD-123319. Time course of theexperiment is shown ( top ); two parts of the trace areexpanded ( middle and bottom ) to demonstrate thefast time resolution. Channel closed level is indicated (C); thepipette holding potential was 30 mV.1 {4 Y: n1 E( N, a
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This notion is also supported by experiments in which the effect of ANGII on [Ca 2  ] i was determined with fura 2 dye(Fig. 4 A ). Figure 4 B summarizes results from four measurements (4 tubulesobtained from 3 rats) showing that addition of ANG II transientlyincreased the [Ca 2  ] i from the control valueof 75 ± 8 to 126 ± 15 nM. Therefore, it is possiblethat the effect of ANG II was mediated by an increase in the[Ca 2  ] i. From our previous studies, weidentified three possible Ca 2  -dependent signaltransduction pathways: PKC, phospholipase A 2 (PLA 2 ), and neuronal (n)NOS. All three pathways have beendemonstrated to regulate either the apical or basolateral K-channelactivity in CCDs ( 33 ). First, we investigated thepossibility that Ca 2  -dependent PKC may mediate the effectof ANG II, because PKC has been shown to mediate the stimulatory effectof ANG II on bicarbonate transport in the proximal tubule( 16 ) and increase basolateral 18-pS K-channel activity inCCDs ( 19 ). Figure 5 summarizes results from five experiments in which the effect of ANG IIon 18-pS K-channel activity was examined in the presence of the PKCinhibitor GF-109203X (5 µM). We confirmed the previous findings thatinhibition of PKC slightly decreased the basal level of channelactivity ( 19 ). However, inhibition of PKC did not blockthe stimulatory effect of ANG II on basolateral K-channel activity, and NP o increased from 0.70 ± 0.15 to1.60 ± 0.25. We next tested the effect of ANG II on channelactivity in the presence of a PLA 2 inhibitor. As shown inFig. 5, inhibition of PLA 2 with AACOCF 3 (5 µM) had no significant effect on channel activity. Furthermore, ANGII could still increase channel activity from 0.93 ± 0.18 to1.66 ± 0.23 in the presence of AACOCF 3. The notionthat the effect of ANG II on basolateral 18-pS K channels was notmediated by a PLA 2 -dependent pathway is also indicated byexperiments in which addition of 10 µM arachidonic acid did notincrease the channel activity (data not shown).. R' E- R: q" Q& z% \
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Fig. 4. A : this original trace shows the effect of 100 nM ANG II on intracellular Ca 2  concentration([Ca 2  ] i ) in the cortical collecting ducts(CCDs). B : data obtained from 4 tubules are summarized toshow the effect of ANG II.0 H( T5 ?9 y, x( F$ n* _

Fig. 5. Effect of 100 nM ANG II on basolateral 18-pS K channelsin the presence of GF-109203X (a PKC inhibitor) andarachidonyltrifluoromethyl ketone [AACOCF 3, aphospholipase A 2 (PLA 2 ) inhibitor]. *Resultwas significantly different from the control value.

After determining that the effect of ANG II was not mediated by PKC- orPLA 2 -dependent pathways, we examined the effect of ANG IIon channel activity in the presence of L -NAME. Because inhibition of NOS significantly decreased channel activity, we selectedpatches in which a high channel activity was observed to examine theeffects of ANG II. Figure 6 is arepresentative recording that demonstrates the effect of ANG II (100 nM) on channel activity in the presence of 0.2 mM L -NAME.Addition of L -NAME decreased channel activity, and NP o decreased from 1.3 ± 0.15 to 0.70 ± 0.1 ( n = 5). Moreover, inhibition of NOS abolishedthe effect of ANG II on channel activity, and ANG II did not increase NP o (control, 0.7 ± 0.1; ANG II, 0.80 ± 0.1). We have previously shown that the stimulatory effect of NO wasmediated by the cGMP-dependent pathway ( 18 ). To determinewhether the effect of ANG II on 18-pS K channels resulted from anincrease in cGMP formation, we examined the effect of ANG II on channelactivity after the CCDs were challenged by 8-bromo-cGMP. Addition of200 µM 8-bromo-cGMP stimulated channel activity and increased NP o from 0.6 ± 0.1 to 1.65 ± 0.2 (Fig. 7 ). Moreover, the effects of ANG IIwere absent in the presence of cGMP. Figure 8 is a representative recording thatdemonstrates the effect of 100 nM ANG II on 18-pS K channels in thepresence of 0.2 mM 8-bromo-cGMP. Before addition of ANG II, NP o was 1.42 ± 0.2 ( n = 6). Addition of 100 nM ANG II did not increase NP o (1.45 ± 0.2). Thus the effect of cGMPand ANG II was not additive, which suggests that the stimulatory effectof ANG II is the result of an increase in cGMP formation in the CCDs.

Fig. 6. Effect of 100 nM ANG II on basolateral K-channel activityin the presence of 0.2 mM N G -nitro- L -arginine methyl ester( L -NAME). Time course of the experiments is shown( top ); three parts of the data indicated by numbers areexpanded ( middle and bottom ) to show fast timeresolution. Pipette holding potential was 30 mV; channel closed lineis indicated (C).
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Fig. 7. A channel recording that demonstrates the effect of 200 µM 8-bromo-guanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) onbasolateral K-channel activity in a cell-attached patch. Time course ofthe experiment is shown ( top ); two numbered areas of thedata are expanded ( middle and bottom ) todemonstrate channel activity at a fast resolution. Holding potentialwas 30 mV; channel closed level is indicated (C).

Fig. 8. A channel recording that shows the effect of 100 nM ANGII on basolateral K-channel activity in the presence of 200 µM8-bromo-cGMP. Experiment was performed in a cell-attached patch withholding potential of 30 mV. Time course of the experiment is shown( top ); two parts of the data indicated by numbers areexpanded ( middle and bottom ) to demonstrate thefast resolution.1 V; R) L! z: r; A: j% B: W
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DISCUSSION

The main findings of the present study are that the stimulation ofAT 1 receptors increases the activity of basolateral 18-pS Kchannels and that the stimulatory effect of ANG II is mediated by acGMP-dependent pathway. A large body of evidence indicates that ANG IIplays an important role in the regulation of Na reabsorption in renaltubules ( 6, 8, 15, 27 ). ANG II stimulates bicarbonate reabsorption in the proximal tubule via a PKC-dependent pathway ( 16 ). In the thick ascending limb, ANG II inhibits theapical 70-pS K channel by increasing 20-hydroxytetraenoic acid release ( 21 ) and decreasing bicarbonate reabsorption by cytochrome P -450 metabolites of arachidonic acid( 8 ).* X3 ?& H9 \4 B% e: R
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The effects of ANG II on epithelial membrane transport are mediated bythe interaction of two types of ANG II receptors: AT 1 andAT 2. It is well documented that both AT 1 andAT 2 receptors are expressed in the kidney ( 4, 9, 36 ). However, AT 1 receptors are most likely to beresponsible for the effects of ANG II on renal tubule transport( 2 ). The highest expression level of AT 1 receptors was identified in the proximal tubules, and the next-highestlevel of AT 1 receptors was found in the thick ascending limb ( 4 ). A large body of evidence has strongly indicatedthat AT 1 receptors are also present in the cortical andouter medullary collecting duct ( 4, 9 ). Also, using RT-PCRtechnique, the presence of AT 2 -receptor mRNA has been foundin the collecting ducts ( 36 ). Therefore, it isconceivable that ANG II plays an important role in regulation of thetransport function in collecting ducts. This speculation has beenconfirmed by our present study: ANG II activates the basolateral 18-pSK channels. Although the effect of ANG II on basolateral K channelsother than the 18-pS channel has not yet been investigated, it ispossible that ANG II may also stimulate the intermediate- andlarge-conductance K channels in the basolateral membrane of CCDs,because all three types of basolateral K channels are activated by cGMP( 10, 32 ). Therefore, it is expected that stimulation ofAT 1 receptors would increase basolateral K conductance./ @+ m* \$ N, C5 g2 w9 {
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Basolateral K channels serve several cell functions in CCDs. First, thechannels are responsible for generating cell-membrane potentials( 7, 33 ). As both K secretion and Na reabsorption in CCDsare electrogenic processes, an alteration in cell-membrane potential isexpected to have an effect on Na absorption. Indeed, it has beendemonstrated that inhibition of the basolateral K conductance reducesNa transport in CCDs ( 29 ). Second, K channels play a rolein basolateral K recycling. Stimulation of Na-K-ATPase has been shownto increase basolateral K conductance in renal tubules includingproximal tubules and CCDs ( 3, 11, 23 ). The couplingbetween Na-K-ATPase activity and basolateral K conductance is importantfor maintenance of a constant intracellular K concentration duringstimulation of Na-K-ATPase activity by a variety of factors. Third,basolateral K channels can provide an alternative route for K to enterthe cells across the basolateral membrane when the cell-membranepotential exceeds the K equilibrium potential ( 28 ).Relevant to the third role of K channels in CCDs is the observationthat K enters the cell across the basolateral membrane via aNa-K-ATPase-independent pathway, presumably via basolateral K channels,when the aldosterone level is high ( 28 ).
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The effects of ANG II on basolateral K channels are mediated byAT 1 receptors, because losartan, a specificAT 1 -receptor antagonist, abolished the stimulatory effectof ANG II on basolateral K channels. Moreover, we have demonstratedthat application of ANG II increases the[Ca 2  ] i in CCDs. The effect of ANG II on the[Ca 2  ] i is generally believed to be mediatedby AT 1 receptors ( 2 ). Therefore, it isconceivable that a Ca 2  -dependent signal transductionpathway is responsible for the effects of ANG II. The ANG II-inducedincrease in [Ca 2  ] i is only a modest 50 nM inthe present experiment. However, it is possible that the increase in[Ca 2  ] i is compartmentalized and is higher inthe close proximity of the basolateral K channels than the amountindicated by the mean increase in [Ca 2  ] i.Moreover, it is also possible that NOS is colocalized with basolateralK channels. Accordingly, stimulation of ANG II receptors could producean elevated NO concentration for activation of the basolateral Kchannels. It has been reported that nNOS and the N -methyl- D -aspartate receptors are physicallylinked by the PSD95-Dlg-zona occludens domain ( 1, 5 ).Furthermore, it is likely that a 50-nM increase in[Ca 2  ] i is sufficient to stimulate the 18-pSK channel. We have previously demonstrated that a 50-nM increase in[Ca 2  ] i significantly stimulates thebasolateral 18-pS K channel in CCDs ( 20 ). It has beendemonstrated that the activity of nNOS is very sensitive to changes in[Ca 2  ] i in the range of 100-200 nM andthat a 100-nM increase in Ca 2  concentration stimulates NOproduction and GC activation by tenfold ( 12 ).Therefore, it is possible that a slight increase in[Ca 2  ] i could be sufficient to activatebasolateral K channels. There are at least threeCa 2  -dependent signal transduction pathways in CCDs

LA 2, PKC, and NOS ( 34 ). The observations thatneither inhibition of PLA 2 nor blocking of PKC had aninfluence on the effects of ANG II excludes the possibility that thesepathways mediate the effects of ANG II on basolateral K channels.

Two lines of evidence suggest that the effects of ANG II on basolateralK channels were mediated by a Ca 2  -dependent NO pathway: 1 ) addition of ANG II elevated the[Ca 2  ] i, and 2 ) inhibition of NOSabolished the stimulatory effect of ANG II on K channels. Figure 9 is a model of a principal cell in theCCD that illustrates a possible mechanism by which ANG II couldincrease the basolateral K-channel activity. The stimulation ofAT 1 receptors increases [Ca 2  ] i,which in turn activates Ca 2  -dependent nNOS and,accordingly, increases NO production. This leads to activation ofsoluble GC, increased cGMP generation, and augmentation ofbasolateral K-channel activity. This hypothesis is supported by ourprevious observation that nNOS was specifically expressed in principalcells of CCDs ( 34 ). Moreover, it has been demonstratedthat soluble GC is present in CCDs ( 22 ). The notion thatcGMP mediates the effects of ANG II on basolateral K channels is alsosupported by the finding that ANG II did not increase channel activityin the presence of the membrane-permeant 8-bromo-cGMP. The effect ofcGMP is most likely mediated by cGMP-dependent protein kinase G (PKG),because PKG has been demonstrated to activate 18-pS K channels( 32 ). This speculation is also consistent with the findingthat although the ANG II-induced increase in[Ca 2  ] i was transient, the stimulatory effectof ANG II on basolateral 18-pS K channels was stable during ourexperimental period (10-15 min). It is possible that when Kchannels are activated by PKG, the channel activity can be maintainedas active for a certain amount of time even if[Ca 2  ] i returns to the control value from itspeak value. A similar finding that NO-cGMP signaling mediates theeffect of ANG II on Na-K-ATPase in the proximal tubule has beenreported ( 37 ).- R1 e+ {- Z/ B; M& S1 W% s
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Fig. 9. A model of a CCD principal cell that illustrates themechanism by which ANG II stimulates the activity of basolateral 18-pSK channels. nNOS, neuronal nitric oxide synthase; PKG, protein kinaseG.
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AT 1 receptors have been shown to be expressed in both theapical and basolateral membranes ( 9 ). Because ourexperiments were carried out in split-open tubules, it is not clearwhether the effect of ANG II on basolateral K channels was the result of stimulation of the basolateral and apical AT 1 receptors.However, it is possible that the effects of ANG II were the result ofstimulation of basolateral AT 1 receptors. The effects ofANG II on basolateral K channels in the regulation of Na reabsorptionand K secretion have not been explored. However, it is possible thatthe stimulatory effect of ANG II on basolateral K channels couldpotentially increase Na reabsorption in CCDs, because hyperpolarizationincreases the electrochemical gradient for luminal Na entering the cellacross the apical membrane. This hypothesis has been supported by amicroperfusion study ( 31 ) in which application of ANG IIstimulated the amiloride-sensitive Na transport in the late distaltubule, which includes the initial CCDs. In addition to stimulation ofbasolateral K channels, cGMP inhibits the 28-pS Na channel in the innermedullary collecting duct (IMCD; Ref. 13 ). Therefore, itis likely that the effect of cGMP on Na transport depends on thenephron segment: cGMP may indirectly stimulate Na absorption byincreasing the driving force for Na in the CCDs and inhibit Naabsorption by blocking the 28-pS Na channels in the IMCD.
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We conclude that ANG II stimulates basolateral 18-pS K channels byactivation of AT 1 receptors and that the stimulatory effect of ANG II on channel activity is mediated by a cGMP-dependent pathway.

ACKNOWLEDGEMENTS& f! J; P% v! o! K

This work was supported by National Institutes of Health GrantDK-47402.1 I: @* x- T2 p! q* o0 y& `
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