Department of Physiology II, Iwate Medical University School of Medicine, Morioka, 020-8505 Japan
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ABSTRACT |
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An ATP-regulated inwardly rectifying K+ channel, whose activity is enhanced by PKA, is present in the plasma membrane of cultured human proximal tubule cells. In this study, we investigated the effects of PKG on this K+ channel, using the patch-clamp technique. In cell-attached patches, bath application of a membrane-permeant cGMP analog, 8-bromoguanosine 3',5'-monophosphate (8-BrcGMP; 100 µM), stimulated channel activity, whereas application of a PKG-specific inhibitor, KT-5823 (1 µM), reduced the activity. Channel activation induced by 8-BrcGMP was observed even in the presence of a PKA-specific inhibitor, KT-5720 (500 nM), which was abolished by KT-5823. Direct effects of cGMP and PKG were examined with inside-out patches in the presence of 1 mM MgATP. Although cytoplasmic cGMP (100 µM) alone had little effect on channel activity, subsequent addition of PKG (500 U/ml) enhanced it. Furthermore, bath application of atrial natriuretic peptide (ANP; 20 nM) in cell-attached patches stimulated channel activity, which was blocked by KT-5823. In conclusion, cGMP/PKG-dependent processes participate in activating the ATP-regulated K+ channel and producing the stimulatory effect of ANP on channel activity.
patch-clamp; human kidney; guanosine 3',5'-cyclic monophosphate; atrial natriuretic peptide; KT-5823
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INTRODUCTION |
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IT IS GENERALLY ACCEPTED THAT the basolateral ATP-regulated K+ channels in the proximal tubule are important for formation of the membrane potential that provides the driving force for electrogenic passive transport across the apical and basolateral membranes (2). Intracellular ATP is considered to act as a modulator coupling the function of these K+ channels to basolateral Na+-K+ pump activity (1, 8, 29, 33, 34). In addition to ATP, several factors affecting channel activity have been reported. Protein phosphorylation is one of the key mediators for channel function. It has been demonstrated that activity of the basolateral K+ channel in Ambystoma proximal tubule cells is subjected to phosphorylation by PKA and PKC (21). Furthermore, the ATP-regulated K+ channel in opossum proximal tubule cells is modulated by PKA (16), PKG (17), PKC, and Ca2+/calmodulin-dependent protein kinase II (CaMKII) (23).
The importance of protein phosphorylation in the regulation of channel activity is also observed in K+ channels of other nephron segments or cloned renal K+ channels. For example, the apical secretory K+ channel in rat cortical collecting duct (CCD), which would be identical to the cloned renal K+ channel, ROMK (15, 22, 26), is modulated by PKA-, PKC- (32), and CaMKII-mediated phosphorylation processes (18). Moreover, the basolateral K+ channels in CCD are regulated by PKG (13, 31)- and PKC-mediated processes (19).
Despite the abundant reports about the ATP-regulated K+ channels along the nephron as mentioned above, these data were based on the experiments using animal species, and properties of the K+ channels in human renal tubule cells have not been sufficiently elucidated. Recently, we identified an ATP-regulated inwardly rectifying K+ channel with an inward slope conductance of ~42 pS in cultured human proximal tubule cells of normal kidney origin and demonstrated that activity of this K+ channel was maintained, at least in part, by PKA-mediated phosphorylation (24). However, there remained the possibility that some mechanisms other than PKA-mediated processes might be involved in regulation of this channel, because a PKA-specific inhibitor only reduced channel activity to ~50% of control (24). In this study, we tested the effect of another protein kinase, PKG, on activity of this K+ channel, using the patch-clamp technique. Furthermore, we examined the effects of a guanylate cyclase-activating peptide, atrial natriuretic peptide (ANP), on channel activity. Although the major sites of ANP action in the kidney were shown to be located in the glomeruli and the inner medullary collecting duct (20, 37), several investigators reported that ANP affected the proximal tubule function (6, 7, 9, 11, 12). In the present study, we demonstrate that cGMP/PKG-dependent processes stimulate K+ channel activity and that ANP also stimulates the activity via the PKG-mediated process in the human proximal tubule cells.
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METHODS |
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Cell culture.
Renal proximal tubule epithelial cells (RPTECs) isolated from
the normal human kidney of a 31-yr-old woman (strain 5899, lot 8F0690)
were purchased from Clonetics (Walkersville, MD). It is guaranteed that
>90% of the cells are positive for -GTP, a marker protein specific
to the proximal tubule (10). These cells were provided as
cryopreserved secondary cultures and maintained in a renal epithelial
cell growth medium (Clonetics) in a humidified atmosphere of 5%
CO2-95% air at 37°C In the experiments, the cells were
dispersed from 70-80% confluence at passages 3-6
with trypsin/EDTA, resuspended in the growth medium, and seeded on
collagen-coated coverslips (Iwaki Glass, Tokyo, Japan) in 24 multiwells
at a density of 2 x 104 cells/well. After a 3- to 7-h
incubation, the coverslips were transferred to an open bath-heating
chamber (Warner, Hamden, CT).
Scanning electron microscopy. RPTECs incubated for 3 h on the collagen-coated coverslips were prefixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 1 h at room temperature. Then, the cells were rinsed with 0.1 M phosphate buffer (pH 7.3) containing 3.3% sucrose and postfixed with 1% OsO4 in 0.05 M phosphate buffer (pH 7.3) containing 6.5% sucrose for 1 h at room temperature. The fixed cells were dehydrated in a graded series of ethanol and subjected to the critical point drying. The dried specimens were coated with OsO4 and viewed in a Hitachi S-4700 scanning electron microscope (Hitachi, Tokyo, Japan).
Solutions. The control bath solution contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES. Patch pipettes were filled with a KCl solution, which contained (in mM) 145 KCl, 1 MgCl2, 1 EGTA, and 1 HEPES. The same KCl solution was employed as the bath solution for inside-out patches. All of these solutions were titrated to pH 7.3 with 5 N NaOH or KOH.
Test substances. MgATP, 8-bromoguanosine 3',5'-monophosphate (8-BrcGMP), cGMP and human ANP (hANP) were purchased from Sigma (St. Louis, MO). A membrane-permeant PKG-specific inhibitor, KT-5823, and a membrane-permeant PKA-specific inhibitor, KT-5720, were from Calbiochem (La Jolla, CA). PKG was from Promega (Madison, WI). KT-5823 and KT-5720 were dissolved in DMSO as stock solutions, whereas others were dissolved in water. The stock solutions were diluted with appropriate amounts of bath solutions and added to the bath. The final concentration of DMSO in the bath ranged from 0.047 to 0.054%, which had no apparent effect on channel activity. These substances were directly added to the bath by hand-pipetting, except for MgATP, which was added to the bottle of KCl bath solution beforehand.
Patch-clamp technique.
Single channel currents were recorded with cell-attached and inside-out
patches applied to the surface membrane of single RPTECs. All patch
experiments were performed at 33°C, which was adjusted by a heater
platform connected with a controller (TC-324B, Warner). This
temperature setting was very suitable for both the gigaseal formation
and cell viability. Patch pipettes were fabricated from glass
capillaries (Warner), with the resistance ranging from 3 to 5 M when
filled with the KCl solution. The pipette holding potential
(Vp) was set at 0 mV for cell-attached patches
and +50 mV for inside-out patches, unless otherwise stated. Current
signals were recorded with a patch-clamp amplifier Axopatch 200B (Axon, Foster City, CA) and stored on a DAT recorder (RD-120TE, TEAC, Tokyo,
Japan). The recorded signals were then low-pass filtered (3611 Multifunction Filter, NF Electronic Instruments, Tokyo, Japan) at 500 Hz and digitized at a rate of 10 kHz through an interface (Digidata
1200A, Axon). The acquired data were analyzed with acquisition/analysis
software (pCLAMP6, Axon) on an IBM-compatible personal computer.
Current traces of downward deflections represented inward currents.
Channel activity was determined by NPo, which was calculated as
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Statistics. Data are expressed as means ± SE from 4-15 patches. Student's t-test or ANOVA in conjunction with Bonferroni t-test was used for statistical comparisons. A P value <0.05 was considered to be significant.
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RESULTS |
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Figure 1 shows a typical scanning
electron microscopic view of a single RPTEC we used in this study. The
cell is spherical in form and attaches to the coverslips. Although
short microvilli are sparsely distributed over the entire plasma
membrane, the apical brush border seen in the proximal tubule cells in
situ is not apparent.
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As we have demonstrated in the previous paper (24), an
inwardly rectifying K+ channel is the most frequently
observed K+ channel in the plasma membrane of RPTECs in
cell-attached patches under the control condition. The current-voltage
(I-V) relationships of this K+
channel are shown in Fig. 2.
Representative current traces, which were obtained from an inside-out
patch under the symmetrical K+ condition, show a typical
inward rectification (Fig. 2A). Although data are not shown,
there was no significant voltage dependency of channel open
probability. Data were pooled from experiments similar to that in Fig.
2A, and an I-V curve was drawn (Fig.
2B, circles). The inward conductance obtained from the slope
between 90 and
30 mV of Vp was 42.5±2.9 pS
(n=12), and the outward slope conductance between +15 and
+60 mV was 7.8±1.5 pS (n=12). An asymmetrical K+ condition was also employed to estimate the
K+ selectivity of this channel. When the internal
K+ concentration was reduced from 145 to 30 mM, the
reversal potential shifted to +31 mV (Fig. 2B, squares).
According to the Goldman-Hodgkin-Katz voltage equation, the calculated
K+-to-Na+ permeability ratio was ~8.
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In our previous study, we showed that the channel is activated by
PKA-mediated phosphorylation (24). To extend our knowledge about the regulation of this K+ channel, the involvement of
the cGMP/PKG pathway was explored. A representative current recording
of the K+ channels in response to a membrane-permeant cGMP
analog, 8-BrcGMP, in a cell-attached patch is shown in Fig.
3A. Application of 8-BrcGMP (100 µM) to the bath caused an increase in the number of active channels compared with control. This result suggests that elevation of
intracellular cGMP activates the K+ channels. Because cGMP
activates PKG, we tested the effect of a membrane-permeant PKG-specific
inhibitor, KT-5823, on channel activity to examine whether PKG is
involved in the regulation of the channel. As the results show in Fig.
3B, bath application of KT-5823 (1 µM) inhibited channel
activity in a cell-attached patch. Summarized data on effects of
8-BrcGMP and KT-5823 on channel activity are shown in Fig.
3C. 8-BrcGMP significantly stimulated, and KT-5823
significantly suppressed, channel activity. These data suggest the
involvement of PKG-mediated phosphorylation processes in activation of
the K+ channel.
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Because the PKA-mediated phosphorylation process can also activate the
K+ channel in RPTECs (24), it is possible that
the stimulatory effect of 8-BrcGMP would have some relationship to the
PKA-mediated process. In fact, it has been demonstrated that cGMP
causes inhibition of phosphodiesterase type 3 (PDE3), which
subsequently increases intracellular cAMP, resulting in stimulation of
PKA activity (4). Therefore, the following experiment was
designed to examine whether the stimulatory effect of cGMP on channel
activity was observed even in the presence of a PKA-specific inhibitor.
A channel recording obtained with a cell-attached patch is shown in
Fig. 4A. A PKA-specific inhibitor, KT-5720 (500 nM), suppressed channel activity observed under
the control condition, and the following addition of 8-BrcGMP (100 µM) restored the activity. The restored channel activity was again
suppressed by KT-5823. These results suggest that channel activation
induced by 8-BrcGMP is not dependent on the PKA-mediated process but
for the most part on PKG-mediated phosphorylation. Similar results were
obtained from another six patches, and the data are summarized in Fig.
4B.
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Next, we examined effects of the cytoplasmic cGMP and PKG on channel
activity in inside-out patches under the symmetrical KCl condition at a
Vp of +50 mV. The bath solution contained 1 mM
MgATP, because this K+ channel requires cytoplasmic ATP to
maintain its activity in inside-out patches (24). Figure
5A shows a representative
current trace in response to cytoplasmic cGMP and PKG in the presence of MgATP. Addition of cGMP (100 µM) alone to the bath had little effect on channel activity, whereas the subsequent addition of PKG (500 U/ml) enhanced it. After the washing out of both cGMP and PKG, channel
activity was reduced to the control level. Data from eight patches were
pooled and summarized in Fig. 5B. There was no statistically
significant difference between the control and cGMP-treated groups,
whereas addition of PKG significantly stimulated channel activity in
the presence of cGMP and MgATP.
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Finally, we tested a guanylate cyclase-activating peptide, hANP, in
cell-attached patches. Addition of hANP (20 nM) to the bath caused a
reversible channel activation, as shown in Fig. 6A. This peptide required a
relatively longer time to express its effect compared with other test
substances. A time course experiment, where five cell-attached patches
were exposed to hANP for up to 5 min, revealed that the hANP-induced
channel activation began at ~2.5 min after addition of hANP and
reached a maximal plateau in 4 min (Fig. 6B). On the basis
of this result, we examined the effect of hANP for ~4-min exposure in
additional six patches. Summarized data from 11 patches are shown in
Fig. 6C. The effect of a PKG-inhibitor, KT-5823, on
hANP-induced channel activation was also explored. This inhibitor
suppressed the channel activated by hANP (Fig.
7A). Furthermore, hANP failed
to reactivate the channel suppressed by KT-5823 (Fig. 7B).
Summarized data are shown in Fig. 7C. It is apparent that
KT-5823 significantly suppresses the effect of hANP, whether this
inhibitor was added before or after hANP. These results strongly
suggest that hANP-induced channel activation is PKG-dependent.
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DISCUSSION |
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The single RPTEC used in this study seemed not to be well polarized. This cell attached to the coverslip, but the sparsely distributed short microvilli, which are far from the apical brush border seen in proximal tubule cells in situ, would suggest the lack of an apparent distinction between the apical and basolateral membrane. In support of this notion, it has been reported that cell-cell interactions were required for the polarization of a basolateral protein in Madin-Darby canine kidney (MDCK) cells (30). The same situation might hold true for the single RPTEC. Thus it is not known whether the inwardly rectifying K+ channel in RPTECs originated from the basolateral or the apical membrane. However, we have previously reported that this K+ channel is pH sensitive and requires PKA-mediated phosphorylation processes to maintain its activity (24). These properties are very similar to those reported previously in the basolateral K+ channel of proximal tubule cells (21). Thus we assume that the K+ channel in single RPTECs would correspond to the basolateral K+ channel in human proximal tubule cells. Nonetheless, we must notice that the properties of this K+ channel are not always identical to those of K+ channels in the native proximal tubule. For example, the activity of the K+ channel in RPTECs is not inhibited by high doses (~10 mM) of ATP (24), which is different from the ATP sensitivity of basolateral K+ channels in proximal tubule cells reported by several investigators (1, 8, 29, 33, 34). Such a difference might result from alterations in channel composition induced by cell culture, although the ATP-sensitivity of human K+ channels in situ is unknown.
Our present data demonstrate that regulation of the K+ channel in RPTECs involve PKG-mediated processes. Channel activity in cell-attached patches was activated by 8-BrcGMP, whereas it was suppressed by a PKG-specific inhibitor. Activation of the channel by 8-BrcGMP was induced even in the presence of a PKA-specific inhibitor. Moreover, although internal cGMP alone in the presence of MgATP had no significant effect on channel activity in inside-out patches, concomitant application of PKG significantly enhanced it. Thus it is suggested that PKG-dependent phosphorylation processes besides PKA-dependent ones enhance K+ channel activity. A similar dual modulation of channel activity has been demonstrated in the inwardly rectifying K+ channel of opossum kidney proximal tubule cells (16, 17).
It has been reported that intracellular cGMP induced not only activation of PKG but also inhibition of activity of PDE3 (4). The latter would lead an increase in cAMP and hence may stimulate the other cyclic nucleotide-dependent protein kinase, PKA. However as mentioned above, 8-BrcGMP stimulated channel activity even in the presence of a PKA-specific inhibitor, suggesting that cGMP-induced channel activation was not due to stimulation of PKA. Furthermore, a direct effect of cGMP on channel activity was also reported, and such a cGMP-gated K+ channel was expressed in the kidney (5, 36). This type of K+ channel contains a cGMP-binding site related to channel activation (5, 36). In immortalized human proximal tubule cells, Hirsch et al. (14) demonstrated the existence of a cGMP-regulated K+ channel, which was inhibited by cGMP without PKG-mediated phosphorylation. In our study, however, direct application of cGMP alone had no significant effect on channel activity in inside-out patches. Taken together, it can be concluded that the stimulatory effect of cGMP on the K+ channel in RPTECs is mainly produced through activation of PKG, which is independent of PKA-mediated processes. However, the site of PKG-mediated phosphorylation in our study is presently unknown. PKG and PKA might share the same phosphorylation site because of a similarity between the substrate-recognition sequences for these two protein kinases (27). It is also possible that PKG would indirectly activate the K+ channel through the phosphorylation of channel-associated proteins rather than the channel protein itself as reported in the maxi-K+ channel in a rat pituitary cell line (35).
Furthermore, channel activation by PKG-mediated phosphorylation was reversible in most cases, as shown in Figs. 3 and 5, suggesting that the phosphatase-mediated dephosphorylation would be involved in this reversibility. Thus channel activity at a given time would be determined by the dynamic balance between protein kinase activity and protein phosphatase activity. The time course of effects of test substances may well also be affected by this balance. If phosphatase activity was potentially high, the effect of a protein kinase inhibitor on the open channels would be rapidly manifested, as seen in Fig. 4A. In contrast, if PKG activity was fairly predominant, a relatively longer time would be required for the expression of the effect of a protein kinase inhibitor, as shown in Fig. 7B. Taken together, it is highly likely that protein phosphatases, as well as protein kinases, are the key factors in regulating channel activity in RPTECs. Further studies will be necessary to clarify what types of phosphatases would be involved in the suppression of the PKG-dependent channel activity.
The present study showed that hANP also activated the K+ channel in RPTECs through PKG-mediated phosphorylation. Although the major sites of ANP action in the kidney are thought to be the glomeruli and the inner medullary collecting duct (20, 37), several investigators have demonstrated the existence of guanylate cyclase-coupled ANP receptors in the proximal tubule (12, 28). In addition, it was reported that ANP caused accumulation of intracellular cGMP in this nephron segment (3, 25). Therefore, it is likely that hANP-induced changes in channel activity would be elicited by binding of this peptide to its specific receptor(s). The slow time course of hANP action observed in our study suggests that the production of an effective concentration of cGMP would take a relatively long time.
As for the effect of ANP on the K+ channel in proximal tubule cells, two reports are now available. One is that ANP inhibited the activity of an apical cGMP-regulated K+ channel in immortalized human proximal tubule cells (14). In contrast, the other report showed that ANP stimulated the activity of an ATP-regulated K+ channel in the surface membrane of opossum kidney proximal tubule cells (17). The present data are consistent with the results shown in the latter report. Considering the role of basolateral K+ channels in the proximal tubule, ANP-induced channel activation seems to be contradictory to the natriuretic property of this peptide. However, some investigators reported that ANP alone did not affect the reabsorption of fluid in the rat proximal tubule, whereas this peptide inhibited the angiotensin II-induced increase in fluid reabsorption (6, 7, 11). Another group demonstrated that ANP suppressed Na+-coupled Pi reabsorption and Na+-H+ exchange but had no effect on Na+-coupled glucose and proline uptake (9). Although the physiological role of ANP in the human proximal tubule is still obscure, one plausible explanation for our finding is that ANP-induced channel activation might prevent the excessive loss of Na+ and water by increasing the driving force for Na+ transport in this nephron even when certain kinds of Na+-coupled transporters would be suppressed by ANP.
In summary, the ATP-regulated inwardly rectifying K+ channel in human proximal tubule cells is under the stimulatory control of PKG-mediated phosphorylation processes, besides PKA-mediated ones. Moreover, PKG-mediated phosphorylation is important for ANP to activate this K+ channel.
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ACKNOWLEDGEMENTS |
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We thank Yasuo Yoshida for excellent technical support of scanning electron microscopy.
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FOOTNOTES |
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This work was supported in part by a grant from the Promotion and Mutual Aid Corporation for Private Schools of Japan.
Address for reprint requests and other correspondence: M. Kubokawa, Dept. of Physiology II, Iwate Medical University, School of Medicine, 19-1 Uchimaru, Morioka, 020-8505 Japan (E-mail: mkubokaw{at}iwate-med.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 4, 2002;10.1152/ajprenal.00023.2002
Received 17 January 2002; accepted in final form 21 May 2002.
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