Signaling and distribution of NPR-Bi, the human splice form of the natriuretic p - 《American Journal of Physiology》美国生理学杂志干细胞之家



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作者：Jochen R. Hirsch, Nikola Skutta, and Eberhard Schlatter作者单位：Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, D-48149Münster, Germany $ P5 w, z9 c9 ~# ~8 Y3 t9 F

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      【摘要】
   Recently, we described a splice variant of the human natriuretic peptidereceptor type B (NPR-Bi) in human proximal tubule cells [immortalized humankidney epithelial cells (IHKE-1) that lacks a functional guanylate cyclasedomain (Hirsch JR, Meyer M, Mägert HJ, Forssmann WG, Mollerup S, HerterP, Weber G, Cermak R, Ankorina-Stark I, Schlatter E, and Kruhøffer M. J Am Soc Nephrol 10: 472-480, 1999). Its signaling pathway doesnot include cGMP, cAMP, or Ca 2 but leads to inhibition of K channels. In patch-clamp experiments, effects of tyrosine kinase receptor blockers on C-type natriuretic peptide (CNP)-mediated depolarizations of membrane voltages ( V m ) of IHKE-1 cellswere tested. The epidermal growth factor (EGF) receptor blocker genistein (10µM) abolished the effect of CNP (0.2 ± 0.4 mV, n = 7), andcomparable results were obtained with 10 µM daidzein ( n = 8).Aminogenistein (10 µM, n = 5) and tyrphostin AG1295 (10 µM, n = 5) had no significant effects. EGF (1 nM) hyperpolarized cells by-5.3 ± 0.8 mV ( n = 5). This effect was completelyblocked by genistein or daidzein. The Cl - channel blockerNPPB (10 µM, n = 5) inhibited the EGF-mediated hyperpolarization.mRNA expression of NPR-B and NPR-Bi shows reversed patterns along the humannephron. NPR-B is highly expressed in glomeruli and proximal tubules, whereasNPR-Bi shows strong signals in the distal nephron. Expression of NPR-Bi in thecortical collecting duct was also confirmed with immunohistochemistry. Inother human tissues, NPR-Bi shows strongest expression in pancreas and lung,whereas in the heart and liver NPR-B is the dominating receptor. In conclusion, CNP inhibits an apical K channel in IHKE-1 cells independently of cGMP and so far this effect can only be blocked by genisteinand daidzein. Tyrosine phosphorylation might be the missing link in thesignaling pathway of CNP/NPR-Bi.
      【关键词】 Ctype natriuretic peptide signal transduction tyrosine kinase patch clamp analysis proximal tubule kidney
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NATRIURETIC PEPTIDES ARE structurally related peptides that bindto different receptors and display a variety of biological actions( 6, 16 ). Although quite a lot isknown about the actions of ANP and the related urodilatin that binds, similar to BNP, to the natriuretic peptide receptor (NPR)-A (GC-A), little is knownabout C-type natriuretic peptide (CNP)( 1, 11, 12 ). CNP, which is found inthe plasma and urine as a 53- and 22-amino acid ring-forming peptide, binds tothe NPR-B (GC-B) receptor, which leads to an increase in intracellular cGMP( 4 ). Recently, a splice variantof the NPR-B receptor was detected and described as NPR-Bi. This receptorcarries a 71-bp insert on the mRNA level, leading to a frameshift and truncated protein when translated( 8 ). Due to the generation ofan early stop codon, the receptor lacks a functional guanylate cyclase domainand does not increase intracellular cGMP when activated by CNP( 8 ). cGMP-independent signalingfor natriuretic peptides has so far only been described for the NPR-C or"clearance" receptor that can influence the adenylate cyclaseactivity through G proteins ( 3, 13 ). We speculated thattyrosine phosphorylation might be the signaling pathway for the NPR-Bireceptor ( 7, 8 ).6 i/ G1 g/ f; }- C: E9 f

It is known that phosphorylation processes regulate the activity ofguanylate cyclase receptors( 15 ). Recently, it could be shown for the guanylate cyclase receptor GC-C, which is bound by guanylin anduroguanylin, that not only serine/threonine-specific phosphorylation stepsregulate guanylate cyclase receptors but also tyrosine phosphorylation( 5 ).4 r6 e0 y5 @! ^# \

In this study, we demonstrate that CNP inhibits K conductance independently of cGMP and that this effect can be blocked only by genisteinand daidzein but not by the structurally related aminogenistein or anothertyrosin kinase receptor blocker tyrphostin AG1295. Furthermore, we show theexpression pattern of NPR-Bi in human tissues, especially in various segmentsof the human kidney.
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MATERIALS AND METHODS
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Cell culture. Immortalized human kidney epithelial (IHKE-1) cells(derived from human embryonic kidneys) were cultured as described previously( 18 ). In short, IHKE-1 cells( passages 162 - 188 ) were grown in 50-ml tissue culture flasks(Greiner, Frickenhausen, Germany) in Dulbecco's modified Eagle's/F-12 medium(1:1) containing 15 mM HEPES, pH 7.3, 1.6 nM EGF, 100 nM hydrocortisone, 83µM transferrin/insulin, 29 nM Na 2 SeO 3, 10 mMNaHCO 3, 20 mM L -glutamine, 1,000 U/lpenicillin/streptomycin, and 1% fetal calf serum. Cells were incubated at37°C in an atmosphere of 5% CO 2 -95% air. After 7 days, theconfluent monolayers were trypsinized with Ca 2 - andMg 2 -free phosphate-buffered saline and 0.05%trypsin-EDTA (Biochrom, Berlin, Germany). Cells grew polarized on glasscoverslips, with the apical surface facing upward.
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Patch-clamp studies. Coverslips with confluent IHKE-1 monolayers were mounted at the bottom of a perfusion chamber on an inverted microscope(Axiovert 10, Zeiss, Oberkochen, Germany). The perfusion chamber wascontinuously perfused at a rate of 10-20 ml/min at 37°C with astandard solution containing (in mM) 145 NaCl, 1.6K 2 HPO 4, 0.4 KH 2 PO 4, 5 D -glucose, 1 MgCl 2, and 1.3Ca 2 -gluconate, pH 7.4. Cells were rinsed for at least 20 min before the electrophysiological measurements.. E* B) C+ Q6 L6 U0 Q
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Membrane voltages ( V m ) were measured with the slowwhole cell patch-clamp technique. For this method, pipettes were filled with asolution containing (in mM) 95 K -gluconate, 30 KCl, 4.8Na 2 HPO 4, 1.2 NaH 2 PO 4,5 D -glucose, 0.73 Ca 2 -gluconate, 1 EGTA, 1.03MgCl 2, and 1 ATP, pH 7.2. To this solution 162 µM nystatin wasadded before use. Patch pipettes had an input resistance of 2.5-12.5M. V m was measured in the current clamp mode of apatch-clamp amplifier (U. Fröbe, Physiologisches Institut,Universität Freiburg, Germany) and recorded continuously on a penrecorder (WeKaGraph WK-250R, WKK, Kaltbrunn, Switzerland)., p/ v) l3 E' y, ]7 V; {- l' U

RT-PCR analysis. Total RNA was isolated using the RNeasy-kit (Qiagen, Hilden, Germany). Isolated total RNA was incubated with 10 U DNase I(Promega, Heidelberg, Germany) at 37°C for 1 h to digest isolated tracesof genomic DNA. RNA and DNase I were then separated by an additional cleanupstep using a new RNeasy column. cDNA first-strand synthesis was performed in atotal reaction volume of 30 µl containing 5 µg total RNA, 10 nM dNTP-Mix(Biometra, Göttingen, Germany), 1 nM p(dT)10 nucleotide primer(Boehringer, Mannheim, Germany), and 200 U molony murine leukemia virusreverse transcriptase (Promega). Of each cDNA first-strand reaction mixture, was then subjected to a50-µl PCR reaction in a UNO II thermo cycler (Biometra) using 20 pmol ofeach primer and 1 U of Taq DNA polymerase (Qiagen). Reactionconditions were as follows: 3 min at 94°C, 30 s at 59°C, and 1 min at72°C, 1 cycle; 30 s at 94°C, 30 s at at the optimal annealingtemperature (OAT), and 1 min at 72°C, 30 cycles; and 30 s at 94°C, 30s at OAT, and 10 min at 72°C, 1 cycle. PCR reaction products were analyzedby agarose gel electrophoresis. Positive signals obtained from PCR experiments were sequenced by SeqLab (Göttingen, Germany). The following PCR primerswere used (listed in 5'- to 3'-direction). The sequence isfollowed by the expected fragment length for the respective sense andantisense primer: NPR-Bi sense: GAC TCT CAC TCC AGC CCT AGT CTC, NPR-Biantisense: TTC AGC GCT TGA CCA TTA GAC TCC, fragment length: 169 bp, OAT =60°C; NPR-B sense: GAG ACG ATT GGG GAT GCT TA, NPR-B antisense: TTC AGC GCT TGA CCA TTA GAC TCC; fragment length: 277 bp, OAT = 60°C; GAPDH sense:CTG CCC CCT CTG CTG ATG, GAPDH antisense: GTC CAC CAC CCT GTT GCT GT, fragmentlength: 614 bp, OAT = 61°C.8 O( F/ C1 Q: p" n1 v) w6 Z2 A! B

Immunocytochemistry. Kryo slices of a human kidney were blocked for 15 min at room temperature with 1% blocking agent (Roche, Mannheim,Germany). The primary polyclonal antibody against NPR-Bi (Immundiagnostik,Bensheim, Germany) was incubated overnight at 4°C in a wet chamber. Afterthree washing steps in PBS, the secondary antibody (goat-anti-rabbit, Vector,Burlingame, CA) was incubated for 45 min followed by three more washing stepsin PBS. The kryo slices were then incubated for 45 min in streptavidin, AlexaFluor 594 Conjugate (Mobitec, Göttingen, Germany) followed by fivewashing steps in PBS. Finally, the slices were covered with mounting mediacontaining DAPI (Vector).

Biochemicals. Human CNP was kindly provided by Dr. Knut Adermann (IPF Pharmaceuticals, Hannover, Germany). Genistein, daidzein, aminogenistein,and tyrphostin AG1295 were supplied by Calbiochem (Bad Soden, Germany). Allother standard chemicals were supplied by Calbiochem, Sigma (Taufkirchen,Germany) or Merck (Darmstadt, Germany).

Statistical analysis. Data are presented as means ± SE, with the number of experiments given in brackets. For statistical analyses,Student's paired and unpaired two-sided t -tests were used. For pairedcomparisons, each effect was compared with its own averaged pre- andpostexperimental controls. A P value 0 l! V' I' C& o$ o/ {5 q/ F
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RESULTS
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To verify if the cGMP-independent CNP-induced depolarization in the humanproximal tubulus cell line IHKE-1 is specifically blocked by genistein orgenerally blocked by a variety of tyrosine kinase receptor blockers, we testedthe epidermal growth factor (EGF) receptor blocker genistein, its"inactive" form daidzein, the structurally related aminogenisteinthat is also an inhibitor of the p56 lck tyrosine kinase, and thePDGF receptor blocker tyrphostin AG1295. In 57 patch-clamp experiments, themembrane voltage ( V m ) of IHKE-1 cells was -42± 1 mV. In seven paired experiments, CNP (10 nM) depolarized V m by 2.8 ± 0.4 mV. In the presence of genistein(10 µM), the CNP-induced depolarization was abolished (0.2 ± 0.4 mV). After the washout of genistein, CNP depolarized V m again by 1.4 ± 0.4 mV ( Fig.1 ). As a negative control, we used daidzein, the inactive form ofgenistein. To our surprise, daidzein (10 µM) reversed the CNP-induceddepolarization ( n = 8). Aminogenistein (10 µM, n = 5) andtyrphostin AG1295 (10 µM, n = 5) had no significant effect on the CNP-induced depolarization ( Fig.1 ).# ]# {) D6 l  O+ v* `$ \8 c

Fig. 1. Effects of genistein ( n = 7), daidzein ( n = 8),aminogenistein ( n = 5), and tyrphostin AG1295 ( n = 5) on theC-type natriuretic peptide (CNP)-mediated depolarization of membrane voltage( V m ) of immortalized human kidney epithelial (IHKE-1)cells. In paired experiments, CNP (10 nM) depolarized V m of IHKE-1 cells by 3 mV. This depolarization was inhibited by theepidermal growth factor (EGF) receptor blocker genistein and its"inactive" form, daidzein, but not by the structurally relatedp56 lck tyrosine kinase blocker aminogenistein or the PDGF receptorblocker tyrphostin AG1295.* P " ^# n: H! E8 F
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To verify the specificity of genistein and daidzein, both were tested ontheir inhibitory effect of the EGF-mediated signaling pathway. In five pairedexperiments, EGF (1 nM) hyperpolarized V m by -5.3± 0.8 mV ( Fig. 2 ). Inthe presence of genistein, this effect was fully inhibited to 1.2 ± 1.1mV and after the washout of genistein also fully reversible (-4.1± 0.9 mV). Daidzein again mimicked the effect of genistein, clearlyshowing that this substance is not inactive in the human proximal tubule cellline IHKE-1 ( Fig. 2 ). Although the CNP-induced depolarization is due to the inhibition of a K channel( 7 - 9 ),the EGF-induced hyperpolarization is due to the inhibition of aCa 2 -dependent Cl - channel since theCl - channel blocker NPPB (10 µM) blocked the EGF effect on V m ( Fig.2 ). Other Cl - channel blockers, such as DIDS andSITS, and Na channel blockers, such as amiloride, had nosignificant effect on the EGF-mediated hyperpolarization (data not shown). EGFalso did not interact with CNP as there was no significant effect of EGF onthe CNP-induced depolarization (2.6 ± 0.4 vs. 2.7 ± 0.6 mV, n = 6). This demonstrates that EGF and CNP act through independentpathways as shown in a simplified cell model in Fig. 3.

Fig. 2. Effects of genistein ( n = 5), daidzein ( n = 5), and NPPB( n = 5) on the EGF-mediated hyperpolarization of V m of IHKE-1 cells. In paired experiments, EGF (1 nM)hyperpolarized V m of IHKE-1 cells by 3-5 mV. Thishyperpolarization was inhibited by the EGF receptor blocker genistein and its"inactive" form, daidzein, and theCa 2 -dependent Cl - channel blockerNPPB. * P

Fig. 3. Simplified cell model of IHKE-1 cell. The basolateral membrane contains theNa -K -ATPase that provides the driving force for theNa -driven transport systems of the luminal membrane. Furthermore,a housekeeping K channel allows K recycling across thebasolateral membrane. The exit pathways for the substrates are left out. Theluminal membrane contains, besides the already mentionedNa -coupled transport systems, an hIK-like K channelthat is most likely responsible for the repolarization of these cells, thusestablishing the driving force for the Na -coupled transportsystems. This Ba 2 -inhibitable K channel isinhibited by CNP cGMP independently through the NPR-Bi receptor. Thisinhibition can be reversed by genistein. Genistein also blocks theEGF-mediated inhibition of a Cl - channel in the samemembrane. Because this Cl - channel is not blocked by SITS orDIDS but NPPB, it is most likely a Ca 2 -dependentintermediate-conductance outwardly rectifying Cl - channel(ICOR).7 C2 S, i0 K* }0 E! Q1 K: I

Because we were interested in the distribution of NPR-Bi and NPR-B in thehuman kidney, we performed RT-PCR on isolated glomeruli and tubules to checkfor their expression pattern along the nephron. Figure 4 shows the interestingresult. Although NPR-B is mainly and strongly expressed in glomeruli andproximal tubules, NPR-Bi is weakly expressed in proximal tubules, stronger inthick ascending limbs, and predominantly expressed in collecting ducts.Because the PCR was performed with the same mRNA/cDNA batches for NPR-B andNPR-Bi and each sample had its own GAPDH control, the data can be seensemi-quantitatively, meaning expression of NPR-B is increasing toward theproximal part of the nephron, whereas that of NPR-Bi is increasing toward thedistal parts of the human nephron ( Fig.4 ). This result is also confirmed by immunocytochemistry, whichshows a clear signal of the antibody against NPR-Bi in the luminal membrane ofthe collecting duct ( Fig. 5 ).Due to the interesting reversed distribution pattern of NPR-B and NPR-Bi inthe nephron, we performed RT-PCR with different human tissue samples tomonitor the distribution of the two human receptors in other tissues. Twosignals of NPR-Bi clearly stuck out compared with NPR-B. The expression inlung and pancreas was dominated by NPR-Bi ( Fig. 6 ). NPR-B expression wasstrongest in the heart and liver. Keeping in mind that lung, pancreas, andcolon are the three tissues mostly affected in cystic fibrosis, we alsoperformed PCR experiments with human colon tissues, confirming that both receptors are equally well expressed (data not shown).

Fig. 4. mRNA expression patterns of natriuretic peptide receptor (NPR)-Bi and NPR-Bin the human nephron amplified by RT-PCR. The intensity of the NPR-Bi signalincreases toward the distal part of the human nephron, being strongest in thecollecting duct, whereas the intensity of the NPR-B signals showed theopposite distribution, being strongest in the glomeruli. As a positivecontrol, GAPDH was amplified for each segment (displayed is only one samplebecause the intensities of the GAPDH signal did not vary). As negativecontrols, all tests were performed in parallel with either no cDNA or no Taq -polymerase.
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Fig. 5. Immunocytochemistry using a polyclonal antibody against NPR-Bi in humankidney slices. A positive signal (red staining) could only be obtained in theluminal membrane of cortical collecting duct cells, confirming the resultfound by RT-PCR. In addition, cell nuclei are stained blue (DAPI).
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Fig. 6. mRNA expression patterns of NPR-Bi and NPR-B in different human tissuesamplified by RT-PCR. Although both receptors are equally well expressed in theheart, brain, placenta, and liver tissue, expression of NPR-Bi is clearlydominant in the lung and pancreas.

DISCUSSION3 s2 N  K" f- [/ t: v/ u+ G

CNP is known to bind the natriuretic peptide B (NPR-B/GC-B) receptor andincrease intracellular cGMP. It is known to display biological actions in thecardiorenal system, bronchial tree and pulmonary vasculature, and theendocrine, gastrointestinal, and neuronal systems( 4 ). Recently, it was shownthat CNP can also act independently of cGMP by inhibiting aCa 2 -dependent, intermediate-conductance, hIK1-likeK channel in human IHKE-1 cells derived from the human renalproximal tubule ( 8 ). Besides the well-known NPR-B receptor, a splice variant (NPR-Bi) was found that is thedominant pathway for CNP in these cells. The NPR-Bi receptor lacks afunctional guanylate cyclase domain, thus inhibiting the hIK1-likeK channel cGMP independently. Furthermore, we were able to showthat neither Ca 2 nor cAMP played any role in theregulation of this K conductance( 8 ). Due to the structure ofthe receptor and a recent report that demonstrated a key role for tyrosinephosphorylation in the activity of the related GC-C receptor( 5 ), we tested various tyrosinekinase blockers. From these inhibitors, only genistein, a known inhibitor ofthe EGF receptor ( 2 ), and itsinactive form, daidzein, were able to block the CNP-induced depolarization ofmembrane voltage. Neither the structurally related aminogenistein nor the PDGFreceptor blocker tyrphostin AG1295 had any significant effects. Althoughgenistein and daidzein also blocked the EGF-mediated hyperpolarization, theydisplayed no direct effect on the hIK1-like K channel itself( 8 ). A direct interactionindependent of tyrosine phosphorylation had been reported for someK channels, a Cl - channel, and theNa -2Cl - -K cotransporter ( 14, 19, 20 ). In IHKE-1 cells, daidzeinwas also not an inactive substance but acted like genistein. So far, these twosubstances are the only blockers capable of inhibiting the CNP-mediated signaling through NPR-Bi, which is most likely related to tyrosine phosphorylation. The hIK1-like K channel apparently plays an important role in these human proximal tubule cells because it can be blockedby ANP, BNP, and urodilatin cGMP dependently through NPR-A/GC-A( 9 ), by CNP cGMP independentlythrough NPR-Bi ( 8 ), and byguanylin, uroguanylin, and STa cGMP dependently through GC-C( 17 ). Furthermore, cGMPinhibits this K channel directly from the extracellular as well asthe intracellular surface ( 10 )as displayed in Fig. 7. Thediverse expression pattern of NPR-B and NPR-Bi in the human nephron clearlyindicates different tasks for CNP through these receptors. ThecGMP-independent pathway seems to play an important role in the humancollecting duct, where NPR-B is not expressed at all and NPR-Bi showed itsstrongest signal on the mRNA and protein level. When the mRNA expressionpattern of NPR-Bi in various human tissues is viewed, it is striking thatsignals from lung and pancreas stick out. The fact that NPR-Bi and NPR-B arealso strongly expressed in the human colon indicates a role for NPR-Bi in cystic fibrosis-related tissues. Regulation of hIK1 in the basolateral membrane of lung or colon cells by tyrosine phosphorylation might be a keyelement in the regulation of Cl - secretion in these cells.Further investigations are needed to clarify the role of NPR-Bi and itsinteraction with hIK1 and CFTR in human cell types such as Calu-3 (lung), T84(colon), and CF-PAC (pancreas).9 }& B* F5 }$ C6 ], v0 n  q) u3 [
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Fig. 7. Simplified cell model of IHKE-1 cell demonstrating the differentcGMP-dependent and -independent pathways used by natriuretic peptides to blockthe hIK-like K channel of the apical membrane. The basolateralmembrane contains the Na -K -ATPase that provides thedriving force for the Na -driven transport systems of the apicalmembrane. Furthermore, a housekeeping K channel in the basolateralmembrane provides the stoichiometry. The exit pathways for the substrates areleft out. The luminal membrane contains, besides the already mentionedNa -coupled transport systems, an hIK-like K channelthat is most likely responsible for the repolarization of these cells, thusestablishing the driving force for the Na -coupled transportsystems. This K channel is blocked directly by cGMP generated byANP, BNP, or urodilatin through the NPR-A/GC-A receptor, by guanylin,uroguanylin, or STa through the GC-C receptor. Extracellular cGMP alsoinhibits this K channel. cGMP is released into the tubular lumeneither by glomerular cells or by a newly described pump in proximal tubulecells. CNP blocks this K channel cGMP independently through NPR-Bimost likely by tyrosine phosphorylation, which can be blocked bygenistein.
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DISCLOSURES2 p; A2 X: e  x0 W2 \+ d
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This work was supported by the Deutsche Forschungsgemeinschaft (Schl277/5-5 to 5-6 and 11-1).

ACKNOWLEDGMENTS& m" M  v8 y$ i/ J8 l; p/ W0 \9 M
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The authors gratefully acknowledge the expert technical assistance of M.Eich, U. Kleffner, U. Siegel, and H. Stegemann.% h2 b/ l5 b1 C& M
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