|
- 积分
- 0
- 威望
- 0
- 包包
- 0
|
作者：Geoffrey E. Woodard, Xiaohong Li, and Juan A. Rosado作者单位：1 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1752; 2 Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029; and 3 Department of Physiology, University of Extremadura, 10071 Cáceres, Spain ! m: ^$ E: a' j, m3 H
! Q/ ^0 h. D% h0 h$ ^8 C
n9 L' u, v9 w
4 L7 U' ^: y3 e/ p
) C. d4 G6 @/ Y: V3 C3 C& f% d
. I6 h, e4 S% z2 J$ Y% P; C H% J
, i: H. \ T( V. `
' c, d* F9 x, [& B. H. G6 r! p r
: H1 w0 w- t5 b9 G+ a' A
3 x" Q, D% m. v$ i 9 l/ j" y0 @5 S1 F1 m
) D0 z* N) Z2 ]4 w3 \9 j / ^" `' Q; J( L9 w4 }- K7 o
【摘要】: l- @5 ]: V* D" h. F1 A
This study investigates the effect of water deprivation on the expression of atrial natiruretic peptide (ANP) 1-28 binding sites in rat kidney. Water deprivation increased the B max of glomerular binding sites for ANP 1-28 and C-type natriuretic peptide (CNP) 1-22 without modifying their affinity, an effect that was prevented in the presence of C-atrial natriuretic factor (C-ANF), suggesting that natriuretic peptide receptor-C (NPR-C) binding sites might be enhanced. Our results indicate that ANP 1-28, CNP 1-22, and C-ANF inhibit cAMP synthesis directly stimulated by forskolin or by the physiological agonists histamine and 5-hydroxytryptamine. The inhibitory effect was found to be significantly greater in water-deprived rats than in controls. Our observations suggest that this effect must be attributed to the 67-kDa NPR-C-like protein, because the 67- and 77-kDa NPR-C-like proteins show high and low affinities for CNP 1-22, respectively, and the enhanced inhibitory effect of CNP on cAMP generation in water-deprived rats was detected at subnanomolar concentrations. In addition, using affinity cross-linking studies we have observed that water deprivation increases the expression of the 67-kDa NPR-C-like protein, and HS-142, which binds to NPR-A and the 77-kDa NPR-C-like but not the 67-kDa protein, reduced ligand internalization without affecting cAMP inhibition by ANP 1-28. Finally, we have found that ligand binding to the 67-kDa NPR-C-like protein is reduced by GTP S, suggesting that this receptor is associated with a G protein in renal glomeruli. The enhanced inhibitory role of natriuretic peptides on cAMP synthesis induced by water deprivation may influence glomerular function in the rat kidney.
6 [5 N( t4 N4 K3 P 【关键词】 Ctype natriuretic peptide Catrial natriuretic factor natriuretic peptide receptorC- M6 M3 B1 S6 A% V" y4 Y
NATRIURETIC PEPTIDES ARE HORMONES involved in the regulation of several physiological functions. The 28-amino acid atrial natriuretic peptide (ANP 1-28 ) is involved in fluid and cardiovascular homeostasis ( 12, 27 ). Two other peptides, the brain and C-type natriuretic peptides (BNP and CNP), have been identified and are involved in renal and vascular functions ( 22, 23, 27, 34, 35 ). In addition, CNP is a local modulator with antiproliferative effects in the vascular cell system ( 22 ). Three types of natriuretic peptides receptors (NPR) have been cloned, NPR-A, -B, and -C ( 28 ). Two of these, designated NPR-A and NPR-B, of 120 kDa, have agonist-dependent guanylate cyclase domains ( 4, 20, 21 ). NPR-A is activated with high affinities by ANP 1-28 and BNP, but hardly binds CNP 1-22, which is known to be a high-affinity agonist of NPR-B ( 18 ).0 \ R) s8 h/ M$ C
6 \' f4 G$ h, {. {3 ]NPR-C is a disulfide-bridged homodimer of 60 kDa units, with a broad range of high-affinity ligands, including ANP 1-28, BNP, CNP 1-22, and the synthetic des[Gln 18,Ser 19,Gly 20,Leu 21,Gly 22 ]ANP 4-23 -amide (C-ANF) ( 13, 18 ). C-ANF is virtually without affinity for NPR-A or NPR-B ( 8, 10, 18, 21 ). The NPR-C function is still not fully understood and might be responsible for the lysosomal clearance of ANP 1-28 ( 1 ). In addition, NPR-C also inhibits adenylate cyclase through the activation of a G i ( 4, 20 ), although this effect has not received full acceptance since the discovery of a cGMP-dependent phosphodiesterase of cAMP that is known to account for the ability of ANP 1-28 to reduce cAMP levels in some tissues ( 16, 21 ). Two NPR-C-like proteins, of 67 and 77 kDa, have been identified in rat glomerular membranes, and their electrophoretic mobilities with and without dithiothreitol suggest that both form disulfide-bridged dimers ( 11 ). Both proteins have high affinities for ANP 1-28 and C-ANF; however, the 67-kDa protein has a high affinity for CNP 1-22, whereas the 77-kDa protein hardly binds this peptide ( 11 ). The 67-kDa protein decreases the cAMP levels in rat glomeruli, an effect that was observed in the absence and presence of isobutylmethylxanthine (IBMX), an inhibitor of cGMP-dependent cAMP phosphodiesterase, suggesting that the 67-kDa protein inhibits cAMP synthesis rather than inducing cAMP degradation ( 11 ). On the other hand, the 77-kDa protein is involved in natriuretic peptide internalization ( 11 ).
+ C5 K3 E4 _! X- h& \/ L1 L6 W/ D. J! d+ Z7 {
If the 67- and 77-kDa NPR-C-like proteins of rat glomerular membranes are indeed functionally distinct receptors, it is likely that their expression will be differentially regulated. On the basis of the fact that both NPR-C-like proteins bind ANP 1-28 but only the 67-kDa protein has high affinity for CNP 1-22, here we show that water deprivation increases the expression of the 67-kDa NPR-C-like protein in rat glomeruli and, therefore, inhibits cAMP synthesis, without affecting ligand binding to the 77-kDa NPR-C-like protein or natriuretic peptide internalization. Furthermore, we show that the NPR antagonist HS-142 binds to the 77- but not the 67-kDa NPR-C-like protein. In addition, we provide further evidence in favor of a role for a G protein in the inhibitory effect of the 67-kDa receptor induced on cAMP synthesis in renal glomeruli.8 `" v" X6 {. H: p- E$ i
+ q6 D3 z' b( V/ m, O( XMATERIALS AND METHODS
A6 \+ v8 Y. P/ n& [) _* q3 ?
1 j- z3 ~- M/ `2 _Materials. Unlabeled peptides and 125 I-tyr-CNP 1-22 (1,300-1,900 Ci/mmol) were from Peninsula Laboratories (St. Helens, Merseyside, UK). 125 I-ANP 1-28, [ 125 I] standard, Hyperfilm 3 H for autoradiography, and cAMP radioimmunoassay commercial kits were from Amersham (Little Chalfont, UK). Bis(sulfosuccinimidyl) suberate (BS 3 ) was from Pierce and Warriner (Chester, UK). GRI-AX film was from Genetic Research Instrumentation (Dunmow, Essex, UK). All other reagents were from Sigma (Poole, UK).
6 q, b2 u5 P2 x
: n: i4 K0 h4 h* YAnimals. Wistar rats (250-300 g) were obtained from a certified commercial supplier (Charles River). Control groups were allowed free access to food and water. Matching groups were kept under identical conditions, except that they received no water for 4 days. Rats were anesthetized with ether and killed by rapid exsanguination.
* \' B# L2 E2 V7 {' C' F$ o# M7 U. c2 i& N% Y
The APSs Guiding Principles in the Care and Use of Laboratory Animals were followed.( p6 v. o! l7 J% x6 Z/ l
' N. i: L! y6 X/ lCompetition experiments in isolated glomeruli. Isolation of rat glomeruli was performed as described previously ( 24 ). Briefly, kidneys were removed and placed in ice-cold Hanks' balanced salt solution (HBSS) containing (in mM) 137 NaCl, 10 HEPES, 5.4 KCl, 0.4 Mg 2 SO 4, 0.34 Na 2 HPO 4, 1.26 CaCl 2, 4.17 Na 2 HCO 3, 0.44 K 2 HPO 4, 0.49 MgCl 2, and 5.56 glucose, pH 7.2, as well as 0.2% (wt/vol) BSA. The cortexes were minced, and glomeruli were isolated by differential sieving. Aliquots of glomeruli were preincubated with 1 mM phenanthroline for 6 min at 4°C and then incubated with 400 pM 125 I-ANP 1-28 or 125 I-CNP 1-22 in the presence of increasing concentrations of unlabeled peptides at 20°C for 10 min. Preliminary experiments showed that specific binding of radioligand reached equilibrium at 10 min (not shown). Incubations were stopped by centrifugation, and 125 I labeling was determined using a Packard Gamma Counter.
+ b$ B6 Y1 z m4 v/ C6 d' I& k1 i; s2 ^. s/ z2 d' v# ^& M
Binding assay of particulate ANP receptors in membranes. Freshly isolated renal papilla was homogenized and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was then centrifuged for 60 min at 4°C at 40,000 g, and the pellet was washed, sonicated, and stored. Aliquots (10 µg) of papillary membrane were incubated for 1 h or 40 min with 200 pM 125 I-ANP 1-28 or 125 I-CNP 1-22 in the absence or presence of increasing concentrations of unlabeled natriuretic peptides in Tris buffer containing (in mM) 50 Tris, 4 MgCl 2, and 1 1,10-phenanthroline, pH 7.6, as well as 0.4% (wt/vol) BSA. Incubation was stopped by filtration. The filter-bound radioactivity was determined in a Packard Gamma Counter. The nonspecific binding was measured in the presence of 1 µM ANP 1-28.
5 A: e; |8 C% T& S) ?5 q7 q+ f W0 e' i
Affinity cross-linking studies. Freshly isolated glomeruli were homogenized and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was then centrifuged for 60 min at 4°C at 40,000 g, and the pellet was washed, sonicated, and stored. Aliquots of glomerular membranes (50 µg) were incubated with 125 I-ANP 1-28 in the presence or absence of the indicated unlabeled peptides and inhibitors at 4°C for 2 h. Preliminary experiments showed that specific binding of the radioligand reached equilibrium after 2 h. The bound 125 I-ANP 1-28 was then cross-linked to its receptor by 500 µM BS 3 for 40 min at 4°C. The cross-linking reaction was stopped, and the samples were resuspended in Laemmli's buffer ( 19 ) and separated by 10% SDS-PAGE. The autoradiographs were exposed for 31 days at -80°C.
: s7 u! I! C1 R3 R/ l3 Z8 E- ^) n' r: a- {5 a+ _
Autoradiographic studies. Kidney slides from male spontaneously hypertensive and Wistar-Kyoto rats in PBS containing (in mM) 120 NaCl, 21.6 Na 2 HPO 4, 8.4 NaH 2 PO 4, pH 7.2, with 1 1,10-phenanthroline were incubated with 100 pM 125 I-ANP 1-28 or 200 pM 125 I-CNP 1-22 (2,000 Ci/mmol) with or without unlabeled peptides at 20°C for 15 min. After incubation, the sections were exposed to Hyperfilm 3 H for 31 days.
! n6 j9 A- |1 D$ W5 u
! ~$ ]8 V! N$ {$ s0 w7 k( @cAMP accumulation. Aliquots of 100 glomeruli (7.6 mg protein) from control and water-deprived rats were suspended in HBSS plus 0.2% BSA with 1 mM IBMX and then incubated for 10 min at 20°C with different agonists in the absence or presence of natriuretic peptides and/or HS-142 as indicated. Incubations were terminated by ice-cold trichloroacetic acid as previously described ( 10, 11 ). Aliquots were then centrifuged at 4,000 g for 10 min, and the supernatants were extracted with ether and radioimmunoassayed for cAMP as previously described ( 10, 11 ).
9 X- \1 A) \9 N
" W5 X }/ s. C+ Y/ n( L2 _cGMP assay. cGMP production in glomerular membranes was determined as described previously ( 36 ). Glomerular membranes were diluted with Tris buffer such that 20 µl of this mixture solution contained 3-5 µg of protein. Guanylate cyclase activity was measured at 37°C in a reaction mixture containing (in mM) 50 Tris·HCl, 4 MgCl 2, 1 IBMX, 1 GTP-Mg 2 , 15 creatine phosphate, and 1 ATP as well as 20 U/ml creatine phosphokinase, pH 7.6. The reaction was started with the addition of the membrane suspension and stopped 20 min later with ice-cold 50 mM sodium acetate (pH 5.8) followed by boiling. The samples were then centrifuged at 4,000 g for 10 min, and cGMP was determined by radioimmunoassay.
* w0 C; M& N! G) g* x1 \6 M" M4 P# R% N: C) g: T' e+ o
Ligand internalization. 125 I-ANP 1-28 internalization was studied in glomeruli from control and 4-day water-deprived rats. Aliquots of 2,400 glomeruli were incubated in HBSS containing 0.2% BSA, 1 mM 1,10 phenanthroline, and 1 nM 125 I-ANP 1-28 (specific activity 500 Ci/mmol) with or without 1 µM ANP 1-28, 10 µM C-ANF, or 30 ng/ml HS-142 for 1 h at 4°C and then for 10 min at 37°C. The incubation was stopped by centrifugation at 1,000 g for 2 min at 0°C, and internalized and surface-bound radioligands were measured. Internalized radioligand was measured after the addition of acetic acid-NaCl [final concentration of 0.2 M acetic acid-0.2 M NaCl (pH 2.5)] for 6 min at 4°C followed by centrifugation at 1,000 g for 2 min at 4°C and analysis of 125 I associated with sedimented glomeruli.
5 ~! h/ O: Q+ i6 @$ B0 l/ n2 w3 Q; [8 Z
Data analysis. Data were analyzed using the LIGAND program. Analysis of statistical significance was performed using Student's t -test. The significance level was P - S5 \, `1 c8 I7 }- n+ ^
- Z( ?8 @9 o8 q2 fRESULTS
- r7 `8 J4 [3 o* Q# \: ]0 k5 \0 X1 b* e
Effects of water deprivation on renal binding of natriuretic peptides. Comparison of autoradiography with the corresponding stained tissue sections revealed a similar anatomic distribution of specifically reversible binding sites for ANP in control and water-deprived rats. 125 I-ANP 1-28 and 125 I-CNP 1-22 bound mostly to glomeruli. Radioligand binding to these structures was virtually abolished in the presence of 1 µM unlabeled ANP 1-28, CNP 1-22, and C-ANF ( Fig. 1; n = 6).# B7 i9 h* T# I5 \0 J0 p. \
7 b! ], A v7 H# S( i! o% ]
Fig. 1. Autoradiographs of renal binding of 100 pM 125 I-labeled atrial natriuretic peptide (ANP) 1-28 and 200 pM 125 I-C-type natriuretic pepetide (CNP 1-22 ) in control and water-deprived rats. Left : sections of kidney from control ( A-D ) or water-deprived ( a-d ) rats were incubated with 100 pM 125 I-ANP 1-28 in the absence ( A and a ) and presence of 1 µM ANP 1-28 ( B and b ), CNP 1-22 ( C and c ), and C-ANF ( D and d ) at 20°C for 15 min. Right : sections of kidney from control ( A-D ) or water-deprived ( a-d ) rats were incubated with 200 pM 125 I-CNP 1-22 in the absence ( A and a ) and presence of 1 µM CNP 1-22 ( B and b ), ANP 1-28 ( C and c ), and C-ANF ( D and d ) at 20°C for 15 min. Autoradiography was performed as described in MATERIALS AND METHODS.
/ L6 p4 q2 r! Y
* R- l" {; t$ {, L& QCompetitive inhibition of 125 I-ANP 1-28 binding by ANP 1-28 was further examined in control and water-deprived rats. As shown in Fig. 2, ANP 1-28 displaced 125 I-ANP 1-28 in a concentration-dependent manner in both control and water-deprived rats. Water deprivation increased the B max of glomerular binding sites for ANP 1-28 without significantly altering their affinity for this peptide ( Fig. 2, Table 1 ). A similar effect was observed when CNP 1-22 binding was tested. Water deprivation increased the B max of the glomerular binding sites for CNP 1-22 without significantly modifying its affinity ( Table 1 ).
; Q& x$ u( x C* p7 q% ^3 v% _3 \4 y; Q4 ~3 `* p
Fig. 2. Competitive inhibition of 125 I-ANP 1-28 binding to isolated glomeruli from control ( A ) and water-deprived ( B ) rats. Binding of 125 I-ANP 1-28 to isolated glomeruli was investigated by incubation of renal glomeruli with 400 pM 125 I-ANP 1-28 in the presence of increasing concentrations of unlabeled ANP 1-28 or CNP 1-22 at 20°C for 10 min as described in MATERIALS AND METHODS. Nonspecific binding was obtained by incubation with 1 µM ANP 1-28. Values are means ± SE of 6 independent experiments./ M( }; _2 d2 T/ W5 B; Q3 e
$ ~# M. w' R2 Z9 t3 [
Table 1. Binding constants for the specifically reversible binding of ANP 1-28 or CNP 1-22 in the glomeruli of control and 4-day water-deprived rats
6 j5 |1 r# n/ [4 }% d' m/ {$ d( ^
9 r7 o1 ]* g, j' o$ N+ gIn contrast, we have found that water deprivation had no significant effects on ANP 1-28 binding to the papilla ( Fig. 3 ), which expresses NPR-A but does not detectably express NPR-C in the rat ( 9 )., h" X p4 ?/ j' T9 s
1 o8 }. h m% w5 ~. x1 Z5 G+ ~9 r3 r
Fig. 3. Competitive inhibition of 125 I-ANP 1-28 binding to papillary membranes from control ( A ) and water-deprived ( B ) rats. Binding of 125 I-ANP 1-28 was investigated after the addition of increasing concentrations of unlabeled ANP 1-28 or CNP 1-22 at 20 o C for 10 min as described in MATERIALS AND METHODS. Values are means ± SE of 6 independent experiments.
, w$ s d! v+ M5 Y
6 F- S+ S7 J8 |' D* ~Interestingly, C-ANF abolished the difference observed in ANP 1-28 glomerular binding in control and water-deprived rats. Thus the specific glomerular binding of 125 I-ANP 1-28 in the absence of C-ANF was 377 ± 26 and 625 ± 99 fmol/mg protein, respectively, in control and water-deprived rats ( P
w8 z; h- k+ F- c) F( L4 g$ I3 |8 n
' `$ @& @0 ]+ Z+ _! T- G! cAffinity cross-linking of ANP receptors in glomerular membranes from control and water-deprived rats. Cross-linking studies using glomerular membranes from control and water-deprived rats revealed that 125 I-ANP 1-28 was covalently incorporated into three bands with apparent molecular masses of 120, 77, and 67 kDa ( Fig. 4 ). The band with a molecular mass of 120 kDa corresponds to NPR-A ( 29 ). In control and water-deprived rats, labeling of the 120-kDa band was completely displaced by incubation with 1 µM unlabeled ANP 1-28, which also displaced the labeling of the 77- and 67-kDa proteins ( Fig. 4 ). The labeling of the 120-kDa protein was partially displaced by increasing concentrations of unlabeled CNP 1-22 ( Fig. 4 ), demonstrating the specificity of the NPR-A receptor. The labeling of the 67- and 77-kDa bands was progressively displaced by increasing concentrations of unlabeled CNP 1-22 ( Fig. 4 ) or C-ANF (not shown). These proteins, therefore, corresponded to the 67- and 77-kDa NPR-C-like proteins in the rat glomerulus. Although the labeling intensity of NPR-A and the 77-kDa NPR-C-like protein was not affected by water deprivation ( Fig. 4 ), that of the 67-kDa NPR-C-like protein was clearly increased by water deprivation ( Fig. 4 ), consistent with the results reported in the previous section, which suggest that water deprivation increases the B max of the 67-kDa NPR-C-like protein./ N, J! O- p# U# X1 R' C9 ]1 r5 H
9 d" B; W( ~$ b5 i4 S/ Z" D
Fig. 4. Autoradiographs of SDS-PAGE from glomeruli of control and water-deprived rats covalently cross-linked to 100 pM 125 I-ANP 1-28. Glomerular membranes were incubated with 125 I-ANP 1-28 in the presence or absence of the indicated unlabeled peptides at 4°C for 2 h. Affinity cross-linked proteins were dissolved in the presence of DTT and then separated by 10% SDS-PAGE. The autoradiographs were exposed for 31 days at -80°C. Each autoradiogram is representative of 3 separate determinations in different rats.
) O) ]" \2 K: R" J3 `
7 w- v$ J4 Z. W6 g4 H* nGlomerular cAMP production in control and water-deprived rats. The basal rate of glomerular cAMP accumulation in the presence of IBMX was 4.8 ± 0.3 and 4.7 ± 0.3 fmol/mg protein in control and water-deprived rats, respectively. Treatment of renal glomeruli with 10 µM forskolin significantly increased glomerular cAMP production in control and water-deprived rats to 14 ± 1.2 and 13.8 ± 0.9 fmol/mg protein, respectively ( P
2 g: Y7 Y* k6 G, |! S6 }% i4 m8 Z" x E9 D0 M1 }7 K8 b8 i' N
Fig. 5. Effect of natriuretic peptides on cAMP generation in glomeruli from control and water-deprived rats. Glomeruli from control and water-deprived rats were stimulated with 10 µM forskolin ( A ), 10 µM histamine ( B ), or 10 µM 5-hydroxytryptamine ( C ) in the absence and presence of ANP 1-28, CNP 1-22, or C-atrial natriuretic factor (C-ANF). cAMP synthesis was measured as described in MATERIALS AND METHODS. Values are means ± SE of 12 separate experiments expressed as percentage of control (agonist-treated glomeruli). * P 1 S% E! a+ Q! ?' C
8 d4 l, k- M. O" ~ q
Effects of HS-142 on ligand binding, internalization, and cyclic nucleotide production. HS-142 is a competitive antagonist of ANP 1-28 binding sites ( 25 ). Consistent with this, we have found that treatment with HS-142 abolishes ANP 1-28 -stimulated cGMP production ( Fig. 6 A ). Our results indicate that HS-142 inhibited the labeling of NPR-A and the 77-kDa NPR-C-like protein by 100 pM 125 I-ANP 1-28, reaching complete inhibition at 40 µg/ml ( Fig. 6 B ). In contrast, the affinity of the 67-kDa NPR-C-like protein for HS-142 was very low because no significant decrease in labeling of this protein was detected even at 100 µg/ml HS-142 ( Fig. 6 B ).
2 {" G' a9 ~- @$ e6 S$ Y" n; {" s9 Z2 m/ l$ h% Q' Q
Fig. 6. Effect of HS-142 on ANP 1-28 -induced cGMP production and specific 125 I-ANP 1-28 binding. A : renal glomeruli were incubated with 1 µM ANP 1-28 in the presence or absence of 40 µg/ml HS-142, and cGMP production was determined as described in MATERIALS AND METHODS. B : renal glomeruli were incubated with 100 pM 125 I-ANP 1-28 in the presence or absence of increasing concentrations of HS-142 as indicated. Affinity cross-linked proteins were dissolved in the presence of DTT and then separated by 10% SDS-PAGE. The autoradiographs were exposed for 31 days at -80°C. Each autoradiogram is representative of 6 separate determinations in different rats.! U1 ~: F9 l" p& t
% ]8 f% w. v5 j: [7 q
Specific binding of 125 I-ANP 1-28 to glomerular sites might be removed by acidic washing, and only the labeling of internalized peptides would remain. Our results indicate that most of the specific binding of 125 I-ANP 1-28 was removed by acidic washing, but a significant acid-resistant component, which could be abolished in the presence of 10 µM C-ANF, remained (not shown). The magnitude of this acid-resistant component was similar in glomeruli from control and water-deprived rats (not shown). Treatment of renal glomeruli with 30 µg/ml HS-142 significantly reduced the acid-resistant component ( Fig. 7 A ) without affecting the inhibitory effect of ANP 1-28 on the histamine-induced increase in cAMP production ( Fig. 7 B ).
6 T2 |0 X+ Q) Y" ^( ]! g T
2 [; a- h+ ^3 j7 ]7 `! U. TFig. 7. Effect of HS-142 on peptide internalization and cAMP production. A : specific binding of 200 µM 125 I-ANP 1-28 was analyzed in the absence or presence of 40 µg/ml HS-142. B : glomeruli from control rats were stimulated with 10 µM histamine in the absence and presence of ANP 1-28 and HS-142. cAMP synthesis was measured as described in MATERIALS AND METHODS. Values are expressed as percentage of control (agonist-treated glomeruli) and are presented as means ± SE of 6 separate experiments. * P 5 Z( q1 \) t& { t2 C
. k7 T% B! Z9 }4 B% H) nEffects of nucleotides on ligand binding. Quantitative in vitro autoradiography demonstrated that increasing concentrations of GTP S progressively inhibited the specific binding of 100 pM 125 I-CNP 1-22 to renal glomeruli ( Fig. 8 A ). As shown in Fig. 8 B, GTP S but not GDP reduced 125 I-ANP 1-28 binding to glomerular sites in a concentration-dependent manner. In addition, GTP S prevented labeling of the 67-kDa NPR-C-like protein by 125 I-ANP 1-28 without significantly affecting the radiolabeling of NPR-A or the 77-kDa protein ( Fig. 8 B ). ATP S had no such selective action. These results confirm that the specific binding site of subnanomolar concentrations of 125 I-CNP 1-22 in the rat kidney is the 67-kDa NPR-C-like protein, whose action is modulated by GTP-binding proteins.9 t+ ^7 l; _/ b `' D
% S) |! j1 R+ }/ E, w6 b) k7 f% |/ s$ M1 z' QFig. 8. Effect of GTP S on natriuretic peptide receptor-C (NPR-C)-like protein labeling by 125 I-ANP 1-28. A : autoradiographs of renal binding of 200 pM 125 I-CNP 1-22 in the rat kidney in the absence ( a ) and presence of 100 µM GTP S ( b ) or 1 µM unlabeled CNP 1-22 ( c ). B : binding of 125 I-ANP 1-28 to isolated glomeruli was investigated by incubation of renal glomeruli with 400 pM 125 I-ANP 1-28 in the presence of increasing concentrations of GTP S or GDP at 20°C for 10 min as described in MATERIALS AND METHODS. Nonspecific binding was obtained by incubation with 1 µM ANP 1-28. Values are means ± SE of 6 independent experiments. C : autoradiographs of SDS-PAGE from glomerular membranes covalently cross-linked to 100 pM 125 I-ANP 1-28 in the absence or presence of increasing concentrations of GTP S and ATP S. Affinity cross-linked proteins were dissolved in the presence of DTT and then separated by 10% SDS-PAGE. The autoradiographs were exposed for 31 days at -80°C. Each autoradiogram is representative of 3 separate determinations in different rats.7 V$ H* S: U$ c6 U" t& v- Y
' }+ B! d* c( E7 v+ U9 iDISCUSSION3 y) H% v7 X& h' O
8 x# Y: A( \9 ^) y6 r/ |% h
Our results indicate that water deprivation increases the expression of the 67-kDa NPR-C-like protein in renal glomeruli, where dense localization of NPR-C has been previously demonstrated by immunohistochemistry and in situ hybridization ( 26, 33 ). As a result, ANP 1-28, CNP 1-22, and C-ANF show a greater inhibitory effect on cAMP production stimulated by forskolin or the agonists histamine and 5-hydroxytryptamine in glomeruli from water-deprived rats compared with those from controls.
. l! F. Z/ f1 r1 n5 G9 j0 V, p( o6 k
The present study supports the idea that the 67-kDa NPR-C-like protein modulates glomerular cAMP synthesis. This hypothesis is based on our observation that subnanomolar CNP 1-22 concentrations inhibited forskolin-induced cAMP synthesis in rat glomeruli. Previous studies have demonstrated that the 67-kDa protein shows high affinity for CNP 1-22 in glomeruli from normal rats ( 11 ), and the present in vitro results were consistent with that conclusion in both control and water-deprived rats; therefore, this protein is the only one that can account for the effects of subnanomolar concentrations of CNP 1-22. cAMP levels were examined in the presence of IBMX, a phosphodiesterase inhibitor, which completely inhibits the cGMP-activated phosphodiesterase that may account for the effects of natriuretic peptides on cAMP levels ( 7 ). Therefore, our findings suggest that natriuretic peptides induce a greater reduction of cAMP levels in glomeruli from water-deprived rats by decreasing the rate of cAMP synthesis more effectively in these glomeruli. Our results indicate that natriuretic peptides induce a greater inhibition of cAMP production stimulated by physiological agonists, such as histamine or 5-hydroxytryptamine, which further suggests the physiological significance of this function.
: \, l7 }- h, b' L+ t
* A3 A6 U( _+ M: I( Y, mThe effects of GTP S further implicate the 67-kDa protein in the control of glomerular adenylate cyclase activity. Both ANP 1-28 and C-ANF inhibit adenylate cyclase activity in rat heart and kidney only if GTP is present ( 2, 5 ). This effect is inhibited by pertussis toxin in association with the ADP ribosylation of a 40-kDa protein, which is probably G i ( 3 ). In agreement with the fact that the ligand affinity of G protein-coupled receptors is reduced by guanine nucleotides ( 15 ), we have found that GTP S inhibited ligand binding to glomerular sites, specifically to the 67-kDa receptor, whereas GDP and ATP S had no effect, suggesting that a G protein may interact with the 67-kDa glomerular protein.
9 b$ V6 m0 q/ |& p4 D \% S
9 L8 J# K; o$ H2 d0 e' nWater deprivation approximately doubled the B max of ANP 1-28 and CNP 1-22 binding sites, corresponding to NPR-C in renal glomeruli, without affecting ligand affinity. Our results indicate that water deprivation did not significantly alter 125 I-ANP 1-28 internalization, which involves the 77-kDa protein because 125 I-CNP 1-22 at subnanomolar concentrations, which only label the 67-kDa receptor, are not internalized, and that low CNP 1-22 concentrations abolish 125 I-ANP 1-28 binding to the 67-kDa receptor without affecting its internalization. This evidence indicates that the 77-kDa NPR-C-like receptor is the only one involved in peptide internalization, and therefore the increased NPR-C B max induced by water deprivation might be explained by an increase in 67-kDa protein expression.
" d" }% [ Z3 ^0 e$ e1 ~6 h
8 h7 `0 S* q7 E- z h, yThe conclusion that the 77-kDa NPR-C-like receptor is the only component involved in ligand internalization is supported by the results obtained using HS-142, an ANP-receptor antagonist. We have found that HS-142 abolishes 125 I-ANP 1-28 binding to NPR-A and the 77-kDa NPR-C-like protein without altering binding to the 67-kDa protein. Consistent with this, HS-142 inhibits cGMP production by NPR-A and ligand internalization but did not modify the inhibitory effect of ANP 1-28 on cAMP production. Two receptors are therefore the candidates for peptide internalization, NPR-A and the 77-kDa protein. The use of C-ANF, which has no affinity for NPR-A, clarifies this issue, because it abolished ligand internalization and therefore presents the 77-kDa NPR-C-like protein as being responsible for peptide internalization in renal glomeruli. In addition, we have found that HS-142 abolished 125 I-ANP 1-28 binding to NPR-A and the 77-kDa NPR-C-like protein; however, HS-142 did not modify ANP 1-28 -induced inhibition of forskolin-dependent cAMP production. This observation confirms that the 77-kDa protein is not involved in regulating the synthesis of cAMP. It is likely that, as reported above, the 67-kDa protein is the only glomerular natriuretic peptide receptor to account for the effects of these peptides on cAMP production in the presence of HS-142.& _0 V( U! V+ Q. s# k$ ]! J! v
6 m* S- x# w& K0 `The increased expression of the 67-kDa NPR-C-like protein in water-deprived rat kidney suggests that this receptor might be involved in the regulation of body fluid. There is a body of evidence suggesting that natriuretic peptides are involved in the regulation of electrolyte balance, extracellular fluid volume, and blood pressure ( 22, 23, 27 ). Water deprivation has been shown to increase the density of glomerular NPRs in rats, which is accompanied by a decrease in plasma ANP ( 14, 17 ). The greater total receptor density observed in water-deprived rats was caused by a striking increase in NPR-C density, whereas NPR-A density was not affected ( 17 ). Consistent with this, our results show an increased expression of NPR-C in water-deprived rats, which is due to the enhanced expression of the 67-kDa NPR-C-like protein. The activation of this protein by natriuretic peptides induces inhibition of cAMP production. A number of studies have reported that vasoactive agents, such as histamine or prostaglandins, increase cAMP generation in messangial cells, which occupy a central position in the renal glomeruli, leading to cell relaxation and an increase in renal filtration and diuresis ( 30, 31 ). Therefore, the enhanced expression of the 67-kDa NPR-C-like protein induced by water deprivation might have a physiological role in the regulation of fluid homeostasis through the inhibition of cAMP synthesis and diuresis.. d o* a1 w3 _& m1 I
* f+ j4 }0 O# O# uIn summary, our observations demonstrate that water deprivation increases the expression of the 67-kDa NPR-C-like protein in rat glomeruli, without affecting the 77-kDa NPR-C-like protein receptor. Our findings support the hypothesis that the 77-kDa protein is involved in peptide internalization, whereas the 67-kDa NPR-C-like protein inhibits adenylate cyclase activity; therefore, water deprivation enhanced the inhibitory role of natriuretic peptides in cAMP synthesis, which is involved in the regulation of renal physiology ( 32, 37 ). These results suggest that the inhibition of adenylate cyclase by water deprivation may be one of the mechanisms through which natriuretic peptides regulate kidney functions. \6 n! ^% a4 `, [' v" G: n
! r) o A: |, J9 ?* B) KACKNOWLEDGMENTS
# H/ O6 V+ r8 m# j# b2 S5 J P4 |$ t0 D2 r
The British Heart Foundation supported this work.3 c$ k& ?% Z. C) S( k2 L- j
【参考文献】
/ i1 N4 N9 b' {$ E" e* \ Almeida FA, Suzuki M, Scarborough RM, Lewicki JA, and Maack T. Clearance function of type C receptors of atrial natriuretic factor in rats. Am J Physiol Regul Integr Comp Physiol 256: R469-R475, 1989.
( D# Z# k6 @, L+ r
+ [$ Z7 v8 o: g4 {& m9 ~4 i% ?3 b7 s6 d; |9 n1 x; h
' m& d6 y: Q; N% L P0 ?
Anand-Srivastava MB and Cantin M. Atrial natriuretic factor receptors are negatively coupled to adenylate cyclase in cultured atrial and ventricular cardiocytes. Biochem Biophys Res Commun 138: 427-436, 1986.# M; f6 ~, _! ?$ r1 u1 i4 d" X% U
8 {' E8 f( I: B% P0 ^1 x. [) r5 a1 e! D: h' a
5 h" o# P7 G9 wAnand-Srivastava MB, Srivastava AK, and Cantin M. Pertussis toxin attenuates atrial natriuretic factor-mediated inhibition of adenylate cyclase. Involvement of inhibitory guanine nucleotide regulatory protein. J Biol Chem 262: 4931-4934, 1987.. u/ N2 k/ k. L, g- J9 J. `
; L- F. ~% v+ w( R3 f% H( p6 Z& c2 Q) P# W
8 _* j* A' N6 x5 w! f
Anand-Srivastava MB and Trachte GJ. Atrial natriuretic factor receptors and signal transduction mechanisms. Pharmacol Rev 45: 455-497, 1993.. B& }9 A! p8 j; C# ]! {
, C9 C: h8 W" q0 s5 t5 s6 d
5 I& r Y( z3 V
! A! p$ d0 q! o- ^9 T: e$ eAnand-Srivastava MB, Vinay P, Genest J, and Cantin M. Effect of atrial natriuretic factor on adenylate cyclase in various nephron segments. Am J Physiol Renal Fluid Electrolyte Physiol 251: F417-F423, 1986.
) k, n7 V T1 t. F! @8 X1 e
+ {" D0 R0 q! x3 Q, i" a
* f+ f9 S' z) x7 C/ Q$ u! C( t' B- `. p
Ballermann BJ, Hoover RL, Karnowsky MJ, and Brenner BM. Physiologic regulation of atrial natriuretic peptide receptors in rat renal glomeruli. J Clin Invest 76: 2049-2056, 1985.
9 M/ A1 D% B- S9 V* b" } w) `$ X$ j# k+ U: g
' E& p4 S- \0 N- w+ v4 ~* ?* q. }# R+ t9 S. I7 \# m
Beavo JA and Reifsnyder DH. Primary sequence of cyclic nucleotide phosphodiesterase isozymes and the design of selective inhibitors. Trends Pharmacol Sci 11: 150-155, 1990.
" F6 p; `5 j+ A! H" C* K& i9 n( C' W, x5 ^
4 J9 a" O0 F# `3 _5 F* R& e( }# z ]: |' H7 C8 O/ v$ S+ z. t
Brown J and Chen Q. Regional expression of natriuretic peptide receptors during the formation of arterial neointima in the rabbit. Circ Res 77: 906-918, 1995.
) V# f) `* j7 m* c. }' D# s6 f( b. z: B% ]0 V( p
$ b, J3 K) J% J" [0 K; j( S8 ]
' n: i! A8 F4 S. t/ W
Brown J, Salas SP, Singleton A, Polak JM, and Dollery CT. Autoradiographic localization of atrial natriuretic peptide receptor subtypes in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 259: F26-F39, 1990. B9 `9 `+ @3 D' V2 }" S8 u
) X1 ~* j! W, z, T! [3 G, {0 M; u
( I: T/ l D7 h, n- c" P4 C
6 d' ^' y& {2 c: HBrown J and Zuo Z. Renal receptors for atrial and C-type natriuretic peptides in the rat. Am J Physiol Renal Fluid Electrolyte Physiol 263: F89-F96, 1992.
6 o+ ^, D9 q+ p: o) i
2 c0 p2 k. g2 l+ h$ z1 C7 N( Y2 `4 ]. h! w. {' Q
) r- X v7 |( Q$ T' L1 D
Brown J and Zuo Z. Receptor proteins and biological effects of C-type natriuretic peptides in the renal glomerulus of the rat. Am J Physiol Regul Integr Comp Physiol 266: R1383-R1394, 1994.
Z: C* S: b# J( d, Z0 r h# u& r; w8 I9 m+ @. V
* y Z/ C1 e! X) n4 L, K% n" Z4 ^1 m& w
De Bold AJ. Atrial natriuretic factor: a hormone produced by the heart. Science 230: 767-770, 1985.7 I( x* U- n5 q! f
5 s, ~2 x5 N8 B N& m
3 `3 e& d$ e+ x n; Y/ {9 v4 a
% B# S; Q( k* ^" F- [; r
Fuller F, Porter JG, Arfsten AE, Miller J, Schilling JW, Scarborough RM, Lewicki JA, and Schenk DB. Atrial natriuretic peptide clearance receptor. J Biol Chem 263: 9395-9401, 1988.6 E1 l4 a5 \* t4 _+ D
# {' s3 t0 @. x1 Z1 Y8 C) ~
6 C/ L- ^3 L- Q4 C
6 [1 X4 J3 I% b- x0 ^Gauquelin G, García R, Carrier F, Cantin M, Gutkowska J, Thibault G, and Schiffrin EL. Glomerular ANF receptor regulation during changes in sodium and water metabolism. Am J Physiol Renal Fluid Electrolyte Physiol 254: F51-F55, 1988.) @% L0 Q" e# L% i9 d- `8 r: S. s
; n8 k1 }8 s, c
4 A! G. n% z5 {+ q. f" }) a
/ P+ c7 @! H/ w9 k' n; NHouslay MD and Milligan G. G-Proteins as Mediators of Cellular Signaling Processes. Chichester, UK: Wiley, 1990. A$ s% q2 N0 Z7 X, I3 r) A! @9 L
5 d" l1 z5 j, J7 U" U5 C6 [- w( c% x, D* R# z) W
) P9 h5 v- z9 a) hKoch B and Lutz-Bucher B. Indirect relationship between vasopressin-induced secretion of ACTH and cyclic nucleotides in cultured anterior pituitary cells. Eur J Pharmacol 158: 53-60, 1989.8 S* F2 E6 n" q# t+ I5 V6 F8 G
; Q5 y/ c7 O2 p/ E
: S( V1 W% `9 X- Y4 s8 Y: H$ g
. F5 F0 L m; G7 g( CKollenda MC, Vollmar AM, McEnroe GA, and Gerbes AL. Dehydration increases the density of C receptors for ANF on rat glomerular membranes. Am J Physiol Regul Integr Comp Physiol 258: R1084-R1088, 1990.4 U, k$ \: O2 W$ J
5 g, u0 w1 a% F* K0 [
" t2 b" S9 ]: \% E& F+ s
' P4 F( D ]6 {- r, D* H# DKoller KJ, Lowe DG, Bennett GL, Minamino N, Kangawa K, Matsuo H, and Goeddel DV. Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science 252: 120-123, 1991." }8 V+ ~7 L; | N5 M7 m) S
- ~. G! ]1 F# x: S+ ^0 k3 x5 F: { ]4 l3 l7 ?8 z4 e9 W7 X9 f
& J' y$ ^6 s! CLaemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970.
3 S+ _$ i1 R& y3 F8 F6 v( P
' d; k" A7 L) G, u: M3 I8 w* a: k' k: I# Z
8 @: B/ m C0 p) v2 {/ t. jLewin ER. Natriuretic peptide C-receptor: more than a clearance receptor. Am J Physiol Endocrinol Metab 264: E483-E489, 1993.
~1 F8 _* Q! F$ {! m. v8 F
/ t6 v8 `! z1 i" _% @, L' L) c$ r- \' m5 a! H% G! X* v
1 z7 v' N4 t* l
Maack T. Receptors of atrial natriuretic factor. Annu Rev Physiol 54: 11-27, 1992.+ z( a4 R8 o: C1 U6 Z$ i4 m" b
/ u$ z! W4 T k5 F; _8 \
, N- c- s3 _" W) L. R# @5 H. a
" o& E+ H4 \% k& ^: RMatsuo H. Discovery of a natriuretic peptide family and their clinical application. Can J Physiol Pharmacol 79: 736-740, 2001.# J% c" K% n& B% _4 N( a
3 a+ f: ?; [3 [' s$ D0 ^
4 V Q1 l8 J) E+ h. n
7 z" V3 M% _/ X& }
Mattingly MT, Brandt RR, Heublein DM, Wei CM, Nir A, and Burnett JC. Presence of C-type natriuretic peptide in human kidney and urine. Kidney Int 46: 744-747, 1994.
* G* I# L& E9 P, x0 x M. c5 e s. y2 c% U+ [# p
% D+ h5 |: K; I c& H
" u Z+ J* ] o0 s' M9 V) T- R
Misra RO. Isolation of glomeruli from mammalian kidneys by graded sieving. Am J Clin Pathol 58: 135-139, 1972.( e6 V8 _# p$ H% D0 t P; J( `
% Y& |! X2 | ?& l) c/ U, g! r: F$ Y; p; `& [' T* ?
* r L& w) Y1 \7 f
Morishita Y, Sano T, Ando K, Saitoh Y, Kase H, Yamada K, and Matsuda Y. Microbial polysaccharide, HS-142-1, competitively and selectively inhibits ANP binding to its guanylyl cyclase-containing receptor. Biochem Biophys Res Commun 176: 949-957, 1991.1 ~8 D9 }& h6 o/ K+ \# s% m
" E# X# o( u) Q( s
8 q6 ^2 Y2 m" Y# a* a
0 B# e# m( i. e* {* |) L( O, yNagase M, Ando K, Katafuchi T, Kato A, Hirose S, and Fujita T. Role of natriuretic peptide receptor type C in Dahl salt-sensitive hypertensive rats. Hypertension 30: 177-183, 1997.
& K. k- m" l5 J. |& U
8 y8 e& a5 r) b& ^6 D. O. |5 _" c
' y6 ?: B9 a6 U* U* E
+ e2 E6 W: t0 W. DNakao K, Ogawa Y, Suga S, and Imura H. Molecular biology and biochemistry of the natriuretic peptide system. I. Natriuretic peptides. J Hypertens 10: 907-912, 1992.
) m; ~$ g% I! e) T! E, v2 i
% C. z4 d1 O! _5 l( [! }
) y. b( m8 T& f( q" D8 Y% p5 D) o. P7 P$ x( b% e. | w' }
Nakao K, Ogawa Y, Suga S, and Imura H. Molecular biology and biochemistry of the natriuretic peptide system. II. Natriuretic peptide receptors. J Hypertension 10: 1111-1115, 1992.
' ~2 c# X! o- v" E( s+ x, _5 L3 [' Y' ~0 i# `0 i! L
_2 W7 E/ E) J7 h" D5 p0 I
: O ~. {. C/ y# A0 J- I# g3 vPotter LR and Hunter T. Guanylyl cyclase-linked natriuretic peptide receptors: structure and regulation. J Biol Chem 276: 6057-6060, 2001.
$ h" S6 W( a8 A
! b, c0 G% O' O# F0 S3 E0 X9 [7 j `' P [+ P# g
8 o' e7 W" @! h9 ]9 H1 ySchlondorff D. The glomerular mesangial cell: an expanding role for a specialized pericyte. FASEB J 1: 272-281, 1987.
" p3 ~3 ^: p0 z) k% A
0 x; o: P0 x; i p- Q
1 a/ i, a, b. ?# a
/ i/ p2 c" D$ }& I: WSedor JR and Abboud HE. Histamine modulates contraction and cyclic nucleotides in cultured rat mesangial cells. Differential effects mediated by histamine H 1 and H 2 receptors. J Clin Invest 75: 1679-1689, 1985.1 l9 U4 a) S$ @% q3 l* X$ I$ l
. t9 {* S& d p x2 ^' N7 p1 n$ R
' G2 j! y5 p( R5 \ A" C: j
% N$ z7 j0 J6 C$ xShenolikar S and Weinman EJ. NHERF: targeting and trafficking membrane proteins. Am J Physiol Renal Physiol 280: F389-F395, 2001.: e* j! z9 z, C/ g
( @2 n$ G( n# q0 Z2 n
e7 q/ r; v* Z7 M' U0 M+ e, m
# R5 N0 b2 p, V) ~, b# X1 k
Shimonaka M, Saheki T, Hagiwara H, Hagiwara Y, Sono H, and Hirose S. Visualization of ANP receptor on glomeruli of bovine kidney by use of a specific antiserum. Am J Physiol Renal Fluid Electrolyte Physiol 253: F1058-F1062, 1987., E8 l) K* A, f) c7 M( w1 K6 y
, D, ^& Y8 e) O; A8 S
/ J3 x3 t1 {* H: Y) P7 E* m+ y9 z! O+ }9 J
Stingo AJ, Clavell AL, Heublein DM, Wei CM, Pittelkow MR, and Burnett JC. Presence of C-type natriuretic peptide in cultured human endothelial cells and plasma. Am J Physiol Heart Circ Physiol 263: H1318-H1321, 1992.
# n) V# _; }* R& D4 _
8 E G% Y6 @$ ~
$ M- S s: b$ [) C
/ s8 O; n3 h7 W/ @2 dSuga S, Nakao K, Itoh H, Komatsu Y, Ogawa Y, Hama N, and Imura H. Endothelial production of C-type natriuretic peptide and its marked augmentation by transforming growth factor-. Possible existence of "vascular natriuretic peptide system." J Clin Invest 90: 1145-1149, 1992.) Q/ w! B, E7 {- B- B
" a Y, u) ~0 W, L" }3 }' h2 Y7 D$ n) g0 O0 J0 }
W2 z% B# y5 y1 FWoodard GE, Zhao J, Rosado JA, and Brown J. A-type natriuretic peptide receptor in the spontaneously hypertensive rat kidney. Peptides 23: 1637-1647, 2002.
* W: ~, `: V. A7 ]# c* Q9 T2 E
- Y' T+ r4 } S& n: n: J* J1 m H& b9 S- R, {2 Z7 g: n0 B) {& b7 [0 A
3 g6 }! Q( u& T# i% X% M8 [ z4 zWright EM, Hirsch JR, Loo DD, and Zampighi GA. Regulation of Na /glucose cotransporters. J Exp Biol 200: 287-293, 1997. |
|
发表于 2009-4-22 08:11
