A-type natriuretic peptide receptor in the spontaneously hypertensive rat kidney

Peptides 23 (2002) 1637–1647

A-type natriuretic peptide receptor in the spontaneously hypertensive rat kidney Geoffrey E. Woodard∗ , Jing Zhao, Juan A. Rosado, John Brown Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK Received 11 January 2002; accepted 3 April 2002

Abstract Renal NPR-A binding characteristics was examined in SHR. Renal ANP binding sites of NPR-A showed a lower maximal binding capacity and higher affinity in SHR than in WKY at all intrarenal sites. Despite the lower Bmax in SHR, both ANP1–28 and ANP5–25 stimulate similar or greater cGMP production in isolated glomeruli. Studies on guanylate cyclase from glomerular and papillary membranes have reported an increased basal and stimulated guanylate cyclase activity in SHR. The present study provides further evidences for altered NPR-A receptors in SHR kidney, which might act as a negative feedback in response to hypertension. © 2002 Elsevier Science Inc. All rights reserved. Keywords: ANP; NPR-A; Hypertension; Kidney

1. Introduction Hypertension results from abnormalities of the control systems, including renal, vascular, cardiogenic, neurogenic, and endocrine mechanisms, involved in the regulation of blood pressure [11]. Kidney is extremely sensitive to changes in blood pressure and responds in several ways to maintain circulatory homeostasis [22]. There is considerable evidence for altered renal hemodynamic and renal sodium handling [23] during the development of genetic hypertension, although, the nature of the abnormalities remains unknown. Since the discovery of the atrial natriuretic peptide (ANP) much attention has been directed towards the involvement of ANP in the development of hypertension. ANP increases the renal glomerular filtration rate and filtration fraction [3]. Abbreviations: ANP, atrial natriuretic peptide; NPR, natriuretic peptide receptor; SHR, spontaneously hypertensive rats; WKY, Wistar–Kyoto rats; HBSS, Hanks’ Balanced Salt Solution; Bmax , maximum binding capacity; IBMX, isobutylmethylxanthine; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; BS3 , bis(sulfosuccinimidyl) suberate ∗ Corresponding author. Present address: National Institutes of Health, National Institute of Diabetes, Digestive and Kidney Disease, Building 10, Room 8C-208, 10 Center Drive, MSC 1754, Bethesda, MD 20824, USA. Tel.: +1-301-402-0391; fax: +1-301-402-0374. E-mail address: [email protected] (G.E. Woodard).

In addition, ANP inhibits sodium and water reabsorption in proximal tubule and collecting duct, suppresses renin secretion, and inhibits vasopressin-mediated water reabsorption [9,27]. Two different ANP receptor subtypes have been identified in kidney, NPR-A and NPR-C. NPR-A exhibits guanylate cyclase activity [4] and shows high affinity for ANP1–28 , less for truncated analogs such as ANP5–25 and no significant affinity for the internally deleted, truncated analogs, such as C-ANP [24]. NPR-C binds a wide range of structural analogs of ANP, including C-ANP and ANP5–25 [12]. NPR-C activates G proteins and also acts as a clearance receptor [17]. The diuretic and natriuretic properties of ANP suggest a key role for ANP in the renal control of blood pressure. Therefore, it is possible that ANP may affect kidney function differently in spontaneously hypertensive rats (SHR) and normotensive Wistar–Kyoto strains of rats (WKY). There are conflicting results about the level of plasma ANP in SHR and WKY rats [10,28] and the density of renal ANP binding sites in SHR is either normal [26] or reduced [1,28] compared to normotensive rats, although they showed a higher affinity for ANP1–28 [1]. This raised the possibility that NPR-A could be more effective in SHR than in normotensive rats. The present study, therefore, examines the binding capacity and guanylate cyclase activity of renal NPR-A in the presence of saturating concentrations of ANP1–28 or ANP5–25 .

0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 1 0 6 - 7

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2. Materials and methods

2.5. Binding assay of particulate ANP receptors in membranes

2.1. Materials SHR and WKY rats were obtained from a certified commercial supplier (Charles River, UK). 3-[125 I]Iodo-28-tyrosyl rat ANP1–28 ([125 I]ANP1–28 ), [125 I]standards, [125 I]labeled cGMP, and Hyperfilm 3 H were from Amersham, Bucks, UK. ANP1–28 , ANP5–25 , and C-ANP were from Peninsula Laboratories (Merseyside, UK). Millipore filter plates were from Millipore. Isobutylmethylxanthine (IBMX), sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), bromophenol blue, V8 protease, bis(sulfosuccinimidyl)suberate (BS3 ), and Tris were from Sigma (Poole, Dorset, UK). All other reagents were of analytical grade.

Freshly isolated glomeruli or renal papilla were homogenized and centrifuged at 1000 × 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 ␮g) of glomerular or papillary membrane preparation were incubated for 1 h or 40 min with 200 pM [125 I]ANP1–28 (200 or 25 pM, respectively) plus 10 ␮M C-ANP, in the absence or presence of increasing concentrations of unlabeled natriuretic peptides, in Tris buffer containing (in mM): 50 Tris, 4 MgCl2 , 0.4% (w/v) BSA,

2.2. Autoradiography Kidney slides from male SHR and WKY rats in PBS containing (in mM): 120 NaCl, 21.6 Na2 HPO4 , 8.4 NaH2 PO4 , pH 7.2, with 1 mM 1,10-phenanthroline were incubated with 100 pM [125 I]ANP1–28 (2000 Ci/mmol) with or without unlabeled peptides at 20 ◦ C for 15 min. After incubation, the sections were exposed to Hyperfilm 3 H for 7–21 days. 2.3. Competition experiments in isolated glomeruli Isolation of rat glomeruli was performed as described previously [16]. 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 Mg2 SO4 , 0.34 Na2 HPO4 , 1.26 CaCl2 , 4.17 Na2 HCO3 , 0.44 K2 HPO4 , 0.49 MgCl2 , 0.2% (w/v) BSA, and 5.56 glucose, pH 7.2. The cortices 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]ANP1–28 plus 10 ␮M C-ANP, to prevent labeling NPR-C [2], with increasing concentrations of unlabeled ANP1–28 or ANP5–25 at 20 ◦ C for 60 min. Preliminary experiments showed that specific binding of radioligand reached equilibrium at 60 min (not shown). Incubations were stopped by centrifugation and [125 I] labeling was determined using a Packard Gamma Counter. 2.4. Radioligand saturation experiments in isolated glomeruli Aliquots of glomeruli in HBSS were preincubated for 6 min at 4 ◦ C with 1 mM phenanthroline, and then incubated with increasing concentrations of [125 I]ANP1–28 plus 10 ␮M C-ANP in the absence or presence of 1 ␮M ANP1–28 at 20 ◦ C for 60 min to reach equilibrium. Incubations and [125 I] labeling was determined as reported earlier.

Fig. 1. Autoradiographs of renal binding of 100 pM [125 I]ANP1–28 in 12-week-old WKY and SHR breeds of rats. Sections of kidney from WKY (A–D) or SHR (E–H) breeds of rats were incubated with 100 pM [125 I]ANP1–28 (200 Ci/mmol) in the absence (A and E) and presence of 1 ␮M ANP1–28 , 1 ␮M ANP5–25 , or 10 ␮M C-ANP as indicated and autoradiography was performed as described under Section 2.

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Table 1 Binding constants for the specifically reversible binding of ANP1–28 to NPR-A in the kidney of 12-week-old WKY and SHR Arcuate arteries

Glomeruli

Outer medullary stripes

Inner medulla

WKY pKa Kd (nM) Bmax (fmol/mg protein)

8.65 ± 0.06 2.2 230 ± 3

8.70 ± 0.08 2.0 300 ± 4

8.34 ± 0.07 4.6 210 ± 4

8.41 ± 0.07 3.9 220 ± 3

SHR pKa Kd (nM) Bmax (fmol/mg protein)

8.77 ± 0.08 1.7 140 ± 10∗∗

8.95 ± 0.06∗ 1.1 160 ± 10∗∗

8.72 ± 0.08∗∗ 1.9 48 ± 7∗∗∗

8.76 ± 0.08∗∗ 1.7 52 ± 7∗∗∗

The maximum binding capacities (Bmax ), dissociation constant (Kd ), and the −log of the apparent association constant (pKa ) were assessed from the competitive inhibition of 100 pM [125 I]ANP1–28 binding by various concentrations of unlabeled ANP1–28 in the presence of 10 ␮M C-ANP. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 compared to age-matched WKY. Data are presented as mean ± S.E. of 10 independent experiments.

Fig. 2. Binding of [125 I]ANP1–28 to cortical and juxtamedullary glomeruli and medulla in 12-week-old WKY and SHR rats. Competitive inhibition of 100 pM [125 I]ANP1–28 binding was investigated after the addition of various concentrations of unlabeled ANP1–28 (䊊) or C-ANP (䊉) as described under Section 2. Data are presented as mean ± S.E. of six independent experiments.

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and 1 1,10-phenanthroline, pH 7,6. 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 ␮M ANP1–28 . 2.6. Determination of cGMP produced in isolated glomeruli and membranes Glomeruli suspensions were preincubated with 1 mM IBMX for 2 min and then incubated for 10 min at 20 ◦ C with the appropriate concentrations of natriuretic peptides or the vehicle as control. The incubation was stopped with trichloroacetic acid followed by centrifugation at 4000 × g for 10 min. The samples were resuspended with 50 mM acetate buffer containing 0.68% Na2 C2 H3 O3 , pH 5.8, and cGMP measured by radioimmunoassay. Guanylate cyclase activity in aliquots of glomerular and papillary membranes (3–5 ␮g of protein) was measured at 37 ◦ C in a reaction mixture containing (in mM): 50 Tris–HCl, 4 MgCl2 , 1 IBMX, 1 GTP–Mg2+ , 15 creatine phosphate, 1 ATP, and 20 U/ml creatine phosphokinase, pH

7.6. The reaction was started by addition of the membrane suspension and stopped 20 min later with 50 mM sodium acetate (pH 5.8) followed by boiling. The samples were centrifuged at 4000 × g for 10 min and cGMP was determined by radioimmunoassay. 2.7. Statistical analysis Data were analyzed using the LIGAND program. Analysis of statistical significance was performed using ANOVA or Student’s t-test. The significance level was P < 0.05.

3. Results 3.1. Competitive inhibition of [125 I]ANP1–28 binding in SHR and WKY Comparison of autoradiography with the corresponding stained tissue sections revealed a similar anatomical distribution of specifically reversible binding sites for ANP in SHR

Fig. 3. Competitive inhibition of [125 I]ANP1–28 binding to isolated glomeruli from WKY and SHR rats. Binding of [125 I]ANP1–28 to isolated glomeruli was investigated by incubation of renal glomeruli with 400 pM [125 I]ANP1–28 plus 10 ␮M C-ANP in the presence of increasing concentrations of unlabeled ANP1–28 or ANP5–25 at 20 ◦ C for 60 min as described under Section 2. Nonspecific binding was obtained by incubation with 1 ␮M ANP1–28 . Values are presented as mean ± S.E. from six independent experiments.

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and WKY. The results in 12-week-old animals were similar to previously reported studies [1]. [125 I]ANP1–28 bound mostly to glomeruli, renal arteries resolved as far distally as the arcuate arteries, stripes in the outer medullae, and the inner medullae. Radioligand binding to these structures was virtually abolished in the presence of 1 ␮M unlabeled ANP1–28 (Fig. 1; n = 6). We further examined the binding characteristics of NPR-A in WKY and SHR rats. As shown in Table 1, both, maximum binding capacity (Bmax ) and dissociation constant (Kd ), were significantly higher in WKY compared to SHR rats (n = 10; P < 0.05). Incubation with C-ANP reduced specific binding of [125 I]ANP1–28 to the glomeruli of SHR and WKY rats (Fig. 2; n = 6). We have found that inhibition of radioligand binding by C-ANP did not differ in SHR and WKY (not shown) as reported previously [1]. As shown in Fig. 2, incubation with 10 ␮M C-ANP inhibited 70–80% of the specific glomerular binding of [125 I]ANP1–28 but did not significantly displace the specific binding of ANP from the inner medullae of either strain (n = 6).

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Table 2 Binding constants for the specifically reversible binding of ANP1–28 to NPR-A in isolated glomeruli and glomerular and papillary membranes of 12-week-old WKY and SHR pKa Glomeruli WKY SHR

Kd (nM)

Bmax (fmol/mg protein)

7.78 ± 0.14 8.14 ± 1.00

16.6 7.24

790 ± 280 250 ± 100∗

Glomerular membrane WKY 9.30 ± 3.34 SHR 9.68 ± 1.45

0.50 0.21

1130 ± 320 216 ± 77∗∗

Papillary membrane WKY 10.01 ± 2.5 SHR 10.09 ± 1.01

0.097 0.081

708 ± 256 434 ± 169(∗ )

The Bmax , Kd , and the −log of the apparent association constant (pKa ) were assessed from the competitive inhibition of 400 pM (in isolated glomeruli) or 100 pM [125 I]ANP1–28 binding by various concentrations of unlabeled ANP1–28 in the presence of 10 ␮M C-ANP. ∗ P < 0.05 and ∗∗ P < 0.01 compared to age-matched WKY. Data are presented as mean ± S.E. of six independent experiments.

Fig. 4. Saturation curve of [125 I]ANP1–28 binding to isolated glomerular cells from WKY and SHR. Isolated glomeruli were incubated at 20 ◦ C for 60 min with increasing concentrations of [125 I]ANP1–28 in HBSS buffer as described under Section 2. Nonspecific binding was obtained by incubation with 1 ␮M ANP1–28 . Values are presented as mean ± S.E. from six independent experiments.

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3.2. Radioligand binding studies in isolated glomeruli Competitive inhibition of [125 I]ANP1–28 binding by ANP1–28 was studied in the presence of 10 ␮M C-ANP in isolated glomeruli from both stains as described under Section 2 (Fig. 3). ANP1–28 displaced [125 I]ANP1–28 in a dose-dependent manner, and only one class of high-affinity binding sites was indicated in both strains. The binding parameters obtained form competitive experiments are summarized in Table 2. We found that Bmax and Kd in SHR rats were lower than in WKY. To further investigate the changes in NPR-A in SHR, ANP5–25 was utilized as competitor of [125 I]ANP1–28 in the presence of 10 ␮M C-ANP. As shown Fig. 3, ANP5–25 displaced [125 I]ANP1–28 binding in a dose-dependent manner and inhibited the binding of [125 I]ANP1–28 in isolated glomeruli more effectively in SHR than in WKY (Fig. 3; n = 6; P < 0.01). Binding of [125 I]ANP1–28 to intact glomeruli membrane was found to be a saturable process in the presence of 10 ␮M C-ANP in both strains (Fig. 4; n = 6). Binding properties

in WKY and SHR were consistent with a single class of sites which binds [125 I]ANP1–28 with pKa = 8.2 ± 0.3 and 8.5 ± 0.4, respectively. 3.3. Radioligand binding in glomerular and papillary membranes Radioligand binding was measured as described under Section 2. As shown in Fig. 5, unlabeled ANP1–28 displaced [125 I]ANP1–28 in a dose-dependent manner in both strains. The Kd was similar in SHR and WKY, but the Bmax was significantly lower in SHR (Table 2; n = 6; P < 0.01). Similar results were obtained when the competition was performed using unlabeled ANP5–25 (Fig. 5; n = 6). ANP5–25 displaced [125 I]ANP1–28 more effectively in the glomerular membranes of SHR than in WKY (P < 0.05). As reported in glomerular membranes, incubation of papillary membranes with unlabeled ANP1–28 displaced [125 I]ANP1–28 in a dose-dependent manner in both strains (Fig. 6; n = 6). The Kd did not differ significantly, but Bmax

Fig. 5. Competitive inhibition of [125 I]ANP1–28 binding to glomerular membranes from WKY and SHR rats. Binding of [125 I]ANP1–28 to glomerular membranes was investigated by incubation with 100 pM [125 I]ANP1–28 plus 10 ␮M C-ANP in the presence of increasing concentrations of unlabeled ANP1–28 or ANP5–25 at 20 ◦ C for 60 min as described under Section 2. Nonspecific binding was obtained by incubation with 1 ␮M ANP1–28 . Values are presented as mean ± S.E. from six independent experiments.

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Fig. 6. Competitive inhibition of [125 I]ANP1–28 binding to papillary membranes from WKY and SHR rats. Binding of [125 I]ANP1–28 to papillary membranes was investigated by incubation with 100 pM [125 I]ANP1–28 plus 10 ␮M C-ANP in the presence of increasing concentrations of unlabeled ANP1–28 or ANP5–25 at 20 ◦ C for 60 min as described under Section 2. Nonspecific binding was obtained by incubation with 1 ␮M ANP1–28 . Values are presented as mean ± S.E. from six independent experiments.

was significantly lower in SHR than in WKY (Table 2; n = 6; P < 0.05). Consistent with the earlier discussion, similar results were obtained by incubation with ANP5–25 (Fig. 6; n = 6). 3.4. cGMP production in isolated glomeruli Although at earlier stages (3 weeks of age), we found that basal rate of glomerular cGMP production was higher in SHR (120 ± 8 pmol/mg protein/min) than in WKY (77 ± 7 pmol/mg protein/min) (P < 0.01), we did not notice any significant difference between the strains at later stages (128.2 ± 13.7 and 65.1 ± 115.9 pmol/mg protein/min for 12- and 18-week-old SHR versus 125.0 ± 12.2 and 62.5 ± 3.2 pmol/mg protein/min for 12- and 18-week-old WKY, respectively). As shown in Fig. 7, stimulation with ANP1–28 increased guanylate cyclase activity in a concentration-dependent

manner. Our results indicate that the maximum increment above basal rate of cGMP production stimulated by ANP1–28 was greater in glomeruli from 3-week-old SHR than WKY (Fig. 7; n = 9; P < 0.01). Both SHR and WKY showed substantial maturational increases in their response to ANP1–28 . As a result, cGMP production was similar at 12 weeks of age (Fig. 7), and at 18 weeks old, the rate of cGMP production stimulated by ANP1–28 was significantly higher in SHR (P < 0.05; not shown). We found that the maximum effect exerted by ANP5–25 was smaller than that observed by ANP1–28 in glomeruli from 3-week-old SHR (P < 0.05), however, it was similar in glomeruli from WKY (Fig. 7; n = 9). The maximum production of cGMP stimulated by ANP5–25 in 3-week-old SHR and WKY glomeruli was similar but became significantly greater in SHR at 12 weeks of age (Fig. 7; n = 6; P < 0.01).

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Fig. 7. ANP1–28 - and ANP5–25 -induced cGMP generation in isolated rat glomeruli from WKY and SHR. Isolated glomeruli from 3- or 12-week-old WKY (䊉) and SHR (䊊) rats were incubated for 10 min with different concentrations of ANP1–28 or ANP5–25 , and cGMP production was determined as described under Section 2. Values are presented as mean ± S.E. of nine separate experiments performed in triplicate.

3.5. cGMP production in membranes Guanylate cyclase activity was further assessed in glomerular and papillary membranes prepared from 12-week-old SHR and WKY as described under Section 2. In glomerular membranes basal guanylate cyclase activity was significantly higher in SHR than in WKY (3.4 ± 0.1 versus 2.0 ± 0.1 pmol/mg protein/min; P < 0.01), in agreement with others [26]. However, in papillary membranes guanylate cyclase activity was found to be similar in SHR and WKY (5.3 ± 0.5 versus 5.6 ± 0.9 pmol/mg protein/min). As shown in Fig. 8, when glomerular membrane suspensions were stimulated with ANP1–28 or ANP5–25 , the rate of accumulation of cGMP was significantly higher in SHR than in WKY (P < 0.01), which is consistent with the observations of others [26]. In our conditions, we have not found significant differences in the EC50 (for ANP1–28 - or ANP5–25 -stimulated guanylate cyclase activity) between the

strains; however, on the basis that ANP5–25 is a weaker agonist, we found that ANP5–25 was less potent than ANP1–28 in both strains (Fig. 8; n = 6). In contrast, in papillary membranes we have not found any differences between SHR and WKY either in the potency or the efficacy of the agonists, although, as reported earlier, ANP5–25 was found to be a weaker agonist than ANP1–28 in both strains (Fig. 9; n = 6).

4. Discussion In the present study, we have investigated NPR-A binding characteristics in SHR in isolated glomerular cells and glomerular and papillary membranes. In agreement with earlier studies by us [1], we have found a reduction in the number of ANP binding sites with higher affinity for ANP1–28 in the glomeruli of SHR compared to WKY rats.

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Fig. 8. ANP1–28 - and ANP5–25 -induced cGMP generation in glomerular membranes from WKY and SHR. Glomerular membranes from 12-week-old WKY () and SHR (䊊) rats were incubated for 10 min with different concentrations of ANP1–28 or ANP5–25 , and cGMP production was determined as described under Section 2. Values are presented as mean ± S.E. of six separate experiments.

Fig. 9. ANP1–28 - and ANP5–25 -induced cGMP generation in papillary membranes from WKY and SHR. Papillary membranes from 12-week-old WKY () and SHR (䊊) rats were incubated for 10 min with different concentrations of ANP1–28 or ANP5–25 , and cGMP production was determined as described under Section 2. Values are presented as mean ± S.E. of six separate experiments.

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ANP receptor subtypes were characterized in kidneys of 12-week-old SHR by autoradiography using C-ANP, which has been considered a relatively specific ligand of the NPR-C. C-ANP competed for about 70% of the ANP binding sites in glomeruli, suggesting heterogeneity of ANP receptors in this tissue. On the other hand, NPR-A was found to be the major receptor in medulla. This proportion did not differ significantly between any groups of rats; however, on the basis that in SHR the NPR-A showed lower Bmax for ANP, it seems that SHR express a lower density of NPR-A than WKY rats. NPR-A in SHR was further investigated by using ANP5–25 , a rather selective ligand of NPR-A [13]. Our data indicates that ANP5–25 binds to NPR-A and compete with [125 I]ANP1–28 more effectively in kidneys of SHR than in WKY. To further examine the binding properties of NPR-A in SHR and WKY, a number of experiments were performed in isolated glomeruli. Under these conditions, saturation and competitive binding experiments revealed a decreased density of ANP1–28 binding sites, and showed that NPR-A has a higher affinity for ANP1–28 and ANP5–25 in SHR. The use of glomerular and papillary membranes provides a different approach to investigate this issue since endogenous agonists and mediators can be eliminated and the synthesis of NPR-A can be prevented. Our results strengthen the hypothesis of a decreased number of NPR-A in SHR with higher binding affinity for ANP1–28 than in WKY. The binding kinetics of NPR-A reported in different cell types is very controversial. In rat oral mucosa, Kd and Bmax values of 3.34 ± 1.35 nM and 2.71 ± 2.21 fmol/mm2 on the tongue epithelium, and 4.09 ± 1.52 nM and 3.45 ± 3.01 fmol/mm2 on the epithelium of the hard palate, respectively, have been reported [19]. In adrenal zona glomerulosa membrane, the Kd and Bmax found were 323 ± 60 pM and 134 fmol/mg proteins, respectively [5]. Finally, NPR-A found in gill of the Atlantic hagfish Myxine glutinosa showed Kd = 15.4 ± 1.6 pM and Bmax = 45.9 ± 3.0 fmol/mg protein [25]. In kidney, a Kd = 421 ± 55 pM and Bmax = 49.2 ± 8.8 fmol/mg protein have been reported in cortical collecting duct cells [15], while we found different Bmax and Kd values for NPR-A in glomerular and papillary membranes from both WKY and SHR rats and also between both strains. Although we have not investigated the nature of the different binding kinetics in different cell types, they might be a result of allosteric regulation by different factors or even the influence of several agents present in the environment of the cells. Guillaume et al., [8] reported that ethanol consumption reduced the Kd and elevated Bmax of renal NPR-A, just opposite to the binding kinetics reported in SHR kidney, showing the protective effect of moderate ethanol consumption on increases of blood pressure. In order to assess the physiological relevance of alterations in NPR-A binding properties, cGMP production in the intact glomeruli from both strains was examined. Our results indicate that, although Bmax of NPR-A has been shown to

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be lower in SHR, production of cGMP was more efficient. This effect was not due to the higher affinity of NPR-A in SHR for ANP1–28 , since we used saturating agonist concentrations. In addition, the production rate of cGMP observed in isolated glomeruli from SHR was not due to a difference in cGMP metabolism by phosphodiesterase since IBMX was present in the medium. However, since the difference in cGMP production between the strains could be due to the difference in synthesized enzyme, or cofactors, whose levels are uncontrolled in whole glomerular cells, we assessed the guanylate cyclase activity in glomerular membranes. Consistent with the earlier discussion, a higher activity of particulate guanylate cyclase was found in membranes from SHR compared to WKY, suggesting that NPR-A function is altered in SHR. Increased cGMP production in SHR has been previously reported [26], but, to our knowledge, this is the first time that a higher rate of cGMP production per receptor is reported. Although speculative, the high rate of cGMP production by NPR-A might be the explanation for the reported prolonged effect of ANP infusions on diuresis, natriuresis, and blood pressure in hypertensive rats [21]. Under our conditions, the potency of the agonists to activate NPR-A guanylate cyclase activity in WKY and SHR is lower than reported by other kinetic studies (EC50 = 5 nM; [6]) or 1 nM described in ventricular myocytes [14]; however, similar results have been reported in different cell types, such as ventricular fibroblasts [14] and granulosa and thecal interstitial cells [18]. The nature of the modification that leads to an exaggerated response to agonist is unknown. Structural divergence between the NPR-A of SHR and WKY may contribute to the more effective reaction of NPR-A to agonist in SHR. Indeed, the characteristic ligand affinities of NPR-A in SHR and WKY support this possibility. The renal excretion of sodium is regulated by redundant control systems [7]. These factors include the renin–angiotensin–aldosterone system, renal nerve activity, plasma and interstitial oncotic pressures, renal perfusion pressure, one or more natriuretic hormones, and the renal prostaglandins [7]. It is known that autonomic nervous system and certain neurohumoral pathways including the hypothalamus–pituitary axis are involved in the regulation of blood pressure. Peng et al. [20] reported that increased levels of ANP in the anterior hypothalamus of SHR may produce local tonic inhibition of noradrenaline release, therefore, reducing excitation of depressor neurons and elevating blood pressure. Consequently, an altered natriuretic peptide system, including the different characteristics of NPR-A, may influence body fluids and blood pressure by a variety of extrarenal as well as renal mechanisms in SHR.

Acknowledgments This work was supported by The British Heart Foundation.

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