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Cardiovascular Research 1997 36(3):363-371; doi:10.1016/S0008-6363(97)00192-2
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Copyright © 1997, European Society of Cardiology

Downregulation of natriuretic peptide C-receptor protein in the hypertrophied ventricle of the aortovenocaval fistula rat

Lesley A Browna, Richard A.D Rutherfordb, Derek J.R Nuneza, John Whartonb, David G Lowec and Martin R Wilkinsa,*

aDepartment of Clinical Pharmacology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Rd, London W12 0NN, UK
bDepartment of Histochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Rd, London W12 0NN, UK
cGenentech Inc., South San Francisco, CA, USA

* Corresponding author: Tel.: +44 181 7403219; Fax: +44 181 7493439; E-mail: mwilkins@rpms.ac.uk

Received 5 March 1997; accepted 12 June 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: This study examined the expression of the C-type receptor for the natriuretic peptide family (NPR-C) in the ventricles of normal and aortovenocaval (AV)-fistula rats, the latter a model of cardiac volume overload producing hypertrophy of both ventricles. Methods: Western blotting with a rabbit anti-NPR-C antibody was used to quantify NPR-C levels in ventricular membranes. NPR-C expression was localised anatomically and measured in frozen sections of cardiac tissue by histochemistry and in vitro autoradiography. Results: Western blot analysis revealed a single band (~120 kDa) in ventricular membranes which was reduced to ~60 kDa after treatment with β-mercaptoethanol. NPR-C immunoreactivity and [125I]rat ANP1–28 binding (displaceable by the NPR-C-specific ligand C-ANP 4–23) were localised to the endocardium. NPR-C protein levels, as measured by all three techniques, were reduced significantly in the hypertrophied ventricles of AV-fistula rats compared to sham-operated animals. Conclusions: Volume-induced cardiac hypertrophy in the AV-fistula rat is associated with downregulation of endocardial NPR-C. This may be one mechanism by which the endocardium regulates the myocardial response to changes in haemodynamic load.

KEYWORDS Rat; Volume overload; Cardiac hypertrophy; Autoradiography; Immunocytochemistry


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Three types of receptor for the natriuretic peptides have been described, NPR-A, NPR-B and NPR-C [1]. NPR-A and NPR-B have a molecular mass of 120–140 kDa and possess integral intracellular guanylyl cyclase activity. These receptors appear to mediate the majority of the biological activities of the natriuretic peptides via guanosine 3',5'-cyclic monophosphate (cyclic GMP) [2]. The third receptor, NPR-C, appears to exist as a homodimer with a molecular mass of 120 kDa on SDS–PAGE which can be reduced to 60 kDa by treatment with β-mercaptoethanol [3]. It has a short intracellular tail and no ability to generate cyclic GMP [3], although in some studies it has been linked to inhibition of adenylyl cyclase activity [4]. NPR-C has less stringent ligand binding requirements than either NPR-A or NPR-B, binding all three natriuretic peptides with similar affinity [5]. This, together with the lack of an obvious second messenger, has led some investigators to propose that NPR-C acts mainly as a ‘clearance’ receptor for the natriuretic peptide family [2].

Recent studies have shown that NPRs are present in the heart [6–10], the major site of ANP and BNP synthesis and secretion into the circulation. Messenger RNAs for NPRs have been demonstrated in rodent and primate hearts using in situ hybridisation [7]and reverse transcription–polymerase chain reaction (RT–PCR) [6, 9, 10]. Furthermore, ANP binding sites have been reported on myocyte membranes [11]and there is evidence that the natriuretic peptides may have direct effects on the heart [12–14]. ANP has been shown to alter intracellular calcium in isolated cardiac myocytes [14]and induce early relaxation of isolated mammalian papillary muscle [13]and, more recently, to influence myocyte volume [12].

Data on the regulation of cardiac NPRs are limited. This is of considerable interest as changes in the level of expression of NPRs may regulate the local myocardial response to natriuretic peptides while allowing secretion from the heart to meet the systemic requirements for these peptides. In a previous study, we examined the changes in the cardiac expression of the messenger RNAs (mRNA) encoding NPRs in the aortovenocaval (AV)-fistula rat [10], a model of 4-chamber cardiac hypertrophy and elevated plasma ANP levels due to chronic volume overload of the heart [15]. In this model, increased cardiac ANP expression was accompanied by a striking reduction of NPR-C mRNA levels (which were below the level of detection of our assay 35 days post-surgery) while the prevalence of transcripts for both NPR-A and NPR-B increased [10]. Because there are examples of a significant dissociation of mRNA abundance and protein expression [16], the current study was undertaken: (i) to establish that cardiac hypertrophy is accompanied by downregulation of NPR-C protein; and (ii) to determine precisely the sites within the heart where these changes take place. Accordingly, we have employed Western blotting, in vitro autoradiography and immunostaining, to measure NPR-C protein levels in hearts from AV-fistula rats.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Animal models
AV-fistula surgery was performed on male Wistar rats (250–270 g; A. Tuck, Battlebridge, Essex, UK) as described previously [10]. Briefly, a 1–1.5-mm fistula was made between the aorta and the inferior vena cava ~10 mm distal to the renal arteries. In sham operations, the abdomen was opened and the aorta and vena cava clamped for 5 min but there was no cutting or suturing of vessels. AV-fistula and sham-operated rats were studied 35 days after surgery (35d), at a time when NPR-C mRNA was below the threshold of detection in our previous study [10]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.2 Tissue preparation
At the experimental time points, the AV-fistula and sham-operated animals were anaesthetised with Hypnorm (fentanyl citrate 0.315 mg/ml and fluanisolone 10 mg/ml; 1 ml/kg administered intraperitoneally; Janssen Pharmaceuticals, Oxford, UK). The patency of the fistula was confirmed by inspection for venous mixing of blood. The hearts were removed, weighed and placed on ice. Both atria were carefully dissected off and the right ventricle (RV) was cut flush from the septum and the left ventricle (LV). The RV and LV+septum were weighed and frozen in liquid nitrogen and maintained at –80°C until used for membrane preparation. For in vitro autoradiography, hearts were mounted on cork mats, surrounded in mounting medium (Tissue-Tek, Miles, Elkhart, IN, USA) and frozen in melting dichlorodifluoromethane (Arcton, 12, ICI, Runcorn, Cheshire, UK) suspended in liquid nitrogen.

2.3 Western blotting
Cardiac membrane samples were prepared from individual ventricles as described previously [17]. The membrane pellets were resuspended in ice-cold Tris–HCl buffer, pH 7.4 (at a protein concentration of approximately 3–6 µg/µl) and stored in aliquots at –80°C. Protein concentration was determined using the BCA method as recommended by the manufacturer (Pierce, IL, USA).

Ventricular membrane suspensions containing 10–150 µg of protein were separated by 8.5% SDS–PAGE using prestained molecular weight markers (high molecular weight range, 36–205 kDa; Sigma, Poole, UK). Proteins were electroblotted at 30 V overnight onto nitrocellulose membranes (ECL-Hyperbond, Amersham International, Amersham, UK) using a Transphor electroblotter (Hoefer Scientific Instruments, San Francisco, CA, USA).

The Western blot protocol employed was that described by Hirokawa et al. [18]using 5% low fat milk and 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) to block the membranes and a rabbit anti-NPR-C antibody raised against a 37-amino-acid sequence from the cytoplasmic domain of bovine NPR-C (peptide R37A) [19]. This region of the sequence is highly conserved between rat, human and bovine NPR-C and bears no resemblance to the other NPR sequences [5]. The specificity of the antibody was tested using recombinant 293 cell lines expressing human NPR-A [20], NPR-B [21]and NPR-C [22]. Briefly, non-ionic detergent lysates were prepared from each cell line and fractionated by SDS–PAGE under reducing conditions and probed with the anti-R37A antibody. As a test of the specificity of the NPR-C immunocomplex in the Western blotting experiments with ventricular membranes, the R37A synthetic peptide antigen (final concentration of 100 ng/ml) was incubated with aliquots of ventricular membrane from a sham-operated animal for 1 h prior to SDS–PAGE and subsequent blotting with the anti-NPR-C antibody. The immunocomplexes were visualised with a goat anti-rabbit IgG conjugated to horseradish peroxidase (ICN Flow, Oxford, UK). Autoradiography was performed after incubation with luminol chemiluminescence reagents as recommended by the supplier (ECL Western blotting detection reagents, Amersham, UK).

The autoradiographs were quantified by optical densitometry using a LKB Ultroscan XL enhanced laser densitometer (LKB, Bromma, Sweden) and the results expressed in arbitrary absorbance units. RV or LV membranes from sham-operated and AV-fistula hearts were analysed in duplicate on the same SDS–PAGE membrane to ensure that experimental and sham-operated samples were handled identically. Upon completion of the Western blotting and ECL detection, the nitrocellulose blots were stained for protein with a 0.1% amido black solution to check for uniform electroblotting.

2.4 Binding studies
Cryostat sections (10 µm) were thaw-mounted onto chrom-alum-gelatin-coated glass slides and stored at –20°C. Sections were pre-incubated at 20°C for 15 min in 30 mmol/l phosphate buffer (comprising 20 mmol/l Na2HPO4·H2O, 10 mmol/l NaH2PO4·2H2O, 123 mmol/l NaCl, pH 7.4) and then incubated at room temperature for 30 min in fresh buffer containing 0.5% BSA, 0.18 mmol/l bacitracin and 250 pmol/l 3-[125I]iodo-tyrosyl rat ANP1–28 ([125I]rANP1–28, 2000 Ci/mmol; Amersham), as previously described [8]. Following incubation the slides were washed twice in buffer at 4°C for 5 min, rinsed in distilled water at 4°C, and dried under a stream of cold air. The effect of prior receptor occupancy was investigated by washing sections in acidified 300 mmol/l NaCl (pH 5.0) for 30 min at 20°C prior to incubation with radioligand in order to remove endogenously bound peptide. Autoradiographic images were produced by co-exposing dry sections and 125I-labelled standards to Hyperfilm-3H (Amersham) for 36 h at 4°C. The autoradiographic film was then developed in Kodak D-19 developer for 4–5 min, fixed in Amfix (Amersham) and rinsed in distilled water.

After film-based autoradiography had defined the general pattern of binding, microautoradiography was performed to obtain greater anatomical resolution of the binding sites [8]. Sections were fixed in Bouin's solution for 1 h at 20°C, washed twice in phosphate buffer (2–3 min) at 4°C, rinsed in cold distilled water, dried under a stream of cold air and dipped in liquid emulsion (LM-1, Amersham) at 43°C. The dried slides were then stored in the dark at 4°C for up to 6 days, developed in Kodak D-19 for 2.5 min at 20°C, fixed and stained with haematoxylin and eosin.

2.5 Quantification of binding studies
A Seescan Symphony Image Analysis System (Seescan, Cambridge, UK) was used to quantify ligand binding to the endocardial endothelium in autoradiographic film images [23]. Standard curves relating optical density values to log concentration of [125I]rANP1–28 bound (amol/mm2) were obtained for each film. Endocardial binding was traced throughout both ventricles wherever it was demonstrated autoradiographically and the average binding density measured by computer-assisted densitometry. Specific binding was determined by subtracting the non-specific binding from the total binding obtained by incubating adjacent sections with 250 pmol/l [125I]rANP1–28 either alone (total) or in the presence of 1 µM unlabelled rat ANP1–28 (non-specific). Specific binding to the NPR-C receptor subpopulation was demonstrated by co-incubation of 250 pmol/l [125I]rANP1–28 with an excess of either the NPR-C selective ligand C-ANP4-23 (1 µmol/l) [2]or the NPR-A/NPR-B antagonist HS-142-1 (500 µg/ml) [23].

2.6 Immunohistochemistry
The anti-R37A (anti-NPR-C) antibody was employed for immunohistochemistry. The avidin–biotin complex (ABC) immunoperoxidase method [24]was used to demonstrate immunoreactivity for the anti-NPR-C antibody (5 µg/ml) and the endothelial marker, von Willebrand factor (vWF, diluted 1:800; Dako code A082) in serial sections of sham-operated and AV-fistula rat hearts. Peroxidase activity was revealed using the diamine–benzidine–glucose oxidase method with nickel enhancement [25]. For the NPR-C sections, controls included the omission of primary antiserum and application of diluted antiserum pre-absorbed with the synthetic peptide antigen, R37A (1 nmol/l–10 µmol/l).

2.7 Statistical analysis
The statistical significance of the changes in NPR-C levels in the Western blots and in vitro autoradiography was determined by the non-parametric Mann–Whitney test using the statistical program Statgrafics (Statistical Graphics Corp, Rockville, MD, USA). The significance level ({alpha}) was set at 0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Development of cardiac hypertrophy in AV-fistula rats
Consistent with previous data [10], chronic volume overload led to marked hypertrophy of both ventricles in the 35d AV-fistula rats. Mean (±s.e.m.) RV/body weight ratios (mg/g) were 0.64 (±0.05) and 1.13 (±0.06) for sham-operated (n = 4) and AV-fistula (n = 7) rats, respectively (P<0.01); mean (±s.e.m.) LV/body weight ratios (mg/g) were 1.98 (±0.06) and 3.1 (±0.08) for sham-operated and AV-fistula rats, respectively (P<0.001).

3.2 Measurement of NPR-C protein levels by Western blotting
Immunoblotting of ventricular membranes from sham-operated rats with the anti-R37A antibody revealed a single band (~120 kDa) which was reduced to ~60 kDa after treatment with β-mercaptoethanol (Fig. 1a, lanes 1–4). To test the specificity of the antibody, membrane protein extracts from transfected 293 cells expressing nprs were separated under reducing conditions before incubation with anti-R37A antibody. Immunoreactive protein ~60 kDa was detected by anti-r37a antibody only in the cell line expressing NPR-C (Fig. 1a, lane 7) and not from cells expressing NPR-A or NPR-B (Fig. 1a, lanes 5 and 6). The intensity of the NPR-C signal increased in a log-linear manner with increasing ventricular membrane protein concentration over a range of 25–100 µg (Fig. 1b). Furthermore, the signal was blocked by co-incubation with an excess of the synthetic peptide antigen R37A (Fig. 1c, lane 3).


Figure 1
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  		Fig. 1 Western blot analysis of NPR-C using anti-R37A antibody. (a) A single band ~120 kDa was detected in ventricular membranes from sham-operated animals (lanes 1 and 2) which was reduced to ~60 kDa after treatment with 5% β-mercaptoethanol (lanes 3 and 4). Blots of lysates from recombinant 293 cells lines expressing NPR-A (lane 5), NPR-B (lane 6) or NPR-C (lane 7) separated under reducing conditions produced an immunocomplex only with those cells expressing NPR-C (lane 7). (b) Protein-dependent increase in NPR-C immunoreactivity in ventricular membranes from sham-operated animals. The signal intensity increased from 25–100 µg of membrane protein (lane 1, 25 µg; lane 2, 50 µg; lane 3, 100 µg; lane 4, 150 µg). (c) A collage of NPR-C immunoblots from representative left ventricles from sham-operated and AV-fistula rats; lanes 1 and 2, LV from two different sham-operated animals; lane 3, sham-operated LV plus the synthetic peptide antigen R37A; lanes 4 to 6, LV from three different AV-fistula rats.

 
Fig. 1c shows typical immunocomplex products obtained with the anti-R37A antibody following SDS–PAGE separation and Western blotting of 50 µg of ventricular membranes from sham-operated (lanes 1 and 2) and hypertrophied AV fistula rat hearts (lanes 4 to 6). Quantitative analysis confirmed that the density of the NPR-C band was significantly decreased in both the RV and LV of 35d AV-fistula rat hearts compared to those from sham-operated animals (Fig. 2).


Figure 2
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  		Fig. 2 NPR-C protein levels, as assessed by Western blot analysis, in the (a) RV and (b) LV of sham-operated and 35d AV-fistula rats. RV and LV membrane protein were separated in duplicate by SDS–PAGE, transferred to nitrocellulose and blotted with anti-NPR-C antibody. Following ECL detection, autoradiographs containing either RV or LV protein from sham-operated or AV-fistula hearts were quantified by densitometry. NPR-C levels were measured in arbitrary absorbance units obtained from the densitometer. Values are mean±s.e.m. (n = 7 rats in each group). *P<0.05 or less compared to values from sham-operated animals.

 
3.3 Measurement of NPR-C protein levels by in vitro autoradiography
Autoradiography demonstrated that the endocardium expressed the highest density of [125I]rANP1–28 binding sites (Fig. 3). An increase in [125I]rANP1–28 binding after preincubation of the sections in acidified NaCl (pH 5.0) was interpreted as demonstrating a significant degree of prior natriuretic peptide receptor occupancy by endogenous ligand. All further incubations were therefore carried out with acid-washed sections. Co-incubation of [125I]rANP1–28 with an excess (1 µM) of rat ANP1–28 or the NPR-C selective ligand, C-ANP4-23, completely inhibited endocardial binding whereas no inhibition was observed in the presence of the non-peptide NPR-A/NPR-B antagonist HS-142-1 (Fig. 3). Endocardial [125I]rANP 1–28 binding was reduced in the hypertrophied ventricles of AV-fistula rats compared to sham-operated rat hearts (Fig. 4). Moreover, there were areas where radioligand binding was not detectable, even though endothelium remained intact throughout the ventricle, as demonstrated by immunostaining for von Willebrand factor (Fig. 5). For each animal, endocardial radioligand binding density was measured at all sites where it was detectable on the film autoradiographs. The density of endocardial binding sites was reduced significantly (P<0.05) in both ventricles of AV-fistula rats compared to sham-operated controls (controls vs. AV-fistula: RV 149.1±5.8 and 118.1±10.8 amol/mm2; LV 148.9±6.1 and 89.1±7.2 amol/mm2). Given that there were regions of intact endothelium in hypertrophied ventricles in which binding sites were not detected, these data represent an underestimation of the extent of the reduction in ligand binding sites in hypertrophied ventricles.


Figure 3
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  		Fig. 3 Autoradiographic localisation and characterisation of [125I]rANP1–28 binding sites in the normal rat heart. Serial cryostat sections of a normal rat heart incubated with 250 pM [125I]rANP1–28 in the absence (a) or presence of 1 µM rat ANP1–28 (b), C-ANP4–23 (c) or 500 µg/ml HS-142-1 (d). NPR-C binding sites are localised to the ventricular endocardium in the RV and LV. Bar=2 mm.

 

Figure 4
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  		Fig. 4 Autoradiographic localisation of [125I]rANP1–28 binding sites in transverse sections of a sham-operated (a) and an enlarged AV-fistula rat heart (b). Compared to the sham-operated ventricular endocardium, binding is markedly reduced in both ventricles of the AV-fistula heart. Bar=2 mm. Arrows show binding in LV.

 

Figure 5
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  		Fig. 5 Microscopic localisation of [125I]rANP1–28 binding sites to the endocardial endothelium in the LV of a sham-operated (a) and AV-fistula rat heart (d). Consecutive sections of ventricle show the distribution of NPR-C immunoreactivity (b and e) and the presence of an intact endocardial endothelium as indicated by immunostaining for von Willebrand factor (c and f). [125I]rANP1–28 binding and NPR-C immunoreactivity is reduced in the endocardium of the AV-fistula heart (d and e) compared to that observed in sham-operated ventricular endocardium (a and b).

 
3.4 Immunostaining
NPR-C immunoreactivity displayed a very similar distribution to that seen with [125I]rANP1–28, being localised to the ventricular endocardial endothelium (Fig. 5). The immunostaining was abolished by pre-adsorption of the antibody with 0.1 µM of the synthetic antigen, R37A. A reduction in NPR-C immunostaining was observed in the ventricles of the AV-fistula heart, consistent with the radioligand binding (Fig. 5). The reduction in radioligand binding and immunostaining did not reflect endothelial damage as an intact endocardial endothelium was demonstrated in the ventricles of AV-fistula rats as well as in the hearts of sham-operated animals by staining for von Willebrand factor (Fig. 5).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This study provides clear evidence for reduced cardiac expression of NPR-C protein in the hypertrophied AV-fistula rat heart and supports and extends our previous observation that myocardial hypertrophy in the AV-fistula rat is associated with a decrease in steady-state NPR-C mRNA levels [10]. Taken together, the data suggest that NPR-C levels are reduced, at least in part, due to reduced de novo synthesis of the protein rather than simply because of internalisation or compartmentalisation of the receptor.

Radioligand binding and immunocytochemistry localised NPR-C to the ventricular endocardium. The binding studies suggest that NPR-C is the predominant receptor subtype in the normal rat heart. This is consistent with the higher prevalence of NPR-C mRNA compared to NPR-A or NPR-B transcripts found in rat cardiac tissue [10]. Indeed, NPR-C is the principal NPR subtype in many tissues [2, 4, 26]. We have not detected significant [125I]rANP binding to myocardium, even after acid-washing to remove bound endogenous ligand. While high-affinity ANP binding sites have been located on myocyte membranes [11]and cardiac fibroblasts [6], others have not observed radiolabelled ANP binding to myocardial tissue by autoradiography [8], suggesting that the level of expression of NPRs in the myocardium is below the level of detection of the autoradiographic technique.

One explanation for the reduction in NPR-C protein in Western blots of tissue from hypertrophied ventricles is that the endocardial NPR-C signal may have been diluted by a disproportionate increase in myocardium in aliquots of tissue from these chambers. However, the consistent reduction in radioligand binding and NPR-C immunoreactivity in hypertrophied ventricles does not support this interpretation.

It is unlikely that the reduction in NPR-C in the endocardium is part of a generalised reduction in expression of this receptor in our animals. NPR-C is expressed most abundantly in the lung and kidney. In previous studies we found no change in NPR-C mRNA in pulmonary and renal tissue from AV-fistula rats (unpublished observations). Although some authors report a reduction in NPRs in vascular membranes from this animal model [27], NPR-C expression in the renal glomerulus is not altered [27, 28]. Although a reduction in pulmonary NPR-C expression has been inferred from clearance studies in dogs with experimental cardiac failure [29]and radioligand binding studies suggest a reduction in NPR-C in platelets from patients with cardiac failure [30], it should be stressed that the animals in our study were overtly healthy and none exhibited cardiac failure. It is our experience and that of others [31]that AV-fistula rats that decompensate do so (and die) in the first week after surgery.

The most striking abnormality in the AV-fistula animals described here is the increase in cardiac mass and we propose that the change in expression of endocardial NPR-C is related to the cardiac hypertrophy. Furthermore, the autoradiographical and immunohistochemical analyses suggest some regional variation in the degree of endocardial NPR-C downregulation in hypertrophied ventricles. Studies of left ventricular wall dynamics have demonstrated significant regional differences in patterns of ventricular wall motion and intra-cavity pressures during contraction–relaxation cycles [32]. These heterogenous myocardial stresses are likely to be amplified by volume overload. An interesting possibility is that the pattern of endothelial NPR-C loss may be a response to these local differences in ventricular wall stress.

Little is known about the biochemical processes regulating NPR-C expression in cardiac myocytes but some insights come from studies of vascular smooth muscle and endothelial cells in culture. Several authors report that NPR-C can be downregulated by increasing intracellular cyclic GMP levels [33, 34]. The hypertrophied AV-fistula rat heart is exposed to increased local and circulating natriuretic peptide levels [10, 15, 35], and so one mechanism by which endocardial NPR-C levels may be downregulated is via natriuretic peptide-stimulated cyclic GMP production. Against this we have been unable to demonstrate guanylyl cyclase-linked receptors in cardiac tissue in our studies. Other investigators have shown that stimulation of β2-adrenoceptors in vascular smooth muscle cells [36], activation of protein kinase A and protein kinase C in endothelial and smooth muscle cells [37]and changes in NaCl [38]lead to reduced expression of NPR-C, suggesting considerable flexibility in the response to regulatory stimuli. Further investigation is needed to determine whether these operate in ventricular endocardium during hypertrophy.

The role of NPR-C in the endocardium, as elsewhere, is still unclear. Unlike the guanylyl cyclase-linked receptors (NPR-A and NPR-B), structure–function analysis of NPR-C has not revealed an obvious signal transduction pathway and it has less stringent requirements for ligand binding than the other two receptors: it binds all three natriuretic peptides with similar affinity, as well as ring-deleted and truncated linear peptides [5]. These observations have given rise to the view that NPR-C functions as a ‘clearance’ receptor [2]. If this is correct, reducing endocardial NPR-C expression would act to impair the local removal of natriuretic peptides and increase local concentration to which the endocardium is exposed. A second mechanism for natriuretic peptide clearance, specifically metabolism by neutral endopeptidase, has been detected in coronary vascular endothelial cells in culture [39]but we have had no success detecting this enzyme immunocytochemically in cardiac tissue, suggesting it is not particularly abundant in vivo and unlikely to offset the effects of endocardial NPR-C downregulation. Moreover, estimates of the Km values for hydrolysis of the natriuretic peptides by neutral endopeptidase are in the micromolar range [40], while the Kd for binding of ANP to NPR-C is in the picomolar range [41].

On the other hand, there is evidence that NPR-C signals through inhibition of adenylyl cyclase [4]. Significantly, anti-R37A antibody has been shown to block ANP-dependent inhibition of adenylyl cyclase activity in particulate fractions of rat heart [19]. ANP has been reported to induce endocardium-dependent early relaxation of isolated mammalian papillary muscle [13]. The NPR subtype mediating this effect was not characterised but our binding studies suggest that the most likely candidate is NPR-C, in which case loss of these receptors from the endocardium might impair ventricular relaxation and predispose to ventricular dysfunction.

Time for primary review 44 days


   Acknowledgements
 
This work was supported by the British Heart Foundation (PG93110). We thank Dr. Yuzuru Matsuda, Tokyo Research Laboratories, Kyowa Hakko Koyyo Co. Ltd., Tokyo 194, Japan, for supplying the non-peptide antagonist HS-142-1.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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L King and M R Wilkins
Natriuretic peptide receptors and the heart
Heart, April 1, 2002; 87(4): 314 - 315.
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