Novel 125I-dendroaspis natriuretic peptide binds natriuretic peptide receptor-A in human ventricle: down-regulation in cardiovascular disease

Gurminder Singh*, Rhoda E. Kuc*, Mark Fidock †, Anthony P. Davenport*. *Clinical Pharmacology Unit, University of Cambridge, Level 6 Centre for Clinical Investigation, Box 110 Addenbrooke’s Hospital, Cambridge, CB2 2QQ, UK. † Department of Target Genomics, Pfizer Global Research and Development, Sandwich.

Natriuretic peptides (NP) are important regulators of blood pressure with circulating plasma levels of atrial-NP (ANP) and brain-NP (BNP) increasing significantly in heart failure patients (Mukoyama et al., 1991). Although NP receptor (NPR) genes are expressed in human heart, radiolabelled NP have not detected specific binding sites in adult human heart. Recently, the novel dendroaspis NP (DNP) was isolated from snake venom (Schweitz et al ., 1992), which is resistant to breakdown by neutral endopeptidases and immunoreactivity has been reported in human cardiovascular tissues (Schirger et al., 1999) . Our aims were to demonstrate saturable and high affinity binding of 125I-DNP to human left ventricle ( LV) and to determine if receptor density is altered with cardiovascular disease.

For saturation experiments LV was obtained with ethical approval from 8 patients transplanted for ischaemic heart disease (IHD) and 6 normal hearts for which there was no suitable recipient. Tissue sections (30μm thick) were pre-incubated with 50mM Tris-HCl buffer (pH 7.2) containing MgCl2 (10mM), EDTA (5mM) at room temperature for 15 mins. Sections were then incubated in buffer containing 2pM–1nM 125I-DNP (custom synthesis) for 1 hr at room temperature. Non-specific binding was determined by including 1µM DNP. Sections were washed and radioactivity counted in a gamma counter. For competition binding experiments, sections of LV (n=4) were incubated with 0.2nM 125I-DNP in the presence of increasing concentrations (2pM–10µM) of competing ligands. Results were analysed using iterative non-linear curve fitting programme (KELL, Biosoft, Cambridge, UK) . Pooled KD, Bmax and Hill slope were expressed as mean ± s.e.mean.

Table 1. Saturation analysis of [125I]-DNP in LV. *P<0.005 Student’s unpaired t-test, compared to normal LV.

125I-DNP bound specifically to diseased and non-diseased LV with comparable subnanomolar affinity (Table 1). Receptor density (Bmax) was ~6 fmol mg-1 protein in normal heart tissue with evidence of significant down-regulation in cardiovascular disease. In competition experiments ANP, BNP and DNP competed for 125I-DNP binding with similar high affinity (KD of 26.75 ± 4.28 nM, 21.46 ± 1.83 nM and 12.15 ± 3.21 nM respectively). CNP and cANF competed with lower affinity (KD of 825.39 ± 174.90 nM and 367.9 ± 77.53 nM respectively). This suggests DNP binds specifically to the NPR-A at the concentrations tested.

This is the first evidence for the characterisation of 125I-DNP binding and demonstration of down-regulation of NPR-A in LV of IHD patients. These data may help explain the lack of response to elevated levels of natriuretic peptides during heart failure.

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