Characterization of the snake venom ligand [125I]-DNP binding to natriuretic peptide receptor-A in human artery and potent DNP mediated vasodilatation.
OBJECTIVE
The natriuretic peptides, ANP and BNP, modulate vascular smooth muscle tone in human conduit arteries. Surprisingly, the natriuretic peptide receptor-A (NPR-A) has not been visualized using radioligand binding in these vessels. A new member of this peptide family, Dendroaspis natriuretic peptide (DNP) identified from snake venom, has been proposed to be present in human plasma and endothelial cells. Also, recently a novel radioligand, [(125)I]-DNP, has been characterized as selective for NPR-A in human heart.


METHODS
Our aims were to investigate expression and function of NPR-A receptors in human mammary artery using [(125)I]-DNP to quantify receptor density, immunocytochemistry to delineate the cellular distribution of the receptor and in vitro pharmacology to compare DNP induced vasodilatation to that of ANP.


RESULTS
Saturable, sub-nanomolar affinity [(125)I]-DNP binding was detected to smooth muscle of mammary artery, with receptor density of approximately 2 fmol mg(-1) protein, comparable to that of other vasoactive peptides. NPR-A immunoreactivity was localised to vascular smooth muscle cells and this was confirmed with fluorescence dual labelling. NPR-A expression was not detected in the endothelium. Like ANP, DNP fully reversed the constrictor response to ET-1 in endothelium intact or denuded mammary artery, with comparable nanomolar potencies.


CONCLUSIONS
This is the first characterization of NPR-A in human mammary artery using [(125)I]-DNP and we provide evidence for the presence of receptor protein on vascular smooth muscle cells, but not endothelial cells. This implies that the observed vasodilatation is predominantly mediated via direct activation of smooth muscle NPR-A.
Singh
G
G
Clinical Pharmacology Unit, University of Cambridge, Centre for Clinical Investigation, Addenbrooke's Hospital, Cambridge, UK.
Maguire
J J
JJ
Kuc
R E
RE
Skepper
J N
JN
Fidock
M
M
Davenport
A P
AP
D053607:Adrenomedullin / Q000494:pharmacology; D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D015740:Calcitonin Gene-Related Peptide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004546:Elapid Venoms / Q000378:metabolism* / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006162:Guanylate Cyclase / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D008323:Mammary Arteries / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D019275:Radiopharmaceuticals / Q000378:metabolism*; D017461:Receptors, Atrial Natriuretic Factor / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents / Q000378:metabolism* / Q000494:pharmacology
C075597:Dendroaspis natriuretic peptide; D004546:Elapid Venoms; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D010455:Peptides; D019275:Radiopharmaceuticals; D014665:Vasodilator Agents; D053607:Adrenomedullin; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; D015740:Calcitonin Gene-Related Peptide