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Solubilization and Molecular Characterization of the Atrial Natriuretic Peptide (ANP) Receptor in Human Platelets: Comparison with ANP Receptors in Rat Tissues^*

ERNESTO L. SCHIFFRIN, FRANCE CARRIER, GAÉTAN THIBAULT and MARIO DESLONGCHAMPS

Clinical Research Institute of Montreal Montreal, Quebec, Canada

Address all correspondence and requests for reprints to: Ernesto L. Schiffrin, M.D., Ph.D., Experimental Hypertension Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7.

We have previously demonstrated the presence of binding sites for atrial natriuretic peptide (ANP) in human platelets. These sites have pharmacological characteristics similar to those of rat vascular smooth muscle. They are subject to regulation by circulating levels of ANP in plasma, varying inversely with the latter after high sodium intake, in arterial hypertension and congestive heart failure. We have now solubilized these platelet receptors with the nonionic detergent Triton X-100 (0.6%). The preparations were incubated with [¹²⁵I] ANP in the presence of increasing concentrations of ANP-(99–126), ANP-(101–126), ANP-(103–126), and ANP-(103–123). The order of potency of these peptides to displace [¹²⁵I]ANP was similar for the solubilized and particulate receptor. Bound [¹²⁵I] ANP was covalently cross-linked to the receptor with 5 mM disuccinimidyl suberate. Autoradiography of the sodium dodecyl sulfate-polyacrylamide gel showed that [¹²⁵I]ANP specifically interacts with a 125-kDa membrane component, some of which may be reduced by 2% mercaptoethanol or 10 mmol/L dithiothreitol to a 70-kDa species. A small proportion of a 70-kDa peptide is also found under nonreiucing conditions. The concentration of ANP-(99–126) that inhibits binding of [¹²⁵I]ANP by 50% to both the 125-kDa and the 70-kDa species was 0.1 nM, while that for ANP-(103–123) was 3 nM. The internally ringdeleted analog Des(Gln¹¹⁶,Ser¹¹⁷,Gly¹¹⁸,Leu¹¹⁹,Gly¹²⁰)ANP-(102–121) or C-ANP displaced with tqual potency ANP binding to the high and low mol wt (Mr) bands, as also found in cultured rat vascular smooth muscle cel.s, but not in the mesemteric arteries these cells are derived from. In the latter, C-ANP displaced only binding from the lower Mr band. These results show that the ANP receptor in human platelets is heterogeneous. There is one nonreducible species with of 125,000 Mr, another of similar Mr containing two disulfide-linked subunits of 70,000 Mr, and, to a lesser extent, a njnreducible 70-kDa species, in agreement with findings in othur tissues in experimental animals.

^* This work was supported by Grants MT-8018 and MT-9112 of the Medical Research Council of Canada (to E.L.S.) and a group grant to the Medical Research Council Hypertension Group (to G.T.).

Predoctoral fellow of the Canadian Heart Foundation.

Received June 18, 1990.