THE PRESENCE OF ATRIAL-NATRIURETIC-FACTOR RECEPTORS OF ANF-R2 SUBTYPE IN RAT PLATELETS - COUPLING TO ADENYLATE-CYCLASE CYCLIC-AMP SIGNAL-TRANSDUCTION SYSTEM

The effect of atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in rat platelet membranes. (ANF- 99-126)-, -(101-126)-, -(103-126)- and -(103-123)-peptide inhibited adenylate cyclase activity in a concentration-dependent manner with an order of potency of ANF-(103-123)-peptide > ANF-(99-126)-peptide > ANF-(101-126)- peptide > ANF-(103-126)-peptide. ANF-(103-123)-peptide and ANF-(99-126)-peptide inhibited the enzyme activity by about 50-55% with an apparent K(i) between 0.1 and 0.5 nM, and ANF-(101-126)-peptide inhibited the enzyme activity by about 35% with an apparent K(i) between 1 and 3 nM. On the other hand, ANF-(103-126)-peptide was the least potent and inhibited the adenylate cyclase activity by about 30% (K(i) approximately 10 nM). The inhibitory effect of ANF on adenylate cyclase was also dependent on the presence of guanine nucleotides and was attenuated by amiloride and pertussis toxin. The stimulatory effects of various agonists such as N-ethylcarboxamideadenosine, prostaglandin E1, isoprenaline and forskolin on adenylate cyclase were also inhibited by ANF to various extents; however, the stimulations were not completely abolished. In addition, I-125-labelled ANF-(99-126)-peptide bound specifically to rat platelet membranes. The binding of I-125-ANF was competitively inhibited in a concentration-dependent manner by the unlabelled peptides which were used for adenylate cyclase inhibition. ANF-(103-123)-peptide, ANF-(99-126)-peptide and ANF-(101-126)-peptide were almost equipotent [IC50 (median inhibitory concentration) = 0.1-1 nM], and ANF-(103-126)-peptide was the least potent (IC50 approximately 10 nM). Scatchard analysis of the data revealed the presence of a single class of binding sites of high affinity (K(d) approximately 120 pM). Affinity cross-linking of I-125-ANF-(99-126)-peptide to its binding sites in rat platelet membranes and analysis by SDS/PAGE followed by autoradiography showed a predominant labelling of a protein band with an apparent M(r) of 66000. These data indicate the presence of only ANF-R2 (low-M(r)) receptors in platelets and suggest that these receptors may be coupled to the adenylate cyclase system.