cyclic-gmp and atrial-natriuretic-factor-26--rat

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 nonreducing 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 ring-deleted analog Des(Gln116,Ser117,Gly118,Leu119,Gly120)ANP -(102-121) or C-ANP displaced with equal potency ANP binding to the high and low mol wt (Mr) bands, as also found in cultured rat vascular smooth muscle cells, 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 nonreducible 70-kDa species, in agreement with findings in other tissues in experimental animals.

Topics: Amino Acid Sequence; Animals; Atrial Natriuretic Factor; Autoradiography; Binding, Competitive; Blood Platelets; Cells, Cultured; Cyclic GMP; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Humans; Iodine Radioisotopes; Male; Molecular Sequence Data; Molecular Weight; Muscle, Smooth, Vascular; Peptide Fragments; Rats; Rats, Inbred Strains; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Solubility

A synthetic peptide corresponding to a sequence of 26 amino acids contained in endogenous rat atrial natriuretic factor (ANF), was infused into one renal artery of anesthetized dogs for a comprehensive in vivo evaluation of the renal and systemic effects of pure ANF. The results proved conclusively that ANF acted directly on the kidney since urine volume and fractional excretion of sodium, potassium, chloride and calcium were elevated in a dose-related manner in the ANF-treated kidney, but were not significantly affected in the contralateral saline-infused organ. The maximum effects achieved with the synthetic ANF were higher than any reported following intravenous administration of crude extracts of rat atria and were similar to those produced by thiazide diuretics. In four of the five dogs studied, renal vascular resistance fell progressively as doses of ANF were increased. Glomerular filtration rate was not significantly elevated during ANF infusion, but was correlated with sodium excretion rates. Even though mean arterial pressure was progressively reduced, there was no significant change in heart rate and no stimulation of renin secretion. Arterial cyclic GMP concentration was higher in the basal state and rose more rapidly than did renal venous levels, indicating that increases in circulating concentrations of arterial cyclic GMP originated from an extrarenal source. Dose-related elevations in urinary cyclic GMP excretion could be explained by increased cyclic GMP filtration, by enhanced production in tubular cells, or by renal tubular secretion. Especially in the saline-infused kidney, there was a clear dissociation between excretion of cyclic GMP and fractional sodium excretion. We conclude that the synthetic ANF increased electrolyte excretion via a direct renal action which was not solely dependent upon changes in renal vasculature, renin secretion or cyclic GMP levels.

Topics: Animals; Atrial Natriuretic Factor; Cyclic AMP; Cyclic GMP; Dogs; Female; Kidney; Kinetics; Natriuresis; Peptide Fragments; Peptides; Rats; Renal Circulation; Renin; Vascular Resistance