NH2-terminal fragment of rat pro-atrial natriuretic factor in the circulation: identification, radioimmunoassay and half-life.
A radioimmunoassay was developed to measure the NH2-terminal counterpart of rat pro-atrial natriuretic factor (pro-ANF) in plasma. Synthetic rat ANF (Asp 11-Ala 37) coupled to bovine serum albumin was used to immunize New Zealand rabbits. The antiserum demonstrated good immunoreactivity towards rat ANF (Asn 1-Arg 98), (Asn 1-Tyr 126), (Asp 11-Ala 37) and even human ANF (Asn 1-Ser 30). The standard curve had an ED80 of 9.5 +/- 2.5 and ED50 of 44.0 +/- 10.5 fmol/tube. Immunoreactive ANF NH2-terminal peptide was measured directly in rat plasma without prior extraction. In fact, extraction of ANF NH2-terminal from plasma by C18 silica gel chromatography revealed inconsistent recovery and a lack of parallelism. Morphine (0.75 mg/100 g), chosen to elicit increased ANF (Ser 99-Tyr 126) secretion, elevated its plasma concentration from 54.1 +/- 3.2 to 190.8 +/- 55.8 fmol/ml after 20 min. At the same time, the immunoreactive NH2-terminal fragment rose from 378 +/- 16 to 1181 +/- 201 fmol/ml. The identity of this immunoreactive material was verified following affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC) of plasma from morphine-treated rats. Molecular sieving and amino acid sequencing demonstrated that it appears to be consistent with or identical to rat ANF (Asn 1-Arg 98). The disappearance rate of ANF (Asn 1-Arg 98) was studied by injecting radioactive material into anesthetized rats. The exponential decay was analyzed by a two-compartment model in which the fast and slow components had a half-life of 2.5 +/- 0.3 and 54.8 +/- 3.9 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Thibault
G
G
Clinical Research Institute of Montreal, Québec, Canada.
Murthy
K K
KK
Gutkowska
J
J
Seidah
N G
NG
Lazure
C
C
Chrétien
M
M
Cantin
M
M
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000097:blood* / Q000276:immunology / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D006207:Half-Life; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D009020:Morphine / Q000008:administration & dosage; D010446:Peptide Fragments / Q000097:blood; D011498:Protein Precursors / Q000276:immunology / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains
D010446:Peptide Fragments; D011498:Protein Precursors; C055659:atrial natriuretic factor prohormone (1-98); D009020:Morphine; D009320:Atrial Natriuretic Factor