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A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps).

1992; Elsevier BV; Volume: 267; Issue: 20 Linguagem: Inglês

10.1016/s0021-9258(19)49658-0

1083-351X

Hugues Schweitz, Paul Vigne, Danielle Moinier, Christian Frelin, Michel Lazdunski,

Receptor Mechanisms and Signaling

Dendroaspis Natriuretic Peptidedescribed previously (15).Cells were grown in M199 medium (GIBCO) supplemented with 20% fetal calf serum (Dutscher, Strasbourg, France), 100 units/ml penicillin, and 100 pg/ml streptomycin.Cultures were dissociated using 0.1% trypsin and passaged weekly.Cells at passages 2-10 were used.Bovine aortic endothelial cells were prepared as described previously (16) and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics.Measurements of cGMP-Confluent cell layers in 12-well Falcon plates were washed twice with an Earle's salt solution (140 mM NaC1, 5 mM KCl, 1.8 mM CaC12, 0.8 mM MgS04, 5 mM glucose buffered at pH 7.4 with 25 mM Hepes/Tris) and further incubated for 6 min in the same solution supplemented with 0.3 mM isobutylmethylxanthine at 37 "C.Cells were then stimulated with the peptides.After 6 min, the incubation solution was aspirated off, and cells were treated with an ethanol, 5 mM EDTA (2:l) solution for 30 min at 4 "C.Extracts were lyophilized, and cGMP was assayed by radioimmunoassay using reagents provided by Amersham.Cell proteins were determined according to Bradford (17) using bovine serum albumin as standard.Data are expressed in pmol of cGMP/6 min/mg of protein.Binding Assays-Confluent layers of aortic myocytes in 12-well Falcon plates were washed twice with an Earle's salt solution and further incubated at 25 "C in the same solution supplemented with 2 mg/ml bovine serum albumin, 0.02% sodium azide, 1 p~ captopril, 1 p~ phosphoramidon, and the desired concentrations of peptide.After 30 min of equilibration, 9 -A N P (9 PM final) was added to each well, and the incubation was allowed to proceed for an additional 80 min.Cells were then washed three times with 1 ml of Earle's salt solution, digested into 0.1 N NaOH, and the cell-associated radioactivity was determined.Means & S.E. are indicated.