Abstract

Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Litho bates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal beta cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P<0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (236% of basal rate, P<0.001) at a concentration of 3 mu M. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 mu M, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIAR-LAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P<0.001 at 3 mu M) but the peptide was cytotoxic at this concentration. (C) 2010 Elsevier Inc. All rights reserved.