Caerulein-related peptides were identified in norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus borealis and the octoploid frog Xenopus amieti using negative ion electrospray mass spectrometry and their primary structures determined by positive ion tandem (MS/MS) mass spectrometry. X. borealis caerulein-B1 (pGlu-Gln-Asp-Tyr(SO(3))-Gly-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains an additional Gly(5) residue compared with X. laevis caerulein and caerulein-B2 (pGlu-Asp-Tyr(SO(3))-Thr-Gly-Trp-Met-Asp-Phe.NH2) contains a Gln(2) deletion. X. amieti caerulein was identical to the X. laevis peptide. In addition, xenopsin, identical to the peptide from X. laevis, together with xenopsin-AM2 (pGlu-Gly-Arg-Arg-Pro-Trp-Ile- Leu) that contains the substitution Lys(3) -> Arg were isolated from X. amieti secretions. X. borealis caerulein-B1, and X. amieti xenopsin and xenopsin-AM2 produced significant (P < 0.05) and concentration-dependent stimulations of insulin release from the rat BRIN-BD11 clonal beta cell line at concentrations >= 30 nM. The peptides did not stimulate the release of lactate dehydrogenase at concentrations up to 3 mu M demonstrating that the integrity of the plasma membrane had been preserved. While their precise biological role is unclear, the caerulein- and xenopsin-related peptides may constitute a component of the animal's chemical defenses against predators. (C) 2011 Elsevier Inc. All rights reserved.