Binding of the VCHSKT peptide to ceramide‐rich and cholesterol‐rich domains of cell membranes in rat pancreatic glands

A peptide with antisecretory effect has been defined and was denoted the antisecretory factor (AF-16) with a sequence of 16 amino acids. A shorter sequence, the AF-6 peptide (VCHSKT, Pepceuticals Ltd, Leicester, England), is readily detected by Time of Flight Secondary Ion Mass Spectrometry (TOF-SIMS), using trifluoroacetic acid (TFA) as ionization catalyzer. Analysis of a reference sample revealed a pseudo-molecular ion at m/z 657 and M+ Na+ at m/z 679. These peaks were also detected in tissue samples of animals exposed to the peptide. The AF-6 peptide was injected intravenously or sprayed into the nostrils of Sprague-Dawley rats, 250-300 g (n = 5). The animals were anesthetized with isoflurane. Pancreas tissue samples were taken after 2 h and were high-pressure frozen in a Lecia EMPACT, sprayed with 0.1% TFA, and analyzed using a TOF-SIMS V instrument (ION-TOF, GmbH, Munster, Germany) equipped with a Bi-3(+)-liquid metal ion gun at the University of Gothenburg. The distribution of the AF-6 peptide could be seen in the pancreas of exposed animals and control animals as high-intensity dots that were heterogeneously distributed in regions with a high intensity of phosphatidylcholine head group m/z 184, indicating a localization to the membrane portion of the pancreatic cells. The peptide was colocalized with a dot-like distribution of galactocylceramide c18:0 and cholesterol. Cholesterol and gal-Cer are known lipid components of specialized membrane areas called lipid rafts. The results indicate that the AF-6 peptide binds to cellular receptors present in such regions of rat pancreatic cells. Copyright (C) 2012 John Wiley & Sons, Ltd.