Vasostatins are the N-terminal chromogranin A peptides 7 approximately 22 kDa. They have been shown to be present in several endocrine tissues and exhibit vasoinhibitory activity in vitro. In a first series of experiments, we investigated the presence and subcellular localization of vasostatins in the bovine splenic nerve. Experimental results, obtained using gradient centrifugation, showed that noradrenaline was enriched 25-fold in the large dense core vesicle fraction, compared with the original homogenate. In the latter fraction, the 7 and 18 kDa peptides were observed following immunodetection with antiserum to chromogranin A1-40 and laser densitometric scanning revealed these two fragments as the major N-terminal fragments. Subsequently, we examined the release of the 7 and 18 kDa peptides from perfused calf spleen during veratridine (20 microM) or 1,1-dimethyl-4-phenylpiperazinium iodide (20 microM) stimulation. In the prestimulation samples, we were not able to detect these peptides, however, following stimulation, the 7 and 18 kDa chromogranin A fragments became apparent. The vasostatin-immunoreactivity, in both bovine chromaffin granule lysate and calf spleen perfusate, elutes at the same retention time on reversed-phase high performance liquid chromatography. The present study demonstrated that vasostatins are present in the large dense core vesicles of sympathetic axons and are released from the nerve terminals in response to stimulation. The release of vasostatins from sympathetic nerves in the spleen suggest an in vivo function for N-terminal chromogranin A products of neuronal origin.

An N-terminal proteolytic processing product of chromogranin A was obtained from bovine chromaffin granules using two steps of C, solid-phase extraction and reversed-phase high-performance liquid chromatography. Electrospray mass spectrometry revealed a protein with a molecular mass of 8632.0 for the fraction showing immunoreactivity against the N-terminus of chromogranin A, which differed by 48 u from that of the N-terminal processing product, vasostatin I (CGA,_,,). Derivatization with mercaptoethanol showed that the peptide had an intact S-S bridge, which is a key structural feature of vasostatin I. Peptide mapping experiments involving reduction/alkylation with vinylpyridine and trypsin digestion were consistent with oxidation of the three methionine residues of vasostatin I to their sulphoxide forms. The oxidation of the methionine residues was found to occur during the C,, solid-phase extraction procedure. The use of freshly prepared T r i s H C l buffer and eluents and flushing the buffer and eluents with nitrogen were shown to result in the isolation of the non-oxidized form of vasostatin I.

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