Stabilization of Vasoactive Intestinal Peptide by Lipids 1

An anionic phospholipid, phosphatidylglycerol (PG), induced vasoactive intestinal peptide (VIP) to adopt a helical conformation, determined by circular dichroism studies. PG inhibited the trypsin-catalyzed, antibody-catalyzed and uncatalyzed cleavage of VIP, measured by radiometric and HPLC methods. Phosphatidylcholine, a neutral lipid, did not alter the circular dichroism spectra of VIP, and it was without detectable effect on the rates of VIP cleavage. Trypsin-catalyzed cleavage of Boc-Ile-Glu-Arg-methylcoumarinamide, a substrate unrelated in sequence to VIP, proceeded at equivalent rates in the absence and presence of PG, which suggests that the phospholipid did not exert a nonspecific inhibitory effect on the enzyme. Study of the kinetics of antibody-catalyzed VIP cleavage indicated that the inhibition by PG was due to decreased affinity for VIP, suggested by observations of increased Km values and unaltered Vmax values. Incorporation of VIP in the liposomes and the liposomal surface permitted maintenance of the peptide in essentially undegraded form at 37°C for 8 days. The longevity of liposomal VIP administered i.v. to mice was increased by about 5-fold compared with aqueous VIP. These observations indicate that certain phospholipids and liposomes can be applied to circumvent the rapid loss of VIP in vitro and in vivo due to degradative processes. The biological actions of neurotransmitters and neuropeptides are terminated rapidly after their release from neurons. In the case of the 28-amino acid neuropeptide VIP, three types of degradative pathways have been identified: 1) cleavage by enzymes such as serine proteases (Caughey et al., 1988; Tam et al., 1990) and neutral endopeptidase (Hachisu et al., 1991), 2) antibody-catalyzed cleavage, which is described to occur in certain disease states (Paul et al., 1991; Gao et al., 1994), and 3) uncatalyzed spontaneous hydrolysis, a process that occurs at comparatively high rates at low VIP concentrations (Mody et al., 1994). These processes are generally assumed to be responsible for the rapid disappearance of VIP from blood (T1/2 5 0.4–1.0 min) after administration of the peptide to experimental animals and humans (Domschke et al., 1978; Mitchell et al., 1982; Hassan et al., 1994). VIP is a remarkably versatile peptide. It is broadly distributed in the CNS and peripheral nervous system, and it exerts diverse biological effects, including smooth muscle relaxation, anti-inflammatory and immunomodulatory effects, regulation of the production of certain hormones, regulation of transport phenomena across exocrine epithelia and modulation of cell proliferation (for reviews see Yanaihara, 1992; Paul and Ebadi, 1993). Deficiencies of VIP have been described in the airways in asthma (Ollerenshaw et al., 1989), in the skin in cystic fibrosis (Heinz et al., 1985), in the penis in impotent males (Crowe et al., 1983) and in the GI tract in ulcerative colitis (Surrenti et al., 1993). Administration of exogenous VIP in patients with such deficiencies could potentially be of therapeutic benefit. Even in instances where a deficit of VIP is not a pathophysiological factor, it may be possible to treat the disease with VIP. For instance, the potent anti-inflammatory effects of VIP (for reviews see Yanaihara, 1992; Paul and Ebadi, 1993) suggest its possible broad utility in controlling tissue inflammation. It is generally held that the rapid degradation of VIP is a major factor limiting the potential clinical applications of this peptide. VIP can be modeled as a cationic amphipathic helix (Musso et al., 1988), which is a common structural motif found in lipid-binding proteins and peptides (Sansom, 1991). In an effect consistent with its predicted lipophilicity, VIP displays reversible and saturable binding to protein-free lipid bilayers (Noda et al., 1994). In the present report, we describe the ability of a negatively charged phospholipid, and of liposomes, to confer helical character to VIP, reduce the uncatalyzed and catalyzed degradation of VIP in vitro and increase the longevity of circulating VIP in mice. These studies suggest that the lipophilicity of VIP can be applied to developing therapeutically useful formulations of the peptide. Received for publication July 2, 1997. Supported by U.S. Public Health Service grants HL44126, AI31268, DE10347 and DE00386. ABBREVIATIONS: BSA, bovine serum albumin; CD, circular dichroism; PG, phosphatidylglycerol; PC, phosphatidylcholine; TCA, trichloroacetic acid; VIP, vasoactive intestinal peptide. 0022-3565/98/2852-0753$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 285, No. 2 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 285:753–758, 1998 753 at A PE T Jornals on M ay 9, 2017 jpet.asjournals. org D ow nladed from Materials and Methods VIP. Synthetic human VIP (HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH2; peptide content 81%, Bachem, Torrance, CA) was labeled with I using chloramine-T. (Tyr–I)VIP was separated by reverse-phase HPLC on a Novapak C18 column (Waters, Milford, MA) using an ISCO HPLC apparatus (Mody et al., 1994). The radiolabeled VIP eluted from the HPLC column at 40% acetonitrile in 0.1% trifluoroacetic acid was diluted with an equal volume of 0.2 M acetic acid containing 0.5% BSA (Sigma, St. Louis, MO, RIA grade) and stored at 280°C in aliquots. Maintenance of the VIP at acidic pH and in the presence of a stabilizing protein is necessary to minimize the uncatalyzed degradation of the peptide (Mody et al., 1994). The specific activity of the peptide was ;2000 Ci/mmol. Unlabeled VIP synthesized at the University of Florida, Gainesville, and purified by preparative reverse-phase HPLC on a C-18 column was used in certain experiments. The peptide content of this preparation was 83%, and its purity was confirmed by amino acid analysis and Nterminal sequencing at the University of Nebraska Protein Structure