Cleavage of vasoactive intestinal peptide at multiple sites by autoantibodies

Vasoactive intestinal peptide (VIP) fragments generated by autoantibodies purified from the blood of two human beings were separated and sequenced. Based on the identity of these fragments, seven peptide bonds cleaved by the antibodies were identified. Six of the seven scissile bonds are clustered in the region of VIP spanning residues 14-22 and were cleaved by antibodies from both human subjects. The seventh scissile bond is located at residues 7-8 and was cleaved by antibodies from one of the subjects. The scissile bonds link amino acid residues with different size, charge, and hydrophobicity. The hydrolytic activity of the antibodies was selective in that they failed to hydrolyze polypeptides unrelated in sequence to VIP (insulin and atrial natriuretic peptide). These observations demonstrate substrate specific hydrolysis by naturally occurring antibodies and expand the range of peptide bonds hydrolyzed by these antibodies.     

Affinity chromatography of catalytic autoantibody to vasoactive intestinal peptide

An autoantibody in human IgG that hydrolyzes vasoactive intestinal peptide (VIP) was identified. The IgG did not hydrolyze VIP unless an unidentified inhibitor was removed by dialysis. The VIP antibody was fractionated by affinity chromatography on immobilized VIP by using IgG without VIP-hydrolytic activity as the starting material. The affinity-purified antibody catalyzed the hydrolysis of VIP (nominal kcat/Km: 1.1 X 10(6) M-1 min-1). The values of Km for the affinity-purified antibody preparation and unfractionated IgG (110 nM and 112 nM) suggested relatively tight antibody-VIP binding. A comparison of the reverse phase HPLC profiles of antibody-treated [Tyr10-125I]VIP with that of synthetic [125I]VIP(1-16) suggested that unfractionated IgG and the affinity-purified antibody cleaved the same peptide bond in VIP (Gln16-Met17).     

Design and development of a vasoactive intestinal peptide analog as a novel therapeutic for bronchial asthma

Analogs of vasoactive intestinal peptide (VIP) were synthesized and screened as bronchodilators with the ultimate goal of enhancing the potency and extending the duration of action of the native peptide. Several design approaches were applied to the problem. First, the amino acid residues required for receptor binding and activation were identified. A model of the active pharmacophore was developed. With knowledge of the secondary structure (NMR) of the peptide, various analogs were synthesized to stabilize α-helical conformations. Having achieved a level of enhanced bronchodilator potency, our approach then concentrated on identification of the sites of proteolytic degradation and synthesis of metabolically-stable analogs. Two primary cleavage sites on the VIP molecule were identified as the amide bonds between Ser²⁵-Ile²⁶ and Thr⁷-Asp⁸. This information was used to synthesize cyclic peptides which incorporated disulfide and lactam ring structures. Analog work combined the best multiple-substitution sites with potent cyclic compounds which resulted in identification of a cyclic lead peptides. This compound, Ro 25-1553, exhibited exceptionally high potency, metabolic stability, and a long duration of action and may be an effective therapeutic for the treatment of bronchospastic diseases. © 1994 John Wiley & Sons, Inc.     

Respirable powder formulation of a shortened vasoactive intestinal peptide analog for treatment of airway inflammatory diseases

The aim of present study was to develop a respirable powder (RP) of a shortened vasoactive intestinal peptide (VIP) analog for inhalation. VIP and C‐terminally truncated VIP analogs were synthesized with a solid‐phase method. A structure‐activity relationship (SAR) study was carried out in terms with binding and relaxant activities of the peptides. Prepared RP formulation of a shortened VIP analog was physicochemically characterized by morphological, in vitro aerodynamic, and pharmacological assessments. The SAR study demonstrated that the N‐terminal 23 amino acid residues were required for biological activity of VIP. Upon chemical modification of VIP(1–23), [R^{15, 20, 21}, L¹⁷]‐VIP(1–23) was newly developed, which had higher binding activity in rat lung and smooth muscle relaxant effect in mouse stomach than VIP(1–23). The [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)‐based RP, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP, exhibited fine in vitro inhalation performance. Airway inflammation evoked by sensitization of antigen in rats was attenuated by pre‐treatment with the [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP at a dose of 50 μg‐[R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/rat as evidenced by a 70% reduction of recruited inflammatory cells in bronchoalveolar lavage fluid. On the basis of these results, [R^{15, 20, 21}, L¹⁷]‐VIP(1–23)/RP might be a promising agent for treatment of airway inflammatory diseases.     

Characterization of vasoactive intestinal peptide receptors by a photoaffinity label. Site-specific modification of vasoactive intestinal peptide by derivatization of the receptor-bound peptide

The biological effects of vasoactive intestinal peptide (VIP) are mediated by binding to a membrane-bound receptor. Probes designed to trap this receptor by binding to it in a covalent way may suffer from a greatly reduced affinity. We report here, for the VIP receptor, the use of a photoaffinity probe obtained by derivatization of receptor-bound VIP with para-azidophenylglyoxal. This method protected the parts of the molecule essential for receptor binding. The VIP derivative thus obtained became covalently linked when irradiated. In the dark, however, it exhibited normal VIP-like behavior and retained its biological activity. This derivatization method might be generally applicable when hormone analogues have to be prepared without loss of receptor affinity. Receptor characterization studies on liver plasma membranes showed the presence of high- and low-affinity binding sites with KD = 0.1 and 5 nM, respectively. Treatment of membranes with dithiothreitol causes loss of high-affinity binding. The high-affinity site, trapped by the photoaffinity probe, resolved into two molecular mass forms, 50 and 200-250 kDa. Reduction of the receptor-probe complex left the 50-kDa form intact, whereas the amount of the 200-250-kDa form greatly diminished. We demonstrate the importance of the presence of disulfide bonds in one of the molecular forms involved in high-affinity binding.     

Circadian regulation of cardiovascular function: a role for vasoactive intestinal peptide

The circadian system, driven by the suprachiasmatic nucleus (SCN), regulates properties of cardiovascular function. The dysfunction of this timing system can result in cardiac pathology. The neuropeptide vasoactive intestinal peptide (VIP) is crucial for circadian rhythms in a number of biological processes including SCN electrical activity and wheel running behavior. Anatomic evidence indicates that SCN neurons expressing VIP are well positioned to drive circadian regulation of cardiac function through interactions with the autonomic centers. In this study, we tested the hypothesis that loss of VIP would result in circadian deficits in heart rate (HR) and clock gene expression in cardiac tissue. We implanted radiotelemetry devices into VIP-deficient mice and wild-type (WT) controls and continuously recorded HR, body temperature, and cage activity in freely moving mice. Under light-dark conditions, VIP-deficient mice displayed weak rhythms in HR, body temperature, and cage activity, with onsets that were advanced in phase compared with WT mice. Similarly, clock gene expression in cardiac tissue was rhythmic but phase advanced in mutant mice. In constant darkness, the normal circadian rhythms in HR were lost in VIP-deficient mice; however, most mutant mice continued to exhibit circadian rhythms of body temperature with shortened free-running period. The loss of VIP altered, but did not abolish, autonomic regulation of HR. Analysis of the echocardiograms did not find any evidence for a loss of cardiac function in VIP-deficient mice, and the size of the hearts did not differ between genotypes. These results demonstrate that VIP is an important regulator of physiological circadian rhythmicity in the heart.     

Vascular effects of pituitary adenylate cyclase activating peptide: a comparison with vasoactive intestinal peptide

The effects of pituitary adenylate cyclase activating peptide (PACAP) on the blood pressure of the anesthetized rat and on the isolated rat tail artery were investigated and compared to those of vasoactive intestinal peptide (VIP). PACAP-38, PACAP-27 and the C-terminal fragment 16–38 caused a dose-dependent decrease in the systemic blood pressure. PACAP-27 and PACAP-38 were equipotent with VIP. The C-terminal fragment 16–38 was much less potent than VIP. The duration of action was longer for equimolar doses of PACAP-38 and PACAP-27 than for VIP and much longer than for PACAP 16–38. PACAP-27 and the phosphodiesterase inhibitor rolipram given in combination produced additive vasodepressive responses. In vitro PACAP-38, PACAP-27, VIP and PACAP 16–38 relaxed the phenylephrine-precontracted rat tail artery. PACAP-38 and PACAP-27 were equipotent with VIP. PACAP 16–38 was much less potent than the full-length peptides. The responses were resistant to atropine and propranolol. Addition of VIP 1 μM to preparations exposed to 1 μM PACAP-38 or -27 did not produce a further relaxation. VIP-like peptides, PACAP in particular, are known to activate adenylate cyclase and to elevate the plasma cyclic AMP (cAMP) concentration. cAMP was found to be a potent vasodepressor in the anaesthetized rat and a potent vasodilator of precontracted blood vessels. On the basis of these results it cannot be excluded that the vascular effects of PACAP are secondary to the effect of elevated levels of extracellular cAMP.     

The vasoactive intestinal peptide (VIP): VI. The 17-norleucine analog of the sequence 14–28

A pentadecapeptide amide with the C-terminal sequence (14–28) of the vasoactive intestinal peptide (VIP), but with the methionine residue in position 17 replaced by l-norleucine, was synthesized. The synthesis was carried out through stepwise chain lengthening, by the in situ technique. The norleucine-containing pentadecapeptide, l-arginyl-l-lysyl-l-glutaminyl-l-norleucyl-l-alanyl-l-valyl-l-lysyl-l-lysyl-l-tyrosyl- l-leucyl-l-asparaginyl-l-seryl-l-isoleucyl-l-leucyl-l-asparaginamide, was as active in relaxing different smooth-muscle preparations as the methionine-containing parent sequence.     

Neuroprotection: a comparative view of vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide

The neuroprotective properties of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) place these peptides in a special category of ligands that have implications for our understanding of pathological conditions as well as a potential basis for therapeutic intervention. It is remarkable that these peptides have a protective impact against such a wide variety of clinical relevant toxic substances. This protective diversity is consistent with the multiple pathways that are activated or inhibited by the action of these peptides. Although knowledge is emerging on the neuroprotective mechanisms of VIP and PACAP, it is already evident that these two peptides are not identical in their action and each peptide has multiple mechanisms that allow for neuroprotective diversity. The multiple intracellular signaling pathways and differing extracellular mediators of neuroprotection contribute to this diversity of action. In this review, examples of neuroprotective actions will be presented that serve to demonstrate the remarkable breadth of neuroprotective processes produced by VIP and PACAP.     

Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors

Bovine t hymic peptide extract (1–100 g/ml) is shown to completely inhibit the binding of [¹²⁵I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [¹²⁵I]VIP binding with a potency that is 4000–7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.     

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.     

Isolation of a low molecular mass vasoactive intestinal peptide binding protein.

A vasoactive intestinal peptide (VIP) binding protein was purified in active form by detergent solubilization of lung membranes, gel filtration, VIP-Sepharose affinity chromatography, reverse phase high performance liquid chromatography, and anion exchange chromatography. The mass of this protein was estimated at 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 17 kDa by gel filtration. The binding of VIP by this protein was inhibited by Mg2+, covalent cross-linking of [Tyr10-125I]VIP to the protein produced two radioactive bands at 22 and 26 kDa identified by electrophoresis, and the purified protein exhibited saturable and high affinity binding of VIP and the related neuropeptide, rat growth hormone releasing factor.     

Prospect of vasoactive intestinal peptide therapy for COPD/PAH and asthma: a review

There is mounting evidence that pulmonary arterial hypertension (PAH), asthma and chronic obstructive pulmonary disease (COPD) share important pathological features, including inflammation, smooth muscle contraction and remodeling. No existing drug provides the combined potential advantages of reducing vascular- and bronchial-constriction, and anti-inflammation. Vasoactive intestinal peptide (VIP) is widely expressed throughout the cardiopulmonary system and exerts a variety of biological actions, including potent vascular and airway dilatory actions, potent anti-inflammatory actions, improving blood circulation to the heart and lung, and modulation of airway secretions. VIP has emerged as a promising drug candidate for the treatment of cardiopulmonary disorders such as PAH, asthma, and COPD. Clinical application of VIP has been limited in the past for a number of reasons, including its short plasma half-life and difficulty in administration routes. The development of long-acting VIP analogues, in combination with appropriate drug delivery systems, may provide clinically useful agents for the treatment of PAH, asthma, and COPD. This article reviews the physiological significance of VIP in cardiopulmonary system and the therapeutic potential of VIP-based agents in the treatment of pulmonary diseases.     

Tuning immune tolerance with vasoactive intestinal peptide: a new therapeutic approach for immune disorders

The induction of immune tolerance is essential for the maintenance of immune homeostasis and to limit the occurrence of exacerbated inflammatory and autoimmune conditions. Multiple mechanisms act together to ensure self-tolerance, including central clonal deletion, cytokine deviation and induction of regulatory T cells. Identifying the factors that regulate these processes is crucial for the development of new therapies of autoimmune diseases and transplantation. The vasoactive intestinal peptide (VIP) is a well-characterized endogenous anti-inflammatory neuropeptide with therapeutic potential for a variety of immune disorders. Here, we examine the latest research findings, which indicate that VIP participates in maintaining immune tolerance in two distinct ways: by regulating the balance between pro-inflammatory and anti-inflammatory factors, and by inducing the emergence of regulatory T cells with suppressive activity against autoreactive T-cell effectors.     

Enteric release of vasoactive intestinal peptide after a peptone meal in the dog

Vasoactive intestinal peptide (VIP) concentrations were measured by radioimmunoassay in plasma from portal and peripheral venous blood obtained from six alert, non-anesthetized dogs before and after gastric infusion of a 10% peptone meal. Mean basal portal and cephalic vein plasma VIP concentrations were $\text{42 ± 11.7}$ and $\text{42 ± 8.0}\text{(S.E.M.) pg/ml}$, respectively. No significant changes in peripheral venous plasma VIP concentrations were noted after the peptone meal throughout the duration of the collection period. In contrast, however, the mean VIP concentration in portal plasma increased promptly after the peptone meal with a peak of $\text{79 ± 8.2}\text{pg/ml}$ ($\text{P < 0.02}$) occurring 8 min after infusion of the meal. This was followed by a gradual decline in portal plasma VIP levels, with a return to prefeeding concentrations at 60 min ($\text{44 ± 6.3}\text{pg/ml}$). Results of these studies demonstrate that following gastric infusion of a peptone meal in the dog, portal, but not peripheral, plasma VIP concentrations increase significantly. Failure to detect augmentation of peripheral vein VIP levels after the meal is probably due to hepatic clearance of VIP.     

Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction.

Wortmannin at nanomolar concentrations is a potent and specific inhibitor of phosphoinositide (PI) 3-kinase and has been used extensively to demonstrate the role of this enzyme in diverse signal transduction processes. At higher concentrations, wortmannin inhibits the ataxia telangiectasia gene (ATM)-related DNA-dependent protein kinase (DNA-PKcs). We report here the identification of the site of interaction of wortmannin on the catalytic subunit of PI 3-kinase, p110alpha. At physiological pH (6.5 to 8) wortmannin reacted specifically with p110alpha. Phosphatidylinositol-4,5-diphosphate, ATP, and ATP analogs [adenine and 5'-(4-fluorosulfonylbenzoyl)adenine] competed effectively with wortmannin, while substances containing nucleophilic amino acid side chain functions had no effect at the same concentrations. This suggests that the wortmannin target site is localized in proximity to the substrate-binding site and that residues involved in wortmannin binding have an increased nucleophilicity because of their protein environment. Proteolytic fragments of wortmannin-treated, recombinant p110alpha were mapped with anti-wortmannin and anti-p110alpha peptide antibodies, thus limiting the target site within a 10-kDa fragment, colocalizing with the ATP-binding site. Site-directed mutagenesis of all candidate residues within this region showed that only the conservative Lys-802-to-Arg mutation abolished wortmannin binding. Inhibition of PI 3-kinase occurs, therefore, by the formation of an enamine following the attack of Lys-802 on the furan ring (at C-20) of wortmannin. The Lys-802-to-Arg mutant was also unable to bind FSBA and was catalytically inactive in lipid and protein kinase assays, indicating a crucial role for Lys-802 in the phosphotransfer reaction. In contrast, an Arg-916-to-Pro mutation abolished the catalytic activity whereas covalent wortmannin binding remained intact. Our results provide the basis for the design of novel and specific inhibitors of an enzyme family, including PI kinases and ATM-related genes, that play a central role in many physiological processes.     

Characterization of a helminthosporic acid analog that is a selective agonist of gibberellin receptor

Helminthosporol, a natural growth regulator isolated from a fungus, stimulates hypocotyl growth and seed germination, similar to gibberellin (GA). We recently reported that helminthosporic acid (H-acid), a synthetic analog of helminthosporol, acts as an agonist of GA receptor. In this study, we showed that a H-acid analog, in which the hydroxymethyl group at the C-8 position of H-acid was converted to a keto group, acts as a selective GA receptor agonist. 1) This analog shows higher hypocotyl elongation activity in Arabidopsis than H-acid does, and induces the degradation of DELLA protein and 2) leads to the formation of the GID1-DELLA complex and 3) regulates the expression of GA-related genes. In addition, 4) its hypocotyl elongation activity was not observed in a atgid1a single mutant, and 5) this analog could promote only the interaction between specific GA receptors and DELLA proteins in vitro. Taken together, our results strongly suggest that the selectivity of the reported H-acid analog depends on the specificity of its GA receptor binding activity.     

Double immunocytochemistry of vasoactive intestinal peptide and cGnRH-I in male quail: photoperiodic effects

Reproduction in Japanese quail is primarily regulated by photoperiod. Vasoactive intestinal peptide (VIP) has been suggested as a transducer of environmental information, especially photoperiodic cues, to the hypothalamo-pituitary-gonadal axis. To investigate the possible interaction of VIP and the reproductive (gonadotropin-releasing hormone, GnRH) system, double-immunocytochemical staining for VIP and cGnRH-I was conducted in sexually mature male quail held under a long-day photoperiod (16L:8D; LD) and in sexually quiescent males held under a short-day photoperiod (8L:16D; SD). VIP-immunoreactive (ir) cells were found primarily in three locations: lateral septal organ (LSO) in nucleus accumbens (Ac), ventral hypothalamus, and infundibular area. VIP-ir cells in LSO displayed characteristics typical of cerebrospinal fluid (CSF)-contacting cells, and co-existed with cGnRH-I-ir cells and beaded fibers. In contrast, VIP-ir cells in the infundibular area did not co-exist with cGnRH-I-ir structures. The number of visible VIP-ir cells in the infundibular area of SD males was significantly lower than that of LD males, while the number of visible VIP-ir cells in Ac/LSO was not altered by photoperiod. A cluster of cGnRH-I-ir cells in the caudalmost septal area was heavily innervated by VIP-ir fibers, which appeared to contact cGnRH-I-ir cells directly at this location. Both VIP- and cGnRH-I-ir fibers heavily innervated the external layer of the median eminence (ME). These data suggest that Ac/LSO, the caudalmost septal area, and ME are possible sites of interaction between the VIP and the GnRH systems.     

Efficient screening for catalytic antibodies using a short transition-state analog and detailed characterization of selected antibodies.

One of the major obstacles to acquiring catalytic antibodies is that it requires labor-intensive procedures to select catalytic antibodies from huge repertories of antibodies. Here, we selected potential catalytic Abs by utilizing their affinity towards a short transition-state analog which contained only the transition-state structural element, and evaluated in detail its efficiency to enrich catalytic Abs. Hybridoma supernatants elicited against a phosphonate derivative, the TSA1, were screened by a three-step screening process: step 1, ELISA for TSA1-BSA; step 2, ELISA for the short TSA4; and step 3, competitive-inhibition by the short TSA2. Only 22. 8% of positive mAbs from step 1 were found to be catalytic. The rate of catalytic Abs increased to 45.7% using screening steps 1 plus 2, and reached 83.3% using all three screening steps. This clearly suggests that our screening protocol is an efficient method to select potential catalytic Abs. Furthermore, we characterized the properties of both the catalytic Abs and the noncatalytic Abs in detail. The catalytic Abs tended to have lower Kd for TSA1 and the short TSA2 than noncatalytic Abs. It was also observed that catalytic Abs showed clear enantiospecificity toward substrate 6 containing d-phenylalanine while noncatalytic Abs did not. The detailed analysis of kinetic and binding parameters for these antibodies gives us further insight into catalytic antibodies.