125I-VIP Binding Research Articles

Caveolae-dependent internalization and homologous desensitization of VIP/PACAP receptor, VPAC2, in gastrointestinal smooth muscle

The main membrane proteins of caveolae (caveolin-1, -2 and -3) oligomerize within lipid rich domains to form regular invaginations of smooth muscle plasma membrane and participate in receptor internalization and desensitization independent of clathrin-coated vesicle endocytosis. We have previously shown that Gs-coupled VIP/PACAP receptors, VPAC2, predominantly expressed in smooth muscle cells of the gut, are exclusively phosphorylated by GRK2 leading to receptor internalization and desensitization. Herein, we characterized the role of caveolin-1 in VPAC2 receptor internalization and desensitization in gastric smooth muscle using three approaches: (i) methyl β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae in dispersed muscle cells, (ii) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and (iii) caveolin-1 knockout mice (caveolin-1−/−). Pretreatment of gastric muscle cells with VIP stimulated tyrosine phosphorylation of caveolin-1, and induced VPAC2 receptor internalization (measured as decrease in 125I-VIP binding after pretreatment) and desensitization (measured as decrease in VIP-induced cAMP formation after pretreatment). Caveolin-1 phosphorylation, and VPAC2 receptor internalization and desensitization were blocked by disruption of caveolae with MβCD, suppression of caveolin-1 with caveolin-1 siRNA or inhibition of Src kinase activity by PP2. Pretreatment with VIP significantly inhibited adenylyl cyclase activity and muscle relaxation in response to subsequent addition of VIP in freshly dispersed muscle cells and in muscle strips isolated from wild type and caveolin-1−/− mice; however, the inhibition was significantly attenuated in caveolin-1−/− mice. These results suggest that caveolin-1 plays an important role in VPAC2 receptor internalization and desensitization.

Vasoactive intestinal peptide–camptothecin conjugates inhibit the proliferation of breast cancer cells

The effects of vasoactive intestinal peptide–camptothecin (VIP–CPT) conjugates were investigated on breast cancer cells and cells transfected with VIP receptors (R). (Ala 2,8,9,19,24.25.27, Nle 17, Lys 28)VIP, (A-NL-K)VIP, was synthesized and Lys 28 was coupled to a linker, N-methyl-amino-ethyl-glycine, L2, which formed a carbamate bond with CPT. The resulting (A-NL-K)VIP-L2-CPT was cytotoxic for MCF7 breast cancer cells, which have VPAC 1-R, with IC 50 values of 380 and 90 nM using the MTT and clonogenic assays, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of 125I-VIP to 3T3 cells transfected with VPAC 1-R with IC 50 values of 1.9, 56 and 126 nM, respectively. In contrast, (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT inhibited specific binding of 125I-Ro25-1553 to 3T3 cells transfected with VPAC 2-R with IC 50 values of 3.9, 3162 and 2690 nM, respectively. (A-NL-K)VIP, (A-NL-K)VIP-L2 and (A-NL-K)VIP-L2-CPT caused increased cAMP after addition to MCF7 cells. 125I-(A-NL-K)VIP-L2-CPT was internalized by MCF7 cells at 37 °C but not 4 °C. These results indicate that (A-NL-K)VIP-L2-CPT is a VPAC 1-R agonist which is cytotoxic for breast cancer cells.

Radiolabeling and in vitro and in vivo Characterization of [18F]FB‐[R8,15,21, L17]‐VIP as a PET Imaging Agent for Tumor Overexpressed VIP Receptors

In an effort to develop a peptide-based radiopharmaceutical for the detection of tumors overexpressed vasoactive intestinal peptide receptors with positron emission tomography, we have prepared a novel [R(8,15,21), L17]-VIP peptide for 18F-labeling. This peptide inhibited 125I-VIP binding to rats lung membranes with high affinity [half-maximal inhibitory concentrations (IC50) of 0.12 nm]. Additionally, [R(8,15,21), L17]-VIP showed higher stability than native vasoactive intestinal peptide in vivo of mice. With N-succinimidyl 4-[18F] fluorobenzoate as labeling prosthetic group, [18F]FB-[R(8,15,21), L17]-VIP was obtained in >99% radiochemical purity within 100 min in decay-for-corrected radiochemical yield of 33.6 +/- 3% (n = 5) and a specific radioactivity 255 GBq/micromol at the end of synthesis. Stability of [18F]FB-[R(8,15,21), L17]-VIP in vitro and in vivo were investigated. Biodistribution of this trace was carried out in mice with induced C26 colorectal tumor. Fast clearance of [18F]FB-[R(8,15,21), L17]-VIP from non-target tissues and specific uptakes by tumors realized higher tumor-to-muscle ratio (3.55) and tumor-to-blood ratio (2.37) 60 min postinjection. Clear difference was observed between the blocking and unblocking experiments in biodistribution and whole body radioautography. [18F]FB-[R(8,15,21), L17]-VIP has demonstrated its potential for diagnosing tumors overexpressed vasoactive intestinal peptide receptors both in vitro and in vivo.

Neuropeptide binding reflects convergent and divergent evolution in species-typical group sizes

Neuroendocrine factors that produce species differences in aggregation behavior (“sociality”) are largely unknown, although relevant studies should yield important insights into mechanisms of affiliation and social evolution. We here focused on five species in the avian family Estrildidae that differ selectively in their species-typical group sizes (all species are monogamous and occupy similar habitats). These include two highly gregarious species that independently evolved coloniality; two territorial species that independently evolved territoriality; and an intermediate, modestly gregarious species that is a sympatric congener of one of the territorial species. Using males and females of each species, we examined binding sites for 125I-vasoactive intestinal polypeptide (VIP), 125I-sauvagine (SG; a ligand for corticotropin releasing factor, CRF, receptors) and a linear 125I-V1a vasopressin antagonist (to localize receptors for vasotocin, VT). VIP, CRF and VT are neuropeptides that influence stress, anxiety and/or various social behaviors. For numerous areas (particularly within the septal complex), binding densities in the territorial species differed significantly from binding in the more gregarious species, and in most of these cases, binding densities for the moderately gregarious species were either comparable to the two colonial species or were intermediate to the territorial and colonial species. Such patterns were observed for 125I-VIP binding in the medial bed nucleus of the stria terminalis, medial septum, septohippocampal septum, and subpallial zones of the lateral septum; for 125I-SG binding in the infundibular hypothalamus, and lateral and medial divisions of the ventromedial hypothalamus; and for the linear 125I-V1a antagonist in the medial septum, and the pallial and subpallial zones of the caudal lateral septum. With the exception of 125I-SG binding in the infundibular hypothalamus, binding densitites are positively related to sociality.

Receptors for VIP and PACAP in Guinea Pig Cerebral Cortex: Effects on Cyclic AMP Synthesis and Characterization by <SUP>125</SUP>I-VIP Binding

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of 125I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of 125I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a B(max) of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of 125I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) > or = PACAP38 approximately PACAP27 approximately guinea pig VIP (gpVIP) > or = mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of 125I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific 125I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [3H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 microM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 microM, produced net increases in cAMP production in the range of 8-9% conversion, whereas 5 microM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC receptors.

Presence of a N-terminal signal peptide in class II G protein-coupled receptors: crucial role for expression of the human VPAC1 receptor

The hVPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) has an N-terminal signal peptide like all other class II G protein-coupled receptors (GPCRs). We determined the role of the signal peptide in expression of human VPAC1 receptor in transfected CHO cells. Three constructs were transfected: Flag30-hVPAC1, a receptor containing an inserted FLAG sequence between Ala30 and Ala31 and fused in the C-terminal position to GFP; Flag30-[delta1–30]-hVPAC1, the same construct as Flag30-hVPAC1 but lacking the 1–30 putative signal peptide (SP) sequence; Flag0-hVPAC1, a receptor containing an N-terminal FLAG sequence and fused in the C-terminal position to GFP. For each construct, we determined 125I-VIP binding, VIP-induced cAMP production, GFP fluorescence and indirect immunofluorescence on nonpermeabilized cells incubated with mouse monoclonal anti-Flag antibodies. The data were consistent with a crucial role of the signal peptide for expression of functional VPAC1 receptors at the cell surface and suggested that the signal peptide is cleaved during the translocation of the receptor to the plasma membrane, probably in the endoplasmic reticulum.

The development of VIP–ellipticine conjugates

The mechanism by which vasoactive intestinal peptide (VIP)–ellipticine (E) conjugates are cytotoxic for human lung cancer cells was investigated. VIP–alanyl-leucyl-alanyl-leucyl-alanine (ALALA)–E and VIP–leucyl-alanyl-leucyl-alanine (LALA)–E inhibited 125I-VIP binding to NCI-H1299 cells with an IC50 values of 0.5 and 0.1 μM, respectively. VIP–ALALA–E and VIP–LALA–E caused elevation of cAMP in NCI-H1299 cells with ED50 values of 0.7 and 0.1 μM. Radiolabeled VIP–LALA–E was internalized at 37 °C and delivered the cytotoxic E into NCI-H1299 cells. VIP–LALA–E inhibited the growth of NCI-H1299 cells in vitro. Three days after the addition of VIP–LALA–E to NCI-H1299 cells, cell viability decreased based on trypan blue exclusion and reduced 3H-thymidine uptake. These results suggest that VIP–E conjugates are internalized in lung cancer cells as a result of VPAC1 receptor-mediated endocytosis.

VIP receptor antagonists inhibit mammary carcinogenesis in C3(1)SV40T antigen mice

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist on mammary carcinogenesis were investigated using the C3(1)SV40T antigen (ag) mice. Ten μg/day VIPhybrid (VIPhyb) administered daily subcutaneously increased significantly the survival of C3(1)SV40Tag mice. At 5.2 months, VIPhyb significantly reduced the mammary tumor burden in C3(1)SV40Tag mice relative to control animals. 125I-VIP bound with high affinity to mouse mammary tumor homogenate. Because (Lys 15, Arg 16, Leu 27)VIP 1–7GRF 8–27 (VPAC 1 selective) but not Ro25-1553 (VPAC 2 selective) inhibited specific 125I-VIP binding to mammary tumor membranes with high affinity, VPAC 1 receptors predominate. By RT-PCR, VPAC 1 receptor mRNA was detected in mammary tumors. By Western blot, a major 60 Kdalton band was detected in mammary tumor extracts using VPAC 1 receptor antisera. By immunocytochemistry, VPAC 1-R immunostaining was detected in the cytosol and plasma membrane but not the nucleus of fixed mammary tumor tissue. Using laser capture microdissected tumor cells and surface enhanced laser desorption/ionization (SELDI) techniques on mammary tumor cells, the proteomic profile was altered in mice treated with VIPhyb. Because VPAC 1 receptor antagonists increase the survival and reduce the tumor burden in C3(1)SV40Tag mice, they may function as chemopreventive agents in mammary cancer.

VIP-ellipticine derivatives inhibit the growth of breast cancer cells

The effects of vasoactive intestinal peptide (VIP)-ellipticine (E) derivatives were investigated on breast cancer cells. VIP-ALALA-E and VIP-LALA-E inhibited 125I-VIP binding to MCF-7 cells with an IC 50 values of 1 and 0.2 μM respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in MCF-7 cells with ED 50 values of 1 and 0.1 μM. VIP-LALA-E caused increased c-fos mRNA in MCF-7 cells. Radiolabeled VIP-LALA-E was internalized at 37 °C and delivered the cytotoxic E into MCF-7 cells. VIP-LALA-E inhibited the clonal growth of MCF-7 cells, decreased cell viability based on trypan blue exclusion and reduced 35S-methionine uptake. These results indicate that VIP-E derivatives function as breast cancer VPAC 1 receptor agonists which inhibit MCF-7 cellular viability.

(N-stearyl, Norleucine<sup>17</sup>)VIPhybrid is a Broad Spectrum Vasoactive Intestinal Peptide Receptor Antagonist

The effects of a (N-stearyl, Norleucine17) vasoactive intestinal peptide hybrid ((SN)VIPhybrid) on cells stably transfected with VPAC,, VPAC2, or PAC1 receptors were investigated. (SN)VIPhybrid inhibited specific 125I-VIP binding to membranes derived from CHO cells transfected with VPAC, or VPAC2 receptors with high affinity (IC50 = 30 and 50 nM). (SN)VIPhyb inhibited specific 125I-PACAP-27 binding to membranes derived from NIH/3T3 cells transfected with PAC1 receptors with high affinity (IC50 = 65 nM). PACAP-27 caused cAMP elevation in NIH/3T3 cells transfected with PAC1 receptors and the increase cAMP caused by pituitary adenylated cyclase (PACAP) was inhibited by (SN)VIPhyb. Also, the increase in cAMP caused by VIP using CHO cells transfected with VPAC1 or VPAC2 receptors was antagonized by (SN)VIPhyb. These results indicate that (SN)VIPhyb is an antagonist for VPAC1, VPAC2, and PAC1 receptors.

VIP receptor antagonists and chemotherapeutic drugs inhibit the growth of breast cancer cells.

The effects of vasoactive intestinal peptide (VIP) antagonists on breast cancer cells were investigated. (N-stearyl, norleucine17)VIP hybrid ((SN)VIPhyb) inhibited specific 125I-VIP binding to MCF7, SKBR3, T47D ZR75-1 and MDA-MB231 cells with high affinity (IC50 values of 0.03-0.06 microM). (SN)VIPhyb, 1 microM, inhibited the ability of 10 nM VIP to cause elevation of cAMP and to increase c-fos mRNA. Micromolar concentrations of (SN)VIPhyb inhibited the proliferation of MDA-MB231 or MCF7 cells using a MTT and clonogenic assay. Using a MTT assay, (SN)VIPhyb enhanced the ability of taxol and doxorubicin to inhibit breast cancer growth. Using nude mice bearing MDA-MB231 xenografts, VIPhyb potentiated the ability of taxol to inhibit proliferation. The results indicate that VIP receptor antagonists increase the ability of chemotherapeutic drugs to kill breast cancer cells.

VPAC1 receptors and lung cancer.

VIP/PACAP are autocrine growth factors for lung cancer. VIP and/or PACAP mRNA is present in most lung cancer cell lines examined. Although mRNA for VPAC2-R is not common, VPAC1-R and PAC1-R mRNA is present in many lung cancer cell lines. 125I-VIP binds with high affinity to lung cancer cells and specific 125I-VIP binding is inhibited with high affinity by (Lys15, Arg16, Leu27)VIP1-7 GRF8-27, the VPAC1-R specific agonist, but not by Ro25-1553(18), the VPAC2-R specific agonist. VIP elevates cAMP and increases c-fos gene expression. The increase in cAMP and c-fos mRNA caused by VIP is inhibited by SN(VH). (SH)VH inhibited the proliferation of NCIH1299 cells in the MTT assay, which is based on cytotoxicity. In a recent cell line screen, (SN)VH inhibited the growth of 51 of 56 cancer cell lines including leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, breast cancer, and prostate cancer (T. Moody, unpublished). It remains to be determined if (SN)VH will be useful for treatment of a wide variety of cancers.

Retinoic acid down-regulates VPAC 1 receptors and TGF-β3 but up-regulates TGF-β2 in lung cancer cells

The effects of retinoic acid (RA) on lung cancer cells were investigated. Both all-trans (t-RA) and 13-cis RA (c-RA) decreased specific 125I-VIP binding to NCI-H1299 cells in a time- and concentration-dependent manner. After 20 hr, 30 μM t-RA decreased specific 125I-VIP binding by 60%. By Scatchard analysis, the density of VIP binding sites but not the affinity was reduced by 42%. NCI-H1299 VPAC 1 receptor mRNA was reduced by 48%. VIP caused a 3-fold elevation in the NCI-H1299 cAMP, and the increase in cAMP caused by VIP was reduced by 38% if the NCI-H1299 cells were treated with t-RA. Using the MTT assay, 3 μM t-RA and 3 μM c-RA inhibited NCI-H1299 proliferation by 60 and 23% respectively. Also, transforming growth factor (TGF)-β2 increased after treatment of NCI-H1299 cells with t-RA whereas TGF-β1 mRNA was unaffected and TGF-β3 mRNA was decreased. These results suggest that RA may inhibit lung cancer growth by down-regulating VPAC 1 receptor and TGF-β3 mRNA but up-regulating TGF-β2 mRNA.

Tryptophan 67 in the Human VPAC1 Receptor: Crucial Role for VIP Binding

The human receptor subtype for VIP and PACAP, referred to as VPAC1 receptor, has a large N-terminal extracellular domain which is critical for VIP binding. We further investigated this domain by mutating 12 amino acid residues which could participate in the formation of a tight bend (W67) or a coiled coil motif. They were changed to alanine (A) and the cDNAs were transiently transfected into Cos cells. All mutants but W67A exhibited Kd values similar to that of the wild-type receptor. For the W67A mutant, no specific 125I-VIP binding could be observed. Mutants at the W67 site were further characterized after stable transfection of epitope-tagged VPAC1 receptor–GFP fusion proteins into CHO cells. W67A, W67E, W67H, and W67K mutants neither bound VIP nor mediated adenylyl cyclase activation by VIP. The W67F mutant mediated stimulation of adenylyl cyclase only at high VIP concentrations. Microscopic analysis and antibody binding experiments showed that all mutants were similarly expressed at the cell surface of CHO cells. Therefore tryptophan 67 in the human VPAC1 receptor plays a crucial role in VIP binding due, in part, to its aromatic moiety.

VIP Activates Gs and Gi3 in Rat Alveolar Macrophages and Gs in HEK293 Cells Transfected with the Human VPAC1 Receptor

We have characterized vasoactive intestinal peptide (VIP) receptor/G-protein coupling in rat alveolar macrophage (AM) membranes and find that pertussis toxin treatment and antisera against Gαi3 and Gαs reduce high-affinity 125I-VIP binding, indicating that both Gαs and Gαi3 couple to the VIP-receptor. The predominant VIP-receptor subtype in AM is VPAC1 and we examined the G-protein interactions of the human VPAC1 that had been transfected into HEK293 cells. VPAC1 has a molecular mass of 56 kDa; GTP analogs reduced 125I-VIP binding to this protein demonstrating that high-affinity binding of VIP to the receptor requires coupling to G-protein. Functional VIP/VPAC1/G-protein complexes were captured by covalent cross-linking and analyzed by Western blotting. The transfected human VPAC1 receptor in HEK293 was found to be coupled to Gαs but not Gαi or Gαq. Furthermore, pertussis toxin treatment had no effect on VPAC1/G-protein coupling in these cells. These observations suggest that the G-proteins activated by VPAC1 may be dependent upon species and cell type.

(N)-Stearyl, Norleucine 17) VIP hybrid inhibits the growth of pancreatic cancer cell lines

The effects vasoactive intestinal peptide (VIP) antagonists were investigated on pancreatic cancer cell lines. (N-Stearyl, Norleucine 17) VIP hybrid ((SN)VIPhyb) inhibited 125I-VIP binding to human Capan-2 cells with an IC 50 value of 0.01 μM whereas VIPhybrid had an IC 50 value of 0.2 μM. By RT-PCR and Northern blot, VPAC 1 receptor mRNA was detected in CAPAN-2 cells. One μM (SN)VIPhyb and 10 μM VIPhyb inhibited the ability of 30 μM VIP to elevate cyclic AMP and increase c-fos mRNA. (SN)VIPhyb, 1 μM inhibited the clonal growth of CAPAN-2 cells in vitro. In vivo, (SN)VIPhyb (10 μg/day s.c.) inhibited CAPAN-2 xenograft growth in nude mice. These results indicate that (SN)VIPhyb is a pancreatic cancer VPAC receptor antagonist.

Vasoactive intestinal peptide (VIP) and VIP receptors: gene expression and growth modulation in medulloblastoma and other central primitive neuroectodermal tumors of childhood.

Vasoactive intestinal peptide (VIP) is a neuromodulator and growth regulator in the developing nervous system. We analyzed 10 primitive neuroectodermal tumor (PNET) cell lines, 29 central PNET (cPNET) and 17 tumors of the Ewing's sarcoma/peripheral PNET family (ESFT) using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern hybridization. Each of the 10 cell lines and 86.2% of cPNET expressed mRNA for VIP receptor 1 (VIPR1) compared to 52.9% of ESFT. VIPR2 was expressed in 75.8% of cPNET, in 28.6% of ESFT and in all 10 cell lines. cPNET demonstrated high-affinity binding of 125I-VIP on quantitative autoradiography and in competitive binding assays. VIP inhibited tumor cell proliferation in a dose-dependent manner in 5 of 7 PNET cell lines. We conclude that VIPR1 and VIPR2 are highly expressed in cPNET and demonstrate that VIP is a growth modulator in these tumors.

(Arg 15, Arg 21) VIP: Evaluation of Biological Activity and Localization to Breast Cancer Tumors

Moody, T. W., J. Leyton, E. Unsworth, C. John, L. Lang and W. C. Eckelman. (Arg 15, Arg 21) VIP: Evaluation of biological activity and localization to breast cancer tumors. Peptides 19(3) 585–592, 1998.—VIP analogs, which contain a single lysine amino acid, were synthesized and evaluated using breast cancer cells. (Arg 15, Arg 20) VIP, (Arg 15, Arg 21) VIP, and (Arg 20, Arg 21) VIP inhibited 125I-VIP binding to T47D cells with high affinity (IC 50 values of 1.2, 1.0, and 0.8 nM, respectively). The VIP analogs elevated cAMP in T47D cells with ED 50 values ranging from 0.1–1 nM. Because (Arg 15, Arg 21) VIP was the most potent at elevating cAMP, it was characterized further. (Arg 15, Arg 21) VIP transiently increased c-fos gene expression in breast cancer cells. N-Succinimidyl-4- 18F (fluoromethly) benzoate was prepared in one chemical step from N-succinimidyl-4-(4-nitrobenzenesulfonyl)oxomethyl)benzoate by adding 18F in acetone at room temperature. This prosthetic group was then reacted with (Arg 15, Arg 21) VIP ((RR) VIP). ( 18F-RR) VIP bound with high affinity to T47D cells and was rapidly internalized. ( 18F-RR) VIP was injected intravenously into nude mice bearing breast cancer xenografts and after 4 h, the density of ( 18F-RR) VIP was elevated in the tumors relative to normal organs. These data suggest that VIP receptors may be used to localize breast cancer tumors.

Pituitary adenylyl cyclase-activating polypeptides and vasoactive intestinal peptide inhibit bone resorption by isolated rabbit osteoclasts.

Nerve fibres present in the periosteum, cortical bone and bone marrow are proposed to be involved in the regulation of bone metabolism by the release of neuropeptides acting locally on bone cells. The present study describes the effects of vasoactive intestinal polypeptide (VIP), shown to be present in the vicinity of bone, and the two related neuropeptides pituitary adenylyl cyclase-activating polypeptide(1-38) (PACAP-38) and PACAP-27 on bone resorption in vitro induced by osteoclasts isolated from 10-day-old rabbits. Bone resorption was measured as the number and total area of pits formed by tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNCs) cultured for 3 days on devitalized slices of bovine cortical bone. All three neuropeptides had significant inhibitory effects on both the number and area of pits formed. At a high concentration (10(-7) M) the mean +/- S.E.M. reductions in the total area resorbed compared with controls were 70.3 +/- 8.2, 45.2 +/- 7.3 and 63.4 +/- 7.2% for PACAP-38, PACAP-27 and VIP, respectively. The corresponding values for the apparent dissociation constants were 0.93 +/- 0.30, 3.2 +/- 1.6 and 0.35 +/- 0.14 nM, respectively. The number of TRAP + MNCs was unaffected by application of neuropeptides. Autoradiography showed the presence of 125I-VIP binding sites on some stromal cells, whereas osteoclasts had no binding sites for 125I-VIP. A high number of 125I-calcitonin binding sites was demonstrated on osteoclasts in the same preparation. The signal transduction pathway remains to be fully elucidated but seems to be partly dependent on changes in intracellular calcium concentrations, since a number of stromal cells responded to application of 10(-8) M PACAP-38 or VIP, and at least partly independent of cAMP accumulation. Trifluoperazine and mellitin, two selective calmodulin inhibitors, failed to block the VIP-induced inhibition of bone resorption. Our results demonstrate a non-toxic and probably stromal cell-derived effect of PACAP-38, PACAP-27 and VIP on isolated rabbit osteoclasts, resulting in a potent inhibition of bone resorption in vitro. The signal transduction pathway for inhibition induced by PACAP-38, PACAP-27 and VIP may be mediated partly by changes in intracellular Ca2+ in stromal cells.

(Stearyl, Norleucine 17) VIP hybrid antagonizes VIP receptors on non-small cell lung cancer cells

The effects of VIP receptor antagonists were investigated using non-small cell lung cancer (NSCLC) cells. By Northern blot and RT-PCR, VIP 1 receptors were detected on NSCLC cell line NCI-H1299. VIPhybrid,(N-Stearyl-Norleucine 17)VIPhybrid ((SN) VIPhybrid) and PTC4495 inhibited 125I-VIP binding to NCI-H1299 cells with IC 50 values of 500, 30 and 5000 nM respectively. (SN) VIPhybrid (1 μM) had no effect on basal cAMP but strongly inhibited the increase in cAMP caused by 10 nM VIP. The order of peptide potency to inhibit cAMP was (SN) VIPhybrid > VIPhybrid > PTC4495. (SN) VIPhybrid was more potent than VIPhybrid at inhibiting NCI-H1299 colony formation. Also, (SN) VIPhybrid was more potent than VIPhybrid at inhibiting NCI-H1299 xenograft formation in nude mice. These data suggest that (SN) VIPhybrid antagonizes VIP 1 receptors on NSCLC cells.