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The VPAC1receptor for vasoactive intestinal peptide (VIP) belongs to the class II family of G protein-coupled receptors and is coupled to Gs protein/adenylyl cyclase. We assessed whether 10 different Ser/Thr residues in human VPAC1receptor intracellular domains play a role in the process of VIP-induced desensitization/internalization by performing a site-directed mutagenesis study. The Ser/Thr residues mutated to Ala include potential G protein-coupled receptor kinase, protein kinase A and protein kinase C targets that are of particular interest for VPAC1receptor desensitization. The data show that when Chinese hamster ovary cells expressing wild-type receptors were pretreated for 5 min with VIP (50 nM), receptor desensitization occurred with a 10-fold right shift of the ED50for adenylyl cyclase activation. When the construct with the widest span of mutations was studied, there was no longer any short-term desensitization. By using constructs with fewer and fewer mutations, we identified Ser447 in the C-terminal tail to be crucial for rapid desensitization. We also showed that Ser447 plays an essential role for VIP-induced VPAC1phosphorylation in Chinese hamster ovary cells. Furthermore, we demonstrated that none of the mutated Ser/Thr residues was involved in down-regulation after a 12-h treatment of cells with 50 nM VIP. Neither were they involved in VIP and VIP-induced receptor internalization as shown using a novel fluorescein-tagged VIP and VPAC1receptor bearing a Flag epitope in the N-terminal domain and a green fluorescent protein at the C terminus. We conclude that Ser447, a likely G protein-coupled receptor kinase target, is crucial for VIP-induced phosphorylation and rapid desensitization of VPAC1receptor.

The VPAC1 receptor for vasoactive intestinal peptide (VIP) belongs to the class II family of G protein-coupled receptors and is coupled to Gs protein/adenylyl cyclase. We assessed whether 10 different Ser/Thr residues in human VPAC1 receptor intracellular domains play a role in the process of VIP-induced desensitization/internalization by performing a site-directed mutagenesis study. The Ser/Thr residues mutated to Ala include potential G protein-coupled receptor kinase, protein kinase A and protein kinase C targets that are of particular interest for VPAC1 receptor desensitization. The data show that when Chinese hamster ovary cells expressing wild-type receptors were pretreated for 5 min with VIP (50 nM), receptor desensitization occurred with a 10-fold right shift of the ED50 for adenylyl cyclase activation. When the construct with the widest span of mutations was studied, there was no longer any short-term desensitization. By using constructs with fewer and fewer mutations, we identified Ser447 in the C-terminal tail to be crucial for rapid desensitization. We also showed that Ser447 plays an essential role for VIP-induced VPAC1 phosphorylation in Chinese hamster ovary cells. Furthermore, we demonstrated that none of the mutated Ser/Thr residues was involved in down-regulation after a 12-h treatment of cells with 50 nM VIP. Neither were they involved in VIP and VIP-induced receptor internalization as shown using a novel fluorescein-tagged VIP and VPAC1 receptor bearing a Flag epitope in the N-terminal domain and a green fluorescent protein at the C terminus. We conclude that Ser447, a likely G protein-coupled receptor kinase target, is crucial for VIP-induced phosphorylation and rapid desensitization of VPAC1 receptor.

Central to the pathophysiology of sickle cell disease are the vaso-occlusive events that lead to tissue damages and life-threatening complications. Lungs are particularly vulnerable to vaso-occlusion because of their specific vasculature. We developed a mouse model of hypoxia/reoxygenation lung injury closely mimicking the lung pathology of patients with sickle cell disease. This model involves the exposure of transgenic sickle cell (SAD) mice to hypoxia (8% oxygen) for 4, 10, and 46 hours followed by 2 hours of reoxygenation. Gene expression profiling of SAD lung tissue pointed to the specific induction of genes involved in the response to ischemic stress and microcirculation remodeling: Hspcb, Hsp86-1, Nfe2l2, Ace, and Fgf7. Hypoxia/reoxygenation also induced a marked increase in bronchoalveolar (BAL) total leukocyte and neutrophil counts, BAL total protein content, and BAL tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-1α, and macrophage inflammatory protein 2 (MIP-2) levels, all indicators of enhanced inflammatory response as compared with control mice. Nitric oxide (NO) was administered to SAD mice. NO (40 ppm) inhalation protected SAD mice from the histopathologic lesions of ischemic/reperfusion lung injury with corresponding normalization and/or modulation of tissue gene expression profiles. Inhaled NO (1) significantly reduced the increase in BAL total protein content, BAL total leukocyte, and neutrophil counts; (2) modulated BAL cytokine network; and (3) did not affect hemoglobin and methemoglobin levels. The present study provides evidences for the beneficial effects of inhaled NO in pulmonary injury induced by hypoxia/reoxygenation in a mouse model of sickle cell disease (SCD) and opens new avenues in drug design based on tissue gene expression profiling.

The human VPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) belongs to the class II family of G protein coupled receptors with seven transmembrane segments. It recognizes several VIP-related peptides and displays a very low affinity for secretin despite >70% homology between VIP and secretin. Conversely, the human secretin receptor has high affinity for secretin but low affinity for VIP. We took advantage of this reversed selectivity to identify a domain of the VPAC1 receptor responsible for selectivity toward secretin by constructing human VPAC1-secretin receptor chimeras. A first set of chimeras consisted of exchanging the entire N-terminal ectodomain or large parts of this domain. They were constructed by overlap PCR, transfected in COS-7 cells, and their ligand selectivity, expressed as the ratio of EC50for secretin/EC50for VIP (referred to as S/V), in stimulating cAMP production was measured. Two very informative chimeras respectively referred to as S144V and S123V were obtained by replacing the entire ectodomain or only the first 123 amino acids of the VPAC1 receptor by the corresponding sequences of the secretin receptor. Whereas S144V no longer discriminated between VIP and secretin (S/V = 1.2), S123V discriminated between the two peptides (S/V = 300) in the same manner as the wild-type VPAC1 receptor. The motif responsible for discrimination was determined by introducing small blocks or individual amino acids of secretin receptor in the 123–144 sequence of the S123V chimera. The data obtained from 14 new chimeras sustained that two nonadjacent pairs of amino acids, Gln135Thr136and Gly140Ser141in the C-terminal end of the N-terminal VPAC1 receptor ectodomain constitute a selective filter that strongly restricts access of secretin to the VPAC1 receptor.

High doses of recombinant human erythropoietin (rhEpo) are required for the treatment of chronic anemia. Thus, it is clear that therapy for chronic anemia would greatly benefit from an erythropoietin derivative with increased erythropoietic activity rather than the native endogenous hormone. In this report, the activity of a human Epo-Epo dimer protein, obtained by recombinant technology, is described and compared with its Epo monomer counterpart produced under identical conditions. Although monomer Epo and dimer Epo-Epo had similar pharmacokinetics in normal mice, the increase in hematocrit value was greater with the dimer than with the monomer. Moreover, in clonogenic assays using CD34+human hematopoietic cells, the human dimer induced a 3- to 4-fold-greater proliferation of erythroid cells than the monomer. Controlled secretion of dimeric erythropoietin was achieved in β-thalassemic mice by in vivo intramuscular electrotransfer of a mouse Epo-Epo plasmid containing the tetO element and of a plasmid encoding the tetracycline controlled transactivator tTA. Administration of tetracycline completely inhibited the expression of the mEpo dimer. On tetracycline withdrawal, expression of the Epo-Epo dimer resumed, thereby resulting in a large and sustained hematocrit increase in β-thalassemic mice. No immunologic response against the dimer was apparent in mice because the duration of the hematocrit increase was similar to that observed with the monomeric form of mouse erythropoietin.

The human VPAC1receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide belongs to the class II family of G-protein-coupled receptors with seven transmembrane segments. Like for all class II receptors, the extracellular N-terminal domain of the human VPAC1receptor plays a predominant role in peptide ligand recognition. To determine the three-dimensional structure of this N-terminal domain (residues 1–144), the Protein Data Bank (PDB) was screened for a homologous protein. A subdomain of yeast lipase B was found to have 27% sequence identity and 50% sequence homology with the N-terminal domain (8) of the VPAC1receptor together with a good alignment of the hydrophobic clusters. A model of the N-terminal domain of VPAC1receptor was thus constructed by homology. It indicated the presence of a putative signal sequence in the N-terminal extremity. Moreover, residues (Glu36, Trp67, Asp68, Trp73, and Gly109) which were shown to be crucial for VIP binding are gathered around a groove that is essentially negatively charged. New putatively important residues for VIP binding were suggested from the model analysis. Site-directed mutagenesis and stable transfection of mutants in CHO cells indicated that Pro74, Pro87, Phe90, and Trp110are indeed important for VIP binding and activation of adenylyl cyclase activation. Combination of molecular modeling and directed mutagenesis provided the first partial three-dimensional structure of a VIP-binding domain, constituted of an electronegative groove with an outspanning tryptophan shell at one end, in the N-terminal extracellular region of the human VPAC1receptor.

The widespread neuropeptide vasoactive intestinal peptide (VIP) has two receptors VPAC1and VPAC2. Solid-phase syntheses of VIP analogs in which each amino acid has been changed to alanine (Ala scan) or glycine was achieved and each analog was tested for: (i) three-dimensional structure by ab initiomolecular modeling; (ii) ability to inhibit 125I-VIP binding (Ki) and to stimulate adenylyl cyclase activity (EC50) in membranes from cell clones stably expressing human recombinant VPAC1or VPAC2receptor. The data show that substituting residues at 14 positions out of 28 in VIP resulted in a >10-fold increase of Kior EC50at the VPAC1receptor. Modeling of the three-dimensional structure of native VIP (central α-helice from Val5to Asn24with random coiled N and C terminus) and analogs shows that substitutions of His1, Val5, Arg14, Lys15, Lys21, Leu23, and Ile26decreased biological activity without altering the predicted structure, supporting that those residues directly interact with VPAC1receptor. The interaction of the analogs with human VPAC2receptor is similar to that observed with VPAC1receptor, with three remarkable exceptions: substitution of Thr11and Asn28by alanine increased Kifor binding to VPAC2receptor; substitution of Tyr22by alanine increased EC50for stimulating adenylyl cyclase activity through interaction with the VPAC2receptor. By combining 3 mutations at positions 11, 22, and 28, we developed the [Ala11,22,28]VIP analog which constitutes the first highly selective (>1,000-fold) human VPAC1receptor agonist derived from VIP ever described.

The basic organization of the human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor (VPAC) 1 promoter was investigated after cloning the 5ʹ-flanking region (1.4 kb) of the VPAC1 gene from a human genomic library. Subsequent functional analysis of various deletions of the 5ʹ-flanking sequence, subcloned upstream of a luciferase reporter gene, was carried out in HT-29 cells. The minimal promoter region identified encompasses the -205/+76 sequence and contains a crucial CCAAT box (-182/-178) and a GC-rich sequence. Moreover a region (-1348/-933) containing a silencer element was identified. We previously showed that the expression of the VPAC1 receptor binding site is strictly dependent upon the enterocytic differentiation of human colon cancer Caco-2 cells [Laburthe, Rousset, Rouyer-Fessard, Couvineau, Chantret, Chevalier and Zweibaum (1987) J. Biol. Chem. 262, 10180-10184]. In the present study we show that VPAC1 mRNA increases dramatically when Caco-2Cl.20 cells differentiate, as measured by RNase protection assays and reverse transcriptase-PCR. A single transcript species of 3 kb is detected in differentiated cells by Northern-blot analysis. Accumulation of VPAC1 receptor mRNA is due to a 5-fold increase of transcription rate (run-on assay) without a change in mRNA half-life (9 h). Stable transfections of various constructs in Caco-2Cl.20 cells and subsequent analysis of reporter gene expression, during the enterocytic differentiation process over 25 days of culture, further indicated that the -254/+76 5ʹ-flanking sequence is endowed with the regulatory element(s) necessary for transcriptional regulation of VPAC1 during differentiation. Altogether, these observations provide the first characterization of the basic organization of the human VPAC1 gene promoter and unravel the crucial role of a short promoter sequence in the strict transcriptional control of VPAC1 expression during differentiation of human colon cancer Caco-2 cells.

We have examined the effect of hydroxyurea (HU), clotrimazole (CLT), magnesium oxide (Mg), and combined CLT+Mg therapies on the erythrocyte characteristics and their response to chronic hypoxia in a transgenic sickle mouse (SAD) model. SAD mice were treated for 21 days with 1 of the following regimens (administered by gavage): control (n = 6), HU (200 mg/d; n = 6), CLT (80 mg/kg/d, n = 5), Mg (1,000 mg/kg/d, n = 5), and CLT+Mg (80 and 1,000 mg/kg/d, respectively, n = 6). Nine normal mice were also treated as controls (n = 3), HU (n = 3), and CLT+Mg (n = 3). Treatment with HU induced a significant increase in mean corpuscular volume and cell K content and a decrease in density in SAD mice. Treatment with the CLT and Mg, either alone or in combination, also increased cell K and reduced density in SAD mice. After 21 days of treatment, the animals were exposed to hypoxia (48 hours at 8% O2) maintaining the same treatment. In the SAD mice, hypoxia induced significant cell dehydration. These hypoxia-induced changes were blunted in either HU- or Mg-treated SAD mice and were completely abolished by either CLT or CLT+Mg treatment, suggesting a major role for the Gardos channel in hypoxia-induced dehydration in vivo.

The expression of subunits of the guanine nucleotide regulatory protein that mediates hormonal stimulation of adenylyl cyclase (Gs) and of the guanine nucleotide regulatory protein that mediates hormonal inhibition of adenylyl cyclase (Gi) was studied during cell migration and differentiation in the rat small intestine crypt-villus axis. Proliferative crypt cells were separated from nonproliferative mature villus cells and the following data were obtained: 1) alpha s subunits were more abundant in crypt cells than in villus cells as evidenced by cholera toxin-catalyzed [32P]NAD ribosylation and Western blotting of this relative molecular weight (M(r)) 42,000 protein; 2) alpha i2- and alpha i3-subunits (M(r) 40,000 and M(r) 41,000, respectively) were preferentially expressed in villus cells as evidenced by pertussis toxin-catalyzed [32P]NAD ribosylation and Western blotting (alpha i1-subunit was not detectable in intestinal epithelium by using these techniques); 3) Western blotting showed a higher expression of the common beta- (M(r) 36,000) subunit of G proteins in villus cells than in crypt cells; and 4) Northern blot analysis using an alpha s-subunit oligonucleotide probe showed a 1.9-kb mRNA that was more abundant in crypt cells than in villus cells. In contrast, alpha i2- and alpha i3-mRNA species (2.3 and 3.5 kb, respectively), analyzed by using specific cDNA probes, were much more abundant in villus cells than in crypt cells. Finally, two beta-subunit mRNA species of 3.3 and 1.8 kb were detectable in intestinal epithelial cells and were more abundant in villus cells than in crypt cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Gila monster venom (1–300 μg/ml) is shown to inhibit completely the binding of [ 125I]VIP to human and rat intestinal epithelial cell membranes. In both models, the venom inhibits [ 125I]VIP binding and stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP and a potency that is 10 000–50 000 times lower than that of the peptide, on a weight basis. At maximal doses, VIP and Gila monster venom do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the venom occurs through VIP receptors. As is the case for VIP, adenylate cyclase activation by Gila monster venom requires the presence of GTP in the incubation medium. Finally, no VIP-like immunoreactivity was detected in the venom using an antiserum raised against mammalian VIP. All these data suggest the presence in the venom of the Gila monster, of a new substance which behaves as a VIP agonist in human as well as rat intestine.

Homozygous β thalassemic mice received 50 U (1,660 U/kg) of recombinant human erythropoietin (rhEpo) 5 days a week for 2 weeks. Hemoglobin increased from 9.2 ± 0.6 g/dL to 10.5 ± 0.4 g/dL (P = .002) and hematocrit increased from 29.2% ± 0.9% to 34.1% ± 1.9% (P = .0014). The β minor/α globin chain synthesis ratio increased slightly but significantly between day -4 (0.75 ± 0.07) and day 4 (0.81 ± 0.04) (P = .01) and reached a minimum ratio (0.67 ± 0.03) on day 15 (P = .001), being parallel to reticulocyte counts and to the incorporated trichloracetic acid (TCA)-insoluble radioactivity, therefore parallel to the erythropoietic output in thalassemic mice, as in normal mice. Erythrocyte defects were improved in β thalassemic mice treated by rhEpo: membrane-associated α globin was significantly decreased (P < .01), thiol group reactivity of ankyrin was significantly improved (P < .05), spectrin alterations were reduced, and deformabil-ity of mouse thalassemic red blood cells was normalized. These results provide experimental criteria for modulating globin chain imbalance necessary for the therapy of human β thalassemia intermedia, and suggest that rhEpo might be of interest to improve the red blood cell mass and reduce erythrocyte alterations in this disease. © 1991 by The American Society of Hematology.

The 5'-flanking region (1.5 kb) of the gene coding for the human VIP1PACAP receptor was isolated, sequenced, and characterized. Transient expression of constructs containing sequentially deleted 5'-flanking sequences of the VIP1PACAP receptor fused to a luciferase reporter gene showed that this sequence was active as a promoter in the intestinal cancer cell line, HT-29, expressing endogenous VIP1PACAP receptor. The shortest DNA fragment with significant promoter activity encompassed the region from ?205 to 76 bp. Deletion of a CCAAT-box sequence in the construction corresponding to ?173 to 76 bp dramatically reduced the promoter activity. The promoter ?205 to 76 bp has a housekeeping gene structure without TATA-box. It contains GC-rich regions characterized by potential Sp1 and AP2 sites and some potential regulatory elements, such as CRE and ATF, and a CCAAT-box sequence (?182 to ?178) crucial for gene transcription.

In order to determine species specificity in growth hormone-releasing factor (GRF) interaction with vasoactive intestinal polypeptide (VIP) receptors, we have tested rat (r) GRF (with a His1 such as in VIP), human (h) GRF and position 1 substituted analogs of hGRF (Ala1, Ac-Tyr1, His1, Phe1, and Trp1 in the place of Tyr1) for their ability to inhibit 125I-VIP binding and to stimulate adenylate cyclase activity in human and rat intestinal epithelial membranes. We show that rGRF has a much higher affinity than hGRF for both human and rat VIP receptors. In humans, the Ki values for inhibiting 125I-VIP binding are 0.5 (VIP), 26 (rGRF), and 830 nM (hGRF). In rats the values are 0.6 (VIP), 46 (rGRF), and 1100 nM (hGRF). This is due in part to the presence of His1 in rGRF since the analog His1 hGRF has a higher affinity than hGRF in man and rat, i.e., Ki = 320 nM and 460 nM, respectively. Studies of adenylate cyclase stimulation reveal that rGRF and His1 hGRF are full VIP agonists in man and rat, whereas hGRF and its other analogs behave as partial agonists in both species. One of the hGRF analogs tested (Ac-Tyr1hGRF) is of great interest since it inhibits 125I-VIP binding to rat intestinal membranes with a Ki = 430 nM but has a negligible intrinsic activity in stimulating adenylate cyclase activity (about 6% of the efficacy of VIP). This analog does inhibit the VIP-stimulated adenylate cyclase activity in a dose-dependent manner, complete inhibition of the VIP (0.01-1 nM) effect being obtained with 30 microM analog. The Schild plot of the inhibitory effect further indicates competitive antagonism. In contrast, Ac-Tyr1hGRF is a partial VIP agonist in humans (about 20% of the efficacy of VIP). These results evidence the important role of His1 for peptide interaction with VIP receptors and provide the first example of a competitive VIP antagonist.

A human leukemic cell line KU 812 was recently established and described as a basophilic cell line. In the present study we show that KU 812 and two of its clones are at least bipotent: in addition to a minor component of basophils, the majority of KU 812 cells belongs to the erythroid cell lineage with a significant percentage (about 15%) of mature hemoglobinized erythroblasts. This terminal differentiation is associated with the synchronized synthesis of the main erythroid proteins, including glycophorins, spectrin beta chain, band 3, and hemoglobin. The predominant hemoglobins are adult, fetal, and Bart's hemoglobin. Adult hemoglobin represented up to 75% of all hemoglobins in the KU 812 F clone in passages containing a high number of mature erythroblasts. Transcripts of all human globin chains were present with ten times less embryonic chain messenger RNA (mRNA) than alpha-, beta- or gamma-chain mRNA. Hemin slightly increased the total hemoglobin production of the cell line, especially gamma-globin chain synthesis, but did not modify the percentage of hemoglobinized cells. Phorbol myristate acetate (PMA) had a complex effect, inducing a proportion of KU 812 cells to adhere to the plastic culture flask. The adherent cell fraction expressed a very low level of specific erythroid proteins, but their ultrastructure was consistent with immature erythroid cells. In contrast, approximately 40% of the nonadherent cells were mature erythroid cells. Cell-sorting experiments showed that this paradoxic effect of PMA is mostly due to cell selection, the more mature cells being unable to adhere. In addition, KU 812 F was found to be sensitive to erythropoietin, which slightly increased its plating efficiency range (from 0% to 50%) in semisolid medium and enhanced hemoglobin accumulation twofold. In binding experiments using 125I erythropoietin, a single class of high-affinity Epo receptors (Kd: 250 pM) was detected by binding with a density of 205 receptors per cell. The KU 812 cell line is therefore a unique model for studying cell commitment toward different hematopoietic lineages and erythroid differentiation.

Bone marrow cells from hematologically normal subjects were obtained by aspiration in 19 cases and by surgery in two cases; erythroid colonies were subsequently cultured from these cells in plasma clots. The hemoglobins synthesized in culture were studied either by incubation of the cells at the 6th-7th day of culture, or at the 13th-l4th day of culture with 3H-leucine, in order to determine whether the reactivation of fetal hemoglobin (Hb F) in vitro was related to the stage of differentiation of the erythroid precursors. Hemoglobins synthesized in culture were specifically purified from other proteins by affinity chromatography on Sepharose-haptoglobin and were analyzed by separation of globin chain on carboxy-methyl-cellulose in urea. In 14-day-old cultures, reactivation of Hb F synthesis was observed in all cases but one (mean value 8.2%, range 2.3% to 17.7%). In the 7-day-old cultures, a lower proportion of Hb F synthesis was observed (mean value 4.2%, range 1.6% to 7.6%).These results indicate that the reactivation of Hb F synthesis is related to the stage of differentiation of the erythroid precursors.

Vasoactive intestinal peptide (VIP)1 receptors in rats and humans recognize peptide histidine isoleucineamide (PHI) with high and low affinity, respectively. We took advantage of this phenotypic difference to identify the domain responsible for the selective recognition of PHI by rat and human receptors which display >80% sequence identity. After transfection of human and rat receptors in COS cells, the ratio of IC50 for PHI/IC50 for VIP (referred to as P/V) in inhibiting 125I-VIP binding was shown to be >1,000 and <40, respectively. Construction of eight rat/human receptor chimerae by overlap polymerase chain reaction and determination of their P/V ratios demonstrated that the critical domain for PHI recognition is present within a sequence comprising part of the first extracellular loop and third transmembrane domain. This domain contains three different amino acids numbered according to human and rat sequences, respectively, e.g. Gln207 (human) versus His208 (rat), Gly211 versus Ala212 and Met219 versus Val220. Site-directed mutagenesis introducing individual, double, or triple mutations in a chimeric construct revealed that all three amino acids were involved in the recognition of PHI. Triple mutations were then introduced in the wild-type receptors i.e. Q207H, G211A, M219V human VIP1 receptor and H208Q, A212G, V220M rat VIP1 receptor, resulting in a complete change in their phenotype from human to rat and from rat to human, respectively. The results demonstrate that three nonadjacent amino acids are responsible for the selective recognition of PHI by human and rat VIP1 receptors.

The intestine is a large endocrine organ, but the dependence of colon cancer on hormones remains unknown. We show here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCT116 and Cl.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells, which do not express NTR1. In SW480 cells, neurotensin is active in the 10^–10 to 10^–6 M concentration range (ED50 = 0.47 nM) while the neurotensin fragment (1–11) is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692, which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54μmol/kg every 24 hr) by osmotic pumps to nude mice that have been xenografted with SW480 cells results in a significant increase of tumor volume, i.e., up to 255% of control at day 20 of treatment. SR 48692 administered alone (1.7 μmol/kg every 24 hr) by daily i.p. injections reduces the development of tumors formed by xenografting SW480 cells in nude mice. A significant mean reduction of tumor volume of 38% is observed during the 22-day period of treatment. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. Our results support the notion that colon cancer growth may be dependent on blood-borne neurotensin and suggest that non-peptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies of colon cancer. Int. J. Cancer 80:448–454, 1999. © 1999 Wiley-Liss, Inc.

The human vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide receptor1 (VPAC1) belongs to the class II subfamily of G protein-coupled receptors. Specific changes by mutagenesis of a strictly conserved threonine (H) into lysine (K), proline (P) or alanine (A) at position 343 of the human VPAC1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of these mutants into Cos cells evoked a 3.5 fold-increase in the cAMP level as compared to cells transfected with the wild-type receptor. In contrast other mutants such as T343C, T343E or T343F were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized cells. Double mutants were then constructed in which the T343K mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production i.e. E36A or D68A. The corresponding double mutants T343K-E36A and T343K-D68A were no longer constitutively activated. A control double mutant (T343K-D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP, was still constitutively activated. Our findings demonstrate that constitutive activation of the VPAC1 receptor can be evoked by specific mutations of T343 at the junction of the second intracellular loop and fourth transmembrane segment. This constitutive activation appears to require the functional integrity of the N-terminal extracellular VIP binding domain.

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP-stimulated cAMP production in intact cells and VIP-stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride-stimulated adenylate cyclase activity in HT29 cells homogenates was not affected byPMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.

The purpose of this work was to study vasoactive intestinal peptide (VIP) receptors and the adenylate cyclase response to VIP upon enterocytic differentiation of the human colon adenocarcinoma cell line Caco-2 in culture. The VIP-stimulated enzyme activity is very low, e.g. 20% above basal activity in undifferentiated cells (day 5) and is enhanced markedly at confluency reaching a maximum, e.g. 270%, above basal activity in fully differentiated cells (day 30). VIP potency is also slightly enhanced, the EC50 of VIP ranging from 0.31 nM at day 5 to 0.07 nM at day 30. Modifications of the adenylate cyclase system are not responsible for the development of VIP response. Indeed, forskolin-stimulated adenylate cyclase activity is unchanged during differentiation supporting no alteration of the enzyme catalytic subunit. The same holds true for NaF and guanosine 5'-(beta, gamma-imido)trisphosphate, indicating a constant activity of the guanine nucleotide regulatory unit which mediates hormonal stimulation of adenylate cyclase (Ns). This is further supported by the similar extent of cholera toxin-catalyzed [32P]ADP-ribosylation of the Ns protein that is observed during differentiation. In sharp contrast, a dramatic increase of VIP receptor concentration is observed ranging from 32 fmol/mg of protein at day 5 to 414 fmol/mg of protein at day 30. This is confirmed by affinity cross-linking experiments showing an increased specific incorporation of 125I-VIP in a major 66,000-dalton component during differentiation. A slight increase in receptor affinity is also observed during differentiation with Kd ranging from 0.39 nM at day 5 to 0.08 nM at day 30. These data indicate that one population of VIP receptors accumulates during Caco-2 cell differentiation, representing the crucial event in the development of adenylate cyclase response to the peptide.

The presence of receptors for insulinlike growth factor I and II in human colonic epithelium is demonstrated. Scatchard analysis of binding data obtained with 125I-insulinlike growth factor I showed an insulinlike growth factor I receptor with a dissociation constant of 8.6 nM and a binding capacity of 0.8 pmol/mg membrane protein. Distinct insulinlike growth factor II receptors labeled with 125I-insulinlike growth factor II were also found with a dissociation constant of 6.9 nM and a binding capacity of 4.7 pmol/mg membrane protein. Two sets of observations make it possible to discriminate between the two types of insulinlike growth factor receptors. (a) Unlabeled insulinlike growth factor I was 3 times more potent than insulinlike growth factor II in inhibiting [125I] insulinlike growth factor I binding. Conversely, unlabeled insulinlike growth factor II was 10 times more potent than insulinlike growth factor I in competing with 125I-insulinlike growth factor II for binding to membranes. Insulin and proinsulin did not compete with either of the tracers. (b) Affinity labeling of membranes followed by sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions, revealed a radioligand-receptor complex of molecular weight 130,000 and 250,000 using 125I-insulinlike growth factor I and 125I-insulinlike growth factor II, respectively. These observations indicate that adult human colonic epithelium is abundantly equipped with two sets of receptors that recognize preferentially either insulinlike growth factor I or insulinlike growth factor II.

The human vasoactive intestinal peptide (VIP) 1 receptor belongs to the new class II subfamily of G protein-coupled receptors. Specific change by mutagenesis of a strictly conserved histidine into arginine at position 178 of the human VIP1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of the H178R mutant into COS cells resulted in a 3.5-fold increase in the cAMP level as compared with cells transfected with the wild type receptor or the vector alone. This increase was proportional to the amount of transfected cDNA. The H178R mutant exhibited an otherwise normal cAMP response to VIP as well as a dissociation constant similar to that of the wild type receptor. Other mutants at position 178 such as H178K, H178A, and H178D were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized cells. Double mutants were then constructed in which the H178R mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production, i.e. E36A or D68A. The corresponding double mutants H178R/E36A and H178R/D68A were no longer constitutively activated. A control double mutant (H178R/D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP was still constitutively activated. Our findings demonstrate that constitutive activation of the VIP1 receptor by mutation of His178 into R requires the functional integrity of the N-terminal extracellular VIP binding domain. They might provide interesting generalities about the activation process of G protein-coupled receptors.

GRF (10 −8–10 −5M) is shown to inhibit competitively the binding of [ 125I]VIP to human and rat intestinal epithelial membranes. The affinity of GRF for VIP receptor is 700–800-times lower than that of VIP in both species. The order of affinity of different peptides is VIP > PHI > secretin > GRF in rat, and VIP > GRF > PHI > secretin in man. The important species specificity of VIP receptors in recognizing PHI and secretin does not occur in the case of GRF. GRF stimulates adenylate cyclase through its interaction with VIP receptors in rat and human membranes. However, while GRF behaves as a VIP agonist in human tissue, it is a partial agonist/antagonist of VIP in the rat.

The soluble pool of α hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, β A hemoglobin chain labeled with [3H]N-ethylmaleimide. This pool of soluble α chains was 0.067 ± 0.017% of hemoglobin in blood of normal adult, 0.11 ± 0.03% in heterozygous β thalassemia and ranged from 0.26 to 1.30% in homozygous β thalassemia intermedia. This elevated pool of soluble α chains observed in human β thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble α chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In β thalassemia intermedia the amount of insoluble α chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [3H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.

Alpha-2 adrenergic receptivity of rat jejunum epithelial cells was studied using the new antagonist radioligand, [3H]RX821002 [( 3H]-2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline). All the parameters of [3H]RX821002 binding were consistent with the labeling of an alpha-2 adrenoceptor. The use of this probe was moreover extremely convenient, because contrarily to [3H]yohimbine and [3H]rauwolscine, [3H]RX821002 displayed in this tissue a very high affinity (Kd = 0.54 +/- 0.12 nM) and a low level of nonspecific binding (5% at 1 nM [3H]RX821002). Competition studies with various antagonists and agonists showed that the labeled sites were alpha-2-selective and stereospecific. Oxymetazoline was much more potent than chlorpromazine or prazosin suggesting that the receptor is of the alpha-2-subtype. Yohimbine and rauwolscine were equipotent, which is also in agreement with the pharmacological definition of this subtype. These two compounds displayed, however, a rather weak affinity (Ki approximately 40 nM), which is somewhat different with what one should expect for a true alpha-2A adrenoceptor. Altogether the competition data indicated that the alpha-2 adrenoceptor from rat jejunal epithelium is neither an alpha-2A, nor an alpha-2B, nor an alpha-2c adrenoceptor and may belong to a fourth subtype.(ABSTRACT TRUNCATED AT 250 WORDS)