Synthesis of the vasoactive intestinal peptide (VIP) : IV. The sequence 14–28

A protected pentadecapeptide with the C-terminal sequence of the vasoactive intestinal peptide (VIP) was prepared by coupling the tetrapeptide derivative t-butyloxycarbonyl- -arginyl-N-benzyloxycarbonyl- -lysyl- -glutaminyl- -methionine azide to the partially deprotected hendecapeptide -alanyl- -valyl-N-benzyloxycarbonyl- -lysyl-N-benzyloxycarbonyl- -lysyl- -tyrosyl- -leucyl- - asparaginyl- -seryl- -isoleucyl- -leucyl- -asparaginamide. The preparation of the protected tetradecapeptide t-butyloxycarbonyl-N-benzyloxycarbonyl- -lysyl- -glutaminyl- -methionyl- -alanyl- -valyl-N- benzyloxycarbonyl- -lysyl-N-benzyloxycarbonyl- -lysyl- -tyrosyl- -leucyl- -asparaginyl- -seryl- -isoleucyl- -leucyl- -asparaginamide is also reported. The protecting groups were removed from samples of the tetradeca- and pentadecapeptides. The resulting free peptides showed, although at high dose levels, increase of visceral blood flow and reduction of blood pressure in the dog, and also relaxation of different smooth muscle preparations, which are the characteristic biological activities of VIP.     

Synthesis of the vasoactive intestinal peptide (VIP) : III. The sequence 7–13

The protected heptapeptide derivative t-butyloxycarbonyl- -threonyl-β-benzyl- -aspartyl- -asparaginyl-O-benzyl- -tyrosyl- -threonyl-nitro- -arginyl- - leucine methyl ester was prepared by stepwise chain lengthening. The protecting groups on the side chains of arginine, tyrosine, and aspartic acid residues were removed by hydrogenolysis and the partially deprotected heptapeptide ester converted to the hydrazide, an intermediate in the synthesis of the (porcine) vasoactive intestinal peptide (VIP).After the removal of the tert-butyloxycarbonyl group, the heptapeptide ester was exposed to the action of trypsin which split off its C-terminal residue, -leucine methyl ester. The hexapeptide was then exposed to chymotrypsin, which cleaved it into an acidic, and a basic fragment. The former was, under the conditions used, indistinguishable on paper chromatography and paper electrophoresis from the tetrapeptide threonyl-aspartyl-asparaginyl-tyrosine which had previously been isolated from natural VIP, of which it comprises the sequence 7–10. Similarly, the basic fragment was indistinguishable from threonyl-arginine, the sequence 11–12 of VIP. This intestinal peptide increases visceral blood flow and reduces blood pressure in the dog, and also causes relaxation of different smooth muscle preparations, e.g., the trachea of guinea pigs. The principal aim of the present synthesis is to provide independent evidence for the sequence of (porcine) VIP.     

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.     

Primary cultures of bovine chromaffin cells synthesize and secrete vasoactive intestinal polypeptide (VIP)

Dispersed cells of the bovine adrenal medulla express immunoreactive vasoactive intestinal polypeptide (VIP) after 24 hours in culture, although VIP could not be detected in extracts of bovine adrenal medulla or cortex. Immunoreactive VIP eluted from a reversed-phrase chromatography column with the same retention time as authentic porcine VIP_1–28. VIP in chromaffin cells in culture appears to be contained in a secretory granule pool, since it, like methionine-enkephalin (met-enk) was released into the medium after exposure of cells to nicotine, carbachol, veratridine and elevated potassium in a dose-dependent manner. Doseresponse curves for VIP and enkephalin release by the above secretagogues were similar but not identical. Enkephalins and VIP may either be contained in separate subpopulations of chromaffin cells or co-stored in the same cells.     

Vasoactive intestinal peptide (VIP) from chicken: Synthesis and properties of the C-terminal hendecapeptide

The hendecapeptide, Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Val-Leu-Thr-NH₂, corresponding to sequence 18–28 of chicken vasoactive intestinal peptide (VIP), was synthesized stepwise, starting with the C-terminal residue. The in situ technique was applied; o-nitrophenyl esters and p-nitrophenyl esters were used for acylation. The product was compared with, and found indistinguishable from, the C-terminal cyanogen bromide fragment of natural chicken-VIP. Some pharmacological properties of the hendecapeptide were also determined. In two separate experiments, the chain of the hendecapeptide was further lengthened to encompass residues 14–28 of chicken-VIP but with leucine and norleucine in place of methionine in position 17. The two pentadecapeptides showed biological activities comparable to those of the C-terminal pentadecapeptide fragment of porcine VIP or its 17-norleucine analog.     

Differential penetration of three anterior pituitary peptide hormones into the cerebrospinal fluid of rhesus monkeys

This paper demonstrates marked differences between blood levels and those in the CSF for three anterior pituitary peptide hormones, prolactin, luteinizing hormone (LH) and adrenocorticotrophin (ACTH) in the rhesus monkey. CSF levels of endogenous prolactin (measured by radioimmunoassay) are about 20% of those in the blood, and this proportion remains constant under conditions of persistent ('steady-state') hyperprolactinaemia (induced by injecting sulpiride). Acutely elevating prulactin, by either an intravenous injection of exogenous ovine prolactin, or sulpiride, resulted in similar rates of entry by prolactin into the CSF, suggesting that retrograde portal flow is not an important mechanism. LH, measured by bioassay, is also present in the CSF, but the CSF/blood ratio is 5-10 times less than for prolactin. Castration, causing blood LH levels to rise, resulted in equivalent changes in CSF, so that the ratio remains constant, though still much lower than for prolactin. There are significant correlations between individual animals in the blood and CSF content of prolactin and LH. In marked contrast, whilst ACTH is found (by cytochemical assay) in the CSF of both intact and adrenalectomized monkeys, no significant change in CSF levels occurs despite 10-fold changes in the plasma of adrenalectomized animals following withdrawal of cortisol. Nor is there any correlation between blood and CSF ACTH levels over a wide range of concentrations. These results show that each of the three peptides studied has a distinct pattern of entry into the CSF from the vascular compartment.     

Molecular conformation of erabutoxin b; atomic coordinates at 2.5 A resolution.

Atomic coordinates determined from a 2.5 Å electron density map are given for erabutoxin b, a sea snake venom postsynaptic neurotoxin. The principal structural features, anti-parallel β pleated sheet, β bulge and β bends are described. The erabutoxin b structure is discussed as structural prototype of this class of homologous curare-mimetic neurotoxins from both land and sea snakes.     

First observation of a beta-turn conformation fused with the oxy-analogue of an alpha-turn: the molecular structure of a model peptide of the C-terminal part of gramicidin A

The molecular structure of N-tert-butyloxycarbonyl-D-leucyl-L-phenylalanyl ethanolamide (t-Boc-D-Leu-L-Phe-EA), a protected analogue of the C-terminal dipeptide of the membrane-active linear antibiotic gramicidin A, has been determined by X-ray diffraction. One of the two independent molecules in the asymmetric unit is characterized by a chain reversal stabilized by an intramolecular, three-centre, double hydrogen bonding. It represents the first experimental evidence for a beta-turn conformation fused with the oxy-analogue of an alpha-turn.     

Isolation of poly(A)-containing RNA on synthetic fluorophlogopite (Mica).

Synthetic fluorophlogopite, an aluminosilicate of the same structure as naturally occurring mineral mica in which potassium ions on the basal surface have been replaced by aluminum ions, has the ability to retain polynucleotides irreversibly. This property of Al³⁺-mica was used for irreversible adsorption of poly(U) and subsequent selective adsorption of poly(A)-containing RNA from rabbit reticulocyte polysomes at high salt concentration and its elution by 50% dimethylsulfoxide. The properties of RNA isolated on poly(U)-Al³⁺-mica were studied by sucrose density gradient centrifugation and by stimulation of globin synthesis in an in vitro protein synthesizing system from wheat germ and from Krebs II-ascites cells. The preparation contained 9s RNA species which corresponds to rabbit globin messenger RNA, and under optimal conditions it stimulated protein synthesis more than 100-fold. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate showed that synthesized product was identical with rabbit globin.     

Effect of chromium(III) on poly(dG-dC) conformation

The interaction of chromium(III) with poly(dG-dC) inhibits the B to Z transition and results in the condensation of the polymer at high Cr/nucleotide ratios. At low Cr/nucleotide ratios chromium(III) enhanced the ability of ethanol to induce the B to Z transition of poly(dG-dC). The effects of chromium(III) on the conformation of DNA may be related to the carcinogenicity of chromium compounds.     

Hormonal regulation of key gluconeogenic enzymes and glucose release in cultured hepatocytes: effects of dexamethasone and gastrointestinal hormones on glucagon action

Hormonal regulation of key gluconeogenic enzymes and glucose release by glucagon, dexamethasone, secretin and somatostatin was evaluated in maintenance cultured rat hepatocytes. (i) Phosphoenolpyruvate (PEP)-carboxykinase activity declined rapidly during the first 24 h in serum- and hormone-free culture with a further slight decay during the following 2 days. Dexamethasone and glucagon independently increased PEP-carboxykinase and acted synergistically when added in combination. Glucose-6-phosphatase activity declining linearly during hormone-free culture was stimulated by glucagon. Dexamethasone itself was without significant effects but completely abolished glucagon action. Fructose-1,6-diphosphatase was maintained at its initial level during the first day under control conditions and declined thereafter. Neither glucagon nor dexamethasone affected total activity or substrate (fructose-1,6-diphosphate) affinity of this enzyme. In short-term experiments on cells cultured under control conditions, protein synthesis-dependent stimulation of PEP-carboxykinase by glucagon and the permissive action of dexamethasone was demonstrated. Glucose-6-phosphatase and fructose-1,6-diphosphatase were not altered by hormones within this period. (ii) Stimulation by glucagon of gluconeogenesis was independent of its action on PEP-carboxykinase. Dexamethasone inhibited glycogenolysis but maintained glucose release at control levels probably by stimulation of gluconeogenesis. When added in combination, the glycogen-preserving action of dexamethasone acutely reduced the glucose release in response to glucagon. Glucagon sensitivity remained unchanged. (iii) The gastrointestinal hormones secretin and somatostatin were ineffective in modulating basal or glucagon-stimulated glucose release and gluconeogenic key enzymes. They are therefore unlikely to play a physiological role in hepatic glucose metabolism.     

Calcium-induced changes in thyroglobulin conformation

Polyethylene glycol solutions (10% w/v) were used to detect the effect of mono- and divalent cations on some properties of thyroglobulin. It is shown that in presence of 10% w/v polyethylene glycol in 0.01 M Tris-HCl, pH 7.5, calcium (less than 0.05 M) modifies the solubility, the sedimentation rate, and the Stokes' radius of thyroglobulin, while monovalent cations up to 0.6 M do not effect any of these properties. These findings can be explained by an increase in molecular compactness of thyroglobulin. Furthermore, it was shown that a synthetic polymer, polyethylene glycol, could be used to detect conformational changes.     

Proteolytic enzymes as probes for the triple-helical conformation of procollagen

Type I procollagen was thermally denatured and partially refolded by cooling to 20°C. The partially refolded protein was then used as a model system for testing proteolytic enzymes as probes for quantitative assay of fully aligned triple-helical molecules. Pepsin and chymotrypsin both digested fully denatured procollagen. However, digestion times of greater than 60 min were required, even with a large molar excess of the proteinases. These enzymes therefore are only useful for examining the folding of procollagen under conditions in which the process occurs at a slow rate. In contrast, trypsin cleaved the collagen domain of denatured procollagen within 2 min. Trypsin did not efficiently remove the precursor specific peptides, and therefore a mixture of chymotrypsin and trypsin was employed as an appropriate proteolytic probe for triple-helical conformation.     

Chiroptical characterisation of polysaccharide secondary structures in the presence of interfering chromophores: Chain conformation of inter-junction sequences in calcium alginate gels

The changes in chain conformation which accompany Ca²⁺-induced gelation of alginate have been investigated by a combined circular dichroism (c.d.) and optical rotatory dispersion (o.r.d.) approach. C.d. changes in the carboxyl n→π^* spectral region, arising predominantly from formation of calcium poly-l-guluronate junctions, were monitored for three alginates of widely differing block composition. The corresponding o.r.d. changes, calculated by Kronig-Kramers trnasform, were subtracted from the observed changes in o.r.d. on gelation, to “unmask” the changes in optical activity of the conformation-sensitive electronic transitions of the polysaccharide backbone. Contributions to the “residual” o.r.d. difference spectra from poly-l-guluronate, poly-d-mannuronate, and heteropolymeric chain-sequences were calculated by solution of simultaneous equations at each wavelength. Results for poly-guluronate sequences are in agreement with previous studies of alginate films by vacuum ultraviolet c.d., and with observed c.d. and o.r.d. changes on addition of calcium ions to homopolyguluronate segments in solution. The much greater changes in backbone optical activity calculated for polymannuronate and heteropolymeric chain-sequences, however, have no counterpart in the behaviour of these sequences in isolation. An explanation is proposed in terms of stretching of interconnecting sequences between calcium polyguluronate junctions in alginate gels, to give a more-extended chain conformation than in free solution.     

Studies on the conformation of secretin: The position of the helical stretch

An analog of the C-terminal tricosapeptide of secretin, with aspartic acid replacing glutamic acid in position 9 and lysine substituted for arginine in position 21, was prepared. The synthesis was carried out in solution by stepwise chain lengthening with the application of the in situ technique. The ord-cd spectra of this new analog closely resemble the spectra of the tricosapeptide with the unaltered secretin sequence and of the analog in which only arginine-21 was replaced by lysine and of secretin itself. The incorporation of aspartic acid instead of glutamic acid-9 resulted in an N-terminal sequence that has a consïderably reduced probability of assuming a helical conformation. The observation that the helix content remained unchanged adds support to a model of secretin in which the helical stretch is near the C-terminus. The role of an acidic residue in position 9 is also discussed.     

The structure and conformation of HC-toxin

Difference nuclear magnetic resonance studies and amino acid oxidase experiments establish the sequence and configuration of amino acids in HC-toxin as cyclo(L-Aoe-D-Pro-L-Ala-D-Ala). HC-toxin adopts the bis-γ-turn conformation in solution previously found for the cytostatic cyclic tetrapeptide chlamydocin.     

Histamine and VIP interactions with receptor-cyclic AMP systems in the human gastric cancer cell line HGT-1

In HGT-1 cells incubated at 20 degrees C for 15 min with 1 mM 3-isobutyl-1-methylxanthine (IBMX), histamine (10(-4)M) increased basal cAMP levels from 2.12 +/- 0.14 to 22.9 +/- 2 pmol per 10(6) cells, with a potency of 6.4 X 10(-6)M. IBMX was added in order to inhibit cAMP degradation by low and high Km cAMP-phosphodiesterases (cAMP-PDE). The use of specific H1, H2 agonists or antagonists indicated that the histamine effect was due to an interaction with typical H2 -receptors that are involved in gastric acid secretion. Cyclic AMP levels were also increased (10-fold) by vasoactive intestinal peptide VIP (3 X 10(-11) - 10(-8)M). Porcine peptide having N-terminal histidine and C-terminal isoleucine amide (PHI) and secretin were respectively 80 and 3600 times less potent than VIP and did not produce additive effect when tested in combinations with VIP. This observation indicates that these two peptides, structurally related to VIP, are acting through the recognition sites for VIP. Combination of VIP and histamine results in additive stimulation on intact cells as well as on membrane-bound adenylate cyclase, suggesting the existence of two cell populations bearing respectively the two sets of receptors. Two other human cancer cell lines originating from nongastric tumors (HT-29 and HL-60) possess only VIP or histamine receptors, respectively, indicating the gastric cellular originality of the HGT-1 cells. It is concluded that HGT-1 cells possess both VIP and histamine H2 receptors with similar pharmacological properties to those characterized in normal human fundic glands (1,2). Therefore, this cell line can be a good model to study drugs used therapeutically during the treatment of patients for gastric ulcer or cancer.