Pancreatic hormones in the nonhibernating and hibernating golden mantled ground squirrel

The effects of hibernation on pancreatic insulin, glucagon, somatostatin, and pancreatic polypeptide was investigated in the golden mantled ground squirrel (Citellus lateralis). During hibernation, pancreatic organ weight decreased to 57% of the nonhibernating weight. The content of all four pancreatic hormones during hibernation was significantly reduced. The concentrations of insulin, pancreatic polypeptide and somatostatin, but not of glucagon, were significantly reduced during hibernation. The maintenance of the pancreatic glucagon concentration during hibernation may be related to its role in counter-regulation and carbohydrate homeostasis during fasting.     

Studies toward the biosynthesis of vasoactive intestinal peptide (VIP)

In view of the potential biological importance of VIP, we have begun to examine the regulation of its biosynthesis. For this purpose we have, as a first step, searched for an enriched source of VIP biosynthesis. By a combination of chromatographic procedures and radioimmunoassays we discovered an as yet unknown source for VIP production, namely a human buccal tumor, containing 0.67 +/- 0.05 ng VIP/micrograms protein which is greater than the richest source in brain (the cerebral cortex). Thus, we decided to use the tumor tissue for VIP-mRNA purification and characterization. To identify VIP-mRNA we are using as hybridization probes, synthetic oligodeoxynucleotides with relatively unambiguous nucleotide sequence complementary to the predicted VIP-mRNA sequence. These probes are synthesized, using the deoxynucleoside phosphoramidite approach, to a length of 17 bases each, and contain all the possible DNA sequences according to the genetic code. These specific probes are then radioactively labelled using the reaction catalyzed by the enzyme polynucleotide kinase and afterwards hybridized to mRNA, which had been resolved on denaturing agarose gels. Employing this approach, we identified a single putative VIP-mRNA band which was then partially purified by sucrose gradient centrifugation. Upon in vitro translation in a rabbit reticulocyte lysate cell free system, this mRNA was found to code for VIP immunoreactive proteins. In conclusion, our studies suggest the existence of high molecular weight precursors to VIP cross-reactive with anti-VIP antibodies, that are coded for by a partially purified mRNA containing VIP sequences.     

Is the vasoactive intestinal peptide (VIP) a prohormone?

The possibility that the vasoactive intestinal peptide (VIP) is a prohormone, which through enzymic fragmentation gives rise to shorter chains with, yet unknown, hormonal activities is suggested by the occurrence of two pairs of adjoining basic residues in its sequence. (A similar pattern can be recognized in proinsulin.) Synthesis of one of the hormone-candidates, -pyroglutamyl- -methionyl- -alanyl- -valyl- -lysyl- -lysyl- -tyrosyl- -leucyl- -asparaginyl- -seryl- - valyl- -leucyl- -threoninamide corresponding to the C-terminal 13-peptide sequences of chicken VIP is reported.     

Expression of vasoactive intestinal peptide (VIP) receptors in human uterus

We show the existence of functional vasoactive intestinal peptide (VIP) receptors in normal human female genital tract (endometrium, myometrium, ovary and Fallopian tube) as well as in leiomyoma (a frequent uterine pathology). The correlation between VIP binding and stimulation of adenylyl cyclase activity for all studied tissues was linear (r = 0.86) suggesting the expression of VIP receptors throughout the human female genital tract. Immunodetection of VIP receptor subtypes gave different molecular weights for VPAC(1) (47 kDa primarily) and VPAC(2) (65 kDa), which may be due to different glycosylation extents. In conclusion, this study demonstrates the expression of both subtypes of VIP receptors and their functionality in human female genital tract, suggesting that this neuropeptide could play an important physiological and pathophysiological role at this level.     

Characterization of vasoactive intestinal peptide (VIP) receptors in mammalian lung

125I-VIP bound specifically to sites on human, rat, guinea pig, and rabbit lung membranes with a dissociation constant (KD) of 60-200 pM and binding site maxima of 200-800 fmol/mg of protein. The presence of a second lower affinity site was detected but not investigated further. High affinity 125I-VIP binding was reversible and displaced by structurally related peptides with an order of potency: VIP greater than rGRF greater than PHI greater than hGRF greater than secretin = Ac Tyr1 D Phe2 GRF. 125I-VIP has been covalently incorporated into lung membranes using disuccinimidyl suberate. Sodium dodecyl sulfate-polyacrilamide gel electrophoresis of labeled human, rat, and rabbit lung membranes revealed major 125I-VIP-receptor complexes of: Mr = 65,000, 56,000, and 64,000 daltons, respectively. Guinea pig lung membranes exhibited two 125I-VIP-receptor complexes of Mr = 66,000 and 60,000 daltons. This labeling pattern probably reflects the presence of differentially glycosylated forms of the same receptor since treatment with neuroaminidase resulted in a single homogeneous band (Mr = 57,000 daltons). Soluble covalently labeled VIP receptors from guinea pig and human lung bound to and were specifically eluted from agarose-linked wheat germ agglutinin columns. Our studies indicate that mammalian lung VIP receptors are glycoproteins containing terminal sialic acid residues.     

Asbestos-related increase in pulmonary levels of vasoactive intestinal peptide (VIP)

Vasoactive intestinal peptide (VIP), leucine-enkephalin (Leu-Enk), dynorphin (Dyn), neurotensin (NT) and substance P (SP) were measured by radioimmunoassay in lung and bronchoalveolar lavage (BAL) fluids of sham operated control rats and rats exposed to asbestos (5 and 10 mg, single intratracheal injections) for 3 and 6 months. Among these peptides, VIP, Leu-Enk and Dyn were the most abundant with 6 to 25 pmoles per g of lung tissue as compared with 0.95 to 1.2 pmoles per g for the other neuropeptides. In the presence of asbestos, VIP levels were selectively increased up to 2.7 times in lung tissue and 4.3 times in BAL fluids. On high pressure liquid chromatography (HPLC), the immunoreactive VIP coeluted with synthetic VIP. It is concluded that this selective increase may be involved in the pathogenesis of asbestos-related diseases. Exposure to asbestos causes chronic inflammatory reactions in the lung which may lead to fibrosis (1) and increase the incidence of pleuropulmonary cancers (2). Little is known concerning the biochemical changes responsible for the deleterious effects of asbestos on pulmonary functions. Previous studies have documented the vast complexity and diversity of lung biochemistry including its ability to metabolize lipids, inactivate certain enzymes and produce physiologically active amines (3-6). Recently, the lung has been recognized as an important source of peptidergic substances. VIP and SP were reported to be localized in nerve terminals of the main airways and in axons of the parasympathetic conducts (7-11). Other neuropeptides including bombesin (12, 13), calcitonin (13, 14) and Leu-Enk (13) were also detected in the lung. However, these latter peptides were mainly confined to diffuse granule-containing cells also known as APUD cells (amine precursor uptake and decarboxylation cells) (15). The role of these neuropeptides in normal lung function and in pulmonary diseases is unknown. However, it has recently been demonstrated that APUD cells proliferate in the rat lung following asbestos inhalation (16) and lung exposure to carcinogens (17, 18). In addition, Moody et al. (19) and Sorenson et al. (20) have observed high levels of bombesin in human cell lines derived from small-cell lung carcinoma. It was then of particular interest to verify if lung exposure to asbestos can induce some changes in the levels of various neuropeptides. In the present study, we report that VIP is significantly increased in the lungs and BAL fluids of rats exposed to asbestos while no significant change in the levels of Leu-Enk, Dyn, NT and SP is observed.     

Interaction of vasoactive intestinal peptide (VIP) with rat lymphoid cells

Receptors for vasoactive intestinal peptide (VIP) have been characterized in rat lymphoid cells. The interaction of [125I] VIP with blood mononuclear cells was rapid, reversible, specific and saturable. At apparent equilibrium, the binding of [125I] VIP was competitively inhibited by native VIP in the 0.01-100 nM range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.050 +/- 0.009 nM and a low binding capacity (2.60 +/- 0.28 fmol/10(6) cells), and a low-affinity class with a Kd = 142 +/- 80 nM and a high binding capacity (1966 +/- 330 fmol/10(6) cells). Secretin, glucagon, insulin and somatostatin did not show any effect at a concentration as high as 100 nM. With spleen lymphoid cells, stoichiometric studies were performed. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.100 +/- 0.033 nM and a low binding capacity (4.60 +/- 1.07 fmol/10(6) cells), and low-affinity class with a Kd = 255 +/- 110 nM and high binding capacity (2915 +/- 1160 fmol/10(6) cells). With thymocytes, no binding was obtained under different conditions.     

Synthesis of vasoactive intestinal peptide (VIP) via the mixed anhydride method

Porcine VIP was synthesized from three segments. The segments, VIP(1-6), VIP(7-13), and VIP(14-28), were synthesized via the Repetitive Excess Mixed Anhydride (REMA) method. The low solubility of the C-terminal segment was greatly improved by a temporary substitution of Asn28 by a beta-t-butyl aspartic acid ester. The segments VIP(1-6) and VIP(7-13) were purified by HPLC and coupled via the mixed anhydride method. The product was purified by gel filtration. VIP was synthesized from VIP(1-13) and VIP(14-28) by the same procedure. After deprotection, Met17-sulfoxide reduction, and purification by ion-exchange chromatography, the product was found to have the expected amino acid composition and biological potency. A HPLC purified sample was compared with several commercial preparations of varying purity.     

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.     

Opiate modulation of the stress-induced increase of vasoactive intestinal peptide (VIP) in plasma

Vasoactive intestinal peptide (VIP) has a variety of extra-intestinal actions which are typical of the body's reaction to stress, such as lipolysis, glycogenolysis and modulation of anterior pituitary hormone secretion. Serial VIP plasma concentrations in patients undergoing major laparotomies were determined. The influence of the mu-receptor agonist, fentanyl, on intra-operative changes was investigated and compared to a control group receiving halothane anesthesia. Plasma levels of typical "stress hormones" cortisol and catecholamines were also monitored for additional information on the extent of perioperative stress. VIP levels increased intraoperatively in the halothane group from 5.9 +/- 4.6 to 15.3 +/- 5.3 pmol/l. Cortisol and catecholamine levels showed a similar increase. The intraoperative VIP increase in the fentanyl group was significantly smaller: 3.5 +/- 1.9 to 7.3 +/- 3.6 pmol/l. Anesthesia itself did not affect VIP concentrations as shown by constant levels during a 30 minute preoperative control period. The observed increases of VIP plasma concentration are thought to reflect a possible role for VIP in the hormonal metabolic response to stress. The attenuation of the increase by fentanyl might be due to a direct opiate action on VIP release.     

Immunohistochemical localization of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat

Summary The indirect peroxidase-antiperoxidase immunohistochemical technique was used to investigate the possible presence of vasoactive intestinal peptide (VIP) in the circumventricular organs of the rat. Considerable numbers of VIP-immunoreactive fibers were seen in the pineal gland. A moderate amount of VIP-immunoreactive fibers was present in the median eminence, the posterior lobe of the pituitary and the area postrema, but only few fibers were found in the organum vasculosum laminae terminalis. No immunoreactivity was observed in the subfornical organ or the subcommissural organ. The circumventricular organs investigated were completely free of VIP-immunoreactive perikarya. In the circumventricular organs, VIP-immunoreactive fibers were visible between the parenchymal cells and in the perivascular spaces. The presence of coarse VIP-immunoreactive terminals in apposition to the portal vessels in the external layer of the median eminence indicates that VIP may be secreted directly into the pituitary portal circulation, thus influencing the anterior pituitary cells. The presence of large VIP-immunoreactive boutons in the posterior lobe of the pituitary suggests a secretion of VIP directly into the systemic circulation. In the pineal gland, a dense innervation by VIP-immunoreactive fibers was found in the peripheral superficial part of organ, with fibers penetrating into its central portion where they mainly terminate near in vicinity of the capillaries. In the area postrema, VIP-immunoreactive material was mainly found at the ventral border of the organ. In addition to the secretion of VIP into the bloodstream via the circumventricular organs, this study provides evidence that VIP exerts specific influence on the cellular elements of these organs.     

Cosecretion of peptide histidine methionine (PHM) and vasoactive intestinal peptide (VIP) in patients with VIP-producing tumors

Regional specific antibodies and chromatography were used to analyze the concentration and molecular forms of vasoactive intestinal peptide (VIP) and peptide histidine methionine (PHM) in plasma from 39 patients with VIP-producing tumors. Plasma VIP concentrations ranged from 29 to 2550 pmol/l and the corresponding PHM immunoreactive values measured with C-terminally directed antibody were 42 to 2100 pmol/l which correlated closely with the VIP concentrations. N-terminal PHM concentrations were significantly higher than the C-terminal values ranging from 92 to 5850 pmol/l and correlated poorly with the corresponding VIP concentrations. Infusion experiments with PHM disclosed that the higher levels of N-terminal immunoreactivity could not be explained by slower metabolic clearance or by degradation to smaller N-terminal immunoreactive forms. N-terminally directed PHM antibody revealed, in addition to intact PHM, a larger immunoreactive form in patient plasma which constituted the major proportion of the total immunoreactivity. In conclusion, VIP and PHM are cosecreted from VIPomas and measurement of PHM, especially N-terminal immunoreactivity, may be useful in this condition.     

Effect of gastroduodenostomy on intestinal vasoactive intestinal peptide (VIP) levels, and VIP binding and VIP stimulation of cyclic AMP in intestinal epithelial cells from rat

The concentration of VIP in duodenum and jejunum as well as the interaction of VIP (binding and stimulation of cyclic AMP accumulation) with epithelial cells from both gut segments were studied in rats after surgical bypass of the pylorus by gastroduodenostomy. Duodenal VIP concentration increased in rats with gastroduodenostomy as compared to sham-operated animals. The binding capacity (but not the affinity) of VIP binding sites and the efficiency (but not the potency) of VIP on cyclic AMP accumulation decreased in the condition of gastroduodenostomy. However, no modifications in either VIP concentration and interaction could be seen at the jejunal level.     

Distribution and dilatory effect of vasoactive intestinal polypeptide (VIP) in human cerebral arteries

Vasoactive intestinal polypeptide (VIP)-containing nerve fibers were demonstrated in human pial arteries by immunocytochemistry. Fine varicose fibers were located in the adventitia close to the media layer. Measurements by radioimmunoassay revealed concentrations of VIP between 0.7 and 2.7 pmol/g in the major arteries at the base of the brain, obtained at autopsy. Isolated human pial arteries, obtained in conjunction with neurosurgery, relaxed in a concentration-dependent manner upon administration of VIP. The relaxation of the vessels amounted to 57 +/- 9% of the contraction elicited by prostaglandin F2 alpha (2.5 microM) with an EC50 value of (8.5 +/- 1.2) X 10(-9) M.     

Action of vasoactive intestinal peptide (VIP) on amphibian adrenocortical function,in vitro

Vasoactive intestinal peptide (VIP) is located in chromaffin cells of the frog adrenal gland and is able to stimulate corticosteroid secretion in amphibians. In the present study we have investigated the possible involvement of prostaglandins, microfilaments and calcium in the mechanism of action of VIP on frog adrenocortical tissue. Rana ridibunda interrenal dice were perifused with amphibian culture medium for more than 10 hours. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. In the presence of indomethacin (5 μM), a specific blocker of prostaglandin biosynthesis, the spontaneous secretion of corticosteroids was markedly reduced (80%) but the stimulatory effect of VIP was not altered. The administration of the microfilament disrupting agent cytochalasin B (50 μM) inhibited both spontaneous and VIP-induced corticosteroid secretion. In the absence of calcium, the spontaneous level of corticosteroid was reduced to about 60% but VIP was still able to stimulate corticosteroid secretion. From these data we conclude that the integrity of the cytoskeleton is required for the secretory response of adrenocortical cells to VIP, whereas neither prostaglandins nor calcium are involved in VIP-induced adrenocortical stimulation.