Pituitary adenylate cyclase-activating peptide (PACAP), a new vasoactive intestinal peptide (VIP)-like peptide in the respiratory tract

Summary Pituitary adenylate cyclase-activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like peptide recently isolated from ovine hypothalami. Nerve fibers displaying PACAP immunoreactivity were found in the respiratory tract of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. A moderate supply of PACAP-immunoreactive fibers was seen in the nasal mucosa of guinea pigs. Few to moderate numbers of PACAP-containing fibers occurred in the tracheo-bronchial wall of rats, guinea pigs, ferrets, pigs, sheep and squirrel monkeys. The fibers were distributed beneath the epithelium, around blood vessels and seromucous glands, and among bundles of smooth muscle. In the lungs, the immunoreactive fibers were observed close to small bronchioli. A few PACAP-immunoreactive nerve cell bodies were seen in the sphenopalatine and otic ganglia of guinea pigs. Simultaneous double immunostaining of the respiratory tract of sheep and ferrets revealed that all PACAP-containing nerve fibers stored VIP. We suggest that neuronal PACAP may take part in the regulation of smooth muscle tone and glandular secretion.     

Homologous regulation of adenylate cyclase-coupled receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes

The effect of agonists on VIP receptor regulation has been investigated in mononuclear human blood leucocytes. VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP accumulation were measured after pretreatment with VIP, PHM-27 or secretin. Pretreatment for 30 min with 0.1 μM VIP caused 28% (S.E.M. = 15) reduction in specific binding, and 52% (S.E.M. = 12) reduction in cyclic AMP accumulation, while 3 h of pretreatment caused 59% (S.E.M. = 10) and 68% (S.E.M. = 12) reduction. Only VIP concentrations at the nanomolar level and higher were shown to have any effect. B_max of the high-affinity receptor was reduced by 66% (S.E.M. = 8) after 30 min, and 95% (S.E.M. = 3) after 3 h of exposure to 0.1 μM VIP. No significant change was observed in receptor affinity, in B_max of the low-affinity receptor, in ED₅₀, or in ED₁₀₀ of VIP-stimulated cyclic AMP accumulation. Pretreatment with PHM-27 (0.1 μM, 3 h) caused 24% reduction in [¹²⁵I]VIP binding and 25% reduction in cyclic AMP accumulation, while no effect was detected after pretreatment with secretin (0.1 μM, 3 h).     

Vasoactive intestinal peptide (VIP) receptors in the canine gastrointestinal tract

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of ¹²⁵I-VIP receptor binding sites in the canine gastrointestinal tract. While the distribution of VIP binding sites was different for each segment examined, specific VIP binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, lymph nodules, and the circular and longitudinal smooth muscle of the muscularis externa. These results identify putative target tissues of VIP action in the canine gastrointestinal tract. In correlation with physiological data, VIP sites appear to be involved in the regulation of a variety of gastrointestinal functions including epithelial ion transport, gastric secretion, hemodynamic regulation, immune response, esophageal, gastric and intestinal motility.     

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.     

Regulation of gap junctional coupling in isolated pancreatic acinar cell pairs by cholecystokinin-octapeptide,vasoactive intestinal peptide (VIP) and a VIP-antagonist

Cholecystokinin-octapeptide (CCK-OP) induces a time- and dose-dependent decrease of gap Junctional conductance in isolated pairs of pancreatic acinar cells. In double whole-cell experiments, the time course could be described by the latency and the half-life time (t _1/2 ) of cell-to-cell uncoupling. The latency shows a biphasic dependence on [CCK-OP] with a minimum of about 50 sec at 10^–9 m CCK-OP. In the presence of vasoactive intestinal peptide (VIP), the biphasic relationship is shifted to lower CCK-OP concentrations. The increase of latency at high concentrations of CCK-OP (> 10⁰⁹ m) was blocked by addition of a VIP-antagonist. t _1/2 decreases monophasically with increasing [CCKOP]. Addition of GTPS to the pipette solution suppresses the [CCK-OP] dependence of the latency and potentiates the uncoupling phase. The kinetic data are discussed in terms of CCK binding to receptors of high and low affinity. Evidence is presented that secretion and cell-to-cell coupling are not related by an all-ornone process, but that for physiological CCK-OP concentrations, gap junctional uncoupling follows secretion.The authors would like to thank Dipl. Biol. F. Mendez for his support in software development for analysis of gap junctional conductance. The work was supported by the Graduiertenkolleg Biochemische Pharmakologie, the Herrmann und Lilly Schilling Stiftung and the Sonderforschungsbereich 156 of the Deutsche Forschungsgemeinschaft.     

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.     

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.     

Septal afferents to the area dentata terminate on vasoactive intestinal polypeptide (VIP)-like immunoreactive,non-pyramidal neurons

Summary Thermic lesions in the medial septum of adult rats result in dark degeneration of terminal boutons in the stratum moleculare and hilus of the area dentata. While most of the degenerating terminals are in synaptic contact with non-reactive cells, part of them end on dendrites of VIP-like immunoreactive neurons.     

Role of vasoactive intestinal polypeptide (VIP) in regulating the pituitary function in man

Intramuscular injection of synthetic VIP (200 micrograms) resulted in a rapid increase in plasma prolactin (PRL) concentrations in normal women, which was accompanied by the 4- to 7-fold increase in plasma VIP levels. Mean (+/- SE) peak values of plasma PRL obtained 15 min after the injection of VIP were higher than those of saline control (28.1 +/- 6.7 ng/ml vs. 11.4 +/- 1.6 ng/ml, p less than 0.05). Plasma growth hormone (GH) and cortisol levels were not affected by VIP in normal subjects. VIP injection raised plasma PRL levels (greater than 120% of the basal value) in all of 5 patients with prolactinoma. In 3 of 8 acromegalic patients, plasma GH was increased (greater than 150% of the basal value) by VIP injection. In the in vitro experiments, VIP (10(-8), 10(-7) and 10(-6) M) stimulated PRL release in a dose-related manner from the superfused pituitary adenoma cells obtained from two patients with prolactinoma. VIP-induced GH release from the superfused pituitary adenoma cells was also shown in 5 out of 6 acromegalic patients. VIP concentrations in the CSF were increased in most patients with hyperprolactinemia and a few cases with acromegaly. These findings indicate that VIP may play a role in regulating PRL secretion in man and may affect GH secretion from pituitary adenoma in acromegaly.     

Immunohistochemical localization of vasoactive intestinal polypeptide(VIP)-containing neurons in the hypothalamus of the Japanese quail,Coturnix coturnix

Summary The localization of vasoactive intestinal polypeptide (VIP) in the hypothalamus of the quail has been studied by means of light- and electron-microscopic immunohistochemistry. Numerous VIP-immunoreactive perikarya are distributed in the caudal portion of the nucleus infundibularis (n. tuberis) and nucleus mamillaris lateralis, and sparse in the preoptic area, nucleus supraopticus and nucleus paraventricularis. Dense localization of immunoreactive-VIP fibers is observed in the external layer of the median eminence, in close contact with the primary portal capillaries. The main origins of these fiber terminals are VIP-immunoreactive perikarya of the nucleus infundibularis. These neurons are spindle or bipolar and extend one process to the ventricular surface and another to the external layer of median eminence. They are CSF-contacting neurons and apparently constitute the tubero-hypophysial tract that links the third ventricle and the hypophysial portal circulation. VIP-reactive neurons in the nucleus mamillaris lateralis also project axons to the external layer of the median eminence, constituting the posterior bundle of the tuberohypophysial tract. Numerous VIP-immunoreactive perikarya occur also in the nucleus accumbens/pars posterior close to the lateral ventricle. They are also CSF-contacting neurons extending a process to the lateral ventricle. There are moderate distributions of VIP-reactive fibers in the area ventralis and in the area septalis.Ultrastructurally, the immunoreactive products against VIP are found in the elementary granules, 75–115 nm in diameter, within the nerve fibers in the median eminence.This investigation was supported by Scientific Research Grants No. 00556196, No. 56360027 and No. 56760183 from the Ministry of Education of Japan to Professor Mikami and Mr. Yamada     

Functional vasoactive intestinal polypeptide (VIP)-system in salt glands of the Pekin duck

Summary In saltwater-acclimated ducks with fully specialized supraorbital salt glands, intracarotid application of acetylcholine (5 nmoles/min/kg b.w.) or porcine vasoactive intestinal polypeptide (pVIP) (240 pmoles/min/kg b.w.) induced secretion from the salt glands at threshold conditions of secretory activity. pVIP-like immunoreactivity could be localized in fibers of the postganglionic secretory nerve ramifying throughout the glandular parenchyma. Both middle-sized arterioles and secretory tubules were innervated, and pVIP-immunoreactive varicose fibers formed peritubular baskets around the basal region of secretory tubules indicating direct innervation of the secretory tissue. pVIP-specific staining could be abolished by preabsorption of the antiserum with peptide extracts of salt-gland tissue. Synthetic pVIP and endogenous VIP from salt glands of the duck co-eluted on the HPLC system, suggesting structural similarity of the peptides. Membrane-binding studies with radioiodinated pVIP revealed the presence of high-affinity binding sites in salt-gland tissue. Affinities of unlabeled pVIP analogues to compete for these binding sites were as follows: pVIP > PHI > pVIP antagonist > secretin > pVIP (10–28) > chicken VIP (16–28). Peptide extracts of salt glands had affinities similar to pVIP. Binding sites could be localized mainly at the apical end of the radially arranged secretory tubules, as demonstrated by receptor autoradiography.It is concluded that, in addition to the classical parasympathetic transmitter acetycholine, VIP serves as neuromodulator/transmitter in cranial parasympathetic control of avian salt-gland secretion by acting on both the arteriolar network and the secretory tubules of the gland.     

Co-existence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man

By immunohistochemistry it was found that PHI- and VIP-like immunoreactivity (-IR) occurred in the same autonomic neurons in the upper respiratory tract, tongue and salivary glands with associated ganglia in rat, guinea-pig, cat, pig and man. VIP- and PHI-like immunoreactivity was also found in similar locations in the human heart. The N-terminally directed, but not the C-terminally directed, PHI antiserum or the VIP antiserum stained endocrine cells in the pig duodenum. This suggests the existence of an additional PHI-like peptide. Ligation of nerves acutely caused marked overlapping axonal accumulations of PHI- and VIP-IR central to the lesion. Two weeks after transection of the nerves, both types of immunoreactivities were still observed in accumulations both in the axons as well as in the corresponding cell bodies. The levels of PHI- and VIP-IR in normal tissues from the cat were around 10-50 pmol/g with a molar ratio of about 1 to 2. Systemic administrations of PHI and VIP induced hypotension, probably due to peripheral vasodilation in both guinea-pig and cat. Furthermore, both PHI and VIP caused an inhibition of the vagally induced increase in respiratory insufflation pressure in guinea-pig. PHI and VIP relaxed the guinea-pig trachea in vitro, suggesting a direct action on tracheobronchial smooth muscle. VIP was about 5-10 times more potent than PHI with regard to hypotensive effects and 2-3-fold, considering respiratory smooth muscle-relaxant effects in the guinea-pig. PHI was about 50-fold less potent to induce hypotension in the cat than in the guinea-pig. Although species differences seem to exist as regards biological potency, PHI should also be considered when examining the role of VIP as an autonomic neurotransmitter.     

Studies on protein kinases in two human rectocolic cell lines by polyacrylamide gel electrophoresis.Effect of the vasoactive intestinal peptide

Electrophoresis and subsequent assay of the enzyme directly onto the gel has allowed a rapid and quantitative characterization of the cyclic AMP-dependent and -independent histone kinases, protamine, phosvitin and casein kinases in HT 29 and HRT 18 cells. The technique has been applied to soluble extracts from cytoplasmic and nuclear fraction prepared in the presence and absence of neutral detergent. A more precise identification of these enzymes has been possible by analysing enzyme fractions obtained after ion-exchange chromatography of the above extracts. The protein kinase equipment of both cell lines was found to be identical (11 major components) but with different relative proportions of several enzymes. In cytoplasmic extracts: VIP activates only the type I, cytosolic, (band 4) and the type II, membrane-bound, (bands 6 and 8) cyclic AMP-dependent histone kinases. These enzymes account, respectively, for 34 and 55% of the total histone kinases in HT 29 and HRT 18 cells. The cyclic AMP-independent histone kinases (band 1,2,5 and 7) also phosphorylate protamine; band 5 was found 3o be much higher (4-fold) in HT 29 cells. In addition, two casein/phosvitin kinases have been identified in both cell lines with phosphorylating activity similar to the total histone kinases. In the nuclear extract two cyclic AMP-independent histone kinases have been found with electrophoretic mobility differing from the cytoplasmic enzymes. Also, two phosvitin/casein kinases specifically nuclear, due to their chromatographical and electrophoretical behaviour, have been characterized.     

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.     

Is vasoactive intestinal polypeptide (VIP) a sleep factor?

Vasoactive intestinal peptide (VIP) was tested in order to determine its hypnogenic properties in cats. VIP was administered intraventricularly in doses of 10 and 100 ng and compared to Ringer controls. In addition the dose of 100 ng was tested in cats pretreated with 150 mg/kg of chloramphenicol (CAP). The results showed that the 100 ng dose of VIP had small but significant REM enhancing properties, but that it did not protect the animals from the specific REM inhibiting properties of CAP. The results suggest that VIP may participate in the regulation of REM sleep.     

Modification of HT 29 cell response to the vasoactive intestinal peptide (VIP) by membrane fluidization

We used liposomes made with phospholipids of fatty acid chain length ranging from C12:0 to C16:0 to modify the cAMP dependent protein kinase (PK) activity of HT 29 cells induced by VIP or forskolin. Both VIP and forskolin effects were inhibited in dilauroylphosphatidylcholine (DLPC) treated cells. PK activity was slightly lowered when cells were treated by dimyristoylphosphatidylcholine (DMPC) liposomes. However neither VIP nor forskolin-induced PK activities were affected with dipalmitoylphosphatidylcholine (DPPC) liposomes. Furthermore, the binding of [125I]VIP to DLPC treated cells was drastically lowered whereas no change was observed when cells were incubated with DMPC or DPPC liposomes. On the other hand, the interaction of HT 29 cells with DLPC vesicles provoked a decrease in membrane cholesterol content with subsequent increase in membrane fluidity. These findings provide evidence that, in HT 29 cells, the mechanisms of VIP-receptor interaction and of adenylate cyclase activation is lipid dependent and is regulated by membrane fluidity.     

Immunocytochemical analysis of calcitonin gene-related peptide and vasoactive intestinal polypeptide in Merkel cells and cutaneous free nerve endings of cats

Summary Calcitonin gene-related peptide (CGRP)-and vasoactive intestinal polypeptide (VIP)-immunoreactivity were observed to coexist in Merkel cells of cats. No differences in peptide content were found between Merkel cells located in epithelia of the hard palate, in hairy and glabrous skin of the upper lip, and in vibrissae follicles. CGRP-and VIP-immunoreactive nerve fibres were also found near CGRP/VIP-immunoreactive Merkel cells. In the vibrissae follicles some CGRP-and VIP-immunoreactive nerve terminals end abutting on the glassy membrane. Other CGRP immunoreactive nerve fibres penetrate the epithelium of the skin and end within it. Electron microscopy of vibrissae follicles revealed that Merkel cell neuntes are not immunostained and that immunostained nerve fibres form unmyelinated bundles before ending freely. Thus, CGRP-and VIP immunoreactive nerve fibres in cat skin do not end as Merkel cell neuntes but as different kinds of free nerve endings.     

Hypermotility induced by vasoactive intestinal peptide in the rat: its reciprocal action to cholecystokinin octapeptide

Intracerebroventricular administration of vasoactive intestinal peptide (VIP) shortened the duration of pentobarbital-induced sleep and produced significant hypermotility in the rat. Although hypermotility induced by methamphetamine was not potentiated by central administration of VIP, L-DOPA-induced hypermotility in pargyline-pretreated rats was markedly enhanced by VIP and this hypermotility was suppressed by simultaneous administration of cholecystokinin octapeptide (CCK-8) in a dose-related manner. Apomorphine-induced hypermotility was also potentiated by VIP. These results suggest that VIP may stimulate postsynaptic dopaminergic receptor, causing an increase in motility, and that a possible reciprocal interaction exists between VIP and CCK-8.     

Renal function during vasoactive intestinal peptide (VIP) infusions in normal man and patients with liver disease

VIP containing nerves are present in the kidney and plasma VIP levels are elevated in cardiac failure and severe liver disease. We studied the effects of intravenous VIP; 6 pmol kg-1 min-1 on 6 normal subjects and 3 patients with liver disease. In normal subjects VIP produced flushing and significant rises in heart rate and pulse pressure but the clearance rates of paraaminohippurate and creatinine did not change significantly. Urine flow fell to about 1/3 and the rate of excretion of electrolytes (except phosphate) fell to about a half of control values. Plasma renin activity rose about 3-fold and there were significant rises in haematocrit and the plasma concentrations of solids, calcium and phosphate. The patients with liver disease responded similarly. Elevated plasma VIP could contribute to salt and water retention in disease states.