The use of region-specific antibodies in the characterization and localization of vasoactive intestinal polypeptide-like substances in the rat gastrointestinal tract

Antisera specific for different regions of porcine VIP have been used in radioimmunoassay and immunohistochemical studies of immunoreactive VIP in rat small and large intestine. Cation exchange chromatography of intestinal extracts separated two major and one minor peak of immunoreactivity. One major peak eluted in a similar position to natural porcine VIP and was read equally by NH₂-terminal-specific, and mid- and COOH-terminal-specific antisera. A second major peak, and the minor peak, eluted earlier than porcine VIP, and were read significantly less well with mid- and COOH-terminal antisera compared with NH₂-terminal-specific antisera. All forms of VIP occurred mainly in extracts of muscle layers of the gut, and no antiserum revealed more than trace amounts of immunoreactivity in mucosal extracts. In immunohistochemical studies all antisera demonstrated fluorescent nerve fibres in the enteric plexuses, circular smooth muscle and lamina propria; some antisera demonstrated nerve cell bodies predominantly in the submucous plexus. NH₂-terminal-specific antisera also demonstrated a sparse population of mucosal endocrine-like cells in the ileum and colon that were not seen with other antisera. It is concluded that VIPergic neurons of the rat gut contain a peptide closely resembling porcine VIP and at least two less basic variants with similar NH₂-terminal antigenic determinants. VIP-like peptides may also occur in endocrine cells, but since these peptides appearto fact that the majority of neuronal VIP in rat gut exists in a form that is both chromatographically and immunochemically distinct from porcine VIP, and may well possess different biological properties.     

Structural characterization of calcitonin gene-related peptide purified from rabbit intestine

Calcitonin gene-related peptide (CGRP) immunoreactive material has been found in extracts of the intestine, however, the structure of intestinal CGRP is not known. Analytical reverse phase HPLC and ion-exchange FPLC revealed one predominant immunoreactive CGRP peak in rabbit intestinal extracts. This material was purified from rabbit intestine by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence and mass spectral analysis of the purified peptide and its chymotryptic fragments were consistent with the structure: GCNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSEAF-amide. Rabbit intestinal CGRP is identical to human CGRP-II in 35 of 37 amino acid residues. Two amino acid differences were detected at position 1, with Gly in rabbit CGRP instead of Ala in human CGRP-II, and at position 35, with Glu instead of Lys, respectively. Rabbit CGRP differed from human CGRP-I by three additional amino acids at positions 3, 22, and 25. This report shows that a CGRP form which closely resembles human CGRP-II, by means of chemical characterization, is the predominant form in rabbit intestine. Rabbit CGRP is the only CGRP form which has Gly as the amino terminal amino acid. Since the amino terminus of CGRP seems to be important for expression of bioactivity, the biological activity of rabbit CGRP may differ from human, rat and porcine CGRP.     

Isolation and characterization of rat vasoactive intestinal peptide

We have used gel filtration, ion exchange chromatography, affinity chromatography and reversed-phase HPLC to isolate vasoactive intestinal peptide from rat intestine. Microsequence analysis of 1 nmole peptide indicated that the sequence was identical to the porcine octacosapeptide VIP. In radioimmunoassay with four antisera and in the turkey pancreas bioassay, rat VIP was equipotent with highly purified preparations of porcine, human and canine VIP. A less basic rat VIP-variant was also isolated and the N-terminal decapeptide region that was sequenced was identical with that of porcine VIP.     

Identification and distribution of immunoreactive peptide YY in the human, canine, and murine gastrointestinal tracts: species-related antibody recognition differences

A radioimmunoassay using two antisera (antibody 80 and antibody 213) from rabbits immunized with porcine peptide YY has been characterized for both sensitivity and specificity. To determine the distribution of peptide YY in the gut, fresh tissue specimens from the human and canine gut were separated into mucosal-submucosal and muscularis externa layers by microdissection. These tissues and transmural specimens from murine gut were acid-extracted and neutralized, followed by radioimmunoassay using each antiserum. Immunoreactive peptide YY in canine and murine gut was present in similar concentration and distribution using each antiserum, with highest concentrations in the mucosal-submucosal layer of the descending colon. Using antibody 213, immunoreactive peptide YY throughout the human gut was measured only at the lower detection limit of the radioimmunoassay. By contrast, using antibody 80, peptide YY in human gut was present in a distribution similar to canine and murine gut. Using antibody 80, one major immunoreactive species was identified with C18 reverse-phase high-performance liquid chromatography in extracts of human, canine, and murine colon. These results suggest species-related antibody recognition differences. The similar concentrations of peptide YY in canine and murine gut determined with the two antisera are consistent with the hypothesis that the amino acid sequences of canine and murine peptide YY are similar to porcine peptide YY. Using antibody 213, the low concentrations of immunoreactive peptide YY found in human gut are consistent with the hypothesis that human and porcine peptide YY have different amino acid sequences. Antisera prepared by immunization with porcine PYY must therefore be carefully characterized prior to studies using human sera or human tissue extracts.     

The distribution and cellular origin of vasoactive intestinal polypeptide in the avian gastrointestinal tract and pancreas

Summary The distribution and origins of vasoactive intestinal peptide (VIP) in the gut and pancreas of the turkey were studied by radioimmunoassay of tissue extracts and by immunocytochemistry. Several antisera were used that vary in their specificity for different regions of porcine or chicken VIP. Radioimmunoassays using NH₂-terminal specific antisera that react almost equally with porcine and chicken VIP's revealed significant amounts of immunoreactive VIP in extracts of pancreas, brain and all regions of the gastrointestinal tract from crop to colon. Highest concentrations (300pmol/g) were found in the colon muscle, and concentrations were generally low (< 20 pmol/g) in the mucosal layers of the small intestine. After ion exchange chromatography of extracts on CM-Sephadex three immunoreactive forms of VIP were separated corresponding to the three molecular forms previously found in mammalian gut extracts. In immunocytochemical studies nerve fibres were found throughout the gut, and in the pancreas. Immunoreactive nerve cell bodies were also identified in the submucous plexus throughout the gut, but were particularly prominent in the oesophagus and pancreas. It has previously been shown that VIP is a strong stimulant of the flow of pancreatic juice in birds whereas the structurally related hormone secretin, which is known to control the flow of pancreatic juice in mammals, is a weak stimulant. It is proposed that in birds VIP might regulate the pancreas, and other aspects of gut function, as a neurotransmitter or neurohormone.     

Porcine brain natriuretic peptide-like immunoreactivity in rat tissues

The presence of immunoreactive porcine brain natriuretic peptide in rat tissues was studied with a specific radioimmunoassay for porcine brain natriuretic peptide-26. The cross-reactivity of the antiserum used was less than 0.001% with rat atrial natriuretic peptide, rat brain natriuretic peptide-32 and rat brain natriuretic peptide-45. Immunoreactive porcine brain natriuretic peptide was detectable in various tissues of the rat, and high concentrations of immunoreactive porcine brain natriuretic peptide were found in the brain and cardiac atrium, with the highest level in the hypothalamus (159±30 fmol/gram wet tissue, mean±SEM, n=4). Reverse phase high performance liquid chromatography showed that the immunoreactive porcine brain natriuretic peptide of the whole brain and heart extracts eluted mainly at an identical position to synthetic porcine brain natriuretic peptide-26. These findings indicate that porcine brain natriuretic peptide-like substance, distinct from rat brain natriuretic peptide, is present in high concentrations in the rat brain and cardiac atrium.     

Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in anuran intestine

Summary Immunocytochemical and radioimmunological techniques with region specific antisera have been used to identify a vasoactive intestinal polypeptide-like material in the anuran intestine. Seven species of Anura were investigated: Bombina bombina, Alytes obstetricans, Rana temporaria, Rana esculenta, Hyla arborea, Hyla crepitans and Bufo bufo.In five of the species (A. obstetricans, R. temporaria, H. arborea, H. crepitans and B. bufo) vasoactive intestinal polypeptide-like immunoreactive mucosal endocrine cells and nerve fibres in all layers of the gut wall, were detected by both immunofluorescence and peroxidase-antiperoxidase methods. In the other two species, R. esculenta and B. bombina, no mucosal endocrine cells were detected although the vasoactive intestinal polypeptide-immunoreactive nerve fibres were plentiful.Radioimmunoassay showed the presence of significant amounts of vasoactive intestinal polypeptide-immunoreactivity in intestinal extracts from all species. The highest quantities were present in those anurans with both immunostained cells and nerves. Gel permeation chromatography showed that most of the vasoactive intestinal polypeptide-like peptide eluted in a position identical to that of natural mammalian (porcine) vasoactive intestinal polypeptide.The results indicate that a vasoactive intestinal polypeptide-like peptide is well represented in the Anura and that it is immunologically very similar to the mammalian peptide.Part of this work was presented at the European Society of Comparative Endocrinology, 1979; see Buchan et al. 1980a     

Comparison of motilin binding to crude homogenates of human and canine gastrointestinal smooth muscle tissue

Pharmacological studies indicate that in man and in rabbit, but not in dog, motilin has a direct influence upon gastrointestinal smooth muscle. In accordance with this hypothesis we have presented direct biochemical evidence for the presence of motilin receptors on rabbit smooth muscle tissue. We have now extended our studies to human and canine tissue. Tissue homogenates were studied in binding experiments with iodinated porcine [Leu13]motilin and iodinated canine motilin. It was ascertained that the iodination procedure had little effect on the biological activity of the porcine analogue. In the human antrum specific binding of the iodinated porcine analogue was only found in the smooth muscle layer. It was absent in mucosal or serosal preparations. At 30 degrees C and pH 8.0, binding was maximal after 60 min of incubation, and was reversed by the addition of unlabeled porcine motilin. Binding was enhanced in the presence of calcium and magnesium ions. At a concentration of 10 mM MgCl2, binding was 220% of the binding observed in its absence. Displacement studies with synthetic porcine [Leu13]motilin or synthetic natural porcine motilin indicated a dissociation constant (Kd) of 3.6 +/- 1.6 nM and a maximal binding capacity (Bmax) of 77 +/- 9 fmol per mg protein. Canine motilin displaced iodinated porcine motilin with an apparent Kd of 2.2 +/- 0.9 nM. Compared to antral binding, receptor density decreased aborally and orally, and was absent in jejunum and ileum. In dog specific binding could not be demonstrated in antral and duodenal tissue, neither with labeled porcine nor with labeled canine motilin. However, labeled canine motilin was equipotent to labeled porcine motilin in binding studies with human tissue: the dissociation constant was 0.9 +/- 0.6 nM. The present studies therefore demonstrate the existence of a specific motilin receptor in the antroduodenal region of the human gut. Apparently, such receptors are not present in the canine gut. Our data support the hypothesis that in the human gastrointestinal tract, the gastroduodenal area is motilin's target region.     

Measurement and chromatographic characterization of vasoactive intestinal peptide from guinea-pig enteric nerves

The material exhibiting immunoreactivity for vasoactive intestinal peptide in guinea-pig enteric nerves has been characterized by high-performance liquid chromatography in three modes: reversed-phase, cation-exchange and gel permeation. In each case a major portion of the material contained in acetic acid extracts of guinea-pig gut showed the same chromatographic properties as the synthetic porcine peptide of defined amino acid sequence. It is therefore concluded that this immunoreactive material is authentic vasoactive intestinal peptide. The study illustrates a number of the problems encountered in attempting to characterize, and measure reliably, peptides in tissue extracts.     

A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors

A peptide that cross reacted with N-terminal, but not C-terminal, antisera to vasoactive intestinal peptide (VIP) was isolated from extracts of intestine from the dogfish Scyliorhinus canicula. Microsequence analysis gave the structure His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ile-Arg-Lys-Gln-Met-Ala-Val-Lys - Lys-Tyr-Ile-Asn-Ser-Leu-Leu-Ala-NH2. C-terminal amidation was determined by HPLC analysis of phenylthiocarbamyl amino acid derivatives after carboxypeptidase Y digestion. The peptide differs at five positions from the porcine octacosapeptide. Dogfish VIP was equipotent with its porcine counterpart in inhibiting binding of 125I-labelled VIP to guinea pig dispersed pancreatic acini, and in stimulating amylase secretion by the same preparation. The data indicate a strong conservation of VIP during evolution and permit identification of residues crucial for bioactivity.     

Motilin receptors in rabbit stomach and small intestine

Motilin receptors in rabbit antral and duodenal smooth muscle tissue were characterized by direct binding technique using 125I-labeled porcine motilin as a tracer ligand. Binding at 30 degrees C was maximal at 90 min, was saturable and partially reversible. Displacement studies with natural porcine motilin, synthetic leucine-motilin or norleucine-motilin indicated a dissociation constant (Kd) of 1.1 +/- 0.3 nM and a maximal binding capacity (Bmax) of 42 +/- 10 fmol/mg protein. Binding was unaffected by glucagon, pancreatic polypeptide and somatostatin, but substance P interfered via an unknown mechanism. By density gradient centrifugation motilin receptors were shown to be present in plasma membranes. Binding could only be demonstrated in preparations from antrum and upper duodenum. These observations provide evidence for a localized target region for motilin in the gastrointestinal tract, and for a direct interaction of motilin with gastrointestinal smooth muscle tissue.     

DISTRIBUTION OF VASOACTIVE INTESTINAL POLYPEPTIDE (VIP) IN THE PORCINE CENTRAL NERVOUS SYSTEM

A sensitive and specific radioimmunoassay for vasoactive intestinal polypeptide (VIP) has been used to determine the regional distribution of the peptide in the porcine CNS. The hippocampus and cerebral cortex contained the highest concentration of VIP, while lower but significant amounts were present in the hypothalamus and neurohypophysis. The immunoreactive VIP in the brain appeared to be identical with the octacosapeptide originally isolated from porcine small intestine, since it was found to have identical chromatographic properties. to dilute in parallel with the standard curves and to react equally with six different VIP antisera independent of extraction procedure used.     

Characterization of complementary deoxyribonucleic acid for precursor of porcine motilin

Cloned cDNAs encoding the precursor protein for motilin and a novel peptide, motilin-associated peptide, were isolated from a library derived from porcine intestinal mucosa mRNA. Nucleotide sequence analysis predicts a precursor protein of 119 amino acids including a signal peptide in direct linkage with the 22 amino acid sequence for motilin, and a 70 amino acid peptide of unknown function. The putative bioactive moieties are separated by Lys-Lys, dibasic residues that serve as substrates for cleavage by proteolytic maturation enzymes in many polyprotein precursors. While there is an abundant literature detailing a spectrum of tissues and cell types which express motilin like immunoreactivity, analysis of mRNA derived from many of these tissues suggests that the mRNA for the mucosal motilin precursor is only transcribed in this tissue. The nature of the immunoreactive material in the central nervous system and other peripheral tissues remains to be determined.     

Antisera raised against eledoisin and kassinin detect immunoreactive material in rat tissue extracts: tissue distribution and chromatographic characterization

Radioimmunoassays were developed for the tachykinins eledoisin (ELE) and kassinin (KAS) using antisera raised in rabbits. The antisera exhibited low (less than 0.1%) cross-reactivities to substance P (SP) and physalaemin (PHY), but crossreacted (with one exception, antiserum K7) to varying extents with neurokinin A (NKA) and neurokinin B (NKB). In the rat, the tissue distribution of the immunoreactive material detected by antiserum (E7) raised against ELE and by another antiserum (K1) raised against KAS both resembled that previously described for SP. Using the highly KAS-specific antiserum K7, no or only very low levels of immunoreactivity could be detected in extracts of various rat tissues. Gel permeation chromatography and ion-exchange chromatography of tissue extracts indicated that all antisera (except K7) detected the same population of immunoreactive molecules. One of the components was chromatographically indistinguishable from NKA. The tissue distribution of this component also resembled that of SP. Another immunoreactive component co-chromatographed with NKB at cation exchange chromatography. Acid tissue extracts, but not neutral tissue extracts, were found to contain immunoreactive components which appeared more basic than NKA and NKB. The total levels of immunoreactivity were higher in neutral than in acid tissue extracts. However, the ratio between the amounts of immunoreactivities in the two types of extracts varied considerably between tissues, indicating that tachykinin immunoreactive components may be present in different relative proportions in various tissues.     

Radioimmunoassay with heterologous antibody (hetero-antibody RIA): utilization of highly cross-reactive antibody present in polyclonal antiserum.

To develop a homologous radioimmunoassay (RIA) for a hormone of a small or rare animal often meets difficulty in collecting a large amount of purified antigen required for antibody production. On the other hand, to employ a heterologous RIA to estimate the hormone often gives poor sensitivity. To overcome this difficulty, a "hetero-antibody" RIA was studied. In a hetero-antibody RIA system, a purified preparation of a hormone is used for radioiodination and standardization and a heterologous antibody to the hormone is used for the first antibody. Canine motilin and rat LH were selected as examples, and anti-porcine motilin and anti-hCG, anti-hCG beta or anti-ovine LH beta was used as the heterologous antibody. The sensitivities of the hetero-antibody RIAs were much higher than those of heterologous RIAs in any case, showing that these hetero-antibody RIA systems were suitable for practical use. To clarify the principle of hetero-antibody RIA, antiserum to porcine motilin was fractionated on an affinity column where canine motilin was immobilized. The fraction bound had greater constants of affinity with both porcine and canine motilins than the rest of the antibody fractions. This fraction also reacted with a synthetic peptide corresponding to the C-terminal sequence common to porcine and canine motilins in a competitive binding test with labeled canine motilin. These results suggest that an antibody population having high affinity and cross-reactivity is present in polyclonal antiserum and indicate that the population can be used in hetero-antibody RIA at an appropriate concentration.     

Idiotypes of Lewis rat antibodies to encephalitogenic peptides of guinea pig myelin basic protein: in vitro and in vivo studies

Lewis rat antibodies raised by immunization with encephalitogenic peptide 68-88 guinea pig myelin basic protein were purified by affinity chromatography and used to immunize rabbits. After exhaustive absorption of the rabbit antisera to remove anti-rat immunoglobulin activity, the antisera retained activity against the immunogen, shown by the ability to block reaction of radioiodinated peptide with the active site of the rat anti-peptide antibodies. Intrastrain idiotypic cross-reactivity was assessed by testing the rabbit antisera against a panel of Lewis anti-peptide antibodies. Each anti-idiotypic antiserum displayed a unique pattern of reactivity with the panel. Similar tests in which a panel of anti-peptide antibodies raised in F344 rats was used demonstrated the presence of interstrain cross-reactive idiotopes. When seven rabbit anti-idiotypic antisera were tested by pretreatment of rats before challenge with encephalitogen for effect in vivo, five were without effect. Of the remaining two, one caused a slight suppression of disease; the other enhanced disease compared to control animals.     

Multiple peptide immunoreactivities in the nervous system of Aeschna cyanea (Insecta, Odonata). An immunohistochemical study using antisera to cholecystokinin octapeptide, somatoliberin, vasoactive intestinal peptide, motilin and proctolin

By use of the indirect immunoperoxidase method, the brain, the suboesophageal ganglion and the corpora cardiaca of the dragonfly Aeschna cyanea have been shown to be immunoreactive to proctolin antiserum and to several mammalian peptide antisera including unsulfated cholecystokinin octapeptide (CCK-8 NS) (Andriès et al. 1989), vasoactive intestinal peptide (VIP), human somatoliberin (hGRF) (Andriès et al. 1984) and motilin antisera. Immunohistochemical studies have been performed on material fixed in a solution of picricacid paraformaldehyde or in Bouin Hollande's sublimate solution. Antisera were applied on alternate sections or, according to the elution-restaining method of Tramu et al. (1978), one after another on the same section. Multiple peptide immunoreactivities appear expressed in the brain and the suboesophageal ganglion. Cells reactive to both hGRF and VIP antisera show also gastrin/CCK-like immunoreactivity and some of them are also detected by motilin antiserum. Besides, some cells immunopositive to CCK-8 NS and motilin antisera do not show hGRF or VIP immunoreactivity. At least, two pairs of protocerebral cells appear immunoreactive to both CCK-8 NS and proctolin antisera. Therefore, the present observations support our previously developed idea (Andriès et al. 1989) that the population of CCK-like cells is heterogenous.     

Rat immunoreactive cholecystokinin (CCK): characterization using two chromatographic techniques

Acid and neutral extracts of rat cerebral cortex and upper small intestine were prepared and the endogenous concentrations of cholecystokinin-like immunoreactivity (CCK-LI) measured by three new CCK-specific radioimmunoassays. The characterization of the immunoreactive CCK molecular forms was undertaken using gel permeation chromatography in the presence of 6 M urea to minimise problems relating to peptide adsorption or aggregation. Reverse-phase high-performance liquid chromatography (HPLC) was also performed on the rat tissue extracts. Rat cortex contained 268 +/- 12 pmol/g CCK-LI, and over 90% resembled the sulphated CCK-8, which was preferentially extracted at neutral pH. In contrast, the rat upper small intestine (97 +/- 8 pmol/g of CCK-LI) contained less than 20% CCK-8, the majority of immunoreactive CCK being of larger molecular size and being preferentially extracted at acid pH. In the small intestine the predominant molecular form(s) was intermediate in size between CCK-33 and CCK-8. Large amounts of CCK-33 and of a molecular form larger than CCK-33 were also detected. It is concluded that post-translational cleavage of CCK differs in rat brain and gut.     

Multiple peptide immunoreactivities in the nervous system of Aeschna cyanea (Insecta,Odonata)

Summary By use of the indirect immunoperoxidase method, the brain, the suboesophageal ganglion and the corpora cardiaca of the dragonfly Aeschna cyanea have been shown to be immunoreactive to proctolin antiserum and to several mammalian peptide antisera including unsulfated cholecystokinin octapeptide (CCK-8 NS) (Andriès et al. 1989), vasoactive intestinal peptide (VIP), human somatoliberin (hGRF) (Andriès et al. 1984) and motilin antisera. Immunohistochemical studies have been performed on material fixed in a solution of picricacid paraformaldehyde or in Bouin Hollande's sublimate solution. Antisera were applied on alternate sections or, according to the elution-restaining method of Tramu et al. (1978), one after another on the same section. Multiple peptide immunoreactivities appear expressed in the brain and the suboesophageal ganglion. Cells reactive to both hGRF and VIP antisera show also gastrin/CCK-like immunoreactivity and some of them are also detected by motilin antiserum. Besides, some cells immunopositive to CCK-8 NS and motilin antisera do not show hGRF or VIP immunoreactivity. At last, two pairs of protocerebral cells appear immunoreactive to both CCK-8 NS and proctolin antisera. Therefore, the present observations support our previously developed idea (Andriès et al. 1989) that the population of CCK-like cells is heterogenous.