N.D. Christofides

Peptide-containing nerves in human urinary bladder

Nerves containing immunoreactive vasoactive intestinal polypeptide (VIP), substance P and two newly discovered peptides, neuropeptide tyrosine (NPY) and PHI (peptide having N-terminal histidine and C-terminal isoleucine), have been found in the human urinary bladder by immunocytochemistry and radioimmunoassay. Somatostatin immunoreactivity was detected by radioimmunoassay. The VIP-immunoreactive nerves were widely distributed in all regions, but were particularly dense beneath the epithelium and in the muscle layer. Scattered intramural ganglia were found to be reactive to VIP antiserum. Higher concentrations of extractable VIP were detected in the trigone than in the dome. VIP- and PHI-immunoreactive nerves were similarly distributed, the latter being less numerous. NPY-immunoreactive nerves were seen mainly in the muscle layer, particularly in the trigonal area. The distribution patterns of VIP- and NPY-immunoreactive nerves resembled those of the previously reported cholinergic and adrenergic nerves, respectively. Many blood vessels were found to be innervated by both types of immunoreactive nerves. Scattered substance P-immunoreactive fibers were occasionally seen, being present in the submucosa and around the detrusor muscles. The significance of these nerves remains to be elucidated.

The distributions of PHI and VIP in porcine gut and their co-localisation to a proportion of intrinsic ganglion cells

VIP and PHI share sequence homology and certain biological actions. Immunocytochemistry and radioimmunoassay were used to see if the two peptides also have similar distributions in the gut of the pig. PHI-immunoreactive fibres were found, like those containing VIP, in all layers of the bowel wall but in lesser numbers. Unlike VIP-immunoreactive nerves, however, which are ubiquitous in the gastrointestinal tract, PHI-containing neurons were numerous in all areas except the fundus, where only few fibres and no ganglion cells were found to be reactive to PHI antibodies. PHI and VIP immunoreactive materials were also quantified by specific radioimmunoassay of tissue extracts. The concentrations of PHI and VIP were similar in all regions of the gut, except in the fundus where the quantities of VIP-immunoreactivity far exceeded those of PHI. The presence of both VIP- and PHI-immunoreactivities in ganglion cells of the sub-mucous plexus allowed investigation of the co-localisation of the peptides. Serial sections through ganglion cells revealed that a major proportion contain both PHI- and VIP-immunoreactivity. Some cells contained VIP alone, or VIP and weak, equivocal immunostaining of PHI, and a sub-population contained no peptide-immunoreactivity. The presence of both VIP- and PHI-immunoreactivities in the same ganglion cell supports the recent reports of the isolation and characterisation, using genetic technology, of their common precursor molecule. The finding of VIP and not PHI in the fundic region suggests the differential expression of the two peptides.

Distribution of galanin immunoreactivity in the genitourinary tract of man and rat

Galanin has been shown to be present in substantial quantities in the human and rat genitourinary tract by radioimmunoassay and immunocytochemistry. The highest concentrations measured by radioimmunoassay were found in the human vas deferens, corpus cavernosum and spongiosum and in the vagina and cervix. In man gel chromatographic analysis showed two molecular forms. The earlier eluting peak was different from porcine galanin standard. There was only one molecular form in the rat which emerged in an earlier position than the porcine standard. Galanin immunoreactive nerve fibres demonstrated in the genitourinary tract were found both in man and rat. They were found within smooth muscle and in close relationship to blood vessels. The presence and distribution of galanin in the genitourinary system suggest the possibility that this neuropeptide could play a role in the regulation of smooth muscle tone, blood flow and motility.

Radioimmunoassay and intramural distribution of PHI-IR in human intestine.

The objective of this study was to develop a radioimmunoassay for PHI and use this to assess its intramural distribution in the human intestine. The antibody was harvested following immunization with porcine PHI conjugated to bovine serum albumin by glutaraldehyde, and the iodinated PHI tracer was prepared by the Iodo-gen method. The assay system showed no cross-reaction with other members of the glucagon-secretin family of peptides and was sensitive to changes of PHI of 2 fmol/tube (95% confidence). High concentrations of immunoreactive PHI were found in the human intestine, exclusively localized in the nonendocrine gut layers, suggesting a possible neuroendocrinological or neurotransmitter role for PHI.

Studies on the distribution of PHI in mammals

We have developed a sensitive and specific radioimmunoassay to PHI and investigated its distribution in four mammalian species (man, cat, guinea-pig and rat). PHI was present in high concentrations, not only in intestine but also in brain, respiratory tract, urogenital tract and other peripheral tissues. Its distribution was similar to that of VIP and in each tissue examined there was always a significant correlation between the concentrations of these two peptides. In a survey of endocrine tumours, PHI was found to be produced only in those tumours that also produced VIP. In addition PHI was only elevated in the plasma of patients that also had high plasma VIP concentrations. This parallel distribution and release was found to be due to the co-synthesis of VIP and PHI in the same pro-hormone peptide. However, the variable ratio of VIP/PHI in different anatomical areas suggest that in these areas there is a different post-translational enzyme processing of the precursor protein.

Peptide histidine isoleucine- and vasoactive intestinal polypeptide-like immunoreactivity coexist in rat hypophysial portal blood☆

Plasma immunoreactive peptide histidine isoleucine (PHI) and vasoactive intestinal polypeptide (VIP) levels were measured by specific radioimmunoassays in urethane-anesthetized rats. Basal levels of plasma PHI-like immunoreactivity (PHI-LI) in the hypophysial portal blood were 414 +/- 180 pmol/l (means +/- S.E.), about 7 times higher than in the peripheral blood. VIP-like immunoreactivity (VIP-LI) was also high in the portal blood (399 +/- 139 pmol/l). The correlation coefficient between PHI-LI and VIP-LI was 0.76. These findings suggest that PHI and VIP are co-released from the median eminence into the hypophysial portal blood in rats.

Comparative distribution of vasoactive intestinal polypeptide (VIP), substance P and PHI in the enteric sphincters of the cat.

In the feline gastrointestinal tract, the neuropeptides, substance P, VIP and PHI were investigated by specific radioimmunoassays and immunocytochemistry. The concentrations of all 3 peptides and the level of peptidergic innervation were significantly less in the anal sphincter than elsewhere, whereas no significant differences were seen between other sphincter and non-sphincter regions.

Ontogeny of PHI in the rat brain

The regional distribution of immunoreactive PHI (IR-PHI) was investigated in rat brain between postcoitum (pc) and day 60 postpartum (pp). IR-PHI was undetectable in all regions of the foetal brain, and only very small amounts were found at day 7 pp. However, there was a dramatic increase thereafter reaching a peak at day 20 pp (e.g. in the hippocampus there was a 12-fold increase in the PHI concentration). Highest concentrations were found in the cortex (40 +/- 5 pmol/g) and hippocampus (35 +/- 8 pmol/g), with lower concentrations in the diencephalon (11 +/- 4 pmol/g) and mesencephalon (10 +/- 3 pmol/g). The brainstem and cerebellum contained very low amounts of IR-PHI. Permeation analysis of brain extracts, on Sephadex G50-superfine, indicated the presence of one major form of IR-PHI which eluted in a similar position to pure intestinal porcine PHI and human intestinal PHI.

PHI-like immunoreactivity co-locates with the VIP-containing system in human lumbosacral spinal cord

Using a specific radioimmunoassay and immunocytochemistry, the quantitative regional distribution and localization of PHI-like immunoreactivity was studied in normal postmortem human spinal cord. The levels of PHI-like immunoreactivity were low in the cervical and thoracic region whereas the lumbar and especially sacral regions showed higher levels, with dorsal sacral concentrations exceeding ventral concentrations. Chromatographic analysis by high pressure liquid chromatography revealed that the PHI-like immunoreactivity in human spinal cord elutes slightly earlier than pure porcine PHI, and may correspond to PHM-27, a PHI-27-like peptide found in human preprovasoactive intestinal polypeptide. Immunocytochemical studies show a distinctive distribution of PHI-like immunoreactive fibres and terminals at the lumbosacral segments. The distribution of PHI-like immunoreactivity is thus similar to that of VIP, and unlike a number of other neuropeptides; with VIP, it may mark a system which has a role in the spinal control of urogenital function in man.

Release of gastrointestinal hormones following an oral water load

The ingestion of 2 different water loads (7.5 and 15 ml/kg) by healthy subjects stimulated the release of plasma motilin, gastrin, pancreatic polypeptide and VIP. Atropine was found to block the release of PP but not the other hormones.