PHI-like Immunoreactivity Research Articles

Involvement of peptide histidine isoleucine in non-adrenergic non-cholinergic relaxation of the rat gastric fundus induced by high-frequency neuronal firing.

The role of peptide histidine isoleucine (PHI) as a neurotransmitter of the inhibitory motor neurones, the physiological role of PHI and vasoactive intestinal polypeptide (VIP) in the non-adrenergic non-cholinergic (NANC) relaxation and the relative amounts of VIP- and PHI-like immunoreactivity (LI) co-released by neuronal activation were investigated in the rat gastric fundus. Longitudinal muscle strips from the rat gastric fundus precontracted by the thromboxane receptor agonist U46619 (0.1 micro M) were studied in organ baths under conditions of muscarinic receptor blockade by atropine (1 micro M) and adrenergic neurone blockade by guanethidine (5 micro M) ("NANC conditions"). Concentration-response curves were plotted for both amplitude and area under the curve (AUC) of the relaxant responses induced by VIP (0.3 nM-0.3 micro M), PHI (0.3 nM-1 micro M) and peptide histidine valine [PHV(1-42); 0.3 nM-1 micro M]. All three peptides were more potent in the curve based on amplitude than in that based on the AUC of relaxation. In addition, VIP was 5.3 and 7 times more potent than PHI and PHV(1-42), respectively, in producing relaxation expressed as amplitude, and 2.7 and 2.8 times, respectively, in producing relaxation expressed as AUC. PHI and PHV(1-42) behaved as partial agonists with respect to VIP in producing relaxation expressed as AUC. Electrical field stimulation (EFS; 120 mA, 1 ms, 4-32 Hz, pulse trains of 2 min) evoked frequency-dependent relaxant responses. Alpha-chymotrypsin (1 u/ml or 3 u/ml), an anti-VIP serum (1:100 or 1:50) and an anti-PHI serum (1:25), slightly reduced the amplitude, but greatly inhibited the AUC of the NANC relaxation induced by EFS (13 Hz) [approximately 72%, 47% and 28% less than that seen in time controls or with normal rabbit serum (1:100 or 1:25), respectively]. EFS (8-32 Hz) caused significant, frequency-dependent increases in the outflow of VIP- and PHI-LI from the strips. The EFS-induced release of VIP-LI was approximately 20% of the PHI-LI release. These findings indicate that PHI is involved in EFS-induced NANC relaxation of the rat gastric fundus, the major physiological role of VIP and PHI is the maintenance of smooth muscle relaxation and VIP is co-released in equimolar amounts mainly with PHI.

Effects of acute and long-term atropine treatment on levels, release and response to VIP and PHI in the submandibular gland of cat and rat.

We have studied the effects of acute and long-term treatment of cats and rats with atropine on the levels, release and effects of two peptides, vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI), that probably co-exist with acetylcholine in the parasympathetic nerves supplying the submandibular gland. Atropine treatment (progressively increasing doses from 2 to 15 mg kg-1 injected s.c.) for 14 days did not alter the contents of VIP- or PHI-like immunoreactivity (-IR) in the cat submandibular gland or in three other tissues (nasal mucosa, trachea and tongue). Acute as well as long-term atropine treatment decreased the vasodilation following low-, but not high-, frequency parasympathetic nerve stimulation. During prolonged stimulation (60 min) there was a decreased vasodilatation response following both acute and long-term atropine treatment. The overflow of VIP-IR and PHI-IR following parasympathetic nerve stimulation was markedly increased by acute, but not by long-term atropine treatment. The VIP- or PHI-induced stimulation of cyclic AMP (cAMP) accumulation in the cat submandibular gland was not altered after long-term atropine treatment. Similarly, treatment of male Sprague-Dawley rats with atropine (20 mg kg-1) or imipramine (20 mg kg-1) for 14 days did not alter the sensitivity to VIP or to PHI of cAMP accumulation in the submandibular gland, nor was there any change in VIP-IR or PHI-IR content. In conclusion, although atropine treatment causes an acute increase in the overflow of VIP and PHI evoked by parasympathetic nerve stimulation, there is no depletion of peptide stores upon long-term treatment, nor is there any change in the effect of exogenous VIP and PHI on cAMP-accumulation.

Immunohistochemical analysis of the ontogeny of peptide histidine isoleucine (PHI)-immunoreactive neurons in the pre- and postnatal rat brain

The development of PHI-like immunoreactivity in the pre- and postnatal rat brain has been investigated by means of the indirect immunohistofluorescence technique. PHI-immunoreactive cells and fibres were first observed at day 19 of gestation, in the suprachiasmatic nucleus. Over ensuing stages of development, PHI-LI appeared in progressively more areas of the central nervous system. In the early postnatal rat, for example, PHI-immunoreactive neurones could be detected in many regions, such as the cingulate, frontoparietal and temporal cortices, and the hippocampus. Later during maturation, such as at day 12 postnatal, the cental amygdaloid nucleus, periaqueductal grey, geniculate nuclei and stria terminalis also exhibited PHI-immunoreactive cells and processes. Around day 21 postnatal, the greatest number of PHI-positive neurones was visible, and this number was approximately maintained in the oldest age studied. Thus, in contrast to the ephemeral appearance of many other peptides during ontogeny, in no PHI-containing regions were cells and fibres visualized at early stages, only to disappear subsequently in older animals. The present study illustrates that the development of PHI-like immunoreactivity is primarily a postnatal process. Its appearance during maturation of the rat brain is consistent with its putative trophic and transmitter roles.

Immunohistochemical studies of the neurochemical markers, CGRP, enkephalin, galanin, γ-MSH, NPY, PHI, proctolin, PTH, somatostatin, SP, VIP, tyrosine hydroxylase and neurofilament in nerves and cells of the human attached gingiva

Punch biopsies were obtained from the buccal gingiva of the lower third molars. Thin nerve fibres, immunoreactive for calcitonin gene-related peptide (CGRP) or substance P (SP), with possible sensory function, were found in the propria often close to the epithelium, sometimes even penetrating into the basal layers. γ-Melanocyte stimulating hormone (γ-MSH)-like immunoreactivity was found in sparsely distributed single cells (except in one specimen containing a dense infiltration), resembling neutrophilic granulocytes of the propria. γ-MSH was present in several single smooth axons and in thick axon bundles of the propria. Surrounding the blood vessels, neuropeptide Y (NPY), tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine amide (PHI) immunoreactive nerve fibres were observed. NPY and TH-positive fibres probably represent sympathetic nerve terminals and VIP/PHI-immunoreactive ones may have a parasympathetic function. Papillae of the propria contained VIP-positive fibres not obviously related to blood vessels. The distribution in papillae of PHI-like immunoreactivity was similar but the PHI-positive reaction was also present in a few cells of the propria, especially near blood vessels. Somatostatin (SOM)-positive reaction occurred in a few dendritic-type cells near or in the epithelium and single nerve fibres close to the epithelium. Several thick axon bundles of the propria contained neurofilament (NF)-immunoreactive material. Some thin NF-fibres were found in the papillae and some seemed to penetrate into the epithelium. No galanin, methionine-enkephalin, parathyroid hormone or proctolin immunoreactive material was found. The rather rich content of several neuropeptides in human attached gingiva, as well as other neurochemical markers, is probably associated with sensory and autonomic functions.

Analysis of peptide histidine-isoleucine/ vasoactive intestinal polypeptideimmunoreactive neurons in the central nervous system with special reference to their relation to corticotropin releasing factor-and enkephalin-like immunoreactivities in the paraventricular hypothalamic nucleus

The distribution of peptide histidine-isoleucine (PHI) and vasoactive intestinal polypeptide (VIP), two peptides derived from the same precursor molecule, was analysed with immunohistochemistry in the central nervous system of the rat, and to a limited extent in some other species including sheep, monkey and man. Special attention was focused on possible cross-reactivity between PHI antisera and corticotropin releasing factor in parvocellular neurons in the hypothalamic paraventricular nucleus projecting to the external layer of the median eminence. (1) Characterization of the PHI and VIP antisera revealed that they recognized different sequences of the peptide molecules. One of the PHI antisera (PHI-N), although mainly N-terminally directed, also probably contained an antibody population directed against the C-terminal amino acid in PHI which is an amidated isoleucine. Rat and human corticotropin releasing factor but not ovine also have an amidated isoleucine in C-terminal position. (2) PHI- and VIP-like immunoreactivity were found with parallel and overlapping distribution in all areas investigated in the rat central nervous system. In many cases coexistence of the two immunoreactivities could be directly demonstrated. PHI neurons were found in some areas so far not known to contain PHI/VIP neurons, including the dorsal septum, the septofimbrial nucleus, the stria terminalis and lamina V of the spinal cord. (3) Using an antiserum directed against the amino acid sequence 111–122 of the VIP/PHI precursor, immunoreactive cell bodies were seen in some areas containing VIP and PHI neurons. PHI- and VIP-like immunoreactivity were expressed in parallel in increasing amounts in the superficial laminae of the dorsal horn after transection of the sciatic nerve [G.P. McGregor et al. (1984) Neuroscience 13, 207–216; S.A.S. Shehab and M.E. Atkinson (1984) J. Anat. 139, 725; S.A.S. Shehab and M.E. Atkinson (1986) Expl Brain Res. 62, 422–430]. (5) The PHI-N antiserum stains large numbers of immunoreactive cells in the parvocellular part of the paraventricular nucleus and these cells are mostly identical with corticotropin releasing factor-positive neurons. Absorption experiments suggested that this PHI-N-like immunoreactivity to a large extent represented cross-reactivity with rat CRF and that earlier demonstration of many PHI-positive neurons in the paraventricular nucleus probably represents an artefact as proposed by F. Berkenbosch et al. ( Neuroendocrinology 44, 338–346). However, some cells did, in fact, contain VIP- as well as PHI-like immunoreactivity as was shown with antisera not cross-reacting with corticotropin releasing factor. Some of these cells in addition contained both corticotropin releasing factor- and enkephalin-like immunoreactivity. (6) PHI-N antiserum seem to cross-react with corticotropin releasing factor also in guinea-pig, cat, monkey and man. In the ovine hypothalamus, however, all PHI as well as VIP antisera stained only small numbers of positive fiber in the external layer of the median eminence and some of these were also corticotropin releasing factor-positive. Corticotropin releasing factor-positive nerve endings formed an extremely dense network in the median eminence. These findings are consistent with the fact that our PHI antisera do not cross-react with ovine corticotropin releasing factor which has arginine in its C-terminal position. (7) Radioimmunoassay revealed that both PHI and VIP could be measured in extracts of the rat median eminence as well as in other brain regions. The present results further underline the great problem with cross-reactivity in immunohistochemical studies, in the present case resulting from similarity of only one amino acid. The use of region specific antibodies may represent one way to reduce the risk for such artefactual results.

Nerve fibres containing peptide histidine isoleucine (PHI) in the respiratory tract.

Recent studies have indicated that vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) are formed by cleavage of a common precursor protein. In the present study we have examined the distribution of nerve fibres displaying PHI-like immunoreactivity in the upper respiratory tract of several mammalian species including man. PHI fibres were found to have the same general distribution as previously described for VIP fibres. These fibres were distributed mainly in the subepithelial connective tissue around both seromucous glands and blood vessels. In the tracheal wall, PHI fibres were also seen in the non-vascular smooth muscle. Sequential immunostaining for PHI and VIP revealed co-existence of the two peptides in the same nerve fibres.

PHI-like immunoreactivity in the nervous system of the cockroach (insect) and Aplysia (Mollusc) with special reference to its relationship to VIP-like immunoreactivity.

Studies in mammals have indicated that PHI (peptide histidine isoleucine) and VIP (vasoactive intestinal polypeptide) share a prepropeptide, and that both peptides are contained in the same neurons. The present study proposes that this relationship may not hold true in protostomian invertebrates. In the nervous system of the cockroach (Insecta, Arthropoda) and Aplysia (Gastropoda, Mollusca) distribution of PHI and VIP was examined by immunocytochemistry. In both animals neurons containing PHI-like immunoreactivity were numerously found and only a small part of them in the cockroach contained VIP-like immunoreactivity. These findings suggest that in these invertebrates, PHI may play a more important regulatory role than VIP. Possible reasons for the difference in the distribution of immunoreactivities for PHI and VIP are discussed.

Neuropeptide immunocytochemistry in protostomian invertebrates, with special reference to insects and molluscs

In some molluscs (Aplysia and Fusitriton) and insects (silkworm and cricket), occurrence and distribution of neuropeptides in the nervous system and gut were studied with following results: (1) in these invertebrates and also in planaria, PP-like immunoreactivity is extensively distributed in neurons and (in insects) in gut endocrine paraneurons. These cells are negative for NPY, the mammalian neuropeptide related to PP in molecular structure. (2) PHI-like immunoreactivity is widely distributed in the neurons of those invertebrates; it occurs also in gut endocrine paraneurons in insects. The PHI-immunopositive cells are immunonegative for VIP and the coexistence of both peptides due to the common precursor in mammals cannot be recognized in these invertebrates. (3) Immunoreactivity for urotensin I, the neuropeptide derived from teleostean urophysial neurons, is widely distributed in the neurons of the invertebrates. In insects (cricket) it occurs in gut endocrine cells.

PHI-like immunoreactivity in the gallbladder and in vitro effect of porcine PHI on smooth muscle of the gallbladder

The occurrence and distribution of PHI-like immunoreactivity in the guinea pig gallbladder has been analysed by radioimmunoassay and immunocytochemistry. Chromatography of gallbladder extracts by gel permeation and high-performance liquid chromatography revealed that guinea pig PHI-like immunoreactivity is of a similar size to that of porcine PHI but may differ in its amino acid sequence. Immunocytochemistry showed PHI-immunoreactivity to be localised to nerves found predominantly in the ganglionated plexus and themucosal plexus of the gallbladder. Pure natural porcine PHI induced a dose-dependent relaxation of the isolated guinea pig gallbladder muscle which was not blocked by antagonists to acetylcholine, catecholamines, histamine, and 5-hydroxytryptamine. PHI may thus be one of the local factors involved in controlling gallbladder function.

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.

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 administration of PHI and VIP induced hypotension, probably due to peripheral vasodilatation 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.

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.

Diarrhoea in vipoma patients associated with cosecretion of a second active peptide (peptide histidine isoleucine) explained by single coding gene.

Peptide histidine isoleucine (PHI), first isolated from pig intestine, is distributed identically to vasoactive intestinal peptide (VIP) in all mammals. 42 patients with high plasma VIP secondary to VIPoma also had very high plasma PHI-like immunoreactivity, in a constant ratio to VIP. None of 125 patients with other endocrine tumours had high levels of either peptide. VIPoma tissue from 20 patients also contained PHI shown by immunocytochemistry to be produced by the same cell as VIP. Messenger RNA(mRNA) from one of these tumours contained the codes for VIP and a separate PHI-like sequence. Human PHI-like sequence differed from porcine PHI in only two aminoacid residues. A single cell thus produces two separate regulatory peptides with apparently similar potencies but different spectra of activity. In normal tissue the constant coproduction of two active neuropeptides by a single neuron provides further evidence against the doctrine of one neuron producing only one neurotransmitter.

Effect of the peptide PHI-27 on prolactin release in vitro.

The present study demonstrates that PHI, a peptide belonging to the glucagon-secretin group and thus structurally similar to VIP, can release prolactin from dispersed rat anterior pituitary cells and also causes release of prolactin from hemipituitaries. PHI-like immunoreactivity has previously been demonstrated in a hypothalamic system with nerve endings in the median eminence, and, taken together, these findings suggest that PHI may represent a physiologic prolactin-releasing factor.

Carboxyl-terminal PHI-like immunoreactivity in intrinsic gastrointestinal nerves: C. Vaillant and G.J. Dockray, Department of Physiology, University of Liverpool, England

Carboxyl-terminal PHI-like immunoreactivity in intrinsic gastrointestinal nerves: C. Vaillant and G.J. Dockray, Department of Physiology, University of Liverpool, England