Jan Fahrenkrug

Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P-, somatostatin-, galanin-, vasoactive intestinal polypeptide- and cholecystokinin-immunoreactive ganglion cells

SummaryBy use of the indirect immunofluorescence technique the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) has been analyzed in cervical and lumbar dorsal root ganglia of untreated and colchicine-treated rats. In addition, lumbar ganglia were examined 2 weeks after transection of the sciatic nerve. The occurrence of CGRP-positive cells in relation to ganglion cells containing substance P-, somatostatin-, galanin-, cholecystokinin (CCK)-, and vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucin (PHI)-LI has been evaluated on consecutive sections as well as using elution-restaining and double-staining techniques.CGRP-LI was observed in many ganglion cells of all sizes ranging in diameter from 15 μm to 65 μm. Thus, this peptide occurs also in the large primary sensory neurons. In contrast to the sensory peptides described to date, CGRP-positive cells constituted up to 50% of all and 70% of the medium-sized neurons, thus being the most frequently occurring peptide in sensory neurons so far encountered. Subpulations of CGRP-positive neurons were shown to contain substance P-, somatostatin-, or galanin-LI and some CGRP-positive neurons contained both substance P- and galanin-LI. In fact, most substance P-, somatostatin- and galanin-positive cell bodies were CGRP-immunoreactive. The coexistence analysis further revealed that galanin and substance P often coexisted and that some cells contained both substance P- and somatostatin-LI, whereas no coexistence between galanin and somatostatin has as yet been seen. VIP/PHI-LI was only shown in a few cells in untreated or colchicine-treated rats. However, after transcetion of the sciatic nerve numerous VIP/PHI-positive cells were observed, some of which also contained CGRP-LI.The present results indicate that a CGRP-like peptide is present in a wide range of primary sensory neurons probably not related to specific sensory modalities. Often this peptide coexists with other biologically active peptides. Taken together these findings suggest that CGRP may have a generalized function.

Distribution of vasoactive intestinal polypeptide in the rat and mouse brain.

Abstract The distribution of cell bodies and nerve fibers that combine with antisera to vasoactive intestinal polypeptide (VIP) was studied by immunohistochemistry in combination with radioimmunoassay in the brain of rat and mouse. The highest concentrations (60pmol/g wet wt) of immuno-reactive VIP were found in the cerebral cortex and in certain limbic structures, whereas the concentrations in the basal ganglia, thalamus, lower brain stem, cerebellum and spinal cord were low ( VIP-immunoreactive fibres had a distribution which on the whole paralleled that of the cell bodies, suggesting that many of the VIP-containing cells project locally. VIP-containing fibres were numerous in the following areas: the entire neocortex, the pyrifom cortex, the entorhinal cortex, the hippocampal complex, the amygdala (the central nucleus in particular), the anterior olfactory nuclei, the nucleus accumbens, ventral pallidum, bed nucleus of stria terminalis, suprachiasmatic nucleus, medial preoptic nucleus, median eminence, lateral geniculate body, pretectum, superior colliculus, periaqueductal gray, and the lateral parabrachial nucleus. Only few, scattered fibres were seen in other parts of the brain stem, in the striatum, thalamus and spinal cord. The cerebellum was devoid of VIP-containing fibres. VIP-containing neurones seem to form predominantly local projections. In addition, some VIP-containing neurones probably also form long projections, such as descending and transcallosal projections from the cortical cells, and projections from the amygdala to preoptic, hypothalamic and basal forebrain areas. The characteristic telencephalic distribution of the neurones that contain VIP suggests a role for this peptide in cortical and limbic functions.

Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin.

The temporal course of changes in peptide expression in the dorsal root ganglia L4 and L5 and in the dorsal horn of the spinal cord has been studied in rats subjected to a sciatic nerve transection at a mid-thigh level following different survival times. Galanin-, substance P-, vasoactive intestinal polypeptide-, peptide histidine-isoleucine- and calcitonin gene-related peptide-like immunoreactivities have been studied both by immunohistochemistry and radioimmunoassay. Galanin messenger ribonucleic acid has also been studied by in situ hybridization in the dorsal root ganglia of normal and lesioned animals. In addition, a group of animals with a sciatic nerve crush was studied to compare possible differences in peptide expression after both types of lesions. The results show that the transection induces an increase in the number of cell bodies expressing galanin-like immunoreactivity in the ganglia, and that the galanin levels rise about 120-fold after three and 14 days of survival. This increase reflected increased synthesis of the peptide, since there was a rise in the galanin messenger ribonucleic acid already at 24 h post-lesion, which was maintained for at least 60 days. In the spinal cord there was an increase of staining in the midportion of the outer layers of the dorsal horn that corresponded to fibers thought to arise from cells of the dorsal root ganglia affected by the transection. Also a depletion of substance P-like and an increase in vasoactive intestinal polypeptide- and peptide histidine-isoleucine-like immunoreactivities in the dorsal root ganglia were confirmed. These changes were shown to be rapidly detectable and were paralleled by similar changes in the dorsal horn of the spinal cord. For calcitonin gene-related peptide the immunohistochemistry was inconclusive, and the radioimmunoassay showed no detectable changes. After nerve crush a transient increase in the number of galanin immunoreactive neurons was observed, as well as a decrease in the number of neurons showing substance P-like immunoreactivity. These changes were most noticeable between six and 14 days of survival. After this, peptide expression seemed to return slowly to normal, that is by day 45 post-crush only a few cells showed galanin-like, and many sensory neurons expressed substance P-like immunoreactivity. The results demonstrate that when primary sensory neurons are peripherally lesioned they respond in a complex manner, altering their normal production of peptides by increasing or decreasing their synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Radioimmunoassay of Vasoactive Intestinal Polypeptide (VIP) in Plasma

Abstract A sensitive and specific radioimmunoassay for vasoactive intestinal polypeptide (VIP) has been developed, which can detect 3.3 pmol × L −1 of the peptide in plasma. Antisera to highly purified porcine VIP coupled to albumin were raised in eight rabbits. The final dilution, the avidity, and the specificity of each antiserum were determined. 125 I-VIP served as label, and highly purified porcine VIP was used as standard. Separation of antibody-bound and free VIP was achieved by plasma-coated charcoal. Nonspecific interference with the assay system was excluded by extraction of plasma samples with ethanol. The reliability of the assay was investigated by recovery experiments, by serial dilution of plasma samples with high concentration of endogenous VIP, and by immunosorption. The within-and between assay reproducibility at a concentration of 18.3 pmol × L −1 was 1.6 and 2.3 pmol × L −1 (1 S.D.), respectively. Median fasting concentration of VIP in plasma from 74 normal subjects was 7.3 pmol × L −1 (range: 0–20.0 pmol × L −1 ).

Vasoactive intestinal polypeptide: measurement, distribution and putative neurotransmitter function.

Vasoactive intestinal polypeptide (VIP) is a highly basic octacosapeptide isolated from porcine small intestine. VIP was originally considered to be a gut hormone, but radioimmunochemical and immunohistochemical studies have revealed that VIP has a widespread distribution localized in neurons. VIP-containing neurons are present in the central nervous system including the cerebral arteries, the digestive tract, the genitourinary tract and the adrenal glands. In the nerve terminals VIP is localized in synaptic vesicles. VIP fulfils a number of criteria to be a neurotransmitter both in central and peripheral nerves. Whilst the role of VIP in the central nervous system is unknown, it is likely that VIP is the mediator of gastrointestinal and pancreatic functions, which are controlled by non-cholinergic, non-adrenergic nerve fibres.

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.

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.

Vasoactive intestinal polypeptide

Recent research has revealed that some of the peptides isolated from the gut are also present in the central nervous system, and, vice versa, several brain peptides are found in the gastrointestinal tract. Among the peptides originally isolated from the porcine intestine is the vasoactive intestinal polypeptide (VIP), discovered as an unexpected side fraction during the purification of secretin. VIP is a highly basic octacosapeptide with a broad range of biological effects including vasodilation, relaxation of smooth muscle cells, and influence on secretory processes in glands.

Pharmacology and functions of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase‐activating polypeptide: IUPHAR Review 1

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP) are members of a superfamily of structurally related peptide hormones that includes glucagon, glucagon‐like peptides, secretin, gastric inhibitory peptide (GIP) and growth hormone‐releasing hormone (GHRH). VIP and PACAP exert their actions through three GPCRs – PAC1, VPAC1 and VPAC2– belonging to class B (also referred to as class II, or secretin receptor‐like GPCRs). This family comprises receptors for all peptides structurally related to VIP and PACAP, and also receptors for parathyroid hormone, corticotropin‐releasing factor, calcitonin and related peptides. PAC1 receptors are selective for PACAP, whereas VPAC1 and VPAC2 respond to both VIP and PACAP with high affinity. VIP and PACAP play diverse and important roles in the CNS, with functions in the control of circadian rhythms, learning and memory, anxiety and responses to stress and brain injury. Recent genetic studies also implicate the VPAC2 receptor in susceptibility to schizophrenia and the PAC1 receptor in post‐traumatic stress disorder. In the periphery, VIP and PACAP play important roles in the control of immunity and inflammation, the control of pancreatic insulin secretion, the release of catecholamines from the adrenal medulla and as co‐transmitters in autonomic and sensory neurons. This article, written by members of the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC‐IUPHAR) subcommittee on receptors for VIP and PACAP, confirms the existing nomenclature for these receptors and reviews our current understanding of their structure, pharmacology and functions and their likely physiological roles in health and disease. More detailed information has been incorporated into newly revised pages in the IUPHAR database (http://www.iuphar‐db.org/DATABASE/FamilyMenuForward?familyId=67).

Gene expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the rat hypothalamus

Pituitary adenylate cyclase activating polypeptide (PACAP) isolated from ovine hypothalamus is considered to be a member of the vasoactive intestinal peptide/glucagon/secretin/growth hormone-releasing hormone family of peptides. Two forms of PACAP, PACAP38 and PACAP27, have been demonstrated in the rat hypothalamus. The PACAP precursor contains another peptide called PACAP-related peptide (PRP), but so far no information on this peptide in tissue exists. We have developed three radioimmunoassays specific for PACAP38, PACAP27 and PRP and demonstrate that all three preproPACAP peptides are expressed in the rat hypothalamus, the PACAP38/PACAP27 ratio being around 60 and the PACAP38/PRP ratio being around 10. HPLC analysis of hypothalamic extract showed that PACAP38 and PACAP27 are found in only one form corresponding to the respective synthetic peptides, whereas PRP eluted in two peaks, the predominant form corresponding to synthetic PRP1-29. The cellular distribution of PACAP38, PACAP27, and PRP and corresponding mRNA in the hypothalamus was determined with immunohistochemistry and in situ hybridization histochemistry. PACAP- and PRP-immunoreactive neuronal perikarya were observed in the medial parvocellular part of the paraventricular nucleus (PVN) in colchicine pretreated rats. Some cell bodies of magnocellular variety were found in the PVN. PACAP mRNA containing cells were observed in moderate numbers in the same parts of the paraventricular nucleus. PACAP- and PRP immunoreactive fibres and varicosities were distributed in the PVN and in the periventricular nucleus. These data show that PACAP38, PACAP27 and PRP are expressed in the parvocellular part of the PVN, implying roles as hypothalamic regulatory peptides.