Regulation of VIP and other neuropeptides by c-Jun in sensory neurons: implications for the neuropeptide response to axotomy.

Peripheral axotomy of adult rat sensory neurons causes induction of the transcription factor c-Jun and increased expression of the neuropeptides vasoactive intestinal polypeptide (VIP), galanin and neuropeptide Y. To determine whether VIP induction is dependent on transcriptional regulation by c-Jun, we exploited the fact that c-Jun and VIP are also induced in cultured sensory neurons. We blocked c-Jun synthesis by microinjecting antisense oligonucleotides and found that VIP expression, determined by quantitative immunofluorescence, was specifically reduced.

Intracellular Microinjection: A Powerful Tool in the Study of Neuronal Gene Expression and Function.

Neurons in primary culture provide a useful experimental system with which to explore the functions of neuronal genes and the mechanisms regulating their expression. Both types of study call for transfection of neurons with plasmid DNA constructs that express either the gene of interest or a reporter gene under the transcriptional control of sequences from the gene being studied. However, the phenotypic heterogeneity and limited cell numbers that are often a feature of primary neuronal culture preparations mean that methods commonly used for bulk transfection and analysis of cell line cultures are inappropriate or difficult to apply.

Repression of preprotachykinin-A promoter activity is mediated by a proximal promoter element.

The rat preprotachykinin-A promoter, which is able to direct reporter gene expression in adult dorsal root ganglia neurons grown in culture, has no detectable activity in HeLa and PC12 cells. DNAase 1 footprinting and electrophoretic mobility shift analyses with HeLa nuclear extract indicated the presence of a protein complex binding to a region of the rat preprotachykinn-A gene promoter between the TATA box and the major transcriptional start site. We demonstrate that the sequence of the preprotachykinin-A promoter spanning nucleotides -47 to +92 functions to repress reporter gene expression in HeLa and PC12 cells but not in adult rat dorsal root ganglia grown in culture, and that this repression is correlated with a protein(s) binding to the element between the TATA box and major transcription initiation site.

An activator element within the preprotachykinin-A promoter.

The rat Preprotachykinin-A promoter (PPT) directs high levels of expression in dorsal root ganglia (DRG) neurons in culture either endogenously or when linked to a receptor construct. It is not active in any of the established tissue culture cell lines which we have analyzed. To search for transcriptional regulators within this promoter we have started to dissect the promoter into individual elements to determine their function.

Neuropeptide expression by newborn and adult rat sensory neurons in culture: effects of nerve growth factor and other neurotrophic factors.

Adult rat dorsal root ganglion sensory neurons in culture require nerve growth factor for synthesis of substance P and calcitonin gene-related peptide but express vasoactive intestinal peptide independently of nerve growth factor. In contrast, the same neurons from newborn rats do not express detectable vasoactive intestinal polypeptide when cultured with nerve growth factor. To further explore the mechanisms regulating neuropeptide expression in these cells, I compared the effects of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor and leukaemia inhibitory factor on substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and somatostatin expression in rat dorsal root ganglion cultures.

The rat vasoactive intestinal polypeptide cyclic AMP response element regulates gene transcriptional responses differently in neonatal and adult rat sensory neurons.

We studied the ability of the rat vasoactive intestinal polypeptide (VIP) cyclic AMP responsive element (CRE) to regulate reporter gene expression through a c-fos promoter in rat sensory neurons transfected in culture by plasmid microinjection. The CRE enhanced the synergistic response of the promoter to combined potassium-evoked depolarisation and forskolin treatment in neonatal but not adult rat neurons. This corresponds to endogenous VIP expression which is induced synergistically by the same stimuli in neonatal but not adult rat neurons.

5'-flanking sequences from the rat preprotachykinin gene direct high-level expression of a reporter gene in adult rat sensory neurons transfected in culture by microinjection.

The rat preprotachykinin-A gene, which encodes substance P, is expressed in response to nerve growth factor in a subpopulation of dorsal root ganglion sensory neurons. To investigate mechanisms regulating preprotachykinin-A transcription, we transfected adult rat sensory neurons in culture by microinjection of plasmids containing genomic DNA sequences linked to a lacZ (beta-galactosidase) reporter gene. Expression of beta-galactosidase was seen in 10-15% of neurons receiving injections of prPPT-betaGAL1, which contained the preprotachykinin transcription start site and 3356 bp of 5'-flanking DNA.

Synergistic regulation of vasoactive intestinal polypeptide expression by cyclic AMP and calcium in newborn but not adult rat sensory neurons in culture.

There is a spontaneous induction of vasoactive intestinal polypeptide (VIP) expression in adult rat dorsal root ganglion sensory neurons when grown in culture. The mechanism of this induction may be the same as that responsible for the increased VIP expression in sensory neurons following peripheral axotomy in vivo. This study investigates the effects of depolarization and cyclic AMP on VIP expression (measured by radioimmunoassay) in cultures of newborn and adult rat sensory neurons.

Neurotoxic damage evokes regenerative responses from adult rat sensory neurones.

When adult rats were pre-treated systemically with the excitotoxin, capsaicin, a subset of their dorsal root ganglion (DRG) sensory neurones responded as if they had been axotomised. Firstly, an increased proportion of capsaicin-sensitive DRG neurones (identified by using a cobalt stain) vigorously extended processes in vitro. Additionally, a subset of small dorsal root ganglion neurones expressed elevated levels of the growth-associated protein-43 (GAP-43) in situ.

Rat dorsal root ganglion neurons in culture express vasoactive intestinal polypeptide (VIP) independently of nerve growth factor.

Expression of vasoactive intestinal polypeptide (VIP) was studied in regenerating adult rat dorsal root ganglion (DRG) neurons in culture. VIP was not detected by radioimmunoassay in newly established cultures but increasing concentrations were observed between 2 and 4 days and VIP remained detectable for more than 2 weeks. Immunocytochemical staining revealed VIP-like immunoreactivity in virtually all neurons.

Intravenous calcitonin gene-related peptide stimulates net water secretion in rat colon in vivo.

We have studied the effect of exogenous calcitonin gene-related peptide on net fluxes of water and electrolytes in the rat small and large intestine. In ligated intestinal loops, intravenous calcitonin gene-related peptide (CGRP) induced colonic fluid secretion but had no effect on the small intestine. Subsequently, using a single-pass perfusion technique, we observed an immediate dose-dependent secretion of water by the rat colon upon intravenous administration of CGRP.

Potent and comparable vasodilator actions of A- and B-calcitonin-gene-related peptides on the superficial subcutaneous vasculature of man.

1. Vasoactivity of human A- and B-calcitonin-gene-related peptides was studied in normal subjects, using reflectance plethysmography to measure relative changes in blood flow produced by superficial subcutaneous injections of the peptides. 2.

Differential expression of alpha-CGRP and beta-CGRP by primary sensory neurons and enteric autonomic neurons of the rat.

Expression of the calcitonin gene-related peptide, alpha-calcitonin gene-related peptide (CGRP), and the homologous beta-CGRP were compared in sensory and enteric nerves of the rat. Analysis of CGRP-like immunoreactivity by cation exchange chromatography and radioimmunoassay showed that in the dorsal root ganglia, dorsal spinal cord and in those peripheral tissues where CGRP-like immunoreactivity is primarily localized to sensory fibres, alpha-CGRP concentrations were three to six times greater than beta-CGRP concentrations. In the intestine, however, beta-CGRP concentrations were up to seven times greater than alpha-CGRP concentrations.

Marked depletion of dorsal spinal cord substance P and calcitonin gene-related peptide with intact skin flare responses in multiple system atrophy.

In view of the presence of neuropeptides in spinal cord autonomic pathways, their regional concentration was studied in post mortem thoracic cord from four cases of multiple system atrophy with progressive autonomic failure (MSA). A marked depletion was observed of substance P, its related peptide substance K, and of calcitonin gene-related peptide (CGRP), particularly in dorsal regions where peptide-containing sensory fibres terminate. As substance P and CGRP in primary sensory fibres are considered mediators of skin flares in Lewis' triple response, histamine-induced skin flares were measured in 12 MSA patients and were found to be preserved.

Calcitonin gene-related peptide is localised to human airway nerves and potently constricts human airway smooth muscle.

In human airways synthetic human sequence calcitonin gene-related peptide (hCGRP), a novel peptide produced by alternative processing of mRNA from the calcitonin gene, caused concentration-dependent contraction of human bronchi (EC50 4.9 X 10(-9) M) and was significantly more potent than substance P or carbachol. The contractile response was unaffected by atropine (2 X 10(-6) M), propranolol (10(-6) M), indomethacin (10(-5) M), tetrodotoxin (3 X 10(-6) M), chlorpheniramine (10(-4) M), cimetidine (10(-5) M), or FPL55712 (10(-4) M) suggesting a direct effect of CGRP on airways smooth muscle.

In vitro production and characterisation of low molecular weight forms of calcitonin gene-related peptide immunoreactivity from rat thyroid.

Rat tissues contained two forms of calcitonin gene-related peptide (CGRP) immunoreactivity of lower molecular weight than CGRP itself. Two immunoreactive products of in vitro degradation of synthetic CGRP by rat tissue homogenates were purified and shown to be chromatographically identical to these naturally occurring moieties. They reacted only with a carboxy-terminal directed CGRP antiserum indicating that they were carboxy-terminal fragments of CGRP.

Occurrence, distribution and origin of peptide-containing nerves of guinea-pig and rat male genitalia and the effects of denervation on sperm characteristics.

A systematic immunohistochemical and radio-immunological survey of the occurrence, distribution and origin of the peptidergic nerve supply in guinea-pig and rat male genitalia is presented. Neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), substance P and CGRP were detected in the genital organs of both species. The densities and distribution patterns of the peptidergic nerves were compared with those of the adrenergic nerves, as revealed by antibodies raised against dopamine-beta-hydroxylase (D beta H) and tyrosine hydroxylase (TH), and the general neuronal component, as revealed by antibodies raised against neurofilament proteins (NF).

Occurrence, distribution and ontogeny of CGRP immunoreactivity in the rat lower respiratory tract: effect of capsaicin treatment and surgical denervations.

The occurrence and distribution of calcitonin gene-related peptide (CGRP) immunoreactivity in the rat respiratory tract were investigated by means of immunocytochemistry and radioimmunoassay using antibodies raised in rabbits to synthetic rat CGRP. Substantial amounts of CGRP immunoreactivity (range 5-37 pmol/g) were detected in all parts of the respiratory tract, the highest being in the stem bronchus. Gel filtration chromatography of extractable CGRP immunoreactivity revealed one single peak, eluting at the position of synthetic rat CGRP.

Capsaicin induces a depletion of calcitonin gene-related peptide (CGRP)-immunoreactive nerves in the cardiovascular system of the guinea pig and rat.

We have demonstrated that calcitonin gene-related peptide (CGRP) immunoreactivity is widely distributed in cardiac and perivascular nerves of the guinea pig and rat. In the guinea pig the number and distribution of CGRP-immunoreactive nerve fibres closely paralleled that of fibres containing substance P, the two immunoreactivities being found invariably to coexist in the same perivascular networks and terminals. In the rat, CGRP-immunoreactive cardiovascular nerves had a similar distribution to those containing substance P, but in contrast to the guinea pig the former were far more numerous.

Calcitonin gene-related peptide immunoreactivity in afferent neurons supplying the urinary tract: combined retrograde tracing and immunohistochemistry.

The innervation of rat and guinea pig urinary tract was examined using immunohistochemistry, radioimmunoassay and True Blue retrograde tracing techniques and was further assessed following both surgical and chemical denervation experiments. Substantial amounts of calcitonin gene-related peptide-like immunoreactivity (range 20-150 pmol/g) were detected in tissue extracts and localised to nerve fibres distributed throughout the urinary tract of both species, these being concentrated in the ureter and base of the bladder. In the guinea pig, the number and distribution pattern of calcitonin gene-related peptide-like immunoreactive nerves appeared to be identical to that of substance P-containing nerves, whereas in the rat the former predominated.

Calcitonin gene-related peptide-immunoreactive nerves in the tongue, epiglottis and pharynx of the rat: occurrence, distribution and origin.

The occurrence, distribution and nature of calcitonin gene-related peptide (CGRP) in the tongue, epiglottis and pharynx of the rat was investigated by immunocytochemistry and radioimmunoassay. Numerous CGRP-containing nerves were found to innervate and terminate freely within the epithelium of the tongue, epiglottis and pharynx. Immunoreactive fibres were also found in the muscle layer and around blood vessels in the tongue, and in motor end plates in the muscle of the epiglottis and pharynx.

The effect of sympathectomy on calcitonin gene-related peptide levels in the rat trigeminovascular system.

The effect of sympathectomy on the calcitonin gene-related peptide (CGRP) level in the rat primary trigeminal sensory neurone was investigated. Six weeks after bilateral removal of the superior cervical ganglion there was a 70% rise in the CGRP content of the iris and the pial arteries, a 34% rise in the concentration in the trigeminal ganglion but no change in the brainstem. The CGRP rise in both end organs suggests that this phenomenon may be common to all peripheral organs receiving combined sensory and sympathetic innervations.