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. Blockade of c-June expression also resulted in reduced neuropeptide Y expression but left galanin, substance P and calcitonin gene-related peptide unaffected. Since in vitro electrophoretic mobility shift assays showed that a nominal cyclic AMP responsive element (CRE) associated with the rat VIP gene could bind c-Jun-containing transcription factor complexes, we next investigated whether VIP expression in sensory neurons might depend on transcription factor binding to the CRE. When a DNA plasmid containing multiple copies of the CRE was injected into newly cultured sensory neurons to sequester transcription factors binding the endogenous CRE, there was a selective reduction in VIP expression. VIP induction in sensory neurons therefore probably results from transcriptional activation by c-Jun acting in combination with other factor(s), possibly acting through the CRE. These results show that c-Jun can regulate transcription of other genes affected by axotomy and imply that it could be a key regulator of the neuronal axotomy response.

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. These results indicate that the tissue-specific expression of the preprotachykinin-A gene could require the interaction of both positive and negative regulatory DNA elements.

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. A DNA element which had previously been suggested to regulate transcription from DNA sequence analysis of the rat PPT promoter occurs at position -200 relative to the major start of transcription within the PPT promoter. The equivalent element from the bovine PPT promoter had previously been proposed to be a cAMP responsive element (CRE). The sequence of this enhancer has similarities with both the AP1 and CRE DNA consensus sequences. We have demonstrated that one copy of this rat PPT element linked to a heterologous basal promoter will enhance transcription in HeLa and PC12 cell lines as well as adult rat DRG neurons grown in culture. It is also demonstrated that the rat PPT element will bind proteins in HeLa nuclear extract distinct from those binding to the well-characterized Gibbon Ape Leukemia Virus (GALV) AP1 or somatostatin CRE sites by gel retardation analysis. This PPT element, when cloned in a heterologous reporter construct, although showing properties of both AP1 and CRE elements, was functionally distinguished from both the somatostatin CRE element and the GALV AP1 enhancer when these elements were tested in the same reporter construct. This PPT element has a constitutive level of activity in adult rat DRG neurons, which is fivefold higher than that driven by the reporter construct promoter. It is also significantly different from the same reporter construct linked to the somatostatin CRE and analyzed in DRG neurons.

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. As with neurons from adult animals, newborn rat sensory neurons required nerve growth factor for synthesis of substance P and calcitonin gene-related peptide. This effect was independent of neuronal survival since most neurons capable of expressing these peptides appeared to survive without added neurotrophic factors. Neurons surviving in the absence of nerve growth factor also expressed vasoactive intestinal polypeptide, suggesting that nerve growth factor suppresses vasoactive intestinal polypeptide expression in immature neurons. However, nerve growth factor withdrawal after eight days' culture failed to cause vasoactive intestinal polypeptide induction which therefore appears to depend on other factors also. Neither ciliary neurotrophic factor nor leukaemia inhibitory factor affected peptide levels when used alone, but both inhibited nerve growth factor-stimulated expression of substance P and calcitonin gene-related peptide in adult rat neurons. They also stimulated vasoactive intestinal polypeptide expression in newborn rat neurons in the presence of nerve growth factor but not to such high levels as those seen under conditions of nerve growth factor deprivation. Neither brain-derived neurotrophic factor nor neurotrophin-3 affected peptide expression significantly. Somatostatin was defected in adult rat neurons, but was unaffected by neurotrophic factors. No somatostatin was detected in newborn rat neurons. These results suggest that in immature animals at least, the increased expression of vasoactive intestinal polypeptide seen in sensory neurons following peripheral nerve injury in vivo, could result from deprivation of target-derived nerve growth factor in combination with increased availability of ciliary neurotrophic factor or leukaemia inhibitory factor from the injured nerve.

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. We conclude that VIP expression in sensory neurons, which is induced by axotomy in vivo, could be regulated through the CRE.

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. Unlike adult neurons, newborn rat sensory neurons, supported in culture with nerve growth factor, did not spontaneously express VIP. However, potassium-evoked depolarization and drugs that increase intracellular cyclic AMP concentrations (forskolin, 8-bromo cyclic AMP, isobutylmethylxanthine) interacted synergistically to stimulate high levels of VIP expression in newborn rat neurons. The contribution of depolarization to this effect could be mimicked by the L-type calcium channel agonist Bay K 8644 and blocked by the channel antagonist nifedipine, implying the involvement of calcium influx through L-type channels. While depolarization and forskolin individually had small effects on VIP content of adult rat sensory neuron cultures, there was no synergism of the kind seen in newborn rat cultures. Immunostaining showed that VIP was localized within approximately 30% of both newborn and adult rat sensory neurons. Thus, a subpopulation of newborn rat sensory neurons exhibit marked plasticity of VIP expression in an integrated response to activation of cyclic AMP- and calcium-dependent signalling pathways. This property is no longer present in mature neurons, however, where VIP expression is regulated by as yet undetermined factors.

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. Deletion analysis showed that expression was directed by 865 bp lying immediately upstream of the transcription start site. Extension of the prPPT-betaGAL1 sequence to include the first intron of preprotachykinin increased beta-galactosidase two- to threefold. Functional promoter and enhancer sequences from the rat prolactin gene failed to direct expression in sensory neurons, indicating neuronal selectivity for preprotachykinin sequences. Expression of prPPT-betaGAL1, measured relative to a construct containing the Rous sarcoma virus promoter, was approximately fivefold higher in neurons than in nonneuronal cells. This suggests selectivity by preprotachykinin 5'-flanking sequences for neuronal expression. However, prPPT-betaGAL1 expression was not restricted to the neuronal subpopulation containing immunoreactive substance P nor was it dependent upon nerve growth factor. Therefore, it does not share all the characteristics of endogenous preprotachykinin expression implying the need for additional regulatory sequences or the involvement of post-transcriptional regulation. Our results show that transfection of differentiated neurons in culture by microinjection has considerable potential in studies of neuron-specific gene expression.

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. Thus, VIP is expressed in regenerating DRG neurons in culture just as it is in vivo following axotomy. However, VIP concentrations in culture were the same whether nerve growth factor was present or not. Therefore, deprivation of retrogradely transported nerve growth factor is unlikely to explain VIP induction by axotomy in vivo.

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. Net secretion of sodium, potassium, and chloride were also raised. The implications of these observations for the possible involvement of high circulation concentrations of CGRP in the watery diarrhea syndrome accompanying medullary thyroid carcinoma are discussed.

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. Injection of 10(-11) mol of either peptide caused an immediate 200% increase in local blood flow and prolonged hyperaemia lasting up to 3 h. The hyperaemic response to 10(-13) mol of each peptide was significantly (P less than 0.01) smaller and shorter in duration than that elicited by 10(-11) mol of the same peptide, and 10(-15) mol of both peptides produced no hyperaemia other than that attributable to needle insertion alone. 3. At all three dosages examined, there were no significant differences between A- and B-calcitonin-gene-related peptides in magnitude or time course of the hyperaemic response. 4. Human A- and B-calcitonin-gene-related peptides are therefore potent vasodilators in man, causing comparable dose-related vasodilatation in the superficial tissues.

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. Only beta-CGRP was detected in the intestines of capsaicin-treated rats. Northern blot and in situ hybridization to alpha-CGRP- and beta-CGRP-specific probes showed that while both alpha-CGRP and beta-CGRP messenger ribonucleic acids occurred in the dorsal root ganglia, only beta-CGRP messenger ribonucleic acid occurred in the intestine, where it was localized to enteric neurons. Receptor binding sites on membranes of rat heart and colon had approximately equal affinities for alpha-CGRP and beta-CGRP. The two peptides were equipotent in increasing the rate and force of atrial contractions but alpha-CGRP was slightly (2.6 times) more potent than beta-CGRP in relaxing colonic smooth muscle. Thus, both alpha-CGRP and beta-CGRP occur in the rat nervous system and are both biologically active. Sensory neurons and enteric neurons have been identified as populations which preferentially express alpha-CGRP and beta-CGRP, respectively.

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. These results provide a new key to the classification and aetiology of autonomic and multiple system degenerations, as well as a model to study the role of sensory neuropeptides in man.

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. CGRP was detected in human airways by radioimmunoassay with highest concentrations in cartilaginous airways. CGRP was localised by immunocytochemistry to both nerves and ganglia in human airways. CGRP, is a potent constrictor of human airways and may have important effects on airway function and be implicated in the pathogenesis of bronchial hyper-responsiveness and asthma.

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. The larger fragment was found to have a molecular mass corresponding to amino acid residues 19-37 of the CGRP molecule.

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). Bilateral transection of the hypogastric nerves, in the guinea-pig, resulted in a decrease of substance P-containing nerves in the vas deferens and of NPY-, PHI- and VIP-containing nerves in the seminal vesicle. Unilateral disconnection of the pelvic nerves caused a decrease of VIP, PHI, substance P and CGRP nerve supply in the ipsilateral vas deferens and cauda epididymidis in the guinea-pig. A marked reduction of noradrenergic and NPY-containing nerves was observed in the vas deferens and sexual accessory glands of rats, chemically sympathectomised by chronic injection of low doses of guanethidine. Conversely, increase of substance P and CGRP immunoreactivities were observed, particularly in the vas deferens. After guanethidine, the cauda epididymidis and vas deferens were distended with spermatozoa, suggesting paralysis of the ducts. Spermatozoa had a decreased percentage of attached cytoplasmic droplets, indicating prolonged retention in the ducts.

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. CGRP immunoreactivity was localized both in mucosal endocrine cells and nerve fibres from the larynx down to the peripheral lung. CGRP-immunoreactive endocrine cells were found singly in trachea and stem bronchi and in groups in intrapulmonary airways. They appeared at a late stage of gestation (17 days), reached a maximum number near term and decreased after birth to maintain a population similar to that of the adult animals by postnatal day 21. Similarly, CGRP-immunoreactive nerve fibres were first identified by day 18 of the gestation period and reached the adult distribution by postnatal day 21. CGRP-immunoreactive nerve fibres were localized among smooth muscle, seromucous glands, beneath and within the epithelium of the airways and around blood vessels. CGRP was also found in sensory ganglia and in motor end plates of the larynx musculature. Neonatal pretreatment with capsaicin caused a marked reduction in CGRP immunoreactivity of nerve fibres in the respiratory tracts as well as a less marked decrease in the population of CGRP-containing endocrine cells of the lung. No change was seen in motor end plates immunostaining. Vagal ligation experiments revealed that CGRP-immunoreactive nerve fibres travelling in the vagus originate mainly from neurons located in the jugular ganglion. Infranodosal right vagal ligation induced a marked loss in CGRP-immunoreactive nerves of the trachea, and of the ipsilateral stem bronchus, but no changes were observed in peripheral lung. By contrast infranodosal left side vagal ligation caused a decrease in CGRP-immunoreactive nerves of the ipsilateral lung and bronchus without affecting the peptide content in the trachea. Left vagal ligation also induced a marked increase in both the intensity of staining and number of CGRP-immunoreactive endocrine cells in the lung. We conclude that CGRP immunoreactivity is localized in both nerve fibres and endocrine cells and is associated principally with the afferent (sensory) innervation of the respiratory tract.(ABSTRACT TRUNCATED AT 400 WORDS)

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. Marked regional variations were observed in the density of the CGRP-immunoreactive innervation in both species. The CGRP-immunoreactive content of tissue extracts was in close agreement with the immunocytochemical findings, the highest levels of CGRP occurring in the mesenteric artery (guinea pig and rat) and inferior vena cava (guinea pig). Following capsaicin treatment of adult guinea pigs and neonatal rats, there was a significant loss of CGRP-immunoreactive nerves in the two species. In the guinea pig, substance P-and CGRP-immunostained fibres were depleted to a similar extent, throughout the cardiovascular system. However, the loss of rat CGRP-immunoreactive nerves was dose-dependent and displayed considerable variation, some perivascular nerve networks appearing less susceptible than others to the action of capsaicin. The results suggest that there may be species differences in the sensitivity of CGRP-containing nerves to capsaicin treatment, but at least the majority of CGRP-immunoreactive cardiovascular nerves may be presumed to be sensory in origin.