Osteotropic effects by the neuropeptides calcitonin gene-related peptide, substance P and vasoactive intestinal peptide

Immunohistochemical phenotypic characterization of skeletal nerve fibers has demonstrated the expression of a restricted number of neuropeptides, including calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP). According to the neuro-osteological hypothesis, such neuropeptides can be released and exert paracrine biological effects on bone cells present close to the nerve endings expressing these signaling molecules. The existence of such interplay is most convincingly shown by the hypothalamic control of bone formation, in the case of leptin stimulation of hypothalamic nuclei mediated by the sympathetic nervous system and inhibitory beta-adrenergic receptors on osteoblasts. In addition to these receptors, osteoblasts and osteoclasts express functional receptors for CGRP, SP and VIP, which can regulate both bone formation and bone resorption. The evidence for these observations is summarized in the present paper.     

Epoxyeicosatrienoic acids are endogenous regulators of vasoactive neuropeptide release from trigeminal ganglion neurons

Epoxyeicosatrienoic acids (EETs) are bioactive eicosanoids produced from arachidonic acid by cytochrome P450 epoxygenases. We previously described the expression of cytochrome P450-2J epoxygenase in rat trigeminal ganglion neurons and that EETs signaling is involved in cerebrovascular dilation resulting from perivascular nerve stimulation. In this study, we evaluate the presence of the EETs signaling pathway in trigeminal ganglion neurons and their role in modulating the release of calcitonin gene-related peptide (CGRP) by trigeminal ganglion neurons. Liquid chromatography tandem mass spectrometry identified the presence of each of the four EETs regio-isomers within primary trigeminal ganglion neurons. Stimulation for 1 h with the transient receptor potential vanilloid-1 channel agonist capsaicin (100 nmol/L) or depolarizing K(+) (60 mmol/L) increased CGRP release as measured by ELISA. Stimulation-evoked CGRP release was attenuated by 30 min pre-treatment with the EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 μmol/L). K(+) stimulation elevated CGRP release 2.9 ± 0.3-fold above control levels, whereas in the presence of 14,15-EEZE K(+)-evoked CGRP release was significantly reduced to 1.1 ± 0.2-fold above control release (p < 0.01 anova, n = 6). 14,15-EEZE likewise attenuated capsaicin-evoked CGRP release from trigeminal ganglion neurons (p < 0.05 anova, n = 6). Similarly, pre-treatment with the cytochrome P450 epoxygenase inhibitor attenuated stimulation-evoked CGRP release. These data demonstrate that EETs are endogenous constituents of rat trigeminal ganglion neurons and suggest that they may act as intracellular regulators of neuropeptide release, which may have important clinical implications for treatment of migraine, stroke and vasospasm after subarachnoid hemorrhage.     

Differential Trk expression in explant and dissociated trigeminal ganglion cell cultures

During embryonic development, expression of neurotrophin receptor tyrosine kinases (Trks) by sensory ganglia is continuously and dynamically regulated. Neurotrophin signaling promotes selective survival and axonal differentiation of sensory neurons. In embryonic day (E) 15 rat trigeminal ganglion (TG), NGF receptor TrkA is expressed by small diameter neurons, NT-3 receptor TrkC and BDNF receptor TrkB are expressed by large diameter neurons. Organotypic explant and dissociated cell cultures of the TG (and dorsal root ganglia) are commonly used to assay neurotrophin effects on developing sensory neurons. In this study, we compared Trk expression in E15 rat TG explant and dissociated cell cultures with or without neurotrophin treatment. Only a subset of TG cells express each of the three Trk receptors in wholemount explant cultures as in vivo conditions. In contrast, all TG neurons co-express all three Trk receptors upon dissociation, regardless of neurotrophin treatment. Neurons cultured in low concentrations of one neurotrophin first, and switched to higher concentrations of another after 1 day, survive and display morphological characteristics of neurons cultured in a mixture of both neurotrophins for 3 days. Our results indicate that wholemount explant cultures of sensory ganglia represent in vivo conditions in terms of Trk expression patterns; whereas dissociation dramatically alters Trk expression by primary sensory neurons.     

Monocytes of allergics and non-allergics produce, store and release the neurotrophins NGF, BDNF and NT-3

INTRODUCTION: Recent studies have shown that neurotrophins (NTs) are involved in inflammatory processes. Elevated plasma levels of NTs were found allergic diseases with the highest levels in allergic asthma. However, the exact cellular sources involved in the regulation and release of neurotrophins in allergic inflammation are still not well defined. OBJECTIVE: The aim of this study was to assess whether monocytes of allergic and non-allergic subjects produce, store and release the neurotrophins NGF, BDNF and NT-3. METHODS: Monocytes of allergic and non-allergic donors were purified by immunomagnetic selection. APAAP-staining for the presence of NTs and their receptors was performed. RT-PCR and Western blot evaluated the production and storage of NTs. Monocytes were incubated and supernatants were collected for measurement of neurotrophic factors after stimulation with lipopolysaccharide (LPS) as inflammatory stimulus. The neurotrophin content in lysates and cell culture supernatants was determined by ELISA. RESULTS: Human monocytes express the neurotrophins NGF, BDNF and NT-3 but also their specific receptors TrkA, TrkB and TrkC. RT-PCR amplification of isolated mRNA demonstrated expression of the examined neurotrophins. Proteins were detectable by Western blot. NTs were found in the monocyte lysates and supernatants at different levels in allergic and non-allergic donors. Cell stimulation with LPS leads to release of NGF and NT3. CONCLUSIONS: Monocytes, produce, store and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.     

Calcitonin gene-related peptide, neurokinin A and substance P: effects on nociception and neurogenic inflammation in human skin and temporal muscle.

Calcitonin gene-related peptide (CGRP) was injected alone and in combination with substance P (SP) or neurokinin A (NKA) into the forearm skin and temporal muscle of human volunteers. In the skin, 50 pmol of CGRP induced a wheal response and a delayed erythema. No pain was recorded. No interaction between CGRP and SP or NKA was observed. In the temporal muscle, 200 pmol of CGRP alone did not induce pain or tenderness but, in combination with SP or NKA, CGRP elicited a significant pain sensation. It is concluded that CGRP may be involved in neurogenic inflammation and that only SP, of the three peptides present in nociceptive C fibers, seems to be of major importance in relation to cutaneous nociception. Simultaneous neurogenic release of CGRP and other neuropeptides in skeletal muscle may induce myofascial pain.     

Transient receptor potential vanilloid 1, calcitonin gene-related peptide, and substance P mediate nociception in acute pancreatitis

The mechanism of pancreatitis-induced pain is unknown. In other tissues, inflammation activates transient receptor potential vanilloid 1 (TRPV1) on sensory nerves to liberate CGRP and substance P (SP) in peripheral tissues and the dorsal horn to cause neurogenic inflammation and pain, respectively. We evaluated the contribution of TRPV1, CGRP, and SP to pancreatic pain in rats. TRPV1, CGRP, and SP were coexpressed in nerve fibers of the pancreas. Injection of the TRPV1 agonist capsaicin into the pancreatic duct induced endocytosis of the neurokinin 1 receptor in spinal neurons in the dorsal horn (T10), indicative of SP release upon stimulation of pancreatic sensory nerves. Induction of necrotizing pancreatitis by treatment with L-arginine caused a 12-fold increase in the number of spinal neurons expressing the proto-oncogene c-fos in laminae I and II of L1, suggesting activation of nociceptive pathways. L-arginine also caused a threefold increase in spontaneous abdominal contractions detected by electromyography, suggestive of referred pain. Systemic administration of the TRPV1 antagonist capsazepine inhibited c-fos expression by 2.5-fold and abdominal contractions by 4-fold. Intrathecal, but not systemic, administration of antagonists of CGRP (CGRP(8-37)) and SP (SR140333) receptors attenuated c-fos expression in spinal neurons by twofold. Thus necrotizing pancreatitis activates TRPV1 on pancreatic sensory nerves to release SP and CGRP in the dorsal horn, resulting in nociception. Antagonism of TRPV1, SP, and CGRP receptors may suppress pancreatitis pain.     

Substance P and calcitonin gene-related peptide intrinsic choroidal neurons in human choroidal whole-mounts

To determine the presence in the human choroid of substance P (SP)-and calcitonin gene-related peptide (CGRP) positive intrinsic choroidal neurons (ICNs), choroidal whole-mounts were processed for indirect immunofluorescence. An antibody to a component of the neuronal cytoskeleton, neurofilament 200 kDa (NF-200), was combined with antibodies to SP and to CGRP (neuropeptides proper to the sensory nervous system). The human choroid possesses numerous SP(+) and CGRP(+) ICNs. These neurons were observed in the suprachoroid, both in isolation and forming microganglia. For both types of ICNs studied, neurons were more numerous in the temporal than in the nasal regions. In both locations, SP(+) and CGRP(+) ICNs were more abundant in the central choroid (the choroid underneath the macular area of the retina), with cell density diminishing outwards to the choroidal periphery. There were no appreciable differences between the two populations of ICNs studied in terms of size, morphology or immunostaining characteristics. In conclusion, given that peripheral sensory innervation could be involved in the regulation of both choroidal blood flow and vascular architecture, the SP(+) and CGRP(+) ICNs described for the first time in the present work may be involved in these mechanisms of vascular regulation.     

Sensory innervation of the ear drum and middle-ear mucosa: Retrograde tracing and immunocytochemistry

Summary The distribution and origin of nerve fibers of presumed sensory nature in the ear drum and middle-ear mucosa of the rat were studied by a retrograde tracing technique in combination with immunocytochemistry.Application of True Blue (TB) on the ear drum or on the middle-ear mucosa labeled nerve cell bodies in the jugular, trigeminal, geniculate and cervical dorsal root ganglia (C₂–C₄). Judging from the number of TB-labeled nerve cell bodies the jugular and trigeminal ganglia contributed the major component to the sensory innervation of the ear drum and the middle-ear mucosa, while the contribution from the geniculate and cervical dorsal root ganglia was relatively minor.The majority of the TB-labeled nerve cell bodies contained calcitonin gene-related peptide (CGRP), whereas minor populations stored substance P (SP) and neurokinin A (NKA). Nerve fibers containing SP, NKA and CGRP were moderate in number in the middle-ear mucosa and few in the ear drum. Double immunostaining revealed that SP invariably coexisted with NKA in nerve cell bodies in the ganglia examined. The SP/NKA-containing nerve cell bodies constituted a subpopulation of those storing CGRP.The findings indicate that several ganglia project to the ear drum and middle-ear mucosa and that many neuropeptides are involved in the mediation of middle-ear sensitivity.     

Sonic hedgehog, secreted by amacrine cells, acts as a short-range signal to direct differentiation and lamination in the zebrafish retina

Neurogenesis in the zebrafish retina occurs in several waves of differentiation. The first neurogenic wave generates ganglion cells and depends on hedgehog (hh) signaling activity. Using transgenic zebrafish embryos that express GFP under the control of the sonic hedgehog (shh) promoter, we imaged the differentiation wave in the retina and show that, in addition to the wave in the ganglion cell layer, shh expression also spreads in the inner nuclear layer. This second wave generates amacrine cells expressing shh, and although it overlaps temporally with the first wave, it does not depend on it, as it occurs in the absence of ganglion cells. We also show that differentiation of cell types found in the inner and outer nuclear layers, as well as lamination of the retina, depends on shh. By performing mosaic analysis, we demonstrate that Shh directs these events as a short-range signal within the neural retina.     

Changes in retinal expression of neurotrophins and neurotrophin receptors induced by ocular hypertension

Open angle glaucoma is defined as a progressive and time-dependent death of retinal ganglion cells concomitant with high intraocular pressure, leading to loss of visual field. Because neurotrophins are a family of growth factors that support neuronal survival, we hypothesized that quantitative and qualitative changes in neurotrophins or their receptors may take place early in ocular hypertension, preceding extensive cell death and clinical features of glaucoma. We present molecular, biochemical, and phenotypic evidence that significant neurotrophic changes occur in retina, which correlate temporally with retinal ganglion cell death. After 7 days of ocular hypertension there is a transient up-regulation of retinal NGF, while its receptor TrkA is up-regulated in a sustained fashion in retinal neurons. After 28 days of ocular hypertension there is sustained up-regulation of retinal BDNF, but its receptor TrkB remains unchanged. Throughout, NT-3 levels remain unchanged but there is an early and sustained increase of its receptor TrkC in Müller cells but not in retinal ganglion cells. These newly synthesized glial TrkC receptors are truncated, kinase-dead isoforms. Expression of retinal p75 also increases late at day 28. Asymmetric up-regulation of neurotrophins and neurotrophin receptors may preclude efficient neurotrophic rescue of RGCs from apoptosis. A possible rationale for therapeutic intervention with Trk receptor agonists and p75 receptor antagonists is proposed.     

Role of capsaicin-sensitive afferents and sensory neuropeptides in endotoxin-induced airway inflammation and consequent bronchial hyperreactivity in the mouse

Substance P (SP) and calcitonin gene-related peptide (CGRP) released from capsaicin-sensitive afferents induce neurogenic inflammation via NK(1), NK(2) and CGRP1 receptor activation. This study examines the role of capsaicin-sensitive fibres and sensory neuropeptides in endotoxin-induced airway inflammation and consequent bronchial hyperreactivity with functional, morphological and biochemical techniques in mice. Carbachol-induced bronchoconstriction was measured with whole body plethysmography 24 h after intranasal lipopolysaccharide administration. SP and CGRP were determined with radioimmunoassay, myeloperoxidase activity with spectrophotometry, interleukin-1beta with ELISA and histopathological changes with semiquantitative scoring from lung samples. Treatments with resiniferatoxin for selective destruction of capsaicin-sensitive afferents, NK(1) antagonist SR 140333, NK(2) antagonist SR 48968, their combination, or CGRP1 receptor antagonist CGRP(8-37) were performed. Lipopolysaccharide significantly increased lung SP and CGRP concentrations, which was prevented by resiniferatoxin pretreatment. Resiniferatoxin-desensitization markedly enhanced inflammation, but decreased bronchoconstriction. CGRP(8-37) or combination of SR 140333 and SR 48968 diminished neutrophil accumulation, MPO levels and IL-1beta production, airway hyperresponsiveness was inhibited only by SR 48968. This is the first evidence that capsaicin-sensitive afferents exert a protective role in endotoxin-induced airway inflammation, but contribute to increased bronchoconstriction. Activation of CGRP1 receptors or NK(1)+NK(2) receptors participate in granulocyte accumulation, but NK(2) receptors play predominant role in enhanced airway resistance.     

Distribution and changes with age of calcitonin gene-related peptide- and substance P-immunoreactive nerves of the rat urinary bladder and lumbosacral sensory neurons

In the distal parts of the urinary tract, nerves containing calcitonin gene-related peptide (CGRP) or substance P (SP) are sensory with their cell bodies located in lumbosacral dorsal root ganglia. These two neuropeptides are recognised as being present in pelvic sensory nerves, and may be involved in the mediation of pain, stretch and/or vasodilatation. We have used indirect immunohistochemical techniques to examine the distribution and regional variation of nerves immunoreactive (-ir) for CGRP and SP in the urinary bladder and in neurons in lumbosacral dorsal root ganglia (L1-L2 & L6-S1) of young adult (3 months) and aged (24 months) male rats. Semi-quantitative estimations of nerve densities were made for CGRP-ir and SP-ir fibres innervating the dome, body and base of the urinary bladder. Quantitative studies were also used to examine the effects of age on the percentage of dorsal root ganglion neurons immunoreactive for CGRP and SP. There were very few immunoreactive axons in the dome and the overall density of innervation increased progressively towards the base of the bladder. The density of innervation in the aged rats revealed a slight reduction in CGRP and SP innervation of the detrusor muscle but was otherwise comparable to the young group. However, immunostaining of the lumbosacral dorsal root ganglia revealed that the percentage of CGRP- and SP-ir neuronal profiles showed a significant (P < 0.05) reduction from (mean +/- S.D) 44.5 +/- 2; 23.3 +/- 2 in young adult to 25.0 +/- 2.9; 14.8 +/- 1.6 in aged rats, respectively. These findings suggest that the involvement of CGRP and SP in urinary bladder innervation is relatively unchanged in old age, but their expression in dorsal root ganglion neurons is affected by age. The afferent micturition pathway from the pelvic region via these lumbosacral ganglia may be perturbed as a result.     

Effect of semax and its C-terminal peptide PGP on expression of neurotrophins and their receptors in rat brain during incomplete global ischemia

Neurotrophins regulate key functions of nervous tissue cells. Analysis of neurotrophin mRNA expression is an appropriate tool to assess therapeutic efficiency of antistroke drugs. We have analyzed the effect of synthetic peptide semax and its C-terminal Pro-Gly-Pro tripeptide on mRNA expression of neurotrophins Ngf, Bdnf, and Nt-3 and their receptors TrkA, TrkB, TrkC, and p75 in rat frontal cortex, hippocampus, and cerebellum after bilateral common carotid artery occlusion. The animals were decapitated at 30 min and 1, 2, 4, 8, 12, and 24 h after the operation. The mRNA expression of neurotrophins and their receptors was assessed by relative quantification using real-time RT-PCR. Our results demonstrated that ischemia caused a significant decrease in gene expression in the hippocampus. Semax and PGP treatment affected the expression of neurotrophins and their receptors predominantly in the frontal cortex and hippocampus of the ischemized animals. In the frontal cortex, Semax treatment resulted in a decrease of mRNA level of neurotrophin receptors, while PGP treatment increased the level of these mRNA. Maximal neuroprotective effect of both peptides was observed in the hippocampus 12 h after occlusion. A decrease of gene expression of neurotrophins and their receptors caused by the occlusion was overcome by Semax and PGP. These results clarify the mechanism of Semax action and reveal certain features of mRNA expression of neurotrophins and their receptors under experimental conditions.     

Distribution of somatostatin receptor 5 in mouse and bullfrog retinas

Somatostatin (SRIF), as a neuroactive peptide in the CNS, may act as a neuromodulator through activation of five specific receptor subtypes (sst(1)-sst(5)). In this work we conducted a comparative study of the expression of sst(5) in mouse and bullfrog retinas by immunofluorescence double labeling. Basically, the expression profiles of sst(5) in the retinas of the two species were similar. That is, in the inner retina sst(5) was localized to dopaminergic and cholinergic amacrine cells, stained by tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) respectively, and cells in the ganglion cell layer, whereas in the outer retina immunostaining for sst(5) was observed in horizontal cells. However, a more widespread, abundant distribution of labeling for sst(5), as compared to mouse retina, was seen in bullfrog retina: strong labeling for sst(5) was diffusely distributed in both outer and inner plexiform layers (OPL and IPL) in the bullfrog retina, but the labeling was only observed in the IPL of the mouse retina. In addition, bullfrog photoreceptors, both rods and cones, but not mouse ones, were labeled by sst(5). In combination with the experiments showing that SRIF-immunoreactivity was mainly found in the inner retina, our results suggest that SRIF, released from SRIF-containing cells in the inner retina, may play a neuromodulatory role in both outer and inner retina mediated by volume transmission via sst(5) in bullfrog retina, while the SRIF action may be largely restricted to the mouse inner retina.     

Neurokinin A and neurokinin B in the human retina

Very recently, the authors found levels of neurokinin (NK) A-like immunoreactivities in the human retina which were more than five times higher than those of substance P (SP). The present study aimed to find out how many of these immunoreactivities can be attributed to NKA and NKB and then the exact distribution pattern of both NKA and NKB was evaluated in the human retina and compared with that of SP. For this purpose, NKA-like immunoreactivities were characterized in the human retina by reversed phase HPLC followed by radioimmunoassay using the K12 antibody which recognizes both NKA and NKB. Furthermore, the retinae from both a 22- and 70-year-old donor were processed for double-immunofluorescence NKA/SP and NKB/SP. The results showed that NKA contributes to approximately two thirds and NKB to approximately one third of the immunoreactivities measured with the K12 antibody. NKA was found to be localized in sparse amacrine cells in the proximal inner nuclear layer, in displaced amacrine cells in the ganglion cell layer with processes ramifying in stratum 3 of the inner plexiform layer and also in sparse ganglion cells. By contrast, staining for NKB was only observed in ganglion cells and in the nerve fiber layer. Double-immunofluorescence revealed cellular colocalization of NKA with SP and also of NKB with SP. Thus, the levels of NKA and NKB are more than three and two times higher than those of SP, respectively. Whereas the distribution pattern of NKA is typical for neuropeptides, the localization of NKB exclusively in ganglion cells is atypical and unique.     

p75 neurotrophin receptor mediates neurotrophin activation of NF-kappa B and induction of iNOS expression in P19 neurons

P19 embryonic carcinoma cells can be differentiated into neurons that form synaptic connections and that produce a variety of neurotransmitters. Results of RT-PCR indicate that P19 neurons express several neurotrophin receptors (p75(NTR), trkB, and trkC, but not trkA) but they do not express any of the four neurotrophins. Consistent with the presence of trkB but not trkA, BDNF causes rapid phosphorylation of MAP kinases ERK1 and ERK2, but NGF does not. Neurotrophins induce translocation of NF-kappaB into the nucleus. All four neurotrophins induce activation of NF-kappaB in a biphasic manner. This effect is apparently mediated by p75(NTR), because an inhibitor of trk receptors, K252a, does not inhibit activation of NF-kappaB. Instead, K252a itself promotes activation of NF-kappaB and this effect is additive with the effect of neurotrophins. Inhibition of reactive oxygen intermediates with PDTC completely abolishes basal activity of NF-kappaB and strongly inhibits activation of NF-kappaB by neurotrophins, indicating an important role of reactive oxygen intermediates in the pathway by which neurotrophins activate NF-kappaB. NF-kappaB is known to promote expression of the iNOS gene. We found that all four neurotrophins increased iNOS mRNA levels, resulting in increased accumulation of iNOS protein. In contrast, none of the neurotrophins stimulated nNOS mRNA or protein synthesis. PDTC abolishes constitutive and neurotrophin-induced expression of iNOS mRNA and protein and abolishes constitutive expression of nNOS mRNA, suggesting that reactive oxygen intermediates promote expression of nNOS.     

Colitis induced by proteinase-activated receptor-2 agonists is mediated by a neurogenic mechanism

Proteinase-activated receptor-2 (PAR2) activation induces colonic inflammation by an unknown mechanism. We hypothesized that PAR2 agonists administered intracolonically in mice induce inflammation via a neurogenic mechanism. Pretreatment of mice with neurokinin-1 and calcitonin-gene-related peptide (CGRP) receptor antagonists or with capsaicin showed attenuated PAR2-agonist-induced colitis. Immunohistochemistry demonstrated a differential expression of a marker for the type-1 CGRP receptor during the time course of PAR2-agonist-induced colitis, further suggesting a role for CGRP. We conclude that PAR2-agonist-induced intestinal inflammation involves the release of neuropeptides, which by acting on their receptors cause inflammation. These results implicate PAR2 as an important mediator of intestinal neurogenic inflammation.