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.     

Neuropeptide (neuropeptide Y,neurotensin, vasoactive intestinal polypeptide,substance P,calcitonin gene-related peptide,somatostatin) immunohistochemistry and ultrastructure of renal nerves

Summary With the use of several region-specific antisera and the peroxidase-antiperoxidase (PAP) technique, several regulatory polypeptides were localized in nerves of the kidney. Neuropeptide Y (NPY)- immunoreactivity (IR), neurotensin (NT)-IR and vasoactive intestinal polypeptide (VIP)-IR occurred at high densities in all segments of the renal arterial system forming a perivascular plexus. Furthermore, NT-IR nerves were particularly frequent at the juxtaglomerular apparatus (JGA). Calcitonin gene-related peptide (CGRP)-IR was mainly concentrated in nerves supplying the hilus arteries and the JGA. Substance P (SP)-IR was predominantly found in large varicosities close to large renal arterial vessels and in the vicinity of the JGA. Somatostatin (SOM)-IR was only observed in single varicosities located at the media-adventitia border of large renal hilus arteries. The peptidergic nerves are correlated to their ultrastructural counterpart. In addition, the distribution patterns and the frequency of the different types of renal peptidergic nerve fibres are evaluated and compared. The functional role of these neuropeptides and their origin within the efferent branch of this part of the peripheral autonomic nervous system is discussed. Furthermore, the implication of some of the neuropeptides studied in afferent renal innervation is also substantiated.Dedicated to Prof. Dr. T.H. Schiebler on the occasion of his 65th birthday     

Neuropeptide (neuropeptide Y, neurotensin, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, somatostatin) immunohistochemistry and ultrastructure of renal nerves

With the use of several region-specific antisera and the peroxidase-antiperoxidase (PAP) technique, several regulatory polypeptides were localized in nerves of the kidney. Neuropeptide Y (NPY)- immunoreactivity (IR), neurotensin (NT)-IR and vasoactive intestinal polypeptide (VIP)-IR occurred at high densities in all segments of the renal arterial system forming a perivascular plexus. Furthermore, NT-IR nerves were particularly frequent at the juxtaglomerular apparatus (JGA). Calcitonin gene-related peptide (CGRP)-IR was mainly concentrated in nerves supplying the hilus arteries and the JGA. Substance P (SP)-IR was predominantly found in large varicosities close to large renal arterial vessels and in the vicinity of the JGA. Somatostatin (SOM)-IR was only observed in single varicosities located at the media-adventitia border of large renal hilus arteries. The peptidergic nerves are correlated to their ultrastructural counterpart. In addition, the distribution patterns and the frequency of the different types of renal peptidergic nerve fibres are evaluated and compared. The functional role of these neuropeptides and their origin within the efferent branch of this part of the peripheral autonomic nervous system is discussed. Furthermore, the implication of some of the neuropeptides studied in afferent renal innervation is also substantiated.     

Innervation of human omental arteries and veins and vasomotor response to noradrenaline, neuropeptide Y, substance P and vasoactive intestinal peptide

Human omental arteries and veins are supplied with nerve fibers containing noradrenaline (NA) and neuropeptide Y (NPY); these two agents probably co-exist in perivascular sympathetic nerve fibers. Substance P (SP)- or vasoactive intestinal peptide (VIP)-containing fibers could not be detected. In studies on isolated omental vessels NA produced constriction. The results of blockade experiments suggest that human omental arteries are equipped predominantly with alpha 1-adrenoceptors and omental veins with a mixture of alpha 1- and alpha 2-adrenoceptors. NPY at a concentration of 10(-7) M or higher had a weak contractile effect on veins and virtually no effect on arteries. NPY at a concentration of 3 X 10(-8) M shifted the NA concentration response curve to the left in arteries (pD2 = 5.8 for NA versus 6.6. for NA in the presence of NPY; P less than 0.001) but not in veins. Both SP and VIP relaxed arteries precontracted with NA or prostaglandin F2 alpha (PGF2 alpha). The potency of SP as a relaxant agent was similar in arteries and veins; the effect of VIP was elicited at lower concentrations in veins than in arteries.     

Cardiovascular effects of calcitonin gene-related peptide in the pithed rat: comparison with substance P

Recently it has been shown that calcitonin gene-related peptide (CGRP) and substance P (SP) are coexisting in central and peripheral nerve endings of sensory neurons. In the present study we compared the vasodepressor and plasma extravasating activity of CGRP with that of SP. Systemic administration of CGRP to pithed, vagotomized rats evoked a dose dependent, long lasting vasodilation accompanied by a parallel rise in heart rate. The tachycardic response to CGRP may indicate a direct positive chronotropic action on the heart since this effect could not be blocked by beta-adrenoceptor blockade. For any equimolar dose the hypotensive effect of CGRP was much larger than that of SP. Both, CGRP and SP, showed a more pronounced decrease in mean arterial blood pressure after elevation of basal blood pressure levels by constant infusion of either phenylephrine, arginine-vasopressin, or angiotensin II. After systemic administration in equimolar doses CGRP was much less effective in producing plasma extravasation than SP. In conclusion, at equimolar doses CGRP is 10 times more potent than SP in producing vasodilatation but it possesses less than a third of the potency of equimolar doses of SP in producing plasma extravasation.     

Quadruple colocalization of calretinin,calcitonin gene-related peptide,vasoactive intestinal peptide,and substance P in fibers within the villi of the rat intestine

Double-labeling immunofluoresenct histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide-containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi.     

Ventilatory effects of substance P, vasoactive intestinal peptide, and nitroprusside in humans.

Animal studies suggest that the neuropeptides, substance P and vasoactive intestinal peptide (VIP), may influence carotid body chemoreceptor activity and that substance P may take part in the carotid body response to hypoxia. The effects of these peptides on resting ventilation and on ventilatory responses to hypoxia and to hypercapnia have been investigated in six normal humans. Infusions of substance P (1 pmol.kg-1.min-1) and of VIP (6 pmol.kg-1.min-1) were compared with placebo and with nitroprusside (5 micrograms.kg-1.min-1) as a control for the hypotensive action of the peptides. Both peptides caused significantly less hypotension than nitroprusside. Substance P and nitroprusside caused significantly greater increases in ventilation and in the hypoxic ventilatory response than VIP. No changes were seen in hypercapnic sensitivity. The stimulation of ventilation and the differential effects on ventilatory chemosensitivity that accompanied hypotension are consistent either with stimulation of carotid body chemoreceptor activity or with an interaction with peripheral chemoreceptor input to the respiratory center, as is seen in animals. The similar cardiovascular but different ventilatory effects of the peptides suggest that substance P may also stimulate the carotid body in a manner independent of the effect of hypotension. This is consistent with a role of substance P in the hypoxic ventilatory response in humans.     

Effect of calcitonin gene-related peptide,neuropeptide Y,substance P,and vasoactive intestinal peptide on interleukin-1beta,interleukin-6 and tumor necrosis factor-alpha production by peripheral whole blood cells from rheumatoid arthritis and osteoarthritis patients

In the present study, we have investigated the in vitro effect of calcitonin-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP) and vasoactive intestinal peptide (VIP) at concentrations of 10(-8), 10(-9) and 10(-10) M on the production of different proinflammatory cytokines or chemokines such as IL-1beta, IL-6 and TNFalpha by peripheral whole blood cells from patients with rheumatoid arthritis, as well as from osteoarthritis patients studied as a control group without immunoinflammatory background. We have found that CGRP, NPY, SP and VIP stimulated significantly the production of those cytokines and chemokines in rheumatoid arthritis patients. In general, the stimulation was higher at the 10(-9) M concentration, with SP and VIP, and in rheumatoid arthritis patients compared to osteoarthritis ones. Neuropeptides did not significantly modify the LPS-induced cytokine production by whole blood cells. The results indicate that physiological concentrations of the neuropeptides studied can modulate the inflammatory and immunological response, stimulating significantly the production of inflammatory cytokines by human whole blood cells in rheumatoid arthritis patients, as well as, in a minor way, in osteoarthritis patients.     

Selective localization of vasoactive intestinal peptide and substance P in human eosinophils

Extracts of purified human eosinophils had a mean concentration of 72 fmol of immunoreactive vasoactive intestinal peptide and 21 fmol of substance P per 10(7) eosinophils, that were significantly higher than the content of immunoreactivity of the same neuropeptides in neutrophils, mononuclear leukocytes, and platelets. In contrast, the lower concentrations of calcitonin gene-related peptide and somatostatin were similar in extracts of all leukocytes. Chromatography of the peptides from eosinophils confirmed their identity with vasoactive intestinal peptide and substance P from neuroendocrine sources. Stores of some neuropeptides may endow eosinophils with unique roles in host defense and hypersensitivity reactions.     

Catabolism of calcitonin gene-related peptide and substance P by neutral endopeptidase.

Calcitonin gene-related peptide (CGRP) and substance P (SP) are released from sensory nerves upon exposure to irritating stimuli. Neutral endopeptidase (NEP), a membrane-bound peptidase, cleaves many peptides including SP, thereby limiting their biological actions. Recombinant NEP cleaved CGRP1 approximately 88-fold less rapidly than it cleaved SP. The slow cleavage by NEP of CGRP compared to SP suggests that this enzyme is likely to have weaker physiologic effects on CGRP than have been demonstrated for SP.     

Effects of substance P and calcitonin gene-related peptide on the pulmonary circulation.

To assess the in vivo effects of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) on the pulmonary vascular bed, the hemodynamic responses to both CGRP and SP were examined in the in situ-perfused lung lobe of open-chest anesthetized pigs. Peptides were infused into the lobar artery under conditions of elevated pulmonary vascular tone by prostaglandin F2 alpha (PGF2 alpha, 20 micrograms/min). Pulmonary airway lobar dynamic compliance (Cdyn) and airway resistance (Re) were computed from simultaneously measured airway pressure and airflow entering the lobe through a Carlens endobronchial divider. PGF2 alpha infusion slightly reduced Cdyn (-20%) and increased Re (+11%) while lobar arterial pressure rose from 14 +/- 1 to 31 +/- 2 mmHg (n = 12). In these conditions, lobar artery infusion of SP (0.5-50 pmol/min) or CGRP (15-5,000 pmol/min) produced a dose-dependent decrease in the pressor response to PGF2 alpha, reaching -54 +/- 3 and -64 +/- 7%, respectively, without alterations in lung mechanics. On a molar basis, SP was more effective than CGRP; its vasodilatory effect was more rapid and of shorter duration. Higher CGRP infusion rates were not studied because of marked systemic hypotension. SP infused at 150, 500, and 1,000 pmol/min significantly reduced Cdyn by 12 +/- 2, 24 +/- 4, and 62 +/- 7%, respectively, but also induced a rise in lobar arterial pressure and a fall in systemic arterial pressure. The results show that both SP and CGRP are potent pulmonary vasodilators. In contrast to CGRP, which did not affect lung mechanics, high infusion rates of SP decreased Cdyn and increased Re.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Distribution of vasoactive intestinal peptide and calcitonin gene-related peptide immunoreactive nerve fibers and binding sites in the hamster seminal vesicle during post-natal development.

The distribution of vasoactive intestinal peptide (VIP)- and calcitonin gene-related peptide (CGRP)-immunoreactive nerves and 125I-labeled VIP- and CGRP-binding sites was studied in the hamster seminal vesicle of 12-, 30- and 60-day-old animals. In addition, the general innervation of the seminal vesicle was examined using the general neuronal marker synaptophysin. Our results show that the densities of the overall (synaptophysin immunoreactive) and CGRP-immunoreactive innervation is constant during the post-natal development of the gland. However, a significant decrease in VIP-containing nerves is observed at the end of puberty. The autoradiographic study revealed that in 12-day-old animals, the epithelium presents VIP binding sites. However, in 30-day-old animals, VIP binding sites are observed in the epithelium of only a few clumps of acini. In 60-day-old animals, the gland is composed of acini with dilated lumina where VIP binding sites are not detected. In all groups studied the epithelium does not exhibit CGRP binding sites. The seminal vesicle muscle layer displays specific binding sites for both VIP and CGRP at all post-natal developmental times, but the density of VIP binding sites is higher in 12- than in 30- and 60-day-old animals. Our results, showing the presence of specific VIP and CGRP binding sites during the development of the hamster seminal vesicle, suggest that these neuropeptides may be involved in the growth and differentiation of the gland.     

Calcitonin gene-related peptide: antinociceptive activity in rats, comparison with calcitonin

The effects of synthetic human calcitonin gene-related peptide (CGRP) on nociceptive response were evaluated in rats by two behavioral tests (tail-flick and hot-plate) and by electrophysiological recording of the firing of thalamic neurons evoked by peripheral noxious mechanical stimuli. CGRP was administered intracerebroventricularly (i.c.v.) and its effects were compared with that of salmon calcitonin (sCT). In the tail-flick test, CGRP (0.25, 2.5 and 5 micrograms/rat) dose-dependently increased response latencies, whereas sCT (0.125, 2.5, 5 and 10 micrograms/rat) did not. Conversely, in the hot-plate test CGRP was effective in enhancing response latencies only at the highest dose of 10 micrograms/rat, while sCT (0.125, 0.25 and 2.5 micrograms/rat) inhibited the hot-plate response dose-dependently. In electrophysiological studies, CGRP (2.5 micrograms/rat, i.c.v.) completely inhibited the evoked neuronal thalamic firing and the same dose of sCT induced only a partial reduction. Furthermore, the antinociceptive effects of CGRP in the tail-flick test and in the electrophysiological studies were not prevented by naloxone. These results demonstrate that central administration of CGRP is effective in inhibiting nociceptive responses and its action like that of sCT does not involve an opioid mechanism. The differences in the antinociceptive profiles of CGRP and sCT suggest that the inhibitory effects of these peptides may involve different neuronal pathways.     

Differential effect of vasoactive intestinal peptide, somatostatin, and substance P on human IgE and IgG subclass production.

We studied the effect of vasoactive intestinal peptide (VIP), somatostatin (SOM), and substance P (SP) on IL-4-stimulated human IgE and IgG subclass production. VIP and SOM, but not SP, inhibited IgE production without affecting IgM or IgA production by mononuclear cells (MNC) from nonatopic donors from 10 pM to 10 nM. These neuropeptides also differentially modulated IgG subclass production. While IgG1 production was not affected by VIP, SOM, or SP, all of the neuropeptides enhanced IgG2 production. By contrast, SOM and SP, but not VIP, inhibited IgG3 production, whereas VIP and SP, but not SOM, enhanced IgG4 production. The effect by neuropeptides was specific since each peptide effect was specifically blocked by each antagonist. To achieve this effect, neuropeptides must be added at the start of the culture and be present throughout the entire culture period. The inhibition of IgE production was not mediated by known inhibitors of IgE production, IFN-gamma or PGE2, because the addition of anti-IFN-gamma mAb (10 micrograms/ml) or indomethacin (0.1 microM) did not overcome the inhibition of IgE production. In contrast to MNC, neuropeptides did not affect IgG subclass production in purified B cells. IgE production was not induced by IL-4 in purified B cells. Neuropeptides also failed to modulate IgG subclass production in cultures of B cells with either T cells or monocytes. However, they modulated IgE production and IgG subclass production in B cells in the presence of T cells and monocytes. In purified B cells, IL-4 plus anti-CD40 mAb induced IgE production which was not inhibited by VIP or SOM. However, VIP or SOM, but not SP, inhibited IgE production in B cells cultured with both T cells and monocytes. Finally, the mechanism of modulation of IgE and IgG4 production was dependent on IL-4-induced switching, since neuropeptides modulated IgG4 and IgE production in surface IgG4-negative (sIgG4-) and sIgE- B cells, respectively. In contrast, modulation of IgG2 and IgG3 production was not due to switching, since neuropeptides did not affect either IgG2 or IgG3 production in sIgG2- or sIgG3- B cells, respectively.     

Distribution and dilatory effect of vasoactive intestinal polypeptide (VIP) in human cerebral arteries

Vasoactive intestinal polypeptide (VIP)-containing nerve fibers were demonstrated in human pial arteries by immunocytochemistry. Fine varicose fibers were located in the adventitia close to the media layer. Measurements by radioimmunoassay revealed concentrations of VIP between 0.7 and 2.7 pmol/g in the major arteries at the base of the brain, obtained at autopsy. Isolated human pial arteries, obtained in conjunction with neurosurgery, relaxed in a concentration-dependent manner upon administration of VIP. The relaxation of the vessels amounted to 57 +/- 9% of the contraction elicited by prostaglandin F2 alpha (2.5 microM) with an EC50 value of (8.5 +/- 1.2) X 10(-9) M.     

Endothelin as a putative sensory neuropeptide in the guinea-pig: different properties in comparison with calcitonin gene-related peptide

Both endothelin-(ET) and calcitonin gene-related peptide- (CGRP) like immunoreactivity (-LI) were present in a variety of organs and neuronal tissue of the guinea-pig as determined by radioimmunoassay (RIA). Neuronal tissues like dorsal root ganglia (DRG) contained by far the highest levels of both ET- (65 +/- 11 pmol/g) and CGRP-LI (34 +/- 5 pmol/g). The tissue levels of ET-LI remained unchanged after 6-hydroxydopamine and capsaicin-pretreatment, while CGRP-LI was markedly reduced after capsaicin. Chromatographic characterization revealed that the main portion of ET-LI in the DRG, right atrium and lung corresponded to synthetic ET-1. Immunohistochemical studies showed the presence of ET-LI in a few neurons of intact DRG and many neurons in DRG cell-cultures, partly co-existing with CGRP-LI. In the neuronal cells of DRG cultures the ratio between the ET- and CGRP-LI was 1:27 compared to 2:1 in intact DRG. 24 h after ligation of the sciatic or vagal nerves no accumulation of ET-LI was observed above the ligation, while CGRP-LI was increased 4-5-fold. Transection (10 days) of the sciatic nerve caused a 85-95% depletion of CGRP-LI in the distal skin, gastrocnemius muscle and trunk below the transection site, while in the proximal portion of the nerve CGRP-LI increased. No effects on ET-LI in these tissues were observed after sciatic nerve transfection. In release experiments on DRG cell cultures. Langendorff heart preparations or perfused guinea-pig lungs, potassium (60 mM), capsaicin or antidromic nerve stimulation evoked a clear-cut increase in the supernatant levels of CGRP-LI, suggesting release, while no effect on the ET-LI concentration was observed in the effluent. Furthermore, anoxia failed to influence the outflow of ET-LI from the heart and lung. It is concluded that ET-1-LI is present in high levels in spinal ganglia and ET-LI occurs in afferent cell-bodies, but in comparison with CGRP, ET shows remarkable inertness upon various experimental conditions including no evidence for axonal transport, loss after denervation or release. The neuronal ET-LI seems to increase under culture conditions, however. The possible function for the high content of ET-LI in the intact guinea-pig peripheral nervous system remains to be elucidated and may mainly be related to a non-neuronal pool considering the relatively low content of ET-LI compared to CGRP in cultured DRG cells.