Peptidergic Nerve Research Articles

Quantitative Evaluation of Synaptophysin-Like Immunoreactive Nerve Terminals or Varicosities in Anterior Pituitary of Normal and Adrenalectomized Rats

Our previous studies have demonstrated the presence of substantial amounts of peptidergic nerve fibres in the anterior pituitary of monkeys, dogs and rats. The nerve fibres increase in number following adrenalectomy and ovariectomy. Furthermore, typical synapses between nerve fibres and anterior pituitary glandular cells have been demonstrated in the dog and rat, strongly suggesting the existence of some direct neural regulation. However, it is difficult to evaluate the functional importance of synapses or nerve terminals in general, unless quantitative data can be provided. The present study therefore aimed to quantify the number of terminals or varicosities in the anterior pituitary of the rat and its changes following adrenalectomy. Nerve terminals or varicosities were identified by their synaptophysin (SYN)-like immunoreactivity. Under the light microscope, SYN-like immunoreactivity appeared mainly as dots, mostly among glandular cells, and some were found distributed along blood vessels. SYN-like immunoreactive dots were found under the electron microscope to be localized in the nerve terminals or varicosities. Synapse-like contracts between the nerve terminals or varicosities and the glandular cells could be identified. The number of SYN-like immunoreactive nerve terminals or varicosities was then counted under the light microscope. There were as many as 12424.2 ± 2941.5 and 20700.4 ± 6128 (mean ± SD) nerve terminals or varicosities per anterior pituitary of normal and adrenalectomized rats, respectively. The increase in number following adrenalectomy was statistically significant. It is concluded that nerve fibres may play an important role in the regulation of the anterior pituitary.

Quantitative evaluation of synaptophysin-like immunoreactive nerve terminals or varicosities in anterior pituitary of normal and adrenalectomized rats.

Our previous studies have demonstrated the presence of substantial amounts of peptidergic nerve fibres in the anterior pituitary of monkeys, dogs and rats. The nerve fibres increase in number following adrenalectomy and ovariectomy. Furthermore, typical synapses between nerve fibres and anterior pituitary glandular cells have been demonstrated in the dog and rat, strongly suggesting the existence of some direct neural regulation. However, it is difficult to evaluate the functional importance of synapses or nerve terminals in general, unless quantitative data can be provided. The present study therefore aimed to quantify the number of terminals or varicosities in the anterior pituitary of the rat and its changes following adrenalectomy. Nerve terminals or varicosities were identified by their synaptophysin (SYN)-like immunoreactivity. Under the light microscope, SYN-like immunoreactivity appeared mainly as dots, mostly among glandular cells, and some were found distributed along blood vessels. SYN-like immunoreactive dots were found under the electron microscope to be localized in the nerve terminals or varicosities. Synapse-like contracts between the nerve terminals or varicosities and the glandular cells could be identified. The number of SYN-like immunoreactive nerve terminals or varicosities was then counted under the light microscope. There were as many as 12424.2 +/- 2941.5 and 20700.4 +/- 6128 (mean +/- SD) nerve terminals or varicosities per anterior pituitary of normal and adrenalectomized rats, respectively. The increase in number following adrenalectomy was statistically significant. It is concluded that nerve fibres may play an important role in the regulation of the anterior pituitary.

Evidence for nodose ganglion as the source of innervation to the anterior lobe of the pituitary gland

Recent studies have provided convincing evidence for the presence of peptidergic nerve fibers in the pituitary anterior lobe in several animal species. This study was aimed at elucidating the origin of this innervation by neuroanatomical tracing, denervation experiments, and immunohistochemistry. Immunohistochemistry against substance P and growth-associated protein 43 revealed a dense fiber plexus within the anterior lobe, and these markers were mostly colocalized. Retrograde tracing with Fluorogold from the pituitary gland stained neurons in the hypothalamus, superior cervical ganglia and the nodose ganglia. None of the Fluorogold-labelled neurons in the hypothalamus or superior cervical ganglion were substance P-immunoreactive, while many of the neuronal cell bodies in the nodose ganglion exhibited substance P immunoreactivity. There were no Fluorogold-labelled neurons in the trigeminal, otic or cervical dorsal root ganglia. Surgical transection of the pituitary stalk or bilateral removal of the superior cervical ganglion did not abolish the anterior lobe nerve fibers, and anterograde tracing with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate from the pituitary stalk failed to stain any nerve fibers within the anterior lobe. Our findings suggest that the nodose ganglion neurons likely innervate the pituitary anterior lobe, while neither hypothalamus nor sympathetic superior cervical ganglion may be a source of this innervation. By showing a distinct neuronal system in the pituitary anterior lobe our findings (i) support the previous functional studies demonstrating a distinct regulation of the morphology of the anterior lobe innervation by hormonal changes, and (ii) suggest that the innervation of the pituitary anterior lobe is a part of the visceral innervation by the vagus nerve rather than a part of the other intracranial innervation. These findings provide a neuroanatomic basis for the reported observations about the neural regulation of the pituitary anterior lobe.

Neuropeptide content and physiological properties of rat cartilage-projecting sensory neurones co-cultured with perichondrial cells

In young rats the cartilaginous epiphyses forming the knee joint are supplied with blood vessels and peptidergic sensory nerve fibres through the perichondrium and cartilage canals. In the present study we show that cartilage-related dorsal root ganglion neurones co-cultured with perichondrial cells develop extensive neurite trees and express calcitonin gene-related peptide (CGRP) and substance P (SP) in in vivo-like proportions using retrograde tracing and immunohistochemistry. Moreover, whole cell patch clamp recordings from these cells showed that the majority is depolarised by application of H +-ions. These results are compatible with the hypothesis that a local imbalance of blood flow and metabolism during normal skeletal maturation may cause tissue acidosis eliciting release of CGRP/SP from sensory nerve endings.

Expression and dynamics of peptidergic nerve fibers in granulation tissue after distance osteosynthesis

The transmitters and/or modulators calcitonin gene-related peptide (CGRP), substance P (SP), neuropeptide Y (NPY) and vasointestinal polypeptide (VIP) are supposed to be involved in bone growth, fracture healing and internal remodeling. Immunohistochemical proof of neuropeptide positive fibers in normal bone let us assume that these substances effect the early phase of fracture healing. Exact time of appearance of neuropeptide positive fibers, localisation in the bone, chemospecifity and mode of genesis are unknown so fare. Research was done on a model of distance osteosynthesis of the rabbit tibia. Primary and secondary antibodies were used for indirect immunohistochemical technique. We put more strength on the concrete stereological calculation of length of the nerve fibers than on a conventional statistical evaluation. After histological preparation of tissue specimens from the interfragmental gap and the bone marrow beside the gap the neuropeptides CGRP, SP and NPY were immunohistochemically expressed. Sprouting of CGRP- and SP-positive nerve fibers has its origin in the bone marrow. A vascularisation in the early state of osteoneogenesis after fracture seems impossible without the nerval peptidergic influence and transmission.

Histological demonstration of voltage dependent calcium channels on calcitonin gene-related peptide-immunoreactive nerve fibres in the mouse knee joint

The afferent (excitability) and efferent functions (release of neuropeptides) of primary afferent nerve fibres are based on Ca 2+-influx. The aim of the present study was to examine the presence of L- and N-type Ca 2+-channels at sensory nerve fibres in the mouse knee joint capsule. Specific fluorescent labelled channel blockers and antisera against these channel subtypes were combined with an immunohistochemical staining for calcitonin gene-related peptide (CGRP), a neuropeptide that is widely distributed in primary afferents. There was a nearly complete colocalisation of CGRP immunoreactivity and the binding of ω-conotoxin GVIA (toxin VIA of Conus geographus or BODIPY-verapamil (BODIPY® FL verapamil, hydrochloride) demonstrating the presence of N-type and L-type Ca 2+-channels, respectively. These data were further confirmed by identical results obtained after an immunohistochemical demonstration of the two channel subtypes at the peptidergic nerve fibres.

Immunohistochemical study of the distribution of adrenergic and peptidergic innervation in the equine uterus and the cervix.

Little is known about neurogenic regulation of uterine contractility in mares. The present study investigated the distribution of adrenergic and peptidergic nerves in the mare uterus. Samples from the uterine horn, body and cervix were collected from 18 cyclic mares for immunohistochemistry. The uterus was well supplied with adrenergic nerves. A large number of tyrosine hydroxylase- and dopamine beta-hydroxylase-immunoreactive nerve bundles and fibres were present in the myometrium and endometrium in all regions of the uterus and cervix. These adrenergic nerve bundles and fibres travelled parallel to the muscle layers and were often associated with blood vessels. The density of peptidergic nerves was less than that of adrenergic nerves, but the pattern of distribution was similar. Neuropeptide Y-immunoreactive nerve fibres were the most abundant, whereas vasoactive intestinal polypeptide- and calcitonin gene-related peptide-immunoreactive nerve fibres were less frequently seen. Substance P-immunoreactive nerve fibres were the most sparse. Peptidergic nerves were distributed among the smooth muscle layers and near endometrial glands and were often associated with blood vessels in all regions of the uterus. The density of peptidergic nerve fibres was similar in the uterine horn and body but was slightly denser in the cervix. These findings indicate that uterine innervation may have an important role in controlling reproductive functions in mares.

Immunohistochemical study of the distribution of adrenergic and peptidergic innervation in the equine uterus and the cervix

Little is known about neurogenic regulation of uterine contractility in mares. The present study investigated the distribution of adrenergic and peptidergic nerves in the mare uterus. Samples from the uterine horn, body and cervix were collected from 18 cyclic mares for immunohistochemistry. The uterus was well supplied with adrenergic nerves. A large number of tyrosine hydroxylase- and dopamine beta-hydroxylase-immunoreactive nerve bundles and fibres were present in the myometrium and endometrium in all regions of the uterus and cervix. These adrenergic nerve bundles and fibres travelled parallel to the muscle layers and were often associated with blood vessels. The density of peptidergic nerves was less than that of adrenergic nerves, but the pattern of distribution was similar. Neuropeptide Y-immunoreactive nerve fibres were the most abundant, whereas vasoactive intestinal polypeptide- and calcitonin gene-related peptide-immunoreactive nerve fibres were less frequently seen. Substance P-immunoreactive nerve fibres were the most sparse. Peptidergic nerves were distributed among the smooth muscle layers and near endometrial glands and were often associated with blood vessels in all regions of the uterus. The density of peptidergic nerve fibres was similar in the uterine horn and body but was slightly denser in the cervix. These findings indicate that uterine innervation may have an important role in controlling reproductive functions in mares.

Increase in calcitonin gene related peptide (CGRP) and decrease in mast cells in dihydroepiandrosterone (DHEA)-induced polycystic rat ovaries.

The polycystic ovary is reported to correspond with a high density in intraovarian nerve fibers and their sympathetic hyperresponsiveness. Peptidergic nerves may also be involved in this process. An interaction between nerve fibers and mast cells is assumed because of nerve growth-factor production by mast cells. Here we investigated CGRP-positive nerve fibers and mast cells in polycystic ovaries induced in immature rats with dihydroepiandrosterone (DHEA). The DHEA treated ovaries contained less corpora lutea than controls (mean +/- SEM: 4.3 +/- 0.6 versus 11.3 +/- 0.9, P > 0.001) and less intact antral follicles (4.7 +/- 0.7 versus 8.1 +/- 1.1; P < 0.05) according to the histometric approach. By immunolabelling more CGRP-positive nerve fibers were found in the DHEA treated ovaries than in controls (mean +/- SEM per one section: 23.2 +/- 5.8 fibers versus 10.3 +/- 0.9 and 171 +/- 44.7 varicosities versus 84 +/- 9.5). This was confirmed by dot blot analysis, showing a significant higher CGRP signal intensity per microgram homogenized ovaries of the DHEA treated group compared to the untreated (P < 0.05). Toluidine-blue-stained mast cells populated the medulla in both groups, yet had strikingly decreased in the DHEA treated ovaries (23.5 +/- 3.9 versus 89 +/- 5.6, P < 0.005). The increase in CGRP-positive nerve fibers and the decrease of toluidine-blue-stained mast cells points to an altered neuroimmune function in DHEA-induced polycystic rat ovaries.

Neuropeptides in the duodenal mucosa of chronic alcoholic heavy drinkers.

Previous studies have shown that patients with chronic alcohol ingestion may show a variety of morphological and functional alterations in the small intestine. In this study, we have focused on the neuroendocrine system in the duodenal mucosa in chronic alcoholics; an area little studied. Twenty-three defined chronic alcoholics admitted to the hospital for detoxification underwent clinical examination, followed by upper gastrointestinal endoscopy and blood tests on average 4 days after the most recent alcohol intake. Biopsy specimens were taken from the distal part of the descending duodenum for both immunohistochemical and routine histological examination. The control group consisted of 25 patients referred for upper endoscopy mainly because of dyspepsia (ulcer, reflux type), but who were otherwise healthy. A normal carbohydrate-deficient transferrin and a history of low alcohol consumption (<40 g/week) were required for inclusion in the control group. The tissue specimens were studied using antisera for the following neuropeptides: cholecystokinin, galanin, gastric inhibitory peptide (GIP), glucagon, motilin, neuropeptide Y, pituitary adenylyl cyclase activating peptide, secretin, serotonin, somatostatin, substance P, vasoactive intestinal polypeptide and protein gene product, as a general marker for neurones and cells of the diffuse neuroendocrine system. The density of nerve fibres was evaluated semi-quantitatively and the number of endocrine cells per unit length of mucosa was counted in sections cut perpendicularly to the mucosal surface. All the different peptidergic nerve fibres in the alcohol group showed higher densities than the corresponding controls. However, this was not a statistically significant difference. A slightly significant increase (P = 0.02) in the numbers of glucagon and GIP cells was seen in the alcohol group. Gastrointestinal symptoms were frequently present (87%) in chronic alcoholics. We suggest that chronic alcohol consumption in man may have a general effect on the peptidergic nerve system and some endocrine cell types in the duodenal mucosa.

The Effect of Unilateral Sympathectomy and Cavity Preparation on Peptidergic Nerves and Cells in Rat Dental Pulp

Recent evidence suggests interactions between primary afferent nociceptors and postganglionic sympathetic efferents in the pathogenesis of inflammation. The effect of unilateral removal of the superior cervical ganglion on the innervation pattern of nerve fibers immunoreactive (IR) to calcitonin gene-related peptide (CGRP), substance P (SP), and neuropeptide Y (NPY), as well as the occurrence of cells in the injured and uninjured rat molar pulp, was investigated. Light microscopic immunocytochemistry demonstrated that the molar pulps contralateral to the sympathectomy contained a NPY-IR nerve fiber network more dense and heavily stained than unoperated control rats. The NPY-IR fibers showed, however, no sprouting after deep cavity preparation. There was no compensatory increase in CGRP- and SP-IR nerve fibers in the dental pulp after unilateral sympathectomy, although a significant increase in cells IR to CGRP and SP was found in the ipsilateral trigeminal ganglion. Unilateral sympathectomy induced a significant increase in cell density both in the inflamed and in the uninflamed dental pulp bilaterally. Our results demonstrate, for the first time, a trophic effect of the sympathetic nerves on cells in the dental pulp, indicating that an imbalance of sympathetic nerves may induce inflammation and pain in teeth.

Influence of neuropeptides on neutrophil adhesion and transmigration through a lung fibroblast barrier in vitro.

The lung is richly supplied with peptidergic nerves that store and secrete substance P (SP), vasoactive intestinal peptide (VIP), and other neuropeptides known to potently modulate leukocyte function in vitro and airway inflammation in vivo. We examined the effect of SP, VIP and the novel sensory neuropeptide secretoneurin (SN), as well as of interleukin (IL)-8, IL-6, IL-1 beta, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF), all associated with acute lung injury, on human neutrophil migration across a 5-mu pore polycarbonate filter system covered by human lung fibroblast monolayers. Additionally, we tested the ability of these neuropeptides to elicit neutrophil adhesion to fibroblast monolayers. SP, but not VIP and SN, may be important in directly influencing neutrophil adhesion to and subsequent migration across a subendothelial barrier of fibroblasts and extracellular matrix towards lung inflammatory sites. The effect was mainly mediated by neurokinin (NK)-1 receptors, as evaluated by a specific NK-1 antagonist, [[(S,S)Pro-Leu(spiro-y-lactam)]9,10, Trp11]substance P (1-11), whereas a specific NK-2 receptor antagonist, [Tyr5, D-Trp6,8,9, Lys10]neurokinin A (4-10), was ineffective. The SP analog septide and the NK-1 receptor agonist ([Sar9 Met(O2)11)SP were comparably effective. Furthermore, the SP effect was concentration and time dependent. However, the other tested neuropeptides might also affect neutrophil recruitment in inflammatory lung by modulating other lung cell functions. Additionally, all tested cytokines stimulated neutrophil transfibroblast migration in vitro, except IL-6. In conclusion, SP in concert with proinflammatory cytokines may regulate neutrophil interstitial accumulation and their traffic to the alveolar space in lung inflammation.

Distribution of peptidergic nerve fibres in the guinea pig trabecular meshwork.

A quantitative analysis of peptidergic nerve fibres located in the trabecular meshwork of the guinea pig has been performed. Our results confirm that this structure contains VIP-, NPY- and substance P-like immunoreactivity as major neurotransmitters. These findings were obtained using immunohistochemical techniques. For this purpose serial sections of the eye were stained by immunohistochemistry for each of three neurotransmitters and stained sections were analysed by quantitative image analysis. Our findings demonstrate that SP-positive, NPY-positive and VIP-positive nerve fibres occupy 11.2, 4.9 and 2.4%, respectively, of the observed area (expressed as conventional units, C.U.) in the trabecular meshwork of the guinea pig eye. It is relevant to emphasize that the area containing these three types of peptidergic nerve fibres appears to be large (18.5 +/- 6.6 C.U.) in proportion to the total observed area. The innervation of the drainage angle of the guinea pig eye has been well described by many authors. This is the first study to report quantitative measurements of three types of peptidergic nerve fibres identified and measured in this area. The presence of these three neurotransmitters in the trabecular meshwork of guinea pig eye suggests their possible participation in aqueous humor regulation.

Tyrosine hydroxylase- and neuropeptides-immunoreactive nerves in canine trachea.

To determine distribution of catecholaminergic and peptidergic nerve fibers in canine tracheas by use of immunohistochemistry. 10 tracheas collected from healthy adult dogs after euthanasia. Structure of the nerve network and distribution of tyrosine hydroxylase (TH)- and 6 types of neuropeptide-containing nerves in canine tracheas were immunohistochemically studied, using neurochemical markers. Intraepithelial free nerve endings with immunoreactivity for calcitonin gene-related peptide (CGRP) and substance P (SP) were observed. Tyrosine hydroxylase-, SP-, vasoactive intestinal peptide (VIP)-, and galanin (GAL)-immunoreactive nerve fibers were observed within and around the submucosal seromucous gland. In the smooth muscle layer, numerous TH- and GAL-immunoreactive nerve fibers, a moderate number of VIP- and neuropeptide Y (NPY)-immunoreactive nerve fibers, and a few SP- and methionine enkephalin (ENK)-immunoreactive nerve fibers were observed. Numerous nerve cell bodies with VIP and GAL immunoreactivity and a few with SP ENK, and NPY immunoreactivity were observed. Many TH-immunoreactive fibers were arranged in a meshwork around blood vessels. Nerves with CGRP-, SP-, VIP-, GAL-, ENK-, and NPY-immunoreactivity were also observed around blood vessels. Complex innervation, including catecholamine- and neuropeptide-containing nerves, which may be related to regulation of muscle contraction and glandular secretion, are found in canine tracheas.

Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-protein nor ATP.

Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K+ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K+ channels in peptidergic nerve terminals. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. The sigma receptor ligands pentazocine and SKF10047 modulated K+ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPbetaS), a G-protein activator (GTPgammaS) or a non-hydrolysable ATP analogue (AMPPcP). Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor protein phosphorylation. This novel transduction mechanism is mediated by membrane proteins in close proximity, possibly through direct interactions between the receptor and channel. This would allow for more rapid signal transduction than other ion channel modulation mechanisms, which in the present case of neurohypophysial nerve terminals would lead to the enhancement of neuropeptide release.

Dihydrocapsaicin treatment depletes peptidergic nerve fibers of substance P and alters mast cell density in the respiratory tract of neonatal sheep

In the present study we administered dihydrocapsaicin (DHC) to neonatal lambs to deplete C-fibers of neuropeptides. We measured the density of substance P (SP)-fibers in nasal septum to assess the effectiveness of the treatment at 3, 9, and 21 days. The numbers of mast cells in the upper and lower respiratory tract were determined at the same time points and histamine content was determined from lung tissue. DHC treatment depleted SP-fibers for up to the 21 day time point. This depletion was estimated as 85% in comparison with controls. In vehicle-treated lambs, the density of SP-fibers decreased progressively with age, but not to the degree of DHC-treated lambs whose SP-fibers were depleted from the initial 3-day measurement. In both, vehicle- and DHC-treated lambs, numbers of mast cells increased progressively with time; however, the density of mast cells was augmented in the entire respiratory tract of DHC-treated animals. Apparently, DHC treatment exerts a single and initial effect in increasing mast cells whereas time maintains a continuous influence; both factors exert their influence independently. Despite large numbers of mast cells in DHC-treated animals, histamine content in the lung had similar levels as controls. Our study provides fundamental data for a better understanding of conditions that may influence defense mechanisms dependent on the mast cell-nerve axis in the respiratory tract.

Morphological relationships of peptidergic and noradrenergic nerve terminals to olivocochlear neurones in the rat

In the rat, the outer hair cells in the cochlea receive direct synaptic input from neurones in the ventral nucleus of the trapezoid body. These so-called medial olivocochlear neurones exert an inhibitory influence on the cochlear neural output. Electrophysiological in vitro studies suggest that the activity of medial olivocochlear neurones may be affected by a variety of neuropeptides as well as noradrenaline, but anatomical confirmation of direct synaptic input is still lacking. We have investigated, at the light microscopical level, the morphological relationships between terminals containing noradrenaline, substance P, cholecystokinin and leu-enkephalin, and medial olivocochlear neurones in the rat. A retrograde tracer was injected into the cochlea to label medial olivocochlear neurones and a double labelling immunocytochemical method was used to visualise the retrograde tracer as well as the neurotransmitters within each brain section. Light microscopical analysis revealed nerve endings containing substance P, cholecystokinin and leu-enkephalin in close apposition to the dendrites of medial olivocochlear neurones, and nerve endings containing dopamine-β-hydroxylase, a marker for noradrenaline, in close contact with the somata as well as dendrites of medial olivocochlear neurones. Although the technique cannot prove the existence of functional synaptic contacts, the results are broadly consistent with electrophysiological data and suggest a direct input to medial olivocochlear neurones from substance P, cholecystokinin, leu-enkephalin and noradrenaline-containing neural pathways. Differences in the densities and spatial distribution of the various neuropharmacological inputs suggest differences in the relative strengths and possible roles of these diverse inputs to the olivocochlear system.

The Inhibitory Effects of Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide on Osteoclast Formation Are Associated with Upregulation of Osteoprotegerin and Downregulation of RANKL and RANK

The presence of a network of peptidergic nerve fibers in the skeleton, expressing several neuropeptides including vasoactive intestinal peptide (VIP), has been demonstrated. This observation, together with our findings in vitro showing that VIP can regulate the activities of osteoblasts and osteoclasts as well as the recruitment of osteoclasts, has suggested the existence of a neuro-osteogenic interplay in bone metabolism. In the present study, the effects of VIP and pituitary adenylate cyclase-activating polypeptide (PACAP), two members of the VIP/secretin/glucagon superfamily, on osteoclast formation and mRNA expression of three key regulatory proteins involved in osteoclast formation have been investigated. VIP, PACAP-27, and PACAP-38, at concentrations of 10−6 M, all significantly inhibited formation of tartrate-resistant acid phosphatase-positive multinuclear cells (TRAP + MNC) in mouse bone marrow cultures stimulated by 1,25(OH)2-vitamin D3 (D3; 10−8 M). By using semiquantitative RT-PCR, it was found that D3 upregulated the mRNA expressions of receptor activator of NF-κB ligand (RANKL) and receptor activator of NF-κB (RANK), whereas the expression of osteoprotegerin (OPG) was downregulated in mouse bone marrow cultures stimulated by D3 for 7 days. Both VIP and PACAP-38 decreased the stimulatory effects of D3 on RANKL and RANK expression, whereas the inhibitory effect of D3 on OPG expression was reversed by VIP and PACAP-38. These observations indicate that the inhibitory effects of VIP and PACAP on osteoclast recruitment are due to regulation of the expression of key proteins involved in later stages of osteoclast differentiation.

Changes in peptidergic nerves in the atrioventricular valves of streptozotocin-induced diabetic rats: a confocal microscopy study.

Several previous studies have described the distribution of neuropeptide Y (NPY)-like and calcitonin gene related peptide (CGRP)-like immunoreactive nerve fibres in the atrioventricular valves of humans and various animals. It has been suggested that peptide-containing nerve fibres might have motor or sensory roles in valvular function. Although there is evidence that diabetic changes occur in the sympathetic (preganglionic and postganglionic), parasympathetic (vagal) and peptidergic nerves of rats, the changes of peptide-containing nerve fibres in the atrioventricular valves of the diabetic rat have not been studied. The distribution, relative density and staining intensity of NPY-like and CGRP-like immunoreactive nerve fibres in the mitral and tricuspid valves were studied in whole mount preparations using confocal microscopy with a computer-assisted image analysis system. Streptozotocin-induced diabetic and control rats were sacrificed at 12 and 24 months. The nerve staining intensity within the tricuspid valve was greater than the mitral valve in both control (P < 0.01) and diabetic (P < 0.001) rats. Nerve density in the anterior leaflet was greater than the posterior leaflet of the mitral valve. However, the anterior leaflet of the mitral and tricuspid valves showed a decreased number of nerve fibres, followed by drastic reduction in the staining intensities for both the peptides studied (P < 0.001) in the long-term diabetic rat. The decrease in the number of nerve fibres that follow the mechanical interruption of nerves raises the possibility that cycles of degeneration may occur. It is suggested that these peptide-containing nerve fibres in the atrioventricular valves may be involved in valvular dysfunction in the diabetic state.

Changes in peptidergic nerves in the atrioventricular valves of streptozotocin-induced diabetic rats: A confocal microscopy study

Several previous studies have described the distribution of neuropeptide Y (NPY)-like and calcitonin gene related peptide (CGRP)-like immunoreactive nerve fibres in the atrioventricular valves of humans and various animals. It has been suggested that peptide-containing nerve fibres might have motor or sensory roles in valvular function. Although there is evidence that diabetic changes occur in the sympathetic (preganglionic and postganglionic), parasympathetic (vagal) and peptidergic nerves of rats, the changes of peptide-containing nerve fibres in the atrioventricular valves of the diabetic rat have not been studied. The distribution, relative density and staining intensity of NPY-like and CGRP-like immunoreactive nerve fibres in the mitral and tricuspid valves were studied in whole mount preparations using confocal microscopy with a computer-assisted image analysis system. Streptozotocin-induced diabetic and control rats were sacrificed at 12 and 24 months. The nerve staining intensity within the tricuspid valve was greater than the mitral valve in both control (P < 0.01) and diabetic (P < 0.001) rats. Nerve density in the anterior leaflet was greater than the posterior leaflet of the mitral valve. However, the anterior leaflet of the mitral and tricuspid valves showed a decreased number of nerve fibres, followed by drastic reduction in the staining intensities for both the peptides studied (P < 0.001) in the long-term diabetic rat. The decrease in the number of nerve fibres that follow the mechanical interruption of nerves raises the possibility that cycles of degeneration may occur. It is suggested that these peptide-containing nerve fibres in the atrioventricular valves may be involved in valvular dysfunction in the diabetic state. Anat Rec 258:277–285, 2000. © 2000 Wiley-Liss, Inc.