Effects of neuropeptides on growth of cultivated rat molar pulp fibroblasts

The effect of the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) on DNA synthesis of dental pulp cells was investigated in cells grown from molar tooth bud explants from 4–6 days old rat pups. A concentration response-assay of the proliferative response of pulpal cells was performed with SP, NPY, NKA, CGRP and VIP (0.01 to 1 nM) in the presence of EGF (10 ng/ml), hydrocortisone (0.4 μg/ml) and 3% FCS, using [³H]thymidine incorporation. The results showed that SP, NKA and CGRP, but not NPY and VIP, increased the cell number in a concentration-dependent manner, with maxima at 10⁻¹⁰ – 10⁻⁹ M (SP, NKA) and 10⁻⁷ M (CGRP). No potentiating effect was noted when cells are simultaneously stimulated with SP and CGRP. The finding that SP, NKA and CGRP have growth regulatory properties on pulpal cells in vitro suggests that sensory neuropeptides may be involved during pulpal development or in wound healing after pulpal injury.     

Secretoneurin, substance P and neuropeptide Y in the oxygen-induced retinopathy in C57Bl/6N mice

In this study, we investigated whether the proangiogenic neuropeptides secretoneurin (SN), substance P (SP), and neuropeptide Y (NPY) contribute to the development of abnormal neovascularization in the oxygen-induced retinopathy (OIR) model in mice. By exposing litters of C57Bl/6N mice to 75% oxygen from postnatal day 7 (P7) until postnatal day 11 (P11) and then returning them to normoxic conditions, retinal ischemia and subsequent neovascularization on the retinal surface were induced. Retinae were dissected on P9, P11, P12-P14, P16 and P20, and the concentrations of SN, SP, NPY and VEGF determined by radioimmunoassay or ELISA. The levels of SN and SP increased in controls from P9 until P16 and from P9 until P14, respectively, whereas the levels of NPY were high at P9 and decreased thereafter until P20, suggesting that NPY may participate in the development of the retina. However, dipeptidyl peptidase IV (DPPIV) and the NPY-Y2 receptor were not detectable in the immature retina indicating that NPY is not involved in the physiological vascularization in the retina. Compared to controls, OIR had no effect on the levels of SN, whereas levels of both SP and NPY slightly decreased during hyperoxia. Normalization of the levels of SP, and to a more pronounced extent of NPY, was significantly delayed during relative hypoxia. This clearly indicates that these three neuropeptides are not involved in the pathogenesis of neovascularization in OIR. Moreover, since there were no differences in the expression of two vessel markers in the retina of NPY knockout mice versus controls at P14, NPY is also not involved in the delayed development of the intermediate and deep vascular plexus in the retina in this animal model.     

Neuropeptide expression in the airways of COPD patients and smokers with normal lung function

Neurogenic mechanisms seem to play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD), as suggested by a number of in vitro data. However, few studies have investigated the presence of neuropeptides in the airways of patients with COPD, and they have yielded conflicting results. The aim of this study is to compare the expression of the neuropeptide substance P (SP), vasoactive intestinal peptide (VIP), and neuropeptide Y (NPY) in the airways of smokers with and without COPD. Surgical lung samples were obtained from 15 smokers with COPD and 16 smokers with normal lung function, who underwent lobectomy for a solitary lung carcinoma. Airway expression and distribution of SP, VIP, and NPY were identified by immunohistochemistry and analyzed by a computerized image analysis system. Compared to smokers with normal lung function, COPD patients exhibited an increased immunoreactivity for SP and VIP, paralleled by a decreased NPY expression in the epithelium and glands, and a decreased expression of all these three neuropeptides in the smooth muscle layer. Therefore, in the present study we have documented a different expression and distribution of the neuropeptides SP, VIP, and NPY in the airways of smokers with and without COPD. These findings suggest a possible involvement of such neuropeptides in the pathogenesis of some changes occurring in COPD.     

Distribution of vasoactive intestinal polypeptide, neuropeptide-Y and substance P and their effects on insulin secretion from the in vitro pancreas of normal and diabetic rats

This study examined the pattern of distribution of vasoactive intestinal polypeptide (VIP), neuropeptide-Y (NPY) and substance P (SP) in the pancreas of diabetic rat to determine whether there are changes in the number and pattern of distribution of these neuropeptides after the onset of diabetes. Moreover, the effect of VIP, NPY and SP on insulin secretion from the pancreas of normal and diabetic rats was also examined. Diabetes mellitus (DM) was induced by a single dose of streptozotocin (STZ) given intraperitoneally (i.p.) (60 mg kg body weight(-1)). Four weeks after the induction of DM, diabetic (n = 6) and normal (n = 6) rats were anesthetized with chloral hydrate and their pancreases removed and processed for immunohistochemistry and insulin secretion. The number of insulin-positive cells in the islets of Langerhans was reduced while that of VIP and NPY increased significantly after the onset of diabetes. The pattern of distribution of VIP, NPY and SP in the nerves innervating the pancreas was similar in both normal and diabetic rats. VIP-evoked large and significant (P < 0.02) increases in insulin secretion from the pancreas of normal and diabetic rats. NPY also induced a marked (P < 0.005) increase in insulin release from pancreatic tissue fragments of normal rat. Stimulation of pancreatic tissue fragments of diabetic rat with NPY resulted in a slight but not significant increase in insulin release. SP induced a large and significant (P < 0.005) increase in insulin secretion from the pancreas of normal rat but inhibited insulin secretion significantly (P < 0.03) from isolated pancreas of diabetic rat. In summary, VIP and NPY can stimulate insulin secretion from the pancreas after the onset of diabetes. The stimulatory effect of SP on insulin secretion is reversed to inhibitory in diabetic rats.     

Dopamine D2 Receptor-Mediated Regulation of Serotonin Extracellular Concentration in the Dorsal Raphe Nucleus of Freely Moving Rats

Abstract: The neuropeptide-inducing activity of neurotrophic factors was tested in cultured cerebral cortical neurons. Brain-derived neurotrophic factor (BDNF) specifically increased contents of the neuropeptides somatostatin (SOM) and neuropeptide Y (NPY), but its effect on contents of cholecystokinin octapeptide and GABA was much less significant. The maximal induction of NPY content (15-fold increase) was achieved by 20 ng/ml of BDNF. These changes were also reproduced at the mRNA level. In contrast, neurotrophin-3 was much less potent at increasing NPY and SOM contents, and nerve growth factor had no effect on them. The expression of mRNA for NPY and SOM was fully dependent on the presence of BDNF in culture but irrelevant to the survival-promoting activity of BDNF, which has been reported previously. Most of the NPY immunoreactivity induced by BDNF was colocalized with glutamate decarboxylase immunoreactivity in cultured cortical neurons. These results suggest that BDNF regulates the peptidergic expression of GABAergic neurons in the cerebral cortex.     

Recombinant Cholinergic Differentiation Factor (Leukemia Inhibitory Factor) Regulates Sympathetic Neuron Phenotype by Alterations in the Size and Amounts of Neuropeptide mRNAs

The cholinergic differentiation factor (CDF) in heart cells is identical to leukemia inhibitory factor (LIF). Recombinant CDF/LIF was shown to alter dramatically neurotransmitter production as well as the levels of several neuropeptides in cultured rat sympathetic neurons. Here it is shown that these changes are likely to be caused by alterations in the mRNA for these proteins and peptides. Growth in 1 nM recombinant CDF/LIF induces mRNA for acetyl CoA: choline-O-acetyltransferase [EC 2.3.1.6; choline acetyltransferase (ChAT)], somatostatin (SOM), substance P, and vasoactive intestinal polypeptide while lowering mRNA levels of tyrosine hydroxylase (EC 1.14.16.2) and neuropeptide Y (NPY). In addition, the sizes of the mRNAs for ChAT, SOM, and NPY are larger after recombinant CDF/LIF treatment.     

Matrix metalloproteinase-13 expression in rabbit knee joint connective tissues: influence of maturation and response to injury.

The hypothesis of the present work was that expression of matrix metalloproteinase-13 (MMP-13, collagenase-3) would be induced during conditions involving important matrix remodeling such as ligament maturation, scar healing and joint instability. Therefore, MMP-13 expression in the medial collateral ligament (MCL) during the variable situations of tissue maturation and healing was assessed. MMP-13 expression in three intra-articular connective tissues of the knee (i.e. articular cartilage, menisci and synovium) following the transection of the anterior cruciate ligament of the knee was evaluated at 3 and 8 weeks post-injury. MMP-13 mRNA (semi-quantitative RT-PCR) and protein (immunohistochemistry and Western blotting) were detected in all of the tissues studied. Significantly higher MCL mRNA levels for MMP-13 were detected during the early phases of tissue maturation (i.e. 29 days in utero and 2-month-old rabbits) compared to later phases (5- and 12-month-old rabbits). This pattern of expression was recapitulated following MCL injury, with very high levels of expression in scar tissue at 3 weeks post-injury and then a decline to levels not significantly different from control values by 14 weeks. Elevated mRNA levels correlated with increased protein levels for MMP-13 in both menisci and synovium following the transection of the anterior cruciate ligament and during medial collateral ligament healing. These results indicate that MMP-13 expression is regulated by a number of variables and that high levels of expression occur in situations when connective tissue remodeling is very active.     

Distribution of NPY and SP and their effects on glucagon secretion from the in vitro normal and diabetic pancreatic tissues

Neuropeptides modulate the function of classic neurotransmitters in the regulation of body function. The role of neuropeptides in the regulation of endocrine secretion from the pancreas of diabetic rat is poorly understood. This study examined the pattern of distribution of neuropeptide-Y (NPY) and substance P (SP) in normal and diabetic rat pancreases. In addition to this, the effect of NPY and SP on glucagon secretion was also examined in the pancreases of normal and diabetic rats. Four weeks after the induction of diabetes, the pancreaseses of normal and diabetic rats were removed and processed for immunohistochemistry and glucagon secretion. The pattern of distribution of glucagon in the pancreas of diabetic rat was conspicuously deranged after the onset of diabetes. The pattern of distribution of NPY and SP was, however, similar in the pancreases of both normal and diabetic rats. Stimulation of normal rat pancreatic tissue with NPY (10(-12) and 10(-9) M) evoked large and significant (P < 0.001) increases in glucagon secretion compared to basal. In contrast to this, NPY inhibited glucagon secretion from the pancreas of diabetic rat. Treatment of pancreatic tissue fragments of normal rat with 10(-9) M SP resulted in significant (P < 0.03) increases in glucagon secretion. SP inhibited glucagon secretion from diabetic rat pancreas. In conclusion, NPY and SP stimulated glucagon secretion from the pancreas of normal rat. In contrast, NPY and SP inhibited glucagon secretion from diabetic rat pancreas.     

Neuropeptide control of thyroid blood flow and hormone secretion in the rat

The effects of peptide HI (PHI), neuropeptide Y (NPY), and substance P (SP) on thyroid blood flow and hormone levels were studied in anesthetized rats. Regional blood flows were determined using radioactive microspheres. No change in heart rate or mean left ventricular pressure occurred during these neuropeptide infusions (0.625 micrograms iv over 2 min). PHI treatment resulted in a four-fold increase in thyroid blood flow. Blood flows to the pancreas and salivary gland also increased during PHI treatment. Infusions of NPY or SP did not significantly alter thyroid blood flow. However, SP decreased blood flow to the spleen and small intestine. These neuropeptides had no effect on blood flows to the adrenal, kidney, brain, heart, and adipose tissues. Following PHI, NPY, and SP infusions, plasma triiodothyronine and thyroxine levels were not different from values in saline-treated rats. This study demonstrates that PHI, like vasoactive intestinal peptide, is a potent thyroidal vasodilator at a dose that does not affect circulating thyroid hormone secretion.     

Involvement of Leukemia Inhibitory Factor in the Increases in Galanin and Vasoactive Intestinal Peptide mRNA and the Decreases in Neuropeptide Y and Tyrosine Hydroxylase mRNA in Sympathetic Neurons After Axotomy

Abstract: In response to axonal injury, noradrenergic sympathetic neurons of the adult superior cervical ganglion (SCG) alter their neurotransmitter phenotype. These alterations include increases in the levels of the neuropeptides, galanin, vasoactive intestinal peptide (VIP), and substance P (SP) and a decrease in the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). Previous studies have indicated that after axotomy in vivo, leukemia inhibitory factor (LIF) plays an important role in increasing the contents of galanin and VIP in the SCG. In the present study, by examining the time courses of the changes in LIF and neuropeptide mRNA and by using LIF null mutant mice, we have determined that LIF alters neuropeptide content in part by increasing levels of peptide mRNA. In addition, LIF also makes a small contribution to the axotomy-induced down-regulation of mRNA encoding TH and neuropeptide Y, both of which are normally expressed at high levels in the SCG. Finally, by using a LIF-blocking antiserum, this cytokine was found to regulate SP expression in an in vitro axonal injury model. Thus, after axotomy, a single factor, LIF, participates in the down-regulation of peptides/proteins involved in normal neurotransmission and the up-regulation of a group of neuropeptides normally not present in the SCG that may be involved in regeneration.     

Bone and joint neuropathy in rats with type-2 diabetes

We have previously demonstrated that Goto-Kakizaki (GK) rats with spontaneous type-2 diabetes and peripheral neuropathy exhibit regional osteopathic changes. In the present study on 18 GK rats and 21 control Wistar rats, the occurrence of the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the autonomic neuropeptide Y (NPY) was analysed in bone and joints, dorsal root ganglia and lumbar spinal cord by immunohistochemistry and radioimmunoassay (RIA). Immunohistochemistry disclosed a predominance of immunoreactivities in vessel-related nerve fibers, although some were also seen in free terminals. While SP, CGRP and NPY in periosteum, cortical bone and synovium was confined to neuronal tissue, the bone marrow in addition exhibited an abundance of NPY-positive megakaryocytes. Apart from this cellular source of NPY, the observations suggest that the three neuropeptides analysed in bone and joints are of neuronal origin. Quantification by RIA showed a significant decrease of NPY in cortical bone (-36%), bone marrow (-66%) and ankle (-29%) of GK rats. CGRP was decreased in the spinal cord (-19%) and dorsal root ganglia (-26%) but was unchanged in bone and joints, as with SP. Given the suggested anabolic role of NPY and CGRP on bone, neuropeptidergic deficit in diabetes may prove to be an important factor underlying the development of regional osteopenia.     

24-hour variation in content and release of hypothalamic neuropeptides in the rat

The tissue content of up to eight neuropeptides, viz bombesin (BOM), cholecystokinin (CCK-8), neurotensin (NT), neuropeptide Y (NPY), peptide histidine isoleucine amide (PHI), somatostatin (SRIF), substance P (SP) and vasoactive intestinal polypeptide (VIP), in rat hypothalami removed at various times of the day, was measured using specific radioimmunoassays. There was significant variation in the content of BOM, CCK-8, NT, PHI, SP and VIP across a 24-h period. The levels of BOM, CCK-8 and NT were lowest around the onset of darkness (1900 h) and rose throughout the night to reach a peak around the time of lights on. Hypothalamic content of all eight peptides fell between 0700 h and 1300 h by an average of 45 +/- 4%. Basal release of these peptides, as well as that in the presence of 48 mM potassium (K+), was measured from hypothalami removed between 0700 and 1900 h and incubated in vitro in a CSF-like medium. Basal secretion of NT significantly increased, whilst that of CCK-8 significantly decreased over the same period. There was no significant change in the basal release of the other neuropeptides. The release in the presence of 48 mM K+ of SP decreased significantly during the day, whilst that of VIP significantly increased. There was also a significant change in the stimulated release of BOM, levels falling during the morning and rising again at 1900 h. 48 mM K+ caused a significant increase in the release of SRIF and SP at all times tested. Whilst 48 mM K+ induced a significantly higher release of CCK-8 and NT in the morning, this stimulus was ineffective in the evening. The contrary was true in the case of BOM, NPY and VIP, where a significant stimulation was induced only at 1900 h. The possible implications of these findings are discussed.     

Autocrine and paracrine regulation of tissue inhibitor of metalloproteinases, transin, and urokinase gene expression in metastatic and nonmetastatic mammary carcinoma cells.

Acquisition of metastatic competence by tumor cells is frequently accompanied by increased expression of extracellular proteases capable of degrading basement membrane and extracellular matrix. However, very little is known about how the genes encoding these enzymes and their inhibitor proteins are regulated in metastatic versus nonmetastatic cells. In this report, we have compared autocrine and paracrine regulation of tissue inhibitor of metalloproteinases (TIMP), transin, and urokinase plasminogen activator (uPA) genes in genetically related nonmetastatic SP1 and metastatic A3a cell lines. Compared to SP1 cells, metastatic A3a cells showed 15-20-fold higher transin, 3-5-fold less TIMP mRNA, and comparable levels of uPA mRNA. A qualitatively similar shift in expression of these genes was rapidly (i.e., 4-8 h) induced in nonmetastatic SP1 cells following the addition of conditioned medium from A3a cells. The gene-regulating activity present in A3a conditioned medium was heat-labile, suggesting that it was protein in nature. The responsiveness of SP1 cells to the factor(s) secreted by A3a conditioned medium was inhibited by cycloheximide. Basic fibroblast growth factor mimicked the effect of the A3a conditioned medium as an inducer of transin expression in the tumor cells. Although medium conditioned by the tumor cells did not affect uPA expression, addition of epidermal growth factor to the tumor cells transiently induced expression of uPA with a biphasic response that differed in SP1 and A3a cells. Initial induction of uPA at 2-4 h was similar for both cell lines, but after 24 h of exposure to epidermal growth factor, SP1 cells showed a net reduction in uPA, whereas metastatic cells returned to the unstimulated levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex differences in neuropeptide distribution in the rat brain

We have investigated possible sex differences in the regional concentrations of neuropeptides in the rat brain. Immunoreactive neurotensin (NT), neurokinin A (NKA), galanin (GAL), calcitonin gene-related peptide (CGRP), substance P (SP) and neuropeptide Y (NPY) were measured by radioimmunoassay in frontal cortex, occipital cortex, hippocampus, striatum, hypothalamus and pituitary in male and female pre- and postpubertal rats. Sex differences were found for NPY (p < 0.001), NT (p < 0.01) and GAL (p < 0.05), in particular in hippocampus, striatum, hypothalamus and pituitary, but not for CGRP, SP and NKA. Results from analysis of neuropeptides in one sex may not be entirely applicable to the other.     

Deficiency of macrophage migration inhibitory factor gene delays healing of the medial collateral ligament: a biomechanical and biological study

The role played by macrophage migration inhibitory factor (MIF) in the process of wound healing is controversial. Besides, there have been no reports that investigated the expression or the role of MIF in the repair process after ligament injury. In this study, we hypothesized that the deficiency in MIF gene might delay ligament healing in mice. The aim of this study was to clarify this hypothesis using MIF gene-deficient mice (MIFKO) and murine model of injury to the medial collateral ligament (MCL). Biomechanical testing showed that the levels of mechanical properties were significantly lower in MIFKO than in wild-type mice (WT) on day 28 after injury. Levels of matrix metalloproteinase (MMP)-2 and -13 mRNA in the healing tissue were significantly lower in MIFKO than in WT on day 28 and on day 7, respectively. Histologically, healing tissues in MIFKO exhibited prolonged hypertrophy, poor vascularity, and prolonged increase in cell number compared with those in WT. Taken together, it was suggested that MIFKO exhibited delayed healing of the MCL, which might be caused by lower mRNA expression of MMP-2 and -13.     

Growth factor modulation of mitogenic responses and proteoglycan synthesis by human periodontal fibroblasts

In order to understand the relationship between specific growth factors and matrix synthesis by periodontal cells, we have investigated the effects of platelet-derived growth factor BB (PDGF-BB), insulin-like growth factor-I (IGF-1), and growth hormone on DNA and proteoglycan synthesis by cultured human gingival and periodontal ligament fibroblasts in vitro. PDGF-BB and IGF-1, but not growth hormone, were mitogenic for both periodontal ligament fibroblasts and gingival fibroblasts, although the periodontal ligament cells responded more strongly. The mitogenic response was accompanied by alterations in expression of matrix proteoglycan mRNA. For both the gingival and periodontal ligament cells, there was a decrease in mRNA for decorin and an increase in mRNA for versican following exposure to IGF-1 and PDGF-BB. Although no change was seen in response to PDGF, biglycan mRNA level was increased by IGF-1 in periodontal ligament fibroblasts. With the gingival fibroblats, biglycan mRNA levels were unaffected by IGF-1, PDGF-BB, or growth hormone. These findings suggest variable responses of fibroblasts to growth factors depending upon anatomical site within the periodontium. Moreover, there appears to be a correlation between cell proliferation and the types of proteoglycan synthesised with decorin expression being suppressed, and versican being increased during fibroblast proliferation. J. Cell. Physiol. 174:353–361, 1998. © 1998 Wiley-Liss, Inc.     

神经肽CGRP、SP、NPY对软骨细胞的影响研究

近年来,由于创伤和感染等多类原因导致的骨折和骨不连等症状均是威胁到人类生命安全及生活质量的一个医学难题,为了更好地提升对于此类患者的治疗效果,有必要明确骨形成和重建等病理生理学情况以及相应的生物学机制。同时,骨组织是一类随细胞外基质的矿化,并根据其自身需求进行修复的动态组织,也是有着血管和神经支配的活性组织,所以,骨折的修复过程当中不仅含机体各类组织和细胞因子间的复杂作用,还和血液供应及神经支配紧密相连。目前,临床关于神经肽的研究逐渐增多,且越来越多的报道表明神经肽类物质能够在骨折愈合及重建过程中发挥出重要作用,原因可能和机体神经发挥相应的调控作用,并刺激骨细胞发生变化等因素有关。本文即据此展开关于神经肽CGRP(降钙素基因相关肽,Calcitonin gene-related peptide)、SP(P物质,Substance P)、NPY(神经肽Y,Neuropeptide Y)对软骨细胞影响情况的综述分析,从而更好地服务临床。