Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study

The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.     

CGRP受体拮抗剂CGRP8-37对甲醛炎性痛大鼠自发痛反应及脊髓后角NOS表达和NO含量的影响

目的:探讨CGRP受体拮抗剂CGRP8-37对甲醛炎性痛大鼠自发痛反应及脊髓后角NOS表达和NO含量的影响.方法:大鼠足底注射甲醛制造炎性痛模型;计数缩足反射次数反映自发痛程度;NADPH-d组织化学法观察脊髓后角NOS表达;硝酸还原酶法测定NO-3/NO-2含量以反映NO含量.结果:足底注射甲醛后,动物出现自发痛反应行为.足底注射甲醛后24 h,双侧脊髓后角NOS表达及NO含量明显增加.预先鞘内注射CGRP8-37可使甲醛诱导的自发性缩足反射次数明显减少,并可明显抑制甲醛炎性痛诱导的脊髓后角NOS表达及NO含量的增加.结论:甲醛炎性痛时,脊髓后角CGRP受体激活可促进NOS活性表达及NO的产生.     

Increased expression in calcitonin-like receptor induced by aldosterone in cerebral arteries from spontaneously hypertensive rats does not correlate with functional role of CGRP receptor

OBJECTIVE: To analyze the effect of aldosterone on the expression of calcitonin gene-related peptide (CGRP) receptor components, calcitonin-like receptor (CL receptor) and receptor activity modifying protein 1 (RAMP1), as well as the effect of this mineralocorticoid on CGRP-mediated vasodilation in middle cerebral arteries from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). RESULTS: CGRP 0.1 nM-0.1 microM induced a concentration-dependent relaxation that was nitric oxide independent and higher in SHR middle cerebral arteries. CL receptor and RAMP1 expression were similar in both strains. The relaxation to CGRP was not modified by aldosterone 1 microM in either strain, although aldosterone 1 microM increased expression of CL receptor without modifying RAMP1 in segments from SHR rats. CONCLUSIONS: CGRP elicits greater vasodilation in middle cerebral arteries from SHR than WKY rats, that is nitric oxide independent, and by mechanism independent of CGRP receptor components expression. Although aldosterone increases the expression of CL receptor in SHR, it does not alter vasodilation to CGRP, since RAMP1 expression is not increased. These results indicate that the increase in CL receptor, without an increase in RAMP1, does not correlate with changes in functional role of the CGRP receptor.     

Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells

The present study was to determine whether clonidine could induce calcitonin gene-related peptide (CGRP) production and the underlying mechanisms. Human umbilical vein endothelial cells were treated with clonidine and the dose–effect or time–effect relationship of clonidine on CGRP production was examined. Youhimbine (a α₂-adrenoceptor blocker) and l-NAME (an antagonist of nitric oxide synthase, NOS) were chosen to explore the role of α₂-adrenoceptor and nitric oxide pathway in the effect of clonidine on endothelial cell-derived CGRP production. The level of CGRP mRNA or protein was detected by Real Time-PCR or radioimmunoassay. Nitric oxide content was measured by nitroreduction assay. The study showed that clonidine was able to induce CGRP mRNA (α- and β-isoforms) expression in a dose-dependent manner in endothelial cells. The effect of clonidine on endothelial cell-derived CGRP synthesis and secretion was attenuated in the presence of youhimbine. l-NAME treatment could also inhibit clonidine-induced CGRP synthesis and secretion concomitantly with the decreased NO content in culture medium. These results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of α₂-adrenoceptor, which is related to the NO pathway.     

Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery

Although airway and pulmonary vessel tone are regulated predominantly by cholinergic and adrenergic impulses, biologically active peptides such as calcitonin gene-related peptide (CGRP) may significantly influence human smooth muscle tone in normal and pathophysiological states. In the present study, the expression of CGRP and its receptor CGRPR-1 and the biological effect of the peptide were investigated in human airways and pulmonary arteries. Immunohistochemistry revealed the presence of CGRP in human airway nerves and neuro-epithelial cells, whereas the receptor was found in epithelial cells and smooth muscle myocytes of the bronchi and in pulmonary artery endothelium. On precontracted bronchi (3-4 mm in diameter) alpha-CGRP (0.01-10 nM) caused a concentration-dependent contraction on epithelium-denuded bronchi, whereas no significant effect was recorded in bronchi with intact epithelium. In pulmonary arteries (2-6 mm in diameter), alpha-CGRP caused a concentration-dependent relaxation of endothelium intact and denuded vessels. Pre-treatment with indomethacin, but not with l-NAME, prevented the relaxation induced by alpha-CGRP in pulmonary arteries suggesting that prostaglandins but not nitric oxide (NO) are involved in the intracellular signal transduction pathway. The effects induced by alpha-CGRP in bronchi and vessels were prevented by application of the antagonist CGRP((8-37)). In summary, the present studies examined the biological function of CGRP in human airways and demonstrated a constrictory effect of CGRP only in epithelium-denuded airway smooth muscle indicating an alteration of CGRP airway effects in respiratory tract pathological states with damaged epithelium such as chronic obstructive pulmonary disease or bronchial asthma.     

Relaxation of myometrium by calcitonin gene-related peptide is independent of nitric oxide synthase activity in mouse uterus

Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(-/-), iNOS(-/-), and nNOS(-/-) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.     

The complex trans-[RuCl([15]aneN4NO]2+ induces rat aorta relaxation by ultraviolet light irradiation.

The aim of the present study was to investigate the possible endogenous storage of photosensitive nitric oxide, and also to examine the relaxant effect of NO released from the compound by UV light irradiation. Aorta was isolated from rats and the endothelium was mechanically removed. Denuded aortic rings pre-contracted with prostaglandin F(2alpha) responded with relaxation to UV light irradiation. The first stimulation produced the greatest response that decreased until complete disappearance. After this, the addition of the compound in the absence of light did not produce any response. However, in the presence of UV light irradiation, the complex trans-[RuCl([15]aneN4)NO]2+ induced 100% relaxation. After incubation with the nitric oxide scavenger, oxyhaemoglobin, this relaxation was completely abolished. In PGF2(2alpha)-pre-contracted aortas, the time to reach maximum relaxation was only 50s. Taken together, these results suggest that preformed endogenous nitric oxide stores exist in the denuded rat aorta, and that they are sensitive to UV light. The photo-induction of the complex trans-[RuCl([15]aneN4NO]2+ induces complete aorta relaxation, which is due to release of nitric oxide in the extracellular medium.     

Quantitative and kinetic characterization of nitric oxide and EDRF released from cultured endothelial cells

Endothelial cells (EC) contribute to the control of local vascular diameter by formation of an endothelium derived relaxant factor (EDRF) (1). Whether nitric oxide (NO) is identical with (EDRF) or might represent only one species of several EDRFs has not been decided as yet (2-5). Therefore, we have directly compared in cultured EC the kinetics of NO formation determined in a photometric assay with the vasodilatory effect of EDRF and NO in a bioassay. Basal release of NO was 16, 4 pmol/min/ml packed EC column. After stimulation with bradykinin (BK) and ATP onset of endothelial NO release and maximal response preceded the EDRF-mediated relaxation. Concentrations of NO formed by stimulated EC were quantitatively sufficient to fully explain the smooth muscle relaxation determined in the bioassay. Our data provide convincing evidence that under basal, BK and ATP-stimulated conditions 1. endothelial cells release nitric oxide as free radical, 2. nitric oxide is solely responsible for the vasodilatory properties of EDRF.     

CGRP modulates mucin synthesis in surface mucus cells of rat gastric oxyntic mucosa

We examined the effects of the calcitonin gene-related peptide (CGRP), including the possible participation of nitric oxide (NO), on mucin biosynthesis in the surface epithelium and remaining deep mucosa as well as the entire mucosa and compared the distribution of CGRP and NO synthase (NOS) using a combination of double immunofluorescence labeling and multiple dye filter. Pieces of tissue obtained from the corpus and antrum were incubated in a medium containing [(3)H]glucosamine and CGRP, with or without the NOS inhibitor. CGRP dose-dependently enhanced [(3)H]glucosamine incorporation into the corpus mucin but had no effect on antral mucin biosynthesis. The CGRP receptor antagonist, CGRP-(8-37), prevented the increase in (3)H-labeled corpus mucin. This stimulation of corpus mucin synthesis disappeared after removal of the surface mucus cell layer. CGRP activated the mucin biosynthesis in the surface mucus cells. In the full-thickness corpus mucosa, CGRP-induced activation was completely blocked by the NOS inhibitor. CGRP-immunoreactive fibers were intertwined within the surface mucus cell layer with type I NOS immunoreactivity. These results show that CGRP-stimulated mucin biosynthesis mediated by NO is limited to surface mucus cells of the rat gastric oxyntic mucosa.     

糖尿病大鼠肠系膜动脉降钙素基因相关肽释放减少以及一氧化氮的作用

我们以前的工作已表明,内毒素可引起降钙素基因相关肽（ＣＧＲＰ）从大鼠肠系膜动脉床释放,此作用部分是通过一氧化氮介导的。我们在离体肠系膜动脉床研究了内毒素引起糖尿病大鼠ＣＣＲＰ释放的改变以及一氧化氮所起的作用。采用ＣＣＲＰ放射免疫分析法测定灌流液中ＣＣＲＰ含量,ＲＴ－ＰＣＲ法测定背根神经节ＣＧＲＰｍＲＮＡ水平。结果显示：内毒素累积灌流引起ＣＧＲＰ浓度依赖性地释放增多,此作用在糖尿病大鼠系膜动脉术明显     

Role of nitric oxide in lower esophageal sphincter relaxation to swallowing.

Studies were performed in the opossum to define the role of the L-arginine-nitric oxide (NO) pathway in lower esophageal sphincter (LES) relaxation to swallowing and vagal stimulation in viv and intramural nerve stimulation in vitro. In vivo, L-NAME, a water soluble NO synthase (NOS) inhibitor, caused antagonism of LES relaxation due to reflex-induced swallowing. L-NAME (20 mg/kg i.v.) reduced the amplitude of swallow induced relaxation from 88% to 28%. LES relaxation due to electrical stimulation of peripheral end of decentralized vagus nerve was also antagonized. The effects of L-NAME were reversed by L-arginine, but not by D-arginine. L-NAME treatment did not antagonize LES relaxation to intravenous administration of isoproterenol. In vitro, NO and sodium nitroprusside (SNP) caused a decrease in the sphincter tone. The relaxing effect caused by NO and SNP was not antagonized by tetrodotoxin or omega-conotoxin. Inhibitors of NO synthase, L-NMMA and L-NNA, caused slight increase in the spontaneous resting LES tone and concentration-dependent antagonism of electrical field stimulation (EFS) induced LES relaxation. L-NNA (10(-4)M) abolished EFS induced LES relaxation at low frequencies (less than 5 Hz) and antagonized the relaxation to a value 20% of the control at 20 Hz. The antagonistic action of L-NMMA and L-NNA was unaffected by D-arginine but was reversed by L-arginine. The inhibitory effect of NO, SNP, or two other putative inhibitory neurotransmitters (VIP and CGRP) on the LES was not antagonized by L-NNA. These studies show that inhibitors of NO synthase selectively antagonize LES relaxation to all three modes of intramural inhibitory nerve stimulation including physiological swallowing. These studies suggest that the L-arginine-nitric oxide pathway is involved in physiological relaxation of the LES.     

eNOS-dependent vascular interaction between nitric oxide and calcitonin gene-related peptide in mice: gender selectivity and effects on blood aggregation

The present study was performed to explore a possible vascular interplay between nitric oxide (NO) and calcitonin gene-related peptide (CGRP). We examined factors affecting CGRP release by the NO donor, nitroglycerin (NTG) and the potential involvement of endothelial NO synthase (eNOS) using eNOS knockout (-/-) vs. wild-type (+/+) mice. In the female eNOS (+/+) mice, but not in males, in vitro NTG (0.73 mM) induced significant increases in the release of CGRP-like immunoreactivity (CGRP-LI) from the aorta and the heart but not from the small intestine. In eNOS (-/-) mice, NTG incubation did not induce any CGRP-LI changes in either gender. These results suggest that NTG-induced CGRP release is eNOS-dependent and tissue- and gender-selective. The functional implication of this NO-CGRP interaction was further examined by testing the anti-aggregatory action of acetylcholine (Ach). Ach-induced platelet inhibition was significantly enhanced by the addition of aorta segments of either gender. However, the female aorta segments exhibited a greater platelet inhibitory effect, which could be reversed by the blockade of either CGRP or eNOS. Our study revealed a novel eNOS-dependent interaction between NO and CGRP, and the possible participation of regulatory peptides in affecting platelet function and possibly cardiovascular protection in females.     

Gastroprotective and vasodilatory effects of epidermal growth factor: the role of sensory afferent neurons

Epidermal growth factor (EGF) has been shown to exert gastric hyperemic and gastroprotective effects via capsaicin-sensitive afferent neurons, including the release of calcitonin gene-related peptide (CGRP). We examined the protective and vasodilatory effects of EGF on the gastric mucosa and its interaction with sensory nerves, CGRP, and nitric oxide (NO) in anesthetized rats. Intragastric EGF (10 or 30 microg) significantly reduced gastric mucosal lesions induced by intragastric 60% ethanol (50.6% by 10 microg EGF and 70.0% by 30 microg EGF). The protective effect of EGF was significantly inhibited by pretreatment with capsaicin desensitization, human CGRP1 antagonist hCGRP-(8-37), or N(omega)-nitro-L-arginine methyl ester (L-NAME). Intravital microscopy showed that topically applied EGF (10-1,000 microg/ml) dilated the gastric mucosal arterioles dose dependently and that this vasodilatory effect was significantly inhibited by equivalent pretreatments. These findings suggest that EGF plays a protective role against ethanol-induced gastric mucosal injury, possibly by dilating the gastric mucosal arterioles via capsaicin-sensitive afferent neurons involving CGRP and NO mechanisms.     

Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells

Pulmonary hypertension is characterized by vascular remodeling involving smooth muscle cell proliferation and migration. Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are potent vasodilators, and the inhibition of aortic smooth muscle cell (ASMC) proliferation by NO has been documented, but less is known about the effects of CGRP. The mechanism by which overexpression of CGRP inhibits proliferation in pulmonary artery smooth muscle cells (PASMC) and ASMC following in vitro transfection by the gene coding for prepro-CGRP was investigated. Increased expression of p53 is known to stimulate p21, which inhibits G(1) cyclin/cdk complexes, thereby inhibiting cell proliferation. We hypothesize that p53 and p21 are involved in the growth inhibitory effect of CGRP. In this study, CGRP was shown to inhibit ASMC and PASMC proliferation. In PASMC transfected with CGRP and exposed to a PKA inhibitor (PKAi), cell proliferation was restored. p53 and p21 expression increased in CGRP-treated cells but decreased in cells treated with CGRP and PKAi. PASMC treated with CGRP and a PKG inhibitor (PKGi) recovered from inhibition of proliferation induced by CGRP. ASMC treated with CGRP and then PKAi or PKGi recovered only when exposed to the PKAi and not PKGi. Although CGRP is thought to act through a cAMP-dependent pathway, cGMP involvement in the response to CGRP has been reported. It is concluded that p53 plays a role in CGRP-induced inhibition of cell proliferation and cAMP/PKA appears to mediate this effect in ASMC and PASMC, whereas cGMP appears to be involved in PASMC proliferation.     

High iNOS mRNA and protein localization during late pregnancy suggest a role for nitric oxide in mouse pubic symphysis relaxation

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The mouse PS remodels in a hormone-controlled process that involves the modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide synthase (NOS) and consequently nitric oxide (NO) generation play important roles in extracellular matrix modification, and may promote cytoskeleton changes that contribute to the remodeling of connective tissue, which precedes the onset of labor. To our knowledge, no studies thus far have investigated inducible nitric oxide synthase (iNOS) expression, protein localization, and NO generation in the mouse PS during pregnancy. In this work, we used a combination of the immunolocalization of iNOS, its relative mRNA expression, and NO production to examine the possible involvement of iNOS in remodeling and relaxation of the mouse IpL during late pregnancy. The presence of iNOS was observed in chondrocytes and fibroblast-like cells in the interpubic tissues. In addition, iNOS mRNA and NO production were higher during preterm labor on Day 19 of pregnancy (D19) than NO production on D18 or in virgin groups. The significant increase in iNOS mRNA expression and NO generation from the partially relaxed IpL at D18 to the completely relaxed IpL at D19 may indicate that NO plays an important role in late pregnancy during relaxation of the mouse IpL.     

Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs

Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to beta-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.     

Involvement of calcitonin gene-related peptide in control of human fetoplacental vascular tone

Calcitonin gene-related peptide (CGRP), one of the most potent endogenous vasodilators known, has been implicated in vascular adaptations and placental functions during pregnancy. The present study was designed to examine the existence of CGRP-A receptor components, the calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1), in the human placenta and the vasoactivity of CGRP in the fetoplacental circulation. Immunofluorescent staining of the human placenta in term labor using polyclonal anti-CRLR and RAMP1 antibodies revealed that labeling specifically concentrated in the vascular endothelium and the underlying smooth muscle cells in the umbilical artery/vein, chorionic artery/vein, and stem villous vessels as well as in the trophoblast layer of the placental villi. In vitro isometric force measurement showed that CGRP dose dependently relaxes the umbilical artery/vein, chorionic artery/vein, and stem villous vessels. Furthermore, CGRP-induced relaxation of placental vessels are inhibited by a CGRP receptor antagonist (CGRP8-37), ATP-sensitive potassium (KATP) channel blocker (glybenclamide), and cAMP-dependent protein kinase A inhibitor (Rp-cAMPS) and partially inhibited by a nitric oxide inhibitor (Nomega-nitro-l-arginine methyl ester). We propose that CGRP may play a role in the control of human fetoplacental vascular tone, and the vascular dilations in response to CGRP may involve activation of KATP channels, cAMP, and a nitric oxide pathway.     

Histamine-induced relaxation in pulmonary artery of normotensive and hypertensive rats: relative contribution of prostanoids, nitric oxide and hyperpolarization

The aim of this study was to determine the relative contribution of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostanoids in histamine-induced relaxation of isolated pulmonary artery from normotensive and hypertensive rats. The hypertension was induced by oral administration of NO synthase inhibitor N(G)-nitro-L-arginine methylester (L-NAME, 50 mg/kg/day) to normotensive rats for 8 weeks. In phenylephrine-precontracted arterial rings the histamine-induced relaxation was significantly reduced in L-NAME-treated rats compared to the controls. Indomethacin (cyclooxygenase inhibitor) and glibenclamide (ATP-sensitive K+-channel blocker) did not inhibit the relaxation response in either control or hypertensive rats. On the other hand, tetraethylammonium (TEA), a K+-channel blocker with a broad specificity, significantly reduced histamine-induced relaxation in the pulmonary artery from both groups examined. The TEA-resistant relaxation was completely abolished by additional administration of L-NAME to the incubation medium. The results indicate that histamine-induced relaxation of the pulmonary artery in both normotensive and hypertensive rats is mediated mainly by nitric oxide, whereas EDHF seems to play a minor role.