Serum Calcitonin Gene-related Peptide Levels Research Articles

Control of the Calcitonin Gene-related Peptide Enhancer by Upstream Stimulatory Factor in Trigeminal Ganglion Neurons

The neuropeptide calcitonin gene-related peptide (CGRP) is a key player in migraine. However, the transcription factors controlling CGRP expression in the migraine-relevant trigeminal ganglion neurons are unknown. Previous in vitro studies demonstrated that upstream stimulatory factor (USF) 1 and USF2 bind to the CGRP neuroendocrine-specific 18-bp enhancer, yet discrepant overexpression results in cell lines, and the ubiquitous nature of the USF cast doubts about its role. To test the functional role of USF, we first demonstrated that small interfering RNAs directed against USF1 and USF2 reduced endogenous CGRP RNA and preferentially targeted the USF binding site at the 18-bp enhancer in the neuronal-like CA77 cell line. In cultured rat trigeminal ganglion neurons, knockdown of either USF1 or USF2 reduced CGRP promoter activity. Conversely, overexpression of USF1 or USF2 increased promoter activity. The activation was even greater upon cotransfection with an upstream activator of mitogen-activated protein kinases and was synergistic in a heterologous cell line. To begin to address the paradox of how ubiquitous USF proteins might direct neuronal-specific activity, we examined USF expression and used a series of adenoviral reporters in the cultured ganglia. Unexpectedly, there was more intense USF immunostaining in neurons than nonneuronal cells. Importantly, the 18-bp USF enhancer driving a minimal promoter was sufficient for neuronal specificity, although it was not the only site that directed neuronal expression. These results demonstrate that USF1 and USF2 are important contributors to neuronal-specific and mitogen-activated protein kinase regulation of the CGRP gene in trigeminal ganglion neurons.

Hypertension and dysregulated proinflammatory cytokine production in receptor activity-modifying protein 1-deficient mice

Calcitonin gene-related peptide (CGRP) is thought to be a prominent neuropeptide in cardiovascular regulation and neuroimmune modulation. There are two isoforms of CGRP (alphaCGRP and betaCGRP), and the main CGRP receptors are probably composed of a calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP)1. However, the physiological functions of CGRP that are mediated through the CLR/RAMP1 receptors remain to be clarified. For an improved understanding of the functions, we generated mice deficient in RAMP1, a specific subunit of CGRP receptors, by a conditional gene-targeting technique. The RAMP1-deficient mice (RAMP1(-/-)) exhibited high blood pressure, with no changes in heart rate. alphaCGRP was found to have a potent vascular relaxant activity compared with betaCGRP in the artery of the WT (RAMP1(+/+)) mice. The activities of both CGRP isoforms were remarkably suppressed in the arteries of the RAMP1(-/-) mice. The LPS-induced inflammatory responses of the RAMP1(-/-) mice revealed a transient and significant increase in the serum CGRP levels and high serum levels of proinflammatory cytokines compared with the RAMP1(+/+) mice. alphaCGRP and betaCGRP equally suppressed the production of TNF-alpha and IL-12 in bone marrow-derived dendritic cells stimulated with lipopolysaccharide. Their inhibitory effects were not observed in the bone marrow-derived dendritic cells of the RAMP1(-/-) mice. These results indicate that CGRP signaling through CLR/RAMP1 receptors plays a crucial role in the regulation of both blood pressure by vascular relaxation and proinflammatory cytokine production from dendritic cells.

Role of neuropeptides in migraine: where do they stand in the latest expert recommendations in migraine treatment?

AbstractMany factors have been implicated in the pathogenesis of migraine headache, including activation of the trigeminovascular system, dysfunction of: cerebral blood vessels, circulating vasoactive substances, mitochondrial energy metabolism, brain oxygenation and metabolism, platelet disorder, alterations in serotonin levels, low levels of brain tissue magnesium, altered transport of ions across the cell membrane, and inheritance and dysfunction of the brainstem periaqueductal gray matter. The headache phase of migraine is associated with cerebral vasodilation and inflammation, presumably mediated by the release of vasoactive substances and neuropeptides including CGRP (calcitonin gene‐related peptide). Increased serum CGRP levels have been detected during migraine and cluster headache. One strategy to treat migraine is to inhibit the release of neuropeptides or to block their receptors. This article briefly reviews some experimental and clinical investigations focused on neuropeptide involvement in migraine. Drug Dev Res 68:294–314, 2007. © 2007 Wiley‐Liss, Inc.

EXOGENOUS CAPSAICIN APPLICATION INDUCES ENDOGENOUS CGRP LIBERATION AND ATTENUATES ACUTE EXPERIMENTAL PANCREATITIS

Introduction: Vasoactive mediators play an important role in the pathophysiology of acute pancreatitis (AP). Calcitonin-gene-related peptide (CGRP) is stored in pancreatic perivascular nerves and influences vasomotion and cytokine release in inflammation. CGRP can be liberated by endo-or exogenous capsaicin. Aim of the study was to evaluate the influence of capsaicin on acute experimental pancreatitis. Methods: AP was induced in rats by glycodeoxycholic acid and caerulein (GDOC, n = 12). The second group received GDOC, caerulein and capsaicin simultaneously (GDOCcaps, n=12). Control animals without AP received either NaCl (control, n=12) or capsaicin (capscontrol, n=12). The following parameters were evaluated after 24h: survival, histology, amylase and CGRP levels (n=6/group). Microcirculation of the pancreas (blood flow, leukocyte adhesion (LEI), n=6/group) were evaluated by intravital microscopy (IVM) after 7h. Results: Control and capscontrol animals showed no mortality, whereas GDOC animals had a 67% mortality. Capsaicin reduced mortality significantly (p < 0.05) to 16% (GDOCcaps). Histology showed no changes in control and capscontrol animals. In the AP groups, inflammation and necrosis were evident. When capsaicin was administered, both, inflammation and necrosis were reduced significantly (p < 0.01) compared to GDOC animals. Serum CGRP levels increased in both capsaicin groups (p < 0.05) indicating a liberation of endogenous CGRP via this pathway. In IVM, erythrocyte velocity was reduced in GDOC animals (p < 0.01) but back to control values after capsaicin application. Capsaicin also increased cell velocity in healthy animals (p < 0.01) compared to controls. LEI was significantly higher in GDOC animals (p < 0.01 vs. control). Application of capsaicin reduced LEI in capillaries and venules (p < 0.05). Conclusion: Capsaicin attenuates severe experimental AP. This is mediated via the liberation of CGRP leading to a vasodilatation and immunomodulation reducing proinflammatory mediators. These results underline the importance of neuropeptide regulations in the early phase of AP. Further studies are necessary to evaluate potential effects of a therapeutic application of capsaicin.

Serum calcitonin gene-related peptide levels in untreated prostate cancer patients

The role of calcitonin gene-related peptide (CGRP) in prostate cancer has not been fully understood. Moreover, the serum CGRP level in prostate cancer patients has never been reported. We measured the serum CGRP levels in untreated prostate cancer patients to elucidate its clinical significance. We used 36 serum samples from prostate cancer patients. All patients had never received any treatment. Serum CGRP was measured by immunoradiometric assay, and we analysed the association between serum CGRP level and clinicopathological factors. Serum CGRP levels in the patients with higher clinical stages and histological grade were significantly higher than in those with lower stages and grade, respectively. But the levels did not correlate with the patient's age, liver or renal functions, serum prostate-specific antigen levels. Serum CGRP levels were significantly elevated in the patients with high grade or high stage untreated prostate cancer patients. Measurement of the serum CGRP may be a useful predictor of staging or grading of prostate cancer in the untreated prostate cancer patients.

Serum substance P and calcitonin gene-related peptide levels in Behcet's disease and their association with disease activity

Substance P (SP) and calcitonin gene-related peptide (CGRP) are neuropeptides that have a role in several cutaneous diseases and inflammations. Aim. To evaluate SP, CGRP and serum interleukin (IL)-8 levels in Behçet's disease (BD) and to explore the relationship of these peptides with BD activity. The study group comprised 30 patients with BD, and 30 healthy individuals acted as controls. Serum levels of SP, CGRP and IL-8 were determined by micro-ELISA test during the active and inactive disease periods of patients with BD. These data were compared with each other and controls. Active and inactive periods of BD were established. The mean +/- SD serum CGRP (ng/ml) and IL-8 levels (pg/ml) in inactive BD (5.87 +/- 2.49 and 0.62 +/- 0.24, respectively) were significantly higher than the control group (4.74 +/- 1.17 and 0.46 +/- 0.11) (P < 0.05 for both). The difference between serum CGRP and IL-8 levels in active BD were also significantly higher than in inactive BD (P < 0.05 for both). Serum SP values (ng/ml) in active BD (18.27 +/- 5.38) were significantly higher than in inactive BD (15.26 +/- 5.74) and controls (12.6 +/- 4.45) (P < 0.05 for all), whereas the difference between the serum SP values in inactive BD and the control group was not statistically significant (P > 0.05). Serum SP and CGRP may have a role in the pathogenesis of BD. In addition, serum IL-8, SP and CGRP levels can be used as laboratory parameters indicating activity in BD.

A Study of the Relationships between Serum Calcitonin Gene-Related Peptide, Sex Hormone, Homocysteine and Coronary Heart Disease in Postmenopausal Women

To investigate the relationships among serum calcitonin gene-related peptide (CGRP), homocysteine (Hcy), female hormone, and coronary arteriopathy in postmenopausal women. Method. In a cross-sectional study, serum CGRP, estrodiol (E2), progesterone（P）and Hcy levels of 144 postmenopausal women with coronary heart disease (CHD) and non-coronary heart disease (NCHD) were measured. Results. The mean serum CGRP level was significantly lower in CHD patients than in NCHD subjects. The mean serum E2 and P level were significantly lower in CHD patients than in NCHD subjects. The mean serum Hcy level was significantly higher in CHD patients than in NCHD subjects. By multivariate logistic regression, the OR of high Hcy level ≥1, p&lt;0.01，which suggests that Hcy is an independent risk factor in the development of coronary arteriopathy. The OR of CGRP, E2 , and P are all≤1, indicating that CGRP , E2 and P are independent protective factor. Conclusion. The results of our study show that Hcy is an independent risk factor in the development of arteriopathy. CGRP and endogenous E2 are independent protective factors in the development of coronary arteriopathy. There are no relationships between Hcy, CGRP, and endogenous E2.

Effects of magnesium sulfate on lipid peroxidation and blood pressure regulators in preeclampsia

Objectives: To investigate the status of lipid peroxidation and serum levels of several vasoactive substances in preeclamptic (PE) pregnant women before and during treatment with magnesium sulfate (MgSO 4). Design and methods: The study population included 16 PE women. Circulating levels of malondialdehyde (MDA), endothelin 1 (ET-1), nitric oxide (NO) metabolites, and calcitonin gene-related peptide (CGRP) were measured before (at admission) and during MgSO 4 treatment (at delivery and 24 h postpartum). Results: At admission systolic and diastolic blood pressures were 157 ± 3 mm Hg and 106 ± 2 mm Hg, respectively, and decreased significantly during treatment at delivery and 24 h postpartum ( P < 0.0001). Before treatment, serum MDA concentrations were 0.383 ± 0.037 μmol/L, and decreased significantly during MgSO 4 administration at delivery and 24 h postpartum ( P < 0.0001). In contrast, serum ET-1 levels at 24 h postpartum were significantly higher as compared with those observed before treatment (79 ± 3 versus 65 ± 2 pg/mL, P = 0.002). Serum NO metabolite concentrations were 26 ± 3 μmol/L, and no significant changes were observed during treatment. Serum levels of CGRP were 50 ± 3 pg/mL at admission, and increased significantly at partum ( P < 0.001). Serum ET-1 correlated negatively with NO metabolites before treatment ( r = −0.69, P = 0.002), but not during treatment. In contrast, ET-1 correlated positively with serum CGRP levels during treatment ( r = 0.73, P = 0.002 and r = 0.71, P = 0.002, at delivery and 24 h postpartum, respectively), but not before treatment. Conclusions: This study demonstrates that MgSO 4 administration to PE pregnant women induced significant changes in lipid peroxidation, production of ET-1 and CGRP.

This month in J Lab Clin Med: Issue highlights for February 2003

This month in J Lab Clin Med: Issue highlights for February 2003

Maternal plasma calcitonin gene–related peptide levels do not change during labor and are not influenced by delivery route

Calcitonin gene-related peptide (CGRP) circulates in maternal circulation throughout pregnancy, and specific receptors for CGRP (CGRPrs) are expressed by human myometrium. Because CGRP induces a dose-dependent relaxation of human myometrium, we examined a role for CGRP in modulation of myometrial smooth muscle contractility during pregnancy and labor. The aim of the present study was to evaluate the changes of maternal serum CGRP levels during parturition, according to the mode of delivery and in relation to cervical dilatation. Circulating CGRP levels were measured in the following groups of healthy women: nonpregnant women, during the follicular phase of the menstrual cycle (n = 19); at term pregnancy (39-40 weeks; n = 24); after elective cesarean delivery (39-40 weeks; n = 20); and at spontaneous vaginal delivery (39-40 weeks; n = 16). In a subgroup of women, blood samples were collected longitudinally throughout labor at various cervical dilatations in the progress of labor (n = 8). Pregnant women at term not in labor had significantly higher CGRP levels than nonpregnant women (P =.021). No significant difference was found between women who delivered vaginally and those who had elective cesarean, and there were no correlations between CGRP plasma levels and cervical dilatation. Parturition is characterized by no significant changes in maternal serum CGRP levels, and no significant correlation exists between plasma CGRP levels and cervical dilatation during labor.

Calcitonin gene- and parathyroid hormone-related peptides in preeclampsia: effects of magnesium sulfate

Objective: To determine whether circulating levels of calcitonin gene-related peptide (CGRP) and parathyroid hormone-related peptide (PTHrP) are altered in preeclampsia, and to assess the effects of magnesium sulfate therapy on circulating levels of these two peptides. Methods: The study population included 25 women with preeclampsia and 25 normotensive controls of similar gestational age. The effects of magnesium sulfate therapy were evaluated in 17 of the 25 preeclamptic women. Circulating levels of immunoreactive CGRP and PTHrP, including calcium, magnesium, and phosphate in the maternal and umbilical cord serum were measured. Results: The frequency of preeclampsia subjects with nondetectable PTHrP (under 3 pg/mL) was significantly higher (92% versus 48%, P < .001), whereas maternal serum CGRP levels were significantly lower (50 ± 19 versus 90 ± 23 pg/mL, P < .001). Similarly, the frequency of newborns with nondetectable PTHrP levels in umbilical serum was significantly higher (68% versus 36%, P < .05), whereas the levels of CGRP were significantly lower (67 ± 17 versus 79 ± 16 pg/mL, P < .05). Magnesium sulfate treatment resulted in a significant increase in maternal circulating CGRP levels (64 ± 17 versus 47 ± 18 pg/mL, P < .05) with no changes in PTHrP. Conclusion: Maternal circulating PTHrP and CGRP concentrations were significantly lower in women with preeclampsia, which may contribute to the development and maintenance of hypertension during pregnancy. Furthermore, magnesium sulfate therapy increased the levels of CGRP in the maternal circulation.

Regulation of calcitonin gene-related peptide expression in dorsal root ganglia of rats by female sex steroid hormones.

Calcitonin gene-related peptide (CGRP), a potent vasodilator primarily synthesized in dorsal root ganglia (DRG) neurons, has been shown to decrease vascular resistance and thus regulate blood flow to a variety of organs in rats. Serum CGRP levels in the human have been reported to increase with pregnancy and decrease postpartum. It has been suggested that female sex steroid hormones play a role in cardiovascular function, but the mechanisms are unknown. In this study, we examined the effects of estradiol-17beta (E(2)) and progesterone (P(4)) on the expression of CGRP in DRG in adult rats both in vivo and in vitro. Ovariectomized (ovx) animals were injected s.c. with 5 microg E(2), 4 mg P(4), or 5.0 microg E(2) + 4 mg P(4) in 0.5 ml sesame oil or with oil only, and groups of 4 rats were killed at 0, 24, or 48 h. DRGs were then removed and analyzed for CGRP mRNA and immunoreactive (i-)CGRP content by Northern blotting and RIA, respectively. Primary cultures of DRG neurons from adult female rats were used to assess the effects of varying doses of E(2) (1, 10, 100 nM), P(4) (10, 100, 1000 nM), or E(2) (10 nM) + P(4) (100 nM) in the absence or presence of nerve growth factor (NGF; 20 ng/ml); and CGRP mRNA content in the cells and i-CGRP in the medium were quantitated at 24 or 48 h after incubation. Results of in vivo studies showed that E(2) caused a significant increase in CGRP mRNA at 24 h (1.8-fold) and in i-CGRP levels both at 24 h (2. 8-fold) and at 48 h (3.4-fold) in DRG of ovx rats. P(4) also stimulated expression of both CGRP mRNA and i-CGRP. In the in vitro studies, either E(2) or P(4) alone or the two in combination were without effect on CGRP expression in cultured DRG neurons at all the doses tested. However, in the presence of NGF, both CGRP mRNA and peptide levels were significantly enhanced by E(2), P(4), and E(2)+P(4) in a time-dependent (2.0- to 2.8-fold at 24 h, 3.0- to 5. 0-fold at 48 h) and dose-dependent manner, with maximal effects achieved at 1.0 nM (E(2)) and 100 nM (P(4)) at 24 h of incubation. In summary, both E(2) and P(4), either alone or in combination, stimulate CGRP peptide synthesis in DRG neurons through increasing CGRP mRNA. The effects of these steroid hormones are mediated through amplifying the NGF-induced synthesis of CGRP in these neurons. Thus, we propose that the cardiovascular functions of female sex steroid hormones may be mediated, at least in part, by the up-regulation of neuronal CGRP synthesis, via NGF-mediated mechanisms.

Calcitonin gene‐related peptide in small cell lung carcinomas

Calcitonin gene-related peptide (CGRP) is a regulatory peptide encoded by the calcitonin gene. CGRP is expressed in increased amounts by the cells of medullary thyroid carcinomas and has been demonstrated by immunohistochemistry to occur in neuroendocrine cells and nerve fibres of lung tissue. Serum CGRP levels were measured in patients with small cell lung carcinomas before treatment (n = 74) and immediately before the second course of chemotherapy (n = 30). In-situ hybridization and immunohistochemistry were performed on tumour tissue and CGRP was extracted from two tumours and characterized by gel chromatography and high pressure liquid chromatography. Serum CGRP levels were elevated in small cell lung carcinomas when compared with healthy controls of similar age and sex (median values 55.0 vs 36.6 pmol/l, P < 0.001), and 27% had levels above the upper normal range. Serum CGRP levels decreased following the initial course of chemotherapy (P < 0.05) but remained elevated when compared to the controls (P < 0.001). In-situ hybridization for CGRP mRNA was positive in three of 17 tumours and immunohistochemistry was positive in seven of 31 tumours investigated. CGRP immunoreactivity extracted from two tumours was characterized by gel chromatography and high pressure liquid chromatography. A major part of the immunoreactivity was demonstrated to represent the intact molecule. We found that patients with small cell lung carcinomas had elevated concentration of serum calcitonin gene-related peptide but only 27% had values above the upper normal range. Serum CGRP is therefore of limited value as a tumour marker. Intact CGRP can be extracted from tumour tissue, but in-situ hybridization and immunohistochemistry showed positive reactions in only a few of the tumours investigated. The elevated serum CGRP levels are therefore likely to be largely of extratumoral origin.

Radioimmunoassay for Calcitonin Gene-Related Peptide and Its Measurement in Sera of Patients with Thyroid Disease.

To investigate serum levels of calcitonin gene-related peptide (CGRP), we developed a sensitive radioimmunoassay (RIA). RIA for CGRP in serum can present some problems: the serum may degradate the tracer during incubation and suppress the antigen-antibody reaction. We avoided these problems by using aprotinin and CGRP-free serum instead of a buffer for the standard curve. We detected serum CGRP in all 39 healthy subjects when CGRP-free serum was not used for the standard curve, but 34 of these subjects had serum CGRP levels below the detection limit (less than 80 pmol/l) when CGRP-free serum was used for the standard curve. We defined the normal range for serum CGRP as below 100.8 pmol/l, which was the maximum level found in the healthy subjects. We studied serum levels of this peptide in patients with thyroid diseases, because the thyroid may be one origin of circulating CGRP. Four of 10 patients with medullary thyroid carcinoma had elevated serum levels of CGRP. Seven of 24 patients with subacute thyroiditis had elevated serum levels of CGRP, but at least one year after clinical recovery, CGRP was undetectable in all. Seven of the 37 patients with hypothyroidism had elevated serum levels of CGRP. None of the patients with hyperthyroidism, adenomatous goiter, thyroid adenoma, or thyroid carcinoma had elevated serum CGRP levels. It is necessary to use a standard curve obtained by the addition of aprotinin and CGRP-free serum to the assay standards to measure serum CGRP levels. Some patients with subacute thyroiditis, hypothyroidism, or medullary thyroid carcinoma had elevated serum CGRP levels.

Circulating concentrations of calcitonin gene-related peptide (CGRP) in normal man determined with a new, highly sensitive radioimmunoassay

A highly sensitive and specific radioimmunoassay for human calcitonin gene-related peptide (CGRP) has been developed and used for determination of serum CGRP levels in 232 normal individuals, 123 females and 109 males, range of age 18 to 79 years. Mean serum concentration was 36.3 pmol/l +/- 6.2 (SD), and serum levels were unrelated to age and sex. The specificity of the assay was confirmed by gel chromatography, followed by HPLC analysis of extracts from normal serum using synthetic alpha- and beta-CGRP as standards.

Evaluation of a Peptide Family Encoded by the Calcitonin Gene in Selected Healthy Pregnant Women

We conducted a longitudinal study on serum levels of peptides encoded by the calcitonin gene before conception, every month during pregnancy, and 24 h and 5 days after delivery in 26 healthy women. Only subjects fulfilling optimality criteria according to the literature were included. Blood samples for ionized calcium, total (tCT) and extractable (exCT) calcitonin, katacalcin, and calcitonin gene-related peptide (CGRP) were collected. We found no significant changes of ionized calcium, tCT, exCT, and katacalcin levels, while CGRP serum levels showed a significant increase during pregnancy and a fall to preconceptional values after delivery. Since variations of calcitonin levels did not occur in our selected pregnant women, we conclude that thyroidal C cell secretion is not increased during pregnancy. Our data suggest that calcitonin is not involved in the modifications of mineral homeostasis occurring in pregnancy. In addition, the variations of CGRP serum levels we found suggest that such a hormone participates in circulation modifications of pregnant women.

ELEVATED SERUM LEVELS OF CALCITONIN GENE‐RELATED PEPTIDE (CGRP) BUT NO EVIDENCE FOR CGRP GENE EXPRESSION IN NON‐SMALL CELL LUNG CARCINOMAS

Calcitonin gene-related peptide (CGRP) is a hormone formed by alternative post-transcriptional processing of the calcitonin gene. It is a neuropeptide localized to discrete regions of the central nervous system (CNS) and in nerve fibres associated with blood vessels. It is also expressed in medullary carcinomas of the thyroid and lung carcinoma cell lines. The latter finding suggests a possible value for CGRP as tumour marker in lung carcinomas. In this investigation of 22 patients undergoing operation for lung tumours, pre and post-operative levels of serum CGRP were measured. Preoperative as well as postoperative serum CGRP levels were significantly elevated when compared to age-matched normals. However, no evidence could be found for CGRP gene expression in tumour tissue from the same patients as judged by immunocytochemistry or in-situ hybridization using CGRP cRNA probes. CGRP has been localized to nerve fibres in relation to pulmonary blood vessels and has been shown to be a potent vasodilator. These findings, and the absence of evidence for synthesis in tumours, as opposed to cell lines derived from lung carcinomas, suggests that the lack of post-operative normalization of serum CGRP concentrations may be related to physiological changes in cardiovascular haemodynamics following surgery. Elevated pre-operative serum CGRP levels may also reflect a consequence of the lung carcinoma leading to increased release of CGRP from sites in the vasculature yet to be determined, but does not indicate synthesis de novo and secretion of CGRP by the tumours.