Expression Of Calcitonin Receptor-like Receptor Research Articles

Abstract A020: Calcitonin receptor-like receptor is expressed in blood vessels in clear cell renal cell carcinoma and upregulated in endothelial cells co-cultured with tumor cells

Abstract Introduction. Clear cell renal cell carcinoma (ccRCC) is a highly vascularized and clinically aggressive kidney cancer, with high rates of metastasis and recurrence after cytoreductive nephrectomy. ccRCC can be treated with anti-angiogenic drugs, however drug resistance usually develops within one year and 5-year survival for metastatic cases is approximately 10%. Calcitonin receptor-like receptor (CLR) is a G-protein-coupled receptor (GPCR) expressed in endothelial cells (EC) in core- (intracellular) and terminally-glycosylated (cell surface-associated) forms. CLR and its three agonists play a role in angiogenesis. In ccRCC, CLR is upregulated in tumor vessels when compared to vessels in adjacent tissues. The aims of this study were to: (1) investigate which types of vessels (blood or lymphatic) in ccRCC tissues express CLR and (2) determine whether ccRCC tumor cells affect expression of this GPCR in EC. Methods. CLR expression was analyzed in sections of ccRCC tissues (primary tumors) from three patients by using immunohistochemistry and our rabbit anti-human CLR antibody LN1436, with controls performed using the antigen (21 amino-acid peptide). Distribution of CLR immunoreactivity was compared to pan-EC (cluster of differentiation 31, CD31) and lymphatic EC-specific (podoplanin, PDPN) marker expression patterns, revealed by using immunohistochemistry on serial sections with mouse monoclonal antibodies JC/70A and D2-40 respectively. Two ccRCC cell lines (786-O and KTCTL-140) were co-cultured with human dermal blood vessel EC (HDBEC) in vitro for 48 hours. Cell types were separated by using magnetic-activated cell sorting (MACS) with anti-CD31 antibody-conjugated microbeads. CLR expression in ccRCC and HDBEC before, during and after co-culture was analyzed by using immunoblotting and LN1436 antibody. Results. In ccRCC tissue sections, CLR immunoreactivity was detected predominantly in blood and not lymphatic vessels. Core- and terminally-glycosylated forms of CLR were expressed in HDBEC, and the expression levels of both were upregulated after co-culture with each of the two studied ccRCC cell lines. Conclusions. These data advance knowledge about CLR expression in ccRCC and suggest that tumor cells may be responsible for its upregulation in EC within the tumor microenvironment. Our findings warrant further investigation of the potential role of the expressed in tumor blood vessels GPCR CLR as a prognostic marker and/or a target for therapy in ccRCC. Citation Format: Matthew A. Morfitt, John Greenman, Anthony Maraveyas, Adrian L. Harris, Leonid L. Nikitenko. Calcitonin receptor-like receptor is expressed in blood vessels in clear cell renal cell carcinoma and upregulated in endothelial cells co-cultured with tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A020.

Abstract B001: Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment

Abstract Introduction. Clear cell renal cell carcinoma (ccRCC) is a highly vascularized and clinically aggressive cancer. In the UK, there are around 9000 diagnoses and 3000 deaths annually. Targeted therapies that inhibit angiogenic regulators, such as bevacizumab (monoclonal antibody which inhibits vascular endothelial growth factor A, VEGF-A), have limited efficacy due to drug resistance. Expression of calcitonin receptor-like receptor (CLR) is upregulated in ccRCC and correlates with disease outcome. The expression of adrenomedullin (AM), peptide agonist of CLR and angiogenic factor, is upregulated in ccRCC. We hypothesized that the CLR signaling axis acts as an alternative pathway that enables resistance to drugs targeting the VEGF-A pathway in ccRCC. In the present study, we aimed to determine whether this signaling axis remains active in VEGF-A-stimulated and bevacizumab-treated human endothelial cells. Methods. Primary human dermal blood vessel endothelial cells (HDBEC; PromoCell) were characterized by immunofluorescence utilizing pan-endothelial (cluster of differentiation 31 and 144, CD31 and CD144, and von Willebrand factor, vWF) and lymphatic-specific (lymphatic vessel endothelial hyaluronan receptor 1, LYVE-1, and prospero homeobox 1, PROX1) markers and confocal microscopy. The activities of CLR agonists (AM; intermedin, IMD; calcitonin gene-related peptide, CGRP; 10−12 - 10−6 M at 10 min) and VEGF-A (50 ng/ml over 30-minute time course) were measured by analyzing phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) using immunoblotting. HDBEC were pre-treated with bevacizumab or human IgG1 at 250 ng/ml and then stimulated with individual CLR agonists at 10−6 M for 10 minutes. Experiments were repeated at least three times. Data were analyzed using Shapiro-Wilk normality, unpaired statistics and appropriate multiple comparisons tests utilizing GraphPad Prism and p<0.05 interpreted as significant. Results. HDBEC were a pure population. AM, IMD and CGRP induced p44/42 MAPK phosphorylation at 10−6 M (p<0.05). For all three CLR agonists and VEGF-A, p44/42 MAPK phosphorylation peaked after 10 minutes of incubation (p<0.05). Bevacizumab treatment at 250 ng/ml was sufficient to inhibit VEGF-A-induced p44/42 MAPK phosphorylation (p<0.05). AM, IMD and CGRP were all able to induce phosphorylation of p44/42 MAPK after bevacizumab treatment (p<0.01). Conclusions. The findings that CLR agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated HDBEC after bevacizumab treatment support our hypothesis. This work provides a foundation for investigating the role of the CLR signaling axis in mechanisms of ccRCC resistance to drugs targeting the VEGF-A pathway. Citation Format: Matthew A. Morfitt, John Greenman, Anthony Maraveyas, Leonid L. Nikitenko. Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B001.

Calcitonin gene-related peptide and its receptor plays important role in nociceptive regulation in the arcuate nucleus of hypothalamus of rats with inflammatory pain

The present study has explored the role of calcitonin gene-related peptide (CGRP) and its receptor in inflammatory pain modulation in arcuate nucleus of hypothalamus (ARC). Our study demonstrated that intra-ARC injection of CGRP induced antinociceptive effects to naïve rats and rats with inflammatory pain, the effect could be inhibited by the selective CGRP receptor antagonist CGRP8–37. Interestingly, the CGRP-induced antinociception effect was decreased in rats with inflammatory pain compared to naïve rats. Similarly, we found that calcitonin receptor like receptor (CLR), a main component of CGRP receptor, had a low decreased expression levels in the ARC regions of rats with inflammatory pain. The CGRP-induced antinociceptive effect was significantly impaired after reducing CLR expression by intra-ARC administration of CLR targeted siRNA. These findings demonstrated that CGRP might play a crucial role in nociceptive modulation in the ARC during inflammatory pain, which was mediated by CGRP receptor in the ARC. This study shed light upon CGRP and its receptor interaction might be valuable strategies for the alleviation of inflammatory pain.

Protective effect of adrenomedullin on hyperoxia-induced lung injury.

To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions. HPMECs were randomly divided into an air group and a hyperoxia group (n=3 each).The HPMECs in the hyperoxia group were cultured in an atmosphere of 92% O2 (3 L/minute) +5% CO2. RT-qPCR and Western blot were used to measure the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB. Other HPMECs were divided into a non-interference group and an interference group (n=3 each), and the mRNA and protein expression levels of ADM, ERK1/2, and PKB were measured after the HPMECs in the interference group were transfected with ADM siRNA. Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB (P<0.05). Compared with the non-interference group, the interference group had significant reductions in the mRNA and protein expression levels of ADM, ERK1/2, and PKB (P<0.05). ERK1/2 and PKB may be the downstream targets of the ADM signaling pathway. ADM mediates the ERK/PKB signaling pathway by regulating CRLR/RAMP2 and participates in the protection of hyperoxia-induced lung injury.

Role of calcitonin gene-related peptide in pain regulation in the parabrachial nucleus of naive rats and rats with neuropathic pain

Researches have shown that calcitonin gene-related peptide (CGRP) plays a pivotal role in pain modulation. Nociceptive information from the periphery is relayed from parabrachial nucleus (PBN) to brain regions implicated involved in pain. This study investigated the effects and mechanisms of CGRP and CGRP receptors in pain regulation in the PBN of naive and neuropathic pain rats. Chronic sciatic nerve ligation was used to model neuropathic pain, CGRP and CGRP 8–37 were injected into the PBN of the rats, and calcitonin receptor-like receptor (CLR), a main structure of CGRP receptor, was knocked down by lentivirus-coated CLR siRNA. The hot plate test (HPT) and the Randall Selitto Test (RST) was used to determine the latency of the rat hindpaw response. The expression of CLR was detected with RT-PCR and western blotting. We found that intra-PBN injecting of CGRP induced an obvious anti-nociceptive effect in naive and neuropathic pain rats in a dose-dependent manner, the CGRP-induced antinociception was significantly reduced after injection of CGRP 8–37, Moreover, the mRNA and protein levels of CLR, in PBN decreased significantly and the antinociception CGRP-induced was also significantly lower in neuropathic pain rats than that in naive rats. Knockdown CLR in PBN decreased the expression of CLR and the antinociception induced by CGRP was observably decreased. Our results demonstrate that CGRP induced antinociception in PBN of naive or neuropathic pain rats, CGRP receptor mediates this effect. Neuropathic pain induced decreases in the expression of CGRP receptor, as well as in CGRP-induced antinociception in PBN.

Adrenomedullin 2 attenuates LPS-induced inflammation in microglia cells by receptor-mediated cAMP-PKA pathway

Inflammation plays a critical role in the development of neurodegenerative diseases. Adrenomedullin 2 (AM2), a member of the calcitonin gene-related peptide family, has been known to have anti-inflammatory effects. Here, we evaluated the anti-inflammatory effects of AM2 in LPS-activated microglia and BV2 cells. The endogenous mRNA and protein expressions of AM2, calcitonin receptor-like receptor (CLR), receptor activity-modifying proteins (RAMPs) including RAMP1, RAMP2 and RAMP3 and the production of inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected by RT-PCR and Western blot. Our results revealed that LPS (1 μg/mL) significantly stimulated CLR, RAMP1, RAMP2 and RAMP3 protein expressions in BV2 microglia cells, but AM2 had a significant decrease. However, the mRNA levels of AM2, CLR, and RAMP1/2/3 were all markedly increased. LPS also induced obvious increases in mRNA and protein levels of the inflammatory mediators (TNF-α, IL-1β, COX2 and iNOS). More importantly, AM2 (10 nM) administration effectively inhibited the mRNA and protein expressions of these mediators induced by LPS and increased the cAMP content in LPS-stimulated BV2 cells. Furthermore, the antagonism with AM2 receptor antagonist IMD17-47, adrenomedullin (AM) receptor antagonist by AM22-52 or the inhibition of protein kinase A (PKA) activation by P1195 effectively prevented the inhibitory role of AM2 in LPS-induced production of the above inflammatory mediators. In conclusion, AM2 inhibits LPS-induced inflammation in BV2 microglia cells that may be mainly through AM receptor-mediated cAMP-PKA pathway. Our results indicate AM2 plays an important protective role in microglia inflammation, suggesting therapeutic potential for AM2 in neuroinflammation diseases caused by activated microglia.

CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis

Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1−/−) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1−/− mice; however, the BM repopulation capacity of Ramp1−/− mice was significantly decreased, and the transplanted Ramp1−/− BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

Adrenomedullin regulates the speed of oviductal fluid flow in cattle.

Unidirectional flow of oviductal fluid from the ovarian to uterine side of the ampulla plays a significant role in successful pregnancy, and is produced by ciliary beating. Various systems regulate ciliary beating, such as paracrine, autocrine, and endocrine. We hypothesized that Adrenomedullin (ADM)-a peptide hormone that acts via its receptors, which are complexes of Calcitonin receptor-like receptor (CRLR) and Receptor activity-modifying protein (RAMP) 2 or 3 - promotes oviductal fluid flow in the ampulla of bovine oviducts. First, we examined the expression of ADM, CRLR, RAMP2, and RAMP3 mRNAs in isolated epithelial cells throughout the estrous cycle, and the localization of ADM receptor protein constituents in the ampulla. RAMP2 expression was significantly higher in the follicular phase. Furthermore, RAMP2 protein was detected only in ciliated cells, whereas CRLR and RAMP3 were detected in all epithelial cells. The effects of ADM and an ADM antagonist on fluid-flow speed were examined using microbeads in ampullary tissue. ADM antagonist decreased bead transport speed, and this decrease was reversed by ADM. In addition, ADM recovered the bead transport speed that decreased in the absence of calcium. Overall, our results suggest that ADM contributes to the regulation of oviductal fluid flow in ampulla.

Effect of Valsartan on Cerebellar Adrenomedullin System Dysregulation During Hypertension.

Adrenomedullin (AM) and its receptors components, calcitonin-receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP1, RAMP2, and RAMP3) are expressed in cerebellum. Cerebellar AM, AM binding sites and receptor components are altered during hypertension, suggesting a role for cerebellar AM in blood pressure regulation. Thus, we assessed the effect of valsartan, on AM and its receptor components expression in the cerebellar vermis of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Additionally, we evaluated AM action on superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity, and thiobarbituric acid reactive substances (TBARS) production in cerebellar vermis. Animals were treated with valsartan or vehicle for 11days. Rats were sacrificed by decapitation; cerebellar vermis was dissected; and AM, CRLR, RAMP1, RAMP2, and RAMP3 expression was quantified by Western blot analysis. CAT, SOD, and GPx activity was determined spectrophotometrically and blood pressure by non-invasive plethysmography. We demonstrate that AM and RAMP2 expression was lower in cerebellum of SHR rats, while CRLR, RAMP1, and RAMP3 expression was higher than those of WKY rats. AM reduced cerebellar CAT, SOD, GPx activities, and TBARS production in WKY rats, but not in SHR rats. Valsartan reduced blood pressure and reversed the altered expression of AM and its receptors components, as well the loss of AM capacity to reduce antioxidant enzyme activity and TBARS production in SHR rats. These findings demonstrate that valsartan is able to reverse the dysregulation of cerebellar adrenomedullinergic system; and they suggest that altered AM system in the cerebellum could represent the primary abnormality leading to hypertension.

Synovial macrophage-derived IL-1β regulates the calcitonin receptor in osteoarthritic mice.

Recent studies have reported that calcitonin gene-related peptide (CGRP) contributes to joint pain. However, regulation of the CGRP/CGRP receptor signalling in osteoarthritis (OA) is not fully understood. To investigate the regulation of CGRP/CGRP receptor signalling by macrophages in the synovial tissue (ST) of OA joints, we characterized the gene expression profiles of CGRP and CGRP receptors in the ST of OA mice (STR/Ort). In addition, we examined whether macrophage depletion by the systemic injection of clodronate-laden liposomes affected the expression of CGRP and CGRP receptors in ST. CD11c(+) macrophages in the ST of STR/Ort and C57BL/6J mice were analysed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to evaluate the expression of interleukin (IL)-1β, CGRP, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in F4/80(+) and F4/80(-) cells. The effects of IL-1β on the expression of CGRP and CLR by cultured synovial cells were also examined. The percentage of CD11c(+) macrophages in the ST of STR/Ort was higher than that in C57/BL6J mice. Notably, the F4/80(+) cell fraction expressed IL-1β highly, whereas the F4/80(-) cell fraction expressed CGRP, CLR, and RAMP1 highly. In addition, expression of the IL-1β and CLR genes was increased in ST, but was decreased upon macrophage depletion, and the IL-1β treatment of cultured synovial cells up-regulated CLR. Taken together, the present findings suggest that synovial macrophages are the major producers of IL-1β and regulators of CLR in OA mice. Therefore, macrophages and IL-1β may be suitable therapeutic targets for treating OA pain.

Role of cerebellar adrenomedullin in blood pressure regulation

Adrenomedullin (AM) and their receptor components, calcitonin-receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMP1, RMP2 and RAMP3) are widely expressed in the central nervous system, including cerebellum. We have shown that AM binding sites are altered in cerebellum during hypertension, suggesting a role for cerebellar adrenomedullinergic system in blood pressure regulation. To further evaluate the role of AM in cerebellum, we assessed the expression of AM, RAMP1, RAMP2, RAMP3 and CRLR in the cerebellar vermis of 8 and 16week old spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. In addition, the effect of microinjection of AM into rat cerebellar vermis on arterial blood pressure (BP) was determined. Animals were sacrificed by decapitation and cerebellar vermis was dissected for quantification of AM, CRLR, RAMP1, RAMP2 and RAMP3 expression using western blot analysis. Another group of male, 16week old SHR and WKY rats was anesthetized, and a cannula was implanted in the cerebellar vermis. Following recovery AM (0.02 to 200pmol/5μL) or vehicle was injected into cerebellar vermis. BP was determined, before and after treatments, by non-invasive plethysmography. In addition, to establish the receptor subtype involved in AM action in vivo, animals received microinjections of AM22–52 (200pmol/5μL), an AM1 receptor antagonist, or the CGRP1 receptor antagonist, CGRP8–37 (200pmol/5μL) into the cerebellar vermis, administered simultaneously with AM or vehicle microinjection. Cannulation was verified post mortem with the in situ injection of a dye solution. Our findings demonstrated that the expression of CRLR, RAMP1 and RAMP3 was higher in cerebellum of SHR rats, while AM and RAMP2 expression was lower than those of WKY rats, both in 8 and 16week old rats. In vivo microinjection of AM into the cerebellar vermis caused a profound, dose dependent, hypotensive effect in SHR but not in normotensive WKY rats. Coinjections of a putative AM receptor antagonist, AM22–52 abolished the decreases in mean arterial pressure (MAP) evoked by AM, showing that AM acts through its AM1 receptor in the vermis to reduce MAP. These findings demonstrate a dysregulation of cerebellar AM-system during hypertension, and suggest that cerebellar AM plays an important role in the regulation of BP. Likewise; they constitute a novel mechanism of BP control which has not been described so far.

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood–brain barrier

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood–brain barrier (BBB). Autoradiography was performed with [3H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.

Expression and distribution of the adrenomedullin system in newborn human thymus.

Adrenomedullin (AM) is a multifunctional peptide endowed with various biological actions mediated by the interaction with the calcitonin receptor-like receptor (CLR), which couples to the receptor activity-modifying proteins 2 or 3 (RAMP2 or RAMP3) to form the functional plasma membrane receptors AM1 and AM2, respectively. In this study, we investigated for the first time the expression and localization of AM, CLR, RAMP2 and RAMP3 in human thymic tissue from newborns and in primary cultures of thymic epithelial cells (TECs) and thymocytes. Immunohistochemical analysis of thymic tissue showed that both AM and RAMP2 are abundantly expressed in the epithelial cells of medulla and cortex, blood vessels and mastocytes. In contrast, RAMP3 could not be detected. In cultured TECs, double immunofluorescence coupled to confocal microscopy revealed that AM is present in the cytoplasmic compartment, whereas RAMP2 could be detected in the cytoplasm and nucleus, but not in the cell membrane. At variance with RAMP2, CLR was not only present in the nucleus and cytoplasm of TECs, but could also be detected in the cell membrane. The nuclear and cytoplasmic localizations of RAMP2 and CLR and the absence of RAMP2 in the cell membrane were confirmed by western-blot analysis performed on cell fractions. AM, RAMP2 and CLR could also be detected in thymocytes by means of double immunofluorescence coupled to confocal microscopy, although these proteins were not present in the whole thymocyte population. In these cells, AM and RAMP2 were detected in the cytoplasm, whereas CLR could be observed in the cytoplasm and the plasma membrane. In conclusion, our results show that the AM system is widely expressed in human thymus from newborns and suggest that both AM1 receptor components CLR and RAMP2 are not associated with the plasma membrane of TECs and thymocytes but are located intracellularly, notably in the nucleus.

Adrenomedullin Expression in the Developing Human Fetal Lung

BackgroundAdrenomedullin (AM) is a vasodilator peptide produced by endothelial and smooth muscle cells in the systemic and pulmonary circulation. It promotes angiogenesis and alveolar growth and has protective effects in...

Intermedin in Paraventricular Nucleus Attenuates Sympathetic Activity and Blood Pressure via Nitric Oxide in Hypertensive Rats

Intermedin (IMD) is a member of calcitonin/calcitonin gene-related peptide family, which shares the receptor system consisting of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs). This study investigated the effects of IMD in paraventricular nucleus (PVN) on renal sympathetic nerve activity and mean arterial pressure and its downstream mechanism in hypertension. Rats were subjected to 2-kidney 1-clip (2K1C) surgery to induce renovascular hypertension or sham operation. Acute experiments were performed 4 weeks later under anesthesia. IMD mRNA and protein were downregulated in 2K1C rats. Bilateral PVN microinjection of IMD caused greater decreases in renal sympathetic nerve activity and mean arterial pressure in 2K1C rats than in sham-operated rats, which were prevented by pretreatment with adrenomedullin receptor antagonist AM22-52 or nonselective nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester, and attenuated by selective neuronal NO synthase inhibitor N(ω)-propyl-l-arginine hydrochloride or endothelial NO synthase inhibitor N(5)-(1-iminoethyl)-l-ornithine dihydrochloride. AM22-52 increased renal sympathetic nerve activity and mean arterial pressure in 2K1C rats but not in sham-operated rats, whereas calcitonin/calcitonin gene-related peptide receptor antagonist calcitonin/calcitonin gene-related peptide 8-37 had no significant effect. CRLR and RAMP3 mRNA, as well as CRLR, RAMP2, and RAMP3 protein expressions, in the PVN were increased in 2K1C rats. Microinjection of IMD into the PVN increased the NO metabolites (NOx) level in the PVN in 2K1C rats, which was prevented by AM22-52. Chronic PVN infusion of IMD reduced, but AM22-52 increased, blood pressure in conscious 2K1C rats. These results indicate that IMD in the PVN inhibits sympathetic activity and attenuates hypertension in 2K1C rats, which are mediated by adrenomedullin receptors (CRLR/RAMP2 or CRLR/RAMP3) and its downstream NO.

The G-Protein–Coupled Receptor CLR Is Upregulated in an Autocrine Loop with Adrenomedullin in Clear Cell Renal Cell Carcinoma and Associated with Poor Prognosis

The G-protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) and its ligand peptide adrenomedullin (encoded by ADM gene) are implicated in tumor angiogenesis in mouse models but poorly defined in human cancers. We therefore investigated the diagnostic/prognostic use for CLR in human tumor types that may rely on adrenomedullin signaling and in clear cell renal cell carcinoma (RCC), a highly vascular tumor, in particular. In silico gene expression mRNA profiling microarray study (n = 168 tumors) and cancer profiling cDNA array hybridization (n = 241 pairs of patient-matched tumor/normal tissue samples) were carried out to analyze ADM mRNA expression in 13 tumor types. Immunohistochemistry on tissue microarrays containing patient-matched renal tumor/normal tissues (n = 87 pairs) was conducted to study CLR expression and its association with clinicopathologic parameters and disease outcome. ADM expression was significantly upregulated only in RCC and endometrial adenocarcinoma compared with normal tissue counterparts (P < 0.01). CLR was localized in tumor cells and vessels in RCC and upregulated as compared with patient-matched normal control kidney (P < 0.001). Higher CLR expression was found in advanced stages (P < 0.05), correlated with high tumor grade (P < 0.01) and conferred shorter overall survival (P < 0.01). In human tissues ADM expression is upregulated in cancer type-specific manner, implicating potential role for adrenomedullin signaling in particular in RCC, where CLR localization suggests autocrine/paracrine mode for adrenomedullin action within the tumor microenvironment. Our findings reveal previously unrecognized CLR upregulation in an autocrine loop with adrenomedullin in RCC with potential application for this GPCR as a target for future functional studies and drug development.

Localization of CGRP Receptor Components, CGRP, and Receptor Binding Sites in Human and Rhesus Cerebellar Cortex

The cerebellum is classically considered to be mainly involved in motor processing, but studies have suggested several other functions, including pain processing. Calcitonin-gene-related peptide (CGRP) is a neuropeptide involved in migraine pathology, where there is elevated release of CGRP during migraine attacks and CGRP receptor antagonists have antimigraine efficacy. In the present study, we examined CGRP and CGRP receptor binding sites and protein expression in primate cerebellar cortex. Additionally, mRNA expression of the CGRP receptor components, calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1), was examined. In addition, expression of procalcitonin was studied. We observed high [(3)H]MK-3207 (CGRP receptor antagonist) binding densities in the molecular layer of rhesus cerebellar cortex; however, due to the limit of resolution of the autoradiographic image the exact cellular localization could not be determined. Similarly, [(125)I]CGRP binding was observed in the molecular layer and Purkinje cell layer of human cerebellum. CLR and RAMP1 mRNA was expressed within the Purkinje cell layer and some expression was found in the molecular layer. Immunofluorescence revealed expression of CGRP, CLR, and RAMP1 in the Purkinje cells and in cells in the molecular layer. Procalcitonin was found in the same localization. Recent research in the biology of cerebellum indicates that it may have a role in nociception. For the first time we have identified CGRP and CGRP receptor binding sites together with CGRP receptor expression through protein and mRNA localization in primate cerebellar cortex. These results point toward a functional role of CGRP in cerebellum. Further efforts are needed to evaluate this.

Intermedin protects against ischaemia and reperfusion injury in human ventricular cardiomyocytes: receptor subtype involvement

Purpose: Early reperfusion following myocardial infarction is essential but results in ischaemia/reperfusion (I/R) injury, whereby myocardial cells die largely due to oxidative stress. Intermedin (IMD; adrenomedullin-2), a novel peptide related to adrenomedullin (AM), is up-regulated in cardiovascular disease and is protective against I/R injury in rodent hearts and human coronary microvascular endothelial cells and cardiac non-myocytes. Protection is dependent on calcitonin receptor-like receptor (CRLR) signalling in association with one of three receptor activity-modifying proteins (RAMPs). IMD has yet to be investigated in human cardiomyocytes for a cardioprotective role. Aims: To identify if IMD and its receptor components are expressed in human ventricular cardiomyocytes; to investigate whether IMD protects these cells against simulated I/R injury and if so, determine receptor subtype involvement. Methods: Left ventricular adult human cardiomyocytes (HCM) were subjected to normoxia or simulated ischaemia for 4 h or I/R (3 h normoxia followed by 1 h reperfusion) all in the absence or presence of IMD. Cell viability was assessed using Trypan blue dye exclusion and oxidative damage by commercial Oxyblot assay. mRNA expression and protein distribution of IMD, AM, CRLR and RAMPs1-3 were assessed by Quantitative Real-Time PCR and indirect immunofluorescence microscopy, respectively. The silencing of the peptides or their receptor components using siRNA knock-down was employed to determine endogenous peptide and receptor subtype involvement in protection against I/R. Results: IMD, AM and their receptor component mRNAs were expressed in HCM; RAMP2 was the most abundant RAMP, while IMD was less abundantly expressed than AM. Ischaemia or I/R reduced the HCM cell count by 40% and 60% respectively (p<0.05 for both). IMD (10-9 mol.L-1) protected against the deleterious effects of ischaemia and I/R injury (∼20% salvaged for both conditions, p<0.05), with preservation of cell structure when examined by phase contrast microscopy and decreased oxidative stress noted on Oxyblot (p<0.001 vs. non-IMD counterpart for ischaemia or I/R). Protection was greatest when IMD was given during reperfusion (p<0.001) and was abolished by silencing CRLR (p<0.05) and RAMP2 (p<0.001). However, silencing endogenous IMD and/or AM expression did not augment extent of I/R injury in HCM. Conclusion: Exogenous IMD is protective against I/R in HCM, attributed to AM1 receptor subtype involvement. IMD administration during cardiac reperfusion could improve levels of morbidity and mortality in patients following myocardial infarction.

RE: “The influence of RAMP1 overexpression on CGRP-induced osteogenic differentiation in MG-63 cells in vitro: An experimental study”

The aim of this study was to elucidate the influence of receptor activity modifying protein 1 (RAMP1) overexpression on the expression and distribution of calcitonin receptor-like receptor (CRLR) in MG-63 cells. Our research also focused on whether RAMP1 overexpression enhanced the promoting effect of exogenous CGRP on osteogenic differentiation in MG-63 cells. We first constructed a eukaryotic expression vector containing human RAMP1 and stably transfected it into MG-63 cells. Real-time PCR and Western blotting were used to determine the expression levels of RAMP1 and CRLR mRNA and protein, respectively. Immunofluorescence analysis was employed to compare the distribution of CRLR in transfected cells. After treatment with CGRP, the extent of osteogenic differentiation was evaluated by simultaneous monitoring of alkaline phosphatase activity, the expression patterns of osteoblastic markers and mineralisation staining. We found that RAMP1 was more highly expressed in the transfected group compared with the control groups (P < 0.01). The CRLR expression was significantly higher than that in the control groups (P < 0.05). In addition, after 7 days of CGRP treatment to induce osteogenic differentiation, the expression of collagen I mRNA was markedly increased in the transfected group (P < 0.05). The transfected group exhibited more granular precipitation in the cytoplasm with alkaline phosphatase staining after 7 and 14 days of differentiation. When stained with Alizarin Red, cells overexpressing RAMP1 were darker and formed many mineralised nodules with clear boundaries and calcium deposition typical of mineralised bone matrix structures at 28 days post-induction of differentiation. The CGRP-induced ALP activity in the RAMP1 overexpression group was significantly higher 3, 6 and 9 days after induction than that in the two control groups (P < 0.05). RAMP1 overexpression promotes CRLR expression, localisation on the cell membrane and enhanced CGRP-mediated differentiation of MG-63 cells. This study contributes to a better understanding of the molecular mechanisms governing CGRP-induced MG-63 differentiation.

Temporo-spatial expression of adrenomedullin and its receptors in the bovine placenta

BackgroundAdrenomedullin (AM) is a potent vasodilator peptide and is also involved in various physiological activities. In humans and rodents, AM is found in the uteroplacental unit and may be responsible for fetal development and maintenance of placental function. This study investigated 1) the mRNA expression patterns of AM and its receptor components (calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) 2 and RAMP3) during pregnancy and 2) mRNA and protein localization of AM, CRLR and RAMPs in the bovine placentome.MethodsFor real-time quantitative RT-PCR, bovine uteroplacental tissues were collected from Day 25, 60, 100, 150, 200 and 250 of gestation and separated into uterine caruncle (CAR), intercaruncular endometrium (ICAR), extra-embryonic membranes on Day 25 and cotyledonary villous after Day 60 (EEM-COT) and intercotyledonary chorion (ICOT). In situ hybridization and immunohistochemistry was performed to investigate the cellular localization of mRNA and protein of AM, CRLR, RAMP2 and RAMP3 in the placentome on Day 56, 150 and 230 of gestation and interplacentomal tissues on Day 56 of gestation.ResultsAM mRNA was highly expressed on Day 200 in EEM-COT, CAR and ICAR. CRLR mRNA was highly expressed on Day 60 in all portions. RAMP2 mRNA was also highly expressed on Day 60 in ICOT and ICAR. In EEM-COT, mRNA expression of CRLR and RAMP2 decreased from Day 150 to 250. RAMP3 mRNA was highly expressed on Day 150 in EEM-COT, ICOT and ICAR. A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells. In interplacentomal tissues, AM was detected in BNCs of fetal membrane and a small part of luminal epithelium, endothelial lineage of blood vessels and glandular epithelium of the endometrium. Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium.ConclusionsOur results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period. Locally produced AM in the BNCs may play a crucial role in regulation of placental vascular and cellular functions during pregnancy.