Expression of the CGRP Family of Neuropeptides and their Receptors in the Trigeminal Ganglion

Edvinsson, Lars; Grell, Anne-Sofie; Warfvinge, Karin

doi:10.1007/s12031-020-01493-z

Expression of the CGRP Family of Neuropeptides and their Receptors in the Trigeminal Ganglion

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Published: 22 February 2020

Volume 70, pages 930–944, (2020)
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Expression of the CGRP Family of Neuropeptides and their Receptors in the Trigeminal Ganglion

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Abstract

The calcitonin gene-related peptide (CGRP) family of neuropeptides, consists of CGRP, adrenomedullin, amylin, and calcitonin. The receptors consist of either calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) which for function needs an accessory protein, receptor activity-modifying proteins (RAMPs). CGRP has a pivotal role in primary headaches but the role of the other members of the CGRP family of peptides in headaches is not known. Here, we describe the expression of these molecules in the trigeminal ganglion (TG) to understand more on their possible role(s). Single or double immunohistochemistry were applied on frozen sections of rat TG using primary antibodies against CGRP, procalcitonin, calcitonin, adrenomedullin, amylin, RAMP1/2/3, CLR, and CTR. In addition, mRNA expression was measured by quantitative qPCR on TGs. CGRP and calcitonin showed rich expression in the cytoplasm of small to medium-sized neurons, and co-localized sometimes. Procalcitonin was observed in the glial cells. Immunoreactive fibers storing both CGRP and calcitonin were also observed. Adrenomedullin immunoreactivity was found in the satellite glial cells and in fibers, probably the myelinating Schwann cells. Amylin was found in the cytoplasm in many TG neurons. Levels of mRNA expression for adrenomedullin, amylin, CLR, RAMP1, RAMP2, RAMP3, and CTR were measured using qPCR. The experiments verified the expression of mRNA in the TG with the exception of CTR, which was above the limit of detection indicating little or no mRNA expression. In addition to the well-known CGRP receptor (CLR/RAMP1) and the receptor for calcitonin—CTR, we propose that other receptors exist in the rat TG: adrenomedullin receptor AM₂ (CLR/RAMP3) in mainly the satellite glial cells, amylin receptors AMY₁ (CTR/RAMP1) in mainly neurons, and AMY₃ (CTR/RAMP3) in the satellite glial cells. It is important to compare peptides and receptors side-by-side in studies to help address questions of actions resulting from cross-reactivity between receptors. Several of the diverse biological actions of the CGRP family of peptides are clinically relevant. Our findings demonstrate the specific ligand and receptor sites in the rat trigeminal ganglion, highlighting recognition mechanisms to facilitate drug development.

The CGRP Family of Neuropeptides and their Receptors in the Trigeminovascular System

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Introduction

The trigeminal ganglion (TG) is primarily a sensory ganglion of the trigeminal nerve (the V^th cranial nerve) that occupies Meckel’s cave at the base of the brain and is surrounded by the dura mater. The neurons within the TG are firmly enveloped by the satellite glial cells (SGCs), demonstrating the close interaction between the neurons and glial cells coupled by gap junctions (Hanani 2005). TG is linked peripherally to the ophthalmic, maxillary and part of the mandibular nerves, and centrally to the trigeminal nucleus caudalis (TNC) and dorsal root C₁-C₃ (Edvinsson 2011).

Calcitonin gene-related peptide (CGRP) is richly located in numerous sites throughout the central and peripheral nervous systems (Russell et al. 2014; Warfvinge and Edvinsson 2019a). A sizeable population of CGRP neurons within the TG signifies a major role for CGRP in trigeminal transmission. About half of all neurons in the TG express CGRP which is visualized by immunohistochemical staining with CGRP antibodies and by in situ hybridization to localize mRNA for CGRP (Eftekhari et al. 2010; Miller et al. 2016). CGRP-positive neurons are predominantly of unmyelinated small-medium diameter which is indicative of cell bodies of C-type sensory pain fibers (Eftekhari et al. 2013). The human CALCA gene codes for both calcitonin and αCGRP via an alternative splicing mechanism in neural tissues (Amara et al. 1982). Both calcitonin and αCGRP are cleaved from larger pro-peptides known as procalcitonin (Pro-CT) and pro-CGRP (Buervenich et al. Full size image

Neuronal soma CT expression displayed a similar pattern as that seen for CGRP (and was co-localized in some cells); granular staining of mainly small to medium-sized neurons and pearl-like staining of C-type fibers. The number of CT-positive cells and pearl-like-positive fibers were, however, less than those seen in CGRP immunohistochemical stained TGs. Yet, the main difference between CGRP and CT immunoreactivity was the CT cytoplasmic granular staining of SGCs, and of Aδ-fibers, which did not occur at CGRP immunohistochemistry.

Procalcitonin (Pro-CT) expression in TG has previously published (Tajti et al. 2011). We here confirm these results. Pro-CT immunoreactivity was observed exclusively in the glial cells, the SGCs, and the myelinating Schwann cells (Fig. 2b). Pro-CT immunoreactivity was found in the soma of the Schwann cells. The myelinated layers of the Schwann cells, surrounding the thick Aδ-fibers, did not express Pro-CT.

AMY was exclusively expressed in the cytoplasm of small- to medium-sized neurons. In some of the cells, the expression was granular, but in others, a general cytoplasmic immunoreactivity was observed.

AM was expressed in the thin cytoplasm of the glial cells, both the SGCs and cells envelo** the neuronal processes, probably Schwann cells. In addition, immunoreactivity was found in blood vessel walls, indicating vascular endothelial staining (not shown).

Receptor Elements Expression (Fig. 3)

CLR and RAMP1 expression has earlier been examined in detail (Eftekhari et al. 2010; Lennerz et al. 2008; Miller et al. 2016). The present paper confirms these results. The receptor components were expressed in the cytoplasm of the neurons (mainly the larger ones) and the SGCs, and the thick fibers, typical for Aδ-fibers.

RAMP2 and RAMP3 were expressed in the nuclei, both in those of neurons and of glia cells. In addition, RAMP3 was expressed in the cytoplasm of the glial cells (possibly both SGCs and Schwann cells envelo** the Aδ-fibers); however, the expression was considerably weaker than the nuclei staining.

CTR was expressed with varying intensity in the cytoplasm of the most neurons and SGCs. In addition, the nuclei of the glial cells (Schwann cells) surrounding the Aδ-fibers showed CTR immunoreactivity. In addition, the Aδ-fibers also expressed CTR, but in a weak manner.

Ligand/Ligand Expression (Fig. 4)

To provide morphological clues for functionality of the CGRP family of peptides in the TG and cross-reactivity, i.e., selectivity of the antibodies used, we performed a series of double immunohistochemistry experiments. In these experiments, we suggest that areas with no co-localization represent areas with no cross-reactivity.

Double staining with CGRP and CT antibodies showed co-expression in the cytoplasm of small-sized neurons. We accordingly suggest that this could indicate cross-reactivity between CGRP and CT antibodies. In addition, CT alone was expressed in the SGCs and thus CT expression in SGCs represent no cross-reactivity.

CGRP and AMY double staining showed co-expression in the cytoplasm of some of the small- to medium-sized neurons and in thin C-fibers; however, some were seen to only express CGRP and not AMY. The number of CGRP-positive cells appeared to be more abundant than those storing AMY.

AM was double stained with CGRP or AMY, respectively (not shown). No co-localization was found in neither with AM plus CGRP, nor with AM plus AMY double immunohistochemistry. AM and CT double staining was not performed since both antibodies were made in mouse.

Receptor/Receptor Expression (Fig. 5)

RAMP1 and CLR are co-expressed mainly in the cytoplasm of larger neurons and in Aδ neuronal fibers (not shown).

RAMP1 was expressed in neurons, in the SGCs and the Aδ fibers. CTR alone was expressed with varying intensity in most neurons and SGCs. RAMP1 and CTR double immunohistochemistry showed co-expression in some neurons, SGCs and fibers, which indicates AMY₁ receptor expression.

RAMP3 and CTR double immunohistochemistry revealed a possible AMY₃ receptor in the cytoplasm of the SGCs.

RAMP3 is expressed in the nuclei, both neuronal and SGCs nuclei. CLR is expressed in the cytoplasm of neurons, in the SGCs and the Aδ fibers. Double immunohistochemistry showed co-expression in the SGCs, which suggests a presence of AM₂ receptor.

Double immunohistochemistry using combinations between CLR, RAMP2, and CTR antibodies could not be performed since all three antibodies were made in rabbit.

Ligand/Receptor Expression (Fig. 6)

The ligands are expressed in different manners: CGRP and CT are expressed in neurons and in pearl-like fibers. In addition, SGCs are CT immunoreactive. AMY was exclusively expressed in the neurons. AM was expressed in the glial cells.

The receptor components show the same diversity in their pattern: CLR and RAMP1 immunoreactivity in the neurons, in the SGCs, and in the thick fibers; RAMP2/3 in the neuron; and SGC nuclei and CTR in most neurons and SGCs.

We have previously shown that no co-localization exists between CGRP and CLR/RAMP1 (Eftekhari et al. 2010). We confirm the results in the present paper (data not shown).

Double CT and CTR immunohistochemistry showed that all CT immunoreactive cells also were CTR positive (data not shown). No co-localization was found in the fibers (CT positive) or in the glial cell nuclei (CTR positive).

CT and RAMP1 double staining revealed co-localization in some neurons. However, some neurons were only CT immunoreactive and some only RAMP1 immunoreactive. The same pattern of immunoreactivity was found using AMY and RAMP1 antibodies; some neurons showed co-localization, some AMY, and some RAMP1 immunoreactivity.

AM is expressed in the glial cells and RAMP2 in the nuclei; consequently, no co-localization was found using double immunohistochemistry.

Distribution of the CGRP Family of Peptides and their Receptors (Fig. 7)

The figure describes, in a schematic drawing, the simplified distribution of the CGRP family of peptides and their receptors. The CGRP receptor (CLR/RAMP1) is mainly expressed in the large neurons and SGCs, the adrenomedullin₂ receptor (CLR/RAMP3) in some SGCs, the amylin₁ receptor (CTR/RAMP1) in large neurons and SGCs, and the amylin₃ receptor (CTR/RAMP3) in some SGCs.

RAMP1 Immunohistochemistry of TG Neuron and Cerebellar Purkinje Cell (Fig. 8)

To give an example on how the cellular distribution of the different receptor components may vary, we compared the expression of RAMP1 in a TG neuron with that of a cerebellar Purkinje cell. The TG neuron expresses RAMP1 in the cytoplasm and the Purkinje cell on the cell surface (results previously published by (Edvinsson et al. 2011; Eftekhari et al. 2010)).

Quantitative PCR (Fig. 9)

Levels of mRNA expression for adrenomedullin, amylin, CLR, RAMP1, RAMP2, RAMP3, and CTR were measured using qPCR. The experiments confirmed the expression of all the genes except CTR (Ct values > 35, see dotted line) indicating little or no gene expression of CTR in TG.

Discussion

This is the first systematic and in detail comparison of the localization and expression of the CGRP family of peptides and their receptors in the trigeminal system. Each of the peptides of the CGRP family exhibits a distinct selection of biological actions (Poyner et al. 2002). CGRP and AMY are the most closely related peptides in terms of amino acid sequence which may cause an overlap in the ability to activate each receptor. Both CGRP and AMY are reported to have effects relating to pain, although, there is still limited data, and it is unclear how much overlap there is, because the peptides are not usually tested in the same study (Iyengar et al. 2017). The receptors themselves are also related and share components (Table 1). The relative potency of CGRP, CT, AMY, or AM to the different receptor complexes is a complicated topic, for example CGRP > AM to RAMP1/CLR, CT > AMY to CTR. Their functional cross-reactivity makes the work challenging (Hay et al. 2018; Hendrikse et al. 2018).

Within the CGRP family of peptides, the N-terminus and C-terminus are the most highly conserved regions with more divergence in the mid-portion of each peptide suggesting an importance in retention of the N-terminal and C-terminal for biological activity (Bower and Hay 2016). The receptor component CTR was cloned in 1991 (Lin et al. 1991), CLR in 1993 (Njuki et al. 1993), and RAMP1 was discovered in 1998 (McLatchie et al. 1998). A sequence database search for expressed sequences similar to RAMP1 identified RAMP2 and RAMP3 (Naot and Cornish 2008).

It is clear that there is a complex interplay among the ligand and receptors, which acquire an experimental system to allow the results to be further validated. However, the examinations in the present study describing the distribution and mRNA levels of the markers might define potential targets of different pathological conditions. So far, immunohistochemical examination of the CGRP family of peptides and their receptors in TG has been performed with CGRP and its receptor components CLR and RAMP1 (Eftekhari et al. 2010). In addition, AMY has been demonstrated in rat TG (Walker et al. 2015). Moreover, Tajti and colleagues found that the SGCs in TG contained Pro-CT which is the pro-peptide form of calcitonin prior splicing to CGRP in neuronal tissue (Tajti et al. 2011). We confirm these results in the present study (Fig. 2b). To our knowledge, immunohistochemistry using RAMP2 and RAMP3 antibodies has hitherto not been shown in the TG or the CNS, except for the rat retina (Warfvinge and Edvinsson 2019b). It is important to compare the peptides side-by-side to help to address questions of which receptor(s) mediate the effects and which are actions as a result to cross-reactivity between receptors. The analysis of the expression of the peptides and the receptors with qPCR revealed their quantitative expression of respective mRNA (Fig. 9). These results support the immunohistochemistry results which provide cellular localization.

The CGRP family of peptides are ligands for closely related family B of G protein-coupled receptors (GPCRs) and share structural homology (Hay et al. 2014). Class B GPCRs are involved in major biological and pathophysiological functions (Hoare 2005). It is now clear that RAMPs can interact with a wider range of GPCRs, e.g., all three RAMPs can interact with the VPAC1 (vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide) receptor. In addition, RAMPs can produce a number of different effects on ligand binding, signal transduction, and receptor trafficking (Hay and Pioszak 2016). Furthermore, Walker and colleagues stated that the results obtained by a GPCR array identified many additional GPCRs within the TG as potential pharmacological targets for migraine which have been previously appreciated (Walker et al. 2015).

Calcitonin Gene-Related Peptide

Antibody treatments towards CGRP itself and towards the CGRP receptor have been approved by regulatory bodies (FDA and EMA) and have reached the clinic as remedies against frequent episodic migraine and chronic migraine (Diener 2019). There are three monoclonal antibodies towards the ligand CGRP and one directed towards the C- and N-terminals of the CGRP receptor. The clinical results have so far been excellent with good efficacy and minor side effects (Edvinsson et al. 2018). Overall, 20% of the patients are super responders (end of headache) while approximately 50% show considerable reductions in migraine headache. The remainders do not respond, and here, we might hypothesize that other molecules or receptors are involved such as amylin.

Since the antibodies do not pass the blood-brain barrier, the efficacy is thought to occur at some parts of the trigeminal system (Lundblad et al. 2015). Importantly, about half of the neurons in the TG store CGRP, and the remainder store CLR/RAMP1, suggesting that CGRP is the key player. CGRP is expressed in many neurons, mainly in small to medium-sized neurons (Figs. 2 and 4). The cellular CGRP is often expressed in or close to the membranes of the Golgi apparatus (Warfvinge, unpublished). In addition, pearl-like CGRP immunoreactivity was detected in fibers that are of the C-type of sensory unmyelinated nerves. The myelinated fibers do not contain CGRP. The receptor components CLR and RAMP1 are expressed in neurons (mainly the larger ones), the SGCs and the thick fibers (typical in Aδ-fibers) (Figs. 3, 5, 6, and 7). CGRP, CLR, and RAMP1 distribution in rat TG have earlier been examined in details (Eftekhari et al. 2010; Lennerz et al. 2008; Miller et al. 2016). The present paper has confirmed these results and extends details of the expression.

Walker and colleagues demonstrated that CTR is expressed in small- to medium-sized neurons and some larger neurons in TG using immunohistochemistry (Walker et al. 2015). In addition, the authors showed that CTR and RAMP1 expressing neurons co-localized. This is in accordance with our results in the present paper using immunohistochemistry. However, by using qPCR, the mRNA expression of the CTR gene was above the limit of detection. The inability to detect the CTR gene by qPCR could be explained by a problem with the reliability of the antibody used. Still, it has been demonstrated that existing incongruent expression between mRNAs and protein can only be unveiled through integrated analyses of both proteins and mRNAs, i.e., to examine more than one aspect of a biological system (Tian et al. 2004).

AM has affinity for CGRP receptors, and CGRP to AMY₁ receptors (Hendrikse et al. 2018) (Table 1). AMY and CGRP are the most closely related peptides in terms of amino acid sequence which may cause an overlap in the ability to activate each receptor. Also, the receptors themselves are related and share components: CLR/RAMP1 (ligand CGRP) and CTR/RAMP1 (ligand AMY). Given the close relationship between AMY and CGRP, since they share the AMY₁ receptor (CGRP = AMY, Table 1), it is worthwhile considering whether amylin is able to trigger migraine. However, the CGRP receptor antibody Erenumab is made to target the N-terminals of CLR and RAMP1, which makes it very specific for the CGRP receptor. The CGRP ligand antibodies do not have such specificity; in fact, they target both αCGRP and βCGRP. Despite this, the antibodies are equally effective.

Adrenomedullin

AM is chiefly found in endothelial cells and was first isolated in 1993 (Kitamura et al. 1993). AM has been found to be generally expressed and to participate in a variety of physiological functions including vasodilation, bronchodilation, growth, and hormone regulation (Ferrero et al. 2018b). Furthermore, AM is involved in several pathophysiological processes such as hypertension, retinopathy, and tumor genesis (Ferrero et al. 2018a).

In 1994, active production of AM in cultured endothelial cells was demonstrated (Sugo et al. 1994). In mammals, endothelial AM immunoreactivity has been inconsistently reported. The reason for this might be that AM in the vascular endothelium is present in low concentrations (Satoh et al. 1996). AM has also been localized in neurons and glial cells (Serrano et al. 2000). In the present study on rat TG, AM was expressed in the glial cells, both the SGCs and cells envelo** the neuronal processes, probably myelinating Schwann cells (Figs. 2 and 6). In addition, immunoreactivity was found in blood vessel walls indicating vascular endothelial staining. Interestingly, immunohistochemistry on rat retina using the same antibodies as reported here, only the vasculature in the inner part of the retina displayed AM immunoreactivity (Warfvinge and Edvinsson 2019b).

We performed double immunohistochemistry with ligand to ligand and ligand to receptor. No co-localization was found between AM and the other members of the CGRP family, even though overlap is known to occur because the peptides share features. The AM receptors consist of CLR and RAMP2 (AM₁ receptor) and CLR and RAMP3 (AM₂ receptor) (Table 1). Double immunohistochemistry with AM and RAMP2 (AM receptor component) antibodies showed no co-localization; AM is mainly expressed in the glial cells and RAMP2 in the nuclei; consequently, no co-localization was observed. The gene expression of CLR, RAMP2, and RAMP3 are all present in the TG. Receptor/receptor immunoreactivity with RAMP3 and CLR showed weak double staining in the glial cells, mainly SGCs (Fig. 5). However, relatively low expression does not automatically translate into little function.

In a study by De Martin and colleagues, the expression of AM and its receptor components in human thymic tissue from newborns were investigated. They demonstrated that AM and RAMP2, but not RAMP3, were largely distributed within the human thymus. RAMP2 was expressed both in the cytoplasm and in the nucleus. They concluded that the AM₁ receptor has an intracellular localization and suggested that AM exerts a control of its functions through the interaction with the receptor component localized in the nucleus (De Martin et al. 2014). We confirm the presence of RAMP2 and RAMP3 in the nuclei of rat TG cells. AM might exert control of its function also in rat TG through interaction with RAMP2 and RAMP3 in the nucleus.

To summarize, we suggest that AM is present in the glial cells and in the vasculature of rat TG. Furthermore, we suggest that an AM functional receptor (AM₂) exists in mainly the SGCs of rat TG. Interestingly, AM did not show any migraine-like effect upon systemic administration in man (Ashina et al. 2017).

Calcitonin

CT was discovered more than 40 years ago (Copp and Cheney 1962) and is a hormone produced by C cells of the thyroid, whose role is to reduce plasma calcium levels and to promote bone formation (Findlay and Sexton 2004). CT is used clinically in the treatment of bone disorders characterized by increased bone resorption, osteoporosis, and hypercalcemia due to malignancy (Findlay and Sexton 2004). However, there is still yet much to learn about the actions and role of CT, in particular to migraine-related structures.

The presence of CT and its receptor in a large number of cell types and tissues suggests multiple physiological roles (Findlay and Sexton 2004). The mediated actions of CT correlate well with the location of CT binding sites. Binding of CT to a specific receptor induces morphological changes in osteoclasts, which results in bone resorption (Chambers and Moore 1983). CT has not been shown to be expressed in the nervous system; however, binding sites for CT are found in many brain structures (Hendrikse et al. 2018). The present study shows CT immunoreactivity in a similar pattern as for CGRP; CT expression in vesicles and pearl-like CT immunoreactivity in C-type of sensory fibers. Hence, we show for the first time that CT is expressed in the nervous system.

The CT receptor CTR acts by itself as a specific CT receptor by reaching the cell surface (Hay et al. 2018). In the present study, it was shown that CTR is expressed in most neurons, SGC, and glial cell nuclei with varying intensity. Although, there was an extremely little or no gene expression of CTR in the TG as well as there was no indication of CTR being present on the cell surface and thereby no indication of activated CTR. However, the expression of CTR resembled that of CLR/RAMP1, the well-documented and recognized CGRP receptor (Hay et al. 2018), and both CLR and RAMP1 are expressed at mRNA levels in TG. To our knowledge, CLR/RAMP1 immunoreactivity has not been shown at cell surfaces of TG. However, we have previously described the distribution of CGRP and its receptor components in rat cerebellum (Edvinsson et al. 2011), where we found the CGRP receptor on the surface of the Purkinje cells (Fig. 8). If this is an effect of functionality and that intracellular CLR/RAMP1 distribution in rat TG is a consequence of no function is not known. However, the literature shows that the CLR/RAMP1 receptor in the TG is a functional receptor even though the distribution of this receptor is not expressed on the surface but intracellularly (Eftekhari et al. 2010). We suggest that the intracellular expression of CTR, as CLR/RAMP1, in rat TG denotes a functional CT receptor, and that CT exerts control over its function through interaction with CTR.

mRNA profiles are often used as surrogates for protein expression. However, mRNA expression can be used to explain at most 40% of the protein expression which is likely a reflection of the underlying biological mechanisms (Tian et al. 2004). We found that the expression of CTR mRNA was above the detection limit (Ct > 35), which could indicate little or no gene expression of CTR in TG. The dissimilar expression between CTR protein and CTR mRNA may be a result of biology of gene expression rather than measurement errors.

Amylin

Human AMY was probably first observed in 1901, and described as hyaline deposits in pancreas of patients with type 2 diabetes (Opie 1901). AMY was isolated in 1987 (Cooper et al. 1987), and it is an endocrine hormone that signals to the brain and acts as a satiety factor. Possibly, AMY may also have other roles (Hay 2017). Research on AMY neuronal deposition has mainly focused on its role in Alzheimer’s disease (Mietlicki-Baase et al. 2017). Recently, it was shown that AMY alters human brain pericyte viability (Schultz et al. 2017). In addition, AMY immunoreactivity has been described in several places along the gut and in some neurons (Hay 2017; Young 2005). We have reported AMY in the TG of cat and that AMY can dilate in vitro as well as in vivo cerebral vessels (Edvinsson et al. 2001). The gene is also expressed in TG.

Walker and colleagues showed the presence of CTR, and furthermore, they suggested that the AMY₁ receptor (CTR/RAMP1) could be a relevant target for CGRP and perhaps a novel way in migraine therapy (Walker et al. 2015). We support the relevance of AMY₁ being a migraine therapy target. In the present study, we show that RAMP1 is expressed in neurons, in the SGCs and the thick fibers (Fig. 5). Furthermore, CTR is expressed with varying intensity in most neurons and SGCs. RAMP1 and CTR double immunohistochemistry showed co-expression in some neurons, SGCs, and fibers, indicating an AMY₁ receptor expression. RAMP3 and CTR double immunohistochemistry revealed a possible AMY₃ receptor in the SGCs.

Findings on thymic epithelial cells using both immunofluorescence and immunogold stainings demonstrated RAMP2 localization in the nucleus, CLR both intracellularly and in the plasma membrane and AM in the cytoplasm. These results agree with our results. The authors suggest that AM activates its receptor in the nucleus to modulate transcription (Castellani et al. 2016). Furthermore, recent finding by Castellani on AM and its receptor distribution strengthens our results and indicates that absence of co-localization does not necessarily mean absence of functional receptors. Moreover, it is possible that the expressional profile might differ in disease (Saeed et al. 2009). Future studies in animal models might unravel unexpected expressional profiles and indicate ways for therapy.

Conclusion

It is important to compare peptides and receptors side-by-side to help address questions of actions resulting from cross-reactivity between receptors. We demonstrate in the present study using qualitative and quantitative methods that calcitonin is expressed in the rat nervous system, i.e., the TG and that possible AM₂, AMY₁, and AMY₃ receptors occur.

Several of the diverse biological actions of the CGRP family of peptides are clinically relevant. Our findings demonstrate the specific ligand and receptor sites in the rat trigeminal ganglion, highlighting recognition mechanisms to facilitate drug development.

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Department of Internal Medicine, Lund University Hospital, Lund, Sweden
Lars Edvinsson & Karin Warfvinge
Department of Clinical Experimental Research, Glostrup Research Institute, Rigshospitalet Glostrup Hospital, Copanhagen, Denmark
Lars Edvinsson, Anne-Sofie Grell & Karin Warfvinge

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Lars Edvinsson
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Anne-Sofie Grell
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Karin Warfvinge
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Correspondence to Karin Warfvinge.

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Edvinsson, L., Grell, AS. & Warfvinge, K. Expression of the CGRP Family of Neuropeptides and their Receptors in the Trigeminal Ganglion. J Mol Neurosci 70, 930–944 (2020). https://doi.org/10.1007/s12031-020-01493-z

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Received: 14 February 2019
Accepted: 30 January 2020
Published: 22 February 2020
Issue Date: June 2020
DOI: https://doi.org/10.1007/s12031-020-01493-z

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Expression of the CGRP Family of Neuropeptides and their Receptors in the Trigeminal Ganglion

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