Research reportTissue-specific mRNA expression of a calcitonin receptor-like receptor during fetal and postnatal development
Introduction
Calcitonin (CT) receptors belong to a subfamily of seven transmembrane G protein-linked receptors which include those of parathyroid hormone/parathyroid hormone-related protein and secretin 8, 13. They are coupled to both the adenylyl cyclase and the phospholipase C pathways. CT exhibits limited structural homology to CT gene-related peptide (CGRP), an alternative splicing product of the CT gene, and to amylin and adrenomedullin (ADM). As a result, overlapping biological actions including inhibition of bone resorption and vasodilation have been attributed to cross-reaction of the different peptides with corresponding receptors [16]. Interactions of amylin and CGRP with CT receptors, and of adrenomedullin and amylin with CGRP receptors, have been recognized 2, 17, 28.
In rat and man, novel highly homologous (90%) CT receptor-like receptors (CRLR) have been identified through molecular cloning 3, 7. Over 50% of the amino acids are identical in cloned CT receptors [8]. Northern blot analysis of rat and human mRNA revealed predominant expression of 4.5 and 5.2 kb transcripts, respectively, in the lung and weaker signals in the heart and kidney 1, 7, 18. Stable transfection of HEK293 cells with the human CRLR encoding cDNA, independently cloned from human lung, revealed CGRP type I receptors in selected clonal transfectants [1]. The results differ from human CRLR expression studies in transiently transfected COS-7 and opossum kidney cells which did not reveal a ligand for CGRP and other homologous peptides such as CT, amylin and ADM [7].
The distribution of [125I]CT and -CGRP binding sites has been extensively studied in membrane homogenates and, by receptor autoradiography, in rat and human tissues. CT receptors are predominantly localized in osteoclasts, renal tubules and in the periventricular region of the brain 6, 9, 15, 19, 20, consistent with in situ hybridization (ISH) analysis of CT receptor mRNA [22]. CGRP binding sites are widely distributed throughout the nervous system with the highest density in the Purkinje cell layer of the cerebellum and the spinal cord 4, 10, 12, 20, 21, 24, 25, 26, 27. With the exception of the spleen, the density of CGRP binding sites is not striking in peripheral organs [23].
In the present study, the developmental distribution of mRNA encoding CRLR was investigated through ISH. The results indicate localization of CRLR mRNA in the rat CNS and in peripheral organs distinct from that of CT receptor encoding mRNA and of the receptor binding sites of CT and CGRP.
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Animals and tissue preparation
Long–Evans rats were bred under controlled light and dark cycles (lights on 02.00–16.00 h) and constant temperature (22°C) with a standard diet (NAFAG 850, Eberle-Nafag, Gossau, Switzerland) and water ad libitum. Timed pregnant rats were used for investigations of prenatal stages. Receptive females were mated between 16.00 and 19.00 h. Gestational day 1 (GD1) was defined as the stage 24 h after the onset of the mating period. As sex determinations were difficult in the early stages of
Specificity of oligonucleotide binding
A similar pattern of distribution was observed when the two 33P-labeled oligonucleotides 1 and 2 were individually hybridized to adjacent brain sections of adult rats (Fig. 1a and b). When the two oligonucleotides were pooled, the signals were further increased (Fig. 1c). Hybridization signals were absent with a 100-fold molar excess of combined unlabeled oligonucleotides (Fig. 1d). In subsequent experiments, the oligonucleotides were pooled to increase sensitivity.
Ontogeny of CRLR
The results obtained are
Discussion
The present study reveals expression of CRLR mRNA during late prenatal development of the rat CNS in the piriform cortex, central and basolateral amygdaloid nucleus, and the amygdalostriatal transition area. Maintained expression of CRLR in these telencephalic areas during postnatal stages and in adult animals suggest an important functional role of CRLR in the CNS. The regional distribution of CRLR mRNA expression in the rat brain is different from that of CT mRNA as well as of [125I]CT and
Acknowledgements
This work was supported in part by the Swiss National Science Foundation (Grant 31-43094.95) and the Kanton of Zurich.
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2013, Neuroscience LettersCitation Excerpt :High-density of CGRP receptor was located by autoradiography of 125I-CGRP in nucleus accumbens, ventral caudate and putamen, lateral amygdaloid nucleus, molecular and Purkinje cell layers of the cerebellar cortex, midbrain and brainstem nuclei [15]. CRLR mRNA was detected in the caudal piriform cortex [7], caudal caudate putamen, amygdala [13] via in situ hybridization, and RAMP1 mRNA throughout the brain in cortex, caudate putamen, olfactory tubercles, amygdaloid complex, hippocampus, cerebellum and ependyma [20]. Recently, CRLR and RAMP1 immunoreactivities were found in the trigeminal ganglia, dorsal root ganglia (DRG), spinal cord and spinal trigeminal nuclei [10], purkinje cells of cerebellum [6] and even nerve fibers of myenteric plexuses of gastrointestinal tract [3].
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