Cloning of a molecule from the rabbit ciliary body which confers responsiveness to calcitonin gene-related peptide

M. I. Rosenblatt, L. Paul, Gerhard Dahl, I. M. Dickerson

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Abstract

Purpose. Calcitonin gene-related peptide (CGRP), a 37 amino acid neuropeptide, when released from nerves within the ciliary body can cause a neurogenic inflammatory response characterized by increased intraocular pressure, breakdown of the blood-aqueous barrier, and increased blood flow. Our laboratory has recently cloned a molecule from the guinea pig Organ of Corti which confers CGRP responsiveness. The goal of these studies was to clone the homologue from the rabbit ciliary body to facilitate a better understanding of the action of CGRP in neurogenic ocular inflammation. Methods. A 101 bp segment of the rabbit molecule was isolated by degenerate reverse transcription polymerase chain reaction (RT-PCR) from rabbit ciliary body mRNA using degenerate primers designed against the cloned guinea pig sequence. Sequence information from this RT-PCR product was used to design specific primers to perform 5′ and 3′ rapid amplification of cDNA ends (RACE). A 500 bp 5′ RACE product and a 1.8 kb 3′ RACE product encoding overlapping fragments were obtained, subsequently cloned, and partially sequenced. Using RT-PCR an amplimer encoding the entire open reading frame for the molecule was isolated, transcribed in vitro, and evaluated for CGRP responsiveness in an oocyte Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) assay. Results. Sequencing of the 3′ and 5′ RACE clones indicates that we have isolated a cDNA encoding a 148 amino acid molecule from the rabbit eye which has an upstream Kozak consensus sequence and is 84% identical at the amino acid level to the molecule conferring CGRP responsiveness isolated from the guinea pig. The transcript from the RT-PCR derived rabbit cDNA displays CGRP responsiveness in a sensitive oocyte CFTR expression assay. Conclusions. We have isolated the rabbit ocular form of a novel molecule which confers CGRP responsiveness in an oocyte CFTR expression assay.

Original languageEnglish
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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ASJC Scopus subject areas

  • Ophthalmology

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