Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) in the cochlea: evidence for specific transcript expression of PAC1-R splice variants in rat microdissected cochlear subfractions

Abstract

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from the hypothalamus, named for its high potency in stimulating adenylyl cyclase in pituitary cells. PACAP acts through the specific receptor PAC1-R to modulate the action of neurotransmitters, and additionally, to regulate cell viability via autocrine/intracrine mechanisms. Evidence has now been obtained that PACAP and multiple splice variants of PAC1-R are expressed in the rat cochlea. mRNA for PACAP precursor protein is found by reverse transcription-polymerase chain reaction (RT-PCR) in microdissected cochlear lateral wall, organ of Corti, and spiral ganglion subfractions. A specific pattern of expression of mRNA for PAC1-R splice variants, which mediate the response to PACAP, has been revealed by RT-PCR and cloning for the cochlear subfractions. Transcript for the short form of PAC1-R is found in all three subfractions. Four additional splice variants -- hop1, hop2, hip, and a novel hop1 splice variant -- are expressed in the lateral wall. For the amino terminus splice region of PAC1-R, a new splice variant has been detected in the organ of Corti, representing a deletion of the first 7 of 21 amino acids detected in the PAC1-R very-short sequence. Overall, from message determinations in cochlear subfractions, there are five PAC1-R splice variants in the lateral wall, two in the organ of Corti and one in the spiral ganglion, indicating multiple possible responses to PACAP and/or mechanisms to modulate the response to PACAP in the cochlea. The variety of PAC1-R splice variants expressed may reflect the diversity in cell function between subfractions that is modulated by PACAP. The neuropeptide and its specific receptor have been immunolocalized in the lateral wall, the source of the largest number of cochlear PAC1-R splice variants. The receptor was targeted by primary antibodies which would elicit immunoreactivity for all splice variants of PAC1-R detected with RT-PCR, and evidence has been obtained with Western blot analysis suggesting that PAC1-R is glycosylated in vivo. Within the lateral wall, PACAP and PAC1-R were immunolocalized primarily to the stria vascularis, with immunoreactivity for both neuropeptide and receptor increasing from the basal to apical cochlear turns. Within the stria, PACAP immunoreactivity was localized to the basolateral extensions of marginal cells, while PAC1-R was clearly associated with tight junctions between the marginal cells close to the endolymphatic compartment. In addition, evidence was obtained that PAC1-R was associated with endothelial cells of the capillaries in the stria vascularis. The large number of splice variants expressed, coupled to the specificity in linkage between PAC1-R splice variants and G-protein-coupled second messenger pathways, could provide a mechanism to closely modulate tight junction integrity in the stria vascularis, impacting the endolymphatic potential.