ABSTRACT: Adenylyl cyclase (AC) signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across AC isoforms, and reverse transcription-polymerase chain reaction (RT-PCR), coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca(2+), the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca(2+). Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate AC activation in the absence of G?s in mammalian cochlear hair cells, the issue of hair-cell G?s mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for G?s/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for G?i have also been detected in the hair cell layer, one with homology to G?i1 and the second with homology to G?i3 of higher vertebrates. Both G?s/olf protein and G?i1/G?i3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. Although a signaling event coupling to G?s/olf and G?i1/G?i3 in the stereocilia is currently unknown, signaling with G?s/olf, G?i3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to G?olf and activation of AC5/6. Co-expression with dopamine D2 receptor, which itself couples to G?i3 and AC5/6, will down-modulate levels of cAMP, thus fine-tuning and gradating the hair-cell response to dopamine D1A. As predicted by the trout saccular hair cell model, evidence has been obtained for the first time that hair cells of mammalian otolithic vestibular end organs (rat/mouse saccule/utricle) express dopamine D1A and D2L receptors, and each receptor co-localizes with AC5/6, with a marked presence of all three proteins in subcuticular regions of type I vestibular hair cells. A putative efferent, presynaptic source of dopamine was identified in tyrosine hydroxylase-positive nerve fibers which passed from underlying connective tissue to the sensory epithelia, ending on type I and type II vestibular hair cells and on afferent calyces.
