IDENTIFICATION OF A(1) ADENOSINE RECEPTORS IN RAT COCHLEA COUPLED TO INHIBITION OF ADENYLYL-CYCLASE

A(1) adenosine receptors (A(1)ARs) are found in a number of tissues in the body where their physiological roles have been identified. In the cochlea, neither the existence of these receptors nor a physiological role of adenosine has been described previously. Membranes prepared f rom rat cochlea demonstrated high affinity and saturable binding to N- 6-2-(4-amino-3-[I-125]iodophenyl)ethyladenosine ([I-125]APNEA), an A(1 )AR agonist, with maximum binding capacity and dissociation constant v alues being 40.5 +/- 0.5 fmol/mg protein and 1.28 +/- 0.03 nM, respect ively. Adenosine analogues competed for [I-125]APNEA binding sites wit h a rank order of potency characteristic of these sites being the A(1) AR. The [I-125]APNEA binding was significantly reduced by pertussis to xin, indicating coupling of these receptors with the G(i) and/or G(o) proteins in cochlear membranes. Photoaffinity labeling of the receptor protein with the A(1)AR agonist N-6-2-(4-azido-3-[I-125] iodophenyl)e thyladenosine showed specific labeling of a 36-kDa receptor protein. A ctivation of the A(1)AR with R-phenylisopropyladenosine (R-PIA) led to inhibition of forskolin-stimulated adenylyl cyclase activity. Amplifi cation of reverse-transcribed RNA derived from cochlear tissue by poly merase chain reaction (using primers for the bovine A(1)AR) yielded a 770-bp product that hybridized to an A(1)AR cDNA probe on Southern blo ts. These data indicate the presence of an inhibitory receptor in the peripheral auditory system, which may play an important role in modula ting auditory functions.