G-PROTEINS COUPLED TO PHOSPHOINOSITIDE HYDROLYSIS IN THE COCHLEAR ANDVESTIBULAR SENSORY EPITHELIA OF THE RAT ARE INSENSITIVE TO CHOLERA AND PERTUSSIS TOXINS

In the cochlear (CSE) and vestibular sensory epithelia (VSE), phosphoi nositides are hydrolyzed in response to stimulation of phospholipase C (PLC) by cholinergic muscarinic and purinergic P-2y agonists. Such re ceptor-mediated activation of PLC is expected to be coupled through gu anine nucleotide-binding proteins (G-proteins). Although several class es of G-proteins have been identified in the inner ear, nothing is kno wn about the type of G-proteins associated with the phosphoinositide s econd messenger system in CSE and VSE. Phosphoinositide hydrolysis was determined by the release of radiolabeled inositol phosphates (InsPs) . Ten mM NaF plus 10 mu M AlCl3 increased basal InsPs accumulation 2-f old in both CSE and VSE of the rat. Release of InsPs was also enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) in saponin-perme abilized tissues. Furthermore, release of InsPs stimulated by both car bamylcholine (CCh) and adenosine 5'-O-[3-thiotriphosphate] (ATP-gamma- S) was significantly inhibited by 100 mu M guanosine 5'-O-[2-thiodipho sphate] (GDP-beta-S). These results strongly suggest the involvement o f G-proteins in the receptor-PLC coupling in CSE and VSE. ADP-ribosyla tion in membrane fractions of CSE and VSE in the presence of cholera t oxin (CTX) or pertussis toxin (PTX) indicated the existence of G(s)- a nd G(i)-type G-proteins. However, neither CTX nor PTX affected basal o r agonist-stimulated release of InsPs. These observations suggest that muscarinic and P-2y purinergic receptors are coupled to PLC via CTX- and PTX-insensitive G-proteins in CSE and VSE.