G-proteins coupled to phosphoinositide hydrolysis in the cochlear and vestibular sensory epithelia of the rat are insensitive to cholera and pertussis toxins

In the cochlear (CSE) and vestibular sensory epithelia (VSE), phosphoinositides are hydrolyzed in response to stimulation of phospholipase C (PLC) by cholinergic muscarinic and purinergic P_2y agonists. Such receptor-mediated activation of PLC is expected to be coupled through guanine nucleotide-binding proteins (G-proteins). Although several classes of G-proteins have been identified in the inner ear, nothing is known about the type of G-proteins associated with the phosphoinositide second messenger system in CSE and VSE. Phosphoinositide hydrolysis was determined by the release of radiolabeled inositol phosphates (InsPs). Ten mM NaF plus 10 μM AlCl₃ increased basal InsPs accumulation 2-fold in both CSE and VSE of the rat. Release of InsPs was also enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP-γ-S) in saponin-permeabilized tissues. Furthermore, release of InsPs stimulated by both carbamylcholine (CCh) and adenosine 5'-O-[3-thiotriphosphate](ATP-γ-S) was significantly inhibited by 100 μM guanosine 5'-O-[2-thiodiphosphate](GDP-β-S). These results strongly suggest the involvement of G-proteins in the receptor-PLC coupling in CSE and VSE. ADP-ribosylation in membrane fractions of CSE and VSE in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicated the existence of G_s- and G_i-type G-proteins. However, neither CTX nor PTX affected basal or 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.