Functional expression of P2X4 receptor in capillary endothelial cells of the cochlear spiral ligament and its role in regulating the capillary diameter

The cochlear lateral wall generates the endocochlear potential (EP), which creates a driving force for the hair cell transduction current and is essential for normal hearing. Blood flow at the cochlear lateral wall is critically important for maintaining the EP. The vulnerability of the EP to hypoxia suggests that the blood flow in the cochlear lateral wall is dynamically and precisely regulated to meet the changing metabolic needs of the cochlear lateral wall. It has been reported that ATP, an important extracellular signaling molecule, plays an essential role in regulating cochlear blood flow. However, the cellular mechanism underlying ATP-induced regional blood flow changes has not been investigated. In the current study, we demonstrate that 1) the P2X4 receptor is expressed in endothelial cells (ECs) of spiral ligament (SL) capillaries. 2) ATP elicits a characteristic current through P2X4 on ECs in a dose-dependent manner (EC₅₀ = 0.16 mM). The ATP current has a reversal potential at 0 mV; is inhibited by 5-(3- bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5- BDBD), LaCl₃, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate (PPADS), and extracellular acidosis; and is less sensitive to α,β-methyleneadenosine 5′-triphosphate (α,β - MeATP) and 2′- and 3′-O-(4-benzoyl-benzoyl) adenosine 5′-triphosphate (BzATP). 3) ATP elicits a transient increase of intracellular Ca²⁺ in ECs. 4) In accordance with the above in vitro findings, perilymphatic ATP (1 mM) caused dilation in SL capillaries in vivo by 11.5%. N^ω-nitro-L-arginine methyl ester hydrochloride (_L-NAME), a nonselective inhibitor of nitric oxide synthase, or 5-BDBD, the specific P2X4 inhibitor, significantly blocked the dilation. These findings support our hypothesis that extracellular ATP regulates cochlear lateral blood flow through P2X4 activation in ECs.