Fluorescence imaging of extracellular purinergic receptor sites and putative ecto-ATPase sites on isolated cochlear hair cells

Abstract

Fluorescence imaging of extracellular adenosine-5′-triphosphate (ATP) binding sites on inner and outer hair cells isolated from the guinea pig organ of Corti was achieved using the fluorescent analog of ATP, 2′- (or-3′)-O-(trinitrophenyl)adenosine-5′- triphosphate (TNP-ATP; 30–75 microM). This analog, which fluoresces on binding to these sites, was pressure applied by micropipette while hair cells were viewed by fluorescence microscopy. Fluorescence imaging revealed a widespread distribution of extracellular binding sites, including the stereocilia, cuticular plate, and the basolateral margins of the cells, but particularly in infracuticular and infranuclear regions. In support of extracellular binding, simultaneous electrophysiological recordings demonstrated that rapid washout of TNP-ATP-induced fluorescence was dependent upon cell integrity. Suramin, a nonselective P2 purinoceptor antagonist, coapplied with TNP-ATP, reduced the fluorescence observed on the stereocilia and apical surface of the cuticular plate only. This implies that binding sites on the apical surface of hair cells are P2 receptors, consistent with previous electrophysiological evidence for localization of P2 receptors to the apical surface of cochlear hair cells (Housley et al., 1992). Binding of TNP-ATP to P2 purinoceptors was confirmed by its antagonism of the inward current elicited by ATP (10 microM) in voltage-clamped hair cells. Fluorescence from the basolateral margin was significantly quenched when TNP-ATP was applied in divalent cation-free solution. Because divalent cations are required for ATPase activity, this finding provides evidence for the presence of ecto-ATPases on the basolateral membrane of hair cells. The divalent cation-free condition had no significant effect on the ATP-gated P2 purinoceptor conductance. We propose that there are two classes of ATP binding sites on cochlear hair cells: apically located P2 purinoceptors gating nonselective cation channels and basolaterally located ecto- ATPases that may be involved in purine turnover.

Purinergic signaling in cochleovestibular hair cells and afferent neurons

“…Part of such discrepancy may be due to the age of animals investigated, since transient expression in certain developmental stages has been shown in each subtype (P2X 2 , P2X 3 , and P2X 7 ). However, among them, consistent results show that the P2X ionotropic channels are localized at the endolymphatic surface [4], especially at the stereocilia [9], and Na + influx through these channels was visualized using fluorescence [5]. More specifically, the P2X 2 receptor subtype was shown to be localized at the stereocilia of guinea pig hair cells [9], and therefore this may be the dominant P2X subtype.…”

“…8 of [29]). Studies using fluorescent imaging of Na + influx confirmed the apical location of ionotropic receptors, facing the endolymphatic compartment [4,5]. To date, there have been few reports on differential expression of P2 receptors along the basilar membrane, i.e., basal, middle, and apical turns.…”

Pathophysiology and Therapeutic Potential of Purinergic Signaling

“…PPADS is a nonselective P2 antagonist, but has the advantage that it associates and dissociates approximately 100 to 10,000 times more slowly than other known antagonists PATHOPHYSIOLOGY AND THERAPEUTIC POTENTIAL OF PURINERGIC SIGNALING (Spelta et al, 2002). The trinitrophenyl-substituted nucleotide TNP-ATP is a very potent antagonist at both P2X 3 and P2X 2/3 receptors (Mockett et al, 1994;King et al, 1997;Virginio et al, 1998;Burgard et al, 2000;Honore et al, 2002). A-317491 is a potent and selective non-nucleotide antagonist of P2X 3 and P2X 2/3 receptors, and it reduces chronic inflammatory and neuropathic pain in the rat (Jarvis et al, , 2004McGaraughty et al, 2003).…”

Competitive Antagonism of Recombinant P2X2/3Receptors by 2′,3′-O-(2,4,6-Trinitrophenyl) Adenosine 5′-Triphosphate (TNP-ATP)

“…However, these compounds are relatively nonselective and exhibit high nanomolar-to-high micromolar affinities for P2X receptors (Bianchi et al, 1999). The ATP analog 2Ј,3Ј-O-(2,4,6-trinitrophenyl) adenosine 5Ј-triphosphate (TNP-ATP) has been used as a probe for more than two decades to label ATP-binding sites on a variety of tissues (Hiratsuka and Uchida, 1973;Watanabe and Inesi, 1982;Mockett et al, 1994). Mockett et al (1994) and King et al (1997) were among the first to describe the antagonist effects of TNP-ATP at P2X receptors.…”

“…The ATP analog 2Ј,3Ј-O-(2,4,6-trinitrophenyl) adenosine 5Ј-triphosphate (TNP-ATP) has been used as a probe for more than two decades to label ATP-binding sites on a variety of tissues (Hiratsuka and Uchida, 1973;Watanabe and Inesi, 1982;Mockett et al, 1994). Mockett et al (1994) and King et al (1997) were among the first to describe the antagonist effects of TNP-ATP at P2X receptors. However, it was not until recently that Virginio et al (1998) described the selective antagonism of P2X 1 , P2X 3 , and P2X 2/3 receptors by low nanomolar concentrations of TNP-ATP.…”