Phenol Red (phenolsulphophthalein sodium salt, 376.4 amu; Aldrich) is a water-soluble pH indicator dye (pH 6.2-8.2, yellow-to-red colour transition), commonly found in culture media (e.g. Dulbecco’s modified Eagle’s medium (DMEM); at 15 mg l^-1 (40 µM)). Here, we report that this compound also inhibits ATP-evoked ion currents at rat P2X (rP2X) receptors heterologously expressed in defolliculated Xenopus laevis oocytes.

Under voltage-clamp conditions (V_h, -30 mV), ATP (applied at its EC₇₀ value) evoked inward ion currents at homomeric P2X receptors, which were inhibited by commercially available Phenol Red with the following potency order (mean pIC₅₀, n = 4): rP2X₁ (5.48) > rP2X₃ (4.60) > rP2X₂ (4.19) > rP2X₄ (< 4.00) – where inhibition was reversed following washout. Purified Phenol Red also inhibited P2X receptors without altering mean pIC₅₀ values (rP2X₁ (5.41) and rP2X₃ (4.67); n = 4). For concentration/ response (C/R) curves to ATP and α,β-meATP, Phenol Red (1-100 µM) inhibited agonist responses at rP2X₁ and rP2X₃ receptors in a non-surmountable manner.

Under whole-cell patch-clamp conditions (V_h, -60 mV), P2X₃-like ATP responses in isolated rat DRG neurons were inhibited by Phenol Red (30 µM). At rat vas deferens, P2X₁-like contractions evoked by either purinergic neurotransmission or exogenous ATP were weakly inhibited by Phenol Red (100 µM). Closer inspection of C/R curves for exogenous ATP revealed two phases – where the initial phase was inhibited by Phenol Red (1, 3 and 10 µM). α,β-MeATP responses in rat vas deferens were inhibited in a surmountable manner by Phenol Red (10, 30 and 100 µM). Electrically evoked purinergic EJPs in mouse vas deferens were not inhibited by Phenol Red (300 µM). Animals were killed by cervical dislocation.

Our results show that Phenol Red (or residual impurity therein) blocks recombinant rP2X₁ and rP2X₃ subtypes in a non-surmountable, yet reversible, manner. This antagonist is also effective at native P2X₃-like receptors in rat tissue. However, Phenol Red was largely ineffective at P2X₁-like electrical (mouse) and mechanical (rat) responses in vas deferens, although P2X₁ plays a critical role in this tissue since purinergic responses disappear in P2X₁ gene-deleted mice (Mulryan et al. 2000). Our results suggest that the P2X₁ subunit mediates purinergic signalling in the vas deferens through the formation of a heteromeric purinoceptor of unknown molecular composition and that homomeric P2X₁ receptors do not predominate in this tissue.