Study of nonspecific cation channel coupled to P_2z purinergic receptors using an acid load technique

-The intracellular pH (pH_i) of rat submandibular cells was measured by 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The cells recovered from ammonium (30 mM) prepulse to their resting pH_i within 10 min. Ethylisopropylamiloride (EIPA), an inhibitor of the Na⁺/H⁺ exchanger, slows the rate of pH_i recovery. ATP (1 mM), in the presence of EIPA, increases the rate of recovery 3.7-fold in the absence or presence of ethylene glycol-bis(β-aminoethyl etheri-N,N,N,N′-tetraacetic acid. The recovery was blocked by the addition of 5 mM Mg²⁺ or 10 μM Coomassie blue. The response was elicited by 2′- and 3′-O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate but not by ADP, UTP, adenyl (β-γ-methylene)-diphosphonate, 2-methylthioadenosine 5′-triphosphate, or muscarinic or β-adrenergic agonists. The purinergic response was also observed when the cells were acidified by sodium propionate and could not be mimicked by the depolarization of the plasma membrane. Aluminum fluoride did not reproduce the response to ATP, suggesting that the observed response does not involve a high-molecular-weight GTP-binding protein. It is concluded that the activation of P_2z receptors, probably by the opening of nonspecific cation channels, increases the permeability to protons in rat submandibular glands.