Interaction of α1D-Adrenergic and P2X7Receptors in the Rat Lacrimal Gland and the Effect on Intracellular [Ca2+] and Protein Secretion

Abstract

To determine whether α(1D)-adrenergic receptors (α(1D)-AR) and P2X(7) receptors interact by determining their effect on ATP release, intracellular [Ca(2+)] ([Ca(2+)](i)), and protein secretion in rat lacrimal gland acini. Exorbital lacrimal glands from male Sprague-Dawley rats were divided into pieces or digested with collagenase to form acini. With the use of an imaging system, [Ca(2+)](i) was measured in acini loaded with fura-2. Adenosine triphosphate (ATP) release was determined using the luciferin-luciferase reaction. Peroxidase secretion, our index for protein secretion, was measured spectrophotometrically. Acini were stimulated with the P2X(7) receptor agonist, (benzoylbenzoyl)adenosine 5' triphosphate (BzATP) or the α(1D)-AR agonist phenylephrine with or without antagonist preincubation. Phenylephrine increased ATP release from pieces in a time-dependent manner. The α(1D)-AR antagonist BMY7378 blocked the BzATP-stimulated increase in [Ca(2+)](i) but not in peroxidase secretion. The P2X(7) antagonist A438079 blocked the phenylephrine-stimulated increase in [Ca(2+)](i) but not peroxidase secretion. The increase in [Ca(2+)](i) caused by phenylephrine and BzATP used simultaneously or sequentially was additive, as was the increase in peroxidase secretion. The inhibition of protein kinase C isoforms or calcium calmodulin kinase II did not alter the BzATP-induced increase in [Ca(2+)](i). The authors conclude that activation of α(1D)-AR releases ATP, which induces P2X(7) receptors to increase [Ca(2+)](i) but not to stimulate protein secretion. P2X(7) receptors in turn activate α(1D)-AR to increase [Ca(2+)](i) but not to stimulate protein secretion. Furthermore, α(1D)-AR compared with P2X(7) receptors use different cellular mechanisms to increase [Ca(2+)](i) and cause protein secretion.