cAMP potentiates ATP-evoked calcium signaling in human parotid acinar cells

Abstract

In salivary acinar cells, intracellular calcium ([Ca2+] i) signaling plays an important role in eliciting fluid secretion through the activation of Ca2+-activated ionic conductances. Ca 2+ and cAMP have synergistic effects on fluid secretion such that peak secretion is elicited following activation of both parasympathetic and sympathetic pathways. We have recently demonstrated that cAMP exerts effects on Ca2+ release, through protein kinase A (PKA)-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP3R) in mouse parotid acinar cells. To extend these findings, in the present study cross-talk between Ca2+ signaling and cAMP pathways in human parotid acinar cells was investigated. In human parotid acinar cells, carbachol stimulation evoked increases in the [Ca2+]iand the initial peak amplitude was enhanced following PKA activation, consistent with reports from mouse parotid. Stimulation with ATP also evoked an increase in [Ca2+]i The ATP-evoked Ca2+ elevation was largely dependent on extracellular Ca2+, suggesting the involvement of the P2X family of purinergic receptors. Pharmacological elevation of cAMP resulted in a ∼5-fold increase in the peak [Ca2+]i change evoked by ATP stimulation. This enhanced [Ca2+]i increase was not dependent on intracellular release from InsP3R or ryanodine receptors, suggesting a direct effect on P2XR. Reverse transcription-polymerase chain reaction and Western blot analysis confirmed the presence of P2X4R and P2X 7R mRNA and protein in human parotid acinar cells. ATP-activated cation currents were studied using whole cell patch clamp techniques in HEK-293 cells, a null background for P2XR. Raising cAMP resulted in a ∼4.5-fold enhancement of ATP-activated current in HEK-293 cells transfected with P2X 4R DNA but had no effects on currents in cells expressing P2X 7R. These data indicate that in human parotid acinar cells, in addition to modulation of Ca2+ release, Ca2+ influx through P2X4R may constitute a further locus for the synergistic effects of Ca2+ and PKA activation.