cAMP potentiates ATP-evoked calcium signaling in human parotid acinar cells.
In salivary acinar cells, intracellular calcium ([Ca(2+)](i)) signaling plays an important role in eliciting fluid secretion through the activation of Ca(2+)-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 Ca(2+) release, through protein kinase A (PKA)-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)R) in mouse parotid acinar cells. To extend these findings, in the present study cross-talk between Ca(2+) signaling and cAMP pathways in human parotid acinar cells was investigated. In human parotid acinar cells, carbachol stimulation evoked increases in the [Ca(2+)](i) and the initial peak amplitude was enhanced following PKA activation, consistent with reports from mouse parotid. Stimulation with ATP also evoked an increase in [Ca(2+)](i). The ATP-evoked Ca(2+) elevation was largely dependent on extracellular Ca(2+), suggesting the involvement of the P2X family of purinergic receptors. Pharmacological elevation of cAMP resulted in a approximately 5-fold increase in the peak [Ca(2+)](i) change evoked by ATP stimulation. This enhanced [Ca(2+)](i) increase was not dependent on intracellular release from InsP(3)R or ryanodine receptors, suggesting a direct effect on P2XR. Reverse transcription-polymerase chain reaction and Western blot analysis confirmed the presence of P2X(4)R and P2X(7)R 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 approximately 4.5-fold enhancement of ATP-activated current in HEK-293 cells transfected with P2X(4)R DNA but had no effects on currents in cells expressing P2X(7)R. These data indicate that in human parotid acinar cells, in addition to modulation of Ca(2+) release, Ca(2+) influx through P2X(4)R may constitute a further locus for the synergistic effects of Ca(2+) and PKA activation.
Brown
David A
DA
Department of Pharmacology & Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York 14642, USA.
Bruce
Jason I E
JI
Straub
Stephen V
SV
Yule
David I
DI
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002217:Carbachol / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D005576:Colforsin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004357:Drug Synergism; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D010306:Parotid Gland / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4
D018679:Cholinergic Agonists; C548226:P2RX4 protein, human; C548227:P2rx4 protein, mouse; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D005576:Colforsin; D000255:Adenosine Triphosphate; D002217:Carbachol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium