Feedback Regulation of ATP-induced Ca2+ Signaling in HL-60 Cells Is Mediated by Protein Kinase A- and C-mediated Changes in Capacitative Ca2+ Entry

Abstract

Extracellular ATP evokes many physiological effects such as platelet aggregation, neurotransmission, inflammation, and muscle contraction in numerous cell types (1). These various effects of ATP are mediated by plasma membrane P 2 purinergic receptors (2). Six subtypes of P 2 purinergic receptors, P 2X , P 2Y , P 2D , P 2T , P 2Z , and P 2U , were identified in pharmacological and functional studies and supported by cloning data (3 -binding proteins are also involved in buffering the cytosolic Ca 2ϩ concentration. We studied the mechanism by which the different patterns of decrease in Ca 2ϩ occur upon stimulation with supramaximal concentrations of ATP in HL-60 cells. Our results suggest that this difference is not due to the cytosolic Ca 2ϩ removal system, but that it is instead mainly due to changes in capacitative Ca 2ϩ entry by actions of PKA and PKC, which are differentially activated by ATP itself. EXPERIMENTAL PROCEDURESMaterials-ATP, UTP, thapsigargin, Triton X-100, Trizma (Tris base), trichloroacetic acid, EGTA, EDTA, sulfinpyrazone, MOPS, sodium fluoride, sodium orthovanadate, sodium pyrophosphate, ␤-glycerophosphate, leupeptin, pepstatin A, aprotinin, phenylmethylsulfonyl fluoride, and IP 3 were purchased from Sigma. Fura-2 pentaacetoxymethyl ester was from Molecular Probes (Eugene, OR), and [ 3 H]IP 3 and

Opposing effects of protein kinase A and C on capacitative calcium entry into HL-60 promyelocytes

“…The opposing regulatory actions of PKA and PKC could provide the cells with an elaborate and highly responsive regulation of capacitative calcium entry in the achievement of calcium homeostasis. Recently, we observed that the elevation of intracellular calcium level elicited by the stimulation of ATP receptors which are coupled to adenylyl cyclase and PLC was modulated in a feedback mode with regulation of capacitative calcium entry by subsequent activation of PKA and PKC [26]. Our present study also suggests that concomitant or sequential activation of various receptors linked to adenylyl cyclase or PLC may very well affect intracellular calcium homeostasis and result in different physiological responses.…”

Phospholipase C-δ1 Is Activated by Capacitative Calcium Entry That Follows Phospholipase C-β Activation upon Bradykinin Stimulation

“…Mn 2ϩ is a good surrogate for Ca 2ϩ ions in these kind of experiments, since it is not pumped out of the cells. Thus, it can be used as a selective tracer for Ca 2ϩ influx (22). As shown in Fig.…”

Purinergic signalling and cancer

“…ATP and UTP activated superoxide formation in HL-60 promyelocytic leukaemic cells [440] and ATP also increased [Ca 2+ ] i in these cells [441,442] probably via P2Y 2 receptors [443,444]. Reduced proliferation was produced by ATP and UTP in both HL-60 promyelocytic and U937 promonocytic human cell lines [445], perhaps via A 3 receptors [446].…”