Differential Regulation of Ca2+ Signaling and Membrane Trafficking by Multiple P2 Receptors in Brown Adipocytes

Abstract

Extracellular ATP triggers changes in intracellular Ca(2+), ion channel function, and membrane trafficking in adipocytes. The aim of the present study was to determine which P2 receptors might mediate the Ca(2+) signaling and membrane trafficking responses to ATP in brown fat cells. RT-PCR was used to determine which P2 receptors are expressed in brown fat cells. Responses to nucleotide agonists and antagonists were characterized using fura-2 fluorescence imaging of Ca(2+) responses, and FM 1-43 fluorescence imaging and membrane capacitance measurements to assess membrane trafficking. The pharmacology of the Ca(2+) responses fits the properties of the P2Y receptors for which mRNA is expressed, but the agonist and antagonist sensitivity of the membrane-trafficking response was not consistent with any P2 receptor described to date. Brown adipocytes expressed mRNA for P2Y(2), P2Y(6), and P2Y(12) metabotropic receptors and P2X(1), P2X(2), P2X(3), P2X(4), P2X(5), and P2X(7) ionotropic receptors. The agonists ATP, ADP, UTP, UDP and 2', 3'-(benzoylbenzoyl) ATP (BzATP) increased intracellular Ca(2+), while 100 microM: suramin, pyridoxal-phosphate-6-azophenyl-2' 4'-disulfonic acid (PPADS), or Reactive Blue 2 partially blocked Ca(2+) responses. ATP, but not ADP, UTP, UDP or BzATP activated membrane trafficking. The membrane response could be blocked completely with 1 microM: PPADS but not by the antagonist MRS2179. We conclude that multiple P2 receptors mediate the ATP responses of brown fat cells, and that membrane trafficking is regulated by a P2 receptor showing unusual properties.