Abstract
The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X7 receptors were detected in these fractions. The location of the P2X7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-beta-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with alpha-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca2+]i) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A2 (PLA2). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. We conclude that P2X7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA2 in response to P2X7 receptor occupancy.
Highlights
The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated
Rafts were isolated after extensive sonication of the plasma membrane followed by centrifugation on a discontinuous sucrose gradient
We show that two populations of purinergic P2X7 receptors are present in the plasma membrane of rat submandibular glands
Summary
The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. The P2X receptors have two transmembrane domains separated by z300 amino acids, which make a large extracellular loop [3]. Both the N and C termini are probably cytoplasmic. The extracellular loop contains several conserved N-glycosylation sites and 10 cysteine residues forming five disulfide bridges [4] These receptors are not coupled to a regulatory protein, but when activated by an agonist they form a cationic channel. The very long (240 amino acids) intracellular C terminus of the P2X7 receptor confers to this protein unexpected properties, such as the capacity to form a pore [7]. This article is available online at http://www.jlr.org
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