Extracellular inorganic pyrophosphate (PP i ) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate PP i via the hydrolysis of ATP released into extracellular compartments by poorly understood mechanisms. Here we report that cultured vascular smooth muscle cells (VSMC) from rat aorta generate extracellular PP i via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog β,γ-methylene ATP (MeATP or AMPPCP) was used to selectively suppress ATP metabolism by eNPPs but not the CD39-type ecto-ATPases. In the absence of MeATP, VSMC generated extracellular PP i to accumulate ≥600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed PP i accumulation while increasing ATP accumulation. Probenecid, which inhibits PP i efflux dependent on ANK, a putative PP i transporter or transport regulator, reduced extracellular PP i accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises ≥50% of the extracellular PP i -generating capacity of VSMC. The accumulation of extracellular PP i and ATP was markedly attenuated by reduced temperature but was insensitive to brefeldin A, which suppresses constitutive exocytosis of Golgi-derived secretory vesicles. The magnitude of extracellular PP i accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to PP i generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix.
