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Vascular calcification is a multifaceted process involving gain of calcification inducers and loss of calcification inhibitors. One such inhibitor is inorganic pyrophosphate ([PP.sub.i]), and regulated generation and homeostasis of extracellular [PP.sub.i] is a critical determinant of soft-tissue mineralization. We recently described an autocrine mechanism of extracellular [PP.sub.i] generation in cultured rat aortic vascular smooth muscle cells (VSMC) that involves both ATP release coupled to the ectophosphodiesterase/pyrophosphatase ENPP1 and efflux of intracellular [PP.sub.i] mediated or regulated by the plasma membrane protein ANK. We now report that increased cAMP signaling and elevated extracellular inorganic phosphate ([P.sub.i]) act synergistically to induce calcification of these VSMC that is correlated with progressive reduction in ability to accumulate extracellular [PP.sub.i]. Attenuated [PP.sub.i] accumulation was mediated in part by cAMP-dependent decrease in ANK expression coordinated with cAMP-dependent increase in expression of TNAP, the tissue nonselective alkaline phosphatase that degrades [PP.sub.i]. Stimulation of cAMP signaling did not alter ATP release or ENPP1 expression, and the cAMP-induced changes in ANK and TNAP expression were not sufficient to induce calcification. Elevated extracellular [P.sub.i] alone elicited only minor calcification and no significant changes in ANK, TNAP, or ENPP1. In contrast, combined with a cAMP stimulus, elevated [P.sub.i] induced decreases in the ATP release pathway(s) that supports ENPP1 activity: this resulted in markedly reduced rates of [PP.sub.i] accumulation that facilitated robust calcification. Calcified VSMC were characterized by maintained expression of multiple SMC differentiation marker proteins including smooth muscle (SM) [alpha]-actin, SM22[alpha], and calponin. Notably, addition of exogenous ATP (or [PP.sub.i] per se) rescued cAMP + phosphate-treated VSMC cultures from progression to the calcified state. These observations support a model in which extracellular [PP.sub.i] generation mediated by both ANK- and ATP release-dependent mechanisms serves as a critical regulator of VSMC calcification. ectonucleotide pyrophosphatase/phosphodiesterase-1; ANK; adenosine 3',5'-cyclic monophosphate; ATP release doi:10.1152/ajpcell.00419.2009.

Extracellular inorganic pyrophosphate ([PP.sub.i]) is a potent suppressor of physiological calcification in bone and pathological calcification in blood vessels. Ectonucleotide pyrophosphatase/phosphodiesterases (eNPPs) generate [PP.sub.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.sub.i] via an autocrine mechanism that involves ATP release tightly coupled to eNPP activity. The nucleotide analog [beta], [gamma]-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.sub.i] to accumulate [greater than or equal to] 600 nM within 2 h while steadily maintaining extracellular ATP at 1 nM. Conversely, the presence of MeATP completely suppressed [PP.sub.i] accumulation while increasing ATP accumulation. Probenecid, which inhibits [PP.sub.i] efflux dependent on ANK, a putative [PP.sub.i] transporter or transport regulator, reduced extracellular [PP.sub.i] accumulation by approximately twofold. This indicates that autocrine ATP release coupled to eNPP activity comprises [greater than or equal to] 50% of the extracellular PP,-generating capacity of VSMC. The accumulation of extracellular [PP.sub.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.sub.i] accumulation in VSMC cultures increased with time postplating, suggesting that ATP release coupled to [PP.sub.i] generation is upregulated as cultured VSMC undergo contact-inhibition of proliferation or deposit extracellular matrix. adenosine 5'-triphosphate release; vascular calcification; ectonucleotide pyrophosphatase; ectoadenosine 5'-triphosphatase

The article comments on two research on cisplatin-induced renal toxicity and the role of magnesium deficiency in the onset of acute kidney injury (AKI). It discusses the different effects of magnesium status on tumor cells and renal cells. It also highlights the importance of the results including the difference in regulation of Abcc6 expression between tumor cells and renal cells and the significance of magnesium supplementation to cisplatin regimens.