Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi

Abstract

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.

Methods to Characterise Enzyme Kinetics with Biological and Medicinal Substrates: The Case of Alkaline Phosphatase

“…In a subsequent study, the authors explored the use of two‐dimensional correlation IR spectroscopy to resolve band overlap [74] . More recently, IR spectroscopy was employed in combination with 31 P NMR spectroscopy to monitor extracellular TNAP and other ectonucleoside triphoshatediphosphohydrolase sequential conversion of ATP to ADP and P i , then to AMP and P i , and finally adenosine and P i , without the formation of PP i during this process [75] . Overall, infrared spectroscopy provides time‐dependent spectra by which one can follow enzyme kinetics.…”

The Purinergic Nature of Pseudoxanthoma Elasticum

“…It is surprising as one might have expected some anti-calcification activity as it competes with ENPP1 for ATP usage, and could possibly have had some influence on PPi production, which seems to depend on the ratio of ENPP1 to ENTPD1 activity [210]. Remarkably, it has recently been shown that hydrolysis of extracellular ATP by VSMCs undergoing chondrocyte-like differentiation generates Pi but not PPi [211], suggesting the loss of pyrophosphate generation and/or an increase in NTPDase activity. While ENPP1 exerts a clear anti-calcifying activity, the role of CD39 in SMC calcification is not obvious and could depend on circumstances.…”

Biomolecules Orchestrating Cardiovascular Calcification

“…Interestingly, Tnap −/− mice still have small crystals that fail to grow, suggesting that TNAP may play a role in modulating crystal growth [47]. Recently, Buchet and colleagues found that TNAP in VSMCs also hydrolyzes exogenous ATP in a sequential manner to yield Pi, leading them to posit that TNAP may directly contribute to calcification by generating the phosphate component of the mineral [50]. However, for this to occur, extracellular ATP concentration would need to be in millimolar concentration, which does occur in the tumor microenvironment [51] and conceivably could occur in the atherosclerotic milieu as well.…”