<p>Glycosaminoglycans (GAGs) and proteoglycans are key components of the extracellular matrix, playing structural and regulatory roles in various biological processes. GAGs are involved in modulating endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. CHO-745 cells, deficient in GAGs, produce significantly higher NO levels than wild-type CHO-K1 cells. Since NO is crucial for vascular function, this study investigated how NO overproduction in GAG-deficient cells affects cytosolic Ca²⁺ transients triggered by P2Y6 receptor (P2Y6R) activation with UDP, a vascular tone regulator. Results showed that GAG-deficient CHO-745 cells experience basal oxidative and nitrosative stress. Although integrin expression was similar, the RGD peptide increased NO production in CHO-K1 but not in CHO-745. P2Y6R activation involved phospholipase C and endoplasmic reticulum (ER) Ca²⁺ stores, processes dependent on surface GAGs. Additionally, store-operated Ca²⁺ entry and Na⁺/Ca²⁺ exchanger (NCX) were involved in UDP-induced Ca²⁺ responses, influenced by NO levels. Excess NO also contributed to ER stress and cell death, particularly in the absence of GAGs. Overall, GAG deficiency leads to oxidative stress, disrupted Ca²⁺ homeostasis, ER stress, and apoptosis, underscoring the importance of GAGs in purinergic signaling and vascular physiology.</p>