Researcher Beware! Decreased TG2 and OCT3 Expression in Vascular Smooth Muscle Cells Upon Culture

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Recently, a protein modification has been described, norepinephrine (NE)‐amidation, in which NE is covalently attached to the γ‐glutamyl moiety of a protein‐bound glutamine by members of the transglutaminase (TG) family. While TG activity is necessary for vascular contraction to NE, the full physiological relevance of NE‐amidation in the vasculature is not known. We hypothesized that vascular smooth muscle cells (VSMCs) maintain a phenotype conducive to characterizing the physiological relevance that the NE‐amidation of proteins serves in NE‐elicited growth and migration. The intensity of immunocytochemical staining for the vascular TGs (TG1, TG2, and TG4) was reduced in cultured compared to freshly dissociated rat aorta and vena cava VSMCs. The organic cation transporter 3 (OCT3), a non‐neuronal NE transporter, localized to the cell surface of freshly dissociated VSMCs, while staining was sparse in cultured VMSCs. While freshly dissociated VSMCs took up a NE‐biotin conjugate, cultured VSMCs lost this ability. Western analysis detected less protein for TG2 and OCT3 in cultured VSMCs versus whole tissue (protein in aortic VSMCs: TG2 = 2.5 ± 0.7, OCT3 = 1.3 ± 0.3 % of protein in whole aorta, p≤0.05). These results demonstrate that VSMCs undergo a phenotypic transformation in culture that is not conducive to the characterization of the physiological responses elicited by NE‐amdiation of proteins.