Norepinephrine (NE) is a biogenic monoamine that plays an integral role in the homeostatic maintenance of blood pressure as a neurotransmitter of the sympathetic nervous system. In certain disease states, such as hypertension, sympathetic output is altered. Therefore understanding all the mechanisms by which NE may regulate function is important. Recently, a novel way that monoamines can signal for physiological effect has been described: the transglutaminase (TG)-mediated, covalent modification of proteins by monoamines. Vascular proteins modified in such a manner by serotonin can elicit biologically relevent effects, such as vascular smooth muscle cell (VSMC) migration, growth, and contraction. In these studies, we hypothesized that NE can also serve as a TG substrate, and that the TG-mediated NE-amidation of proteins signaled for a physiologically relevant, biological effect in the vasculature, such as vascular contraction, or VSMC migration and proliferation. We investigated this hypothesis in rat aorta (RA) and vena cava (RVC) tissues.We found that both RA and RVC tissues express multiple TGs. Moreover, we found that at least two of these TGs, namely TG1 and TG2, were active in the VSMCs of these tissues. These TGs were able to incorporate NE into vascular proteins, as visualized with a NE-biotin conjugate. This incorporation was sensitive to the TG inhibitor cystamine, suggesting that this was a TG-dependent event. Furthermore, NE-biotin could be competed off with cold, unbiotinylated NE, suggesting that NE and NE-biotin were handled by TGs in a similar fashion. TG protein expression appeared to be downregulated in cell culture. As an intracellular source of NE is necessary for NE-amidation of proteins to occur, we investigated a mechanism by which NE may gain entry into VSMCs. We found that RA and RVC VSMCs expressed the organic cation transporter 3 (OCT3)--a monoamine transporter capable of transporting NE--on their cell surface. The canonical transporter of NE, NET, did not appear to be expressed by VSMCs. Furthermore, in whole RA tissues, NE uptake was found to be sensitive to OCT3 inhibition. Although we also measured NE uptake in the RVC, we were not able to block its uptake with any of the inhibitors used. Interestingly, OCT3 expression and NE uptake appeared to be downregulated in VSMC cultures.To understand the role the NE-amidation of proteins plays in vascular function, potential NE-amidation substrates were identified with mass-spectrometry and NE-biotin. Several candidate substrates, such as several small GTPases and cytoskeletal proteins such as α-actin, were identified. Confirmation that α-actin served as a TG substrate was done with immunoprecipitation. We investigated the role that NE-amidation of proteins plays in vascular contraction. The TG inhibitor cystamine inhibited and eventually abolished NE-induced contraction in the RA and the RVC. RVC tissues seemed to be more sensitive to this inhibition. Contraction to the non-receptor mediated contractant, was slightly reduced at concentrations of cystamine that abolished NE-induced contraction. Taken together, these results suggest that the NE-amidation of proteins occurs in the vascular, and may be important in NE-induced contraction.
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