gms | German Medical Science

Background: Catecholamines are well known neurotransmitters of the sympathetic nervous system regulating numerous physiological or pathological processes in the body. Tyrosine-hydroxylase (TH) catalyzes the first step of catecholamine biosynthesis. Common TH activity measurements are time-consuming (e.g. HPLC), require extensive enzyme work-up, or demand special handling (e.g. tritium release method). Therefore, we aimed to establish a simple but specific TH activity assay suitable for primary cell culture modified according to Vermeer et al . [1]

Methods: Human and murine induced TH cells (iTH) were generated and human synovial cells of osteoarthritis (OA) and rheumatoid arthritis patients (RA) were cultivated as described previously [2]. Cell lysates were stabilized using protease inhibitor cocktail. Vermeers protocol [1] was established with recombinant murine and human TH and modified by using alpha-methyl-para-tyrosine (ampt), a specific an highly potent TH inhibitor [2], as negative control. Furthermore, whole cell lysates (100 µl) were applied to the reaction mix (100 µl) containing L-tyrosine, sodium periodate, tetrahydrobiopterin, and Fe²⁺. Kinetic measurement was performed in a plate reader (490 nm) for 20 min. Specific TH activity in cell lysates was calculated by subtracting the area under curve of lysate plus ampt from area under curve of whole lysate without ampt, in order to exclude effects caused by tyrosinase-specific tyrosine conversion. The thus calculated TH activity values were controlled by HPLC.

Results: Specific TH activity measurement could be established using recombinant TH proteins and ampt. Generated human and murine iTH showed high TH activity, which was confirmed by HPLC. These cells can serve as positive control for future experiments. Specific TH activity of both OA and RA mixed synovial cells was higher under hypoxia compared to normoxia confirming previous immunohistological and HPLC results.

Conclusion: This study shows that specific TH activity can be determined in murine and human primary cell cultures in a simple plate reader assay using whole cell lysates and a specific TH inhibitor. This enables the high-throughput analysis of primary material.