Nitric oxide (NO) dependent cellular signaling in cultured lymphatic smooth muscle cells

Lymphatics use both phasic and tonic contractions of lymphatic muscle. The cellular and molecular mechanisms by which lymphatic muscle performs these dual tasks are still unknown. Recently, we demonstrated that cGMP and PKG (protein kinase G) are involved in the NO modulation of rat thoracic duct contractility. We aim to study the NO dependent signaling mechanisms in cultured lymphatic muscle cells. Rat mesenteric lymphatic muscle cells (RMLMC) and rat thoracic duct lymphatic muscle cells (RTDLMC) were used in this study. Cells were maintained in DMEM media supplemented with 10% fetal bovine serum and were incubated at 37°C and 5% CO2. Cells were treated with a NO donor SNAP (S‐Nitroso‐N‐Acetyl‐D,L‐Penicillamine, @ 10, 25, 50, 100 or 200 μM) for 4 and 8 hours in different wells (n=6 in each treatment) separately. The sGC (soluble guanylate cyclase sGC) isoforms α, and βand PKG 1α and PKG 1β were measured by Western blotting using anti‐sGC α or anti‐sGC βor anti‐PKG 1α or anti‐PKG 1β antibodies. The expression of sGC α, sGC β, PKG 1α and PKG 1β proteins were significantly upregulated in 200 μM SNAP‐treated RMLMC and RTDLMC. Both dose and time dependent effects of SNAP on the expression of these proteins were observed. These findings demonstrate the usefulness of using cultured lymphatic muscle cell lines in the study of the involvement of sGC and PKG on NO dependent signaling. Supported by NIH HL70308