EXOGENOUS NITRIC OXIDE (NO) MODULATES THE G‐PROTEIN COUPLED SIGNALING PROTEINS IN CULTURED LYMPHATIC SMOOTH MUSCLE CELLS

Abstract

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) 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 two NO donors SNAP (S‐Nitroso‐N‐Acetyl‐D,L‐Penicillamine) and DETA NONOate (Diethylenetriamine NONOate) at 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) isoforms α and β, PKG 1α, and PKG 1β were measured by Western blotting using anti‐sGC α, anti‐sGC β, 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 25–200 μM SNAP and DETA NONOate treated RMLMC. Both dose and time dependent effects of SNAP and DETA NONOate on 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