title: Cyclic guanosine monophosphate and the dependent protein kinase regulate lymphatic contractility in rat thoracic duct.
abstract: We have previously demonstrated a principal role for nitric oxide (NO) in the endothelium/shear-dependent regulation of contractility in rat thoracic duct (TD). In this study we tested the hypothesis that cyclic guanosine monophosphate (cGMP) and the dependent protein kinase (PKG) are central to the intrinsic and extrinsic flow-dependent modulation of lymphatic contractility. Lymphatic diameters and indices of pumping in isolated, cannulated and pressurized segments of rat TD were measured. The influences of increased transmural pressure (1-5 cmH2O) and imposed flow (1-5 cm H2O transaxial pressure gradients) on lymphatic function were studied before and after: (1) inhibition of guanylate cyclase (GC) with and without a NO donor; (2) application of stable cGMP analogue; and (3) inhibition of the cGMP activation of PKG. Additionally, Western blotting and immunofluorescent tissue staining were used to analyse the PKG isoforms expressed in TD. We found that the GC inhibitor ODQ induced changes in TD contractility similar to NO synthase blockade and prevented the relaxation induced by the NO donor S-nitroso-N-acetylpenicillamine. The cGMP analogue, 8-(4-Chlorophenylthio)-guanosine 3,5-cyclic monophosphate sodium salt (8pCPTcGMP), mimicked the extrinsic flow-induced relaxation in a dose-dependent manner, whereas treatment with the cGMP/PKG inhibitor, guanosine 3,5-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-, Rp-isomer, triethylammonium salt (Rp-8-Br-PETcGMPS), eliminated intrinsic flow-dependent relaxation, and largely inhibited extrinsic flow-dependent relaxation. Western blotting demonstrated that both PKG-Iα and -Iβ isoforms are found in TD, with ∼10 times greater expression of the PKG-Iα protein in TD compared with the aorta and vena cava. The PKG-Iβ isoform expressed equally in TD and vena cava, both being ∼2 times higher than that in the aorta. Immunofluorescent labelling of PKG-Iα protein in the wall of rat thoracic duct confirmed its localization inside TD muscle cells. These findings demonstrate that cGMP is critical to the flow-dependent regulation of TD contractility; they also indicate an important involvement of PKG, especially PKG-Iα in these processes and identifies PKG protein as a potential therapeutic target.
authors.affiliation: O. Y. Gasheva: Department of Medical Physiology, College of Medicine, Cardiovascular Research Institute Division of Lymphatic Biology, Texas A&M Health Science Center, 702 SW H.K. Dodgen Loop, Temple, TX 76504, USA. ogasheva@tamu.edu.
authors.forename: Olga Yu
authors.initials: OY
authors.lastname: Gasheva
authors.forename: Anatoliy A
authors.initials: AA
authors.lastname: Gashev
authors.forename: David C
authors.initials: DC
authors.lastname: Zawieja
mesh_terms: D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D042601:Lymphatic Vessels / Q000378:metabolism* / Q000502:physiology; D008297:Male; D009119:Muscle Contraction; D009569:Nitric Oxide / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013897:Thoracic Duct / Q000378:metabolism / Q000502:physiology*
chemical_list: C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D007527:Isoenzymes; D010069:Oxadiazoles; D047428:Protein Kinase Inhibitors; D011810:Quinoxalines; D009569:Nitric Oxide; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D006152:Cyclic GMP