Cyclic guanosine monophosphate and the dependent protein kinase regulate lymphatic contractility in rat thoracic duct

Abstract

Key points• Nitric oxide plays a principal role in the lymphatic endothelium/shear-dependent regulation of contractility in rat thoracic duct.• 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.• Both PKG-Iα and -Iβ isoforms are found in the thoracic duct, with ∼10 times greater expression of the PKG-Iα protein compared with the aorta and vena cava.• Functional data demonstrate that cGMP is critical to the flow-dependent regulation of thoracic duct contractility.• These findings indicate an important role for PKG, especially PKG-Iα in these processes and identifies the PKG protein as a potential therapeutic target. AbstractWe 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 cmH 2 O) and imposed flow (1-5 cm H 2 O 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 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.

Mechanical forces and lymphatic transport

“…Several studies have identified that the mechanism for flow-mediated modulation of the lymphatic contractile cycle requires endothelial production of NO (Gashev et al, 2002; Mizuno et al, 1998; Shirasawa et al, 2000; von der Weid et al, 1996; von der Weid et al, 2001). Presumably the action of NO is activation of soluble guanylate cyclase, accelerated production of cGMP, and activation protein kinase G (Gasheva et al, 2013; Kurtz et al, 2014a). Interestingly, in aged rats (22 months old), shear stress-induced relaxation of lymphatic smooth muscle is only partially inhibited by blockade of NO synthase, unlike the 9-month old controls (Nagai et al, 2011).…”

Reduction of contractility and reactivity in isolated lymphatics from hemorrhagic shock rats with resuscitation

“…Because lymphatic pumping plays a vital role in the regulation of interstitial fluid volume 12 , therefore, the treatment of enhancing lymphatic pump function may be beneficial to hemorrhagic shock.…”

The Position- and Lymphatic Lumen-Controlled Tissue Chambers to Study Live Lymphatic Vessels and Surrounding Tissues ex Vivo

“…We used a welldescribed experimental technique for measuring function in isolated lymphatic vessels. 4,11,[25][26][27] One deviation from the previously described protocol was the use of DMEM/F-12 in place of albumin containing physiological salt solution.…”

“…28 Figure 1, panel E, demonstrates the screen capture of the diameter tracking computer program with an example of a MLV inside mesenteric tissue and the diameter tracking window in analog mode. We used cardiac pump analogies to define systole and diastole in reference to the lymphatic contractile cycle, 8,27,29 and the enddiastolic and end-systolic points in the diameter tracings were recorded for each 5-min interval for the transmural pressure of 1, 3, and 5 cm H 2 O and for imposed flow gradients of 0, 1, and 5 cm H 2 O (at transmural pressure of 5 cm H 2 O). This allowed us to determine transmural pressure-and wall shear stresssensitivity of MLVS within tissue explants.…”

“…For these experiments, we used previously described techniques to measure contractile and pumping activity of isolated lymphatic vessels. [25][26][27]30 We performed isolated MLV experiments using DMEM/F-12 and recorded their activity under the same conditions as for MLVs situated within tissue explants. This allowed us to directly compare function of MLVs in tissue chambers and isolated MLVs.…”