Involvement of histamine in endothelium-dependent relaxation of mesenteric lymphatic vessels

Abstract

Objectives: The knowledge of the basic principles of lymphatic function, still remains, to a large degree, rudimentary and will require significant research efforts. Recent studies of the physiology of the MLVs suggested the presence of an EDRF other than NO. In this study, we tested the hypothesis that lymphatic endothelium-derived histamine relaxes MLVs.

Methods: We measured and analyzed parameters of lymphatic contractility in isolated and pressurized rat MLVs under control conditions and after pharmacological blockade of NO by L-NAME (100 μM) or/and histamine production by α-MHD (10 μM). Effectiveness of α-MHD was confirmed immunohistochemically. We also used immunohistochemical labeling and Western blot analysis of the histamine-producing enzyme, HDC. In addition, we blocked HDC protein expression in MLVs by transient transfection with vivo-morpholino oligos.

Results: We found that only combined pharmacological blockade of NO and histamine production completely eliminates flow-dependent relaxation of lymphatic vessels, thus confirming a role for histamine as an EDRF in MLVs. We also confirmed the presence of HDC and histamine inside lymphatic endothelial cells.

Conclusions: This study supports a role for histamine as an EDRF in MLVs.

Keywords: EDRF; histamine; lymphatic endothelium; mesenteric lymphatic vessels.

Figure 1

Effect of combined pharmacological blockade of nitric oxide and histamine production on flow-dependent relaxation of mesenteric lymphatic vessels. A – lymphatic tone index; B – contraction amplitude; C- contraction frequency; D – fractional pump flow. Control – control MLVs after overnight culture; L-NAME – same control vessels after subsequent acute pharmacological blockade of NO production by the nitric oxide synthase non-specific blocker, L-NAME (100 μM); a-MHD – MLVs after overnight pharmacological blockade of histamine production by HDC specific blocker, α-MHD in 10 μM; a-MHD + L-NAME – same α-MHD-treated vessels after subsequent acute pharmacological blockade of NO production by nitric oxide synthases non-specific blocker, L-NAME in 100 μM. * indicates significant differences (P < 0.05) between lymphatic contractile parameters within each treatment group at no-flow and at any imposed flow condition. + indicates significant differences (P < 0.05) between lymphatic contractile parameters in any treatment groups and control within any given imposed flow condition.

Figure 2

Representative epifluorescence (Panels A–I) and confocal fluorescence (Panels J–O) images of rat isolated mesenteric lymphatic vessels. CD31 (PECAM-1) is stained in green to indicate lymphatic endothelial cell junctions and therefore to delineate location of lymphatic endothelial cells and lymphatic valves (lv). A–C: Histamine (stained in red) in control MLVs cultured overnight. D–F: Histamine (stained in red) in α-MHD-treated MLVs cultured overnight. G–I: Axial view of MLVs stained for histidine decarboxylase (stained in red). J–L: Cross sectional view of MLVs stained for histidine decarboxylase (stained in red). M–O: Cross sectional view of MLVs stained for histidine decarboxylase (stained in red). M - presence of HDC signal inside lymphatic valves in control MLVs; N - scrambled morpholino constructs were not able to change HDC signal; O - specific HDC-targeted morpholino constructs were able to deplete HDC signal. Merged images (Panels C, F, I and L) demonstrate localization of histamine or histidine decarboxylase inside the lymphatic endothelial cells. Insert (between Panels H and I) demonstrates representative western blot analysis of histidine decarboxylase in MLVs. Scale bar on Panel A represents 100 μm and applies to images on Panels A–L, scale bar on Panel M represents 50 μm and applies to images on Panels M–O.