Cyclic Adenosine Monophosphate Protects Endothelial Barrier Function Independent of Inhibiting 20-kd Myosin Light Chain-Dependent Tension Development

During episodes of inflammation, polymorphonuclear leukocyte (PMN) transendothelial migration has the potential to disturb vascular barrier function and give rise to intravascular fluid extravasation and edema. However, little is known regarding innate mechanisms that dampen fluid loss during PMN-endothelial interactions. Using an in vitro endothelial paracellular permeability model, we observed a PMN-mediated decrease in endothelial paracellular permeability. A similar decrease was elicited by cell-free supernatants from activated PMN (FMLP 10−6 M), suggesting the presence of a PMN-derived soluble mediator(s). Biophysical and biochemical analysis of PMN supernatants revealed a role for PMN-derived 5′-adenosine monophosphate (AMP) and its metabolite, adenosine, in modulation of endothelial paracellular permeability. Supernatants from activated PMN contained micromolar concentrations of bioactive 5′-AMP and adenosine. Furthermore, exposure of endothelial monolayers to authentic 5′-AMP and adenosine increased endothelial barrier function more than twofold in both human umbilical vein endothelial cells and human microvascular endothelial cells. 5′-AMP bioactivity required endothelial CD73-mediated conversion of 5′-AMP to adenosine via its 5′-ectonucleotidase activity. Decreased endothelial paracellular permeability occurred through adenosine A2B receptor activation and was accompanied by a parallel increase in intracellular cAMP. We conclude that activated PMN release soluble mediators, such as 5′-AMP and adenosine, that promote endothelial barrier function. During inflammation, this pathway may limit potentially deleterious increases in endothelial paracellular permeability and could serve as a basic mechanism of endothelial resealing during PMN transendothelial migration.

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Cyclic Adenosine Monophosphate, Inositol 1,4,5-trisphosphate, Calcium, and Phosphorylated Myosin Light Chain Regulation Through M2 and M3 Muscarinic Receptors of Scleral Fibroblast Cells in Rat Myopia Model

Open Access Macedonian Journal of Medical Sciences ◽

10.3889/oamjms.2021.6752 ◽

2021 ◽

Vol 9 (B) ◽

pp. 890-899

Author(s):

Nanda Wahyu Anandita ◽

Nurdiana Nurdiana ◽

Endang Sri Wahyuni ◽

Hidayat Sujuti

Keyword(s):

Animal Models ◽

Muscarinic Receptor ◽

Light Chain ◽

Rattus Norvegicus ◽

Myosin Light Chain ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Fibroblast Cells ◽

Inositol 1,4,5 Trisphosphate ◽

M3 Muscarinic Receptor

AIM: This study aims to investigate the concentration of cyclic adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate (IP3), calcium (Ca2+), and the expression phosphorylated myosin light chain (MLC) in Rattus norvegicus scleral fibroblast cells. METHODOLOGY: This study utilized an in vitro experimental study by applying Rattus norvegicus scleral fibroblast cell culture. The cultured cells were divided into control and lens-induced myopia (LIM) groups. The control and LIM culture groups were each divided into five groups, namely, negative control, 0.1 μM acetylcholine, 0.1 μM himbacine, 0.1 μM methoctramine, and 0.1 μM 4-DAMP group. The cAMP, IP3, and Ca2+ concentration were analyzed in the 0th, 5th, 10th, 20th, and 30th. The phosphorylated MLC expression was analyzed using confocal microscope. RESULTS: In the LIM group, the highest cAMP concentration is visible at the 10th min on the himbacine group (0.304 ± 0; p = 0.043) and on the 4-DAMP group (0.346 ± 0; p = 0.043). The highest IP3 concentration is found on the LIM group at the 20th min in comparison to the control group (2503.6 ± 11 vs. 2039.2 ± 2.1; p = 0.046). The highest Ca2+ concentration belongs to the 4-DAMP treatment group from the 5th to the 30th min. The highest average phosphorylated MLC expression value in the LIM group is shown by the 0.1μM 4-DAMP treatment (184.2 ± 37.9c au). CONCLUSION: The regulation of cAMP, IP3, Ca2+, and phosphorylated MLC on the M2 and M3 muscarinic receptor of the scleral fibroblast cells of myopia animal models differs from normal animal models which may be due to interactions of M2 and M3 muscarinic receptor as compensation reaction or crosstalk on myopia induction.

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