The Change And Regulation Of Intracellular Trafficking And Signaling Of Beta-adrenergic Receptors In Rat Pulmonary Microvascular Endothelial Cells

Drowning sea lung injury includes the decrease of lung function and the damage of lung tissue structure. The leading cause of death was pulmonary edema after seawater drowning. Pulmonary endothelium serves as a semi-selective barrier between the plasma and interstitial to circulatory cells, micromolecules, and bioactive agents. Maintenance of this semi-selective barrier represents an important physiological process for vessel wall homeostasis and lung function. Sea water drowning will injury the endothelium and results in the increase of permeability of endothelial cells that contributes to edema in lung injury. Thus reducing the hyperpermeability of microvascular endothelial cells is an important part of treating lung injury.Beta-adrenergic receptors (β-ARs) play a critical role in the regulation of the permeability and function of vascular endothelial cells in both normal and pathological conditions. Allβ-ARs subtypes, namelyβ₁-,β₂-, andβ₃-AR, have been described as having a role in the vasculature. A number of studies have demonstrated that the increased endothelial permeability that occurs after the administration of inflammatory mediators could be mediated by activation ofβ₂-AR. However, the activation ofβ₂-AR cannot completely reverse the endothelial hyper-permeability induced by inflammatory mediators. Few specific therapies are available to attenuate the endothelial hyper-permeability, and current therapies are often unsuccessful. Nevertheless, new insights into the mechanisms underlying the cell-surface expression ofβ-ARs offer potential novel targets and strategies for pharmacological intervention.The intracellular trafficking ofβ-ARs is a critical event in regulating its function. After it is synthesized, folded, and assembled in the endoplasmic reticulum (ER), theβ-ARs is transported to the Golgi apparatus, where it is post-translationally modified. It is then transported to the plasma membrane. Onceβ-ARs arrive at the plasma membrane, they may undergo internalization to the endosome upon stimulation by their ligands. The internalized receptors may then be transported to the lysosome for degradation or recycled back to the plasma membrane. Thus, the amount ofβ-ARs at the plasma membrane is determined by the overall balance ofβ-ARs export to the cell surface, internalization, recycling, and degradation. Because most studies onβ-ARs trafficking have focused on the events involved in its internalization, recycling, and degradation, the export ofβ-ARs from the ER through the Golgi to the cell surface and regulation of receptor function by these processes remain poorly understood.Intracellular protein trafficking between organelles is coordinated by Rab proteins, which are members of the Ras superfamily of monomeric GTPases. Recently, more than 60 Rab GTPases have been identified in the human genome. Nearly all of these enzymes are involved in vesicle formation, motility, dococking, or fusion, events critical to endococytosis and exocytosis in endothelial cells. Endothelial cells express Rab 1–9, 11, 13, 14, 15, 18, 22, and 30. Rab1 is one of the most extensively studied Rab GTPases involved in the regulation of vesicular protein transport between intracellular organelles. Rab1 is localized in the ER and Golgi, and regulates antegrade protein transport specifically from the ER to the Golgi and among the Golgi compartments. It is known that Rab1 regulates the transport of G protein–coupled receptors (GPCRs) from the ER to the cell surface in HEK293T cells and cardiocytes. These findings suggest that an alteration in Rab1 function might play an essential role in regulation of the cell-surface expression ofβ-ARs and subsequently change endothelial permeability.In the present study, we determined whether modified ER-to-Golgi transport could alter the cell-surface expression and function of endogenousβ-ARs in the rat pulmonary microvascular endothelial cells (RPMVECs). The role of Rab1-mediated reduction in endothelial permeability throughβ-ARs was also investigated.Methods:1. RPMVECs were cultured by peripheral lung tissue-sticking method. Histological sections from peripheral lung tissue pieces used for cell culture were examined, CD34, lectin from Bandeiraea simplicifolia and factorâ…§related antigen in the cultured cells were quantified by immunocytochemical staining. In addition, the cell morphology and ultrastructure were observed with inverted optical microscope and transmission electron microscope respectively. 2. Cell-surface expression ofβ-ARs in RPMVECs was measured by intact cell ligand binding after RPMVECs were treated with sea water and infected with wild-type Rab1, dominant-negative mutant Rab1N124I driven by lentivirus.3. The cell-surface expression and subcellular distribution ofβ-ARs in RPMVECs were revealed by fluorescence microscopy after cells were infected with Rab1WT, dominant-negative mutant Rab1N124I and Rab1siRNA driven by lentivirus.4. The activation of ERK1/2 induced by the agonists ofβ-AR was determined by Western blot analysis using phospho-specific ERK1/2 (ERK1/2-P) antibodies after cells were infected with Rab1WT and dominant-negative mutant Rab1N124I driven by lentivirus.5. RPMVECs mono-layers were seeded on cell co-cultured dishes. The permeability of RPMVEC mono-layers was measured by diffusion of biotinylated bovine serum albumin (biotin-BSA) and biotin-BSA concentrations were determined by enzyme-linked immunosorbent assay after RPMVECs were treated with sea water and infected with wild-type Rab1 driven by lentivirus.Results:1. Histological sections showed that tissue pieces were scissored from periphery lung lobes accurately.â…§factor related antigen and CD34 expression detected by immunofluorescence was positive, and BSI binding test was positive, too. Transmission electron microscope showed lots of protuberance on cell membrane and pinosomes in cytoplasm.2. The cell-surface expression of totalβ-ARs andβ₂-AR was significantly augmented by 38.3% and 49.0%, respectively, in RPMVECs infected with Rab1WT lentivirus as compared with that from RPMVECs infected with control lentivirus. Cell-surface expression ofβ₁-AR was not significantly altered by Rab1. In contrast,β₂-AR expression at the cell surface was markedly attenuated by 49.2% in RPMVECs infected with the dominant-negative mutant Rab1N124I lentivirus. It is noteworthy that the cell-surface expression ofβ-ARs after treated with sea water in RPMVECs infected with Rab1WT lentivirus was significantly augmented compared with cells infected with control lentivirus.3.β₂-AR-GFP was mainly localized at the cell surface in cells infected with control lentivirus. In contrast,β₂-AR-GFP accumulated in the perinuclear regions and colocalized with the ER marker-pDsRed2 and could not be transported to the cell surface in cells infected with Rab1N124I and Rab1 siRNA.4. Lentivirus-mediated Rab1WT expression significantly enhanced ERK1/2 activation in response to stimulation with ISO in RPMVECs compared with cells infected with control lentiviruses. In contrast, expression of dominant-negative Rab1N124I markedly reduced ISO-induced ERK1/2 activation compared with cells infected with control lentiviruses. ISO-mediated ERK1/2 activation in the absence or presence of the antagonists was similarly inhibited in cells infected with Rab1N124I compared with cells infected with control lentiviruses. In contrast, ISO-mediated ERK1/2 activation in the absence or presence of the antagonists was selectively augmented in cells infected with Rab1WT compared with cells infected with control lentiviruses. It was found that lentivirus-mediated Rab1WT expression mainly enhancedβ₂-AR-mediated ERK1/2 activation.5. Although the expression of Rab1WT did not directly influence the permeability of RPMVECs, the monolayer permeability of RPMVECs infected with Rab1WT lentivirus and stimulated with ISO was significantly abated compared with the monolayer permeability of RPMVECs infected with control lentivirus. Importantly, consistent with Rab1 function in modulation ofβ-ARs signaling, lentivirus-mediated Rab1WT expression attenuated permeability mainly through activation ofβ₂-AR.Conclusion:1. These data indicate that manipulation of Rab1 function modifies the cell-surface expression and the subcellular distribution of endogenousβ₂-ARs in RPMVECs.2. These data indicate that Rab1 modulates not onlyβ₂-ARs trafficking but also their signal transduction3. These data indicate that manipulation of Rab1 function modifies the cell-surface expression of endogenousβ₂-ARs in RPMVECs and subsequently regulates of the monolayer permeability of RPMVECs.