Protective effect of Growth Hormone-Releasing Hormone agonist in bacterial toxin-induced pulmonary barrier dysfunction

Rationale: Antibiotic treatment of patients infected with G(-) or G(+) bacteria promotes release of the toxins lipopolysaccharide (LPS) and pneumolysin (PLY) in their lungs. Growth Hormone-releasing Hormone (GHRH) agonist JI-34 protects human lung microvascular endothelial cells (HL-MVEC), expressing splice variant 1 (SV-1) of the receptor, from PLY-induced barrier dysfunction. We investigated whether JI-34 also blunts LPS-induced hyperpermeability. Since GHRH receptor (GHRH-R) signaling can potentially stimulate both cAMP-dependent barrier-protective pathways as well as barrier-disruptive protein kinase C pathways, we studied their interaction in G H RH agonist-treated HLMVEC, in the presence of PLY by means of siRNA-mediated protein kinase A (PKA) depletion. Methods: Barrier function measurements were done in HLMVEC monolayers using Electrical Cell substrate Impedance Sensing (ECIS) and VE-cadherin expression by Western blotting. Capillary leak was assessed by Evans Blue dye (EBD) incorporation. Cytokine generation in broncho-alveolar lavage fluid (BALE) was measured by multiplex analysis. PKA and PKC-alpha activity were assessed by Western blotting. Results: GHRH agonist JI-34 significantly blunts LPS-induced barrier dysfunction, at least in part by preserving VE-cadherin expression, while not affecting inflammation. In addition to activating PKA, GHRH agonist also increases PKC-alpha activity in PLY-treated HLMVEC. Treatment with PLY significantly decreases resistance in control siRNA-treated HLMVEC, but does so even more in PKA-depleted monolayers. Pretreatment with GHRH agonist blunts PLY-induced permeability in control siRNA-treated HLMVEC, but fails to improve barrier function in PKA-depleted PLY-treated monolayers. Conclusions: GHRH signaling in HLMVEC protects from both LPS and PLY-mediated endothelial barrier dysfunction and concurrently induces a barrier-protective PKA-mediated and a barrier-disruptive PKC-alpha-induced pathway in the presence of PLY, the former of which dominates the latter.