Proteomic Analysis of the Role of the Adenylyl Cyclase-cAMP Pathway in Red Blood Cell Mechanical Responses

Abstract

Red blood cell (RBC) deformability is modulated by the phosphorylation status of the cytoskeletal proteins that regulate the interactions of integral transmembrane complexes. Proteomic studies have revealed that receptor-related signaling molecules and regulatory proteins involved in signaling cascades are present in RBCs. In this study, we investigated the roles of the cAMP signaling mechanism in modulating shear-induced RBC deformability and examined changes in the phosphorylation of the RBC proteome. We implemented the inhibitors of adenylyl cyclase (SQ22536), protein kinase A (H89), and phosphodiesterase (PDE) (pentoxifylline) to whole blood samples, applied 5 Pa shear stress (SS) for 300 s with a capillary tubing system, and evaluated RBC deformability using a LORRCA MaxSis. The inhibition of signaling molecules significantly deteriorated shear-induced RBC deformability (p < 0.05). Capillary SS slightly increased the phosphorylation of RBC cytoskeletal proteins. Tyrosine phosphorylation was significantly elevated by the modulation of the cAMP/PKA pathway (p < 0.05), while serine phosphorylation significantly decreased as a result of the inhibition of PDE (p < 0.05). AC is the core element of this signaling pathway, and PDE works as a negative feedback mechanism that could have potential roles in SS-induced RBC deformability. The cAMP/PKA pathway could regulate RBC deformability during capillary transit by triggering significant alterations in the phosphorylation state of RBCs.

Keywords: capillary transit; cytoskeletal proteins; phosphorylation; red blood cell deformability; shear stress.

Figure 1

EI–SS curves before and after 5 Pa SS exposure. The graphs demonstrate deformability changes by SQ22536 (A,B), H89 (C,D), pentoxifylline (E,F), and gadolinium (G,H) compared to the control. Panels (A,C,E,G) show deformability curves before SS exposure and panels (B,D,F,H) show deformability curves after SS exposure. Data are represented as means ± SD, n = 12, * p < 0.05.

Figure 2

Ratio of the elongation index (EI) values measured after and before the application of 5 Pa shear stress. * p < 0.05 control vs. SQ, θ p < 0.05 control vs. pentoxifylline, # p < 0.05 control vs. H89, and γ p < 0.05 control vs. gadolinium. Data under 0.95 Pa are not plotted. Data are represented as means ± SE, n = 12.

Figure 3

The categorization of proteins according to their molecular function (A) and protein class (B). The majority of the proteins are protein-modifying enzymes and cytoskeletal proteins (B) and have functions of binding and catalytic activity (A).

Figure 4

The changes in the phosphorylation status of RBCs due to different treatments. Tyrosine and serine phosphorylation are shown in green (488 nm) in panels (A,B), respectively.

Figure 5

The changes in the phosphorylation of RBC membrane proteins due to different treatments. Phosphorylation at tyrosine and serine residues of cytoskeletal proteins are depicted in panels (A,B) and (C,D), respectively. Panels (A,C) are representative images. Data are represented as means ± SD, n = 10, * p < 0.05.

Figure 6

A schematic illustration for the phosphorylation events through cAMP signaling mechanisms. Shear stress in blood flow could be sensed via Piezo1 and GPCRs that trigger cAMP signaling or crosstalk between signaling pathways. Closed lines show an inhibition and black arrows represent a phosphorylation event. GPCR: G protein-coupled receptors, PDE: Phosphodiesterase, PKC: Protein kinase C.