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Journal of Trauma-Injury Infection & Critical Care:
Original Articles

The Roles of Cyclic Adenosine Monophosphate- and Cyclic Guanosine Monophosphate-Dependent Protein Kinase Pathways in Hydrogen Peroxide-Induced Contractility of Microvascular Lung Pericytes

Williams, Mallory MD; Kerkar, Sid BA; Tyburski, James G. MD; Steffes, Christopher P. MD; Carlin, Arthur M. MD; Wilson, Robert F. MD

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Abstract

Background : Sepsis and posttraumatic inflammatory processes are accompanied by definite changes in microvascular permeability, particularly in the lung. These permeability changes may occur because of damaged regulatory mechanisms at the level of the capillary wall. Pericytes are adventitial cells located within the basement membrane of capillaries. These cells contain multiple cytoplasmic processes that envelope endothelial cells, and are consequently thought to stabilize capillary walls and participate in microcirculation and endothelial cell permeability. Data from this laboratory and other laboratories have confirmed that pericytes are contractile cells, adding to the evidence that pericytes may influence or help regulate capillary permeability. We have already determined that hydrogen peroxide (H2O2) causes dose-dependent relaxation in microvascular lung pericytes (MLPs) at 10 minutes and, conversely, dose-dependent contraction at 30 minutes. It is the aim of this study to determine the mechanism of this biphasic contractile response. Specifically, we will determine whether cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-dependent protein kinase intracellular pathways are responsible for the hydrogen peroxide-induced contractility of MLPs.

Methods : Rat MLPs were isolated by previously published protocol and cultured on collagen gel matrices. MLPs were pretreated with either ODQ, a soluble guanylate cyclase inhibitor (100 μmol/L), for 15 minutes; GKIP, a protein kinase G inhibitor (100 μmol/L), for 1 hour; SQ22536, an adenylate cyclase inhibitor (100 μmol/L), for 15 minutes; or H89, a protein kinase A inhibitor (10 μmol/L), for 1 hour. Hydrogen peroxide was then introduced to each MLP culture at 10 μmol/L, 100 μmol/L, and 1 mmol/L. After each of these treatments, the surface area of the collagen gels was digitally quantified at 10 and 30 minutes.

Results : SQ22536 attenuated both relaxation at 10 minutes and the contraction seen at 30 minutes for all concentrations of H2O2. H89 caused a marked basal relaxation and prevented the cells from contracting at 30-minute exposures to all concentrations of H2O2. Both ODQ and GKIP attenuated the relaxation at 10 minutes but had no affect on the later contraction.

Conclusion : The cGMP-dependent protein kinase pathway is a mechanism for H2O2-induced relaxation of MLPs. Up-regulation of cAMP and cGMP is responsible for early H2O2-induced relaxation and late contraction. Protein kinase A (cAMP-dependent protein kinase pathway) may be an important intracellular signaling protein in the H2O2-induced contraction of MLPs or may be unable to down-regulate cAMP once inhibited. This evidence further supports the concept that there are separate intracellular pathways that regulate divergent cellular responses. This idea parallels the clinical concept of reversible and irreversible dysfunction of cellular processes in shock, and that the cellular dysfunction is initiated by separate intracellular pathways.

Sepsis and posttraumatic inflammatory processes are accompanied by definite changes in microvascular permeability, particularly in the lung. 1–5 Multiple pathways exist in the systemic inflammatory response syndrome (SIRS), with mediators at selected times provoking divergent responses. 1 An ideal example of this is the lung microvasculature. Mediators of sepsis and SIRS cause vasodilation and constriction responses in the microvasculature leading to edema and inflammation. 1 These permeability changes may occur because of damaged regulatory mechanisms at the cellular level of the capillary wall. 3 We therefore are focused on microvascular lung pericytes (MLPs), contractile adventitial cells located in the basement membrane of capillaries, and their interaction with endothelial cells. MLPs contain multiple cytoplasmic processes that envelope endothelial cells, and are consequently thought to stabilize capillary walls and to participate in microcirculation and endothelial cell permeability. 2–4 The role of MLPs as a contractile cell has been established, 2,3,6–11 providing the initial evidence for their participation in regulating microvascular permeability.

The main sequelae of SIRS, septic shock, and acute respiratory distress syndrome are increased microvascular permeability leading to pulmonary edema. 1–4 More commonly seen are general anesthesia and surgery, which are also associated with increased lung water. The microvascular barrier through which fluid shifts occur is the capillary basement membrane composed of the endothelial cell surrounded by the pericyte on the abluminal surface. Pericyte-endothelial cell interaction is possible through tight junctions, peg-and-socket connections, adhesion plaques, and gap junctions. 6 These specific structures provide the capability for communication and coordination. This is further evidence that pericytes may influence or help regulate pericyte permeability.

Hydrogen peroxide (H2O2) is a known product of inflammatory cells, often in millimolar concentrations in the microenvironment of the cell. 12 H2O2 exposure stimulates protein kinase C, phospholipase A2 activity, arachidonic acid release, and increases in intracellular cyclic adenosine monophosphate (cAMP) levels (without affecting cyclic guanosine monophosphate [cGMP] levels). Rahman et al. have shown that H2O2 alters histone acetylation/deacetylation, and activates NF-κB and AP-1, leading to the release of the proinflammatory cytokine interleukin-8 in human alveolar epithelial cells. 13

Our laboratory has shown that pericytes undergo dose-dependent H2O2-induced biphasic relaxation at 10 minutes and then contraction at 30 minutes (Fig. 1). 5 This H2O2-induced biphasic response was completely attenuated with catalase. 5 It is now our goal to define the mechanism of H2O2-induced pericyte relaxation and contraction. This may also in part explain increases in pulmonary microvascular permeability during sepsis. We started by examining whether the cAMP-dependent protein kinase or cGMP-dependent protein kinase pathway is responsible for the H2O2-induced biphasic contractile responses seen in microvascular lung pericytes.

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© 2003 Lippincott Williams & Wilkins, Inc.

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