Ca(2+)-inhibitable adenylyl cyclase and pulmonary microvascular permeability

Abstract

Intracellular mechanisms responsible for endothelial cell disruption are unknown, although either elevated cytosolic Ca2+ ([Ca2+]i) or decreased adenosine 3',5'-cyclic monophosphate (cAMP) promotes permeability. Recent identification that Ca(2+)-inhibitable adenylyl cyclase establishes an inverse relationship between [Ca2+]i and cAMP in macrovascular endothelial cells provided a possible mechanism of development of permeability. However, these data utilized an in vitro model; lacking was evidence supporting 1) expression of Ca(2+)-inhibitable adenylyl cyclase in pulmonary microvascular endothelium and 2) Ca2+ inhibition of adenylyl cyclase and cAMP content as a paradigm for inflammatory mediator-induced permeability in the intact circulation. We therefore addressed these issues in microvascular endothelial cells derived from rat lung and in an isolated perfused rat lung preparation. Results demonstrate expression of a Ca(2+)-inhibitable adenylyl cyclase in microvascular endothelial cells. Furthermore, data suggest that Ca2+ inhibition of adenylyl cyclase is necessary for development of microvascular permeability in the intact circulation. We conclude Ca2+ inhibition of cAMP represents a critical step in genesis of microvascular permeability in the intact pulmonary circulation.

Microtubule Motors Regulate ISOC Activation Necessary to Increase Endothelial Cell Permeability

“…2, the endoplasmic reticulum was translocated toward the plasma membrane following rolipram treatment, suggesting that endoplasmic reticulum localization may be critical for I SOC activation. To determine whether microtubules regulate endoplasmic reticulum localization within PMVECs, organelle distribution was assayed in control and nocodazoletreated PMVECs using the endoplasmic reticulum-specific probe diOC 6 . As shown in Fig.…”

“…D and E, microtubule organization determines endoplasmic reticulum disposition. Distribution was analyzed using the endoplasmic reticulum-specific probe diOC 6 in untreated PMVECs (D) and in cells that were first treated with nocodazole (E; 10 mol/liter for 60 min) prior to observation. The arrowheads in D show the cell borders, demonstrating that the endoplasmic reticulum has collapsed toward the cell center in cells without intact microtubules.…”

“…During inflammation, neurohumoral inflammatory agonists increase endothelial cell cytosolic calcium ([Ca 2ϩ ] i ), and this rise in [Ca 2ϩ ] i reorganizes the cytoskeleton, decreases cell-cell and cell-matrix adhesion, and increases centripetally directed tension, all of which contribute to intercellular gap formation that allows for an increase in paracellular permeability (1)(2)(3)(4). Neurohumoral inflammatory agonists activate G q proteins and may promote calcium entry through either receptor-operated or store-operated calcium entry channels; however, it is the activation of store-operated calcium (SOC) 3 entry channels that is most important for increasing endothelial cell permeability (5)(6)(7).…”

Mechanisms regulating cAMP-mediated growth of bovine neonatal pulmonary artery smooth muscle cells

“…Expression of PKCstimulated adenylyl cyclase (type II) was tested generally as previously described (4). Briefly, total RNA was extracted from cell monolayers with Qiagen RNeasy (Qiagen, Santa Clarita, CA) and subjected to first-strand synthesis with reverse transcriptase (GIBCO BRL) and oligo(dT) primer after treatment of RNA with DNase I (GIBCO BRL).…”

“…Immunostaining was generally as previously described (4,17). To test for adenylyl cyclase expression, the slides were incubated in 0.3% H 2 O 2 in metha-L1011 SMOOTH MUSCLE CELL GROWTH AND CAMP on December 5, 2006 ajplung.physiology.org nol for 30 min to decrease endogenous peroxidase activity.…”