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Regulation of Pulmonary Microvascular Endothelial Cell Cyclic Adenosine Monophosphate by Adenylyl Cyclase*: Implications for Endothelial Barrier Function FREE TO VIEW

Troy Stevens, PhD; W.J. Thompson
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*From the Department of Pharmacology, University of South Alabama College of Medicine, Mobile, AL.

Correspondence to: Troy Stevens, PhD, Department of Pharmacology, University of South Alabama College of Medicine, MSB 3130, Mobile, AL 36688-0002

Chest. 1999;116(suppl_1):32S-33S. doi:10.1378/chest.116.suppl_1.32S-a
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Pulmonary microvascular endothelial cells (PMVECs) form a more restrictive barrier to solute and macromolecular transfer than do pulmonary artery endothelial cells (PAECs), although mechanisms responsible for enhanced barrier properties in PMVECs are unknown. Recent studies in PAECs indicate inflammatory mediators that stimulate store-operated Ca2+ entry decrease cyclic adenosine monophosphate (cAMP) content due to Ca2+ inhibition of adenylyl cyclase activity, an effect that promotes barrier disruption. PMVECs similarly express a Ca2+-inhibited adenylyl cyclase but basal and agonist-evoked Ca2+ entry is reduced in these cells compared with PAECs. Present studies therefore examined the role of store-operated Ca2+ entry in regulation of PMVEC adenylyl cyclase activity and cAMP content.

Cytosolic Ca2+, cAMP turnover, and total cAMP were measured in intact rat PMVECs and PAECs in response to activation of store-operated Ca2+ entry using thapsigargin (1 nmol to 10 μM). Both cell types responded to thapsigargin with a slowly developing increase in cytosolic Ca2+ that reached a peak within approximately 3 min and was followed by a sustained plateau. At each dose of thapsigargin, the peak and plateau responses were decreased in PMVECs compared with PAECs, but effective concentration (50%) values were similar (approximately 30 nmol). Basal cAMP turnover was lower in PMVEC (0.107 ± 0.012% cAMP conversion) than in PAEC (2.810 ± 0.094% cAMP conversion), and total cAMP content was increased (PMVEC = 93.6 ± 4.3 vs PAEC = 42.2 ± 2.1 pmol/mL/106 cells). Thapsigargin (1.5 μM for 2.5 min) did not influence cAMP turnover in PMVEC but decreased cAMP turnover 40% in PAEC.

Studies were repeated in the presence of rolipram, a cAMP phosphodiesterase 4 inhibitor, to determine whether increased cAMP turnover would unmask Ca2+ inhibition of adenylyl cyclase activity in RPMVECs. Rolipram (40 μM) increased cAMP turnover in PMVEC (0.775 ± 0.045% cAMP conversion) but did not influence cAMP turnover in PAEC (2.900 ± 0.109% cAMP conversion). In the presence of rolipram, thapsigargin inhibited cAMP turnover 40% in PMVEC and 22% in PAEC.

Taken together, the data indicate that in the presence of increased cAMP turnover, as occurs with rolipram treatment in PMVEC and basally in PAEC, activation of store-operated Ca2+ entry inhibits adenylyl cyclase activity and decreases cAMP content. However, under basal conditions, store-operated Ca2+ entry does not inhibit adenylyl cyclase activity in PMVECs since constitutive enzyme activity is low. We speculate the low basal cAMP turnover, elevated cAMP content, and insensitivity of adenylyl cyclase to inflammatory mediator-evoked Ca2+ entry serve to enhance PMVEC barrier function.




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