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Regulation of Pulmonary Microvascular Endothelial Cell Cyclic Adenosine Monophosphate by Adenylyl Cyclase^*

Implications for Endothelial Barrier Function

^* 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

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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This Article Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Article Archive Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stevens, T. Articles by Thompson, W.J. Search for Related Content PubMed PubMed Citation Articles by Stevens, T. Articles by Thompson, W.J.