Short‐term or long‐term treatments with a phosphodiesterase‐4 (PDE4) inhibitor result in opposing agonist‐induced Ca2+ responses in endothelial cells

Abstract

We previously reported that agonist-induced rises in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in human umbilical vein endothelial cells (HUVEC) were inhibited after a short-term (2 min) pre-treatment with cAMP-elevating agents. The aim of this work was to study the effects of longer term (8 h) pre-treatment with dibutyryl-cAMP (db-cAMP) or rolipram, a specific inhibitor of phosphodiesterase-4 (PDE4), on [Ca(2+)](i), cAMP levels and PDE activity and expression in HUVEC. [Ca(2+)](i) changes were measured in isolated HUVEC by Fura-2 imaging. Intracellular cAMP levels and PDE4 activity were assessed by enzyme-immunoassay and radio-enzymatic assay, respectively. PDE expression was measured by northern and western blot analysis. Long-term pre-treatment of HUVEC with rolipram or db-cAMP significantly increased ATP-, histamine- and thrombin-induced [Ca(2+)](i) rises. Short-term pre-treatment with rolipram was associated with an increase in cAMP, whereas long-term pre-treatment was associated with a decrease in cAMP. Long-term pre-treatment with rolipram or db-cAMP induced a significant increase in PDE4 activity and the expression of 74 kDa-PDE4A and 73 kDa-PDE4B was specifically enhanced. All these effects were suppressed by cycloheximide. Our data suggest that sustained inhibition of PDE4 by rolipram induced an increase in PDE4 activity, possibly as a compensatory mechanism to accelerate cAMP degradation and that PDE4A and PDE4B were implicated in the regulation of [Ca(2+)](i). Thus, isozyme-specific PDE4 inhibitors might be useful as therapeutic agents in diseases where [Ca(2+)](i) handling is altered, such as atherosclerosis, hypertension and tolerance to beta-adrenoceptor agonists.