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

Abstract

Background and purpose: We previously reported that agonist‐induced rises in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_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 [Ca²⁺]_i, cAMP levels and PDE activity and expression in HUVEC. Experimental approach: [Ca²⁺]_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. Key results: Long‐term pre‐treatment of HUVEC with rolipram or db‐cAMP significantly increased ATP‐, histamine‐ and thrombin‐induced [Ca²⁺]_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. Conclusions and implications: 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 [Ca²⁺]_i. Thus, isozyme‐specific PDE4 inhibitors might be useful as therapeutic agents in diseases where [Ca²⁺]_i handling is altered, such as atherosclerosis, hypertension and tolerance to β‐adrenoceptor agonists.