1. ¹Departmento de Farmacoloxía, Facultade de Farmacia, Universidade de Santiago de Compostela, Santiago de Compostela, Spain
2. ²Département Pharmacologie et Physico-Chimie, Université Louis Pasteur—Strasbourg I, CNRS UMR 7175, Illkirch, France

Correspondence: Dr C Lugnier, Départment Pharmacologie et Physico-Chimie, Faculté de Pharmacie, Université Louis Pasteur—Strasbourg I, CNRS UMR 7175, 74 rte du Rhin, BP 60024, Illkirch F-67401, France. E-mail: claire.lugnier@pharma.u-strasbg.fr

³These authors contributed equally to this work.

Received 8 November 2007; Revised 15 January 2008; Accepted 30 January 2008; Published online 3 March 2008.

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 -adrenoceptor agonists.