Short-term or long-term treatments with a phosphodiesterase-4 (PDE4) inhibitor result in opposing agonist-induced Ca(2+) responses in endothelial cells.
OBJECTIVE
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.


METHODS
[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.


RESULTS
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.


CONCLUSIONS
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.
Campos-Toimil
M
M
Departmento de Farmacoloxía, Facultade de Farmacia, Universidade de Santiago de Compostela, Santiago de Compostela, Spain.
Keravis
T
T
Orallo
F
F
Takeda
K
K
Lugnier
C
C
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000096:biosynthesis / Q000235:genetics; D003513:Cycloheximide / Q000494:pharmacology; D003627:Data Interpretation, Statistical; D042783:Endothelial Cells / Q000187:drug effects*; D005456:Fluorescent Dyes; D016257:Fura-2; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D058988:Phosphodiesterase 4 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020889:Rolipram / Q000494:pharmacology
D005456:Fluorescent Dyes; D058988:Phosphodiesterase 4 Inhibitors; D010726:Phosphodiesterase Inhibitors; D011500:Protein Synthesis Inhibitors; D003994:Bucladesine; D006632:Histamine; D003513:Cycloheximide; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram; D002118:Calcium; D016257:Fura-2