Regulation of cyclic AMP in rat pulmonary microvascular endothelial cells by rolipram-sensitive cyclic AMP phosphodiesterase (PDE4).
We report here studies on the regulation of the metabolism of adenosine 3',5'-monophosphate (cAMP) in established and primary cultures of rat pulmonary microvascular endothelial cells (RPMVEC). Inhibition by rolipram, a selective inhibitor of cAMP phosphodiesterase (PDE) of the PDE4 gene family, was required to achieve maximal cAMP accumulation induced by direct or receptor-mediated adenylate cyclase activation when measured by [3H]-adenine prelabeling. Rolipram increased cAMP accumulation more effectively than did forskolin, isoproterenol, or adenosine derivatives alone, although extensive synergy was seen with combined agents. High-affinity PDE4 inhibitors, but not low-affinity or non-selective inhibitors, were effective inducers of cAMP accumulation in intact cells. The maximum effects (i.e. intrinsic activities) of these agents in the intact cell did not correlate with their in vitro PDE4 inhibitory affinities. RPMVEC were shown to express almost exclusively the PDE4 gene family isoforms A6 and B3. Guanosine 3',5'-monophosphate hydrolysis, observed in other types of endothelial cells was not found in early or late passage RPMVEC. Reverse transcription-polymerase chain reaction identification of mRNAse supported these conclusions with the exception that PDE2 and PDE4D mRNA isoform transcripts were present. These studies also support the conclusion that the mechanism of rolipram reversal of rat lung ischemia-reperfusion-induced permeability involves PDE4 inhibition in the microvascular endothelial cells of the lung.
Thompson
W Joseph
WJ
Department of Pharmacology, University of South Alabama College of Medicine, Mobile, AL 36608, USA. jthompson@cellpathways.com
Ashikaga
Takashi
T
Kelly
John J
JJ
Liu
Li
L
Zhu
Bing
B
Vemavarapu
Lakshimi
L
Strada
Samuel J
SJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004347:Drug Interactions; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008168:Lung / Q000166:cytology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D051381:Rats; D020889:Rolipram / Q000494:pharmacology*; D013997:Time Factors
D010726:Phosphodiesterase Inhibitors; D005576:Colforsin; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram; D007545:Isoproterenol