Regulation of cyclic AMP in rat pulmonary microvascular endothelial cells by rolipram-sensitive cyclic AMP phosphodiesterase (PDE4)

Abstract

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 [${}^3\text{H}$]-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.

Introduction

Cyclic nucleotide phosphodiesterase(s) (CN PDEs) (EC 3.1.4.17) have a central role in the regulation of endothelial cell (EC) barrier permeability [1], [2], [3], [4], [5], [6]. CN PDEs constitute a complex system of 11 gene families with multiple subfamilies that hydrolyze cAMP and guanosine 3′,5′-monophosphate (cGMP) [7], [8]. Various inhibitors, selective for different PDE isozymes, have been shown to differentially affect tissue responses depending upon isoform expression [9], [10]. For example, selective inhibitors of the PDE2 and PDE3 families of isozymes in the lung, but not inhibitors of PDE1, 4, or 5 enzymes, attenuate acute hypoxia-induced vasoconstriction [11], [12]. Zaprinast, a relatively selective PDE5 inhibitor, reverses chronic pulmonary hypertension in rats [13]. Selective inhibitors of the PDE4 gene family isoforms consisting of subfamilies A–D have cell-specific actions [14], [15] over other PDE inhibitors. Selective inhibition of PDE4 isoforms provides the basis for a new class of agents to treat chronic obstructive pulmonary disease (COPD) and asthma [9].

Decreased cAMP and increased calcium levels in some types of ECs have been associated with increased permeability in several studies [3], [16], [17], [18]. Tumor necrosis factor α-induced permeability is associated with an activation of PDE4 and PDE2 and concomitant decreased cAMP levels in conduit vessel ECs [3]. Conversely, agents that increase EC cAMP levels are known to prevent barrier injury in several lung injury models [19], [20], [21], [22], [23], [24], [25], [26], [27].

Since pulmonary microvascular EC CN PDEs have not been characterized previously, the studies reported here were directed toward defining the functional isoforms of pulmonary microvascular EC PDE families in comparison to other ECs. PDE4 isoforms were the dominant enzymes in these ECs. The results of a portion of these studies have been published in abstract form [28].