Biology Reference
In-Depth Information
3 Targeting Phosphodiesterases in PAH
The rationale for targeting cyclic nucleotide phosphodiesterases (PDEs) in PAH is
based on the favorable effects achieved by increasing the intracellular concentration
of cAMP and cGMP, and the idea that the amplitude and duration of their increase in
concentration can be enhanced by decreasing their degradation. Tissue-related
variations in distribution and physiological function of PDEs make them good
pharmacological targets for PAH, i.e., by tailoring cyclic nucleotide signaling in
a pulmonary-specific manner (Bender and Beavo
2006
). For example, since PDE5
and other components of the cGMP-signaling pathway are abundant in the lung, this
helps explain the interest and success of PDE5 inhibitors in effectively promoting
pulmonary vascular smooth muscle relaxation (Corbin et al.
2005
). Activities of
both cAMP-PDE and cGMP-PDE are increased in the PA from experimental
models of PAH, which is also true in PASMCs isolated from PAH patients (Maclean
et al.
1997
; Murray et al.
2007
; Wharton et al.
2005
). The increased PDE activity has
important consequences: unless a PDE inhibitor is present, cAMP accumulation in
PAH-PASMCs in response to agonists that increase cAMP synthesis, for example,
forskolin or beraprost (an analogue of prostacyclin) is attenuated compared to that of
control-PASMCs (Murray et al.
2007
).
In the lung, the main PDEs that control cyclic nucleotide levels were initially
shown to be PDE3 (cGMP-inhibited, cAMP-specific) and PDE5 (cGMP-specific);
inhibitors for each of these PDEs relax isolated preconstricted human and rat PAs
(Cohen et al.
1996
; Dent et al.
1994
; Maclean et al.
1997
; Phillips et al.
2005
; Pyne
et al.
2007
; Rabe et al.
1994
). Real-time polymerase chain reaction (PCR) data
(Fig.
1a
), although confirming that PDE3A and PDE5A are the highest expressed
PDE isoforms in PASMCs, also detect mRNA expression of PDE1A, 1C, 2A, 3B,
4A, 4B, 4C, 4D, 7A, 7B, 8A, 9A, 9B, 10A, and 11A, thereby suggesting that a
number of other PDEs could contribute to the maintenance of low pulmonary
vascular tone. In all patients with PAH-PASMCs whom we studied, we found
increased expression of PDE1A, PDE1C, PDE3B, PDE5A, and PDE7A (PDE3A
was only increased in IPAH patients, Fig.
1b
): the increase in expression correlated
with an increase in the relative contribution of PDE1 and PDE3 to total cAMP-PDE
activity in PAH-PASMCs compared to control (Fig.
1c
). PDE activity was deter-
mined using 1
m
M cAMP as substrate through a two-step radioassay procedure
(Keravis et al.
2005
; Thompson et al.
1974
). Substrate and protein were incubated
over a period of time that PDE activity was linear. To identify the contribution of
specific PDEs, activity assays were performed in the presence of specific PDE
inhibitors [PDE1, 30
m
M vinpocetine, 30
m
M 8-methoxy-methyl-3-isobutyl-1-
methylxanthine (8-MM-IBMX); PDE2, 10
m
M erythro-9-(2-hydroxy-3-nonyl)-
adenine (EHNA) in the presence of excess cGMP; PDE3, 10
m
M milrinone; and
PDE4, 10
m
M rolipram] and with or without Ca
2+
/calmodulin in the presence of
EGTA. In addition to being therapeutic targets for PAH, the upregulation of these
PDE isoforms may provide insights into the mechanisms of the disease (Murray
et al.
2007
). In this chapter, we review the basic research and clinical data that
