Biology Reference
In-Depth Information
relaxation elicited (Harris et al.
1989
). This result demonstrated (1) that PDE3
inhibition contributes to relaxation and (2) that inhibition of PDE3 is similar in the
cell-free system and in the intact cell. On the contrary, although PDE4 inhibition
contributed significantly to relaxation, no obvious correlation was obtained. This
indicates that the IC50 for inhibition of isolated PDE4 does not quantitatively reflect
the effect of PDE4 inhibition in the intact tissue. This result was surprising and the
lack of correspondence of the effect of an inhibitor on isolated PDE4 with the effects
of the inhibitor on PDE4 in a tissue, respectively, was a challenging phenomenon for
optimising the effects of new compounds targeting PDE4 function. Interestingly, a
much better correlation was obtained if, instead of enzymatic IC50, the IC50 for
competition with binding of radioactive rolipram to the PDE4 was used. This raised
the hypothesis that the binding competition is the better measure for the affinity of
the inhibitor to PDE4 enzyme in the tracheal muscle (discussion in Sect.
4.5
).
4
Isolated Cellular Systems
4.1 Human Platelets Offer Easy Determination of PDE3
and PDE5 Inhibitors
Human platelets offered at least four important advantages as an analytical
tool. These cells and their enzyme content were of (1) human origin, (2) easy to
prepare and (3) represented a homogeneous cell preparation. Moreover, aggrega-
tion induced by ADP or collagen was an established routine measurement, and
theophylline and papaverine completely inhibited aggregation indicating a relevant
function of PDEs. This important regulatory function was further improved by
inhibitors of aggregation such as forskolin, adenosine or ß2-agonists representing
AC activators. Hidaka had already shown in 1976 that two prominent peaks of PDE
activity, which were cGMP specific (peak I) and one cAMP specific (peak III),
respectively, could be separated from platelets (Hidaka and Asano
1976
). Later
peak III was identified as cGMP-inhibited and cAMP-hydrolysing PDE3, which
was undistinguishable from soluble PDE3 in cardiac tissue (MacPhee et al.
1986
).
Thus, the crude extracts of platelets offered two easily distinguishable PDEs, and in
addition, a common function related to both PDEs could be measured. The corre-
spondence of the effect of a PDE inhibitor on the isolated enzymes and its effect in
the intact platelet could be confirmed in a perfect correlation of both data sets
(Schudt et al.
1991c
). The relationship between an increase in platelet cGMP, which
is followed by an increase in cAMP due to cGMP-inhibition of cAMP breakdown at
the catalytic site of PDE3 was first demonstrated in 1990 (Maurice and Haslam
1990
). The presence of a third PDE fraction was indicated in the elution profiles and
identified as PDE2 (Schudt et al.
1991a
,
b
,
c
), and the interaction of a cGMP-
stimulated and a cGMP-inhibited PDE could be studied functionally (Dunkern and
Hatzelmann
2005
).
