A. Smolenski et al., FUNCTIONAL-ANALYSIS OF CGMP-DEPENDENT PROTEIN-KINASE-I AND PROTEIN-KINASE-II AS MEDIATORS OF NO CGMP EFFECTS/, Naunyn-Schmiedeberg's archives of pharmacology, 358(1), 1998, pp. 134-139
NO and cGMP have emerged as important signal transduction mediators of
the effects of certain hormones, inter-/intracellular signals, toxins
and drugs. However, a major challenge is to define relevant criteria
for determining which of the many NO and/or cGMP effects are dependent
on cGMP-dependent protein kinases (cGKs). Important criteria include
that: (1) the cell types/tissues investigated contain at least one for
m of cGK which is activated by the cGMP-elevating agent in the intact
cell system; (2) specific activators/inhibitors of cGKs mimic/block th
e effects of cGMP-elevating agents in the intact cell system; and (3)
the cGMP effect is absent or blunted in cGK-deficient systems, or can
be reconstituted by the introduction of active cGKs. Previously, analy
sis of cGK activity in intact cells has been very difficult. However,
the analysis of vasodilator-stimulated phosphoprotein (VASP) phosphory
lation by polyclonal antibodies and newly developed monoclonal antibod
ies, each of which specifically recognize different phosphorylation si
tes, allows the quantitative measurement of cGK activity in intact cel
ls. With the use of these methods, the properties of certain cGK mutan
ts, cGK activators (cGMP, 8-Br-cGMP, 8-pCPT-cGMP) as well as various '
'specific cGK inhibitors'' (KT 5823, Rp-8Br-PET-cGMPS, Rp-8-pCPT-cGMPS
, H8 and H89) were investigated. Although these ''specific cGK inhibit
ors'' have been widely used to establish or rule out functional roles
of cGKs, very few studies have actually addressed the efficiency/speci
ficity of such compounds in intact cells. Our results demon strate tha
t these inhibitors are useful tools only when used in combination with
other experimental approaches and biochemical evidence.