Activation of soluble guanylate cyclase in the presence of purified human aldehyde dehydrogenases
Matteo Beretta
2
Alexander Kollau
2
Michael Russwurm
1
Doris Koesling
1
Wing Ming Keung
0
Kurt Schmidt
2
Bernd Mayer
2
0
Department of Pathology, Harvard Medical School
,
Boston, 02115, MA
,
USA
1
Department of Pharmacology and Toxicology, Ruhr-Universitat Bochum
,
44780 Bochum
,
Germany
2
Department of Pharmacology and Toxicology, Karl-Franzens-Universitat Graz
,
8010 Graz
,
Austria
<supplement><title><p>3<sup>rd</sup>InternationalConferenceoncGMPGenerators,EfectorsandTherapeuticImplications</p></title><note>Me tingabstracts-AsinglePDFcontainingal abstractsinthisSup lementisavailable<ahref="htp:/www.biomedcentral.com/content/files/pdf/1471-2210-7-S1-ful.pdf">here</a>.</note></sup lement> This abstract is available from: http://www.biomedcentral.com/1471-2210/7/S1/P7
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Mitochondrial aldehyde dehydrogenase (ALDH2) was
reported to catalyze bioactivation of nitroglycerin (GTN),
resulting in the formation of a nitric oxide (NO) like
species activating soluble guanylate cyclase (sGC).
However, the reaction product is thought to be nitrite, which
does not activate sGC, so the link between GTN
metabolism and sGC activation is not clear yet.
Since we found no link between ALDH2-catalyzed GTN
metabolism and mitochondrial nitrite reduction (Kollau
A., Beretta M. & Mayer B.; unpublished), we investigated
the possibility that an activator of sGC is produced
directly in the course of ALDH2 reaction by co-incubation
of purified human ALDH2 with purified sGC in the
presence of GTN. In the absence of ALDH2, GTN had no effect
on sGC activity (0.28 0.07 mol/mg/min). In the
presence of 25 g of ALDH2 there was a biphasic stimulation
of cGMP formation with an EC50 (half-maximally
effective concentration) of ~1 M GTN and a maximum at 10
M GTN (1.66 0.22 mol/mg/min). The effect of
ALDH2 was dependent on the protein concentration,
with a linear increase from 1 to 100 g (2.10 0.23 mol/
mg/min) and saturation between 100 and 250 g of
ALDH2. ALDH2-dependent sGC activation was inhibited
by 1 mM chloral hydrate (41.5% of control) and by 100
M daidzin (18.9% of control). The effect of ALDH2 on
cGMP formation was almost completely inhibited by the
NO scavenger oxy-haemoglobin, the superoxide
generator flavin adenine dinucleotide, and the heme site sGC
inhibitor ODQ (0.1 mM, each).
The cytosolic ALDH isoform (ALDH1) also metabolized
GTN to 1,2- and 1,3-GDN (dinitroglycerin) and triggered
sGC activation. In line with the about 1,000-fold lower
substrate affinity of ALDH1, significantly higher GTN
concentrations were required for sGC activation. There was a
linear increase in cGMP formation by sGC co-incubated
with 50 g of ALDH1 and 10 M to 1 mM GTN. At the
highest GTN concentration tested (1 mM), sGC activity
was 4.25 0.15 mol/mg/min. As observed with
ALDH2mediated sGC activation, the response to ALDH1 was
dependent on the protein concentration with a linear
increase from 5 to 100 g ALDH1. The effect of ALDH1 on
cGMP formation was inhibited by 1 mM chloral hydrate
(57% of control) but not by 100 M daidzin.
These results suggest that both ALDH1 and ALDH2
catalyze GTN bioactivation by direct formation of NO or a
NO-like activator of sGC.
(...truncated)