Research Report 2000 - MDC

New modes of calcium signaling
Maik Gollasch, Matthias Löhn, and
Michael Fürstenau are making
exciting advances in electrophysiology.
During a Humboldt fellowship at the
University of Vermont, Gollasch
worked with Mark Nelson and studied
local calcium transients termed
calcium sparks. These sparks are
apparently caused by opening of
clustered ryanodine receptors in the
sarcoplasmic reticulum. Gollasch’s
team has investigated caveolae,
cholesterol/sphingolipid-rich
invaginations of the plasma membrane
which colocalize with both the
subsarcolemmal occurrence of
calcium sparks and the junctional
sarcoplasmic reticulum. They have
found that a transient elevation in
calcium at the inner mouth of a single
L-type calcium channel within
caveolae induces simultaneous
activation and opens several
ryanodine receptors to generate a local
calcium spark. They are the first to
show that a caveolae-calcium
signaling pathway may regulate
cellular functions via local ryanodine
receptors in the sarcoplasmic
reticulum. The first illustration of a
calcium spark in man (from a
coronary artery vascular smooth
muscle cell) is shown in the figure.
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Christian Maasch, Carsten Lindschau,
Gabi Alexander and others are using
green fluorescent protein technology
to observe protein kinase Cα targeting
in vascular smooth muscle cells. They
recently showed that local changes in
calcium regulate PKCα translocation.
The targeting mechanism is dependent
on the C2 and P domains of the
enzyme. Their observations are the
first to show that localized calcium
changes determine the spatial and
temporal targeting of PKCα.
Gene therapy
The group has relied on antisense
strategy in a series of exciting studies
aimed at treating ischemia-reperfusion
injury occuring after organ
transplantation and they have used a
rat renal transplant model. Duska
Dragun has recently completed a
study in which she showed that the
antisense treatment is very effective in
the prevention of ischemia-reperfusion
injury in transplantation. A chronic
isograft transplantation study has also
been completed.
Patient-oriented research
Hermann Haller, Volker Homuth,
Friedrich C. Luft, and Gerd Wallukat
have collaborated with obstetricians in
Berlin to elucidate new mechanisms
responsible for endothelial damage in
preeclampsia. In a cell culture system
of endothelial cell monolayers, the
group showed that a factor from the
serum of preeclamptic women
stimulates increased cell-layer
permeability. This process involves
PKC signaling, principally PKCα and
PKCε. The permeability increase was
blocked with antisense against the
mRNA of these enzymes. In
subsequent studies, the team showed
that women with preeclampsia
produce agonist autoantibodies
directed at the AT1 receptor. The
antibodies are directed at the second
extracellular loop. Colocalization
studies have demonstrated the
necessary specificity. The subsequent
signaling proved to be a PKCmediated
pathway. Very recently, the
group has shown that the antibodies
are capable of making vascular
smooth muscle cells produce tissue
factor. The work of the four
investigators was awarded the
Galenus von Pergamon prize for 1999.
Figure 33: Calcium sparks are localized
intracellular events released through ryanodine
receptors. The sparks then control excitationcontraction
coupling. The confocal line-scan
image of a fluo-3-loaded human coronary
artery vascular smooth muscle cell is shown.
The fluoresence time course of the sparks was
determined over the time indicated by the two
arrows.