Stamler. JS: Redox signaling: Nitrosylation and related target interactions of nitric oxide. Cell. 78: 931-936, .... PA, Nelson. Ri, Singer JW, Bursten. S: Protec-.
Inhibition Dependent Adenosine
of Nitric Oxide Calcium Influx Monophosphate: KENICHIRO Third Medicine
Abstract.
Cell
destruction
calcium
mechanisms
that
are
not
fully
II,
through
(Ca1)
defined,
but
proteins
from
thrombin
and
adenylyl
cyclase
understand signaling
effects
may
of adenylyl of adenylyl
Hypoxic
and
occurs
in
renal
production
(1-4).
severity to cell
epithelial
the
through
a variety
Ga1,
cooperate
to
injury
and
cell
to define
the
Ca influx
injury
frequently
activation
two
to these
and
events
intracellular
have
subsequent High
insults,
been
the
epithelial
calcium implicated
of experimental sysentry of extracellular
activation of proteases levels of NO may injure
of mechanisms,
including
eration and nitrosylation of proteins increased Ca levels or NO production
the
free
and cells
radical
gen-
(5-7). Evidence that the contribute to cell injury
Received
August
versity
of
Florida,
FL 32610. Dr. Jared Grantham disposition
20, 1996. Accepted to
of
this
Dr. Box
R.
Tyler
100224
served
December
Miller,
Internal
JHMHC.
1600
as Guest
Editor
MedicinefNephrology,
SW Archer
and supervised
manuscript.
Road,
Uni-
Gainesville,
the review
and
final
Copyright
of the American Society of Nephrology (C) 1997 by the American Society of Nephrology
influx
was
is modulated to
the
production.
phospholipase
was
authors
but by an
insensitive
conclude
and
that
by
indicated
Ca influx,
The
extent
by
by activa-
NO
that
by NO-dependent
that
blocked inhibited
regulates
mechanism cyclase.
on
which
inhibiting
that
bradykinin-stimulated
adenylyl
and
by
depends
and
kinase,
bra-
to
that
independent
increased
NO
Soc
Nephrol
8: 558-568,
by
preventing
the
macologically
the
mech-
Ca
of Ca across
is referred
including
NO
Ca
hormones
NO
cell
influx,
whereas
influx
pathway
the
cell
cGMP
the
(12,14).
in part
by
of
Ca-calmodulin,
eNOS, and
cy-
Ca entry In
reduce
reconstitute may
signaling NO
activ-
guanylyl
cyclase
in the
by three NOS,
(NOS)
(11,12,14-16).
in Ca1 or NO
of normal
to release
stimulates
space
In
factors,
to peptide
synthase
which
and
CRAC.
other
stimulates
or guanylyl
is controlled
or
in Ca1 due
Consequently,
or derangement
Ca influx
influx,
of NO or cGMP
increases
forms
of Ca
Release
in response
extracellular
readdition injury,
level,
from
(10).
A rise NO
Ca
event,
by
in turn
of NOS
of
modulated
production
stimulates
inhibitors
production
Ca
(1 1-14).
the
from
constitutive
regulated
by
in Ca1 phar-
1P3-mediated
to stimulate
be
production
raises
systems,
NO
be reduced
release
membrane
may
(!P3
or autocoids) This
the
release-activated influx
stores
Increased
activation
can
or rise
and
is required
production
of intracellular
ischemic
injury
by
the cell
stores
to as Ca cells,
the
cell
production
a phospholipase-C
intracellular
clase.
that
in NO
is regulated
stores,
epithelial
ity.
finding
(1,2,8,9).
influx
from
injury (J Am
1997)
increase
Intracellular and
Ca
production
contributes to increased Ca influx and cell injury, cell may be reduced by agents that activate adenylyl cyclase.
1046-6673/0804-0558$03.00/0 Journal
by
influx
these
30, 1996.
Ca
was
cyclase
production
by
increased that
influx
protein
cGMP-independent
into
Correspondence
NO
the 0 protein
C, also increased
intracellular
(Ca1) in
blocks
of Gaq.
is supported
of
Ca guanylyl
of cAMP-dependent
anisms,
injury,
of
initiated
Ga,3
by a mechanism
and
cyclase.
signals
thrombin.
increased
synthase
adenylyl
cAMP
inhibition
To
The Departments
of the
and
The
of NO
Expression
of the
injury.
cell
that
NO,
activators
and
components Ca influx
cGMP.
of
mech-
protein-dependent
or
In response
cell with derangements.
for
distinct of cell
0
pathogenesis of cell injury in a number tems. Ca-mediated cell injury involves Ca into metabolic
proteins
inflammation
increases
These
activators
this type of injury the levels of autoas thromboxane, thrombin, Ang II, and
are elevated.
NO
rises
tion
on bradykinin-stimulated
of
During such
distinct through
through
which
and
inhibitors
have
of the
of different for thromboxane
dykinin-stimulated
II act
receptors
in ameliorating
cyclase
clotting cascade. coids and peptides
the
contribute
setting
that
act
Japan;
Florida.
separation
NO
receptors
settings,
by
cyclase
ischemic
the
bradykinin
reduce
mechanisms
systems
role
cell
of experimental
of 0
MILLER
Kumainoto,
and NO in cultured renal epithelial cells that stably overexpress Gaq and Ga,3 were studied. This system allowed for the receptors
injury
as bradykinin,
members
0
in
The
cell
activation
such
cells
a rise
production.
families
These
of tissue
during
These
act through
system
the
setting
bradykinin and Ang Gaq families, whereas
in epithelial
In a number
Gainesville,
involve
substances
families.
NO
may
different
for and
of Florida,
with
NO
thrombin.
thromboxane
Gaq and Ga , regulate Ca1 and
the
for and
Receptors of the Ga1
University
NO
and
R. TYLER
University,
in the
increased
and
Kumamoto
is associated
in Ca
they
receptors
functions. members
anisms.
and
rises
thromboxane, 0
occurs and
trigger
protein-coupled Ang
frequently
TOMITA,
Medicine,
Pharmacology,
inflammation
intracellular
KIMIO
of Internal
and
injury
and
KITAMURA,
Department
Synthase Activity and Nitric Oxidein Renal Epithelial Cells by Cyclic Implications for Cell Injury
early
Ca
the Ca stages
occur
of
through
pathways. synthetic
and
bNOS
the
inducible
enzymes, that
are form,
Inhibition
iNOS its
The
(5,17,18). level
of
lipopolysaccharide cifically that
ofiNOS usually
(LPS).
induces
G
activity
expression,
iNOS
in renal
iNOS
ceptors
that
These
activate
levels. Substances act through
epithelial
will
cells,
mean
Ga13
are
spe-
GaqQ29L
of
for
re-
expression
and
to
activate
cyclase.
These
terized
signaling
phospholipase
receptors
have
ubiquitously
cell
through
line,
iNOS
thromboxane naling The
identified
(20,2
and
1
that
Ga,3
thrombin
and and
Gaq
,
and
agents
tissue
injury
steps
in these
found
models,
that
inhibited
are which
showed
processes
reduces
study
renal
In
NO
MgCl,,
in a
and
for
their
of
the
effects
of
cells
that
of Gaq
and
cell or
influx
titative
was
that
and
re-
(Boston,
(Eugene,
purchased supplies
OR).
MA). Tissue
Fura-2
cyclase
Ca
influx
medium,
serum,
from Molecular were
from GIBCO (Grand Island, NY). Tissue culture plastic were from Falcon (Oxnard, CA). Other chemicals were from
Sigma (St. (Pittsburgh,
Louis, PA).
MO)
or
(molecular
biology
grade)
from
Fisher
MCT
cells,
a gift
DMEM/Ham’s formed
when
an SV4O-transformed Eric
F-12 cells
studies.
in lysis
leupeptin. and
125
lysed
was
of Ca
buffer
(20
2 mM
mol
PMSF,
in a glass
dounce
at 1 200 X g to
centrifuged
Influx
fluorometrically
by measuring Mn
100%
in Fura-2-loaded
containing
rate
portion
increase
over
was
of the
quench
control
To
calculate
by Mn and
digitonin the tangent
or Neo cells.
was
One
cell
the cells
by measuring and
quan-
different
by exposing
and 75 pg/ml curve
the
to allow
and
determined
MnCl,
of intracellular
(12.14).
treatments
was
determined
Mn
untreated
nm
different
0.5 mM
of Ca influx
360
fluorescence
value
(SLM 8000at 510 nm. Ca
the rate of quench
at
among quench
( I 2,14). to the
presented
tracing
as fold
is considered
“n.”
whole
Activity crude
and
as conversion
NOS
activity
Bredt
and time
with
of NOS cells was
measured
Snyder
(35).
j.d of PBS
7.8
mM
glucose,
for
15 mm.
homogenates,
MCT
(pH
and
were
grown
the medium 7.3)
1 pCi
At time
cells
containing [3H]arginine
0, bradykinin
was 4.2
activity
was
Whole
cell
(l0
of the
technique
of
in 24-well
clusters.
At
removed mM
and
M and 2 x I0 M), or L-NAME the incubation was continued for
(10 and
NOS
to [3H]citrulline.
by a modification
of the experiments,
400
cell
of [3H]arginine
was
M), (5
an
and
KCI,
replaced
1 mM
incubated
forskolin
l0 additional X
CaC1,, at 37#{176}C
and
IBMX
M) were added 10 mm. The
reactions were stopped by adding 10 ,.d of 5-M TCA, and were held on ice for 30 mm. The supernatant was gently
(Dowex AG8 200-400); mM HEPES (pH 5.5).
from
neomy-
for
the plates removed,
extracted twice with 2 volumes of ether, and neutralized with 1.5 ml of 25 mM HEPES. Each reaction was then added to a 2-ml column
Cell Culture were
and
in
from En-
and G-4l8
on ice
smol
measured
of Fura-2
Measurement
were obtained from DuPont-New
was
quench
to medium
one
Ga13.
was purchased
culture
used
2 mM mercaptoethanol,
homogenate
extracellular
the
initial
led to
Ca
comparisons
types, The
findings, cyclase
adenylyl
scraped
inhibitor)
Measurement
by
have
induction,
and 25
trypsin
was quantitated
Fura-2
the
Nuclear
Probes
suffi-
expression
GaqQ2(,[ were
cells in suspension in a dual excitation microfluorometer C). Excitation was at 360 and 380 nm, and emission
we
Materials
gland
Ga,3
aprotinin,
soy
Intracellular
and Methods DNA modifying enzymes WI) and radionucleotides
a chain
was
Extracts
at 4#{176}C. The
measured
Restriction and Promega (Madison,
metal-
in G protein expressing
in PBS
TIU/ml
In both
Materials
pZem-
activity
pH 8, 150 mM NaCI,
Fluorescent
cells
the
cells
nuclei.
studies,
production,
express
remove
in MCT a mechanism
activation cGMP
0.2
percentage
and iNOS
using
by a mouse
of promoter
line
and
sig-
of cell
affect
washed
10 j.tg/ml
common
past
influx
through
cell injury
production,
epithelial
range
they
in MCT
expressing
Ga,
MCT cells expressing the mammalian exwithout an insert were used as controls
of Cell were
homogenizer
produc-
protein kinase (3 1). These that activation of the adenylyl
epithelial
on NO
Ca
of Ga
system
that
(24-30).
by activation cAMP-dependent with the fact
system
suggests
of
Ga13 was cloned was a gift of Silvio
(14,19,33).
mM NaHEPES
that
receptors Ga13,
in a wide
hormone-dependent
quired coupled the
effective
cell
cells
559
Expression
is driven
increases
neomycin-resistant vector pZem-228 cells)
expressed level
Influx
expression
(14,19,33). GaqQ2(L
expression
basal
A clonal
pooled
Cells
may contribute to cell injury (20). of cell injury can be reduced by administration
These
(33).
Preparation
are
mechanisms
Because
activate
regusecond
which
increases
(14,19).
The
measurable
activity
(Neo
of agents such as PGE2, PGE2 agonists, melanocyte-stimulating hormone, and phosphodiesterase inhibitors that raise cAMP.
to cause
Pooled pression
adenylyl
cell
promoter.
Cell and
previously PCR, and were
mammalian
lothionein
Mammalian
Ca
and throm-
we
by different
cDNAs
The
NO-Dependent
vectors,
(34).
cm-resistant
well-charac-
Gaq
).
Recently,
influx
induction
and
pathways severity
Ca
inhibit less
a chains, Gaq. whose effector
(22,23).
increase
and other
for thrombin
two 0 protein CJ3, and Ga,3, not been
epithelial
tion
C
activate
Receptors
expressed
and Gaq
renal
also
pathways.
boxane A2 activate lates phospholipase messenger
thromboxane, and thrombin domain, 0 protein-coupled
and described RNA by
where
and
such as bradykinin, seven transmembrane
receptors
Ga,3
228,
and
constructs,
were MCT cell
Gutkind
cient
NO
Activity
Constructs cDNA
from
capable
ligands
iNOS
cDNA The
demonstrating
that
increase
by
bacterial
that
systems
results
Ga13
and
showed
signaling
( 19).
primarily
cytokines
We recently
protein-dependent
inducing
is regulated by
of NOS
Neilson (50:50)
were
mouse (32).
plus
approximately
5%
proximal
Cultures FBS. 90%
tubule
were
cell line,
maintained
in
Experiments
were
per-
confluent.
Serum
was
removed 18 to 24 hours before experiments to minimize growth factors. Cell lines were maintained in G-4l8, removed 48 h or two passages before experiments.
effects of but it was
in a scintillation
protein the
mean
flavin
with
counter.
The counts
and are expressed
as CPM/mg
of triplicate
For measurement (100
eluted
2 mM EDTA,
j.d) contained adenine
in each prot/min.
well
were Each
wells ± SEM. of NO activity in cell homogenates, 50 p.g ofcell
dinucleotide
M), tetrahydrobiopterin
2 ml of solution
(10 (2 X l06
containing
and 2 mM EGTA;
homogenate,
20 mM
20
and counted normalized
point
for
represents
each reaction HEPES
M), flavin mononucleotide M), /3-reduced nicotinamide
pH 7.4, (l0 ade-
560
Journal
nine
of the American
dinucleotide
phosphate
Society
of Nephrology
(2 X l0
M),
EDTA
(5
I0
X
M),
CaCl, (5 X l0 M), arginine (2 X l0 M), [3Hlarginine (approximately 180,000 cpm); and l0 M cAMP (8-Br-cAMP) and protein kinase A (10 U of catalytic subunit in 3 mM DTT). where indicated. The reactions were carried out for 30 mm at 37#{176}C and were stopped by the addition of 400 l of stop solution containing 20 mM HEPES pH 5.5, 2 mM EDTA, and 2 mM EGTA and by placement on ice. [3H} citrulline
was
separated
by
column
above. Reactions were performed expressed as fmol citrulline/mg
Measurement 10
in triplicate, prot/min.
as
and
the
results
was removed ml
of
from
fresh
10-cm
serum-free
dishes
medium
ofcells
were
and was replaced
containing
experimental
cGMP
Dowex
was
isolated
and alumina
ilized,
acetylated,
column
were
were mm.
Each
(36).
bar
ml of medium
in buffer,
at 4#{176}C. The
plasma
centrifuged,
counted
8.0
chromatography.
resuspended
cGMP antibody overnight precipitated with bovine samples
from
Values
represents
I 2%
supernatant
are expressed the mean
was lyoph-
incubated
with
anti-
complex
polyethylene was removed,
as fmol
value
sequential
The eluate and
antigen-antibody
and
the
by
of triplicate
The cells
protl20
Hormone-dependent in Fura-2
easier
Experimental Multiple
groups program
comparisons
Newman-Keuls
were from
compared GraphPad
plates.
among
test. P values
groups
cGMP-sensitive
nLL
=
c\r1
c
C tj:
0
esI
the
levels
with
+1 +1
)
I
2
5)
r1
the
addition
of
results
results
cyclase)
of
Ca
Ca
influx
forskolin-inhibited
Similar
These
+
component
larger,
Ga,3).
z
either
were
demonstrate
was
was
NO
forskolin
to control
cGMP
with
at a step
substantially
restored
or
obtained
that
Ca influx
inhibits
influx
(Figure
cAMP
2C,
analogues.
(activation
of adenylyl
at or before
the production
of NO.
-‘
.-
significance. the NO and
..“
. ‘)
C
‘O
Ca influx, but did not achieve statistical in the cells that express Ga13, in which
clD L,
C
U-
O
>
*
.a ) a C.) n
+1 +1 +1
*
C
c,
I
C
0
z
U #{149}0 s
2Ce
z
.
ai
.-c
(NC
)I
-‘‘-C#
-
(
.n
.5
z
NO express
and
Neo
Gaq
cells
isoforms. and
a minimal
of
the
effect
adenylyl
basal
and
that
express
have
substantially
The
Bradykinin
express
on
cells
that
express
Neo
NOS Ga13.
forskolin
NOS
activity
cells,
activities
activity but it
GaqQ2O9L,
with
cells
to iNOS,
of the
it has no significant the
in the
stimulates
that
In
of iNOS
attributable
be a composite
and
to a much
activity
cells
but
level
is increased
NOS
system
MCT
Ga13 Gaq
bradykinin-stimulated
Ga13,
studied. bNOS,
is primarily
the
of
cAMP-
(eNOS,
in the
cyclase
GaqQ2O9L.
were
production.
it may
and
of NO
Consequently,
cells
that
NOS
Activation
inhibits
both
whole
cells
in
effect
on cells
forskolin
activity
the inhibitory
does not bradykinin.
require
The
mechanism
and
effect
previous
NOS
also
that
inhibits
(Figure
4). These
results
of forskolin
on NOS
activity
activation
+1+1
I
U
and
and
by
activity
activation
of 0 proteins
or NOS
by
,n
ri *
cells
Ga,3,
bition
Q-:’-: +1+1+1
expressing
Ga13
protein
C\1
o=
2T
nase
have
adenylyl
using
cyclase
cell
protein
is reduced
kinase,
protein kinase acute activation
or others)
of NOS
by
activity
Consequently,
:
of
further
of cAMP-dependent
homoge-
kinase
with
bradykinin
is not whole
kinase and
effects
on
that
activation
activity
inhi-
of cells
8-Br-cAMP
inhibit
and
NOS
cAMP-dependent
NOS
NOS (Ga1,
for
in extracts
cells,
do not
proNeo
by 8-Br-cAMP
required
However,
in the
8-Br-cAMP different
70%
indicating
is sufficient to reduce of various G proteins
by cAMP.
expressing GaqQ2O9L. as cAMP-dependent protein
cc
activation
explored
and the catalytic subunit of cAMP-dependent As shown in Figure 5, NOS activity in the
of cAMP-dependent activity and that
...
which was
cAMP-dependent
Gaq.
2Ce
and
NOS
Neo
activity
express
that
in the
cells
‘.-
*
2
Ga13
in the
8-Br-cAMP tein kinase.
n .
cells (19).
express
nates r1
+
.
of iNOS
degree
reduces
C
z
-‘
but
has
oo C-
LI
‘C
iNOS),
Expressing activation
influx
protein
whereas
0 I-
the
express
that
*
*
Ca
activity,
dependent
5) .2 5) 0a t)
inhibits
NOS
-u
in Cells whether
activity.
protein in cells
ki-
expressing
-
c
C C .. c
)n .
U
k.
from
Neo
expression
of
cells
or cells
is not or protein different
expressing
Ga,3.
The
reason
clear, but it may be due to high kinase-C activity in these cells or to accessory
proteins
or
cofactors
for
NOS.
._:
cGMP
g
,..
-Q)
-
C, -.
.
GaqQ2O9L
for this difference phospholipase-C
11
.
n
.
troo
C’JC)
0
5.)
To
.
>izOO
cells
Production determine in a way
in
whether that
Cells cGMP
is consistent
Expressing
Ga13
is regulated with
its regulation
and
by NO of Ca
Gaq in MCT influx,
Inhibition
A.
of NOS
Activity
and
NO-Dependent
Ca
Influx
563
BK
Ca
H4
Thr
TXA2
B.
BK
Ca Thr
TXA2
BK
U
-4---
Ca
Thr TXA2
Figure cells
3. Schematic are shown
Ga1 and Gaq. protein
Potential a chains are
overexpression
representation
in A, cells
that
showing overexpress
the regulation GaqQ2L
activation of Ga,, Gaq, denoted with ‘. Arrows
of G protein
a chains.
In B, where
of NO are shown
and Ca influx in B, and
cells
in MCT that
cells
and Ga13 by thrombin (Thr) or thromboxane show signaling pathways. Heavy arrows
GaqQ2(L
S
expressed,
release
stimulated
overexpress
of Ca from
Gcs,3
with
A, (TXA,) show signaling intracellular
bradykinin
are shown
(BK).
are also shown. pathways that stores
Control
in C. Bradykinin
and
The are
Ca influx
(Neo) activates
expressed activated
G by
are increased
compared with A. However, the importance of the NO- and cGMP-dependent pathways in modulating Ca influx is relatively small, as demonstrated by the findings that their inhibition has little effect on Ca influx. In C. where Ga13 is expressed, iNOS expression is increased, with consequent increases in NO, cGMP, and Ca influx. In these cells, the increased Ca influx can be blocked by inhibition of these pathways and
by cAMP,
which
inhibits
iNOS
activity.
564
Journal
the American
of
Society
of Nephrology
300 C) C
E 0.
C)
200
ci)
Cont.
C
w
BK
V////
Forsk
0 >
C,)
I 00
w z
0
Neo Figure 4. Whole arginine-deficient
a13
aq
in Neo cells and cells expressing GaqQ2O9L and Ga13. Cells were pre-incubated for 15 mm in They were then incubated with bradykinin (BK) l0 M or bradykinin (lO_8 M), and forskolin (l0 M) and IBMX (l0 M), for an additional 10 mm. Parallel samples were incubated with L-NAME (5 mM). The reactions were stopped by the addition of TCA, were extracted with ether, and [3H]citrulline was separated from [3H]arginine by column chromatography (35). Each sample
was
cell assay of NOS activity medium and [3H] arginine.
corrected
for recovery
by the addition
L-NAME-inhibitable
arginine-citrulline
providing
of NOS
a measure
cGMP
production
was
ofapproximately
conversion
activity
activity.
in cpmlmin/mg
measured
under
1200
The activity
conditions
similar
to
Ca influx and NO production in the Neo that express Ga,3 or Gaq Figure 6). In the
control
cGMP
bradykinin dykinin
and
NO.
not
observed
is
83583,
or
level
of
cells,
Stimulation
forskolin.
cGMP
production in
In
production
presence
of
cells
is elevated
that
cells
(approximately
cGMP,
like that of NO, is negligible
or NO.
L-NAME, in
pattern is similar
These
to
agents
in
NO,
inhibit
cells
and
production
the
Neo
that
guanylyl cells
cells
and
express
the
levels
NOS
in
the in
a chains,
activated
by
simultaneously,
A2 contribute
to cell
from ential
each other. regulation
for these
of
and
as well
as others,
Our
higher.
receptors
approach
of
wild-type
or mutant
pathways
they
in the cGMP
by
do not block Ca influx is
cell
functions
were
distinct selective ways
are
Ga,3
such
as
and the
shown
or Gaq.
expressing and cell
are
G
thrombin
acti-
to separate
a chains
selectively
The effects
specific
to differ-
protein
the
chain
the signaling
expressed
a chains
individual
a chains
in Figure
cells
events.
and those of related and tissue injury,
on have
The results are summarized or Ga13-dependent signaling
schematically
on these
a
components of either
activates
ofthe
because
0
of individual Overexpression
and path-
3.
and
cells
we studied bradykinin-stimulated cGMP production, and the effects
cyclase
two
When
difficult
a single
analysis signals.
neomycin-resistant
cause its levels in inflammation
by
receptors.
pathways
effects (14,19,31,33). activation of Gaq
In control,
These
are activated
protein-coupled
control. are
Gaq.
Additionally, they may be subject other signaling processes.
to
inhibited
0
Ga13
by
by
injury,
pathways
these
is blocked
substances
raise cAMP ameliorate cell cells in which individual
expression
many
vated
adenylyl
and thromboxane
the
permits the separation of receptor-generated
Discussion thrombin
of the signaling by
responds
Although 5
by which agents that studied renal epithelial
components
The
GaqQ2o9L
activities
GaqQ2L
by which
represent
and thromboxane receptors (20). Multiple parallel signaling pathways corresponding to different 0 protein a chains are
cGMP
are
cyclase
mechanism injury, we
production, the mechanisms
shown
from the total activity,
LY-
bradykinin.
LY-83583, and forskolin, these agents because the effect of Gaq to increase of this mechanism (14).
To understand
values
protein
increase
express
Ga,3.
The
bra-
to bradykinin
production
cyclase express
that
that
guanylyl
cGMP
that
that
of
solution.
was subtracted
activated
basal
all inhibit
absence but
the
the
forskolin
cells,
that
and
in the
in the
L-NAME, Ca influx, independent
or
and
in the cells
demonstrate and
that
presence production
that
results
bradykinin Neo
the
of cGMP
Ga,3,
in response
and
to the stop
with L-NAME
both
by
above
fourfold),
LY-83583,
by
L-NAME,
express
substantially
control
production
is stimulated
of cGMP the
the
of [‘4Clcitrulline
in the samples
protein.
those used to study cells and in the cells (Neo)
cpm
Bradykinin compounds and because
that
express
Ca influx, of activation
NO of
was
be-
studied are increased it provides
a
Inhibition
of NOS
Activity
and
NO-Dependent
Ca
Influx
565
80 C)
E
70
C
60
E 0.
C)
ci)
50
C
Cont.
I-
w
40
0
PK-A
ci) >
30
C,) C
ci) 0?
20
LU
z
10 0
Neo Figure
5. Measurement
cells
and
cells
of cytosolic
NOS
GaqQ2(L
and
expressing
[3Hlcitrulline
in the absence
[3H]citrulline
was
reliable
Ca signal
influx
requires
mediated
by Gaq.
activation found is
in these
of that
Ca influx
guanylyl
dependent
on
expression
can
In these
intracellular
be modulated and
and
and
cGMP
that
NO
production
influx
by a mechanism
cAMP. cAMP
Additional production
could
the
cells,
ity)
and
activation
processes
In
but
many
of have
NO
through
production. Ca influx
be
increased
is regulated
levels
of NO
they
those
or in the early
(12).
In
influx
some
is difficult
cell
may
explored
stages types,
of
with
those
be
activity
in post-nuclear
by measurement
subunit
of protein
extracts
of conversion
kinase
A. The
of Neo
of [3Hjarginine
reactions
hormone-dependent
full found
of induction the
NO-sensitive
to demonstrate.
Under
influx
induction in the of
cells, are
were
to
stopped,
iNOS
that
of
and low expression levels of NOS (Figure 3). cluding renal epithelial cells, vascular smooth
be
and
cell
proximal
of Ca of Ga,3
In tissues inmuscle cells,
tubules,
tions,
Ca
(2,9).
Excessive
can
synergistically pression
shape,
blockers enter
ATP
phospholipases radicals
(2).
cells
Ca1
NO
by
ability
of
activity
under
transporters
host
tissues,
mechanism that
including
act sup-
activation
generation cells
to
condi-
effects
mitochondria,
ischemic
(40).
injury
pathologic
selective toxic
and
of mul-
metabolism,
of ischemic
multiple by
Ca and
Regardless
control and
a specific,
to injure
proteases,
the
that
that
have
production
and The
may
in cell
Ca1, it is an important
alters
severity
to
could
iNOS.
rises (2,8,9,39).
junctions,
the
demonstrating
with of
tight
of
activation Ga13
to induce
systems
reduce
and
response
to increased
signaling
Amplification
cellular
pathologic
its elevation
as thrombox-
act through
of situations
leads
and
cell
volume,
Ca channel Ca
that
messenger
the
would
of intracellular
iNOS.
that
cytokines
release
induction
of
the
scenario such
the
agents
could enhance
This
to induce
in a number
essential
influx.
in-
expression
could
of substances
with
is
for receptors
INOS
and
of iNOS
to interfere
is valuable
increase
Ga,3,
LPS
with
ability
second cell
Ca
a component
synergistically
of the mechanism
but
of
Additionally,
Ca
circumstances
(the Neo control cells), the NO-sensitive component influx may be small because of low signaling activity
may
the
express of
Ga13
tiple
signals
activate
and
expression
lipopolysaccharide)
Ca
as a consequence
inflammation.
NO
that
presence that
cytokines
by
by cytokines
component
normal
and
iNOS
like ligands
bacterial
on
sustained
Ca,
Ca signals
small
of iNOS, cells
the
or thrombin
The
agents
and
component
Ca
(38).
other
effects
ane
act
/3 cells,
( I 8). Presumably,
cytokines
similar
require
inhibof Gaq
pancreatic
Ga,3,
as
NO-sensitive
in these
In these
activate
have
the
and
by cytokines
that
that
by
studies
of Ca
duced (such
increased
involved
cGMP.
regulation
found
NOS
GaqQ2O9L.
activity
hepatocytes,
We
to inhibition
in these
through
for with
Ga13
but
not sensitive
a component by NO
compatible
degree
kinases
been
required
with
are
was
is
of the Ga1s, such as inhibition of could result in increased iNO activ-
MAP
not
tissues,
influx compared
that
Ca
and
NO-cGMP-Ga,3-dependent
to a smaller
effects (which
and
for NO
and the catalytic
component of Ca influx was inhibited by cAMP. cAMP ited Ca influx by inhibiting NO production. Expression increased
Ga,3
assayed
stores by
cGMP
of hormone-stimulated
NO
of Ga13,
expressing
were
M 8-Br-cAMP
(14,31,33). from
cyclase
the component
cells
extracts
aq
chromatography.
cells
of Ca
in MCT Cell
of l0
by column
release
activity Gr,3.
or presence
isolated
a13
of free to
regulate
of
oxygen their
C 566
Journal
of the American
Society
of Nephrology
a phosphodiesterase
thelial
cell
a number dins E
of
of the
lyl
inhibitor,
injury
clinical
and
ing
6. Measurement
GaqQ2o9L.
Cells
the beginning and
of cGMP
were of the
replaced
with
in MCT
serum-deprived
cells
expressing
24 h before
experiments,
the
culture
fresh medium containing M), NaNO2 (NO 15 mM),
Ga1,
and
medium vehicle bradykinin
was
At
flow
and
the
gastric
removed
mm,
the
medium
was
harvested
and
cGMP, and cell protein was measured. cGMP/mg prot/20 mm. Each value triplicate
frozen
for
Values
measurement
are expressed
represents
the
mean
of as fmol
±
SEM
of
experiments.
phritis
(25).
creased.
PGE,,
sumed
to result
from
Na,K-ATPase could
flow
be
ATP
production
by
whether
variable
Under
normal
absent,
NO
tive
in tissue when
conditions,
that
siveness
of
and
of
increased injury,
those
achieved
tions,
NO
is toxic
(5,7).
High
intestinal epithelial and damage DNA occur teins,
Ca
to
eNOS
tumor
levels
cell monolayers, (7,41). These
that
raise
cell
melanocyte-stimulating
cAMP
injury deter-
(25,29,45).
to epithelial
cells. is
are
by constitu-
regulated
by
reduces
mucus
production
With
Ca
adheof
to inflammation higher
or bNOS.
Under
these
condi-
microorganisms,
the
and
permeability
such
mechanisms
tissue
some
but
hormone,
not
production that
observations,
type
that
that cell
affects
first
injury
and
add
evidence
cell
injury-
occur
through
Agents
cyclase that
are
Ca
regardless
with
in counteracting in
re-
inhibit
required
influx
death
that
may
it simultaneously step
re-
mechanism
reducing Ca
their cAMP
presumably
this
redundant.
pathways
stimulates
Ga,3,
of
adenylyl
for
throm-
cAMP.
injury
Nevertheless,
is an important to
and
of signaling
a mechanism
and
and
by
cell
(18).
receptors
cell
pathway
and
by
effect
iNOS
our findings
another
complex
production activity
through
cytokines
Tissue
NO NOS
that
raise
inflammation
function.
receptor
and
extent
NO
as
cAMP
activity (45). In a cells, MSH acting
as thromboxane
agents by
cells,
induces
by
by stimulating
all
cell
the
of
such
be a tissue-specific
and
of
are
through
to influx of
the
NO
pro-
mechanisms
a variety
of
pathologic
conditions.
host
of
of pro-
Acknowledgments The authors discussions,
for
as misoprostol
to
to inhibit
system,
of
cAMP
such
iNOS
influx.
injury
lead
cells,
inhibits
that
duce
that
reduce ATP production, toxic effects of NO could nitrosylation
also Ca
duction
times
of
To these
preserve in
induction
influx
severity
cAMP
increased or
many
increase
that
low
to levels cells,
of NO
epithelial production
in response
is elevated with
the
levels
(7,1 1,12).
activity
induction
duces
may
in-
the ability
In insulinoma
of iNOS
that,
be blocked
blocks
cyclase
in is
system
inhibition
cells
by activation
explain
and 264.7
induction However,
indicate Ca
will
cyclase
adenylyl
substances
stimulate
effects
of LPS
expression
partially
(25).
muscle
results
elimi-
influx,
increases
tract
through production of peroxynitrite, or by other mechanisms (1,42).
Agents agonist)
GI
NOS NO
than tissues
and
the
that
regulates
bin
may
and in MCT
be
can
expression
at low
on the blood
and
is magnified
iNOS
adenylyl
the
blocks
in smooth
These
(28);
effects
effect
this
where
of the
(29).
inhibitors
reduce and NO
or bNOS)
NO
granulocytes,
cells
or tissue
(eNOS
setting,
because
this
flow
the lethal
iNOS expression cell line, RAW
cAMP
in these
renal
by mecha-
of glomerulone-
inhibited
inflammation
analogues inhibit macrophage-like
cAMP
blood
and
Ga13,
activate
to reduce
cytokines
includ-
reduce
failure
is acutely
system,
ability
proinflammatory
NOS
flow
iNOS
by
depend wherein
injury,
or beneficial when
reduced
in Ca1.
blood
is controlled
of NOS
In this
epithelial
is harmful
production
forms
(5,7).
INOS
NO
and
of NO are complex and of NO. In whole animals,
however,
is pre-
initiated
a rise
nated as a variable, NOS inhibitors (1 ,7). The level of iNOS expression mines
in hypoxia
swelling
exacerbated
is an important injury;
swelling
However,
The cellular effects type and the level
exacerbate
Cell
decreased
activity.
mechanism
cell
(2).
activity
and Ca influx
substances
of
of
(27);
of insults,
(44);
in a model
reduces
express
activity
by
is compromised
NOS
The
NOS
after
(30).
cyclase
that
of rejection suppression
renal
independent
by (and
is independent
drugs
Pentoxyphylline
cells,
cells
a variety
of macrophages
of the adenylyl the
against
of ischemic
to be
permeability
that
immune
in
reduce
Misoprostol
incidence
involves
mucosa
in mice
through
volume
probably
appear
infiltration
In Neo
(BK l0 (BK lOK M and L-NAME 5 x l0 M), bradykinin and LY-83583 (BK 10-8 M and LY 2.5 X l0 M), and forskolin (Forsk l0 M and IMBX 2 x l0 M) and was returned to the incubator. After 20 kinin
that
the
adeny-
IFN-y
and
cAMP
(43).
a mechanism
in models
reduce
stimulate TNF,
cellular
anti-inflammatory
injury
in sepsis
(control), bradyand L-NAME
raise
in
Prostaglan-
in endothelial
reduce by
nonsteroidal
nisms
the experiments.
agonists)
transplantation
tubular
Figure
that
epi-
reactions
situations. of IL-l,
rearrangement
PGE1
preventing
misoprostol)
increases
protect
ccq
Agents
cytoskeletal
blood
a13
and
inflammation-induced
kidney
Neo
(26).
of
inflammatory
production
blocking
other
capable
experimental
(PGE,_3
reduce
macrophages
I!
and
E series
cyclase
are
and of suppressing
(a PGE1
or pentoxyphylline,
measuring
tance. DK39298,
thank Craig
This
cGMP, work
Paul Sternweis
Tisher
was
F32-GM15359,
for
and
and Shmuel
persistent
Paul
supported
McLeroy
for
by grants
GM31954,
Muallem
encouragement,
the
expert
from
for helpful
Susan
the
American
DuBois
technical NIH Heart
assis-
DK41726, Associa-
Inhibition
tion grant
with
funds
from
from
the
Florida
the National
Affiliate
Kidney
(RTM),
Foundation
and
by a training
20.
(KK).
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Offermanns
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