From the
Nitric oxide synthase (NOS) isoforms are discovered in an
increasing variety of cell types with different roles in signaling. The
inducible NOS (i.e. iNOS or NOS II) is expressed in cardiac
myocytes in response to specific cytokines. Independent of iNOS
induction, however, receptor-dependent signaling is modulated by a
constitutive nitric oxide (NO) synthase isoform in these cells
(Balligand, J. L., Kelly, R. A., Marsden, P. A., Smith, T. W., and
Michel, T.(1993) Proc. Natl. Acad. Sci. U. S. A. 90,
347-351). We now show that cardiac myocytes constitutively
express the endothelial isoform of NO synthase (ecNOS or NOS III).
Transcripts for NOS III were detected by Northern blot in myocyte
extracts using as a probe a polymerase chain reaction-generated cDNA
amplified with isoform and species-specific primers. In subcellular
fractionation experiments, a calcium-sensitive NO synthase activity was
present primarily in the particulate fraction, coinciding with the
distribution of NOS III analyzed by protein immunoblotting. The
localization of NOS III within cardiac myocytes was further
demonstrated by immunohistochemistry. The functional role of NOS III
was explored by analyzing the effects of NOS inhibitors on single
myocyte L-type calcium current and contractility. Inhibition of NOS
blocked the attenuation by carbamylcholine of the increases in both
parameters induced by
Three NO
Nitric oxide regulates
cardiac muscle function in cardiac myocytes in
vitro(12, 13, 14, 15) , in isolated
cardiac muscle preparations (16), and in hearts examined in situ in experimental animals (17). Even in the absence of NOS II
induction, inhibition of NO synthase modifies the contractile
responsiveness of cardiac myocytes to autonomic nervous system
agonists, implying the presence of a constitutively active NO synthase
isoform in heart muscle(12, 17) . However, the specific
cell type(s) producing NO to alter cardiac myocyte function in vivo or in vitro has not been definitively identified, nor has
the identity of the isoform of NOS constitutively expressed in cardiac
myocytes previously been determined.
In this study, we characterize
the constitutive isoform of NO synthase in cardiac myocytes as that
originally identified in endothelial cells (10) (i.e. NOS III)
and provide evidence for its role in mediating muscarinic cholinergic
and
Total cellular homogenates from approximately
2
For cell
fractionation experiments, homogenates were prepared as described
above. Aliquots were ultracentrifuged for 1 h at 100,000
The NOS III
PCR product was purified by agarose gel electrophoresis, cloned into
pBluescript (Stratagene), and used to transfect DH5a Escherichia
coli. The nucleotide sequence was determined for cDNA inserts from
three positive clones on both strands by the dideoxy chain termination
technique using a Sequenase kit (U. S. Biochemical Corp.).
For Western blotting,
aliquots of whole homogenates or subcellular fractions of ventricular
myocytes were run on a 12% SDS-polyacrylamide gel and electroblotted
onto nitrocellulose. Equal transfer among lanes was verified by
reversible staining with Ponceau red. Western analysis was performed as
previously described (7) using a mouse monoclonal antibody
raised against a polypeptide (residues 1030-1209) of the human
NOS III protein (Transduction Laboratories).
All three NOS isoforms characterized to date depend upon
calmodulin activation, but NOS I and NOS III are distinguished from NOS
II by their calcium sensitivity for the binding of activator calmodulin
and enzymatic activity within the physiological range for intracellular
calcium(6) . A constitutive NO synthase activity was detected
and quantified in whole extracts of freshly isolated adult rat
ventricular myocytes, and this activity was dependent upon the presence
of calcium (Fig. 1A). Duplicate extracts were assayed
both in the presence of a maximally activating concentration of calcium
(1 mM) and in a nominally calcium-free buffer containing EGTA
(2 mM). This calcium-dependent NO synthase activity was about
one-tenth of the calcium-independent NO synthase activity we measured
in isolated adult ventricular myocytes after induction of NOS II
activity with interleukin-1
The three isoforms of NOS are encoded by
different genes(1, 4, 5, 10) . We have
shown previously that NOS II is expressed after cytokine stimulation in
purified primary isolates of adult ventricular myocytes but cannot be
detected in untreated cells(7) . We developed isoform- and
species-specific oligonucleotide probes to amplify cDNA for the
constitutive isoforms of NOS in cardiac myocytes by reverse
transcription-PCR. Amplimers specific for the rat NOS I or NOS III were
designed according to the previously published rat brain cDNA sequence
(1) and a partial rat endothelial cDNA sequence deposited in GenBank
(accession no. RNU02534), respectively. The results of the
amplification reactions using RNA from purified (typically 95-98%
homogenous) ventricular myocytes are shown in Fig. 2. No PCR
product was obtained from freshly isolated ventricular myocytes when
amplified with primers specific for either rat brain (NOS I) or
inducible NOS (NOS II). Products amplified with these primers were
present in the appropriate positive controls, namely rat brain cDNA
(for NOS I) and cDNA from rat cardiac myocytes treated with cytokines
(for NOS II), as previously reported(7) . Using amplimers
specific for NOS III, a single 324-bp product was amplified from
reverse-transcribed RNA from purified ventricular myocytes but not from
negative controls consisting of samples processed in parallel
containing either no cDNA or RNA samples processed in the absence of
reverse transcriptase. Cloning and nucleotide sequence analysis of the
PCR product (three independent clones, sequenced on both strands)
revealed a sequence 84 and 91% identical to the originally described
bovine endothelial NOS III isoform at the nucleotide and amino acid
levels, respectively. Of note, the 324-bp-specific product was
amplified from cytokine-treated cardiac myocytes in which a NOS II
product was also identified, as previously reported(7) .
Previous studies have
demonstrated a calcium-sensitive NO synthase activity in human cardiac
muscle (28) and in isolated cardiac myocyte preparations from
different species(12, 29) . In the present study, we
identify NOS III (ecNOS) as the isoform that is constitutively
expressed in adult ventricular myocytes at the transcript and protein
levels, including immunohistochemical localization in single cells in vitro and in vivo. Endothelial cells from the
heart microvasculature strongly expressed NOS III in situ, as
detected by immunohistochemistry.
Since its original
cloning from endothelial cells, NOS III has also been identified in
hippocampal pyramidal neurons(11) . Among other cell types (for
review, see Ref. 6), NOS I has also recently been localized in fast
twitch skeletal muscle cells(3) . We excluded the presence of
NOS I in ventricular myocytes by Northern analysis and reverse
transcription-PCR using species-specific probes. NOS II is expressed in
response to several inflammatory stimuli in a variety of cell types,
including neonatal and adult rat cardiac
myocytes(7, 9) . Together with our previous
data(7) , this study shows that ventricular myocytes can express
both NOS II and III (Fig. 2) and that specific cytokines
down-regulate the abundance of NOS III transcript in ventricular
myocytes. Determination of whether this is mediated through changes in
the half-life of the NOS III transcript, as previously shown in aortic
endothelial cells(23) , will await further experiments.
Overall, our data further support the concept of a more widespread
tissue distribution and diverse signaling roles for NOS III beyond the
paradigm originally described in the vascular wall.
-adrenergic stimulation. We conclude that
NO-dependent parasympathetic signaling is mediated by NOS III in
cardiac myocytes.
(
)synthase isoforms have been
identified to date, and although originally classified according to the
tissue of origin, all three have been subsequently found in disparate
cell types. The constitutive isoform first identified in brain (ncNOS)
(1) has recently been found to be expressed in skeletal muscle cells
(2, 3). The inducible isoform, or iNOS, was first characterized in a
macrophage cell line (4, 5) and is now known to be
expressed in a wide variety of cells after stimulation with
inflammatory mediators(6) , including cardiac
myocytes(7, 8, 9) . The constitutive isoform
originally characterized in endothelial cells (ecNOS) (10) has
now been identified in rat hippocampal pyramidal cells(11) . A
numerical nomenclature recently proposed by Nathan and Xie (6) identifies ncNOS, iNOS, and ecNOS as NOS I, NOS II, and NOS
III, respectively, and will be used here.
-adrenergic regulation of voltage-dependent calcium current
and contraction in isolated ventricular myocytes.
Cell Culture
Purified adult rat
ventricular myocytes were prepared as previously described(18) ,
plated on laminin, and cultured for 24 h, unless specified otherwise.
Measurement of NO Synthase
Activity
Myocyte NOS activity was quantified by measuring
the conversion of L-[H]arginine to L-[
H]citrulline, as previously
described(7) .
10
myocytes were prepared by sonication in a
buffer containing 0.5 mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 1 mM tetrahydrobiopterin, 1 mM leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, and 20
mM Tris-HCl (pH 7.4, 37 °C). After centrifugation of the
homogenates (1,000
g, 5 min, 4 °C) and
determination of the protein content (Bradford assay), aliquots
(approximately 80 µg) were added to 200 µl of buffer containing
50 mM Tris-HCl (pH 7.4), tetrahydrobiopterin (10
µM), dithiothreitol (1 mM), calmodulin (10
µg/ml), FAD (4 µM), FMN (4 µM), NADPH (1
mM), L-arginine (2 µM), purified L-[
H]arginine (2
10
cpm, Amersham Corp.) and incubated for 60 min at 37 °C in the
presence (1 mM CaCl
) or absence of calcium (i.e. nominally calcium free, with 2 mM EGTA).
Parallel reactions were analyzed in the presence of 1 mML-N-monomethylarginine, an inhibitor of NO synthase.
The reaction was stopped by addition of ice-cold sodium acetate (20
mM, pH 5.5) containing 1 mML-citrulline, 2
mM EDTA, and 0.2 mM EGTA, and the L-[
H]citrulline content was determined
by anion-exchange chromatography. The calcium-sensitive activity was
calculated by subtraction of the activities measured in the presence
and absence of calcium after normalization for protein content from at
least three separate experiments, each in duplicate.
g. The supernatant (cytosolic fraction) was kept on ice for
subsequent assay, and the pellet was resuspended in a buffer containing
50 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, 0.1 mM EDTA, 2 mM
-mercaptoethanol, protease inhibitors (as
above), 10% glycerol, and 1 M KCl. After a second
ultracentrifugation (100,000
g, 30 min), the second
pellet was resuspended in the same buffer containing 20 mM CHAPS, but without KCl, and sonicated; aliquots were assayed as
above. About 70% of the total activity was recovered in both fractions.
PCR Cloning and Sequencing of NOS III mRNA from
Cardiac Myocytes
The reverse transcription reaction was
primed with random hexamers and done as previously
described(7) . For PCR amplification (35 cycles), the following
oligonucleotide primers were used: 5`-GGGCCAGGGTGATGAGCTCTG-3` (sense)
and 5`-CCCTCCTGGCTTCCAGTGTCC-3` (antisense) for NOS III;
5`-GAGGCACCCCAACTCTGTG-3` (sense) and 5`-TCCTGATTCCCGTTGGTGTGG-3`
(antisense) for NOS I; for NOS II, the primers were as described in
Ref. 7. An annealing temperature of 60 °C was used.
Northern and Western Blotting
Northern
blots were performed as previously described(7) . The 324-bp NOS
III cDNA insert was P radiolabeled using random
hexanucleotide primers according to the manufacturer's protocol
(Boehringer Mannheim). Blots were washed in 0.2
SSC and 0.1%
SDS up to 65 °C and then autoradiographed at -70 °C with
intensifier screens for 12-14 h.
Immunohistochemical Analysis
Cryostat
sections of heart tissue or cytospin preparations of suspensions of
freshly isolated myocytes and cultured myocytes that had been cultured
in defined medium for 24 h were fixed in buffered 2% paraformaldehyde
for 5 min, followed by 10 min in 100% methanol. After rinsing,
immunostaining was performed by sequential application of primary
antibody (mouse anti-human NOS III, 5 µg/ml (see above)), goat
anti-mouse IgG (1/50, Steinberger Monoclonals, Inc.), mouse
peroxidase-anti-peroxidase complex (1/100, Steinberger Monoclonals,
Inc.), followed by labeling with the chromogen diaminobenzidine (Sigma)
and HO
. The slides were washed with water,
counterstained with hematoxylin, dehydrated, and mounted for light
microscopy. For negative controls, mouse myeloma IgG (Sigma) was used
as the primary antibody, and the samples were processed identically.
Measurements of Calcium Current and
Contraction
Adult rat ventricular myocytes were isolated as
described above. All cells selected for electrophysiological study had
a resting potential of -75 mV or more negative in buffer
containing 5.4 mM [K]
. Unloaded cell
shortening was recorded with a video edge detector(12) . The
nystatin-perforated (19) patch-clamp technique in the whole cell
configuration (20) was used to allow simultaneous recordings of
single L-type Ca
current (I
)
along with cell shortening. The membrane-ruptured patch-clamp technique
was used to internally dialyze the cell with methylene blue. Transient
outward current (I
), which interferes with the
measurement of I
, was blocked by
4-aminopyridine (3 mM, in nystatin-perforated experiments) or
by using Cs
to substitute for K
during internal dialysis.
and interferon
(7) .
Figure 1:
NO synthase
activity and subcellular distribution in adult rat ventricular
myocytes. A, whole cell extracts from purified cardiac
myocytes were assayed for the enzymatic conversion of L-[H]arginine to L-[
H]citrulline in the presence and
absence of calcium (see ``Experimental Procedures''), and the
calculated calcium-sensitive activity is compared with background
levels obtained from boiled extracts (left) or extracts
incubated in the presence of the NO synthase inhibitor L-N-monomethylarginine (L-NMMA) (right). B, the same extracts were fractionated by
ultracentrifugation (see ``Experimental Procedures''), and
the activity, assayed as described above, is compared in the cytosolic
and particulate fractions.
The NO synthase isoforms are also distinguished on the basis of
their subcellular distribution. NOS III is found primarily in the
particulate subcellular fraction of endothelial
cells(21, 22) , but in most tissues, NOS I and NOS II
are cytosolic(6) , although a recent report has described
association of NOS I with skeletal muscle membranes(3) . To
examine the pattern of distribution of constitutive NOS activity in
cardiac myocytes, whole cell extracts were resolved into cytosolic and
particulate fractions by ultracentrifugation and assayed for NOS
activity (Fig. 1B). Most of the calcium-sensitive NO
synthase activity segregated with the particulate fraction. Therefore,
both the calcium dependence and subcellular localization of the cardiac
myocyte constitutive NOS activity suggested that the responsible NOS
isoform was either NOS I or NOS III. We pursued molecular cloning and
immunohistochemical analyses to definitively identify the isoforms
expressed by these cells.
Figure 2:
Identification of transcripts for NO
synthase isoforms in adult rat ventricular myocytes by reverse
transcription-PCR. Poly(A) RNA from purified adult rat
ventricular myocytes or total RNA from rat brain was reverse
transcribed with random hexamers and amplified by PCR with rat-specific
oligonucleotide primers for NOS I (ncNOS), NOS II (iNOS), or NOS III
(ecNOS) and analyzed by agarose gel electrophoresis and ethidium
bromide staining. lanes 1-4, samples from freshly
dissociated, untreated ventricular myocytes; no PCR product was
obtained from the control sample in the absence of reverse
transcription (lane 1); a 324-bp single band was amplified
from cDNA with NOS III (ecNOS) primers (lane 2); with the same
cDNA, NOS I (lane 3)- or NOS II (lane 4)-specific
amplimers gave no product. Lane 5, cDNA from rat brain; NOS I
primers amplified the expected 587-bp product (positive control). lanes 6-7, cDNA from purified myocytes treated for 18 h
with recombinant human interleukin-1
(2 ng/ml) and recombinant
mouse interferon
(500 units/ml); a 324-bp band was obtained with
NOS III (ecNOS) primers (lane 6) and a 217-bp band with the
iNOS primers (lane 7, Ref. 7).
To
quantitate the abundance of the corresponding transcripts, the cloned
NOS III cDNA was used as a probe in Northern analyses of total RNA from
purified primary isolates of adult cardiac myocytes under high
stringency hybridization conditions. A transcript of a size (4.6
kilobase pairs) consistent with that of NOS III was detected (Fig. 3). Notably, the abundance of the transcript for NOS III
was markedly decreased after 24 h of treatment of adult myocytes with
cytokines, similar to the effect described in other cell types (Refs.
10 and 23 and Fig. 3).
Figure 3:
Identification of NOS III transcripts in
adult rat ventricular myocytes by Northern analysis. Total RNA from
purified ventricular myocyte primary isolates was analyzed by Northern
blot using the 324-bp NOS III (ecNOS) cDNA fragment obtained by PCR
(see Fig. 2) as a probe. Lane 1, RNA from untreated
ventricular myocytes. lane 2, RNA from ventricular myocytes
treated for 24 h with recombinant human interleukin-1 (2 ng/ml)
and recombinant mouse interferon
(500
units/ml).
Using a monoclonal antibody directed
against human NOS III, we detected a protein that was consistent in
size (130 kDa) with NOS III in whole extracts of purified myocytes by
Western analysis (Fig. 4). Quantitative comparison of the
abundance of NOS III protein in subcellular fractions of cardiac
myocytes revealed that most of the protein was present in the
particulate fraction, consistent with the distribution of NOS enzymatic
activity as described above.
Figure 4:
Detection of NOS III protein by Western
analysis in extracts of adult rat ventricular myocytes. Equal amounts
of protein from whole extracts or subcellular fractions of purified
myocytes were analyzed by Western blotting using a monoclonal antibody
specific for NOS III (ecNOS) (see ``Experimental
Procedures''). The abundance of the specific 130-kDa protein is
shown for lanes1-5. Lanes 1 and 4, whole extract of ventricular myocytes; lanes 2 and 3, cytosolic and particulate fractions of ventricular
myocytes, respectively; lane 5, human endothelial cell extract
(positive control).
The same antibody was used for the
immunohistochemical localization of NOS III in single ventricular
myocytes in culture and in sections of rat ventricular myocardium (Fig. 5). No staining was detectable above background when the
primary antibody was omitted or nonspecific mouse myeloma IgG was used.
With the NOS III-specific antibody, a strong signal was detected in
endothelial cells from the microvasculature in myocardial sections. In
addition, a distinct signal was observed in cardiac myocytes both in
primary isolates and in situ.(
)No other
cell type stained positively.
Figure 5:
Immunohistochemical detection of NOS III
protein in freshly isolated primary isolates of adult rat ventricular
myocytes and sections of adult rat ventricular myocardium.
Immunostaining of sections of rat heart tissue (A and B) and freshly isolated (C and D) and
cultured (E and F) rat ventricular myocytes. A, rat heart myocytes in situ show distinct
immunostaining with anti-NOS III (ecNOS); note strongly positive
adjacent capillaries (arrow). C, cytospin
preparations of unpurified fresh rat heart cell suspensions show
prominent immunostaining of both myocytes and endothelial cells. E, purified rat ventricular myocytes prepared, as previously
described (18) and plated on laminin and cultured for 24 h, show
prominent immunostaining with anti-NOS III (ecNOS). B, D, and F, no labeling is seen with control IgG either
in rat heart tissue or in freshly isolated or cultured myocytes
(respectively). Original magnification (200), except for C (400
) is shown.
We have shown previously that NO
antagonists and inhibitors of NO synthase attenuate the effect of
muscarinic cholinergic agonists on the spontaneous beating rate of
neonatal rat cardiocytes(12) . The regulation by NOS III of both
L-type calcium current and contractility in response to cholinergic and
adrenergic agonists was further studied in single adult rat ventricular
myocytes using NO antagonists and a NO synthase inhibitor (Fig. 6). Carbamylcholine, a muscarinic cholinergic agonist that
is resistant to degradation, attenuated the isoproterenol-stimulated
increase in I and amplitude of unloaded cell
shortening in paced ventricular myocytes (Fig. 6A).
After extracellular perfusion (Fig. 6B) or internal
dialysis (Fig. 6C) of the cell with methylene blue, the
antagonistic effect of carbamylcholine on
-adrenergic stimulation
of both I
and the amplitude of unloaded cell
shortening was totally abolished. Internal dialysis with the NO
synthase inhibitor L-N-monomethylarginine also
completely abrogated carbamylcholine's effect on I
(Fig. 6D). A similar reversal
of carbamylcholine's effect on contractility was also observed
after NO blockade with hemoglobin (Fig. 6E).
Figure 6:
NO antagonists block the
carbamylcholine-induced attenuation of L-type calcium current (I) and cell shortening in adult rat
ventricular myocytes stimulated with isoproterenol. Simultaneous
recordings of I
and amplitude of shortening
obtained in single rat ventricular myocytes are shown. Panels A and B, superimposed traces showing changes in cell
shortening (in arbitrary units, aU, top) and I
(bottom) under control (a),
isoproterenol alone (ISO, 1 µM) (b), and
isoproterenol and carbamylcholine (CCh, 1 µM) (c). Results illustrated in panels A and B were obtained in the absence and presence of 10 µM methylene blue in the extracellular buffer, respectively. Panel C, superimposed current traces recorded under control (a), ISO (1 µM) (b), and CCh (1
µM) (c) in the presence of ISO in a cell
internally dialyzed with methylene blue (20 µM). Panel
D, the same experiment as in C, in a cell internally
dialyzed with L-N-monomethylarginine (1 mM). Panel E, graphic representation of mean changes in cell
shortening amplitude, relative to the maximum response to isoproterenol
(1 µM, 100%), in the presence of ISO alone, isoproterenol
with carbachol (1 µM, ISO + CCh),
isoproterenol with carbachol in the presence of methylene blue (10
µM, ISO + CCh + MB), or hemoglobin (10
µM, ISO + CCh + HB). *, p <
0.05 compared with ISO + CCh. Note that ISO significantly
increased I
and contraction and that both
actions were attenuated by CCh (panel A, n = 11).
Methylene blue and hemoglobin block the attenuating effects of CCh on
force of contraction (panels B and E) and I
(panels B and C, n = 4 and 3 cells, respectively), as does L-N-monomethylarginine (panel D, n = 7 cells).
The
regulation of voltage-dependent calcium channels by sympathetic
agonists has been clearly demonstrated as a control mechanism of the
rate and force of contraction in the mammalian
ventricle(24, 25) . The mechanism by which acetylcholine
modulates the adrenergic effect on cardiac action potential differs
according to the region of the heart, with attenuation of
-adrenergic agonist increases in I
being
predominant in the mammalian ventricle(26, 27) . In this
study, we provide evidence that the cholinergic attenuation of
-adrenergic increases in I
in ventricular
myocytes involves nitric oxide, as shown by the abolition of
carbamylcholine's effect with methylene blue and hemoglobin, both
blockers of NO, and L-N-monomethylarginine, a NO
synthase inhibitor. That this has important functional consequences is
shown by a quantitatively similar effect of methylene blue and
hemoglobin on the cholinergic attenuation of the contraction of the
same cells after isoproterenol stimulation.
(
)However,
given the low percentage of non-myocytes (typically 2-5%,
including endothelial cells) in the preparations of purified cardiac
myocytes used in this study, it is unlikely that any substantial
component of the NOS signal detected by Northern and Western analysis
came from endothelial contaminants. The expression of type III NOS in
cardiac myocytes themselves is clearly demonstrated by
immunohistochemical detection in single cells.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.