(Hypertension. 1995;25:421-430.)
© 1995 American Heart Association, Inc.
Articles |
Effects of Interleukin-1ß and Nitric Oxide on Cardiac Myocytes
From the Hypertension and Vascular Research Division, Henry Ford Health Sciences Center, Detroit, Mich.
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Abstract |
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Abstract Using cultured neonatal ventricular myocytes, we investigated whether nitric oxide (NO) directly influences myocyte growth. Treatment of myocytes with phenylephrine stimulated growth, as indicated by increases in atrial natriuretic factor, brain natriuretic peptide (BNP) mRNA and BNP secretion, activator protein 1 activity (activation of early-response genes), and total cellular protein content. NO was stimulated by treatment of myocytes with interleukin-1ß (IL-1ß) or was generated by the NO donor nitroglycerin, and its effects on total protein content and BNP secretion were measured. Treatment of cardiocytes with 3.4 nmol/L IL-1ß for 24 hours stimulated NO (nitrite) production by threefold, which resulted from an increase in the inducible isoform of NO synthase mRNA. Dexamethasone inhibited IL-1ß induction of nitrite production, whereas the protein kinase C inhibitor staurosporine had no effect. IL-1ß had no effect on either basal or phenylephrine-stimulated protein content but inhibited phenylephrine-stimulated BNP secretion. Nitroglycerin (10-7 to 10-3 mol/L) dose-dependently increased NO production; however, only the highest dose (10-3 mol/L) reduced basal and phenylephrine-stimulated total protein content and BNP secretion. cGMP, a second messenger of NO, had no effect on either basal or phenylephrine-stimulated BNP secretion or total protein content. In conclusion, our data indicate that BNP mRNA is stimulated by phenylephrine as shown previously for atrial natriuretic factor. Although both BNP and total protein content are increased by phenylephrine, these effects are not inhibited by NO. However, IL-1ß inhibits phenylephrine-stimulated BNP secretion but not total protein content, suggesting that regulation of BNP secretion can be dissociated from total protein synthesis during myocyte growth.
Key Words: brain natriuretic peptide • heart hypertrophy • interleukins • cytokines • nitric oxide • muscle, cardiac
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Introduction |
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Nitric oxide (NO) is generated from the L-arginine (L-Arg)–NO pathway by the enzyme NO synthase (NOS), which exists in at least two isoforms: one is constitutive and Ca2+-calmodulin dependent (cNOS), and the other is inducible (iNOS) and independent of calcium. iNOS can be induced by cytokines (eg, interleukin-1ß, [IL-1ß]) in vascular smooth muscle cells1 2 and cardiac myocytes3 and may have important physiological and pathological effects. In this respect, Roberts et al3 reported that IL-1ß decreases the beating rate of neonatal myocytes; this effect correlates with induction of NOS, a subsequent rise in NO production, and increases in cGMP. Similarly, Balligand et al4 reported that induction of an autocrine NO-signaling pathway depresses the contractile response of rat cardiac myocytes to ß-adrenergic stimulation.
In addition to its effect on cardiac myocyte contractility, NO has been postulated to play a role in cardiac growth. One in vivo study5 suggests that nitroso-containing vasodilators can decrease left ventricular mass by nonhemodynamic mechanisms, whereas others6 7 suggest the opposite, ie, that inhibition of the endogenous NO system decreases left ventricular mass. In contrast, stimulation of guanylate cyclase by NO blocks proliferation of vascular smooth muscle cells in vitro.8 9 Although these studies suggest that nitroso-containing vasodilators may oppose cell growth under certain conditions, they do not describe the biochemical or molecular basis for these effects. Regarding the studies on cardiac hypertrophy, it is uncertain whether the effects of NO occur at the level of the cardiac myocyte or in other cell types.
Cardiac myocyte growth can be induced by
1-adrenergic stimulation.10 Features of
this well-characterized model include induction of (1) early response
genes, including the proto-oncogenes c-fos and
c-jun11 ; (2) muscle-specific genes, such as
ß-myosin heavy chain12 and myosin light
chain-213 ; (3) the cardiac hormone atrial natriuretic
factor (ANF)13 ; and (4) myocyte growth assessed as
increases in RNA and total protein.12 Hypertrophy is
accompanied in vivo by enhanced expression of the genes encoding ANF
and brain natriuretic peptide (BNP), and upregulation of these genes is
considered a marker of hypertrophy.14 Our studies examined
phenylephrine regulation of BNP and ANF mRNA, BNP secretion, and total
protein content. We further investigated whether IL-1ß induction of
NO and the NO donor nitroglycerin influence the growth of cardiac
myocytes after 1-adrenergic stimulation. We also
measured nitrite production as an index of NO generation in ventricular
myocytes treated with either nitroglycerin or IL-1ß and characterized
the NOS mRNA associated with this particular cell type. The functional
significance of NO production in ventricular myocytes was assessed by
determining whether NO production could inhibit
phenylephrine-stimulated protein content and BNP secretion.
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Methods |
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Preparation of Ventricular Myocytes and Cell Culture
Our protocol was approved by the Henry Ford Hospital Care of Experimental Animals Committee and is in accordance with US federal guidelines. Six litters of 1- to 2-day-old neonatal Sprague-Dawley rats (Charles River, Kalamazoo, Mich) were killed, and their hearts were removed. Primary cultures of neonatal ventricular myocytes were prepared as described previously.15 Nonmyocyte contamination was minimized by differential attachment at a preplating stage and by culturing myocytes in medium containing 0.1 mmol/L bromo-deoxyuridine. Ventricular myocytes were plated at a density of 1x105 cells/cm2 in Dulbecco's modified Eagle medium (DMEM, GIBCO/BRL) and cultured as described.15 Test compounds were added for the times indicated in "Results." Phenylephrine stimulated beating of the cells.
RNA Isolation and Analysis
Total RNA was analyzed by Northern blot. GAPDH mRNA was used as
an internal control as described previously.15 To detect
iNOS mRNA, a partial rat cDNA clone for iNOS was generated by use of
primers derived from the mouse macrophage NOS cDNA sequence. The sense
and antisense primers correspond to sequences at 1467 to 1493 and 1892
to 1915 bp, respectively.16 Total RNA from
IL1-ß–treated ventricular myocytes was reverse-transcribed into cDNA
with random oligohexamers (Boehringer Mannheim), and the cDNA was
amplified by the polymerase chain reaction (PCR) for 30 cycles
(denaturation at 95°C for 1 minute, annealing at 55°C for 2
minutes, and extension at 72°C for 3 minutes). Amplification resulted
in a cDNA of the expected size (448 bp), and the identity of the
fragment was verified by restriction mapping and partial sequencing.
For detection of constitutive NOS RNA (cNOS or endothelial cell NOS
[eNOS]), a full-length bovine cDNA probe was provided by Dr William
Sessa of Yale University.17 ANF and BNP cDNAs and
hybridization and washing conditions were described
previously.15 Nylon filters were exposed to Kodak X-Omat
AR film for 1 to 4 days. Densitometry (model GS-670, Bio-Rad) was used
to quantitate mRNA and normalize it to GAPDH mRNA. Appropriate
unsaturated film exposures were used for densitometry, whereas darker
exposures were used to make figures if necessary.
Transfection and Luciferase Assay
Freshly isolated ventricular myocytes were transiently
transfected by electroporation. 
73coll-Luciferase contains the
activator protein 1 (AP-1) site (phorbol ester response element, TRE,
which binds protein products of the jun and fos
gene families) from the collagenase gene cloned upstream of luciferase.
In this construction, the endogenous AP-1 site in the pUC vector
backbone has been deleted.18 73coll-Luciferase (10
µg) and RSV–ß-galactosidase (2 µg, Rous sarcoma virus enhancer
and promoter), an internal control, were used per
12x106 cells. Twelve to 24x106
cells and plasmid DNA were suspended in 0.4 mL phosphate-buffered
saline (PBS) supplemented with 0.1% glucose and electroporated in a
1-mL cuvette at 280 V and 250 µF with a Bio-Rad Gene Pulser. After a
5-minute incubation at room temperature, an additional 0.4 mL of
PBS-glucose was added. Cells were plated at a density of
2x105 cells/cm2 in DMEM containing 10% fetal
bovine serum and 0.1 mmol/L bromo-deoxyuridine
(2x106 cells per well of a six-well plate). After
18 hours, the medium was changed to serum-free DMEM. After 24 hours,
stimulatory agents were added for 3 or 6 hours. After treatment, cells
were washed with PBS and incubated with 0.25 mL 1x reporter lysis
buffer (Promega) at room temperature for 15 minutes. The cellular
lysates were frozen in liquid nitrogen, thawed, centrifuged to remove
debris, and then assayed for either luciferase (Luciferase Assay
System, Promega) or galactosidase (Galacto-Light chemiluminescent
assay, Tropix) activity in an OptoComp 1 luminometer (MGM Instruments
Inc). The manufacturers' protocols were followed for both assays.
Luciferase activity was normalized to ß-galactosidase and reported as
the level of increase (onefold, twofold, etc) versus untreated control.
For each experiment, each treatment was done in triplicate or
quadruplicate and each sample was assayed in duplicate.
Radioimmunoassay for BNP and cGMP
BNP secretion was measured from aliquots of medium by
radioimmunoassay (Peninsula Laboratories) as previously
described.15 Intracellular cGMP was assayed 0 to 24 hours
after treatment. At each time point, secretion medium was removed from
myocyte cultures, and fresh serum-free medium containing 1 mmol/L
3-isobutyl-1-methylxanthine was added. After the plates were incubated
at 37°C for 30 minutes, the medium was removed and the cells were
lysed with ice-cold methanol. cGMP content was analyzed by
radioimmunoassay (Biomedical Technologies) as previously
described.19 The lowest level of cGMP detectable in this
assay is 4 fmol.
Nitrite Production
Nitrite production, an index of NOS activity, was measured in
myocyte medium by the Greiss reaction.20 The plates were
read with an enzyme-linked immunosorbent assay plate reader at 505 nm.
Nanomoles of nitrite were determined by comparison to a standard curve
of NaNO2. The lowest level of NO2 detectable in
this assay is 5 nmol/mL.
To exclude the possibility that nitrite production was a function of fibroblast and endothelial cell contamination of our cultures, the following experiment was performed. A mixed population of cells taken before the preplating stage that consisted of myocytes, endothelial cells, and fibroblasts was treated with IL-1ß. Nitrite production from these cells was lower than that of the myocyte-enriched cultures (14.00±1.41 compared with 25.77±2.46 nmol NO2 per 106 cells, respectively). Thus, IL-1ß–induced nitrite production is greater in cultures enriched for myocytes than in mixed-cell cultures. This is consistent with a recent report.21
Measurement of Protein Content
Protein content was determined by incorporation of
[3H]leucine into trichloroacetic acid (TCA)–insoluble
material. Myocytes were grown under serum-free conditions for 24 hours,
after which they were treated with the appropriate compound for 24
hours in medium containing 2.5 µCi [3H]leucine (6.2
TBq/mmol=168 Ci/mmol, DuPont-NEN). Protein was precipitated by addition
of ice-cold 10% TCA and vacuum-filtered through GF/C filters
(Whatman). Filters were washed three times with 5 mL of 5% TCA and
70% ethanol and counted in 3 mL scintillation fluid (Insta-gel XF,
Packard Instrument Co) with a scintillation counter.
Statistical Analysis
Data are reported as mean±SEM, and the differences between
treatments were analyzed by a paired t test by Bonferroni's
adjustment for multiple comparisons. A value of P<.05 was
considered significant unless otherwise indicated in the figure
legends.
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Results |
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Effects of Phenylephrine on BNP and ANF mRNA and AP-1 Activity
We first addressed whether BNP mRNA was regulated by phenylephrine as has been shown for ANF mRNA. Fig 1A shows that phenylephrine stimulated BNP mRNA by 2.2±0.2-fold (n=9) at 24 hours and by 2.7±0.3-fold at 48 hours (n=7). Elevated BNP mRNA was seen as early as 6 hours after treatment (M.C.L., unpublished observations, 1994). To ensure that phenylephrine was working through the
-adrenergic receptor, cells were treated simultaneously with
phenylephrine and 1 µmol/L prazosin (an 1-adrenergic
receptor antagonist). Fig 1B shows that prazosin inhibited
phenylephrine stimulation of BNP mRNA. Also, the intracellular
signaling system activated by phenylephrine was studied by use of the
protein kinase C inhibitor staurosporine (0.01 µmol/L), which
inhibited the effect of phenylephrine (Fig 1B).
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We verified our in vitro model by examining the effects of phenylephrine on ANF mRNA. Fig 1C shows that ANF mRNA was stimulated at both 24 and 48 hours of treatment.
Phenylephrine previously was shown to stimulate the early response genes c-jun and c-fos and AP-1 activity. To verify our model further, we determined whether AP-1 activity was stimulated by phenylephrine by use of a luciferase reporter gene containing an AP-1 binding site.17 Fig 1D indicates that phenylephrine activated AP-1 binding (as measured by an increase in luciferase activity) by 1.9±0.06-fold and 2.1±0.12-fold at 3 and 6 hours, respectively, which compared favorably with phorbol ester (phorbol 12-myristate 13-acetate [PMA]) treatment (2.4±0.15-fold and 3.0±0.24-fold increases at 3 and 6 hours, respectively).
Preliminary experiments were done to determine whether phenylephrine stimulated intracellular and secreted BNP in addition to BNP mRNA. In response to 24 hours of phenylephrine, secreted BNP increased from 19.8±1.4 to 39.8±3.9 ng per 106 cells, and intracellular BNP increased from 90.6±16 to 199.6±21 ng per 106 cells (n=8 separate wells). Thus, the increase in BNP secretion reflected mRNA increases, and BNP secretion was monitored in subsequent experiments.
Effects of IL-1ß on Cardiac Myocytes
We next examined the effect of IL-1ß on NO production in cardiac
myocytes. Fig 2, top, shows the time course of NO
stimulation. Levels of nitrite in the medium were undetectable until 9
hours and by 24 hours were four times higher than those of untreated
myocytes (23.8±0.6 versus 5.8±0.1 nmol NO2 per
106 cells). To characterize the NOS isoform involved
in this response, we treated cells with IL-1ß plus either
nitro-arginine (N-Arg), an NO synthesis inhibitor; dexamethasone, a
known inhibitor of iNOS; cycloheximide, a protein synthesis inhibitor;
or staurosporine, a protein kinase C inhibitor (Fig 2, bottom). The
addition of N-Arg, dexamethasone, and cycloheximide reduced
IL-1ß–stimulated nitrite production by 57.2±11.4% (n=4),
47.6±6.6% (P<.025, n=6), and 43.5±18.2% (n=4),
respectively. Surprisingly, the addition of staurosporine did not
inhibit but tended to potentiate nitrite production (n=5,
P=.2).
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indicates control;
, IL-1ß. Bottom, Bar graph showing the effect of
10-6 mol/L dexamethasone (DEX), 20 µg/mL cycloheximide
(CYCLO),10-3 mol/L nitro-arginine (N-ARG), and
10-8 mol/L staurosporine (STAUR) on IL-1ß–stimulated
nitrite production over 24 hours. For IL+DEX, n=6 (where n is the
number of separate experiments); IL+CYCLO, IL-1ß plus cyclosporine
(n=4); IL+N-ARG, IL-1ß plus nitro-arginine (n=4); and IL+STAUR,
IL-1ß plus staurosporine (n=5). *P<.05 compared with
production from IL-1ß–stimulated control.To determine whether the stimulation of NO production by IL-1ß treatment resulted from the induction of iNOS mRNA, we extracted total RNA from treated cells and analyzed it by Northern analysis with a partial iNOS cDNA probe. iNOS mRNA increased 3 hours after IL-1ß treatment and persisted for at least 24 hours (Fig 3A). Densitometry revealed that iNOS mRNA, corrected to GAPDH, was induced 9 to 11 times control levels by 3 to 24 hours in this particular experiment. In seven separate cardiocyte preparations, iNOS mRNA was induced 23.8±11.4-fold after 24 hours of treatment. To test the specificity of our iNOS cDNA probe, RNA from bovine pulmonary artery endothelial cells, a rich source of cNOS mRNA, was hybridized with our probe, and no band was detected (Fig 3B). When this blot was rehybridized with an eNOS probe, mRNA for eNOS was readily detectable.
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In our next series of experiments, we studied the effect of IL-1ß on protein content and BNP secretion in control and phenylephrine-stimulated myocytes. Fig 4 shows that phenylephrine stimulated total protein by 49.1±8.24% (P<.001), whereas IL-1ß had no effect on either basal or phenylephrine-stimulated protein content. Fig 5A shows that phenylephrine stimulated BNP secretion by 62.9±16.3% (P<.001, n=22). Interestingly, IL-1ß alone had no effect on BNP secretion but abolished phenylephrine-stimulated secretion (P<.017, phenylephrine versus phenylephrine plus IL-1ß). Preliminary studies show similar results with ANF secretion (P.H., O.A.C., and M.C.L, unpublished observations, 1994). The decrease in BNP secretion was accompanied by increased NO levels (P<.001 for phenylephrine versus phenylephrine plus IL-1ß, Fig 5B). However, inhibition of NO synthesis with N-Arg did not reverse the inhibitory effect of IL-1ß, suggesting that NO was not responsible (23.4±3.6 ng BNP per 106 cells for phenylephrine plus IL-1ß versus 25.6±4.5 ng BNP per 106 cells for phenylephrine plus IL-1ß plus N-Arg, n=4).
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Effects of Nitroglycerin on Protein Content and BNP Secretion
Because IL-1ß can have many effects on cells in addition to
stimulation of NO production (see "Discussion"), we repeated our
experiments with the NO donor nitroglycerin. When myocytes were treated
with various concentrations of nitroglycerin for 0 to 24 hours, NO
accumulated in a time- and dose-dependent fashion (Fig 6,
left). At 10-7 and 10-5 mol/L
nitroglycerin, there was a delay before nitrite was detectable;
thereafter, nitrite accumulated to values of 8.99±1.39 and 19.35±0.06
nmol NO2 per 106 cells. NO produced by
10-5 mol/L nitroglycerin was equivalent to that produced
by IL-1ß. In contrast, treatment with 10-3 mol/L
nitroglycerin resulted in the immediate detection of nitrite, which
reached 148.18±2.39 nmol NO2 per 106
cells at 24 hours. Fig 6, middle and right, shows that
10-3 mol/L nitroglycerin reduced basal protein content (by
47.4±4.6%, P<.017, n=6) and phenylephrine-stimulated
protein content (Fig 6, right; P<.017 for phenylephrine
versus phenylephrine plus 10-3 mol/L nitroglycerin, n=6).
Inhibition of endogenous NO with N-Arg also had no effect on total
protein (2279±350 cpm leucine incorporated for phenylephrine versus
2735±345 cpm for phenylephrine plus N-Arg, n=12).
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, 10-5 mol/L NG; and
, 10-7 mol/L NG. For some values, the error bars
are contained within the symbols. Middle and right, Values
represent protein content expressed as mean percent change from
control (mean±SEM) in myocytes treated with 10-3 to
10-5 mol/L either alone or in combination with 50 µmol/L
phenylephrine (PE) for 24 hours. Total cellular protein was determined
by [3H]leucine incorporation. Each experiment was
performed in triplicate wells; number of separate experiments is as
follows: PE, n=18; each experiment involving 10-5 mol/L
NG, n=12; and all other experimental procedures, n=6.
**P<.017; ***P<.001 compared with control;
++P<.017 compared with PE alone. P<.025 is
considered significant.In the next series of experiments, phenylephrine stimulated BNP secretion by 84.21±32.14% (Fig 7, P<.017, n=8). Nitroglycerin (10-5 mol/L) did not affect either basal or phenylephrine-stimulated BNP secretion, whereas 10-3 mol/L nitroglycerin reduced basal secretion by 82.3±2.7% (P<.001, n=5), consistent with its effects on protein content. Likewise, phenylephrine-stimulated BNP secretion was also inhibited by 10-3 mol/L nitroglycerin (P<.017 for phenylephrine versus phenylephrine plus nitroglycerin).
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Effects of cGMP on BNP Secretion and Total Protein Content
To investigate a role for cGMP in the actions of nitroglycerin and
IL-1ß, we examined the time course of cGMP production in myocytes
treated with either nitroglycerin (10-5 and
10-3 mol/L) or IL-1ß. At 6, 9, 12, and 24 hours of
treatment, IL-1ß increased intracellular cGMP approximately three
times (Fig 8). Of interest, there was no detectable
increase in intracellular cGMP in cells treated with either
10-5 or 10-3 mol/L nitroglycerin during the
time course examined.
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, IL-1ß; Since IL-1ß treatment was associated with a rise in intracellular cGMP and prevented phenylephrine-stimulated BNP secretion, we examined whether a stable analogue of cGMP could mimic the actions of IL-1ß. Fig 9A shows that dibutyryl cGMP (10-3 mol/L) had no effect on either basal or phenylephrine-stimulated BNP secretion. This suggests that cGMP does not mediate the inhibitory effect of IL-1ß on phenylephrine-stimulated BNP secretion. Although we did not detect an increase in cGMP in response to nitroglycerin, we wished to test its effect on total myocyte protein. Fig 9B shows that dibutyryl cGMP did not change total cellular protein in control and phenylephrine-stimulated cells.
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Discussion |
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Because the
-adrenergic agonist phenylephrine stimulates ANF
secretion and mRNA and is used to induce myocyte growth in vitro, we
hypothesized that phenylephrine would also stimulate the ventricular
natriuretic peptide BNP. Our data are the first to show that
phenylephrine stimulates BNP mRNA and secretion of immunoreactive BNP.
The increase in BNP mRNA was similar to that of ANF mRNA (Fig 1). The
fact that phenylephrine induction of mRNA was not greater probably
reflects differences in our culture conditions versus those of other
laboratories (eg, time in serum-free medium13 22 ).
Nonetheless, enhanced BNP gene expression seems to be a marker of
hypertrophic growth, as are the ANF,13 22 myosin light
chain-2,13 23 and ß-myosin heavy chain
genes.12 The effect of phenylephrine was maintained for at
least 48 hours, suggesting that the signaling mechanisms were
persistently activated, which was consistent with our previous studies
on PMA stimulation of BNP mRNA15 ; however, one difference
was that phenylephrine increased BNP mRNA to a lesser extent than did
PMA. This may be related to differences in transcriptional versus
posttranscriptional regulation of BNP mRNA by phenylephrine and PMA. On
the basis of our studies with the inhibitor staurosporine,
phenylephrine stimulation of BNP mRNA was in part a function of
activation of protein kinase C, although other kinases most likely were
also involved. Involvement of protein kinase C in the regulation of BNP
mRNA is also consistent with previous reports on regulation of ANF
synthesis and secretion24 25 26 27 as well as other genes
upregulated during hypertrophy.28 29
Our studies demonstrate that IL-1ß induces iNOS mRNA in ventricular
myocytes, confirming a study published while the present article
was in review.21 IL-1ß stimulated iNOS mRNA as early as
3 hours and preceded detectable nitrite levels in the medium by 6
hours, similar to studies with vascular smooth muscle
cells9 and ventricular myocytes.21 The
increase in NO production elicited by IL-1ß was partially blocked by
cotreatment with N-Arg, confirming the involvement of the
L-Arg–NO pathway. The partial inhibition results from the
fact that our culture medium (DMEM) contains 0.48 mmol/L L-Arg.
Dexamethasone also partially inhibited the effects of IL-1ß, as has
been demonstrated previously.30 Although protein synthesis
has been shown to be necessary for cytokine induction of NOS in
macrophages,31 the protein synthesis inhibitor
cycloheximide was unable to completely inhibit NO production. This
result is consistent with recent work, which shows that low
concentrations of cycloheximide stimulate iNOS mRNA through a
transcriptional mechanism.32 As cycloheximide stimulates
the activity of the transcription factor nuclear factor
(NF)-
B33 and because there are several NF-B binding
sites in the promoter of the iNOS gene,34 35 it is
possible that one effect of cycloheximide is to partially activate the
iNOS gene in myocytes. If, on the other hand, cycloheximide inhibits
the synthesis of other important transcription factors involved in iNOS
induction, as occurs in macrophages,31 then the partial
inhibition of IL-1ß–stimulated nitrite production by cycloheximide
in myocytes is probably a function of both positive and negative
influences on transcription factors. The intracellular signaling
pathway used by IL-1ß in cardiac myocytes is unclear, although a
number of mechanisms have been proposed for other cell
types.36 37 38 39 A recent study indicates that the cytoplasmic
portion of the receptor is required for signaling,37 but
no protein directly coupling the receptor with regulatory molecules
involved in gene transcription, such as NF-B, has been described.
Preliminary studies in our laboratory show that NF-B is activated in
myocytes after IL-1ß treatment and that the tyrosine kinase inhibitor
genistein abrogates IL-1ß–stimulated NO production (M.C.L.,
unpublished observations, 1994). Because the interleukin receptor is
not a tyrosine kinase, the possibility exists that a nonreceptor
tyrosine kinase may be involved in IL-1ß signaling.
In our studies, IL-1ß inhibited one marker of growth, BNP secretion,
but not the increase in total cellular protein, excluding the
possibility that the cytokine was having generalized cytotoxic effects
or preventing phenylephrine from binding to its receptor. One possible
explanation of our results is that the signaling pathways activated by
phenylephrine and IL-1ß are interfering with each other and
dissociating BNP synthesis and secretion from protein synthesis
accompanying myocyte growth. Studies have shown that cytokines can
interfere with both 1-adrenergic40 and
ß-adrenergic4 41 responses, perhaps by uncoupling the
receptor from its G protein–mediated signaling pathway. Regarding
cytokine signaling, there is evidence both for and against involvement
of protein kinase C38 and/or phosphatases39
as intracellular mediators. In ventricular myocytes, the protein kinase
C inhibitor staurosporine did not prevent the effects of IL-1ß,
suggesting no requirement for protein kinase C in the induction of iNOS
(Fig 2, bottom), consistent with recent reports.2 21
However, staurosporine inhibited phenylephrine-stimulated BNP mRNA (Fig 1B) and secretion (M.C.L., unpublished observations, 1994), suggesting
the involvement of protein kinase C or another staurosporine-sensitive
kinase, as shown for the effects of PMA on BNP mRNA.15
Since IL-1ß opposes phenylephrine-stimulated BNP secretion, it is
possible that IL-1ß is either inhibiting a particular isoform of
protein kinase C or activating a phosphatase, thus interfering with the
phenylephrine signaling pathway for synthesis and secretion of BNP.
Such a mechanism does not seem to be involved in phenylephrine
stimulation of protein synthesis.
The effects of NO on cardiac myocyte growth were investigated by examining the effect of IL-1ß and nitroglycerin on total cellular protein content. Phenylephrine treatment of ventricular myocytes stimulated total protein by 49%. Reports in the literature demonstrate increases in total protein from 20% to 60% in myocytes stimulated for 24 hours with stretch42 43 or norepinephrine.44 45 IL-1ß induction of NO was unable to inhibit the accumulation of protein under basal or phenylephrine-stimulated conditions. Since IL-1ß has been shown to stimulate growth-inhibitory compounds (eg, prosta- glandin E2)46 and growth-promoting compounds (eg, platelet-derived growth factor)47 in vascular smooth muscle cells, its lack of effect on total cellular protein in our studies may therefore be attributed to a net balance between the production of stimulatory and inhibitory growth factors. To circumvent this possible problem, we repeated our studies with the NO donor nitroglycerin. Since only the highest concentration of nitroglycerin (10-3 mol/L) reduced protein content and BNP secretion under both basal and phenylephrine-stimulated conditions, this concentration of nitroglycerin seems cytotoxic. Indeed, this concentration is three orders of magnitude greater than that achieved during therapy, and a number of recent studies describe deleterious actions of high levels of NO.48 49 50 51 52 One possible explanation of our results is that the large amounts of NO produced by 10-3 mol/L nitroglycerin stimulated ADP ribosylation of components of the protein synthetic machinery or exocytosis, thus damaging cell function.49 50 51 52 The ineffectiveness of more pharmacologically relevant doses of nitroglycerin (10-5 mol/L) on protein synthesis suggests that NO does not oppose growth, a result confirmed by the inability of N-Arg to potentiate phenylephrine-stimulated protein synthesis. The lack of an effect of 10-5 and 10-4 mol/L nitroglycerin is in contrast to the findings of Garg and Hassid,8 who reported that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibits DNA synthesis and proliferation of rat aortic vascular smooth muscle cells. SNAP also inhibits mitogenesis of fibroblasts by a cGMP-independent mechanism.53 It is unclear at present whether the difference between our study and those cited is due to the NO donor used.
Despite stimulating similar levels of NO, IL-1ß and nitroglycerin were disproportionate in their effects on cGMP production. Treatment with IL-1ß maximally stimulated cGMP at 9 to 12 hours, in agreement with Roberts et al3 and Corbett et al,54 who used cultures of cardiac myocytes and islets of Langerhans, respectively. In contrast, stimulation of cGMP was not observed after nitroglycerin treatment. Certainly, the lack of effect of nitroglycerin on cGMP production in our study would support the idea that the efficacy of NO donors in stimulating guanylate cyclase is variable55 and tissue specific.56 57 58 Although we cannot discount the possibility that the peak in cGMP production may have occurred within minutes after treatment with nitroglycerin, as occurs in vascular smooth muscle cells,57 the stable cGMP analogue dibutyryl cGMP did not influence either total cellular protein or BNP secretion in our studies, emphasizing that neither NO nor cGMP affects myocyte growth in our model. However, the role of these compounds on other cell types within the heart (eg, fibroblasts, endothelial cells, and smooth muscle cells) cannot be discounted and may explain the reports in the literature that attribute growth-inhibitory properties to these compounds in vivo4 or in vitro.8 9 53 Despite the fact that NO did not affect the amount of total cellular protein, we have shown that IL-1ß can disrupt myocyte function; in particular, IL-1ß prevents phenylephrine stimulation of BNP secretion. Severe hypotension and impaired myocyte contractile function are important problems in endotoxic shock resulting from overproduction of NO by macrophages.3 41 59 60 Our results would suggest the possibility that excess cytokines produced during endotoxic shock or perhaps locally in damaged heart tissue could act directly on myocytes to induce NO and impair myocyte function. In fact, preliminary studies in our laboratory indicate that iNOS mRNA is induced in the infarcted left ventricle as early as 6 hours after ischemic injury (D. Wang et al, unpublished observations, 1994). Thus, specific inhibition of iNOS activity in myocytes, resulting in decreased NO and cGMP, might produce a positive inotropic effect to maintain blood pressure.
In conclusion, our studies indicate that phenylephrine stimulates BNP mRNA and secretion in ventricular myocytes, as previously demonstrated for ANF, and stimulates total cellular protein. Also, IL-1ß induces iNOS mRNA, NO production, and cGMP in myocytes, but neither IL-1ß, NO, nor cGMP affects basal or phenylephrine-stimulated total cellular protein levels. On the basis of these particular studies, we have to reject our initial hypothesis and conclude that NO does not oppose growth of neonatal cardiac myocytes in vitro. In contrast, IL-1ß but not NO or cGMP inhibits phenylephrine stimulation of BNP. Since activation of the IL-1ß signaling pathway disrupts phenylephrine-stimulated BNP secretion but not protein synthesis, we conclude that distinct intracellular signals are involved in the regulation of different markers of cardiac myocyte growth.
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Acknowledgments |
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This work was supported by National Institutes of Health grants HL-28982 (O.A.C.) and HL-03188 (M.C.L.). We thank Jodi Sitkins and Kim Sarcino for excellent technical assistance.
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Footnotes |
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Reprint requests to Dr Margot C. LaPointe, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202.
Received May 19, 1994; first decision August 9, 1994; accepted November 2, 1994.
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