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Am J Physiol Heart Circ Physiol 287: H14 –H21, 2004;
10.1152/ajpheart.01235.2003.
Invited Review: Point-Counterpoint
Endocardial endothelium in the avascular frog heart: role for diffusion
of NO in control of cardiac O2 consumption
Alexandra Adler,1 Harer Huang,1 Zipping Wang,1 Joseph Conetta,2
Ellen Levee,2 Xiaoping Zhang,1 and Thomas H. Hintze1
1
Department of Physiology and 2Department of Comparative Medicine,
New York Medical College, Valhalla, New York 10595
nitric oxide; Western blot analysis; Triton X-100; mitochondria; Griess reaction
THE ENDOCARDIAL ENDOTHELIUM (EE) plays a vital role in the
regulation of myocardial performance and electrical activity as
recently reviewed by Brutsaert (3). Located between luminal
blood and the underlying cardiac muscle, the EE synthesizes
and releases nitric oxide (NO) in amounts sufficient to regulate
myocardial contraction under basal conditions (4, 24). NO is
also known to regulate O2 consumption under a variety of
conditions (2, 12, 15, 22, 25). This regulation of myocardial
metabolism may be one of NO’s most significant roles. We
have proposed that endothelial NO synthase (eNOS), which is
the most highly expressed isoform of NOS in vascular tissue
under physiological conditions, contributes to the control of
tissue O2 consumption by NO (16, 25).
As a model to determine the significance of the diffusion of
NO in the control of cardiac cell O2 consumption, the frog
heart is of much interest because it has no coronary circulation
and can therefore be used to study the interaction between the
Address for reprint requests and other correspondence: T. H. Hintze, Dept.
of Physiology, New York Medical College, Valhalla, NY 10595.
H14
EE and the myocardium without interference from the vascular
endothelium (13, 24). This striking absence of the coronary
vasculature is characteristic of all amphibians (13) and may be
a consequence of a change in respiratory patterns from water
environment to air (8, 19). The frog ventricle is highly trabeculated and has a much higher endothelial surface-to-myocardial volume ratio than higher vertebrates (24). The EE of the
avascular frog heart is unique because it is the only endothelial
barrier between blood and the myocardial environment and is
the only source of NO. Thus the EE mediates the inotropic
effect of luminal cholinergic stimuli via the NO-cGMP pathway (11).
In 1997, Sys et al. (24) suggested that the EE is the sole NO
source in the heart of the frog and that NO produced by the EE
may act as a short-distance, bidirectional messenger between
EE cells and myocytes (11). Brutsaert (3, 4) has also suggested
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Adler, Alexandra, Harer Huang, Zipping Wang, Joseph Conetta, Ellen
Levee, Xiaoping Zhang, and Thomas H. Hintze. Endocardial endothelium in the
avascular frog heart: role for diffusion of NO in control of cardiac O2 consumption.
Am J Physiol Heart Circ Physiol 287: H14 –H21, 2004; 10.1152/ajpheart.01235.
2003.—We investigated the role of nitric oxide (NO) in the control of myocardial
O2 consumption in the hearts of female Xenopus frogs, which lack a coronary
vascular endothelium and in which the endocardial endothelium is the only source
of NO to regulate cardiac myocyte function. Hence, frogs are an ideal model in
which to explore the role of diffusion of NO from the endocardial endothelium (EE)
without vascular endothelial or cardiac cell NO production. In Xenopus hearts we
examined the regulation of cardiac O2 consumption in vitro at 25°C and 37°C. The
NO-mediated control of O2 consumption by bradykinin or carbachol was significantly (P ⬍ 0.05) lower at 25°C (79 ⫾ 13 or 73 ⫾ 11 nmol/min) than at 37°C
(159 ⫾ 26 or 201 ⫾ 13 nmol/min). The response to the NO donor S-nitroso-Nacetyl penicillamine was also markedly lower at 25°C (90 ⫾ 8 nmol/min) compared
with 37°C (218 ⫾ 15 nmol/min). When Triton X-100 was perfused into hearts, the
inhibition of myocardial O2 consumption by bradykinin (18 ⫾ 2 nmol/min) or
carbachol (29 ⫾ 4 nmol/min) was abolished. Hematoxylin and eosin slides of
Triton X-100-perfused heart tissue confirmed the absence of the EE. Although
endothelial NO synthase protein levels were decreased to a variable degree in the
Triton X-100-perfused heart, NO2 production (indicating eNOS activity) decreased
by ⬎80%. It appears that the EE of the frog heart is the sole source of NO to
regulate myocyte O2 consumption. When these cells are removed, the ability of NO
to regulate O2 consumption is severely limited. Thus our results suggest that the EE
produces enough NO, which diffuses from the EE to cardiac myocytes, to regulate
myocardial O2 consumption. Because of the close proximity of the EE to underlying myocytes, NO can diffuse over a distance and act as a messenger between the
EE and the rest of the heart to control mitochondrial function and O2 consumption.
Invited Review: Point-Counterpoint
DIFFUSION OF NO TO MITOCHONDRIA
MATERIALS
Bradykinin (BK), carbachol, S-nitroso-N-acetyl penicillamine
(SNAP), N␻-nitro-L-arginine methyl ester (L-NAME), and Triton
X-100 were purchased from Sigma (St. Louis, MO).
Animal tissue. Female Xenopus frogs were obtained from NASCO
(Ft. Atkinson, WI). All experimental protocols were approved by the
Institutional Animal Care and Use Committee and conform to the
National Institutes of Health Guide for the Care and Use of Laboratory Animals and American Physiological Society “Guidelines for the
Use and Care of Laboratory Animals.” Before death for in vitro
studies, the frogs were anesthetized with dissolved 3-aminobenzoic
acid ethyl ester (1.7 g/l), and the heart was removed and cut into 8 or
12 pieces.
Preparation of myocardial tissue and measurement of O2 consumption. The frog hearts were separated, freed of connective tissue and
fat, and cut into pieces weighing 20 – 40 mg. Tissue was incubated in
Krebs bicarbonate solution containing (in mmol/l) 118 NaCl, 4.7 KCl,
1.5 CaCl2, 25 NaHCO3, 1.2 KH2PO4, 1.1 MgSO4, and 5.6 glucose at
37°C and bubbled with 21% O2-5% CO2-74% N2 (pH 7.4) to
equilibrate for at least 1.5 h. At the end of the incubation period, two
pieces of tissue were placed in a continuously stirred chamber with 3.0
ml of air-saturated Krebs bicarbonate solution containing 10 mmol/l
HEPES (pH 7.4). The chambers were sealed using Clark-type platinum O2 electrodes (Yellow Springs Instruments) that were connected
to O2 monitors (model YSI 5331) to continuously measure the O2
concentration in the buffer. The drop of O2 levels in the buffer is
caused by and is directly related to uptake of O2 by the tissue. This
methodology has been used by us (1) and by others (16, 17) in
previous studies.
Effect of BK and carbachol on myocardial O2 consumption at 37°C
and 25°C. The effect of cumulative doses of the B2 kinin receptor
agonist BK (10⫺8–10⫺4; Sigma) and the muscarinic receptor agonist
carbachol (10⫺8–10⫺4; Sigma) on myocardial O2 consumption was
examined in tissue from frog hearts (n ⫽ 9 and 6, respectively). O2
consumption studies were performed at two temperatures, 37°C and
25°C, to determine the effect of temperature on the ability of NO to
compete with O2 at its cytochrome c oxidase binding site and thus
confirm its effect on mitochondrial respiration. These responses were
also examined in the presence of the NOS inhibitor L-NAME (10⫺4
mol/l; Sigma) to verify the role of NO production by NOS in the
regulation of myocardial O2 consumption.
AJP-Heart Circ Physiol • VOL
Effect of SNAP on myocardial O2 consumption at 37°C and 25°C.
Cumulative doses of the NO donor SNAP (10⫺7–10⫺4 mol/l) on
myocardial O2 consumption were examined in frog heart tissue (n ⫽
7) to test the sensitivity of the frog heart to NO. These responses were
also examined in the presence of L-NAME and at both temperatures.
Effect of Triton X-100 on myocardial O2 consumption. To remove
the source of the NO production in the Xenopus hearts, Triton X-100
(0.5% in saline), a detergent, was used. Triton X-100 dissolves lipids
and therefore the endocardial endothelium of the heart, which has
been shown to be the sole source of myocardial NO (24). The heart
was exposed and Triton X-100 was introduced through the apex of the
heart using a 23-gauge needle. The heart was perfused for 2 min and
then removed and used for O2 consumption studies as described
above. The effect of Triton X-100 on myocardial O2 consumption was
examined in frog heart tissue (n ⫽ 4 each) in the presence of
cumulative doses of BK, carbachol, and SNAP.
Hematoxylin and eosin slides. Hearts were removed from Xenopus
frogs (n ⫽ 10) or perfused with Triton X-100 (n ⫽ 4) and preserved
in a 10% formalin solution for 48 h to visualize the disruption of the
EE by Triton. Hematoxylin and eosin slides were then prepared using
standard methods from the Manual of Histologic and Special Staining
Technics as well as an H2500 microwave processor (Energy Beam
Sciences; Agawam, MA).
Immunoblotting. Myocardial tissue (both with and without Triton
X-100, n ⫽ 5 each) was snap frozen in liquid nitrogen and stored at
⫺80°C. For preparation of extracts, tissue was pulverized in liquid
nitrogen, followed by homogenization in 5 vol of lysis buffer (0.05 M
Tris 䡠 HCl, pH 7.2, 1 mM EDTA, 10 mM dithiothreitol, 1 mg/ml
PMSF, 100 ␮g/ml leupeptin, 100 ␮g/ml soybean trypsin inhibitor, and
20 ␮g/ml aprotinin) for 15 s ⫻ 3 at 4°C and sonication for 1 min. The
resulting lysate was centrifuged at 10,000 g for 10 min at 4°C and
stored at ⫺80°C before use. Protein concentrations of supernatants
were measured using a standard protein assay (Bio-Rad Laboratories).
Samples of tissue lysate (75 ␮g of protein) were loaded onto
individual lanes of a 8% polyacrylamide gel containing the detergent
SDS prepared using standard techniques. Proteins were separated
according to their molecular size. Proteins in the gel were transferred
from the gel to polyvinylidene difluoride membranes (Amersham
Pharmacia Biotech) using a semidry transfer cell (Bio-Rad). Membranes were blocked for 1 h with 5% milk-PBS to block all other free
protein-binding sites on the membrane. Purified polyclonal antibody
to eNOS (1:1,000 dilution, Affinity Bioreagents) and inducible NOS
(1:1,000 dilution, Affinity Bioreagents) in 1% milk-PBS was added,
and incubation continued at 4°C overnight to allow the antibody to
bind to its respective proteins. After incubation with a secondary
anti-rabbit antibody (1:5,000 dilution, Chemicon) conjugated to
horseradish peroxidase, sites of antibody-antigen reaction were visualized using Super Signal West Pico Chemiluminescent Substrate
(Pierce; Rockford, IL), followed by exposure to X-ray film (Kodak;
Rochester, NY).
Quantification of proteins. The relative intensities of the bands in
the exposed film were determined by scanning on a documentation
and analysis system (Alphaimager 2000, Imgen Technologies; Alexandria, VA), followed by analysis with Image software. Band intensity correlates directly with amounts of the original protein present in
the gel.
Effect of BK and Triton X-100 on nitrite production in heart tissue.
Frog heart tissue sections, ⬇30 – 60 mg/section (wet wt), perfused
with Triton X-100 (n ⫽ 5) or control (n ⫽ 5), were incubated and
bubbled with 95% O2-5% CO2 in Krebs for 35 min at 37°C in 5-ml
plastic tubes that contained BK (10⫺4 M) or L-NAME plus BK (Griess
reaction). At the end of the incubation time, the tubes were removed
from the tissue bath, and sulfanilamide (450 ␮l of a 1% solution) and
N-(1-naphthyl)ethylenediamine (50 ␮l of a 0.2% solution) were added
to each tube for diazotization of sulfanilic acid by NO. After 5–10 min
of incubation at room temperature for full color (pink) development,
the supernatant was removed from each tube. Formation of NO was
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that the endothelial cell NOS-NO-cGMP signaling pathway
could be involved in the control of trans-EE-permeability.
Thus it appears that NO must diffuse from the EE of the frog
heart to affect myocardial performance. In our study, we
removed the EE in hearts from Xenopus frogs using a detergent
to elucidate the role of the EE as an NO source. It has also been
shown previously that eNOS is located only in the EE of the
frog heart (24). Given the close proximity of the EE and
underlying myocytes, eNOS activity in the EE directly affects
myocardial contractility (3, 24). Our studies examined the role
of eNOS in hearts both with and without an intact EE. Activity
of eNOS in hearts without an EE was, however, greatly
decreased. Thus it seems that an intact EE is necessary for the
proper function of eNOS. The ability of NO to diffuse across
the heart has been highly debated. Although some have suggested that NO can diffuse (11, 21), others have disagreed (25)
and claimed that local production/concentrations of NO are
important. The aim of our work was to examine the possibility
that NO can diffuse from one cell type to another to regulate
myocardial O2 consumption in the absence of the confounding
effects of multiple sources of NO, i.e., myocyte NOS or a
vascular eNOS.
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Invited Review: Point-Counterpoint
H16
DIFFUSION OF NO TO MITOCHONDRIA
measured as nitrite. Nitrite release was measured with a spectrophotometer (Uvikon 930 spectrophotometer, Kontron Instruments) as the
increase in absorbance at 540 nm and compared with known concentrations of nitrite. Absorbance was measured and converted to a
straight line with the use of linear regression analysis (y ⫽ ax ⫹ b,
R ⬎ 0.99). We have described these methods recently (26, 27).
Statistical analysis. The data are shown as means ⫾ SE. Comparisons between groups were made using one-way ANOVA, followed
by correction for multiple comparisons with Bonferroni’s t-test using
statistical software. Statistical significance was accepted at P ⬍ 0.05.
RESULTS
Fig. 1. Inhibition of myocardial O2 consumption by bradykinin
(10⫺4–10⫺8 mol/l) and carbachol (10⫺4–10⫺8 mol/l) in Xenopus. There was a significant, concentration-dependent decrease
in myocardial O2 consumption at 37°C (Œ) and 25°C (}) in
response to bradykinin (A) and carbachol (B). The decrease was
significantly less at 25°C compared with 37°C. *P ⬍ 0.05, 25°C
vs. 37°C.
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Baseline O2 consumption in myocardial tissues from Xenopus hearts at 25°C and 37°C. Baseline myocardial O2 consumption was half the rate at 25°C [263 ⫾ 22.8 nmol
O2 䡠min⫺1 䡠g⫺1 (n ⫽ 10)] compared with 37°C [459 ⫾ 45.9
nmol O2 䡠min⫺1 䡠g⫺1 (n ⫽ 7); P ⬎ 0.05]. These baselines were
not affected by L-NAME [426 ⫾ 50.1, 303 ⫾ 39.8 nmol
O2 䡠min⫺1 䡠g⫺1 (n ⫽ 8 each) at 25°C and 37°C, respectively,
P ⬎ 0.05].
Effect of BK on myocardial O2 consumption at 37°C and
25°C. BK (10⫺8–10⫺4 mol/l) caused concentration-dependent
decreases in myocardial O2 consumption (Fig. 1A) at 37°C
(n ⫽ 9) (from 53.8 ⫾ 3.6 to 159 ⫾ 25.7 nmol/min) and at 25°C
(n ⫽ 6) (from 24.0 ⫾ 5.4 to 77.7 ⫾ 12.7 nmol/min). The
response to BK at 25°C was significantly less than that at 37°C
(P ⬍ 0.05). The addition of L-NAME significantly inhibited the
response to BK at both temperatures. The response to BK with
L-NAME was, however, not significantly different at the two
temperatures.
Effect of carbachol on myocardial O2 consumption at 37°C
and 25°C. Carbachol (10⫺8–10⫺4 mol/l) caused concentrationdependent decreases in myocardial O2 consumption (Fig. 1B)
at 37°C (n ⫽ 6) (from 24.8 ⫾ 14.5 to 201.0 ⫾ 13.0 nmol/min)
and at 25°C (n ⫽ 7) (from 26.8 ⫾ 7.1 to 72.7 ⫾ 11.0
nmol/min). The response to carbachol at 25°C was significantly less than at 37°C (P ⬍ 0.05). The addition of L-NAME
significantly inhibited the response to carbachol at both temperatures. The response to carbachol with L-NAME was, however, not significantly different at the two temperatures.
Effect of SNAP on myocardial O2 consumption at 37°C and
25°C. SNAP (10⫺7–10⫺4 mol/l) caused concentration-dependent decreases in myocardial O2 consumption at (Fig. 2) 37°C
(n ⫽ 7) (from 22.1 ⫾ 82.9 to 218 ⫾ 16.3 nmol/min) and at
25°C (n ⫽ 3) (from 9.49 ⫾ 2.6 to 89.5 ⫾ 8.4 nmol/min).
L-NAME did not significantly alter the response to SNAP at
any temperature.
Effect of Triton X-100 on myocardial O2 consumption. The
addition of Triton X-100 (0.05%) caused a significant decrease
Invited Review: Point-Counterpoint
DIFFUSION OF NO TO MITOCHONDRIA
H17
Fig. 2. Inhibition of myocardial O2 consumption by S-nitroso-Nacetyl penicillamine (SNAP; 10⫺4–10⫺7 mol/l) in Xenopus. There
was a significant, concentration-dependent decrease in myocardial
O2 consumption at 37°C (Œ) and 25°C (}) in response to SNAP.
The decrease was significantly less at 25°C compared with 37°C.
*P ⬍ 0.05, 25°C vs. 37°C.
15.3 to 112 ⫾ 14.0 nmol/min; without Triton X-100: from
22.1 ⫾ 8.3 to 218 ⫾ 16.3).
Hematoxylin and eosin slides. Hematoxylin and eosin slides
indicate that Triton X-100 removed the layer of endothelial
Fig. 3. Addition of the detergent Triton X-100 abolished the
regulation of O2 consumption by bradykinin (10⫺4–10⫺8 mol/l)
and carbachol (10⫺4–10⫺8) at 37°C. A: in the presence of
bradykinin, regulation of O2 consumption was significantly
decreased by the addition of Triton X-100 (}) compared with the
control (■). B: in the presence of carbachol, regulation of O2
consumption was significantly decreased by the addition of
Triton X-100 compared with control. *P ⬍ 0.05 with Triton
X-100.
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in the control of O2 consumption by tissue from Xenopus hearts
at 37°C in the presence of either BK or carbachol (Fig. 3, A and
B). Triton X-100 altered O2 consumption in the presence of
SNAP (10⫺7–10⫺4 mol/l) (with Triton X-100: from 50.2 ⫾
Invited Review: Point-Counterpoint
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DIFFUSION OF NO TO MITOCHONDRIA
cells around the lumen, the EE. Fig. 4, left, shows control
tissue, whereas Fig. 4, right, shows tissue after perfusion with
Triton X-100. The endothelial cells with the addition of Triton
appear ripped, whereas the control cells are intact.
Effect of Triton X-100 on eNOS protein. Levels of eNOS
protein decreased in only some of the hearts that were perfused
with Triton X-100 (Fig. 5). Although Triton X-100 ripped the
EE, eNOS protein could still cling to underlying tissue. The
eNOS would be inactive, however, because of the lack of an
intact cell membrane.
Effect of BK and Triton X-100 on nitrite production in heart
tissue. Tissue perfused with Triton X-100 had significantly
lower nitrite production in response to BK compared with
control tissue (Fig. 6) (Griess reaction). L-NAME inhibited the
production of nitrite in response to BK in the control tissue
(BK: 81.4 ⫾ 23.7% change compared with control; BK ⫹
L-NAME: 23.7 ⫾ 19.2% change compared with control).
L-NAME had no effect on the production of nitrite in response
to BK in the tissue perfused with Triton X-100 (BK: 13.9 ⫾
8.9% change compared with control; BK ⫹ L-NAME: 13.1 ⫾
5.8% change compared with control).
tissues of female Xenopus frogs at 37°C. The abolition of these
responses after the addition of L-NAME, an NOS inhibitor,
indicates a role for NO in the regulation of mitochondrial
respiration in the heart of frogs. When O2 consumption was
examined at 25°C, the response to BK and carbachol was
significantly decreased. Importantly, the regulation of O2 consumption in response to BK and carbachol was nearly abolished in tissue perfused with Triton X-100, a detergent that
strips away the EE. Removal of the EE was confirmed with the
DISCUSSION
The results of the present study indicate that BK and carbachol significantly decrease O2 consumption in the myocardial
Fig. 5. Levels of endothelial nitric oxide (NO) synthase (eNOS) protein was
decreased only in some of the Triton X-100-perfused hearts. This suggests that
eNOS protein still clings to the disrupted EE. N, normal.
AJP-Heart Circ Physiol • VOL
Fig. 6. Addition of the detergent Triton X-100 abolished the ability of
bradykinin (10⫺4 M) to stimulate NO production (measured as nitrite) in frog
heart tissue. Nitrite production in the normal tissue (solid bar) was nearly eight
times higher than in the Triton X-100-perfused tissue (hatched bar). *P ⬍ 0.05,
bradykinin vs. bradykinin ⫹ Triton X-100.
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Fig. 4. ⫻40 magnification of four sections:
two control (left) and two Triton X-100
(right). The endocardial endothelium (EE) of
the control hearts is intact, as indicated by
the smooth outline of the tissue with regularly spaced, horizontally oriented nuclei.
The EE of the tissue perfused with Triton
X-100 appears ripped with nuclei and perhaps cell membranes remaining stuck to the
underlying tissue.
Invited Review: Point-Counterpoint
DIFFUSION OF NO TO MITOCHONDRIA
AJP-Heart Circ Physiol • VOL
These receptors may, however, exist in other parts of the heart,
particularly the EE (7). BK caused a significantly smaller
reduction in myocardial O2 consumption in hearts perfused
with Triton X-100 compared with control hearts, suggesting
that an intact EE is vital for the activity of BK.
Carbachol, a muscarinic receptor agonist that stimulates
endogenous NO production through the activation of eNOS,
was used as well. Carbachol also caused a significantly smaller
reduction in myocardial O2 consumption in hearts previously
perfused with Triton X-100. These results again support the
conclusion that the EE is the sole source of NOS and NO and
that NO must therefore diffuse to other tissues in the heart to
regulate O2 consumption. The decreased effect of carbachol or
BK in Triton-perfused tissue is due to decrease eNOS activity.
Because there are no BK receptors on frog cardiac myocytes, the use of BK after Triton X-100 does not directly
address the possibility that frog myocytes make NO. However,
this is not the case with carbachol, whose actions, like those of
ACh (Vagusstoff), on the heart were originally described by
Loewi in 1903 (20). Furthermore, Gattuso et al. (11) have
shown more recently that ACh regulates cardiac contraction
through NO production.
In our study, the exogenous NO donor SNAP reduced tissue
O2 consumption to a similar degree in the control and Triton
X-100-perfused heart tissue. The loss of the ability of BK and
carbachol to reduce O2 consumption is therefore not a result of
an impairment of the ability of NO to modulate tissue O2
consumption (i.e., altered mitochondrial sensitivity to NO) but
rather a reduction in biologically active NO. This is due to the
loss of the EE in the Triton X-100-perfused heart tissue.
Hematoxylin and eosin slides provided qualitative evidence
for removal of the EE after treatment with Triton X-100. The
slides, examined at ⫻40 magnification, showed the physical
effect of Triton X-100 on EE cells. In control hearts, the EE
appears as a smooth layer of cells lining the lumen and
covering the sinusoids. This smooth endothelial layer appears
distorted in the heart perfused with Triton X-100. Although the
nuclei remain, they are clumped and not located in intact cells.
In our study, with Western blot analysis, we found that tissue
perfused with Triton X-100 did not, on average, significantly
lower eNOS levels. It seems likely that although the EE is
destroyed, eNOS protein, like the cell nuclei, may still cling to
remaining tissue. These conclusions were reinforced in studies
using the Griess reaction to measure nitrite production in the
buffer. Nitrite production was decreased by 85.4% in the Triton
X-100-perfused tissue compared with the control tissue. This
suggests that, whereas eNOS protein may still cling to remaining cells in the lumen of the frog heart, it is inactive after
treatment with Triton X-100. Our data suggest that intact
endocardial endothelial cells are necessary for eNOS activity.
Therefore, when the EE is distorted and cells are no longer
intact, eNOS is inactive, and NO production does not occur.
The lack of effect by BK in the Triton X-100-perfused tissue
also supports the concept that the EE is the only NO source in
the frog heart.
In the present study, we have clearly shown that the stimulation of endogenous NO production failed to reduce tissue O2
consumption in the presence of Triton X-100. Previous work
performed in frogs has suggested that they are ideal models in
which to study the EE. It is known that NOS is only found in
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use of hematoxylin and eosin slides of tissue in the presence
and absence of Triton X-100. Western blot analysis indicated
that eNOS protein levels were variably decreased by Triton
X-100. Activity of eNOS (and NO production), however, was
significantly reduced in Triton X-100-perfused tissue, suggesting its singular role in NO production in the frog heart. The
effect of SNAP was not altered by the addition of Triton
X-100, suggesting that the underlying myocytes were still
functional and that the EE is the sole source of NO in the frog
heart. Thus NO must diffuse from the EE to other parts of the
heart (i.e., myocytes) to regulate myocardial O2 consumption.
Our data indicate that pretreatment with L-NAME had no
effect on baseline O2 consumption in the frog hearts. We have
interpreted this previously to suggest that the basal release of
NO is small in the absence of an agonist that stimulates NO
production in our preparation. Our data also suggest that the
ability of endogenous NO to regulate O2 consumption was
greatly reduced in tissues studied at 25°C compared with those
studied at 37°C. Such a reduction leads to reduced ability of
BK, for example, to regulate myocardial mitochondrial O2
consumption. The effect of the exogenous NO donor SNAP
was significantly decreased at 25°C as well, demonstrating that
the tissue is less sensitive to NO at a lower temperature. The
role of temperature is of interest in amphibians because they
are cold-blooded and their enzyme activity varies over a range
of temperatures, the Q10. The reduction in NO bioactivity at
25°C may be dependent upon the competitive relationship
between NO and O2 for cytochrome c oxidase. At 25°C,
biochemical work by the heart is decreased, intramitochondrial
PO2 is higher (5, 6, 25), and NO does not compete as well for
the binding site of O2 as it would at a higher temperature (i.e.,
high O2 consumption). Therefore, the effect of BK on the
control of O2 consumption at 25°C is predictably less than it
would be at 37°C, as we have shown.
One of the most exciting findings in this study was that the
control of O2 consumption by endogenous NO was nearly
abolished in tissue from hearts perfused with Triton X-100.
The addition of Triton X-100 significantly decreased the ability
of BK or carbachol to regulate O2 consumption. Triton X-100
has previously been studied and shown to affect cardiac function and to destroy EE cells (24). This finding suggests that the
EE may be the sole source of NO in the frog heart and that
when it is removed, control of O2 consumption by BK and
carbachol is severely limited. Examination of hematoxylin and
eosin slides verified the absence of viable endothelial cells in
hearts perfused with Triton X-100. The EE of the control tissue
appeared intact while the EE of the perfused tissue with Triton
was disrupted. To better understand our findings, Western blot
analysis for eNOS and nitrite, the hydration product of NO,
production studies were performed. Although levels of eNOS
protein were variably reduced, production of nitrite (an indicator of eNOS activity) was significantly reduced in the Triton
X-100-perfused tissue. From these findings, it seems likely that
NO was produced solely in the EE and that NO diffuses from
the EE to myocytes to regulate myocardial O2 consumption.
BK, the B2 kinin receptor agonist, stimulates endogenous
NO production through the activation of eNOS. This production is blocked in the presence of L-NAME. BK has previously
been studied in frog hearts, and it has been shown that there are
no BK receptors present in frog myocardial cells (14). Therefore, BK cannot modulate NO production in frog myocytes.
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Invited Review: Point-Counterpoint
H20
DIFFUSION OF NO TO MITOCHONDRIA
POINT-COUNTERPOINT: ROLE OF NO IN THE HEART:
CONTROL OF BEATING OR BREATHING
The study by Paulus and Bronzwaer proposes that the
reduction in systolic time, the early onset of diastole, and
increased compliance caused by NO in cardiac myocytes result
in reduced systolic tension or pressure development and can be
misconstrued as a reduction in cardiac contractility. This occurs whether NO is produced by endothelial cells subsequent to
stimulation by substance P or is given as an NO donor.
Furthermore, the source of NO may be either inducible NOS or
eNOS and would in the main have a beneficial effect on cardiac
contraction. Coupled with a reduction in vascular tone and
aortic impedance, the reduction in systolic function may also
lead to increased ejection, even in a failing heart. In the
presence of adrenergic stimulation, the reduction in systolic
time would cause a reduced systolic contraction and appear as
a negative inotropic effect. If the NO were from an endogenous
source, then an NOS inhibitor would appear to increase inoAJP-Heart Circ Physiol • VOL
tropic state and increase systolic tension development, an
observation made in both experimental animals and in humans
with heart failure. This interesting view of the literature by
Paulus and Bronzwaer provides an alternative explanation for
the paradoxical conclusion that what is otherwise a physiological important substance at low concentrations is an etiologic
agent in the development of cardiac dysfunction at the same
concentrations. By redefining the mechanism of reduced cardiac contraction by NO from a negative inotropic effect to an
early onset of cardiac relaxation, at the same inotropic state, the
onerous implications of reduced contraction to the development of disease are lessened. It should be remembered that
catecholamines enhance the velocity of cardiac cell relaxation
by a cAMP-associated mechanism, and this is considered of
substantial physiological significance at high heart rates and to
maintain filling during cardiac dysfunction. However, catecholamines are notoriously O2 wasting and the early relaxation caused by NO may serve to reduce this O2 wasting effect,
which is especially important in the diseased heart.
The mechanism by which NO causes an early onset of
cardiac relaxation is most probably an increase in cGMP and
phosphorylation of troponin I (TNI) (21a). This phosphorylation of TNI initiates relaxation regardless of the intracellular
calcium levels, thus at any given inotropic state. A similar
although undefined process encompassing the interaction of
cAMP and ACh on cardiac contraction was discussed by
Greengard (15a) as the “Yin-Yang” hypothesis of cardiac
contractions as early as the 1970s. The theory proposed by
Paulus and Bronzwaer maybe an explanation for those early
findings. The signaling mechanisms involved are not the focus
of the discussion provided rather, the focus is on basic and
clinical implications of this mechanism.
These can be summarized as follows: 1) the phosphorylation
of TNI by cGMP results in an early onset of cardiac relaxation
abbreviating isometric contraction without affecting the velocity of contraction; 2) the phosphorylation of TNI results in
increased distensibility; 3) the interaction of this mechanism
with catecholamines results in a reduction of cardiac isovolumic contraction without a change in inotropic state; 4) the
inhibition of catecholamine-induced contraction is O2 sparing;
and 5) coupled with a reduction in impedance, stroke volume
can be maintained or enhanced without increasing O2 consumption even in the presence of catecholamines and cardiac
failure. Importantly, diastolic dysfunction is thought to be an
early hallmark of heart failure (16), perhaps reduced NO
production is partially responsible. These are important consequences with substantial implications for our understanding of
the role of NO in the genesis and treatment of cardiovascular
disease.
In relation to our study, we found it most interesting that
infusion of substance P even into the human heart resulted in
abbreviation of systole and increased compliance (16). The
source of NO was attributed to the endothelial cells. Furthermore, the mechanism of action of angiotensin-converting enzyme inhibitors, perhaps the most useful drugs in the treatment
of heart failure in particular and cardiovascular disease in
general (7a), was at least in part attributed to this mechanism of
action and a kinin-mediated release of NO from the endothelium. With the use of the same reasoning, the effects of
L-NMMA on the failing heart of patients in the presence of
high adrenergic tone could also be explained by interfering
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mance of mammals (24). The frog ventricle is highly trabeculated: it has a much higher endothelial surface-to-myocardial
volume ratio than higher vertebrates. Unlike the hearts of
mammals, the frog heart has no coronary circulation and can
therefore be used to study the interaction between the EE and
the myocardium without interference (13). NO produced by the
EE modulates intramyocardial cGMP levels and contractility
and therefore acts as a short-distance messenger (bidirectional)
between EE cells and myocytes, which are separated only by a
thin layer of extra cellular material (13).
The findings of the present study support our previous work
indicating that BK activates eNOS to regulate cardiac O2
consumption and that this does not occur in the eNOS knockout mouse heart (16, 18). More importantly, we demonstrated
previously the transfer of NO from dog coronary microvessels
coincubated with eNOS knockout mouse heart regulates tissue
O2 consumption (21). Those studies and the current one support the concept that NO can diffuse over a distance to regulate
myocardial O2 consumption (25), just as NO can diffuse from
a single layer of endothelial cells to regulate vascular tone (9).
We have used the frog heart to explore the possibility that
NO can diffuse from one cell to another to regulate myocardial
O2 consumption. Control of O2 consumption was found to be
temperature dependent in the frog heart. We have also shown
that the EE in the frog heart produces enough NO to regulate
myocardial O2 consumption (21). Triton X-100 abolished the
ability of an agent that stimulates NO production to regulate O2
consumption in the cardiac tissue, suggesting that eNOS is
present only in the EE cells. Hematoxylin and eosin slides
confirmed the ability of Triton X-100 to destroy the EE cells.
Although levels of eNOS protein were variable between control and Triton X-100-perfused tissue, NO production was
reduced in the Triton X-100-treated tissue, suggesting that
eNOS cannot function without an intact EE and that the EE is
the sole eNOS source in the frog heart. This suggests that NO
must diffuse from the EE to other parts of the heart, such as
myocytes, and in this way, regulate O2 consumption. The
avascular frog heart is a unique model in which the roles of the
EE and NO as a messenger can be studied without the confounding effects of the vascular endothelium or the presence of
myocyte NOS.
Invited Review: Point-Counterpoint
DIFFUSION OF NO TO MITOCHONDRIA
GRANTS
This study was supported by National Heart, Lung, and Blood Institute
Grants PO-1 HL-41023, HL-60142, and HL-61290 (to T. H. Hintze) and
American Heart Association Grant 02-35493T (to X. Zhang).
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with the effects of the production of NO from the endothelium
on the myocardium. Thus the conclusions by Paulus and
Bronswaer support our contention that endothelium-derived
NO may diffuse to the adjacent cardiac cells to modulate
cardiac contractile function in this case or mitochondrial function as discussed in our studies. This by no means indicates that
the only source of NO in the normal or diseased heart is the
endothelium. Several studies have proposed focal concentrations of NO based on immunohistochemical localization of
NOS isoforms in cardiac myocytes (1a). The question to be
addressed is not the potential of myocytes to make NO but the
concentration gradient over which NO is diffusing. If endothelial eNOS is downregulated and cardiac myocyte NOS, whatever the isoform, is upregulated then the cardiac isoform may
the primary contributor. When isoform swithching occurs in
the disease process is an area in need of some perspective and
for future studies. Our hypothesis that NO regulates cardiac
myocyte O2 consumption by regulating mitochondrial function
(25) would be complimentary with the proposal that NO
regulates cardiac contractile function associated consumption
of O2 even in the face of O2 wasting effects of excess
catecholamines.
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