A role for nitric oxide in hypoxia-induced activation of cardiac KATP channels in goldfish (Carassius auratus)
Department of Biological Sciences, Wellesley College, Wellesley, MA 02481, USA
* Author for correspondence (e-mail: jcameron{at}wellesley.edu)
Accepted 11 August 2003
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Summary |
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Key words: nitric oxide, hypoxia, ATP-sensitive K+ channels, KATP, K+ channels, goldfish, Carassius auratus
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Introduction |
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Mounting evidence suggests that KATP channels play a beneficial
role in cellular response to hypoxia and ischemia in mammals
(Cohen et al., 2000;
Grover and Garlid, 2000
;
O'Rourke, 2000
;
Yamada et al., 2001
). The
precise mechanism by which cardiac KATP channel activation protects
the heart is unclear, and the question is complicated by the fact that the
relative roles of sarcolemmal vs mitochondrial KATP
channels in cardioprotection are still being debated
(Gross and Fryer, 1999
;
Rajashree et al., 2002
;
Sato et al., 2000
). These
observations, however, invite speculation as to the role potentially played by
these channels in the hearts of aquatic ectotherms, which may be regularly
exposed to environmental hypoxia. Sarcolemmal KATP channels are
found at high densities in the hearts of goldfish
(Ganim et al., 1998
), and
those of the closely related crucian carp Carassius carassius are
activated during chemical anoxia and metabolic inhibition
(Paajanen and Vornanen, 2002
).
In addition, mitochondrial KATP channels have been shown to
influence cardiac performance during anoxia in the yellowtail flounder
(Limanda ferruginea; MacCormack
and Driedzic, 2002
). It has been hypothesized that KATP
channel activation under metabolically stressful conditions, whether in the
heart or in the brain, could contribute to the enhanced tolerance of
environmental hypoxia exhibited by many ectothermic vertebrates
(Cameron and Baghdady, 1994
;
Pek-Scott and Lutz, 1998
).
To the extent that KATP channels are acknowledged to play a
cardioprotective role during hypoxia in mammalian myocardium, identification
of the signaling pathway by which channel activation occurs has become a
priority. Within the past ten years, investigators have begun to appreciate
the diversity of roles played by nitric oxide (NO) in a number of
physiological processes within the heart
(Kelly et al., 1996). NO is
thought to exert a cardioprotective effect during ischemia in mammals
(Dawn and Bolli, 2002
;
Rakhit et al., 2001
). In
addition, NO has been reported to activate KATP channels in
mammalian ventricular myocytes (Moncada et
al., 2000
; Sasaki et al.,
2000
), and the protective effects of NO have been linked directly
to mitochondrial KATP activation
(Ockaili et al., 1999
;
Wang et al., 2001
).
Among non-mammalian vertebrates, information on the distribution of NO and
its importance to cardiovascular function has begun to emerge. Nitric oxide
synthase (NOS) has been localized in the hearts of teleost fish, including the
goldfish (Bruning et al.,
1996). There is evidence that NO acts as a modulator of peripheral
circulation in zebrafish (Danio rerio;
Fritsche et al., 2000
), eel
(Anguilla anguilla; Pellegrino et
al., 2002
) and most recently in the icefish Chionodraco
(Pellegrino et al., 2003
). NO
has been found to play a major role in regulating ventricular performance in
the working eel heart, acting through a cGMP-dependent mechanism
(Imbrogno et al., 2001
).
Finally, in relation to hypoxia, microvascular NOS production was found to be
enhanced after exposure to low oxygen conditions in the epaulette shark
(Hemiscyllium ocellatum; Renshaw
and Dyson, 1999
).
The purpose of this study, then, was to examine the potential role of NO in
cardiac KATP channel activation during hypoxia in a teleost species
known to be particularly tolerant of depleted environmental oxygen. Our
hypothesis was that the hypoxia-induced release of NO from cardiac myocytes or
vascular endothelium activates sarcolemmal KATP channels in
goldfish, a response that may enhance tolerance of hypoxia in this species.
Preliminary results of this work have appeared in abstract form
(Zia et al., 2001).
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Materials and methods |
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Intracellular studies
For conventional intracellular recording, hearts were quickly removed,
mounted in a tissue bath and superfused with normoxic saline solution at a
rate of 15 ml min-1. This solution contained: 150 mmol
l-1 NaCl, 5 mmol l-1 KCl, 1.2 mmol l-1
NaH2PO4, 1.2 mmol l-1 MgSO4, 1.8
mmol l-1 CaCl2, 10 mmol l-1 K-Hepes and 10
mmol l-1 glucose; pH was maintained at 7.6 and temperature held at
21±1°C.
Standard glass microelectrodes filled with 3 mol l-1 KCl were connected to a differential amplifier (WPI, Sarasota, FL, USA); spontaneous intracellular action potentials recorded from the most superficial layer of ventricular muscle were digitized (PowerLab; ADInstruments, Castle Hill, NSW, Australia) and analyzed using appropriate software (Chart, Peak Parameters; ADInstruments). Parameters measured were action potential amplitude, resting membrane potential, slope of action potential upstroke phase and action potential duration at various levels of repolarization. Data were exported to a Microsoft Excel spreadsheet for further analysis (see below).
Oxygen partial pressure in the temperature-controlled tissue bath was monitored with an electrode (DO-051; Cameron Instruments, Port Aransas, TX, USA) connected to an OM-200 oxygen meter. Moderate, substrate-free hypoxia (6.1±0.2 kPa) was induced by switching the superfusion solution from air-bubbled saline (20 kPa) to a glucose-free solution gassed with 100% N2; the new condition was reproducible, reversible and achieved in the tissue bath within 1 min. Electrophysiological responses to the acute onset of hypoxia were assessed first in the absence of any drug. Isolated hearts were allowed to equilibrate in the bath for 20 min prior to control recordings; experimental recordings were collected after 10 min exposure to hypoxia and again 20 min after a return to normoxic solution.
Drugs
Drugs, including the KATP channel antagonist 2,3-butanedione
monoxime (BDM; 60 µmol l-1) and the nitric oxide (NO) donor
S-nitroso-N-acetylpenicillamine (SNAP; 100 µmol
l-1), were purchased from Sigma-RBI (St Louis, MO, USA). BDM has
been shown to block surface KATP channels directly, by mechanisms
independent of channel dephosphorylation, and without affecting current
amplitude (Smith et al.,
1994). Following the initial period of equilibration in normoxic
saline, the effects of 5 min exposure to SNAP on ventricular action potential
parameters were recorded. In other experiments, BDM was made up in hypoxic
solution and applied to the tissue bath; recordings were made after 10 min, as
described above. Dosages were chosen from among those recently employed in
comparable studies using mammalian or teleost tissues.
Single-channel studies
Isolation of cardiac myocytes
Cell isolation procedures were extensively modified from those of Karttunen
and Tirri (1986), as
previously described (Ganim et al.,
1998
). All solutions used in this procedure were kept on ice at pH
7.6 and bubbled with 100% O2; all reagents were obtained from
Sigma.
After removal from the animal, the heart was bathed in a Ca2+-containing saline buffer: 137 mmol l-1 NaCl, 4.6 mmol l-1 KCl, 3.5 mmol l-1 NaH2PO4, 1.2 mmol l-1 MgCl2, 11 mmol l-1 glucose, 10 mmol l-1 K-Hepes and 0.025 mmol l-1 CaCl2. The atrium was tied off and an olive-tipped cannula (27 gauge) was inserted into the ventricle through the opening of the bulbus arteriosus. The ventricle was then perfused with an identical but nominally Ca2+-free saline solution for 5 min, followed by 25 min perfusion with an enzyme buffer containing 50 ml Ca2+-free saline, 35 mgcollagenase (Type I) and 25 mg bovine serum albumin (Fraction V). The heart was then removed from the perfusion apparatus and carefully torn into small pieces. After 10 min of swirling agitation, the supernatant containing single free-floating cells was removed. Fresh buffer was added, and agitation continued for an additional 10 min. After removal of the second supernatant fraction, the remaining tissue was gently triturated with a Pasteur pipette and agitated for 5 min, leaving a third fraction from which most of the cells used in this study were obtained. This procedure produced a high yield (80-90%) of viable, spindle-shaped myocytes.
Patch clamp
Isolated single myocytes were placed into a 7.5 ml tissue bath
(21±1°C) mounted on the stage of an inverted microscope (Nikon) and
superfused with normal saline or a pipette solution containing: 140 mmol
l-1 KCl, 5 mmol l-1 K-Hepes and 1 mmol l-1
EGTA. Single-channel recording was carried out using the cell-free
(inside-out) and cell-attached patch-clamp techniques described by Hamill et
al. (1981), as modified in our
laboratory for use with teleost myocytes
(Ganim et al., 1998
).
Recording electrodes of 2-4 M
resistance were prepared using 1.6
mm-diameter borosilicate glass capillary tubing (Drummond Scientific,
Broomall, PA, USA), pulled with a Sutter P-97 micropipette puller,
heat-polished on a custom microforge and positioned with a three-dimensional
hydraulic micromanipulator (Narishige, Tokyo, Japan). Appropriate suction
(10-30 cm water) to form a 1-3 G
seal was applied with a 60 ml syringe.
A List EPC-7 amplifier was used to monitor channel activity; output currents
were low-pass filtered at 1 kHz. The cell-free patches were bathed
symmetrically in equimolar potassium solution
([K+]i=[K+]o= 140 mmol
l-1; EK=0). In order to avoid channel rundown,
most experiments were conducted within 10-20 min of patch excision.
Data analysis
All single-channel data were stored using a Dagan DAS-900 videocassette
recorder. In most cases, data were also digitized on line using an ITC-18
computer interface (Instrutech, Port Washington, NY, USA), then filtered and
stored using commercial software (Acquire; Bruxton Corp., Seattle, WA, USA).
Subsequent analysis of stored records was accomplished using TAC/TACFit
software for Macintosh (Bruxton Corp.); event detection was carried out over
the first 10 s of data recorded after the switch from 0 voltage to a new
holding potential. The degree of channel activity before and after
experimental manipulation was assessed and plotted through determination of
the overall number of active channels in the patch, the overall probability
that a channel would be found in the open state (Po;
Ganim et al., 1998) and
through an analysis of channel closed- and open-lifetime distributions. For
this latter determination, a duration histogram was used to display the
relative frequency of dwell times between the transition from any one current
level within the recording to any other. Most patches contained at least 3-10
active channels, as reflected by multiple discrete levels of current within
the patch-clamp record.
Drug protocols
All pharmacological agents were obtained from Sigma-RBI and included ATP (2
mmol l-1), BDM (60 µmol l-1), SNAP (100 µmol
l-1), the stable and cell-permeable cGMP analog 8-bromoguanosine
3',5'-cyclic monophosphate (8-Br-cGMP; 200 µmol
l-1), an inhibitor of NO-sensitive guanylate cyclase
[1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ); 20 µmol l-1]
and the NO synthase inhibitor N-nitro-L-arginine methyl
ester (L-NAME; 50 µmol l-1).
Drugs were made up in the bath solution and administered either to the intracellular side of the membrane patch in the cell-free configuration or to the extracellular milieu in the case of cells studied using the cell-attached configuration. In most cases, drugs were made up as stock solutions and frozen in aliquots maintained at -70°C until just before use. These were added directly to the tissue bath. Other agents, in particular those made up in hypoxic solution (see below), were contained in separate reservoirs and entered the bath through a multi-barrel rapid infusion system (Warner Instruments, Hamden, CT, USA).
As in the intracellular experiments, ion channel responses to the acute onset of moderate hypoxia were first determined in the absence of any drug. After a brief period of equilibration under normoxic conditions (>20 kPa), hypoxic solution (6.0±0.3 kPa; N2-gassed and pH=7.6, as above) was rapidly infused into the tissue bath, and KATP channel activity over a range of depolarizing and hyperpolarizing potentials was assessed after 10 min, 20 min and 30 min using the cell-attached patch configuration. After each experimental challenge, the myocytes were again exposed to normoxic solution to test for current run-down, myocyte damage and reproducibility of results.
To determine their effects on the KATP channel response to hypoxia in intact cells, BDM, ODQ and L-NAME were made up in the N2-gassed hypoxic solution and were administered to the tissue as described for hypoxic solution alone. In other experiments, cells were exposed to each of these drugs under normoxic conditions. Channel activity in response to a range of holding potentials was recorded at 10 min, 20 min and 30 min after each experimental manipulation.
To help characterize the KATP channels under study and to determine their response to agents thought to influence nitric oxide pathways, we also recorded channel response to ATP, SNAP, 8-Br-cGMP and 8-Br-cGMP + BDM in cell-free, inside-out patches. Thus, these agents were applied directly to the intracellular surface of the patch. In each case, the initial 15 min stabilization period was followed by administration of the drug directly to the tissue bath; channel responses were recorded within 3 min and again at 10 min after drug exposure.
Statistical analysis
Data obtained from all experiments were transferred to Microsoft Excel
spreadsheets; paired t-tests and analysis of variance for repeated
measures (ANOVA with post-hoc Fisher PLSD and Scheffé F-test;
Statview) were used to determine statistical significance (P<0.05)
among group means; results were expressed as means ±
S.E.M.
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Results |
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The effect of hypoxia on APD was reversible upon the reintroduction of normoxic solution (20 kPa) to the tissue bath (Fig. 1, washout). Values for APD were collected 20 min after washout of the hypoxic solution; oxygen levels had returned to pre-experimental levels within 3 min.
To determine whether the hypoxia-induced reduction in myocardial APD was due to the activation of KATP channels, isolated hearts were exposed to hypoxia in the presence of the KATP antagonist 2,3-butanedione monoxime (BDM). At the concentration tested (60 µmol l-1), this agent prevented hypoxia-related shortening of APD (Fig. 2; N=6).
|
Effects of NO
To indirectly assess the potential for NO to activate KATP
channels in goldfish myocardium, the effects of the NO donor SNAP on action
potential parameters were monitored in isolated hearts. At a concentration of
100 µmol l-1, SNAP significantly reduced both APD50
(by 26.9%) and APD90 (by 12.1%)
(Fig. 3; N=8). There
were no significant effects of SNAP on any other action potential
parameter.
|
Single-channel studies
Characterization of goldfish cardiac KATP channels
Sarcolemmal KATP channel activity was first observed in isolated
myocytes using single-channel recording techniques in the cell-free
(inside-out) configuration (Hamill et al.,
1981). The channels showed some inward rectification, and the
current/voltage (I/V) relationship reflected a linear slope
conductance of 61 pS, a value similar to that previously reported by us for
goldfish (Ganim et al., 1998
)
and by others for KATP channels in mammalian heart
(Noma, 1983
;
Kakei et al., 1985
). Channel
activity was entirely abolished by the application of 2 mmol l-1
ATP.
Cell-attached patch-clamp experiments
Effects of hypoxia. Cardiac KATP channel activity was
also monitored using the cell-attached, patch-clamp configuration
(Hamill et al., 1981).
Following an initial equilibration period, intact, individual myocytes were
exposed in the tissue bath to moderately hypoxic solution (6.0±0.3
kPa), and channel activity was recorded at 10 min intervals over the following
30 min. In response to hypoxia, a slowly developing increase in
KATP channel activity was observed in seven of eight preparations.
Specific hypoxia-induced changes included an increase in the frequency of
KATP current transitions within 10 s of the application of
depolarizing potentials (Fig.
4), an increase in the number of active channels in the patch
(Fig. 5, inset), as well as
elevated open-state probability (Po;
Fig. 5, normoxia vs
hypoxia). Current amplitude and conductance were not affected. The overall
increase in channel activity was maximal after 20 min exposure to hypoxia and
did not increase significantly thereafter. The effect of hypoxia on the
channels was reversible, in that KATP activity declined again upon
return to the normoxic solution (Fig.
5, washout).
|
|
Effects of BDM, ODQ and L-NAME on hypoxia-induced changes. To determine whether the hypoxia-induced enhancement of channel activity could be inhibited by the KATP channel antagonist BDM, this agent was made up in the N2-gassed solution and administered simultaneously with hypoxic solution (N=4). At the concentration tested (60 µmol l-1), BDM prevented any significant increase in channel activity during hypoxia. Channel activity at 10 min, 20 min and 30 min, as determined by the number of active channels per patch, by overall Po (Fig. 5) and by channel closed- and open-lifetime distribution, was not significantly changed.
To further determine whether hypoxia-induced changes in KATP channel activity were mediated by a cGMP-dependent mechanism, the guanylate cyclase inhibitor ODQ was administered simultaneously with the onset of hypoxia (N=6). ODQ also prevented the significant increase in KATP activity observed with hypoxia alone (Fig. 5).
To clarify the role of NO in the hypoxia-induced elevation of KATP channel activity, the NO synthase inhibitor L-NAME was also administered simultaneously with the low oxygen solution (50 µmol l-1; N=4). After 20 min, channel activity was significantly elevated relative to normoxia; however, the extent of the increase was much less than that noted with hypoxia alone (Fig. 5).
To assess the influence of these agents when given alone, additional patches were treated with either BDM, ODQ or L-NAME under normoxic conditions. In four experiments each, exposure to BDM induced a consistent but not significant decline in KATP channel activity. At the concentrations tested, neither ODQ nor L-NAME induced any significant change in KATP channel Po (Fig. 5).
Cell-free patch-clamp experiments
Effects of NO. The inside-out patch clamp configuration was
employed to determine the effects of the NO donor SNAP on sarcolemmal
KATP channel activity. When applied directly to the intracellular
surface of the membrane, SNAP significantly increased KATP channel
activity, as measured by Po
(Fig. 6, left) and by the
number of open channels per patch (N=6). The NO-induced augmentation
of channel activity was usually observed within the first minute of exposure
to SNAP and was maximal after 3 min.
|
Effects of 8-Br-cGMP. In four experiments, the effects on KATP channel activity of the stable cGMP analog 8-Br-cGMP were monitored. Under normoxic conditions, this agent induced a significant increase in overall Po in all preparations (Fig. 6, right). When 8-Br-cGMP and BDM were applied simultaneously, however, there was no significant change in channel activity, suggesting that BDM blocks KATP activation by cGMP (Fig. 6; N=4).
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Discussion |
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KATP channels and hypoxia
Previous work in our laboratory has established that sarcolemmal
KATP channels show reduced ATP sensitivity in animals acclimated at
low temperatures, such that overall channel activity is enhanced
(Ganim et al., 1998). In the
present study, we observed a significant shortening of action potential
duration during hypoxia in the isolated goldfish ventricle
(Fig. 1), a response that was
inhibited by the KATP channel antagonist BDM
(Fig. 2). Hypoxia-induced APD
shortening in cardiac muscle is well established in the mammalian heart and
has been repeatedly ascribed to the activation of sarcolemmal KATP
channels (Nakaya et al., 1991
;
Nichols and Cukras, 2001
;
Shigematsu and Arita, 1997
).
Moreover, we showed that single KATP channels are activated during
hypoxia (Figs 4,
5), and that this response is
also sensitive to inhibition by BDM (Fig.
5). A similar response by sarcolemmal KATP channels in
trout heart might provide a mechanism for the protective effects of anoxic
myocardial preconditioning reported for that species
(Gamperl et al., 2001
), a
phenomenon widely attributed in mammals to KATP channel activation
(Grover and Garlid, 2000
;
O'Rourke, 2000
). Mitochondrial
KATP channels in the heart may promote hypoxia tolerance in the
yellowtail flounder (Limanda) by stabilizing intracellular
Ca2+ concentration (MacCormack
and Driedzic, 2002
).
Activation of cardiac KATP channels during moderate hypoxia has
not previously been demonstrated in ectotherms using single-channel recording
techniques. During complete metabolic inhibition induced by sodium dithionate
and/or iodoacetic acid, KATP channel activity has been induced in
cardiac myocytes of the closely related crucian carp (C. carassius),
even in the presence of 4 mmol l-1 ATP
(Paajanen and Vornanen, 2002).
In this species, however, severe chemical anoxia using oxygen scavengers or
cyanide (Vornanen and Tuomennoro,
1999
) first induces a large inward current that prolongs APD and
overwhelms the current through the KATP channel, reducing its
cardioprotective influence (Paajanen and
Vornanen, 2002
). In mammals, the cell-attached patch-clamp
configuration and a hypoxia regime similar to that employed in the present
study have been used to demonstrate a potentially cardioprotective
KATP current in guinea pig myocytes
(Liu et al., 2001
).
While myocardial ischemia may be a prominent component of the
pathophysiology of heart disease in humans, ATP/ADP ratios would not
ordinarily be expected to vary significantly in the healthy mammalian heart.
Aquatic ectotherms, however, may regularly be exposed to acute or chronic
depletion of environmental oxygen, and many are remarkably tolerant of
complete anoxia. Hochachka
(1986) and Hochachka et al.
(1996
) have proposed two
principal strategies for extending tolerance of hypoxia in such species. One
mechanism involves the matching of ATP requirements to reduced rates of ATP
synthesis by simply arresting metabolism. Alternatively, an organism might
remain active, compensating for reduced ATP-dependent ion pumping by
increasing the energetic efficiency of metabolic processes. One way to manage
this would be to decrease membrane permeability to Ca2+; thus,
despite decreased ATP turnover rates, ionic and electrical gradients across
the cell membrane would not be lost and intracellular Ca2+
accumulation could be avoided. An adaptive strategy that increases tissue ATP
levels or blocks uncontrolled Ca2+ influx might lessen the effects
of `leaky' membranes and promote hypoxia tolerance. Specific measures that
could be expected to diminish Ca2+ entry would include the
inhibition of sarcolemmal Ca2+ channels
(Wasser, 1996
) and the
activation of outward potassium currents, hyperpolarizing the cell and
blocking Ca2+ influx through voltage-gated channels
(Nilsson, 2001
;
Pek-Scott and Lutz, 1998
). It
is possible that KATP channel activation in critical tissues such
as heart or brain could, as one component in a suite of protective mechanisms
(Hochachka and Lutz, 2001
),
extend tolerance of hypoxia in those species most capable of survival under
such conditions (Cameron and Baghdady,
1994
; Lutz and Prentice,
2002
).
Nitric oxide
The role of endogenous NO generation in the regulation of myocardial
energetics in mammals has been studied extensively in recent years
(Kelly et al., 1996). Each of
the three principal isoforms of nitric oxide synthase (NOS) has been localized
within the heart and within individual cardiac myocytes
(Hare and Stamler, 1999
).
Indeed, NOS has been shown to be upregulated in cardiac myocytes during
ischemia, serving a cardioprotective function
(Wang et al., 2002
).
Subcellular localization of two isoforms in particular, NOS1 (nNOS) and NOS3
(eNOS), allows for varied effects of NO on cardiac ion channels in different
parts of the cell and even opposing effects on the heart with regard to
increased vs decreased contractility
(Barouch et al., 2002
).
Many recent studies suggest a cardioprotective role for NO during ischemia
in the mammalian heart (Dawn and Bolli,
2002; Nandagopal et al.,
2001
; Rakhit and Marber,
2001
). A variety of mechanisms by which NO release could protect
the heart during ischemia has been proposed; one possibility is that NO binds
to a heme moiety at the active site on soluble guanylate cyclase, leading to
an increase in the intracellular concentration of the second messenger cGMP.
This molecule could in turn influence the activity of specific Ca2+
or K+ channels, reducing myocardial contractility, oxygen
consumption and energy demand (Rakhit and
Marber, 2001
). KATP channels have been proposed as a
downstream effector in this NO-dependent cardioprotective pathway
(Dawn and Bolli, 2002
).
Relevant to the present study, NO has been reported to activate
KATP channels in mammalian vascular smooth muscle
(Miyoshi et al., 1994;
Murphy and Brayden, 1995
), as
well as sarcolemmal (Moncada et al.,
2000
) and mitochondrial
(Sasaki et al., 2000
) channels
in ventricular myocytes. The latter investigators used whole-cell recording
techniques to show that SNAP activated mitochondrial but not sarcolemmal
KATP channels in rabbit ventricular myocytes
(Sasaki et al., 2000
). This is
in contrast to the present observations in goldfish (Figs
3,
6) and to those of others using
a different NO donor and guinea pig myocytes
(Moncada et al., 2000
) or
endogenous NO and intact hearts (Chen et
al., 2000
).
The cardioprotective effects of NO have been associated with the acute or
chronic activation of mitochondrial KATP channels in mammals
(Eells et al., 2000;
Ockaili et al., 1999
;
Wang et al., 2001
). There is
evidence, however, that some forms of SNAP-induced cardioprotection in mammals
may indeed involve the mitochondria, yet remain entirely independent of
KATP channel activation (Rakhit et al.,
2000
,
2001
). Mitochondrial
KATP channels have been shown to exist in the fish heart
(MacCormack and Driedzic,
2002
), and NO in turn has been found to play a prominent role in
regulating peripheral vascular resistance and heart function in a variety of
teleosts (Fritsche et al.,
2000
; Imbrogno et al.,
2001
; Pellegrino et al.,
2002
,
2003
). Vascular NOS production
is enhanced during hypoxia in elasmobranchs
(Renshaw and Dyson, 1999
),
just as it is in mammals (Wang et al.,
2002
). The present results with the NOS inhibitor
L-NAME (Fig. 5)
indicate that a hypoxia-induced increase in NO production in goldfish heart
could activate sarcolemmal KATP channels (Figs
3,
6), potentially enhancing
tolerance of oxygen stress. The data also show that if this is the operative
mechanism for KATP channel activation, NOS must be localized within
individual myocytes, as endothelial cells were not present during patch-clamp
experiments. Even more NOS would be available in the intact heart, in light of
the fact that the endocardial endothelium in fish has a much greater surface
area relative to the volume of the ventricle than does the equivalent tissue
in mammals (Imbrogno et al.,
2001
).
In mammals, the hypoxia-induced activation of sarcolemmal KATP
channels is thought to involve NO and to be a cGMP-dependent mechanism
(Chen et al., 2000;
Han et al., 2001
). Prior
experiments with teleost fish involving the stable and lipid soluble cGMP
analog 8-Br-cGMP and the guanylate cyclase inhibitor ODQ indicate that an
NO-cGMP-dependent pathway is also operative in the vasculature and the heart
of the eel Anguilla anguilla
(Imbrogno et al., 2001
;
Pellegrino et al., 2002
). In
the present study, Fig. 5
indicates that the hypoxia-induced activation of sarcolemmal KATP
channels in goldfish can be blocked by ODQ. In addition,
Fig. 6 illustrates that the
effect of SNAP on these channels can be mimicked by 8-Br-cGMP and that this
response is prevented by the KATP channel antagonist BDM. These
observations are in accord with those in mammals, where 8-Br-cGMP has been
shown to mirror the effects of NO on sarcolemmal KATP channels,
decreasing APD by a mechanism that could be blocked by a KATP
antagonist (Chen et al., 2000
).
On the other hand, 8-Br-cGMP appears to have little effect on mitochondrial
KATP channels (Sasaki et al.,
2000
).
Tolerance of environmental hypoxia has evolved independently in a number of
different teleost lineages (Hochachka and
Lutz, 2001). Few species have been thoroughly investigated in this
regard, and some observations suggest that there are broad interspecific
differences in terms of the precise suites of defense mechanisms employed
among various taxa. The evidence increasingly supports the possibility,
however, that certain mechanisms arose early in the vertebrate line and serve
to extend the hypoxia tolerance of specific tissues in a variety of living
species. While a full evolutionary analysis of hypoxia adaptations in fishes
must await further investigation, the present data support the hypothesis that
KATP channels in goldfish play a cardioprotective role in hypoxia,
just as they do in mammals (Grover and
Garlid, 2000
). These results further suggest that a
hypoxia-induced enhancement of NO production in the heart of this species
opens sarcolemmal KATP channels via a cGMP-dependent
mechanism.
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Acknowledgments |
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References |
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