Articles in PresS. Am J Physiol Heart Circ Physiol (August 22, 2008). doi:10.1152/ajpheart.00623.2008
Role Played by Acid Sensitive Ion Channels in evoking the Exercise Pressor
Reflex
Shawn G. Hayes1, Jennifer L. McCord1, Jon Rainier3, Zhuqing Liu4
and Marc P. Kaufman1
Heart and Vascular Institute, Penn State College of Medicine1,
Department of Chemistry3, University of Utah and Department of
Chemistry, University of Pennsylvania, Philadelphia4
Running title: A-317567 and the exercise pressor reflex
1
Copyright © 2008 by the American Physiological Society.
Abstract
The exercise pressor reflex arises from contracting skeletal muscle
and is believed to play a role in evoking the cardiovascular responses to
static exercise, effects which include increases in arterial pressure and
heart rate. In part, this reflex is believed to be evoked by metabolic and
mechanical stimulation of thin fiber muscle afferents. Lactic acid is known
to be an important metabolic stimulus evoking the reflex. Until recently,
the only antagonist for acid sensitive ion channels (ASICs), the receptors
to lactic acid, was amiloride, a substance which is also a potent
antagonist for both epithelial sodium channels (ENaCs) as well as voltage
gated sodium channels. Recently, a second compound, A-317567, has
been shown to be an effective and selective antagonist to ASICs in vitro.
Consequently, we measured the pressor responses to static contraction of
the triceps surae muscles in decerebrate cats before and after popliteal
arterial injection of A-317567 (10 mM solution; 0.5 ml). We found that this
ASIC antagonist significantly attenuated by half (P < 0.05) the pressor
responses to both contraction and to lactic acid injection into the
popliteal artery. In contrast, A-3175657 had no effect on the pressor
responses to tendon stretch, a pure mechanical stimulus, and to popliteal
arterial injection of capsaicin, which stimulated TRPV1 channels. We
2
conclude that ASICs on thin fiber muscle afferents play a substantial role
in evoking the metabolic component of the exercise pressor reflex.
Key words: group III and IV muscle afferents, cats, neural control of
the circulation, lactic acid, mechanogated channels, TRPV1 channels.
Introduction
The exercise pressor reflex arising from contracting skeletal muscles
is one of the mechanisms responsible for the cardiovascular adjustments
to static exercise (5; 20). These reflex adjustments include increases in
arterial blood pressure, heart rate and ventilation (22). The afferents arm
of the exercise pressor reflex is composed of group III and IV muscle
afferents(20; 22) andis thought to be evoked by both mechanical and
metabolic stimuli within the contracting muscles (14-16).
One of the metabolic by-products of muscle contraction that is
thought to play a role in evoking the metabolic component of the
exercise pressor reflex is lactic acid. Several lines of evidence suggest that
lactic acid plays such a role. The most direct evidence is that lactic acid
injected into the arterial supply of muscle stimulates group III and IV
muscle afferents as well as evokes a reflex pressor response (25-27).
Another line of evidence is that lactic acid concentrations in the muscle
interstitium are increased by contraction (19). Finally, blunting lactic acid
production with either dichloroacetate or glycogen depletion decreases
the exercise pressor reflex (9; 28).
3
An important requirement of showing that a substance plays a role
in evoking the metabolic component of the exercise pressor reflex is to
demonstrate that this component is attenuated by blockade of its
receptor on group III and IV muscle afferents. Recently, this demonstration
was done using amiloride (4; 12). However amiloride has many effects in
addition to blocking the receptor for lactic acid. Two of the most
troublesome are that amiloride blocks epithelial sodium channels (ENaCs)
in lower concentrations than those needed to block ASICs and that it
blocks voltage gated sodium channels in concentrations possibly higher
than those needed to block ASICs. Blocking either ENaCs or voltage
gated sodium channels would interfere with an afferent’s ability to
generate action potentials, thereby preventing it from evoking reflex
effects regardless of the stimulus used. Because either of these effects
could occur using amiloride we are still uncertain if ASICs play a role in
evoking the reflex. To determine whether ASICS play a role in evoking the
exercise pressor reflex we used A-317567 to block this receptor to lactic
acid on sensory nerve endings in skeletal muscle. A-317567 has been
reported to have no effect on ENaCs (8)and in the doses that we used
did not prevent reflex responses to stimuli other than lactic acid.
Methods
4
All procedures were reviewed and approved by the Institutional
Care and Use Committee of the Pennsylvania State University, Hershey
Medical Center.
Surgical preparation. Adult cats of either sex (n = 12, 2.9 ± 0.2 kg,
range: 2.3 – 3.2 kg) were anesthetized with a mixture of 5% isoflurane and
oxygen. The right jugular vein and common carotid artery were
cannulated for the delivery of drugs and fluids and the measurement of
arterial blood pressure, respectively. The carotid arterial catheter was
connected to a pressure transducer (model P23 XL, Statham) to monitor
blood pressure. Heart rate was calculated beat-to-beat from the arterial
pressure pulse (Gould Biotach). The trachea was cannulated, and the
lungs were ventilated mechanically (Harvard Apparatus). Arterial blood
gases and pH were measured by an automated blood gas analyzer
(model ABL-700, Radiometer). PCO2 and arterial pH were maintained
within normal range by either adjusting ventilation or intravenous
administration of sodium bicarbonate (8.5%). A temperature probe was
passed through the mouth to the stomach. Temperature was continuously
monitored and maintained at 37–38°C by a water-perfused heating pad.
The left common iliac artery and vein were isolated, and snares
were placed around these vessels to trap the A-317567 in the circulation
of the leg (see below). The left triceps surae muscles, left popliteal artery,
and tibial nerve were isolated. The cat was placed in a Kopf stereotaxic
5
frame and spinal unit. The left calcaneal bone was cut, and its tendon
was attached to a force transducer (model FT-10C, Grass) for
measurement of the tension developed during stretch and static
contraction of the left triceps surae muscles. The knee joint was secured to
a post.
The cats were decerebrated at the mid-collicular level under
isoflurane anesthesia. Dexamethasone (4 mg) was injected intravenously
just before the decerebration procedure to minimize brain edema. The
left common carotid artery was tied off to reduce bleeding. All neural
tissue rostral to the mid-collicular section was removed and the cranial
vault was filled with agar.
Experimental protocol. The effect of A-317567 (10 mM solution; 0.5
ml) and its structural analog ZL-1 (10 mM solution; 0.5 ml) (Figure 1) on the
reflex pressor and cardioaccelerator responses to four stimuli were
assessed: 1) static contraction of the left triceps surae muscles, 2) stretch
of the left calcaneal tendon, which in turn stretched the triceps surae
muscles, 3) injection of lactic acid (24mM; 0.5-1.0 ml) into the left popliteal
artery, and 4) injection of capsaicin (2 µg) into the left popliteal artery. In
a three kilogram cat, the amount of A-317567 injected into the popliteal
artery corresponded to a dose of about 1.7 µM/ Kg. Contraction and
tendon stretch lasted 60 s. The triceps surae muscles were contracted
statically by electrical stimulating the left tibial nerve (40 Hz, 25 µs, 2 times
6
motor threshold); stretch was induced by turning a rack-and-pinion that
was attached to the calcaneal tendon. Baseline tension was set at 0.3–0.5
kg. Arterial blood pressure and heart rate were recorded for 60 s before
and during static contraction or tendon stretch. Injections of lactic acid
and capsaicin were accomplished by gently inserting a 30-gauge needle
into the popliteal artery and then injecting the compounds over
approximately 10 s into the vasculature of the triceps surae muscles.
Before injecting lactic acid into the popliteal artery, we paralyzed the cat
by injecting intravenously rocuronium bromide (0.5–0.7 mg/kg). These
maneuvers were repeated at 10 and 45 minutes after injection of A317567 into the left popliteal artery. Immediately before injecting A317567, we tightened the snare placed around the left common iliac
artery and vein. A-317567 was then injected into the popliteal artery and
the snares were maintained for 10 minutes, after which they were
released and the hind limb was freely perfused. At this point, the triceps
surae muscles were contracted followed by stretch and injection of lactic
acid. A-317567 attenuated the exercise pressor response by 10 minutes
and was no longer effective 45 minutes after its injection.
Data analysis. Mean arterial blood pressure and heart rate values
are expressed as means ± SEM. Baseline mean arterial blood pressure and
heart rate were measured immediately before a maneuver, and peak
mean arterial blood pressure and heart rate were measured during
7
injection of lactic acid and capsaicin as well as during the 60 s of tendon
stretch or static muscle contraction. For statistical analyses, data from
experiments using A-317567 and ZL-1 were pooled. Statistical comparisons
were performed either with a one-way repeated-measures ANOVA or
two-way repeated-measures ANOVA. If significant main effects were
found with an ANOVA, post-hoc tests were performed with the StudentNeuman-Keuls test between individual means. We used Bonferroni and
the Dunnett’s post hoc tests to determine significant differences between
time course means. The criterion for statistical significance was P < 0.05.
Results
We examined the effects of injecting A-317567 and ZL-1 (both 0.5
ml; 10 mM) into the popliteal artery on the pressor-cardioaccelerator
responses to four stimuli, namely static contraction of the triceps surae
muscles, stretch of the calcaneal tendon, which in turn stretched the
triceps surae muscles, lactic acid injection (0.5 ml; 24mM) into popliteal
artery, and capsaicin (0.5 ml; 2 µg) into the popliteal artery. The blockade
of ASICs by A-317567 or ZL-1 was short lasting (about 15 minutes) and
therefore not every stimulus was tested in every cat. Because A-317567
and ZL-1 produced similar effects on the cardiovascular responses to
8
each maneuver the data were pooled. The 10 mM concentration of A317567 or ZL-1 was used because in three cats a ten-fold smaller
concentration (i.e., 1mM) in 0.5 ml had no effect on either the pressor
response to either lactic acid injection or static contraction of the triceps
surae muscles. For example, the pressor responses to lactic acid injection
before, 10 minutes and 45 minutes after A-317567 averaged 32 ± 6 mmHg,
30 ± 6 mmHg, and 35 ± 5 mmHg, respectively (n=3). These very low
concentration injections also acted as vehicle controls because the same
volume of the vehicle (0.5 ml) was injected as with the larger (10mM)
doses.
Lactic Acid Injection. In each of the 12 cats tested, we found that
A-317567 (n=5) and ZL-1 (n=7) attenuated the pressor response to lactic
acid injection into the popliteal artery. On average, the pressor responses
were decreased by almost 75% by the ASIC antagonist (P < 0.05). The
interval between injection of A-317567 and second lactic acid injection
was ten minutes. On average, the pressor response to lactic acid was fully
restored 45 minutes after injection of the ASIC antagonist (Figures 2 and 3;
Table 1). The cardioaccelerator responses to lactic acid injection were
small and variable and were not significantly decreased by A-317567
(Table 1).
9
Capsaicin Injection. In six cats, we found that A-317567 (n=3) and
ZL-1 (n=3) had no effect on the pressor-cardioaccelerator responses to
capsaicin injection into the popliteal artery (Figure 2).
Tendon Stretch. In eight cats, we found that A-317567 (n=3) and ZL1 (n=5) had no effect on the pressor-cardioaccelerator responses to
stretching the calcaneal tendon (Figures 2 and 4; Table 1). The ASIC
blockers had no effect on either the magnitude of the pressor response or
its onset latency. Specifically, the increase in blood pressure from the
onset of tension development had a latency of 4.2 ± 1.5 seconds before
A-317567 and 4.0 ± 1.5 seconds after A-317567 (P>0.05; n=8). The tension
time indices calculated before, 10 minutes after and 45 minutes after
injection of A-317567 were not significantly different from each other (P
>0.05; Table 2).
Static Contraction. In each of 9 cats tested, we found that A-317567
(n=4) and ZL-1 (n=5) attenuated the peak pressor response to static
contraction decreasing it on average by about 60% (P <0.05). The interval
between injection of A-317567 and the second static contraction was ten
minutes. On average, the pressor response to static contraction was fully
restored 45 minutes after injection of the ASIC antagonist (Figures 2 and 4;
Table 1). The cardioaccelerator responses to static contraction were small
and variable and were not significantly decreased by A-317567 (Table 1).
10
The ASIC blockers did not decrease the magnitude of the pressor
response within the first 5 seconds of contraction. For example, at 2
seconds after the onset of tension development the mean pressor
response was 4.1±1.6 mm Hg before ASIC blockade and 3.3 ± 0.8 seconds
afterwards. Likewise, at 5 seconds after the onset of tension development
the mean pressor response was 11.8 ±2.8 mm Hg before ASIC blockade
and 9.6 ± 3.1 mm Hg afterwards (Figure 5A and B). The ASIC blockers did
not change the onset latency of the pressor response (Figure 5A).
Specifically, the increase in blood pressure from its onset of tension
development had a latency of 1.9 ± 0.4 seconds before A-317567 and 2.1
± 0.3 seconds after A-317567 (P>0.05; n=8). The tension time indices
calculated before, 10 minutes after and 45 minutes after injection of A317567 were not significantly different from each other (P >0.05; Table 2).
Last, in three cats we found that A-317567 injected intravenously
into the jugular vein (0.5 ml, 10mM) had no effect the pressor responses to
static contraction. Specifically, the change in mean arterial pressure
during static contraction was 22.0 ± 1.5 mm Hg before i.v injection of A317567 and 22.5 ± 0.3 mm Hg afterwards.
Discussion
A-317567 is an ASIC antagonist that is structurally dissimilar to either
amiloride or its analogues (8). We have shown that popliteal arterial
injection of A-317567 markedly attenuated the pressor responses to both
11
static contraction of the triceps surae muscles and lactic acid, but had no
effect on the pressor responses to either tendon stretch or capsaicin
injection. Our findings are consistent with the hypothesis that A-317567
antagonized acid sensitive ion channels on group III and IV afferents, but
had no effect on either TRPV1 channels or mechanogated channels.
Previous evidence suggests that the dose of A-31756 used in our
experiments did not antagonize epithelial sodium channels (ENaCs) (8).
Dube et al found in mice that intraperitoneal injection of A-317567, in
doses that blocked withdrawal responses to nociceptive stimuli, had no
effect on urine output, and sodium and potassium excretion (8). The
strong possibility that A-317567 did not antagonize ENaCs distinguishes this
compound from amiloride, which blocks both ENaCs and ASICs. In fact,
the concentration of amiloride needed to block ENaCs is 10-100 fold
lower than that needed to block ASICs (1; 17).
Use of amiloride as an antagonist to ASICs has a second
complication in addition to its blockade of ENaCs. Specifically, amiloride
in relatively high concentrations appears to block voltage gated sodium
channels (3; 12; 18), an effect that impairs the ability of sensory nerves to
generate action potentials. This complication was not a factor in our past
studies because the dose of amiloride used was an order of magnitude
lower than that needed to block voltage gated sodium channels (12, 21).
Moreover, if this complication occurred in the present experiments, one
12
would expect to see an A-317567-induced attenuation of the pressor
responses to capsaicin injection and to tendon stretch, effects which did
not occur.
Acid sensitive ion channels found in dorsal root ganglion cells are
encoded by three genes and two splice variants (2). A-317567 probably
does not distinguish between the various ASICs. Nevertheless, the ASIC3,
which is also known as DRASIC, stands out because it is for the most part
only found on dorsal root ganglion cells, whereas the other ASICs are
distributed throughout the spinal cord and brain (33). In vitro, the ASIC3
opens when exposed to pHs of 7.4-7.0, proton concentrations that are
found in the interstitium of skeletal muscle when it contracts (13). These
findings led us to speculate that ASIC3 on the endings of group III and IV
muscle afferents were blocked in our experiments by A-31756.
In humans, the role played by lactic acid in evoking the metabolic
component of the exercise pressor reflex is controversial. In particular, this
controversy has arisen from the study of the responses to exercise by
patients with McArdle’s disease, a pathological condition in which
individuals produce little lactic acid because of a myophosphorylase
deficiency. Two studies using McArdle’s patients have shown that the
pressor and muscle sympathetic nerve responses to static exercise are
almost non-existent (10; 24), whereas two others have shown that these
responses to handgrip are preserved (31; 32). In humans, substantial
13
evidence has accumulated that both the pressor and muscle
sympathetic nerve responses to static exercise are caused by the exercise
pressor reflex (23; 30), and as a consequence these responses are
important indications of its presence or absence. Our past (12; 21) and
present findings demonstrate that lactic acid does play a role in the
generation of the metabolic component of the exercise pressor reflex,
although we can offer no insights into the controversial findings in humans
about this issue.
Our findings suggest that ASIC blockade inhibited the discharge of
metaboreceptors most of which respond to contraction with a latency of
at least 5 seconds, but had little, if any, effect on the discharge of
mechanoreceptors, most of which respond to contraction with a latency
of a second or less (15). In a previous study, we found that amiloride, an
ASIC antagonist, did not significantly attenuate the contraction-induced
increase in renal nerve sympathetic activity for the first ten seconds of the
contraction period (21). Similarly, in the present study we found that A317567 attenuated the peak pressor responses to contraction, but had no
effect on the pressor responses to tendon stretch. Moreover, the pressor
response to the first five seconds of the contraction period was not on
average attenuated by ASIC blockade (Figures 4 and 5); likewise, the
onset latency of this response was not changed either. Based on these
findings, we offer the speculation that A-317567 attenuated input from
14
metaboreceptors, most of which are innervated group IV afferents (15;
16). Usually group IV afferents do not respond much during the first five
seconds of contraction, a period of time which usually does not allow
metabolic by-products of contraction to accumulate to threshold levels.
Findings from isolated dorsal root ganglion cells taken from
transgenic mice suggest that ASICs play little role in non-noxious
mechanosensation but may play an important role in transducing pain.
For example, the currents generated by wild type dorsal root ganglion
cells responding to mechanical stimuli were no different from those
generated by ASIC2 and 3 knockout dorsal root ganglion cells (6). In
addition, ASIC 3, but not ASIC 1, knockout mice failed to develop chronic
pain caused by repeated intramuscular injection of acid (29). Finally, A317567 has been shown in rats to be a more effective blocker than
amiloride of post-operative pain, which in turn was thought to be caused
by proton release from the wound site (8).
ASICs in dorsal root ganglion cells are believed to be
heteromultimeric (2). Not only can different ASICs combine to form
channels on sensory nerves, but ASICs can combine with ENaCs to form
channels as well (7). This latter type of combination might be especially
relevant to acid sensing by thin fiber afferents in skeletal muscle.
Specifically, the δENaC protein is thought to confer proton sensitivity to
multimeric channels (7), and in combination or in isolation with the ASIC 3
15
protein might determine proton sensitivities to lactic acid generation by
exercising muscles.
In conclusion, we have shown that A-317567 injected into the
arterial supply of hind limb muscle attenuated the reflex pressor responses
to lactic acid and static contraction. Injection of this purported ASIC
antagonist had no effect, however, on the reflex pressor responses to
either capsaicin injection or to tendon stretch. The former stimulus is an
agonist for TRPV1 channels and the latter activated mechanogated
channels, which can be blocked by gadolinium injection into the arterial
supply of the hind limb muscles (11). A-317567 in doses that appear to
block ASICs appears to have little effect on ENaCs. Consequently, the
combination of its apparent blocking effect on lactic acid-evoked pressor
reflex and its lack of effect on ENaCs(8), A-317567, is at the present time,
the compound of choice with which to determine the role played by
ASICs on thin fiber muscle afferents in reflexly activating the
cardiovascular system during exercise.
Acknowledgements
We thank Jennifer Probst for her technical assistance. This work was
supported by NIH grant HL30710.
16
Table 1. Effects of A-317567 injected into the popliteal artery on the cardioaccelerator
and pressor responses to lactic acid, static contraction, capsaicin and tendon stretch.
Change HR
Baseline HR
Change MAP Baseline MAP
Lactic Acid (n=12)
Before
11 ± 4**
145 ± 14
38 ± 5**
121 ± 7
10 Minutes
0 ± 2†
157 ± 10
13 ± 3†
133 ± 9
45 Minutes
7 ± 2*
163 ± 11
39 ± 6**
131 ± 9
Static Contraction (n=9)
Before
10 ± 3**
129 ± 17
36 ± 5**
122 ± 12
10 Minutes
4 ± 2*
148 ± 14
17 ± 2†
134 ± 11
45 Minutes
10 ± 2**
140 ± 13
28 ± 3**
132 ± 11
Capsaicin (n=6)
Before
15 ± 10
149 ± 19
34 ± 8
122 ± 6
10 Minutes
8±3
164 ± 13
40 ± 11
129 ± 8
45 Minutes
10 ± 9
174 ± 11
37 ± 10
132 ± 11
Tendon Stretch (n=8)
Before
5 ± 2*
142 ± 21
23 ± 5**
117 ± 13
10 Minutes
3±1
153 ± 17
24 ± 4**
124 ± 11
45 Minutes
4 ± 2*
156 ± 17
22 ± 3**
126 ± 10
Asterisks denote significance from corresponding baseline value (* P< 0.05; ** P< 0.01).
Daggers denote significance from value before A-317567 injection († P< 0.05).
17
Table 2. The tension time indices for static contraction and tendon stretch during reflex
experiments.
Tendon Stretch Static Contraction
Before A-317567
146 ± 16
143 ± 17
10 minutes
165 ± 18
135 ± 13
45 minutes
163 ± 14
119 ± 9
There were no significant differences (P>0.05) between corresponding means before and
10 or 45 minutes after A-317567 injections (10mM; i.a.). All values are expressed in
kg•s.
18
Figure 1. Two compounds, A-317567 and its structural analog ZL-1,
were used in the present study. The two compounds are structurally
identical
except
for
their
connectivity
about
their
respective
tetrahydroisoquinoline rings. As illustrated, the cyclopropane of A-317567 is
attached to position 7 of the isoquinoline while the cyclopropane in ZL-1 is
connected at position 6. Both compounds were thoroughly characterized
and their structures assigned using a combination of chemical intuition
and spectroscopy (NMR (1H and 13C), IR, and MS). For the purposes of this
work, A-317567 and ZL-1 behaved identically.
Figure 2. Effects of A-317567 on the pressor responses to arterial
injection of lactic acid (0.2-0.5 ml; 24mM), static contraction, arterial
injection of capsaicin (1-2 ug, 0.25 ml) and stretch of the calcaneal
tendon. Note each maneuver was performed before giving A-317567, 10
minutes after giving A-317567, and 45 minutes after giving A-317567. Filled
bars represent changes in mean arterial pressure above baseline.
Asterisks (*) signify significant difference (P< 0.05) between baseline and its
corresponding
peak
effect.
Horizontal
brackets
signify
significant
difference (P< 0.05) between pressor response before injection and that
either 10 or 45 minutes after giving A-317567.
Vertical brackets signify
standard errors.
19
Figure 3. Pressor (MAP) and heart rate (HR) responses to popliteal
arterial injections lactic acid (0.2-0.5 ml; 24mM) (Panel A-before giving A317567; panel B-10 minutes after giving A-317567, and panel C-45 minutes
after giving A-317567). Vertical arrow represents the beginning of the
injection. Horizontal bar indicates 10 seconds.
Figure 4. Pressor responses to static contraction and stretch of the
calcaneal tendon before giving A-317567; 10 minutes after giving A317567, and 45 minutes after giving A-317567. Note the top trace is muscle
tension (T) and the lower trace is arterial blood pressure (BP).
Figure 5. Pressor responses to static contraction at the onset of
tension development before and 10 minutes after ASIC blockade. Panel
A: Muscle tension and BP responses to static contraction before (upper
traces) and 10 minutes after (lower traces) A-317567 injection. Dashed
vertical lines represent three time points: onset of tension development
(0), 5 seconds and 10 seconds. Horizontal dashed lines represent BP at 200
and 160 mm Hg. Note that in the first five seconds of contraction Panel B:
Changes in mean arterial pressure during contraction at 2 seconds and 5
seconds after the onset of tension development. Filled circles represent
before A-317567 and open circles represent after A-317567. Note there
were no significant differences in the pressor responses at either 2 or 5
20
seconds
21
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27
Figure 1
NH
NH
NH2
N
7
A-317567
NH2
6
N
ZL-1
Figure 5
A
0
5
B
10
Before A‐317567
3
Tension
(kg)
0
200
BP 160
(mm Hg)
100
3
Tension
(kg)
0
200
BP 160
(mm Hg)
100
After A‐317567