Vol. 281, Issue 4, H1771-H1777, October 2001
Parallel metabotropic pathways in the heart of the toad,
Bufo marinus
Narelle J.
Bramich,
Helen M.
Cousins,
F. R.
Edwards, and
G.
D. S.
Hirst
Department of Zoology, University of Melbourne, Victoria, Australia
3010
 |
ABSTRACT |
This study examined the transduction pathways
activated by epinepherine in the pacemaker region of the toad heart.
Recordings of membrane potential, force, and intracellular
Ca2+ concentration ([Ca2+]i) were
made from arrested toad sinus venosus. Sympathetic nerve stimulation
activated non-
-, non-
-adrenoceptors to evoke a membrane depolarization and a transient increase in
[Ca2+]i. In contrast, the -adrenoceptor
agonist isoprenaline (10 µM) caused membrane hyperpolarization
and decreased [Ca2+]i. The phosphodiesterase
inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) mimicked the
isoprenaline-evoked membrane hyperpolarization. Epinephrine
(10-50 µM) caused an initial membrane depolarization and an
increase in [Ca2+]i followed by membrane
hyperpolarization and decreased [Ca2+]i. The
membrane depolarizations evoked by sympathetic nerve stimulation or
epinephrine were abolished either by the phospholipase C
inhibitor U-73122 (20 µM) or by the blocker of
D-myo-inositol 1,4,5,-trisphosphate-induced Ca2+ release, 2-aminoethoxydiphenyl borate (2-APB, 60 µM). Neither U-73122 nor 2-APB had an affect on the membrane
hyperpolarization evoked by -adrenoceptor activation. These results
suggest that in the toad sinus venosus, two distinct transduction
pathways can be activated by epinephrine to cause an increase in heart rate.
cardiac; inositol trisphosphate; cAMP
| |
INTRODUCTION |
STIMULATION OF THE
SYMPATHETIC nerves innervating mammalian and amphibian hearts
causes an increase in heartbeat rate (12, 21). These
responses are generally assumed to result from the activation of
postjunctional -adrenoceptors by catecholamines, with such
-adrenoceptors being linked to the activation of adenylate cyclase
and production of cAMP (13). However, catecholamines released from sympathetic nerves innervating amphibian and mammalian cardiac pacemaker tissue appear to combine with a distinct set of
adrenoceptors linked to a pathway that does not involve cAMP (4,
6, 8). In both mammals and amphibians, the adrenoceptors linked
to the cAMP-independent pathway are likely to have a junctional location residing within the neuroeffector cleft and are stimulated readily by a neurally released transmitter. Exogenously applied transmitters do not readily activate junctional receptors but easily
activate extrajunctional receptors that are located outside the
neuroeffector cleft and are linked to the cAMP-dependent pathway (4, 6, 8). A comparison of the differences between
neurally released and exogenously applied catecholamine is most easily studied in amphibians where, in contrast to mammals, the junctional and
extrajunctional adrenoceptors are pharmacologically distinct, junctional receptors being non--, non--adrenoceptors
and extrajunctional receptors being -adrenoceptors. In amphibians,
the differences between the activation of the two types of
adrenoceptors are most apparent in pacemaker preparations in which the
discharge of pacemaker action potentials has been prevented by organic
Ca2+ antagonists. In these arrested preparations, the
activation of -adrenoceptors causes membrane hyperpolarization as
does the elevation of cAMP. In contrast, sympathetic nerve stimulation initiates a membrane depolarization that does not appear to involve the
elevation of cAMP (4). The adrenoceptors activated either by a neuronally released transmitter or high concentrations of rapidly
applied epinephrine cannot be blocked with either - or -adrenoceptor antagonists (6, 19). However, such
receptors can be blocked with dihydroergotamine (6). This
indicates that both neuronally released epinephrine and bath-applied
epinephrine can activate a set of dihydroergotamine-sensitive receptors
that are non--, non--adrenoceptors. When activated, these
receptors cause the release of Ca2+ from intracellular
stores and initiate positive chronotropic and inotropic responses
(5). Furthermore, transient increases in intracellular
Ca2+ concentration ([Ca2+]i)
accompany membrane depolarization evoked by stimulation of the
junctional non--, non--adrenoceptors in arrested preparations (9). In many other preparations, transmitter-evoked
release of Ca2+ from intracellular Ca2+ stores
is triggered by the production of D-myo-inositol
1,4,5,-trisphosphate [Ins(1,4,5)P3] (2).
First, this study examined the differences between the responses
produced by -adrenoceptor activation and those produced by
sympathetic nerve stimulation and the activation of non--,
non--adrenoceptors in arrested preparations of toad sinus venosus.
Second, the hypothesis was tested that, in the toad sinus venosus, the
stimulation of non--, non--adrenoceptors results in the
activation of a pathway involving the elevation of
Ins(1,4,5)P3.
| |
METHODS |
The procedures described have been approved by the animal
experimentation committee at the University of Melbourne. Toads (Bufo marinus) were anesthetized by immersion in a solution
of 0.5% tricaine methanesulfonate (Thomson and Joseph, Norwich, UK) in
tap water. Preparations consisted of the sinus venosus and two atria,
with the ventricle and truncus arteriosus removed. Both the left and
right vagosympathetic trunks were dissected free back to the
sympathetic chains and left intact with the sinus venosus. Preparations
were pinned flat in a shallow recording chamber, the base of which
consisted of a microscope coverslip coated with Sylgard silicone resin
(9) and continuously perfused with physiological saline
solution composed of (in mM) 115 NaCl, 3.2 KCl, 20 NaHCO3,
3.1 NaH2PO4, 1.8 CaCl2, 1.4 MgCl2, and 16.7 glucose gassed with 95% O2-5%
CO2 at a rate of 6 ml/min (bath volume, 1.5 ml) unless
otherwise stated. In some experiments, force generated by the tissue
was also measured by placing a fine platinum hook attached to a tension
transducer through the sinus venosus. The discharge of pacemaker action
potentials was prevented by adding nifedipine (10 µM) to the
perfusion fluid (6). Experiments were carried out at room
temperature (22-25°C). The sympathetic nerves were stimulated by
passing the sympathetic trunks through a pair of platinum ring
electrodes (stimulation currents, 10-60 nA; pulse width, 1.0 ms).
Sympathetic nerves were stimulated at 10 Hz for 1 s. Pilot experiments
using long trains of sympathetic nerve stimulation revealed that a 1-s
train of sympathetic stimuli is maximal. That is, there is no change in
the amplitude, duration, or complexity of response with increasing the
duration of sympathetic stimuli (up to 30 s). Drugs were added to
the preparation by changing the inflow line from the control solution
to one containing the appropriate concentration of drug. When
epinephrine and isoprenaline were perfused onto the sinus venosus, the
perfusion rate was increased to 12 ml/min. Membrane potential
recordings were made using conventional techniques with fine glass
microelectrodes (resistance, 120-240 M
) filled with 0.5 M KCl.
In some experiments, concurrent measurements of membrane potential and
relative changes in [Ca2+]i were made from
pinned sinus venosus preparations loaded with the fluorescent
Ca2+ indicator fura 2. This was achieved by incubation of
the sinus venosus preparations in HEPES-buffered physiological saline
solution containing the membrane permeable form of fura 2, fura
2-acetoxymethyl ester (fura 2-AM, 10 µM), and a low concentration of
Ca2+ (0.1 mM). The dispersal of fura 2-AM into the muscle
cells was aided by the addition of Pluronic F127 (0.01%) to
the buffer solution. After incubation for 2.5 h at 22°C,
preparations were then warmed to 32°C for 30 min before washing in
fura 2-AM-free physiological saline solution for 40 min. The ratio of
the fluorescence recorded at 510 nm after excitation with 340-nm light
to that recorded after excitation with 380-nm light
(F340/380) was taken as a qualitative indicator of
[Ca2+]i.
All values are expressed as means ± 1 SE. Each n value
represents the mean result from a different animal. Where indicated, the statistical significance of the difference between two means was
determined using a Student's t-test.
Drugs used were fura 2-AM (Molecular Probes; Eugene, OR), epinephrine
bitartrate, dihydroergotamine tartrate, isoprenaline hydrochloride, nifedipine hydrochloride, propranolol
hydrochloride, 3-isobutyl-1-methylxanthine (IBMX) (Sigma; St.
Louis, MO), U-73343, U-73122 (Sapphire Bioscience; New South Wales,
Australia), and 2-aminoethoxydiphenyl borate (2-APB, Calbiochem;
Croydon, Victoria, Australia). All drugs were dissolved in distilled
water except nifedipine and 2-APB, which were dissolved in absolute
alcohol, and fura 2-AM, U-73343, and U-73122, which were dissolved in
dimethyl sulfoxide (ICN Biomedicals; Aurora, OH). In all experiments,
solutions containing nifedipine were made daily and protected from
light using aluminum foil wrapping.
| |
RESULTS |
Effects of isoprenaline, epinephrine, and sympathetic nerve
stimulation on membrane potential and
[Ca2+]i in the arrested
sinus venosus.
Membrane potential recordings were made from the sinus venosus, the
pacemaker region of the toad heart. In the presence of nifedipine (10 µM), the membrane potential was stable and lay in the range of 
31
to 39 mV, mean 35 ± 1 mV (n = 25 hearts).
In arrested preparations of toad sinus venosus, trains of sympathetic
nerve stimuli (10 impulses at 10 Hz) caused a complex membrane
depolarization that typically displayed oscillations (Fig.
1A, trace a)
(4, 6). These depolarizations had amplitudes of 25.2 ± 2.1 mV (n = 7) and were initiated after a latency
(time elapsed between the start of the stimulation train and the
attainment of 10% of the peak amplitude) of 1.27 ± 0.04 s.
When the [Ca2+]i was monitored
simultaneously, it was apparent that the depolarizations produced by
sympathetic nerve stimulation were associated with an increase in
[Ca2+]i (Fig. 1A, trace
b) (5). Stimulation with 10 impulses delivered at 10 Hz evoked an increase in [Ca2+]i with an
amplitude of 0.15 ± 0.03 F340/380 (n = 5).
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Comparison of the effects of sympathetic nerve
stimulation, isoprenaline, and 3-isobutyl-1-methylxanthine (IBMX) on
membrane potential and intracellular Ca2+ concentration
([Ca2+]i) recorded from the toad sinus
venosus in the presence of nifedipine (10 µM). A: effects
of sympathetic nerve stimulation (10 impulses at 10 Hz, 1.0 ms/60 V) on
membrane potential (trace a) and
[Ca2+]i (trace b). Stimulation of
the sympathetic nerves caused a membrane depolarization and an increase
in [Ca2+]i that were oscillatory in nature.
B: effects of the bath application of isoprenaline (10 µM,
30 s) on membrane potential (trace a) and
[Ca2+]i (trace b). Isoprenaline
caused a slowly developing membrane hyperpolarization associated with a
decrease in [Ca2+]i. C: IBMX (0.5 mM) applied to the sinus venosus caused a similar membrane
hyperpolarization. The resting membrane potential was 34, 35, and
32 mV in A, B, and C, respectively.
F340/380, ratio of the fluorescence recorded at 510 nm
after excitation with 340-nm light to that recorded after excitation
with 380-nm light.
|
|
In contrast, in arrested preparations, the activation of
-adrenoceptors evoked a membrane hyperpolarization (Fig.
1B, trace a) (4). Rapid perfusion of
isoprenaline (50 µM) over the sinus venosus for 30 s evoked a
membrane hyperpolarization that had a mean nadir of 3.5 ± 0.1 mV
and lasted ~5 min (n = 3). This membrane hyperpolarization was associated with a decrease in
[Ca2+]i of 0.12 ± 0.02 F340/380 (Fig. 1B, trace b;
n = 3) (10). This hyperpolarization was
mimicked by activating adenylate cyclase or by inhibiting
phosphodiesterases (4). Thus the addition of the
phosphodiesterase inhibitor IBMX (0.5 mM) to the solution bathing the
sinus venosus evoked a membrane hyperpolarization with a similar
magnitude to that evoked by -adrenoceptor activation (Fig.
1C). The ionic basis of the membrane potential change
produced by isoprenaline or by IBMX detected in arrested amphibian
pacemaker cells remains unclear. However, it is clear that
-adrenoceptor agonists and agents that cause the elevation of cAMP
produce similar sequences of membrane potential changes (4,
13) but ones quite different to those produced by sympathetic
nerve stimulation.
The rapid perfusion of epinephrine (10 µM) onto the arrested sinus
venosus evoked an initial transient membrane depolarization of 9.7 ± 1.2 mV followed by a hyperpolarization with peak nadir of 3.3 ± 0.6 mV (n = 10; Fig.
2, A, trace a, and
B, trace a). The depolarization was associated
with an increase in [Ca2+]i, whereas
hyperpolarization was associated with a fall in
[Ca2+]i (Fig. 2A, trace
b, n = 5) (10). On some occasions,
membrane potential oscillations were superimposed on the falling phase of the depolarization (Fig. 5B, trace a). These
oscillations had a similar frequency to those evoked by sympathetic
nerve stimulation. The membrane hyperpolarization, which resembled that
produced by either isoprenaline or IBMX, was abolished by pretreating
the tissues with the -adrenoceptor antagonist propranolol (1 µM; Fig. 2B, trace b) suggesting that it resulted
from -adrenoceptor activation. In the presence of propranolol (1 µM), applied epinephrine evoked only a depolarization that was
abolished by dihydroergotamine (10 µM; Fig. 2B,
trace c) (6). This effect of dihydroergotamine was not due to its action as an -adrenoceptor antagonist because membrane depolarizations persist in the presence of other
-adrenoceptor antagonists (6). These results suggest
that the rapid bath application of epinephrine results in the
activation of non--, non--adrenoceptors like that produced by
sympathetic nerve stimulation as well as the activation of
-adrenoceptors (4, 6, 9).
View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of epinephrine on the membrane potential and
[Ca2+]i recorded from the arrested toad sinus
venosus. The exogenous application of epinephrine (50 µM, 30 s)
caused a membrane depolarization (A, trace a, and
B, trace a) associated with an increase in
[Ca2+]i (A, trace b).
The initial membrane depolarization was followed by a small
hyperpolarization (A, trace a, and B,
trace a) and a subsequent decrease in
[Ca2+]i (A, trace b).
The addition of propranolol (1 µM) to the perfusion fluid abolished
the membrane hyperpolarization but had no effect on the amplitude of
the initial membrane depolarization (B, trace
b). The membrane depolarization produced by epinephrine was
abolished after the addition of dihydroergotamine (10 µM;
B, trace c). The resting membrane potential was
33 mV (in A) and 36 mV (in B).
|
|
Effect of phospholipase C inhibition on responses to epinephrine
and sympathetic nerve stimulation in arrested sinus venosus.
Previous experiments show that sympathetic nerve stimulation and
applied isoprenaline initiate distinct sequences of membrane potential
changes presumably by activating different metabolic pathways.
Sympathetic nerve stimulation is associated with an increase in
[Ca2+]i resulting from Ca2+ store
release that does not involve the activation of cAMP (4, 9). Very often Ca2+ store release results from the
activation of an Ins(1,4,5)P3-dependent pathway
(2). The possibility that such a pathway mediated
responses resulting from the activation of non--,
non--adrenoceptors was investigated by examining the effects of
inhibiting phospholipase C (PLC) with U-73122 (3,
23) on responses evoked by sympathetic nerve stimulation.
Responses evoked by sympathetic nerve stimulation (10 impulses at
10 Hz, n = 5) were abolished with U-73122 (20 µM).
However, the effect of this agent may have resulted in part, if not
entirely, from effects on transmitter secretion. In parallel sets of
experiments, U-73122 (20 µM) abolished the responses to vagus nerve
stimulation (n = 3). To avoid this complication,
non--, non--adrenoceptors were activated by the rapid application
of epinephrine. Figure 3A,
traces a and b, shows that the PLC inhibitor U-73122 (20 µM) abolished the membrane depolarization produced by
applied epinephrine (n = 5). However, the membrane
hyperpolarization resulting from the activation of -adrenoceptors
was unchanged.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of the phospholipase C (PLC) inhibitor U-73122 on
the membrane potential change evoked by stimulation of non--,
non--adrenoceptors with applied epinephrine in the toad sinus
venosus. A: control response evoked by the exogenous
application of epinephrine (50 µM, 30 s) to the arrested toad
sinus venosus (trace a). Epinephrine evoked a membrane
depolarization and a subsequent membrane hyperpolarization. After the
addition of the PLC inhibitor U-73122 (20 µM) to the physiological
saline solution, the membrane depolarization produced by epinephrine
(50 µM) was abolished leaving a membrane hyperpolarization
(trace b). B: membrane potential changes evoked
by the exogenous application of epinephrine (50 µM) before
(trace a) and after (trace b) the
addition of the inactive compound U-73343 (20 µM). Note that the
membrane depolarization and the afterhyperpolarization both persisted
in the presence of U-73343. All recordings were performed in the
presence of nifedipine (10 µM). The resting membrane potential in all
records was 32 mV. The calibration bars refer to all traces.
|
|
This indicates that U-73122 neither blocks -adrenoceptors nor
interferes with the cAMP-dependent pathway. The simplest explanation is
that U-73122 inhibits the depolarization evoked by the activation of
non--, non--adrenoceptors by preventing PLC activation. As a
further control, the effects of the closely related compound U-73343
(3) on the responses to applied epinephrine were examined. U-73343 (20 µM), which does not block PLC, had no effect on the amplitude of either the depolarization or the afterhyperpolarization produced by added epinephrine (P > 0.05, n = 5; Fig. 3B, traces a and
b).
In a number of tissues, it has been shown that, in addition to
inhibiting PLC, U-73122 may also cause the release of Ca2+
from intracellular stores (16, 18). To examine whether the effects of U-73122 were due to Ca2+ store depletion, the
effect of U-73122 on responses evoked by application of caffeine (10 mM) was examined. It has previously been shown (5) that
the rapid bath application of high concentrations of caffeine causes a
membrane depolarization that is oscillatory in nature (Fig.
4A, trace a). This
is associated with an oscillatory increase in force production of the
sinus venosus (Fig. 4A, trace b).
These changes in membrane potential and force are due to the release of
Ca2+ from ryanodine-sensitive stores (5).
U-73122 (20 µM) had little effect on the underlying membrane
depolarization or increase in force production evoked by caffeine;
however, it abolished the oscillations in both membrane potential and
force (Fig. 4B). Therefore, part of the effects of U-73122
on responses evoked by sympathetic nerve stimulation may have been due
to its action on Ca2+ stores rather than PLC inhibition.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of PLC inhibition on the changes in membrane
potential and force evoked by caffeine in the toad sinus venosus.
A: effect of the rapid perfusion of caffeine (10 mM) on
membrane potential (trace a) and force (trace b)
recorded from the arrested toad sinus venosus. Caffeine caused an
initial membrane depolarization followed by oscillations in membrane
potential. Such changes were associated with an oscillatory change in
force production. B: responses evoked by caffeine (10 mM) in
the presence of U-73122 (20 µM). U-73122 abolished the oscillatory
changes in membrane potential (trace a) and force production
(trace b) but had little effect on the underlying membrane
depolarization and increase in force. The resting membrane potential in
A, trace a, and B, trace a,
was 36 mV. All recordings were performed in the presence of
nifedipine (10 µM). The time calibration bar refers to all traces.
|
|
To further examine the possibility that Ins(1,4,5)P3
production was necessary for the response produced by the activation of
non--, non--adrenoceptors, the effects of inhibition of
Ins(1,4,5)P3-induced Ca2+ release by 2-APB (60 µM) (1, 17) were examined on both membrane potential and
force changes evoked by sympathetic nerve stimulation. In control
solution, stimulation of the sympathetic nerves evoked a membrane
depolarization of 19 ± 5 mV (n = 5). The addition
of 2-APB (60 µM) to the physiological saline solution (30 min) had no
effect on either the resting membrane potential or the basal force
production. However, 2-APB significantly reduced the amplitude of the
nerve-evoked membrane depolarization (4 ± 2 mV, P = 0.01, n = 5, Fig.
5A). Washout of 2-APB for 60 min partially restored the response to sympathetic nerve stimulation
(15 ± 3 mV). In four experiments, the effect of 2-APB (60 µM)
on force changes evoked by sympathetic nerve stimulation was also
examined. Before the addition of 2-APB, sympathetic nerve stimulation
evoked an increase in basal force of 0.28 ± 0.10 mN. After the
addition of 2-APB, the increase in force production evoked by
sympathetic nerve stimulation was significantly reduced (0.03 ± 0.01 mN; P = 0.02). At 60 µM, 2-APB had no effect on
responses evoked by vagus nerve stimulation (n = 3),
suggesting that it was not inhibiting transmitter release.
View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of the inhibition of
D-myo-inositol 1,4,5,-trisphosphate
[Ins(1,4,5)P3]-induced Ca2+ release with
2-aminoethoxydiphenyl borate (2-APB) on the membrane potential change
evoked by stimulation of non--, non--adrenoceptors with
sympathetic nerve stimulation and applied epinephrine in the toad sinus
venosus. A: membrane potential responses to sympathetic
nerve stimulation (10 impulses at 10 Hz) before (trace a)
and after (trace b) the addition of 2-APB (60 µM, 30 min).
It can be seen that 2-APB reduced the membrane depolarization to
~10% of the amplitude recorded in control solution. B:
effect of 2-APB (60 µM) on responses evoked by the rapid bath
application of epinephrine (10 µM). In control solution, epinephrine
caused a transient membrane depolarization, with oscillations on its
falling phase and a subsequent membrane hyperpolarization (trace
a). After the addition of 2-APB (60 µM) to the physiological
saline (trace b), epinephrine failed to cause a membrane
depolarization; however, a membrane hyperpolarization persisted. All
recordings were performed in the presence of nifedipine (10 µM). The
resting membrane potential was 37 mV (in A) and 35 mV
(in B).
|
|
The effect of 2-APB was also examined on responses evoked by the bath
application of epinephrine. If 2-APB was acting specifically to inhibit
Ins(1,4,5)P3-induced Ca2+ release, it would be
expected that 2-APB would abolish the membrane depolarization resulting
from the activation of non--, non--adrenoceptors but have no
effect on the membrane hyperpolarization resulting from
-adrenoceptor activation. Epinephrine (50 µM) evoked an initial
membrane depolarization of 7 ± 2 mV followed by a membrane hyperpolarization of 4 ± 1 mV (n = 4) in
amplitude (Fig. 5B, trace a). The addition of
2-APB (60 µM, 30 min) significantly reduced the amplitude of the
membrane depolarization (1 ± 1 mV; P = 0.04) but
had no effect on the membrane hyperpolarization (5 ± 1 mV; P = 0.09; n = 4; Fig. 5B,
trace b).
Although 2-APB (60 µM) has previously been shown to have no
effect on either the production of cAMP or Ca2+-induced
Ca2+ release, 2-APB has been shown to partially inhibit the
reuptake of Ca2+ into Ins(1,4,5)P3-sensitive
Ca2+ stores when used at high concentrations (
100 µM)
(17). To examine whether 2-APB was depleting
intracellular stores due to the inhibition of Ca2+
reuptake, the effect of 2-APB on changes in membrane potential and
force evoked by caffeine was examined. Both the membrane depolarization and increase in force evoked by caffeine persisted in the presence of
2-APB (60 µM, n = 6, Fig.
6). In addition, both the oscillations in
membrane potential and force production also persisted in the presence
of 2-APB.
View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of inhibition of Ins(1,4,5)P3-induced
Ca2+ release on the changes in membrane potential and force
evoked by caffeine in the toad sinus venosus. A: effect of
the rapid perfusion of caffeine (10 mM) on membrane potential
(trace a) and force (trace b) recorded
from the arrested toad sinus venosus. Caffeine caused oscillatory
changes in membrane potential and force. B: responses evoked
by caffeine (10 mM) in the presence of 2-APB (60 µM). It was evident
that 2-APB had little effect on the amplitude of responses and did not
effect the oscillations in membrane potential (trace a) or
force (trace b) evoked by caffeine. The resting membrane
potential in A, trace a, and B,
trace a, was 38 mV. All recordings were performed in the
presence of nifedipine (10 µM). The time calibration bar refers to
all traces.
|
|
| |
DISCUSSION |
This study suggests that two distinct transduction pathways, one
involving cAMP and the other involving Ins(1,4,5)P3, are present and can be activated by catecholamines in the toad sinus venosus. In the toad sinus venosus, just as in mammalian pacemaker preparations, applied catecholamines most readily activate
-adrenoceptors to elevate cAMP. This pathway results in the
phosphorylation-dependent and -independent modulation of
voltage-dependent channels involved in pacemaking activity (11,
13). The resulting tachycardia is associated with an increase in
the amplitude of both pacemaker action potentials (4, 6)
and the [Ca2+]i transient that accompanies
each action potential (5).
In contrast, responses evoked by sympathetic nerve stimulation
resulting from activation of non--, non--adrenoceptors are not
associated with an increased influx of Ca2+ through
voltage-dependent Ca2+ channels (5) and do not
involve cAMP (4). However, such responses are likely to
involve a different complex metabotropic pathway; both the sympathetic
tachycardias recorded from beating preparations and the membrane
depolarizations recorded in arrested preparations begin after a long
latency and last many seconds even when triggered by only a few
sympathetic stimuli (4, 9). In arrested preparations,
stimulation of the sympathetic nerves evokes an oscillatory membrane
depolarization and a concomitant increase in
[Ca2+]i (9), both of which have
temporal characteristics similar to the sympathetic tachycardia
recorded from beating preparations (6). This suggests that
a similar transduction pathway is activated in beating and arrested
preparations of toad sinus venosus. We have recently shown that both
the membrane depolarization and the increase in
[Ca2+]i evoked by sympathetic nerve
stimulation in arrested preparations result from the release of
intracellularly stored Ca2+ (9). Similar
responses also resulting from the activation of non--,
non--adrenoceptors can be produced by rapid bath application of
epinephrine to the arrested pacemaker tissue (Fig. 2) (4, 6). Responses evoked by the activation of these receptors were inhibited by prevention of Ins(1,4,5)P3-induced
Ca2+ release. This was achieved by either preventing
the formation of Ins(1,4,5)P3 with the PLC inhibitor
U-73122 or by directly preventing Ins(1,4,5)P3-induced
Ca2+ release with 2-APB (Figs. 3 and 5). This suggests that
the responses involve the activation of PLC and the formation of
Ins(1,4,5)P3, which triggers the release of
Ca2+ from intracellular stores. The subsequent activation
of the Na+/Ca2+ exchanger found in cardiac
muscle (7, 14) would provide a net inward current, which
could account for both the depolarization in arrested preparations and
the tachycardia in beating preparations (15).
How these findings relate to mammalian pacemaker cells remains unclear.
In the mammalian heart, both neuronally released and added
norepinephrine activate -adrenoceptors. However, the responses to
neuronally released and added norepinephrine differ in much the same
way as they do in the amphibian heart (8). Clearly, one
explanation could be that the -adrenoceptors under sympathetic nerve
terminals are linked to PLC, whereas those at a more distant location
are linked to adenylate cyclase. However, we know of no example where a
-adrenoceptor is linked to PLC. Indeed in mammalian hearts, this
pathway is usually linked to an -adrenoceptor (20, 22).
Whatever the case, it is clear that in both amphibians and
mammals, the same transmitter activates separate metabotropic pathways. One pathway is activated by the local high concentrations of
transmitter achieved after nerve activity. In amphibians, this
pathway appears to involve the second messenger
Ins(1,4,5)P3, which releases Ca2+ from an
intracellular store to increase heart rate. The other pathway, which
involves cAMP, is activated by a more diffuse action of transmitter
such as that occurring during a rise in the concentration of
catecholamine circulating in the blood.
| |
ACKNOWLEDGEMENTS |
This work was funded by the National Health and Medical Research
Council of Australia. We thank them for their financial support.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: N. J. Bramich, Dept. of Zoology, Univ. of Melbourne, Victoria, Australia 3010 (E-mail: narelleb{at}unimelb.edu.au).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 31 October 2000; accepted in final form 5 June 2001.
| |
REFERENCES |
1.
Ascher-Landsberg, J,
Saunders T,
Elovitz M,
and
Phillippe M.
The effects of 2-aminoethoxydiphenyl borate, a novel inositol 1,4,5-trisphosphate receptor modulator on myometrial contractions.
Biochem Biophys Res Commun
264:
979-982,
1999[Web of Science][Medline].
2.
Berridge, MJ.
Inositol trisphosphate and calcium signalling.
Nature
361:
315-325,
1993[Medline].
3.
Bleasdale, JE,
Thakur NR,
Gremban RS,
Bundy GL,
Fitzpatrick FA,
and
Smith RJ.
Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils.
J Pharmacol Exp Ther
255:
756-768,
1990[Abstract/Free Full Text].
4.
Bramich, NJ,
Brock JA,
Edwards FR,
and
Hirst GDS
Responses to sympathetic nerve stimulation of the sinus venosus of the toad.
J Physiol (Lond)
461:
403-430,
1993[Abstract/Free Full Text].
5.
Bramich, NJ,
and
Cousins HM.
Effect of sympathetic nerve stimulation on membrane potential, [Ca2+]i, and force in the toad sinus venosus.
Am J Physiol Heart Circ Physiol
276:
H115-H128,
1999[Abstract/Free Full Text].
6.
Bramich, NJ,
Edwards FR,
and
Hirst GDS
Sympathetic nerve stimulation and applied transmitters on the sinus venosus of the toad.
J Physiol (Lond)
429:
349-375,
1990[Abstract/Free Full Text].
7.
Campbell, DL,
Giles WR,
Robinson K,
and
Shibata EF.
Studies of the sodium-calcium exchanger in bull-frog atrial myocytes.
J Physiol (Lond)
403:
317-340,
1988[Abstract/Free Full Text].
8.
Choate, JK,
Edwards FR,
Hirst GDS,
and
O'Shea JE.
Effects of sympathetic nerve stimulation on the sino-atrial node of the guinea-pig.
J Physiol (Lond)
471:
707-727,
1993[Abstract/Free Full Text].
9.
Cousins, HM,
and
Bramich NJ.
Effect of sympathetic nerve stimulation on membrane potential, [Ca2+]i and force in the arrested sinus venosus of the toad, Bufo marinus.
J Physiol (Lond)
505:
513-527,
1997[Abstract/Free Full Text].
10.
Cousins, HM,
and
Bramich NJ.
Parallel metabotropic factors, cAMP and IP3, in the cane toad heart.
Proc Aust Neurosci Soc
7:
70,
1996.
11.
Difrancesco, D,
and
Tortora P.
Direct activation of cardiac pacemaker channels by intracellular cyclic AMP.
Nature
351:
145-147,
1991[Medline].
12.
Gaskell, WH.
On the augmentor (accelerator) nerves of the heart of coldblooded animals.
J Physiol (Lond)
5:
46-48,
1884.
13.
Hartzell, HC.
Regulation of cardiac ion channels by catecholamines, acetylcholine and second messenger systems.
Prog Biophys Mol Biol
52:
165-247,
1988[Web of Science][Medline].
14.
Hume, JR,
and
Uehara A.
"Creep currents" in single frog atrial cells may be generated by electrogenic Na/Ca exchange.
J Gen Physiol
87:
857-885,
1986[Abstract/Free Full Text].
15.
Huser, J,
Blatter LA,
and
Lipsius S.
Intracellular Ca2+ release contributes to automaticity in cat atrial pacemaker cells.
J Physiol (Lond)
524:
415-422,
2000[Abstract/Free Full Text].
16.
Jin, WZ,
Lo TM,
Loh HH,
and
Thayer SA.
U-73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells.
Brain Res
642:
237-243,
1994[Web of Science][Medline].
17.
Maruyama, T,
Kanaji T,
Nakade S,
Kanno T,
and
Mikoshiba K.
2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release.
J Biochem (Tokyo)
122:
498-505,
1997[Abstract/Free Full Text].
18.
Mogami, H,
Lloyd Mills C,
and
Gallacher DV.
Phospholipase C inhibitor, U73122, releases intracellular Ca2+, potentiates Ins(1,4,5)P3-mediated Ca2+ release and directly activates ion channels in mouse pancreatic acinar cells.
Biochem J
324:
645-651,
1997.
19.
Morris, JL,
Gibbins IL,
and
Clevers J.
Resistance of adrenergic neurotransmission in the toad heart to adrenoceptor blockade.
Naunyn Schmiedebergs Arch Pharmacol
317:
331-338,
1981[Web of Science][Medline].
20.
Otani, H,
Otani H,
and
Das DK.
1-Adrenoceptor-mediated phosphoinositide breakdown and inotropic responses in rat left ventricular papillary muscles.
Circ Res
62:
8-17,
1988[Abstract/Free Full Text].
21.
Randall, WC.
Neural Regulation of the Heart. New York: Oxford University Press, 1977.
22.
Schmitz, W,
Scholz H,
Scholz J,
and
Steinfath M.
Increases in IP3 precedes -adrenoceptor-induced increase in force of contraction in cardiac muscle.
Eur J Pharmacol
140:
109-111,
1987[Web of Science][Medline].
23.
Smith, RJ,
Sam LM,
Justen JM,
Bundy GL,
Bala GA,
and
Bleasdale JE.
Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness.
J Pharmacol Exp Ther
253:
688-697,
1990[Abstract/Free Full Text].
Am J Physiol Heart Circ Physiol 281(4):H1771-H1777
0363-6135/01 $5.00
Copyright © 2001 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
