Intracellular cAMP: the “switch” that triggers on “Spontaneous
Transient Outward Currents” generation in freshly isolated myocytes
from thoracic aorta.
S. Hayoz, J.-L. Beny and R. Bychkov
S.Hayos et al., Intracellular cAMP: the “switch” that triggers STOCs
Key words: cAMP, STOCs, forskolin, ATP, aorta
Sébastien Hayoz1 , Jean-Louis Bény1 ,Rostislav Bychkov1.
1Department of Zoology and Animal Biology, University of Geneva, Sciences III
Correspondence: Rostislav Bychkov: Department of Zoology and Animal Biology,
University of Geneva, Sciences III, 30 Quai Ernest Ansermet, CH-1211 Geneva
4, Switzerland. Telephone number: +41 22 379 30 67 .
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Articles in PresS. Am J Physiol Cell Physiol (December 27, 2006). doi:10.1152/ajpcell.00522.2006
Copyright © 2006 by the American Physiological Society.
Abstract: STOCs (Spontaneous Transient Outward Currents) have been reported
in resistance and small arteries but have not yet been found in thoracic aorta. Do
thoracic aorta myocytes possess cellular machinery that generates STOCs? It
was found that majority of aortic myocytes do not generate STOCs. STOCs were
generated in 8.7% of freshly isolated aortic myocytes. Myocytes that did not
generate STOCs we have called “silent” myocytes and myocytes with STOCs
have been called “active”. STOCs recorded in “active” myocytes were voltage
dependent and were inhibited by ryanodine, caffeine and charybdotoxin.
Forskolin was reported to increase STOCs frequency in myocytes isolated from
resistance arteries. Forskolin (10µM) triggered STOCs generation in 35.1% of
“silent” aortic myocytes. In 36.8% percent of “silent” myocytes forskolin did not
trigger STOCs but increased the amplitude of charybdotoxin sensitive outward-
net current to 136.1±8.5% at 0 mV. Membrane permeable 8BrcAMP triggered
STOCs generation in 38.7% of “silent” myocytes. Forskolin or 8BrcAMP-triggered
STOCs were inhibited by charybdotoxin. 8BrcAMP also increased open
probability of BKCa-channels in BAPTA-AM pretreated cells. Our data
demonstrate that, in contrast to resistance arteries, STOCs are present just in
minority of myocytes in the thoracic aorta. However, cellular machinery that
generates STOCs can be “switched” on by cAMP. Such an inactive cellular
mechanism could modulate the contractility of the thoracic aorta in response to
Key words: Thoracic aorta myocytes, STOCs, cAMP, Foskolin, BKCa-channels
Page 2 of 28
Physiological properties of blood vessels exhibit great variations among
vascular beds. Segmental differences of the functional anatomy and vasomotor
reactivity in the same vessel are documented in the vasculature (23,13,8).
However, there are few data that allow comparing cellular mechanisms of
vascular reactivity in resistance and conduit arteries.
We reported that myocytes in the rings of mouse thoracic aorta reacted to
the steps of stretch by several patterns of Ca2+ discharges (9). It was shown that
thoracic aorta generated myogenic component in response to applied stretch of
the vascular wall. This mechanism, previously attributed to resistance arteries,
could modulate the degree of aorta contraction during imposed stretch.
Activation of the KCa viewed from this point of view could oppose stretch-induced
constrictions of conduit vessels.
Ca2+-dependent K+-channels are expressed to different degrees in arterial
tree (1, 10). They play a pivotal role in vascular responses. An increase in KCa
channel current hyperpolarizes membrane potential and lowers global
intracellular Ca2+, which exerts a vasorelaxing influence (5,6,12,14). Localized
cellular events known as Ca2+ sparks activate 10–100 nearby sarcolemmal Ca2+-
sensitive K+ (KCa) channels to cause an outward K+ current (20,11), previously
referred to as ‘‘spontaneous transient outward current’’ (STOC) (2). Frequency
modulation of Ca2+ sparks, and consequently STOCs, can continuously regulate,
as a negative feedback element, the membrane potential of smooth muscle cells
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and arterial tone in resistive arteries (20,22). However, STOCs were not reported
in myocytes from main conduit artery thoracic aorta.
We therefore characterized STOCs in mouse thoracic aorta and then
explored the possible effects of forskolin and cAMP on STOCs. Specifically, our
results provide direct evidence that the level of intracellular cAMP can switch on
STOCs generation in “silent” aortic myocytes, which did not exhibit any STOCs
activity. This switch provides a feedback mechanism to regulate the degree of
aorta contractility in response to physiological demand.
Mouse aorta preparation and isolation of single smooth muscle cells
All animal handling was in accordance with institutional guidelines
established by the ‘Swiss Academy of Medical Sciences’ and the ‘Helvetic
Society of Natural Sciences’: animal experimentation authorization
Male C57BL/6 mice that were 3 to 4 weeks old were anesthetized with 2-
bromo-2-chloro-1, 1, 1-trifluoroethane. Smooth muscle cells were isolated as
described previously (5). The thoracic aorta was removed and cleaned from fat
and connective tissue, Aorta was placed in low Ca2+ solution containing in (mM):
NaCl 137, KCl 5.4, K2HPO4 0.44, NaH2 PO4 0.42, MgCl2 6H2O 2, NaHCO3 4.17,
CaCl2 2H2O 0.2, EGTA 0.05, glucose 11, HEPES 10, pH was adjusted to 7.4 with
NaOH. Aorta was incubated for 40 minutes at 37oC in the low Ca2+ solution
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containing 2 mg ml-1 elastase type (IV) and collagenase (type IA-S) 1 mg ml-1.
Vascular smooth muscle cells were isolated by careful shaking of the tissue, then
placed on cover-slips and stored at 4oC.
Patch Clamp recording
Membrane currents were recorded at room temperature (20°C) using nystatin
perforated patch and whole-cell configuration with a patch amplifier (Axopclamp
200B). The patch electrodes from borosilicate capillary glass were pulled using a
Shutter instrument (Model P-2000, USA). They had resistance of 4-7 MΩ. Patch
pipettes were filled with (in mM): KCl 130, HEPES 10, EGTA 2 (pH=7.4) for the
whole cell experiments. The same solution without EGTA was used for the
perforated patch clamp experiments. Nystatin (Sigma, Deisenhofen, FRG) was
dissolved in DMSO and diluted into the pipette solution to give a final
concentration ranging from 50 to 100 µg ml-1. The aortic smooth muscle cells
were bathed in a solution containing (in mM): NaCl 130, KCl 5.6, MgCl2 6H2O 1,
CaCl2 2H2O 2, HEPES 8, Glucose 10 (pH 7.5). ATP and other chemicals were
added to the bath solution. The linear voltage ramps were applied from the
holding potential of -60 mV with 500 ms duration and voltage varied from -100 to
100 mV. Voltage step pulses were applied from the holding potential of -60 mV
with the 10 mV increment between -100 and 100 mV. The large amplitude and
low open probability (Po) of the KCa channel permitted the measurement of single
KCa channel currents with the use of the perforated-patch configuration of the
whole cell voltage clamp. To observe single KCa channel currents, Ca2+ sparks
and hence STOCs were prevented by BAPTA-AM, which decreased intracellular
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Ca2+ level and consequently inhibited STOCs. Cells were clamped at 0 mV. NPo
was calculated over 7-minutes intervals as
j=1tj · j/T, where tj is the time spent
with j = 1, 2, . . . N channels open, N is the maximum number of channels
observed, and T is theduration of the recording (7 min).
Charybdotoxin, EGTA (Ethylene glycol-bis (β-aminoethyl ether)-tetraacetic
acid), elastase (type IV from porcine pancreas), collagenase (type IA-S), BAPTA-
AM (1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl
ester), ryanodine (Ryanodol 3-(1H-pyrrole-2-carboxylate), 8-Br-cAMP (8-bromo-
adenosine 3’-5’cyclic monophosphate), PKI 14-22 amide (Myristoylated Ptoteine
Kinase A Inhibitor Amide 14-22, cell permeable) and forskolin were obtained from
Sigma Chemical (Buchs, Switzerland).
Data treatment and Statistics
Each set of data was expressed as a mean±SEM. All presented
experiments were repeated at least five times. We employed Wilcoxon Two
Sample test to compare data sets. Pairs of data sets represented measurements
of the current amplitude, slope values, half width, open time of single BKCa
channels. A value of P<0.05 was considered as statistically significant.
Mean±STOCs decay was fitted with Boltzmann function: Amplitude (pA)=(A1-
A2)/(1+exp(x-x0)/dx)+A2 where A1- is initial Y value A2– is final Y value, x0 is the
center and dx is the width. Data sets of dwell time BKCa channels were fitted
with exponential decay first order function: Y(number of counts)=Y0+A1exp(-x/t1)
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where Y0 is the Y offset, A1 is initial Y value, t1 is decay constant. Histogram
distributions of the single BKCa channels were fitted with two-peak Gaussian
Characterization of STOCs in freshly dissociated mouse aortic smooth
Spontaneous transient outward currents (STOCs) were found only in 8.7%
of freshly dissociated aortic smooth muscle cells contrary to 60-80% reported for
resistive arteries (22). Myocytes that did not generate STOCs in the control were
named “silent” myocytes when myocytes with STOCs were named “active”.
Outward net currents recorded in perforated patch configuration were elicited by
voltage steps or voltage ramps from holding potential of -60 mV varying from -
100 mV to 100 mV. Aortic smooth muscle cells with or without STOCs had
similar current voltage relations at control conditions. Charybdotoxin (100 nM)
inhibited outward K+-current to 41.4±10% and to 51.8.2±7.9% of the control in
cells with and without STOCs measured at the holding potential of 0 mV.
Cumulative application of 4AP (2mM) plus TEA (5mM mM) further inhibited the
outward current to 12±3.1% and to 9.9+1.5% in cells with and without STOCs
correspondingly (n=7, n=7). STOCs were not found in aortic smooth muscle cells
dialyzed with 2 mM EGTA in the whole-cell configuration (n=35). BKCa–channels
could be expressed differently in “silent” and “active” myocytes. To test this
hypothesis ionomycin Ca2+ ionophore (10 µM) was added to the bath solution to
activate all BKCa-channels (Fig. 1 Panel F and Panel G). Rise of intracellular Ca2+
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increased the amplitude of the outward net currents and shifted activation
threshold of the current voltage relation to from 10±1.3 mV to -39±2.1 mV (n=9)
in both “silent” and “active” myocytes. Ionomycin increased amplitude of the
outward net current but inhibited STOCs in “active” myocytes (n=4). This effect is
explained by the fact that ionomycin masks or inhibits local Ca2+ events by
global intracellular Ca2+ rise. Recorded K+-currents were normalized to cell
capacitance. Average capacitance of single aortic myocyte was 11±0.6 pF
(n=25). Ionomycin increased amplitude of the outward K+ currents from 18.4±1.5
pA/pF to 26.3±1.8 pA/pF in “active” myocytes and from 18.1±1.4 pA/pF to
27.6±2.5 pA/pF in “silent” myocytes measured at 20 mV. Iberiotoxin (100 nM)
decreased inonomycin stimulated current to 13.1±1.5 pA/pF (n=7) in “active” and
to 11.4±1.8 pA/pF (n=15)” in “silent” myocytes indicating that both types of cells
expressed similar quantity of BKCa channels.
STOCs were recorded in “active” aortic myocytes at steady-state holding
potentials varying from -30 mV to 20 mV (Fig. 1 Panel A). Individual STOCs had
variable amplitude and duration through all imposed voltages. To compare
STOCs under different experimental conditions, STOCs were regrouped in data
sets for each imposed voltage and aligned to the beginning of the transient
KCa2+-current as illustrated by the example shown in panel B (Fig. 1 Panel B).
Average-STOCs current was calculated from all regrouped STOCs in each data
set. Same method was applied to analyze STOCs in all experiments. Average-
STOCs current obtained from STOCs recorded at different holding potentials
were superimposed in one plot. Average-STOCs current showed increase of
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peak amplitude with the increase of imposed voltage (Fig. 1C) (n=5). Data set of
distribution of mean peak amplitudes of STOCswas plotted against
corresponding data set of mean durations in semi- logarithmic scale (Fig. 1E)
(n=4). The plot shows that STOCs amplitude correlates to duration as
exponential function. STOCs duration varied from 23.4±2.1 ms to 76.3±5.2 ms at
a holding potential of -20 mV and increased to a maximum of 100.5±7.4 ms for
STOCs recorded at 30 mV (n=5). Normalized to the peak amplitude average-
STOCs showed similar kinetics with a closed half-width of 40.5±1.2 ms and slope
of 8.6±0.4 indicating that high amplitude STOCs can be obtained as a sum of
STOCs of smaller amplitude. Thus, STOCs recorded in thoracic aorta
correspond to STOCs reported in other vascular beds. They are randomly
generated with varying amplitude and duration, when STOCs with high amplitude
could be represented as the simple sum of elementary STOCs.
We performed the following experiments to investigate the
pharmacological properties of STOCs in aortic smooth muscle. Charybdotoxin
(100 nM) (n=4) and iberiotoxin (100 nM) (n=3), the specific blockers of BKCa
channels, inhibited STOCs (Fig. 2 Panel A and Panel B). Caffeine (1 mM)
increased transiently outward current with a peak amplitude of 124.4±25.8 pA
and duration of 14.6±3.2 seconds, and temporally inhibited STOCs by depleting
intracellular stores of the aortic myocytes (Fig. 2 Panel D) (n=5). After removal of
caffeine from the bath, STOCs recovered within 1-3 minutes. Ryanodine (50 µM,
n=6) a Ca2+ spark inhibitor and BAPTA-AM (30µM) membrane permeable Ca2+
chelator (n=4) completely blocked generation of STOCs (20,12 ).
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We tested the action of forskolin on freshly isolated myocytes since
forskolin was reported to increase STOCs in resistance vessels (20). Forskolin
(10 µM) increased STOCs frequency from 2.1±0.2 Hz to 2.9±0.3 Hz in “active”
myocytes (8.7%). In “silent” aortic myocytes (36.8%) forskolin increased the
outward net current to 136.1±8.5% at 0 mV. Charybdotoxin (100 nM) decreased
the amplitude of the forskolin-elicited outward current to 103.2±4.1% (pA).
Another population of myocytes developed a leak inward current with the
reversal potential of 0 mV. Interestingly, forskolin triggered STOCs generation
with a delay of 9.5±0.7 minutes in the third population of “silent” myocytes
(35.1%) (Fig. 3 Panel A). Low amplitude forskolin-elicited STOCs appeared as
single events with frequency of 0.5 Hz. STOCs frequency increased as a function
of time to 3.5±0.4 Hz. However, frequency did not increase progressively from
the beginning of forskolin-elicited STOCs generation to the point when STOCs
reached their maximum amplitude. Myocytes had variable frequency evolution of
STOCs. Maximum frequency of forskolin-elicited STOCs could be reached at the
time when STOCs amplitude was half of the maximum or at the time when
STOCs amplitude just reached maximum. Evolution of STOCs frequency and
amplitude could reflect progressive recruitment and/or cooperative functioning of
“functional unites” generating STOCs. Iberiotoxin (100 nM) inhibited forskolin-
elicited STOCs (n=3) (Fig. 3 Panel D).
Three averages of forskolin-triggered STOCs were calculated from data
sets of regrouped STOCs obtained from three consecutive time-laps of
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continuous recordings (n=5). The time-lap to assemble STOCs was chosen with
relation to the evolution of STOCs amplitude and duration: at the beginning, in
the middle and in the end of the recording, where STOCs amplitude reached
maximum. Peak amplitude of the forskolin-triggered average STOCs increased
gradually from 47.2±9.1 pA to 134.1±13.2 pA at the end of the recording (Fig.
3B). Average-STOCs normalized to the peak (Fig. 3 inset in Panel B) showed
that the half width calculated for the STOCs recorded in the beginning was
28.7±1.4 ms and increased to 65.3±0.4 ms for STOCs that reached maximum
amplitude. Slope of the average-STOCs decay also increased from 12.2±0.7 to
17.9±0.2. The data set of distribution of mean amplitudes of forskolin-triggered
STOCs was plotted against the data set of mean durations in semi-logarithmic
scale (Fig.3 Panel C) (n=4). The plot shows that STOCs amplitude increased
with their duration, in an exponential manner, from the beginning of STOCs
generation to the time when STOCs reached maximum amplitude.
Effect of 8BrcAMP on thoracic aorta myocytes
The effect of the membrane permeable analog of cAMP 8BrcAMP was
tested on isolated aortic myocytes in the next series of experiments. Addition of
8BrcAMP (10µM) to the bath solution triggered STOCs generation with a delay of
10.5±1.3 minutes in 38.7% of “silent” aortic myocytes (Fig. 4 Panel A). STOCs
did not appear spontaneously in “silent” myocytes maintained at different holding
potentials for the period of 20 to 30 minutes of recording (Fig. 4 Panel B). In
45.2% of cells, 8BrсAMP increased the outward net current to 147.1±12.2% at 0
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mV. Charybdotoxin (100 nM) decreased the amplitude of the 8BrcAMP-elicited
outward current to 87.5±9.3%. In the remaining myocytes 8BrcAMP activated
Three averages of 8BrсAMP-triggered STOCs were calculated from data
sets of regrouped STOCs obtained from three consecutive time-laps of
continuous recordings (n=5). The time-lap to assemble STOCs was chosen with
relation to the evolution of STOCs amplitude and duration: at the beginning, in
the middle and in the end of the recording, where STOCs amplitude reached
maximum. Peak amplitude of the cAMP-triggered average STOCs increased
from 48.5±9.3 pA at the beginning of STOCs generation to 104.1±13.2 pA (Fig.
4C). Mean-STOCs currents were normalized to their peak amplitudes (Fig. 4
inset in the Panel C). Half width of normalized average-currents increased from
27.6±2.3 ms to 80.1±3.2 ms. Decay slope of the average-STOCs, at their
appearance, increased from 11.7±0.6 to 30.8±1.9 when STOCs reached
maximum amplitude. The data set of the distribution of mean amplitudes of
cAMP-triggered STOCs was plotted against the data set of mean durations in
semi- logarithmic scale (Fig.4 Panel D) (n=4). The plot showed that STOCs
amplitude increased with duration, in an exponential manner.
In the next series of experiments aortic myocytes were pretreated with
myristoylated PKI 14-22 amide (10 µM) for 1 hour to demonstrate that PKA-
mediates triggering of STOCs in “silent” cells. 8BrcAMP did not stimulate STOCs
generation in myocytes pretreated with PKI-14-22 amide (n=10) (Fig. 4 Panel D).
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Effect of 8BrcAMP on BKCa channels
The amplitude of STOCs elicited by forskolin and 8BrcAMP in “silent”
myocytes gradually increased with time. This increase could be due to an
elevated open state probability (Po) of KCa channels caused by PKA
phosphorylation of the channel, as demonstrated in resistance arteries (20).
To test the effects of 8BrcAMP on BKCa-channels, single-channel currents
through BKCa channels were recorded from isolated aortic myocytes in the whole-
cell mode, using the perforated-patch-technique. Cells were pretreated with a
membrane permeable calcium chelator, BAPTA-AM (30 µM), for 10-15 minutes
to buffer intracellular Ca2+ changes. BAPTA-AM decreased the amplitude of the
whole cell outward net current to 71.2±4.3 at 0 mV (n=6). Single KCa channels
were identified by their characteristics: large single channel conductance of
189±16 pS calculated from 0 mv to 30 mV (Fig. 5 inset in the Panel A), voltage
dependence, and sensitivity to blocking by charybdotoxin. 8BrcAMP increased
single BKCa channel activity (measured as NPo) from 0.086±0.012 to 0.18±0.02
(n=7) measured over 7 minutes at 0 mV. The single current amplitude was not
affected by 8BrcAMP. The 8BrcAMP-dependent increase in NPo did not appear
to be result of an elevation in intracellular Ca2+ since intracellular Ca2+ was
buffered by BAPTA -AM and no 8BrcAMP-elicited STOCs were observed.
The study reports the cellular signaling pathway that can switch on
generation of spontaneously transient outward currents (STOCs). STOCs were
Page 13 of 28
reported previously in cerebral resistance arteries, coronary arteries and the
mesenteric artery (2,20,5,22,12). Activation of the KCa channel by the Ca2+ spark
pathway was suggested to oppose pressure-induced constrictions of resistance
arteries (12). STOCs were found to be affected during vascular dysfunction and
to be one of the key players in heart failure (19,3). Taken together, these data
indicate that STOCs represent one of the key-regulatory elements in the
STOCs were identified and analyzed, to answer the question: do thoracic
aorta myocytes possess a functional organization like myocytes in resistance
arteries? The primary conclusion drawn from our data suggested that STOCs are
not important for thoracic aorta function. They were present only in a small
population of freshly isolated myocytes in contrast to myocytes isolated from
resistance arteries. However, close investigation revealed that thoracic aorta
myocytes possess “silent” but functional cellular machinery that generates
STOCs which can be switched on by an increased level of intracellular cAMP.
STOCs recorded in control conditions in thoracic aorta myocytes had the same
pharmacological properties as STOCs found in smaller arteries. They were
inhibited by ryanodine Ca2+-sparks inhibitor and they were affected by caffeine.
That indicates that generation of Ca2+-sparks elicited STOCs are produced
by the mechanisms reported previously. The fact that only a low number of
freshly isolated myocytes have STOCs supports general assumption that smooth
muscle cells lining the vessels represent a non homogeneous population (10,1,
8). It is possible to suggest that only a small proportion of thoracic aorta
Page 14 of 28
myocytes express ryanodine receptor-channels (RyR) or that only these
myocytes have functional units composed by RyR receptors co-localized with
BKCa-channels to produce STOCs. Another explanation for the low number of
myocytes with active STOCs suggests that large number of thoracic aorta
myocytes have colocalized RyR receptors and BKCa channels, but their is a
mechanism that inhibits activity of functional unite. Such a mechanism has been
proposed recently to explain the high level of STOCs observed in RyR type 3
deficient myocytes and suggests that RyR3 receptor inhibits release of Ca2+ from
RyR1/2 (15). Alternatively, a proportion of Ca2+ sparks could not be able to
activate KCa channels. KCa channel sensitivity to Ca2+ sparks could be modified
by the modulation of the basal level of intracellular Ca2+like it was shown in the
case of fractional Ca2+ spark uncoupling in newborn cerebral artery myocytes
(16). Sparks generated in “silent” myocytes should be completely uncoupled
from BK-channels according to this hypothesis. Increased basal level of cAMP is
triggering STOCs by coupling sparks and BK-channels. Prolonged depolarization
of the membrane could favor rise of intracellular calcium and thus couple some
fraction of sparks with BK-channels. STOCs were not observed in “silent”
myocytes even when cell were exposed to potentials positive to 0 mV for 20-30
minutes. Administration of myocytes to low concentration of ionomycin also did
not favor coupling between sparks and STOCs. Direct measurements of sparks
in mouse aortic myocytes will clarify the question: do aortic myocytes generate
sparks uncoupled from BK -channels activity or do sparks appear after the rise of
basal level of cAMP and trigger STOCs generation.
Page 15 of 28
Forskolin was able to switch on generation of STOCs in “silent” aortic
myocytes that did not produce STOCs at any imposed voltage for more than 10
minutes after the beginning of the recording. It was suggested that activation of
adenylyl cyclase increases the intracellular cAMP concentration. When cAMP
reaches a threshold level STOCs generation is consequently switched on. This
hypothesis was supported by the fact that a membrane permeable analog of
cAMP reproduced the same effect as forskolin. Increased open probability of
BKCa-channels by cAMP amplified the effect of Ca2+-sparks on STOCs and could
explain the continuous increase of STOCs amplitude and duration. Activation of
A-kinase could be enhanced and modulated by intracellular Ca2+. It was reported
that at a low concentration of Ca2+, higher concentration of cAMP was required
for activation of the KCa-channel, when intracellular Ca2+ increases lower
concentration of cAMP was sufficient for activation (18). Inhibition of A-kinase
activity prevented cAMP stimulated STOCs generation indicating direct
implication of PKA in the chain of events that switches on STOCs generation in
When can Spontaneous Transient Outward Currents (STOCs) be
switched on in vivo in thoracic aorta and why is their activity critical for the
vascular network? One possibility could be that cellular membrane stretch can
switch on the adenylyl cyclase/cAMP/PKA pathway (17,7). STOCs viewed in this
scope prevent thoracic aorta from prolonged contraction during the myogenic
response triggered by pulsatile pressure. This cellular mechanism could regulate
the degree of thoracic aorta dilation in systolic phase. Thereby representing a link
Page 16 of 28
between aortic stiffening and exposure of resistance vessels to elevated
mechanical strains in vascular beds artificially vasodilated by medication. Indeed,
exposure of resistance vessels to highly pulsatile pressure and flow during
treatment of vascular dysfunctions was suggested to provoke microvascular
damage and coronary arteries dysfunction (4,24,21). Intracellular cAMP that
switches on STOCs generation in thoracic aorta myocytes could reduce pulsatile
pressure and thus prevent microvascular damage.
In conclusion: aortic myocytes possess inactive cellular machinery
responsible for STOCs generation that can be switched on by cAMP. The
presence of “silent” cellular mechanism suggests that contractility of the aorta
could be modulated in response to the physiological demand by recruitment of
inactive functional domains.
Sources of Funding
This work was supported by the Swiss National Foundation of Scientific
Research (3100170-100098/1) and Founds Clara.
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Figure 1. Spontaneous Transient Outward Currents (STOCs) recorded in freshly
isolated thoracic aorta myocytes. Panel A: Steady state recording of STOCs
recorded at holding potentials indicated in the left upper part of each trace.
Constant component of steady-state current was subtracted to 0 pA. Panel B:
Superimposed at the beginning STOCs represent a typical data set of STOCs
extracted from steady state recording of -10 mV holding potential and used to
calculate average-STOCs. Panel C: Average-STOCs, calculated from the data
sets obtained from 5 myocytes, represent a family of STOCs recorded at different
voltages, indicated on the panel. Panel D: Average-STOCs presented in panel C
were normalized to their peak amplitudes to compare half width and half time
decay. Panel E: Two data sets were obtained from STOCs: mean peak
amplitudes and mean duration. Distribution of mean±SEM of peak amplitude was
plotted against the distribution of mean±SEM of mean duration in semi-
logarithmic scale. Panel F: Outward K+-currents elicited by voltage steps applied
from the holding potential of -60 mV to 20 mV in “silent” myocytes (left traces).
Current voltage relations elicited in “silent” myocytes by voltage ramps from the
holding potential of – 60 mV varying from -100 mV to 100 mV (right graph). Panel
G: Outward K+-currents elicited by voltage steps applied from the holding
potential of -60 mV to 20 mV in “active” myocytes (left traces). Current voltage
relations elicited in “active” myocytes by voltage ramps from the holding potential
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of – 60 mV varying from -100 mV to 100 mV (right graph). Outward K+-currents
were recorded in control, after application of ionomycin (10 µM) and after
application of ionomycin plus iberiotoxin (100 nM).
Figure 2. Representative trace of the steady state currents recorded at holding
potential of -30 mV. Application of Iberiotoxin (100 nM) (Panel A), Charybdotoxin
(100 nM) (Panel B), BAPTA-AM (30µM) (Panel C), Caffeine (1mM) (Panel D),
Ryaonidine (50 µM) (Panel E) and is indicated by line.
Figure 3. Panel A: Representative trace of the steady state current recorded in
“silent” myocytes at holding potential of -30 mV before and after application of
forskolin. Extracellular application of forskolin (10 µM), indicated by the line,
triggered STOCs firing. Panel B: Superimposed average-STOCs calculated from
the data sets obtained from 5 recordings at sequential time laps demonstrate
STOCs evolution at the beginning, in the middle and in the end of the recording.
The inset graph shows shown the same average-STOCs normalized to their
peak amplitude. Panel C: Graph represents distribution of mean forskolin-
triggered STOCs amplitude plotted against mean forskolin-elicited STOCs
duration (n=4). Panel D: Action of iberiotoxin (100 nM) on forskolin-triggered
STOCs. Application of forskolin and iberiotoxin is indicated by line.
Figure 4. Representative trace of the steady state current recorded in “silent”
myocytes at a holding potential of -30 mV, before and after application of
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8BrcAMP. Extracellular application of 8BrcAMP (indicated by the line) triggered
STOCs generation. Panel B: Traces represent recordings of the steady state
currents at different imposed voltages in “silent” myocytes before and after
application of 8BrcAMP. Panel C: Superimposed average-STOCs calculated
from the data sets of 8BrcAMP-elicited STOCs extracted from 5 recordings at
sequential time laps demonstrate STOCs evolution at the beginning, in the
middle and in the end of the recording. The inset graph shows average-STOCs
currents normalized to their peak amplitude. Panel D: Graphic represents
distribution of the mean 8BrcAMP-triggered STOCs amplitude plotted against
distribution of their mean duration (n=4). Panel E: Steady-state recording
represents action of 8BrcAMP on PKI 14-22 (PKA inhibitor) pretreated cells.
Inhibition of PKA activity prevented stimulation of STOCs by 8BrcAMP.
Figure 5. Panel A: Amplitude histogram of the single BKCa channels before
(closed triangles) and after 8BrcAMP (closed circles) application. Single channels
were recorded in the presence of calcium chelator BAPTA-AM. Data were fitted
with two peak Gaussian function with peaks at 0.45 pA and 6.25 pA at the control
and with peaks at 0.45 pA and 6.2 pA after the 8BrcAMP application. In the inset
is shown the current voltage relation of BKCa channels amplitude. Data were
fitted with linear function with a slope of 189±16 pS. Panel B: Open time of the
BKCa channels in the control (closed triangles) and after the application of
8BrcAMP (closed circles). Data sets were fitted with exponential decay function
first order with t=4.1±0.2 (control) and t=7.9±0.3 (8BrcAMP). Panel C.
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Represents two traces of the single BKCa channel currents recorded before
(control) and after 8BrcAMP application.
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