Spontaneous transient outward currents (STOCs) 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 the 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 (10 μM) 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 the 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 physiological demand.
- thoracic aorta myocytes
- BKCa channels
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 (8, 13, 23). 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 (11, 20), 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 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 31.1.1008/2129/0.
Male C57BL/6 mice that were 3 to 4 wk 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) 137 NaCl, 5.4 KCl, 0.44 K2HPO4, 0.42 NaH2 PO4, 2 MgCl2 6H2O, 4.17 NaHCO3, 0.2 CaCl2 2H2O, 0.05 EGTA, 11 glucose, 10 HEPES, pH was adjusted to 7.4 with NaOH. Aorta was incubated for 40 min at 37°C in the low Ca2+ solution containing 2 mg/ml elastase type (IV) and 1 mg/ml collagenase (type IA-S). Vascular smooth muscle cells were isolated by careful shaking of the tissue and then placed on coverslips and stored at 4°C.
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). They had resistance of 4–7 MΩ. Patch pipettes were filled with (in mM) 130 KCl, 10 HEPES, 2 EGTA (pH 7.4) for the whole cell experiments. The same solution without EGTA was used for the perforated patch-clamp experiments. Nystatin (Sigma, Deisenhofen, Germany) was dissolved in DMSO and diluted into the pipette solution to give a final concentration ranging from 50 to 100 μg/ml. The aortic smooth muscle cells were bathed in a solution containing (in mM) 130 NaCl, 5.6 KCl, 1 MgCl2 6H2O, 2 CaCl2 2H2O, 8 HEPES, 10 glucose (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 Ca2+ level and consequently inhibited STOCs. Cells were clamped at 0 mV. NPo was calculated over 7-min intervals as ∑j=1N tj·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 the duration of the recording (7 min).
Charybdotoxin, EGTA, elastase (type IV from porcine pancreas), collagenase (type IA-S), BAPTA-AM, ryanodine, 8-bromo-adenosine 3′-5′cyclic monophosphate (8BrcAMP), PKI 14–22 amide (myristoylated protein kinase A inhibitor amide 14–22, cell permeable), and forskolin were obtained from Sigma (Buchs, Switzerland).
Data treatment and statistics.
Each set of data was expressed as means ± SE. 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, and open time of single BKCa channels. A value of P < 0.05 was considered as statistically significant. Means ± 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) where Y0 is the Y offset, A1 is initial Y value, and t1 is decay constant. Histogram distributions of the single BKCa channels were fitted with two-peak Gaussian function.
Characterization of STOCs in freshly dissociated mouse aortic smooth muscle cells.
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 to 100 mV. Aortic smooth muscle cells with or without STOCs had similar current-voltage relationships 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 (2 mM) plus TEA (5 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, F and G). Rise of intracellular Ca2+ increased the amplitude of the outward net currents and shifted activation threshold of the current-voltage relationship from 10 ± 1.3 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 to 26.3 ± 1.8 pA/pF in active myocytes and from 18.1 ± 1.4 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 to 20 mV (Fig. 1A). 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 Fig. 1B. Average STOCs current was calculated from all regrouped STOCs in each data set. The same method was applied to analyze STOCs in all experiments. Average STOCs currents obtained from STOCs recorded at different holding potentials were superimposed in one plot. Average STOCs current showed increase of peak amplitude with the increase of imposed voltage (Fig. 1C; n = 5). Data set of distribution of mean peak amplitudes of STOCs was plotted against corresponding data set of mean durations in semilogarithmic 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 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, A and 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 s and temporally inhibited STOCs by depleting intracellular stores of the aortic myocytes (Fig. 2D; n = 5). After removal of caffeine from the bath, STOCs recovered within 1–3 min. Ryanodine (50 μM, n = 6), a Ca2+ spark inhibitor, and BAPTA-AM (30 μM), a membrane-permeable Ca2+ chelator (n = 4), completely blocked generation of STOCs (12, 20).
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 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 min in the third population of silent myocytes (35.1%; Fig. 3A). Low-amplitude forskolin-elicited STOCs appeared as single events with a 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. 3D).
Three averages of forskolin-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 forskolin-triggered average STOCs increased gradually from 47.2 ± 9.1 to 134.1 ± 13.2 pA at the end of the recording (Fig. 3B). Average STOCs normalized to the peak (Fig. 3B, inset) 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 semilogarithmic scale (Fig. 3C; 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. The addition of 8BrcAMP (10 μM) to the bath solution triggered STOCs generation with a delay of 10.5 ± 1.3 min in 38.7% of silent aortic myocytes (Fig. 4A). STOCs did not appear spontaneously in silent myocytes maintained at different holding potentials for the period of 20 to 30 min of recording (Fig. 4B). In 45.2% of cells, 8BrcAMP increased the outward net current to 147.1 ± 12.2% at 0 mV. Charybdotoxin (100 nM) decreased the amplitude of the 8BrcAMP-elicited outward current to 87.5 ± 9.3%. In the remaining myocytes, 8BrcAMP activated leak-like currents.
Three averages of 8BrcAMP-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. 4C, inset). Half width of normalized average currents increased from 27.6 ± 2.3 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 semilogarithmic scale (Fig. 4D; 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 h 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. 4D).
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 min 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 to 30 mV (Fig. 5A, inset), 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 min 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 STOCs. STOCs were reported previously in cerebral resistance arteries, coronary arteries, and the mesenteric artery (2, 5, 12, 20, 22). 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 (3, 19). Taken together, these data indicate that STOCs represent one of the key regulatory elements in the cardiovascular system.
STOCs were identified and analyzed to answer the question: do thoracic aorta myocytes possess 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+ spark inhibitor and they were affected by caffeine. That indicates that generation of Ca2+ sparks-elicited STOCs is 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 nonhomogeneous population (1, 8, 10). It is possible to suggest that only a small proportion of thoracic aorta myocytes express ryanodine receptor channels (RyR) or that only these myocytes have functional units composed by RyR receptors colocalized 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 there is a mechanism that inhibits activity of functional unity. 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 (16). 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 (15). 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 cells were exposed to potentials positive to 0 mV for 20–30 min. 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.
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 min 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. However, when intracellular Ca2+ increases lower concentration of cAMP was sufficient for KCa 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 silent myocytes.
When can 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 (7, 17). 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 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, 21, 24). 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.
This work was supported by the Swiss National Foundation of Scientific Research (3100170–100098/1) and Founds Clara.
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