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Am J Physiol Lung Cell Mol Physiol 275: L145-L154, 1998;
1040-0605/98 $5.00
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Vol. 275, Issue 1, L145-L154, July 1998

Spontaneous transient outward currents and delayed rectifier K+ current: effects of hypoxia

C. Vandier1, M. Delpech2, and P. Bonnet2

1 Unité Mixte de Recherche (UMR) Centre National de la Recherche Scientifique 6542, Physiologie des Cellules Cardiaques et Vasculaires, Faculté des Sciences, 37200 Tours Cedex; and 2 UMR Centre National de la Recherche Scientifique 6542, Physiologie des Cellules Cardiaques et Vasculaires, Faculté de Médecine, 37032 Tours Cedex, France

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Single smooth muscle cells of rabbit intrapulmonary artery were voltage clamped using the perforated-patch configuration of the patch-clamp technique. We observed spontaneous transient outward currents (STOCs) and a steady-state outward current. Because STOCs were tetraethylammonium sensitive and activated by Ca2+ influx, they were believed to represent activation of Ca2+-activated K+ channels. The steady-state outward current, which was sensitive to 4-aminopyridine, was the delayed rectifier K+ current. In cells voltage clamped at 0 mV, we found that STOCs were not randomly distributed in amplitude but were composed of multiples of 1.57 ± 0.56 pA/pF. The mean frequency of STOCs was 5.51 ± 3.49 Hz. Ryanodine (10 µM), caffeine (5 mM), thapsigargin (200 nM), and hypoxia (PO2 = 10 mmHg) decreased STOCs. The effect of hypoxia on STOCs was partially reversible only if the experiment was conducted in the presence of thapsigargin. Hypoxia and thapsigargin decrease steady-state outward current. Thapsigargin and removal of external Ca2+ abolished the effect of hypoxia, suggesting that hypoxia decreases steady-state outward current by a Ca2+-dependent mechanism.

pulmonary artery smooth muscle cells; calcium ion regulation; sarcoplasmic reticulum; potassium ion

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

THE MECHANISM BY WHICH hypoxia causes pulmonary vasoconstriction has not been elucidated. Studies of the hypoxic response reported that hypoxia directly acts on smooth muscle cells (18, 25) and on membrane K+ channels. Indeed, hypoxia inhibited delayed rectifier K+ channels (2, 21) and activated Ca2+-activated K+ channels (2, 5). Also, hypoxia inhibited and activated L-type Ca2+ currents, respectively, in proximal and distal pulmonary artery smooth muscle cells (8).

The Ca2+ present in the sarcoplasmic reticulum (SR) seems to play an important role in the hypoxic response of pulmonary smooth muscle cells (21, 23, 25). Several investigators suggested that hypoxic mobilization of Ca2+ from internal stores was the initial event induced by hypoxia (21, 23, 25). Salvaterra and Goldman (23) showed that hypoxia induced a later hypoxic response phase that consists of an activation of Ca2+ influx, in part, through channels other than L-type Ca2+ channels. This late hypoxic phase was completely blocked by thapsigargin (23). Then external Ca2+ and both the release of Ca2+ from SR and the depletion of this Ca2+ appear to be important in the hypoxic response.

Recently, we showed in rabbit pulmonary artery rings that hypoxia, which has no effect on resting tone of rabbit intrapulmonary artery rings, increased the amplitude of the norepinephrine phasic-induced contraction in the absence of external Ca2+ (27). This effect was blocked by ryanodine. This suggested an important role of the Ca2+ present in the SR for the hypoxic response of rabbit pulmonary artery cells.

We have examined the effect of hypoxia on spontaneous transient outward currents (STOCs), which reflect Ca2+ release of superficial SR (4), and on outward current activated by a ramp protocol having a low speed of depolarization. We show here that hypoxia decreases the delayed rectifier K+ current via a Ca2+-dependent mechanism and decreases STOCs in intrapulmonary artery smooth muscle cells. Both effects appear to result from a depletion in Ca2+ concentration of superficial SR.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Cell isolation. All animal experiments were conducted according to the ethical standards of the Ministère Français de l'Agriculture. Rabbits of either sex (2-3 kg) were killed by cervical dislocation. Before the left and right proximal intrapulmonary arteries (external diameter approx  2-3 mm) were dissected, the lung was removed and the pulmonary arteries were perfused with a cold physiological salt solution (PSS) that contained 10 µM sodium nitroprusside. Vessels were opened along their longitudinal axis and incubated in Ca2+-free solution for 10 min. They were then cut into small pieces and placed in 5 ml of a Ca2+-free solution containing 191 U/ml collagenase (CLS 2; Worthington Biochemical), 0.22 U/ml pronase E (Sigma), and 3 mM dithiothreitol at 37°C for 20-23 min on a tridimensional agitator. The tissue was washed and placed in Ca2+-free solution without enzymes and gently agitated for 40 min. The tissue was then strongly agitated with a polished wide-bore Pasteur pipette to release the cells. Cells were stored at 4°C and used between 2 and 10 h after isolation. PSS solution contained (in mM) 138.6 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 11 glucose; pH was adjusted to 7.4 with NaOH. The Ca2+-free solution was a PSS solution without added Ca2+.

Electrophysiology. For electrophysiological recording, the cells were placed in a 1-ml volume bath and continuously superfused by gravity at the rate of 4 ml/min from reservoirs. The reservoirs could be switched manually to allow addition and removal of different solutions to the bath as required. Total solution exchange in the bath was reached ~1.5 min after switching from control solution. Cell membrane currents were recorded with a List EPC-7 patch-clamp amplifier (List Electronics, Darmstadt, Germany). Patch pipettes were pulled from borosilicate glass capillaries and had resistance of 4-5 MOmega . The head stage ground was connected to an Ag-AgCl pellet that was placed in a side bath filled with the pipette solution, connected to the main bath via an agar bridge containing 3 M KCl. The junction potentials between the electrode and the bath were canceled by using the voltage pipette offset control of the amplifier. The capacitances of the pipette and the cells were canceled. The series resistance was also canceled at 50-85%.

We used the perforated-patch technique to record whole cell membrane currents (22), with amphotericin B included in the patch pipette at 240 µg/ml. Briefly, pipette tips were filled by dipping the tip of the pipette into the pipette solution, and then the pipette was backfilled with pipette solution containing amphotericin B. After the gigaseal between the pipette and the cell was realized, the electrical access to the cytoplasm was monitored by applying -10-mV pulses for 10 ms from a holding potential of -60 mV and monitoring the capacitive transient. This current was filtered at 5 kHz and sampled at 50 kHz. Typically, access was gained within 10 min and was stable within 30 min. All the experiments started after these 30 min. The pipette solution contained (in mM) 122 glutamic acid, 25 KCl, 1 MgCl2, 10 HEPES, and 1 EGTA; pH was adjusted to 7.2 with KOH. In 94 cells, final access resistance was estimated to be 28 ± 8 MOmega (range 10-50 MOmega ). Only cells with series resistance <30 MOmega were kept.

The voltage-clamp protocol used to evaluate the cell current-voltage (I-V) characteristics was a voltage ramp of 0.03 mV/ms from -90 to 0 mV. The holding potential was -60 mV. Data were sampled at 670 Hz and filtered at 150 Hz. I-V relationships were also generated in voltage-clamped cells held at a membrane potential of -60 mV and then were stepped in 10-mV increments to command potentials between -90 and 0 mV. The voltage steps were 400 ms in duration, with 5-s intervals between steps. The data were sampled at 5 kHz and filtered at 1 kHz. The voltage-ramp protocol was checked by comparing the I-V relationships obtained from the voltage ramp and the current measured at the end of the 400-ms voltage steps (n = 11). The results were identical. To characterize STOCs, the membrane potentials of the cells were held at a maintained voltage of 0 mV. The STOCs were recorded using a DAT recorder (DTR-1204 recorder; Biologic) for later analysis. Then STOCs were sampled at 1 kHz and filtered at 200 Hz for analysis. Voltage-clamp protocols were generated, and the data were captured with a PC using a labmaster TL1-125 interface (Scientific Solutions) and pClamp 5.5.1 software (Axon Instruments). The analysis was realized using pClamp and Origin software (Microcal Software, Northampton, MA).

O2 tension. The external solution was equilibrated with either air (PO2 ~150 mmHg) or N2 (PO2 <10 mmHg) in a reservoir. PO2 in the output of the recording chamber was monitored with an O2-sensing electrode (oxymeter, 781 Strahkelvin Instruments). The interval of time between the switching of bath perfusion to a measured drop in PO2 was ~3 s, and saturation of the bath at PO2 of ~10 mmHg was achieved within ~50 s.

Chemicals. Stock solutions of sodium nitroprusside (10 mM) and ryanodine (20 mM) were prepared in distilled water and then diluted in PSS at an appropriate concentration. 4-Aminopyridine (4-AP), caffeine, tetraethylammonium chloride (TEA), and cadmium (Cd2+) were prepared daily in PSS. Anthracene-9-carboxylic acid (9-AC; 1 mM) was dissolved in DMSO, which was present at 0.05% after dilution. Stock solution of thapsigargin (2 mM) was prepared in DMSO, which was present at 0.01% after dilution. When we used 9-AC and thapsigargin, the same percentage of DMSO was added to all of the experimental solutions. All chemicals were from Sigma (St. Quentin Fallavier, France).

Statistics. Data are expressed as means ± SD. We used Wilcoxon's test and randomization test for paired comparisons. Differences were considered significant at P < 0.05. Statistical analysis was realized using Stat, a software developed in this laboratory by Dr. J. Y. Le Guennec.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Membrane currents recorded in perforated patch. Membrane currents were recorded in single smooth muscle cells from the intrapulmonary artery in PSS solution. Ten step depolarizations between -90 and 0 mV from a holding potential of -60 mV elicited both an outward steady-state current and STOCs (Fig. 1A). The steady-state outward current was not always evident when STOCs were superimposed upon it. TEA (1 mM) in the bath solution abolished STOCs, and the steady-state outward current was then clearly visible. It was an outward current that activated near -30 to -40 mV, and its amplitude increased with depolarization. The addition of 1 mM 4-AP to the bath solution containing 1 mM TEA almost totally abolished the steady-state outward current (n = 11). These two types of outward currents were observed in the same cell in response to a voltage ramp from -90 to 0 mV (Fig. 1B). With the voltage-ramp protocol, we clearly see that the STOCs and steady-state outward current activated at about the same voltage of -30 to -40 mV.


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  		Fig. 1.   Effects of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on steady-state outward current and on spontaneous transient outward currents (STOCs) elicited by either voltage steps (A) or voltage ramps (B). In control, depolarization of an intrapulmonary artery cell from -90 to -40 mV induced a steady-state inward current of small amplitude. When the cell was depolarized further than -40 mV, the steady-state current was outward, and amplitude varied irregularly with spontaneous peaks in which the maximal value could reach ~250 pA and 100 ms duration (STOCs). The same phenomenon was observed when the same cell was depolarized from -90 mV up to 0 mV by the voltage ramp protocol. In both protocols, 1 mM TEA in the bath solution abolished STOCs. In presence of 1 mM TEA and 1 mM 4-AP, the steady-state outward current was also almost totally suppressed. Arrows in A and B and dashed line in B indicate the 0 current level.

The effect of 9-AC, a chloride channel blocker, was tested on four cells with the same voltage-clamp protocols. Step depolarizations between -90 and 0 mV from a holding potential of -60 mV elicited the two outward currents. 9-AC (1 mM) had no effect on the steady-state outward current or on STOCs (Fig. 2A). Changing the bath solution to one that contained 1 mM 9-AC and 1 mM 4-AP almost totally abolished the steady-state outward current but did not affect the STOCs. Similar results were obtained from the same cell in response to the voltage-ramp protocol (Fig. 2B). The steady-state outward current was an outward current activated at -30 to -40 mV, blocked by 1 mM 4-AP, and not affected by TEA or 9-AC. Characteristics that correspond represented the delayed rectifier K+ current that has already been observed in these cells (9, 20, 21, 26).


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  		Fig. 2.   Effects of anthracene-9-carboxylic acid (9-AC) and 4-AP on steady-state outward current and STOCs elicited by voltage steps (A) or voltage ramps (B) in the same cell. Cell was first depolarized by steps of 10 mV, which induced steady-state outward current and STOCs (A: control). 9-AC (1 mM) had no effect on STOCs. Addition of 1 mM 9-AC and 1 mM 4-AP to the bath solution had little effect on STOCs, but the steady-state outward current was totally suppressed. Similar observations were made when the cell was depolarized by voltage ramps (B: -90 to 0 mV membrane potential). Arrows in A and B and dashed line in B indicate the 0 current level.

STOCs, which were observed in >75% of the cells recorded with the perforated-patch technique, were activated around -30 to -40 mV, and their amplitude increased with depolarization (Figs. 1 and 2); they were abolished by 1 mM TEA and insensitive to 1 mM 4-AP and 1 mM 9-AC, suggesting that STOCs represented a Ca2+-activated K+ current rather than a chloride current.

STOCs were evaluated in 19 cells, with an example represented in Fig. 3. The membrane potential was held at 0 mV, and the amplitude and frequency of STOCs were measured over 60-s periods. STOC amplitude was normalized according to cell capacitance. The smallest STOC that we could distinguish had an amplitude of 20 pA (corresponding to 0.76 pA/pF); below this value, we could not accurately differentiate STOCs from the membrane noise. These 19 cells show that, although STOCs were not of uniform size, their amplitudes were not randomly distributed. All-point amplitude histograms of membrane current revealed peaks that represented STOC size to be composed of multiples of 1.57 ± 0.56 pA/pF (n = 19). The frequency of STOCs was 5.51 ± 3.49 Hz (n = 19).


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  		Fig. 3.   Example of the nonuniformity of STOC amplitude in a cell voltage clamped at 0 mV. A: continuous recording of membrane current. Average frequency of STOCs was 6.41 Hz in this example. Bottom trace shows the selected region on an expanded time scale. B: all-point amplitude histogram of the record shown in A. Distribution of the STOC amplitudes as a function of the number of events recorded distinguished four classes of STOCs shown by four peaks of amplitudes indicated by asterisks (*). We demonstrated that the amplitude interval between each peak of each class was constant and of 1.57 ± 0.56 pA/pF (n = 19). Arrow in A indicates the 0 current level.

In 30% of cells when the membrane potential was maintained at 0 mV for >10 min, we observed a decrease of STOC activity with time. In 70% of cells, STOC activity was stable for 25 min. To evaluate this rundown, five cells were held at 0 mV for 25 min, and we measured the frequency of STOCs during 60 s, after 4 min, after 14 min, and after 24 min. At these three different times (4, 14, and 24 min), the frequencies of STOCs were 1.29 ± 0.58, 0.96 ± 0.53, and 0.55 ± 0.51 Hz, respectively. At these three different times, the frequencies were not significatively different.

Role of extracellular Ca2+ in STOC activity. To investigate the role of external Ca2+ in STOCs, we first changed the bathing solution (PSS) to a Ca2+-free solution containing 1 mM EGTA in three cells. In Fig. 4A, the cell was voltage clamped with the voltage-ramp protocol. This protocol elicited STOCs and the steady-state outward current. Removal of extracellular Ca2+ for 10 min totally abolished STOCs at all ramp voltages. This suggested that Ca2+ influx was indispensable for STOC activity. The removal of extracellular Ca2+ was also associated with an increase in steady-state outward current (Fig. 4A).


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  		Fig. 4.   Importance of extracellular Ca2+ for STOC activity. A: typical trace of current elicited by a voltage-ramp protocol in a cell recorded first in control conditions (control) and then after exposure to 0 Ca2+ solution (0 Ca). Removal of the external Ca2+ abolished STOCs and increased the steady-state outward current. B: in a different experiment, the cell, whose membrane potential was held at 0 mV, was superfused by saline solution containing 1 mM Cd2+. Cd2+ (1 mM) reversibly decreased the frequency of STOCs. It also reversibly depressed the steady-state outward current. Arrows in A and B and the dashed line in A indicate the 0 current level.

In three cells, we tested some properties of the L-type Ca2+ current to see if this was one pathway for Ca2+ to enter the cell and activate STOCs. The L-type Ca2+ current of rabbit pulmonary artery is abolished by external Cd2+ (7). Figure 4B shows a cell voltage clamped at 0 mV for 6 min. During the first minute, the cell was bathed with PSS solution, and it showed STOCs. When the bath solution was changed to a solution that contained 1 mM Cd2+, the frequency of STOCs rapidly decreased and was stable after 2 min in this solution. The effect of Cd2+ was reversible, and the activity of STOCs recovered in PSS solution after a 2-min wash. Thus, under these conditions of 0-mV voltage clamp with 1 mM external Cd2+, the STOCs were not totally abolished, and the mean frequency of STOCs decreased from 0.51 ± 0.16 Hz in PSS solution to 0.16 ± 0.05 Hz in solution with Cd2+ (n = 3). In these cells, Cd2+ also provoked a reversible decrease of the steady-state outward current.

Role of intracellular Ca2+ stores in STOC activity. To investigate the role of intracellular Ca2+ stores, we tested the effects of caffeine, ryanodine, and thapsigargin on STOCs.

To test the effect of caffeine on STOCs, eight cells were voltage clamped at 0 mV in the presence and in the absence of external caffeine. Figure 5A shows a cell voltage clamped at 0 mV, which showed STOCs in the PSS solution. The activity of STOCs was stable during this 2 min in PSS solution. External application of 5 mM caffeine rapidly induced a large transient increase in STOCs that summated to give a peak outward current of 19 ± 16 pA/pF (n = 8) ~30 s after the beginning of the application of caffeine. After this time, the amplitude of the outward current decreased and was stable ~1 min after the beginning of the application of caffeine. After this period, the STOCs were totally abolished. The effect of caffeine was slowly reversible, and STOCs began to reappear after 2 min in PSS solution and fully recovered after 4 min.


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  		Fig. 5.   Role of intracellular Ca2+ stores on STOCs. A: in a cell held at 0 mV, 5 mM caffeine rapidly elicited an increase in STOCs that summated to form a transient peak of current. Then STOCs were progressively abolished. After the washout of caffeine, STOCs reappeared. B: in a different cell, also held at 0 mV, 10 µM ryanodine slowly decreased STOCs. Arrows in A and B indicate the 0 current level.

To confirm the involvement of ryanodine-sensitive Ca2+ pools in the activation of STOCs, 10 µM ryanodine was applied in three cells voltage clamped at 0 mV. Figure 5B shows that the application of 10 µM external ryanodine decreased STOCs gradually but not completely in 12 min. The effect of ryanodine was not reversible on the time scale of these experiments. The slow reduction in STOCs is consistent with the gradual depletion of the Ca2+ pool by this agent (12, 17).

To test whether STOCs were linked to the accumulation of Ca2+ into the SR by Ca2+-ATPase, we used thapsigargin, which is known to inhibit this Ca2+-ATPase in pulmonary artery (10). Seven cells were held at 0 mV to test the effect of thapsigargin. Figure 6 shows a cell with STOCs and a steady-state outward current in PSS solution. When the bath solution was changed to one that contained 200 nM thapsigargin, the amplitude and the frequency of STOCs gradually decreased. Finally, after 7 min of superfusion of thapsigargin, STOCs were totally abolished. The inhibitory effect of thapsigargin was not reversible within the time scale of these experiments. Together these results suggested that STOCs require Ca2+ in the SR and that Ca2+-ATPase, which sequesters Ca2+ into the SR, also has an important role.


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  		Fig. 6.   Role of sarcoplasmic reticulum Ca2+-ATPase in STOCs. Record illustrates the application, in the bath solution, of 200 nM thapsigargin to a cell held at 0 mV. Effect of thapsigargin was similar but faster than that of ryanodine (compare Figs. 5B and 6). Arrow indicates the 0 current level.

Effects of hypoxia on steady-state outward current. The effect of hypoxia was tested on 11 cells. Ramp depolarization between -90 and 0 mV from a holding potential of -60 mV elicited the two outward currents. For eight cells, 10 min of hypoxia decreased both STOCs and the amplitude of the steady-state outward current (Fig. 7A). Upon reoxygenation, the effect of hypoxia was partially reversible on the steady-state outward current (data not shown) but was not reversible on STOCs (see Fig. 9). For three cells (which did not present STOCs), hypoxia increased the amplitude of the steady-state outward current or had no effect (data not shown).


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  		Fig. 7.   Effects of hypoxia on steady-state outward current and on STOCs elicited by voltage ramps. A: when the cell was superfused by saline solution containing 1.8 mM Ca2+, 10 min of hypoxia decreased STOCs and depressed the amplitude of steady-state outward current. B: when the cell was bathed in 0 Ca2+ solution, 10 min of hypoxia increased the amplitude of steady-state outward current. Arrows and the dashed lines in A and B indicate the 0 current level.

When we changed the bath solution to Ca2+-free solution for 10 min, we first observed an increase in the steady-state outward current as already described in Fig. 4A, and then hypoxia never decreased the steady-state outward current (Fig. 7B), but, in contrast, hypoxia increased in amplitude. This effect was observed in all six cells tested and was clearly seen for membrane potential positive to -20 mV. For two cells, hypoxia also decreased the amplitude of the steady-state outward current for membrane potential between -40 and -20 mV.

Seven cells were voltage clamped with the voltage-ramp protocol in the presence and in the absence of external thapsigargin. Figure 8 shows that the amplitude of the outward current was decreased in the presence of external solution containing thapsigargin (200 nM) during ~10 min. This effect was observed in all of the cells tested. When the bath solution was further changed to a hypoxic solution containing thapsigargin (200 nM), 10 min of hypoxia increased the amplitude of steady-state outward current in four of the seven cells (Fig. 8C). This effect, which was partially reversible, was similar to the effect of hypoxia observed in Ca2+-free solution (Fig. 7B). In three cells, hypoxia had no effect on the amplitude of the steady-state outward current.


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  		Fig. 8.   Effects of thapsigargin on steady-state outward currents elicited by voltage ramp in normoxia and in hypoxia. Traces of currents elicited by a voltage-ramp protocol in a cell recorded in control conditions (trace A, PO2 = 145 mmHg) and after exposure to 200 nM thapsigargin (trace B, PO2 = 145 mmHg). In the same experiment and in the presence of thapsigargin, 10 min of hypoxia increased the amplitude of steady-state outward current (trace C, PO2 = 10 mmHg). Arrow and the dashed line indicate the 0 current level.

Effects of hypoxia on STOCs. To test the effect of hypoxia on STOCs, cells were superfused with a solution equilibrated with 100% N2. Figure 9 shows STOCs in a cell held at 0 mV before and after a period of hypoxia. When the cell was in normoxia, a PO2 of ~150 mmHg, the activity of STOCs was stable for the 5 min of recording. Changing the bath solution to a hypoxic PSS solution decreased the PO2 to ~10 mmHg, and then we observed a decrease in the frequency of STOCs. After 10 min of hypoxia, STOCs had almost totally disappeared. Next, the solution was reoxygenated by changing the bath solution to normoxic PSS solution for 10 min. Upon reoxygenation, STOCs did not recover within the time scale of these experiments. To evaluate the effect of hypoxia and reoxygenation on STOCs, we measured the frequency of STOCs in eight cells for 60-s periods after 4 min in control conditions, after 9 min of hypoxia, and after 9 min of reoxygenation. The frequency decreased from 0.37 ± 0.46 Hz in control to 0.12 ± 0.22 Hz in hypoxia and to 0.07± 0.19 Hz upon reoxygenation. Compared with normoxia, the frequencies were significantly lower during hypoxia and after reoxygenation. This effect was not due to a run-down phenomenon since, in control experiments, we did not observe a significant decrease in STOC frequency over a similar time scale (see above).


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  		Fig. 9.   Effect of hypoxia-reoxygenation on STOCs. A: membrane currents recorded from a cell voltage clamped at 0 mV. B: variation of PO2 in the experimental chamber during the superfusion of normoxic physiological salt solution (PSS) and PSS equilibrated with N2 (see METHODS). When the PO2 in the chamber decreased from ~150 mmHg (normoxia) to 10 mmHg (hypoxia), STOC frequency decreased progressively. Even after return to normoxia (PO2 150 mmHg), this effect was not reversible. Arrow in A indicates the 0 current level.

Seven cells were voltage clamped at 0 mV during hypoxia and reoxygenation in the presence of thapsigargin. Figure 10 shows that, during 5 min in a normoxic PSS solution, STOC activity was stable. The application of 200 nM thapsigargin to the bath solution in normoxia decreased the amplitude and frequency of STOCs before totally abolishing them after 8 min. The PO2 in the bath solution was then decreased from 150 mmHg to ~10 mmHg in the continued presence of thapsigargin. This had no visible effect on STOCs, and during 10 min of hypoxia no STOCs were observed. Upon reoxygenation in the presence of thapsigargin, STOCs rapidly reappeared but with smaller amplitudes and lower frequencies than those seen in the initial control condition. In five of the seven cells held at 0 mV and exposed to hypoxia in the presence of thapsigargin, we observed STOCs upon reoxygenation.


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  		Fig. 10.   Effect of hypoxia-reoxygenation on STOCs in the presence of thapsigargin. A: membrane currents recorded from a cell voltage clamped at 0 mV. B: PO2 in the experimental chamber during the superfusion of normoxic PSS (PO2 value of 150 mmHg) and PSS equilibrated with N2 (PO2 <20 mmHg). Thapsigargin (200 nM) abolished STOC activity, and hypoxia had no further effect. Upon reoxygenation (PO2 recovers to 150 mmHg), we observed a resumption of STOC activity. Arrow in A indicates the 0 current level.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Membrane currents recorded in perforated patch. The steady-state outward current was an outward current activated at -30 to -40 mV, blocked by 1 mM 4-AP, and not affected by TEA or 9-AC. These characteristics correspond to the delayed rectifier K+ current [IK(dr)] that has already been observed in these cells (9, 20, 21, 26). Removal of extracellular Ca2+ was associated with an increase of the amplitude of IK(dr). This could be explained by a decrease in a Ca2+ inward current or by a screening of surface charges (11).

STOCs, which were observed in >75% of the cells recorded with the perforated-patch technique, were activated around -30 to -40 mV. Their amplitude increased with depolarization, and they were abolished by 1 mM TEA and were activated by Ca2+ influx, suggesting that STOCs represented a Ca2+-activated K+ current. Furthermore, 1 mM 9-AC had no effect, suggesting that STOCs were not chloride currents (13).

Importance of external Ca2+ in STOC activity. In Ca2+-free solution, STOCs totally disappeared, suggesting that a Ca2+ influx was necessary. Similar results were found in rabbit cerebral artery and in jejunal smooth muscle cells (3, 15). However, removal of external Ca2+ did not suppress STOCs in ear artery smooth muscle cells (3). This could be explained by a difference in the arrangement of the Ca2+ stores (3).

The fact that at 0 mV and with Cd2+ we still observed STOCs suggests that the L-type Ca2+ current was not indispensable. The decrease in STOC activity in the presence of Cd2+ may instead result from an inhibition of Ca2+ leak current, a residual Ca2+ window current of L-type Ca2+ current, Na+-Ca2+ exchange current (16), or a screening of charge surface (11). Bychkov et al. (6) showed that Ca2+ entry through a reverse-mode Na+-Ca2+ exchanger determines Ca2+ store refilling and then regulates STOC activity. Because the Na+-Ca2+ exchanger current is blocked by 1 mM Cd2+ (16), this could explain our results.

Importance of internal stores of Ca2+ in STOC activity. Caffeine at concentrations >1 mM decreases the threshold of Ca2+-induced Ca2+ release (14) before depleting Ca2+ stores. The initial increase in STOC activity induced by 5 mM caffeine could be due to the increase in internal Ca2+ from SR resulting in Ca2+-induced Ca2+ release; then, after the SR was emptied, the STOCs would be abolished. Ryanodine at 10 µM, a concentration used to deplete the internal store (12, 17), gradually decreased STOCs. The fact that ryanodine did not completely abolish STOCs suggested that only a part of the Ca2+ from the SR responsible for STOCs was released through the ryanodine receptor, and we cannot exclude that Ca2+ was also released through inositol 1,4,5-trisphosphate-induced Ca release.

The internal Ca2+ store dependence of STOCs was confirmed by the inhibiting action of thapsigargin. Thapsigargin (200 nM) totally abolished STOCs in rabbit pulmonary smooth muscle cells. At this concentration, thapsigargin was also known to abolish STOCs in rat cerebral arterial smooth muscle cells (19). Thapsigargin is known to block Ca2+-ATPase of SR in pulmonary artery (10) and then to deplete Ca2+ stores (24).

Are STOCs activated by graded release of Ca2+ from the SR? At 0 mV, the amplitudes of STOCs were not uniform, and the step increment of 1.57 ± 0.56 pA/pF suggested that Ca2+, which activated STOCs in these cells, is uniformly released from the SR and particularly from superficial SR (4). In cells clamped at 0 mV with a 4-137 mM K+ gradient, the unitary conductance for Ca2+-activated K+ channel would be 86 pS, and with 1 µM intracellular Ca2+, the open probability would be 0.25 (1). If we assume that the rise in Ca2+ close to the channel was 1 µM, then 20 channels would be required to open and to induce a uniform interval of 1.57 ± 0.56 pA/pF. The maximum amplitude of STOCs would then represent the activation of 160 channels. Nelson et al. (19) showed that STOCs are activated by spontaneous release of Ca2+ (Ca2+ sparks) from the SR close to the sarcolemma. They suggested that one spark activates 13 Ca2+-activated K+ channels, which is close to the value that we estimate.

Effect of hypoxia on IK(dr). In the presence of external calcium, hypoxia decreased the amplitude of IK(dr). This effect was abolished by removal of Ca2+ in the external solution. This suggested that hypoxia decreases IK(dr) via a Ca2+-dependent mechanism. Because 10 min in Ca2+-free solution also depletes Ca2+ from superficial SR, this source of Ca2+ was also important in the effect of hypoxia on IK(dr). This was confirmed by the inhibitory action of thapsigargin (which was applied 10 min before) on the effect of hypoxia. Indeed, thapsigargin is known to deplete SR Ca2+ stores (24).

These results are consistent with those of Salvaterra and Goldman (23), who showed that thapsigargin blocked the elevation of internal Ca2+ concentration induced by hypoxia. Also, Post et al. (21) showed that hypoxia could release Ca2+ of SR, which induced a decrease in the amplitude of IK(dr). However, this release of Ca2+ may induce an increase in STOCs in our cells, but we always observed a decrease in these currents. This could be explained by the use of the perforated-patch technique instead of the whole cell configuration of the patch-clamp technique. The mechanism to explain this difference needs to be demonstrated. Indeed, with the perforated-patch technique, Ca2+ homeostasis is less modified by intrapipette dialysis (22). Also, Post et al. (21) didn't observe STOCs in their cells.

Next, we suggested that the decrease in IK(dr) was blocked by a depletion in Ca2+ concentration of SR.

Effect of hypoxia on STOCs. Ten minutes of hypoxia decreased the activity of STOCs, and this effect was not reversible. The effect of hypoxia may be due to an inhibitory action of hypoxia on the Ca2+-activated K+ channel, on the Ca2+-release channels of the SR, on Ca2+ influx, or on Ca2+-ATPase of the SR. An inhibitory action of hypoxia on Ca2+-activated K+ channel and on L-type Ca2+ current was not likely because hypoxia had no effect or activated the Ca2+-activated K+ channel (2, 5, 21), and the L-type Ca2+ current was mostly inactivated at 0 mV (7). It was shown that Ca2+ release by SR was activated and not inhibited by hypoxia (23, 25). Then the more likely action by which hypoxia decreased STOCs was an inhibitory action on the Ca2+-ATPase of the SR, leading to a decrease in Ca2+ concentration of superficial SR. Indeed, thapsigargin is known to block Ca2+-ATPase of the SR in pulmonary artery (10) and then to deplete Ca2+ stores (24).

However, because the presence of thapsigargin allows the STOCs to return after hypoxia, this might implicate a different mechanism of action. More experiments are needed to discover such new mechanisms.

In conclusion, we showed that hypoxia decreases both STOCs and IK(dr) by a Ca2+-dependent mechanism. Thapsigargin and removal of external Ca2+ abolished the effect of hypoxia on IK(dr), suggesting that hypoxia decreases IK(dr) by a Ca2+-dependent mechanism that depends on the Ca2+ concentration of SR.

    ACKNOWLEDGEMENTS

We thank Dr. Ian Findlay for helpful criticisms on the manuscript. We thank Dr. Dominique Thuringer for useful discussions and for helping in isolating cells, Dr. Claire Malécot for critical reading of the paper, Maryse Pingaud for technical assistance, Gilles Pinal for building some electronic devices, and Chantal Boisseau for secretarial assistance.

    FOOTNOTES

This work was supported by le Ministère de l'Enseignement Supérieur et de la Recherche and la Fondation pour la Recherche Médicale.

Address for reprint requests: P. Bonnet, UMR CNRS 6542, Physiologie des Cellules Cardiaques et Vasculaires, Faculté de Médecine, 2 bis, Boulevard Tonnelé, B.P. 3223, 37032 Tours Cedex, France.

Received 27 May 1997; accepted in final form 20 March 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(1):L145-L154
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


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