Division of Pediatric Pulmonology and Critical Care Medicine, Departments of 1 Pediatrics, 2 Physiology, and 3 Medicine, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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To study developmental changes in intracellular calcium handling in pulmonary artery smooth muscle cells (PASMCs), cells were isolated from distal and proximal pulmonary arteries from rabbits at different developmental stages: juvenile (4-6 wk old), newborn (<48 h), and full-term fetal. Isolated PASMCs were studied using the calcium-sensitive dye fura 2. Cells from each age group responded to caffeine with an increase in calcium; however, ryanodine (50 µM) only increased calcium in fetal distal PASMCs. The ryanodine-induced increase was due to influx of extracellular calcium because it was blocked by removal of extracellular calcium or by diltiazem. The calcium-sensitive potassium (KCa) channel blocker iberiotoxin produced a transient increase in calcium in the fetal distal PASMCs, which could be inhibited by prior application of ryanodine. Conversely, the ryanodine response was inhibited if iberiotoxin was given first. With the use of electrophysiology and confocal microscopy, fetal PASMCs were shown to exhibit spontaneous transient outward currents and calcium sparks, respectively. These observations suggest that ryanodine-sensitive release of calcium from the sarcoplasmic reticulum and KCa channels act together to control intracellular calcium only in fetal distal PASMCs.
intracellular calcium; calcium sparks; spontaneous transient outward currents; calcium-sensitive potassium channels; perinatal
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INTRODUCTION |
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AT BIRTH THE PULMONARY VASCULATURE undergoes a dramatic and unparalleled change in resistance. In the fetus, oxygen tension is low, pulmonary blood flow is low, and pulmonary vascular resistance is high. At birth, with the establishment of the air-liquid interface and the increase in oxygen tension, the pulmonary arteries immediately dilate, and pulmonary vascular resistance drops significantly. Over the first several days of life, perinatal pulmonary vasodilation is sustained and progressive. In the absence of disease, this change in resistance is maintained for the life of the animal (12, 25).
Several birth-related stimuli contribute to perinatal pulmonary vasodilation including rhythmic distension of the lung, establishment of an air-liquid interface, the rise in oxygen tension, the release of nitric oxide (NO) from the pulmonary endothelium, and the increase in shear stress as flow increases (1, 2, 8, 13, 20). A key step in the vasodilatory pathway common to many of these factors is an activation of potassium channels in the smooth muscle and/or endothelial cell membrane (3, 9, 26, 29, 30), specifically the calcium-sensitive potassium channel (KCa channel). Pulmonary artery smooth muscle cell (PASMC) KCa channel activation results in membrane hyperpolarization and closing of voltage-dependent calcium channels, leading to a decrease in intracellular calcium and thus vasodilation.
While KCa channel activation plays a critical role in mediating perinatal pulmonary vasodilation, the subcellular pathway leading to KCa channel activation and vasodilation remains incompletely understood. Evidence from vascular (18, 21, 22) and other smooth muscle cell types (4, 16, 27, 33) indicates that vasodilation may result from increasing frequency of small localized ryanodine-sensitive spontaneous bursts of calcium released from intracellular stores (calcium "sparks"). These calcium sparks activate plasmalemmal KCa channels and produce characteristic large transient outward currents (21, 22). These currents have been termed "spontaneous transient outward currents" (STOCs). In simultaneous confocal microscopy and patch-clamp experiments (22), a direct temporal correlation was demonstrated between intracellular calcium sparks and STOCs, with 96% of calcium sparks being associated with a detectable STOC. STOCs can be inhibited by the KCa channel blocker iberiotoxin or by ryanodine, which blocks release of calcium sparks from the sarcoplasmic reticulum (18, 21). Charybdotoxin (another KCa channel-blocking agent)-sensitive STOCs have also been observed in ovine fetal PASMCs (24).
Work by Gollasch et al. (14) suggests that relatively immature systemic arterial smooth muscle cells do not exhibit calcium sparks, possibly due to the arrangement of ryanodine receptors, and that these neonatal arteries do not respond to inhibitors of KCa channels and ryanodine receptors. However, evidence that ryanodine-sensitive calcium release (and thus possibly calcium sparks) may play a role in the regulation of perinatal pulmonary vascular tone derives from whole animal studies (26), demonstrating that infusion of ryanodine attenuated perinatal pulmonary vasodilation in response to inhaled NO. Therefore, if NO acts in part through a ryanodine-sensitive mechanism, we hypothesized that calcium release from the sarcoplasmic reticulum may facilitate pulmonary vasodilation. Moreover, since perinatal pulmonary vasodilation in response to an increase in oxygen tension, NO, and shear stress is biologically imperative in the neonatal period, the contribution of the ryanodine-sensitive mechanism may be greatest in the period of development around parturition and decreases with postnatal maturation. In this study, the hypothesis that a ryanodine-sensitive mechanism contributes to intracellular calcium concentration ([Ca2+]i) has been tested using freshly isolated smooth muscle cells from fetal, newborn, and juvenile (4-6 wk old) rabbit pulmonary arteries.
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METHODS |
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Cell preparation. Proximal (first and second order, extralobar) and distal (fourth and fifth order, intralobar) arteries were dissected from full-term fetal rabbits (27 days gestation), newborn rabbits (<48 h old), and juvenile rabbits (4-6 wk old).
Single cells were enzymatically dispersed using a papain and collagenase digestion protocol (salts and enzymes were obtained from Sigma, St. Louis, MO). Briefly, arteries were incubated for 15 min at 37°C in a solution of 0.5 mg/ml papain plus 1 mg/ml dithioerythritol plus 1 mg/ml albumin, then transferred using a wide-bore pipette to a solution of 0.5 mg/ml collagenase (1:1 mixture of collagenases H and F) plus 1 mg/ml albumin and incubated at 37°C for 8 min. The arteries were then washed twice in an albumin-containing solution before gentle trituration in an albumin-free solution. To maintain the low oxygen state of the fetal environment, cells from fetal animals were prepared and stored in a relatively hypoxic solution (O2 tension of 21-30 mmHg; composition given in Solutions and drugs used). To confirm that this hypoxic isolation did not affect the results of the experiment, some cells were also isolated from juvenile animals in hypoxic solution (as indicated in RESULTS). All cells were studied in identical conditions within 2 h of preparation.Conventional calcium imaging. After trituration of the digested tissue (see above), suspensions of single PASMCs were transferred to a perfusion chamber (1 ml volume) on the stage of an inverted microscope (Nikon Eclipse TE200). The cells were loaded with dye by incubation with 10 nM fura 2-AM plus 2.5 µg/ml Pluronic acid (Molecular Probes, Eugene, OR) for 20 min in calcium-free solution (composition given below) followed by a 20-min wash in a calcium-containing recording solution (see composition below) before start of the experiment. Ratiometric imaging was performed using excitation wavelengths of 340 and 380 nm, and emission wavelength of 510 nm. Images were recorded with an intensified charge-coupled device camera (Photonic Science, Robertsbridge, UK) using Axon Instruments (Foster City, CA) image capture and analysis software. Calcium calibration was achieved by measuring a maximum (with 1 µM ionomycin) and a minimum (with 10 mM EGTA) from each cell [assuming a dissociation constant (Kd) of 220 (28)].
Confocal calcium imaging. Cells were loaded with the calcium-sensitive indicator fluo 3 by a 20-min incubation in 5 µM fluo 3-AM plus 2.5 µg/ml Pluronic acid (Molecular Probes) followed by a 20-min wash. All measurements were made within 30 min of the end of the wash. The cells were scanned with a Bio-Rad MRC1000 laser scanning confocal microscope using a ×60 oil immersion objective. Images were acquired using the line scan mode of the confocal microscope (as described in Refs. 5 and 7). This mode repeatedly scans a single line through a cell. A scan speed of 6 ms/line was used. To generate the line scan image, the digitized data from each pass through the cell are pseudocolored to represent relative change in fluorescence compared with the level in the cell while quiescent. These colored lines are displayed horizontally, and each subsequent line (pass through the cell) is added beneath the preceding line to form a composite representation with the two dimensions x-time. In these images, time passes in the vertical direction running from top to bottom, position along the scan line is given by the horizontal displacement, and the change in fluorescence intensity is given by the color change. To minimize the possibility of laser damage affecting the calcium handling of the cells, each cell was scanned for the same brief duration, 18 s total (6 consecutive line scan images of 512 lines at 6 ms/line were recorded from each cell along a single line). Sparks were analyzed using custom-written (IDL; Research Systems, Boulder, CO) analysis programs.
Electrophysiology.
After trituration of the digested tissue, suspensions of single
PASMCs were transferred to a perfusion chamber (500 µl volume) on the
stage of an inverted microscope (Nikon Eclipse TE300) for amphotericin
perforated patch-clamp studies (23). After a brief period
to allow partial adherence to the bottom of the recording chamber,
fire-polished microelectrodes of resistances between 3 and 4 M
containing a solution of (in mM) 140 KCl, 1.0 MgCl2, 10 HEPES, and 0.1 EGTA, as well as 100 µg/ml amphotericin B (pH 7.2 with
KOH) were positioned above single cells, with coarse manipulators and
micromanipulators. Electrodes were lowered onto the surface of a cell,
at which point slight negative pressure, applied by mouth, formed a
high-resistance seal with the membrane, electrically isolating the
small area of membrane under the electrode. Electronic compensation of
whole cell capacitance provided an estimate of the cell size (in pF).
Recordings were not made until access resistance was <5 M
to reduce
voltage errors. Series resistance were compensated as necessary.
Changes in current amplitude were recorded while holding the cell at a
fixed membrane potential of
20 mV. This potential was chosen because
it was close to the average resting membrane potential of these cells.
All patch-clamp studies were done at room temperature.
Solutions and drugs used. Cells were prepared, stored, and loaded with dye in the following calcium-free solution (in mM) 10 HEPES, 10 glucose, 55 NaCl, 80 sodium glutamate, 5.6 KCl, and 2 MgCl2 (pH 7.4 with NaOH). Calcium imaging and electrophysiology were performed using a calcium-containing recording solution containing (in mM) 10 HEPES, 10 glucose, 135 NaCl, 5.6 KCl, 1.8 CaCl2, and 2 MgCl2. All salts were obtained from Sigma. Fluorescent dyes and ryanodine were obtained from Molecular Probes, iberiotoxin was obtained from Alomone Labs (Jerusalem, Israel), and diltiazem was obtained from Calbiochem (La Jolla, CA).
Statistical methods. Results are presented as means ± SE. Statistical significance was tested with the Student's t-test (paired or unpaired as appropriate), with a significance level of P < 0.05.
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RESULTS |
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Ryanodine (50 µM) was added to isolated smooth muscle
cells from proximal and distal regions of the pulmonary
vasculature at three different developmental ages (full-term fetal,
newborn, and juvenile 4-6 wk old). Ryanodine caused an increase in
intracellular calcium in distal PASMCs isolated from fetal arteries
(Fig. 1). The response was
rapidly developing and transient, with a duration of 90 ± 12 s (n = 76 cells; Fig. 1). There was no response to ryanodine in cells from distal pulmonary arteries from newborn or juvenile rabbits (Fig. 2). There was
also no response observed in cells from proximal pulmonary arteries
from the fetal, newborn, or juvenile rabbits (Fig. 2). Caffeine
produced a transient increase in calcium in all cell types (Fig. 2).
Fetal cells were isolated in a low-oxygen environment to mimic the
fetal environment. To confirm that this did not affect the results, the
ryanodine and caffeine experiment was also repeated in juvenile cells
isolated in a low-oxygen environment. The isolation conditions did not affect the cells' response to caffeine or the lack of response to
ryanodine in these cells (Fig. 2).
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Fifty micromolar ryanodine had no effect in distal PASMCs that had been exposed to a zero-calcium bathing solution (n = 12). Because prolonged exposure to zero calcium may empty the intracellular stores, time was minimized between exchange of bath solution to zero calcium and application of ryanodine (<20 s; in all experiments, ryanodine was added directly to the bath to optimize drug delivery). To confirm that zero calcium did not empty the intracellular stores, cells were treated with ionomycin or caffeine at the end of the study period. Calcium increased to 1,202 ± 204 nM (n = 12) with ionomycin and 923 ± 196 nM (n = 10) with caffeine in the presence of zero extracellular calcium. Ryanodine (50 µM) also had no effect on intracellular calcium in fetal distal PASMCs in the presence of the voltage-dependent calcium channel blocker diltiazem (10 µM). Diltiazem did not inhibit the fetal distal PASMC response to caffeine {baseline [Ca2+]i 133 ± 7 nM; +diltiazem (10 µM) 143 ± 7 nM; +ryanodine (50 µM) 151+7 nM; +caffeine (2 mM) 900 ± 116 nM}.
To establish whether the KCa channel was involved in
the ryanodine response in fetal distal PASMCs, the cells were exposed to the KCa channel blocker iberiotoxin (100 nM)
before addition of ryanodine (50 µM). Iberiotoxin alone induced a
transient increase in calcium (783 ± 168 nM, n = 4). In some cells the iberiotoxin response appeared to have a
sustained component (see, e.g., Fig. 3); however, on average this was not
found to be statistically significant
([Ca2+]i 169 ± 83 nM;
n = 4). After addition of iberiotoxin, there was no
response to ryanodine (Fig. 3). In separate experiments (Fig. 4), cells were first exposed to
ryanodine, which produced a transient increase in calcium as described
above. Then, in the continued presence of ryanodine,
iberiotoxin had no effect.
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With the use of electrophysiology, STOCs were observed in fetal distal
PASMCs, and Fig. 5 is a representative
tracing of a current recording showing STOCs. STOCs were present in all
fetal distal PASMCs studied (n = 4 cells from 4 different animals) but not in any newborn (n = 4 cells)
or juvenile (n = 4 cells) distal PASMCs studied.
Calcium sparks were detected in 15 of the 76 fetal distal PASMCs
studied as measured using line scan confocal microscopy (an example is
shown in Fig. 6).
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DISCUSSION |
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These studies show that fetal distal PASMCs exhibit calcium sparks and STOCs and that application of ryanodine causes an increase in the [Ca2+]i. The increase in calcium was prevented either by removal of external calcium or by application of the KCa channel blocker iberiotoxin. Ryanodine caused an increase in cytosolic calcium in the fetal, but not in newborn or juvenile, animals, suggesting developmental regulation of the spark/STOC mechanism.
The present study provides evidence that the ryanodine-induced increase in cytosolic calcium in fetal PASMCs results from an entry of extracellular calcium as opposed to release of calcium from the sarcoplasmic reticulum. First, the concentration of ryanodine used in the present study has previously been shown to block vascular smooth muscle calcium sparks and STOCs (21). Similarly, in cardiac muscle, micromolar concentrations of ryanodine block release of calcium from the sarcoplasmic reticulum (5-7), whereas lower (nanomolar) concentrations of ryanodine may promote release from the sarcoplasmic reticulum by locking the ryanodine receptor in a subconductance state (32). However, this effect was not observed in channels isolated from vascular smooth muscle (15). Thus the concentration of ryanodine used in the present study argues against release of calcium from the SR as the source of the increase in cytosolic calcium. The observation that removal of extracellular calcium suppressed the ryanodine-induced calcium transient in fetal PASMCs provides further evidence that influx of extracellular calcium underlies the increase in fetal PASMC cytosolic calcium. Removal of external calcium did not prevent the ionomycin or caffeine response, indicating that the limited exposure to zero calcium did not deplete the intracellular stores. Therefore, the observed increase in intracellular calcium in response to ryanodine in the fetal cells is due to an influx of extracellular calcium. The increase in cytosolic calcium in response to ryanodine was also prevented by the voltage-dependent calcium channel blocker diltiazem (consistent with data from other smooth muscle cells in Refs. 21 and 31). It is, therefore, likely that calcium enters the fetal PASMCs via voltage-dependent calcium channels in response to ryanodine.
In other smooth muscle types, calcium sparks activate KCa channels causing STOCs. KCa channel (STOC) activation hyperpolarizes the cells, causing voltage-operated calcium channel closure and preventing calcium entry (for review see Refs. 4, 16, 17). The results presented in this study indicate that this spark/STOC mechanism is present and controls [Ca2+]i in isolated fetal pulmonary vascular smooth muscle cells. STOCs were observed in patch-clamp experiments with fetal distal PASMCs but not in cells isolated from newborn or juvenile animals.
The frequency of calcium sparks we observed in fetal rabbit pulmonary artery cells (~20% of cells having sparks) is comparable to that observed by Nelson et al. (21) in adult rat cerebral artery cells using the same line scan method. This method only scans ~1% of the cell volume; therefore, it is reasonable to assume that many sparks are missed and the actual frequency is much higher. In contrast, neonatal cerebral arteries exhibit an extremely low spark frequency, as Gollasch et al. (14) only observed four sparks in what they estimated to be >7,000 cells. These discrepant results suggest an interesting tissue-specific developmental difference between pulmonary and cerebral arteries. It is clear from these data that pulmonary arteries of the fetal rabbit are capable of producing calcium sparks, and these sparks may play a role in mediating perinatal pulmonary dilation.
Caffeine releases calcium from the intracellular stores (15) while ryanodine prevents release from the stores. The fact that all cell types responded to caffeine implies that they all have a functional sarcoplasmic reticulum that is loaded with calcium. Ryanodine blocks calcium sparks in smooth muscle (21). Only cells in which the resting [Ca2+]i is controlled by the hyperpolarizing influence of calcium sparks will respond to the removal of this influence by ryanodine, with an influx of extracellular calcium.
Iberiotoxin blocks KCa channels. In vascular smooth muscle, blockade of KCa channels with iberiotoxin produces an increase in calcium (and vasoconstriction) that is prevented by voltage-dependent calcium channel antagonists (18, 19, 21). In the present study, addition of iberiotoxin to fetal distal PASMCs produced a calcium transient. This confirms that the KCa channel is active under basal conditions, keeping the membrane potential and intracellular calcium low. Because charybdotoxin (another KCa channel blocker) causes a similar increase in basal cytosolic calcium in ovine fetal PASMCs (11), it is likely that this observation is not limited to the rabbit pulmonary vasculature.
The observation that the iberiotoxin transient is blocked by prior application of ryanodine is evidence that calcium release from intracellular stores plays a central role in regulating KCa channel activity. In pressurized systemic arteries, both ryanodine and iberiotoxin cause depolarization and vasoconstriction (21). In the presence of ryanodine, iberiotoxin has no effect on membrane potential or vessel diameter. The present study not only extends the observation that ryanodine prevents the action of iberiotoxin to fetal pulmonary artery smooth muscle but also shows that it can be seen in measurements of intracellular calcium in single isolated smooth muscle cells.
To further confirm the hypothesis that calcium sparks and KCa channels are working together to control intracellular calcium in these cells, it was demonstrated that the ryanodine transient was inhibited by prior addition of iberiotoxin. This indicates that functional KCa channels are required for the mechanism of action of ryanodine in fetal distal pulmonary vasculature. It is proposed [consistent with the previously proposed smooth muscle mechanism (17)] that, in the presence of KCa channel blockade, calcium sparks are unable to activate the KCa channel and cause membrane hyperpolarization. Blockade of the KCa channels effectively removes the influence of the calcium sparks; thus addition of ryanodine can have no further effect on membrane potential or intracellular calcium.
The transient nature of the response to ryanodine and iberiotoxin in these cells was surprising. The present study proposes that, in fetal distal PASMCs, ryanodine blocks production of calcium sparks, which otherwise cause KCa channel activation and STOCs. Ryanodine therefore removes a tonic hyperpolarizing stimulus. Removal of this hyperpolarizing signal would cause a depolarization of the membrane, opening of the voltage-dependent calcium channels, and calcium influx (16, 21, 31). Intuitively, removal of a tonic hyperpolarizing signal should result in a sustained increase in [Ca2+]i. The underlying reason for the short duration of the observed elevation in [Ca2+]i is unknown, although it may be a function of observing the response in a single cell and not in thousands of electrically coupled cells, as would be the case in an intact artery. It is possible that there is an additional compensatory mechanism that repolarizes the cell, prevents further influx of calcium, or increases removal or sequestration of calcium to compensate.
This study identifies a significant change in the intracellular calcium handling of PASMCs at birth. Specifically, cells isolated from fetal pulmonary arteries exhibit STOC activity and a transient increase in intracellular calcium in response to ryanodine, whereas those isolated from older (newborn and juvenile animals) did not. These observations are consistent with previous work in ovine tissue, which demonstrates a shift in the potassium channel controlling the PASMC membrane potential from a KCa channel in the late-gestation fetus to a voltage-gated potassium channel with postnatal maturation (24). Recent molecular data provide further support for the present findings by showing that Kv2.1 channel mRNA and protein expression increases with maturation (10). Thus the ryanodine response is present only at the developmental stage where the KCa channel plays an important role in the regulation of pulmonary vascular tone. The present study did not address whether cells from newborn or adult animals exhibited calcium sparks. However, it is not clear what role sparks could play in these cells in the absence of a contribution of the KCa channel to membrane potential and vascular tone.
In conclusion, this study demonstrates an interesting developmental change in calcium handling from fetal to newborn distal pulmonary artery cells. The role of a ryanodine-sensitive mechanism (probably involving calcium sparks and STOCs) in controlling intracellular calcium is much greater in the fetal distal pulmonary arteries than in more proximal tissue or in cells from newborn or older animals. The developmental change in the expression of this mechanism suggests a key adaptation of the fetal distal pulmonary arteries. The presence of calcium spark activation of KCa channels may prove to be critical in the initiation of the perinatal pulmonary vasodilation of the distal pulmonary arteries. These arteries are uniquely adapted to quickly respond to an increase in oxygen tension and NO. It is clear, however, that some alternate mechanism takes over to maintain this vasodilation. Older postnatal pulmonary arteries are adapted to respond rapidly to decreases in oxygen tension rather than to increases to match ventilation to perfusion. One could speculate that the loss of the calcium spark/STOC mechanism might, in part, explain this shift. The demonstration of the presence of the calcium spark/STOC mechanism in these fetal cells may represent a novel therapeutic target for the treatment of perinatal pulmonary vascular disease.
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ACKNOWLEDGEMENTS |
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This work was supported by American Heart Association Northland Affiliate Grants-in-Aid (to V. A. Porter and D. N. Cornfield) and by National Heart, Lung, and Blood Institute Grants RO1-HL-60784 (to D. N. Cornfield) and R29 HL-59182-01 (to H. L. Reeve).
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FOOTNOTES |
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V. A. Porter is a Parker B. Francis Fellow in Pulmonary Research.
Address for reprint requests and other correspondence: V. A. Porter, Div. of Pediatric Pulmonology and Critical Care, Univ. of Minnesota School of Medicine, 420 Delaware St. SE, Minneapolis, MN 55455 (E-mail: porte030{at}tc.umn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 February 2000; accepted in final form 4 May 2000.
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