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1 University Department of
Pharmacology, The pulmonary circulation changes rapidly at
birth to adapt to extrauterine life. The neonate is at high risk of
developing pulmonary hypertension, a common cause being perinatal
hypoxia. Smooth muscle K+ channels
have been implicated in hypoxic pulmonary vasoconstriction in adults
and O2-induced vasodilation in the
fetus, channel inhibition being thought to promote
Ca2+ influx and contraction. We
investigated the K+ currents and
membrane potentials of pulmonary artery myocytes during development, in
normal pigs and pigs exposed for 3 days to hypoxia, either from birth
or from 3 days after birth. The main finding is that cells were
depolarized at birth and hyperpolarized to the adult level of
newborn pig; pulmonary artery remodeling; porcine pulmonary artery; hypoxia
AT BIRTH there is a rapid fall in
pulmonary vascular resistance, accompanied by rapid structural
remodeling involving the entire pulmonary arterial tree, from hilum to
capillary bed (16). During this time the pulmonary vasculature appears
to be excessively reactive, and experimental studies showed that
structural remodeling is accompanied by changes in pharmacological
properties (20, 36). In several species endothelium-dependent and
endothelium-independent relaxation are less effective at birth and
mature rapidly during the first 2 wk of life (16). Before birth,
pulmonary blood is characteristically hypoxemic and raising fetal blood
O2 tension lowers pulmonary
vascular resistance due to pulmonary vasodilation (1). It was recently
shown that O2-induced pulmonary
vasodilation in fetal lambs could be inhibited by blockers of
K+ channels and that changing from
an hypoxic to a normoxic environment caused enhancement of the
macroscopic K+ current recorded
from fetal lamb pulmonary artery smooth muscle cells (11, 29). In adult
pulmonary artery smooth muscle cells, K+ channels determine the resting
membrane potential (12, 22, 33) and both acute (22, 25, 34) and chronic
(28) exposure to hypoxia cause
K+-channel inhibition and membrane
depolarization. Depolarization opens voltage-gated
Ca2+ channels, leading to
increased Ca2+ influx and
contraction.
We hypothesized that the hyperreactivity of the newborn pulmonary
vasculature might reflect a relatively low level of smooth muscle
K+-channel activity because of
fetal hypoxemia, which could give rise to excessive smooth muscle cell
depolarization. Postnatal adaptation and/or remodeling of the
pulmonary artery might then be regulated by subsequent changes in the
relative expression or activity of
K+-channel subtypes, triggered by
the increased pulmonary arterial O2 tension at birth. Failure of
the pulmonary vasculature to adapt to extrauterine life results in
persistent pulmonary hypertension of the newborn. This is a common
cause of morbidity and mortality and can be initiated by exposure to
hypoxia in the perinatal period (15). We therefore investigated
developmental changes in the electrophysiological properties of
pulmonary artery smooth muscle cells from the fetus through to adult
life and how these changes were influenced by exposure to chronic
hypobaric hypoxia (50.8 kPa) for 3 days during the neonatal period. The
pig was chosen as the experimental model because the structural and
functional maturation of its pulmonary vasculature, as well as its
alteration by chronic hypobaric hypoxia, is similar to that in humans
(17, 18).
Animals
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
40
mV within 3 days. Hypoxia prevented the hyperpolarization when present
from birth and reversed it when present from the third postnatal day.
The mechanism of hyperpolarization is unclear but may involve a
noninactivating, voltage-gated K+
channel. It is not caused by increased
Ca2+-activated or delayed
rectifier current. These currents were small at birth compared with
adults, declined further over the next 2 wk, and were suppressed by
exposure to hypoxia from birth. Hyperpolarization could contribute to
the fall in pulmonary vascular resistance at birth, whereas the low
K+-current density, by enhancing
membrane excitability, would contribute to the hyperreactivity of
neonatal vessels. Hypoxia may hinder pulmonary artery adaptation by
preventing hyperpolarization and suppressing
K+ current.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Lungs from adult pigs were obtained from a local abattoir immediately after death and transported to the laboratory on ice. Porcine fetal lungs were purchased from Selbourne Biological Services (Selbourne, UK).
Tissue Preparation and Cell Isolation
The heart and lungs were removed together and placed in chilled physiological salt solution (PSS) composed of (in mM) 119 NaCl, 4.7 KCl, 2.5 CaCl2, 25 NaHCO3, 0.026 Na2EDTA, 1.2 KH2PO4, 1.2 MgSO4, and 5.5 glucose, gassed with 95% O2-5% CO2, with pH adjusted to 7.4 with NaOH. In all cases, second-order branches off the main intrapulmonary arteries were dissected out and cleaned of connective tissue using a dissecting microscope. Vessels were always obtained from the same anatomic position within the lung, so that variation in K+-channel distribution along the length of the pulmonary arterial tree (3) would not distort the results. Vessels were cut into rings or strips <1 mm in length. Smooth muscle cells were isolated from each preparation as previously described (9), using a low-Ca2+ dissociation medium (DM) composed of (in mM) 110 NaCl, 5 KCl, 15 NaHCO3, 0.16 CaCl2, 2 MgCl2, 0.5 NaH2PO4, 0.5 KH2PO4, 10 glucose, 15 HEPES, 0.04 phenol red, 0.49 EDTA, and 10 taurine, equilibrated with 95% air-5% CO2 and adjusted to pH 7.0 with NaOH. For most experiments vessel strips were washed in DM and then placed in fresh DM containing 0.25 mg/ml papain (catalog no. 76218, Fluka Chemicals) and 0.02% bovine serum albumin (fraction V, fatty acid and globulin free; Sigma, Poole, UK) and stored overnight at ~6°C. The next morning, 0.2 mM dithiothreitol (Sigma) was added to the enzyme solution containing the tissue, which was then warmed to 37°C for 10 min. After the tissue was transferred to fresh, enzyme-free DM, single cells were then released by gentle trituration and stored in DM in a refrigerator until required for experiments. Examples of cells isolated from fetal, newborn, and 3-day-old normoxic piglets are shown in Fig. 1.
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Because after removal from the animals, vessels were maintained in
normoxic solutions and the cell isolation method involved an overnight
incubation, we were concerned that properties of cells from fetal,
newborn, or hypoxic animals might change before they were recorded. We
therefore examined cells from newborn and fetal animals that were
isolated shortly after the vessels were removed, by incubating the
tissue at 37°C for 30-60 min in DM containing 0.25 mg/ml
papain, 0.02% bovine serum albumin, and 0.2 mM dithiothreitol,
followed by trituration. This method was not used routinely, because
the yield of viable cells was less reproducible. Nevertheless, the
cells had properties comparable to those isolated with the overnight
method. Importantly, the K+
current activated by voltage steps to 40 mV (16 ± 2 pA/pF at 40 mV;
n = 3 cells) was similar to that in
cells obtained by overnight digestion, and the cells were similarly
depolarized (see RESULTS); two cells
had resting potentials of 0 and 5 mV, values observed in newborn
cells obtained by overnight digestion but never in cells from older
animals. In addition, contractile responses of vessel rings to
K+ and pharmacological agents were
unchanged by overnight incubation in the refrigerator. Thus the cell
isolation method did not appear to compromise the results.
Electrophysiology
Cells were transferred to a 400-µl experimental chamber mounted on the stage of an inverted microscope, maintained at room temperature (22-25°C), and superfused at ~0.5 ml/min with PSS composed of (in mM) 124 NaCl, 5 KCl, 15 NaHCO3, 1.8 CaCl2, 1 MgCl2, 0.5 NaH2PO4, 0.5 KH2PO4, 10 glucose, and 15 HEPES, gassed with 95% O2-5% CO2 and adjusted to pH 7.3 with NaOH. The whole cell configuration of the patch-clamp technique was used to measure the resting membrane potential under current clamp and macroscopic K+ currents under voltage clamp. An Axopatch 1A or 200A patch-clamp amplifier (Axon Instruments, Foster City, CA) was used. Patch pipettes (1-2 M
) were pulled from filamented borosilicate glass capillaries (Clark Electromedical Instruments, Pangbourne, UK) and
filled with recording solution composed of (in mM) 130 KCl, 1 MgCl2, 1 EGTA, 20 HEPES, and 0.5 Na2GTP, pH adjusted to 7.2 with KOH. The junction
potential between the pipette and bath solution (2-4 mV) was
canceled before pipette-cell contact. Reported voltages were not
corrected for junction potential errors arising on formation of the
whole cell configuration. The input resistance was measured under
voltage clamp from the step change in current induced by a 10-mV
hyperpolarizing step applied from 80 mV. Cell capacitance and
series resistance were estimated from the capacity transient at the
leading edge of the current response to the same voltage step. Series
resistance averaged 10.8 ± 0.4 M (n = 104 cells) and
was routinely compensated by 80-90%. Because
K+-current amplitudes rarely exceeded 500 pA, voltage
errors caused by series resistance would have been no more than
1-2 mV in the worst case. Voltage commands were generated with
pCLAMP data acquisition software (versions 5.5 and 5.7; Axon
Instruments), through a Labmaster TM-40 (Scientific Solutions) or
Digidata 1200 (Axon Instruments) interface. Currents were filtered at
0.5-5 kHz, digitized on-line at 1-16 kHz, and stored on disk
using pCLAMP (versions 5-6). Data were analyzed using pCLAMP and
Origin (Microcal, Northampton, MA) software. Current records were not
leak subtracted unless stated.
Statistical Analysis
Data are given as means ± SE, and error bars in Figs. 2, 5, 6, and 7 represent SE. Trends across multiple groups of cells from different developmental stages were tested by one-way ANOVA, with Bonferroni's correction applied for post hoc comparisons between pairs of groups within the series. Cells from hypoxic animals were compared with the relevant normoxic controls using a two-tailed, unpaired t-test. Statistical significance was assumed if P < 0.05.Drugs and Chemicals
Stock solutions of tetraethylammonium chloride (TEA, 1 M; Fluka), quinine sulfate (1 mM; Merck) and 4-aminopyridine (4-AP, 10 mM; Sigma) were prepared daily in PSS, the pH of the 4-AP solution being adjusted to 7.3 before dilution. Glibenclamide (10 mM; Sigma) was dissolved in DMSO. DMSO was present at 0.1% after dilution, which by itself had no effect on the K+ currents recorded. Fresh experimental solutions were prepared daily by dilution in PSS. Drugs were applied either to the PSS superfusing the bath or by microsuperfusion from a nearby flow pipe.| |
RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Passive Membrane Properties
The cell capacitance and input resistance were calculated for voltage-clamped cells from the current response to a 10-mV hyperpolarizing step applied from a holding potential of80 mV,
at which most voltage-gated channels are closed. Capacitance is
directly related to membrane surface area, whereas the input resistance
measured at these potentials is indicative of the intrinsic membrane or "leak" conductance. As shown in Fig.
2A, the
input resistance varied on average between 5 and 10 G but there were
no significant differences among any of the groups of cells studied.
There was some variation in cell capacitance, as shown in Fig.
2B. In cells from animals allowed to
develop in a normoxic environment, capacitance was almost twofold
higher at day 14 compared with any
other age group (P < 0.001), although there were no significant differences among the other
control groups, which had mean capacitances of ~10-15 pF. The
capacitance of cells isolated from animals exposed to an hypoxic
environment from birth until the third day of life was higher than that
of the normoxic controls (P < 0.01).
In contrast, if the animals were initially maintained in a normoxic
environment after birth, followed by exposure to hypoxia between
days 3 and 6, cell capacitance was not affected.
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The resting membrane potential was measured under current clamp as the
zero-current potential. In most cells, the resting potential was stable
over several minutes of recording, but occasionally a progressive
hyperpolarization was observed after the whole cell configuration was
formed. This may have reflected the gradual appearance of a background
ATP-sensitive K+ current (see
K+ Currents), but because
hyperpolarization was infrequently observed it was not investigated
further. Resting potentials of cells from different age
groups were compared using measurements made shortly after establishing
the whole cell configuration. The mean resting potentials lay in the
range from 17 to 50 mV. As shown in Fig. 2C, smooth muscle cells isolated from
fetal and newborn animals were significantly depolarized in comparison
with cells from older animals. By day
3 the resting potential had reached the adult level of
39 ± 4 mV (n = 13 cells),
because there were no significant differences among cells from 3-day,
6-day, 10-day, 14-day, or adult animals. Cells isolated from animals
exposed to an hypoxic environment were also significantly depolarized
in comparison with their age-matched controls. This was true whether
hypoxia was present during the first 3 days of life
(P < 0.001) or during days 3-6
(P < 0.05). The resting potentials
of cells isolated from animals exposed to hypoxia were not
significantly different from the newborn or fetal cells.
K+ Currents
Glibenclamide-sensitive current.
At least three types of K+ current
could be identified in porcine pulmonary artery smooth muscle cells
after several minutes of dialysis with an ATP-free pipette solution.
Figure 3A
shows voltage-clamp records of the compound current activated in an adult cell by steps to various potentials, applied from a holding potential of 80 mV. Voltage steps induced an instantaneous
change in current followed by a time-dependent current, which increased to a plateau by the end of the 100-ms step and became more pronounced with increasing depolarization. The extracellular application of 10 µM glibenclamide abolished the instantaneous component, with little
effect on the time-dependent current. This is illustrated in Fig.
3A, which shows a series of currents
recorded before and after glibenclamide was added as well as the
glibenclamide-sensitive component obtained by digitally subtracting
records in the presence of glibenclamide from those in its absence. The
glibenclamide-sensitive current appeared to be time independent and
linearly dependent on the test potential between 90 and
20 mV but exhibited inward rectification at more positive
potentials, as illustrated in Fig. 3B.
As expected for a K+ current, the
reversal potential was close to 82 mV (Fig.
3B), the equilibrium potential for
K+ in the conditions used. The
glibenclamide-sensitive current developed progressively after dialysis
with ATP-free pipette solution, suggesting that it resulted from ATP
washout. The properties of this current suggest that it was carried by
ATP-sensitive K+
(KATP) channels and that its
gradual activation was responsible for the progressive
hyperpolarization described in Passive Membrane Properties. We did not systematically
investigate this current in different age groups because it was
infrequently observed. It was, however, pronounced in several adult
cells but generally difficult to resolve in cells from younger animals.
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Voltage-activated
K+ current.
The glibenclamide-resistant current was sensitive to inhibition by a
number of pharmacological agents known to block
K+ channels. Figure
4A shows
the influence of 10 mM TEA, 1 mM 4-AP, and 10 µM quinine on outward
currents recorded from a 6-day cell superfused continuously with 10 µM glibenclamide. The currents were activated by a series of voltage
steps applied from 80 mV to more positive potentials. These
drugs were tested because they previously proved helpful in
distinguishing between different components of voltage-activated
K+ current in pulmonary artery
smooth muscle cells of other species (9, 12, 22, 28, 33). Thus
millimolar TEA predominantly inhibits large-conductance
Ca2+-activated
K+
(BKCa) channels, which
frequently give rise to a noisy component of current at positive
potentials (5, 9). As seen in Figs. 3 and 4 the current at
positive potentials often appeared noisy, and in the presence of TEA
the noise was reduced. In contrast, 4-AP primarily inhibits
voltage-gated delayed rectifier
(KV) channels. Quinine is a nonselective drug but at 10 µM blocks delayed rectifier current in rabbit pulmonary artery myocytes while having little effect
on a noninactivating, voltage-gated
K+ current that was shown to be
important in maintaining the resting membrane potential (12, 22). At
the concentrations used, all three drugs caused pronounced inhibition
of the compound current activated by depolarizing steps over a wide
range of test potentials (Fig. 4B),
implying that the current was predominantly carried through
K+ channels. In agreement with
this, there was essentially no outward current at positive potentials
when the K+ in the pipette
solution was replaced with Cs+
(n = 6; newborn, 3 day, and 6 day).
There may have been some overlap in the types of channel inhibited by
the three drugs. This is suggested by Fig.
4C, which plots the amplitude of the current sensitive to block by each drug as a function of the test potential. The drug-sensitive current was estimated by subtracting the
current amplitude in the presence of each drug from that in the absence
of drug. The current versus voltage relationship was similar for all
three drug-sensitive currents, although there appeared to be less 4-AP
block at the most positive potentials.
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80 to 40 mV for each cell studied, by normalizing the current amplitude reached at the end of the step against cell capacitance. Figure 6A compares
the mean outward current density for cells from all groups of animals.
Current density was similar in fetal, newborn, 3-day-old, and 6-day-old
animals, at ~20 pA/pF, but between days 6 and
14 after birth there was a significant (P < 0.01) decline to only 7 ± 2 pA/pF (n = 9 cells).
Interestingly, current density increased again in the adult cells
(P < 0.01), to a value larger than observed in any of the
immature groups. Exposing animals to an hypoxic environment for the
first 3 days of life resulted in a 50% reduction of
K+-current density compared with animals
allowed to develop normally for 3 days
(P < 0.05). In contrast, if animals
were allowed to develop normally for 3 days, followed by 3 days in an
hypoxic environment before being tested,
K+-current density was not
significantly different from that in the age-matched controls.
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(1) |
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80 mV) and
plotted as a function of the prepulse potential, as shown in Fig.
6C for cells from newborn, 3-day-old normoxic, and 3-day-old hypoxic animals. To compare the voltage dependence of inactivation in all groups, inactivation curves were fit
by a Boltzmann function of the form
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(2) |
50 and
80 mV during a subsequent voltage ramp to negative potentials.
Figure 7A
shows examples of current records obtained in response to a voltage
ramp to 100 mV, applied after clamping the cells at 0 mV for 5 min. In some records, such as that shown for a newborn cell,
IKN was clearly
absent; no current remained after 5 min at 0 mV, and there was a linear
relationship between current and voltage during the subsequent ramp. In
others, such as that shown for a 6-day cell,
IKN was present;
a measurable current remained after 5 min at 0 mV, and it rectified
outwardly during the subsequent voltage ramp, indicative of
K+-channel closure (12). With the
use of this protocol, evidence for the presence of
IKN was found in
cells from all age groups, but in each group it varied widely in
amplitude, sometimes being very small or absent with rectification not
clearly distinguishable. It therefore proved difficult to quantify and
compare this current in different age groups.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The major finding of this study is that pig pulmonary artery smooth
muscle cells are depolarized at birth and hyperpolarize to the adult
level of approximately 40 mV within 3 days after birth.
Hyperpolarization was prevented by exposing animals to an hypoxic
environment for the first 3 days of life and was reversed by exposure
to hypoxia later during the perinatal period. Variations in resting
potential among the cell groups were not caused by differences in leak
current, because they were not accompanied by differences in the input
resistance, measured at potentials at which most ion channels are
closed. In pulmonary artery myocytes from other species, the resting
potential is largely determined by a
K+ conductance in parallel with a
leakage conductance, with general agreement that the
K+ channel involved is voltage
gated (3, 12, 22, 24, 33). K+
current activated by 100-ms depolarizing steps, which reflected KV and
BKCa channels, was found to vary
during development and to be influenced by chronic hypoxia but not in a
way that can fully explain the observed pattern of resting potentials.
The porcine cells contained a noninactivating component of
voltage-gated K+ current, similar
to IKN found
previously to regulate resting potential in rabbit pulmonary artery
smooth muscle. Our results suggest that variation in this current may
contribute to postnatal hyperpolarization, although the evidence is not
clear-cut and other mechanisms cannot be ruled out. Variations in
resting potential could alternatively reflect changes in inward
current, through Ca2+ or
Cl
channels, for example,
or in the balance between outward and inward currents.
K+ Currents in Developing Porcine Pulmonary Arteries
Two distinct outward K+ currents were activated by depolarizing steps. The time-independent component was identified as KATP current because of block by glibenclamide (10, 26). The time-dependent component was carried by KV and BKCa channels, on the basis of its sensitivity to TEA, 4-AP, and quinine and its similarity to the time-dependent currents recorded from pulmonary artery myocytes of other species (12, 13, 24, 28, 33). Developmental changes in KATP current were unlikely to be responsible for the postnatal hyperpolarization, because it contributes little to the resting potential of pulmonary vascular muscle from other species (10, 12, 22, 33) and became visible only after dialysis from ATP-free pipette solution. Moreover, in the same neonatal pig model, the KATP channel opener levcromakalim relaxed all pulmonary arteries with no change in responsiveness over the first week of life or after chronic hypoxia (7).The density of total outward current activated by brief depolarizing
steps decreased between birth and day
14 but then increased dramatically in the adult. In
addition, hypoxia significantly reduced current density when presented
from birth. These variations may reflect changes in channel expression
or open probability; chronic hypoxia inhibited the expression of
K+-channel 
-subunits in
cultured rat pulmonary artery myocytes (32). However, the low current
density at day 14 is at least partly
explained by a large membrane capacitance. Variations in current
density were not associated with shifts in the voltage dependence of
current activation or inactivation or in the sensitivity to
K+-channel blockers. This suggests
general variation in K+-channel
activity rather than in specific channel types. The activation threshold lay close to the normal resting potential of 40 mV, whereas maximal inactivation required more positive potentials. In the
steady state, a sustained outward "window" current could therefore persist near, and influence, the resting potential. Such a
window current would be reduced in cells from hypoxic 3-day animals,
and this could explain their failure to hyperpolarize normally. On the
other hand, the postnatal decline in current density is predicted to
cause depolarization, not hyperpolarization as observed, implying that
the K+ current activated by brief
voltage steps was not a major contributor to resting potential.
Although the voltage dependence of
K+-current inactivation remained
constant during development, the proportion of current that inactivated
in the steady state did not. Prepulses to 30 mV or more positive
potentials caused complete inactivation in newborn cells but not in
cells from older animals. Moreover, hypoxia from birth, or from 3 days
after birth, significantly reduced the proportion of noninactivating
current compared with age-matched controls. These variations in
noninactivating current correlate with resting potential, implying a
contribution to postnatal hyperpolarization and its inhibition by
hypoxia. The noninactivating current appeared similar to
IKN in rabbit
pulmonary artery myocytes, which activates slowly (~1.5 s) and
contributes little to the K+
current activated during 100-ms depolarization (12, 22). Like
IKN, the
noninactivating current in porcine cells persisted after 5 min at 0 mV,
although with a small and highly variable amplitude, even among cells
from a single age group, making it difficult to analyze. Nevertheless,
IKN appeared to
be present in most cells from 3-day-old or older animals but in few
newborn cells, although differences in
IKN amplitude
between these age groups, or between hypoxic and control groups, failed
to reach significance. The changes in resting potential observed after birth or exposure to hypoxia would, however, require only small changes
in membrane current (~2 pA), which would be difficult to detect.
Although we cannot conclude with certainty that changes in
IKN were
responsible for the depolarized potentials at birth, the postnatal
hyperpolarization, and the prevention and/or reversal of
hyperpolarization by hypoxia, we propose that they were a contributing factor. More selective approaches are needed to determine how particular K+ channels vary during
development. Unfortunately, little is known about the molecular
properties of the channel subunits encoding K+ currents in pulmonary artery
smooth muscle, although a recently cloned Kv9.3 subunit was proposed to
underlie IKN
(23). It should be possible to assess the developmental regulation of
specific channel types in the future, when knowledge of their molecular characteristics improves.
Cell Phenotypes
Our results could have been influenced by variation in the predominant cell type present in the vessel wall at different times during development. Smooth muscle cells in pulmonary arteries are phenotypically heterogeneous and change rapidly in shape and cytoskeletal composition during the postnatal period (14). However, cells were always isolated from the same place in the vessel wall, and there was as much variability in morphology between preparations from the same age group as between different age groups, presumably associated with the dissociation technique itself. We aimed to record from spindle-shaped cells, as indicated in Fig. 1. However, when we deliberately recorded from more rounded cells, we found no obvious differences in resting potential or K+-current density, suggesting that the cell types studied from each age group were comparable. Consistent with this, cell capacitance, and hence membrane surface area, was similar in most age groups. The striking increase in capacitance at 14 days of age compared with any other time suggests an increased cell size. This is in accord with morphological studies showing a larger mean diameter in cells freshly isolated from the same arteries at 17 days compared with 6 days (P < 0.001; S. Hall, personal communication). If a specific membrane capacitance of 1 µF · cm2
is assumed, the 30 pF found at 14 days predicts a membrane surface area
of 3,000 µm2, twice as large as
at any other time.
Implications for Adaptation of Pulmonary Circulation in Neonates
Changes in smooth muscle membrane potential have pronounced effects on pulmonary vascular tone. Depolarization causes contraction (8), mainly because of increased Ca2+ influx through voltage-gated Ca2+ channels but also because of enhanced D-myo-inositol 1,4,5-trisphosphate synthesis (6, 19) and intracellular Ca2+ release. Depolarization may therefore contribute to the high pulmonary arterial resistance found in the fetus and newborn. The finding that 3 days after birth the resting potential had already reached the adult level suggests that changes in membrane potential are an important feature of adaptation to extrauterine life; they occurred simultaneously with rapid pulmonary artery dilation (18). That cells from newborn piglets exposed to chronic hypoxia remained or became depolarized, as they were in utero, further suggests a role for membrane potential, because pulmonary arteries from the same animals failed to dilate normally and endothelium-dependent and endothelium-independent relaxations were impaired (30). In clinical practice, perinatal hypoxia is a common cause of persistent pulmonary hypertension. The pulmonary arteries fail to dilate normally, and morbidity and mortality are high.Smooth muscle depolarization may help to explain the lack of endothelium-dependent vasodilation seen normally at birth (20), caused in part by a lack of smooth muscle responsiveness to cytoplasmic cGMP (30). cGMP-dependent vasodilators are less effective in a number of conditions promoting smooth muscle depolarization, apparently because of interference at a step after cGMP formation (27). Because the vasodilator action of cGMP in pulmonary arteries has been proposed to involve K+-channel activation (3, 35), the low K+-current density observed in neonatal cells may also contribute to their lack of cGMP responsiveness. On the other hand, responses to endothelium-dependent vasodilators and NO donors increased between birth and 10 days of age (20, 36), whereas K+-current density did not.
The low density of K+ current in smooth muscle cells from animals up to 2 wk old might enhance vessel reactivity, because the cells would be less able to oppose the depolarizing action of vasoconstrictor agonists. Consistent with this hypothesis, K+-channel blockers enhanced pulmonary vasoconstriction in dogs in response to endothelin (ET)-1 (4). This peptide may be involved in postnatal adaptation, because plasma ET levels and smooth muscle ETA-receptor density are both high at birth and exposure to hypoxia from birth prevents the normal reduction in plasma ET during the postnatal period and increases ETA-receptor density (21). Sympathetic vasoconstrictor nerves, the main nerves innervating human and porcine pulmonary arteries at birth, increase in density with age (31) and may also play a role during development. Moreover, the walls of distal pulmonary arteries are prematurely innervated in babies with pulmonary hypertension (2). Thus the low K+-current density at birth and its subsequent decline during the postnatal period, combined with the changing resting potential, could have an important influence on contractility and responsiveness to vasoactive agents, thereby contributing to the susceptibility of infants to pulmonary hypertension.
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ACKNOWLEDGEMENTS |
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The authors thank D. C. Ellershaw and members of S. G. Haworth's laboratory for help in preparing tissues and cells.
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FOOTNOTES |
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The authors are grateful to the British Heart Foundation for supporting this work. A. M. Evans is a Wellcome Trust Career Development Fellow.
Address for reprint requests: A. M. Gurney, Dept. of Physiology and Pharmacology, Univ. of Strathclyde, Royal College, 204 George St., Glasgow G1 1XW, Scotland, G1 1XW.
Received 8 December 1997; accepted in final form 21 May 1998.
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REFERENCES |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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