Division of Pediatric Pulmonology and Critical Care Medicine, Departments of 1Pediatrics, 2Surgery, and 3Physiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
Submitted 1 April 2003 ; accepted in final form 24 June 2003
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ABSTRACT |
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fetus; cytosolic calcium; potassium channel
Recent studies (9) from our laboratory have demonstrated that fetal PA SMC respond directly to changes in PO2. Fetal PA SMC respond to acute hypoxia with an increase in [Ca2+]i. In response to an acute increase in PO2 (acute normoxia), fetal PA SMC [Ca2+]i decreases (26). O2 causes a decrease in PA SMC [Ca2+]i via activation of a cGMP-sensitive kinase that mediates localized Ca2+ release from a ryanodine-sensitive intracellular Ca2+ store (24). Localized Ca2+ release results in activation of the Ca2+-sensitive K+ (KCa) channel and PA SMC membrane hyperpolarization, which leads to vasodilation (21).
Interestingly, the response of PA SMC to changes in PO2 is developmentally regulated. Fetal, but not adult, PA SMC respond to an acute increase in PO2 with a decrease in [Ca2+]i (23, 26). In contrast, adult PA SMC respond to acute hypoxia with a more rapid and greater increase in [Ca2+]i than fetal PA SMC (8). The differential response to PO2 between fetal and adult PA SMC may derive, in part, from changes in the predominant ion channel population that controls resting membrane potential (Em). In the fetus, the KCa channel determines Em, and, therefore, through subsequent effects on voltage-gated Ca2+ channels, basal levels of PA SMC [Ca2+]i. In contrast, the Em and basal [Ca2+]i of PA SMC from the adult pulmonary circulation is mediated by voltage-sensitive K+ channels (Kv) (25). Thus fetal PA SMC are uniquely well adapted to respond to an acute increase in PO2 and thereby enhance postnatal adaptation of the pulmonary circulation, whereas adult PA SMC are adapted to respond to an acute decrease in PO2 and thereby match ventilation and perfusion to prevent intrapulmonary shunting and subsequent hypoxemia (36).
In some newborn infants, pulmonary vascular resistance remains elevated after birth, resulting in a clinical syndrome termed persistent pulmonary hypertension of the newborn (PPHN). PPHN is characterized by extrapulmonary right-to-left shunting of blood across the ductus arteriosus or patent foramen ovale causing severe hypoxemia (16). Infants with PPHN respond incompletely to perinatal pulmonary vasodilator stimuli. Evidence suggests that adverse intrauterine stimuli, such as chronic hypoxia or hypertension (19), can decrease endothelial nitric oxide (NO) synthase (eNOS) gene and protein expression, NOS activity, and limit perinatal NO production, thereby contributing to the pathophysiology of PPHN (2, 30, 35). A recent study (7) from an animal model of PPHN demonstrated a decrease in KCa channel gene expression in whole lung tissue of fetal lambs with chronic intrauterine pulmonary hypertension. It remains unknown whether 1) the decrease in fetal lung KCa channel gene expression is sufficient to compromise fetal pulmonary vascular oxygen sensing; and 2) chronic intrauterine pulmonary hypertension directly affects PA SMC oxygen sensing.
Because PA SMC KCa channel activity determines resting membrane potential (25) and mediates the response of fetal PA SMC to an acute increase in PO2 (24), as well as NO (29), we hypothesized that chronic intrauterine pulmonary hypertension has direct effects on PA SMC O2 sensing. To test this hypothesis, we used fluorescence microscopy to study the effect of an acute increase in PO2 on [Ca2+]i of PA SMC isolated from fetal lambs with chronic intrauterine pulmonary hypertension.
To determine any changes in K+ channel activity due to chronic intrauterine pulmonary hypertension, the effect of K+ channel antagonists (5) was studied. PA SMC isolated from normotensive and hypertensive fetal ovine PAs were treated with iberiotoxin, a selective KCa channel antagonist, and 4-aminopyridine (4-AP), a Kv antagonist on PA SMC [Ca2+]i. The effect of chronic intrauterine hypertension on intracellular calcium homeostasis was addressed by treating PHTN and normotensive PA SMC with thapsigargin, an inhibitor of sarcoplasmic reticulum (SR) ATPase, and ryanodine, a stimulant of Ca2+ release from ryanodine-sensitive intracellular Ca2+ stores (17). KCa channel mRNA levels were determined with the use of quantitative internally controlled RT-PCR.
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METHODS |
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Solutions. Recording solutions consisted of (in mM) 10 HEPES, 10 glucose, 135 NaCl, 5.6 KCl, 1.8 CaCl2, and 1.2 MgCl2. All solutions were made with the use of nanopure distilled water. Osmolality was adjusted to 300 mosM, and pH adjusted to 7.4.
RT-PCR. Arterial tissue was removed from the lung. Tissue was taken from pulmonary arteries that were greater than or equal to fourth generation. Pulmonary arterial tissue was suspended in liquid N2 and ground to powder with a prechilled mortar and pestle. Total RNA was extracted with the use of the guanidium thiocyanate-phenol-chloroform method (Trireagent; Sigma). After homogenization, the samples were processed according to the reagent instructions and the RNA was dissolved in diethyl pyrocarbonate-treated water and stored at -70°C. Optical density was measured to determine the RNA concentration. One microgram of RNA was added to 11 µl of First Strand cDNA Synthesis reagent (Pharmacia) with random hexamers as primers in a final volume of 33 µl. Two microliters of this RT reaction were added to each PCR reaction. Oligonucleotide primers used to amplify Kv 2.1 cDNA were based on the human sequence (3) and were (forward) 5' ACAGAGCAAACCAAAGGAAGAAC 3' and (reverse) 5' CACCCTCCATGAAGTTGACTTTA 3'. The use of these primers yielded a PCR product consistent with that expected for a fragment of the human Kv 2.1 mRNA. The fragment size was 385 base pairs. The identity of the product was confirmed with sequence analysis. Oligonucleotide primers used to amplify KCa cDNA were based on the human sequence (34) and were (forward) 5' CTACTGGGATGTTTCACTGGTGT 3' and (reverse) 5' TGCTGTCATCAAACTGCATA 3'. The use of these primers yielded a PCR product consistent with that expected for a fragment of the human KCa mRNA. The fragment size was 446 base pairs. Identity of the product was confirmed with sequence analysis.
18S rRNA was analyzed concurrently in RT-PCR as an internal control. 18S cDNA was amplified with a Quantum-RNA primer/competimer set (Ambion) to act as an internal control for the quantitation of relative expression of the ethidium bromide-stained bands. This control band appears as 324 base pairs. Because 18S rRNA is far more abundant than the mRNA under study, the 18S amplification reaction was modulated by the addition of "competimers." These competimer primers are modified to block extension by DNA polymerase. When combined with the functional primers for 18S cDNA the amplification efficiency is reduced. Pilot experiments determined the correct ratio of primers/competimers, cycle number, and RT input to yield multiplex PCR products that are all in the linear range of amplification. The PCR cocktail consisted of 1x PCR buffer (Perkin Elmer) with 1.5 mM Mg2+, 10 pM each Kv 2.1 primer, 10 nM 2-deoxynucleotide 5'-trisphosphate mixture, 20 pM of 18S primer mixture (ratio of 1:9) of 18S primers/competimers, 1 U AmpliTaq polymerase and water to make 50 µl. PCR was performed in an MJ Research thermocycler with a heated lid and 0.2-ml thin-walled tubes. The PCR reaction was 2 min at 90°C, followed by 28-32 cycles of 1 min at 94°C, 1 min at 54°, 2 min at 72°, then an extension of 2 min at 65°. Samples without RT were evaluated in PCR; the products were absent. The identity of the band was confirmed by sequencing the product (>91% homology with known sequences). Densitometry was used to quantify the RT-PCR product (NIH Image software; Scion, Frederick, MD) and the internal control 18S rRNA to correct for variability in lane loading. Each gel contained PCR product from both hypertensive and control animals. The relative density of the 18S ribosomal and potassium channel PCR products were compared in each individual gel. PCR was run two times on each RNA sample.
Western blot analysis for KCa protein. Cultured smooth muscle cell monolayers were rinsed with cold PBS and scraped from their 25-mm coverslips in RIPA buffer with protease inhibitor cocktail and 0.1% Triton X-100. The homogenate was sonicated briefly (2 s) on ice and then centrifuged at 1,000 g for 5 min. The supernatant protein concentration was determined with the bicinchoninic acid protein assay (Pierce). Protein (75 µg) was combined with SDS-PAGE reducing sample buffer and electrophoresed in a 4-20% gradient gel. The proteins were electroblotted onto polyvinylidene difluoride membrane (Bio-Rad). Skim milk (6%) in 20 mM Tris-buffered saline (TBS) was used for blocking and washing the membranes. Antibody against KCa channel (Alomone) was diluted 1:200 in milk-TBS. In blocking experiments the antibody was incubated 1 h as recommended with specific antigen provided with the antibody and then diluted 1:200. The membranes were rotated in the solutions overnight at 4°C and washed. Second antibody was anti-rabbit IgG horseradish peroxidase conjugate (Jackson), 1:3,000 in milk-TBS with 0.01% Tween 20, rocked for 2 h at room temperature. After being washed, the membranes were incubated 10 min at room temperature with Super Signal West Pico chemiluminescent reagent (Pierce) and the membrane was exposed to Kodak X-omat fs-1 film. The KCa channel band was seen at 125 kDa, and antibody binding to these bands was blocked by antigen preincubation.
Animals. The procedures used in these studies were previously reviewed and approved by the Animal Care and Use Committee at the University of Minnesota Medical School. Fetal sheep were used in this study. At the time of tissue procurement, fetal ages ranged from 135 to 140 days gestation (term is 147 days). Ewes with time-dated pregnancies were fasted for 24 h and were sedated with pentobarbital sodium (10 g total dose). Fetal lambs were rapidly delivered through a hysterotomy, and an injection of pentobarbital sodium was given in the umbilical artery to prevent spontaneous breathing. After thoracotomy, the lungs were isolated, and samples were preserved for RNA analysis by freeze clamping and storage at -70°C.
Experimental model of chronic intrauterine pulmonary hypertension. Surgical ligation of the ductus arteriosus was performed as previously described (20, 28). Mixed-breed (Columbia-Rambouillet) pregnant ewes between 126 and 128 days gestation (term = 147 days) were fasted for 24 h before surgery. Ewes were sedated with intravenous pentobarbital sodium (total dose: 2-4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. The ewes were kept sedated but breathed spontaneously throughout the operation. Under sterile conditions, the gravid uterus was delivered through a midline laparotomy. The fetal lamb's left forelimb was withdrawn through a small hysterotomy. A skin incision was made under the left forelimb after local infiltration with lidocaine (2-3 ml, 1% solution). A left thoracotomy exposed the heart and great vessels. The ductus arteriosus was visualized. A 2-0 silk suture was placed around the ductus arteriosus and tied. The ribs and skin were reapproximated. The hysterotomy was closed, and the uterus was returned into the maternal abdominal cavity. The ewes recovered rapidly from surgery and were generally standing in their pens within 6 h. Food and water were provided ad libitum. After 7-12 days, animals were euthanized rapidly after high-dose maternal and fetal infusions of pentobarbital sodium, and the lung tissue was harvested as described above.
Drugs used. Thapsigargin and 4-AP were obtained from Sigma. Fura 2-AM was obtained from Molecular Probes (Eugene, OR) and diluted in DMSO. Iberiotoxin and ryanodine were obtained from Alomone Laboratories (Jerusalem, Israel). Ionomycin and diltiazem were obtained from Calbiochem (La Jolla, CA). All drug solutions were adjusted to pH 7.4 before use and solubilized in normal saline.
Ca2+ imaging. To assess dynamic changes in [Ca2+]i in individual PA SMC, we used the Ca2+-sensitive fluorophore fura 2-AM (Molecular Probes). Subconfluent fetal PA SMC on 25-mm2 glass coverslips were placed on the stage of an inverted microscope (Nikon Diaphot). Cells were loaded with 100 nM fura 2-AM plus 2.5 mg/ml Pluronic acid (Molecular Probes) for 20 min in Ca2+-free solution, followed by a 20-min wash in calcium-containing solution before the start of the experiment. Ratiometric imaging was performed with the use of excitation wavelengths of 340 and 380 nm and an emission wavelength 560 nm. Imaging was performed with an ICCD camera (Photonic Science; Robertsbridge, UK) using Axon Instruments (Foster City, CA) image capture and analysis software. Ca2+ calibration was achieved by measuring a maximum (with ionomycin 1 mM) and a minimum (with EGTA 10 mM) for each cell. PO2 was controlled by aerating the recording solution reservoir with either 21% O2 with balance N2 or 100% N2. PO2 was monitored throughout with the use of an O2 electrode (Microelectrodes; Bedford, NH). Further control of PO2 was obtained by aerating the stage microincubator with 21% O2 with balanced N2 or 100% N2. pH was 7.40 ± 0.05 and did not change during the experiments. Intracellular free Ca2+ was calculated assuming a dissociation constant of 220 (31). For each experiment, 5-12 cells were visualized and cytosolic Ca2+ measurements were made from individual cells.
Measurement of [Ca2+]i. [Ca2+]i measurements were made after stable baseline Ca2+ values were obtained in either hypoxic (PO2 25 mmHg) or normoxic (21% O2; PO2
120 mmHg) recording solution. The recording solution was superfused over the cells at a rate of
2 ml/min in all experiments. Measurements of [Ca2+]i were made for at least 10 min after a change in conditions or addition of a drug. KCl (60 meq, 60 K) was superfused over the cells at 2 ml/min at the conclusion of the experiment to ensure cellular viability.
Basal [Ca2+]i and the effect of an acute increase in PO2 on [Ca2+]i in chronically hypoxic PHTN and control fetal PA SMC. After stable baseline values in hypoxic recording solution were obtained, normoxic recording solution was superfused over the cells while [Ca2+]i was measured in PHTN (n = 96) and control (n = 117) PA SMC.
Effect of potassium and voltage-operated Ca2+ channel antagonists on basal [Ca2+]i in hypoxic PHTN and control fetal PA SMC. In separate experiments, [Ca2+]i was measured in control and PHTN PA SMC during the application of the following: 1) iberiotoxin (10-9 M) in PHTN (n = 17) and control (n = 51) PA SMC; 2) 4-AP (10-3 M) in PHTN (n = 47) and control (n = 38) PA SMC; and 3) diltiazem (10-5 M) in PHTN (n = 19) and control (n = 29) PA SMC.
Intracellular Ca2+ stores in hypoxic PHTN and control fetal PA SMC. After obtaining stable baseline values in hypoxic recording solution, the recording solution was changed to hypoxic solution without added Ca2+ for 2 min. Cells were then treated with thapsigargin (10-6 M) while [Ca2+]i was measured in PHTN (n = 27) and control (n = 30) PA SMC. In a separate series of experiments, cells were treated with ryanodine (5 x 10-5 M), whereas [Ca2+]i was measured in PHTN (n = 29) and control (n = 29) PA SMC.
Basal [Ca2+]i and the effect of an acute increase in PO2 on [Ca2+]i in serum-starved chronically hypoxic PHTN PA SMC. SMC from PHTN lambs were isolated and maintained in primary culture under hypoxic conditions as described above. In separate sets of experiments, the medium over the cells was changed from 10% to 0.1% fetal bovine serum for 1 or 7 days. Ca2+ imaging was performed while the conditions were changed to acute normoxia (n = 30 cells).
Statistical analysis. A two-way ANOVA with repeated measures and a Student-Newman-Keuls post hoc test were used to assess the differences between and among groups in each experimental protocol. Values are expressed as means ± SE. P values <0.05 were considered significant.
In each individual RNA experiment, the ratio of K+ channel to 18S rRNA content from hypertensive animals was compared with the ratio of K+ channel to 18S rRNA content in control animals. Total RNA was isolated from four control animals and six PHTN animals. The Student's t-test was used to assess differences between experimental groups. P values <0.05 were considered significant.
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RESULTS |
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Effect of potassium and voltage-operated Ca2+ channel antagonists on basal [Ca2+]i in PHTN and control fetal PA SMC. In control cells, iberiotoxin (Fig. 3) caused an increase of 224 ± 25 nM. In PHTN fetal PA SMC, iberiotoxin had no effect. In control fetal PA SMC, 4-AP caused an increase of 15 ± 3 nM, whereas in PHTN cells, 4-AP caused an increase of 32 ± 3 nM. In control PA SMC, diltiazem caused [Ca2+]i to decrease by 34 ± 4 nM from baseline, whereas in PTHN PA SMC [Ca2+]i increased by 21 ± 5 nM.
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Intracellular Ca2+ stores in hypoxic PHTN and control fetal PA SMC. Ryanodine caused an increase in [Ca2+]i in both control and PHTN fetal PA SMC. In control PA SMC, ryanodine increased [Ca2+]i by 147 ± 28 nM, while in PHTN PA SMC [Ca2+]i increased by 347 ± 46 nM (Fig. 4A). In control fetal PA SMC, thapsigargin increased [Ca2+]i by 45 ± 12 nM. In PHTN fetal PA SMC, thapsigargin had no effect on [Ca2+]i (Fig. 4B).
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Basal [Ca2+]i and effect of acute increase in PO2 on [Ca2+]i in serum-starved chronically hypoxic PHTN PA SMC. Serum deprivation had no effect on basal [Ca2+]i or on the response to an acute increase in PO2.
KCa subunit and Kv channel RT-PCR. KCa mRNA expression was decreased in PHTN, compared with control fetal PA SMC as determined by quantitiative, internally controlled RT-PCR (Fig. 5, A and B). The ratio of
KCa RNA to 18S RNA levels was 1.562 ± 0.136 in controls (n = 4) and 1.41 ± 0.103 in animals with PHTN (n = 6 animals). Kv channel mRNA expression in PHTN PA SMC was not significantly different from control PA SMC.
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KCa channel protein expression. Consistent with the observed effect on mRNA expression, Western blot analysis of KCa channel expression demonstrated decreased KCa channel protein (125-kDa band) in the PHTN PA SMC compared with controls (Fig. 6).
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DISCUSSION |
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Previous studies have demonstrated that perinatal pulmonary vasodilator stimuli act, at least in part, through activation of the pulmonary vascular KCa channel. In specific ventilation (33) and shear stress (32), an acute increase in PO2 (6) and NO (29) causes perinatal pulmonary vasodilation through activation of the KCa channel. From a teleologic perspective, the normal perinatal pulmonary circulation is uniquely well adapted to respond to an acute increase in PO2 as fetal, but not adult, PA SMC respond to an acute increase in PO2 with a decrease in [Ca2+]i (26). The inability of PA SMC derived from fetal lambs with chronic intrauterine pulmonary hypertension to respond to an acute increase in PO2 is consistent with previous reports of a decrease in pulmonary KCa channel expression in this model (7). In addition, recently published electrophysiology data indicate that the predominant potassium channel contributing to Em shifts from the KCa channel to the Kv channel in PA SMC from lambs with chronic intrauterine pulmonary hypertension (22). Moreover, the present observation is consistent with the incomplete response to vasodilator stimuli that characterizes the clinical presentation of persistent pulmonary hypertension (16).
To address the possibility that the attenuated Ca2+ response in hypertensive PA SMC results from increased Ca2+-ATPase activity in the SR, cells were treated with thapsigargin. In normotensive fetal PA SMC maintained under hypoxic conditions, thapsigargin caused a substantial increase in [Ca2+]i, implying that Ca2+-ATPase activity plays a role in the maintenance of basal Ca2+ stores. In contrast, thapsigargin had no effect on the basal [Ca2+]i of hypertensive PA SMC maintained under hypoxic conditions. Thus, in PHTN SMC, the SR Ca2+-ATPase activity is diminished. Consequently, the absence of the normoxia-induced decrease in [Ca2+]i in PHTN SMC is unlikely to result from augmented Ca2+-ATPase activity. Further studies are necessary to determine the significance of the relatively diminished role of Ca2+-ATPase activity in the maintenance of basal [Ca2+]i in PA SMC from animals with chronic intrauterine pulmonary hypertension.
To further investigate the effect of chronic intrauterine hypertension on intracellular Ca2+ handling, PA SMC from hypoxic normotensive and hypertensive animals were treated with ryanodine, which causes release of calcium from ryanodine stores in the SR (14, 17). Ryanodine caused an increase in both normotensive and PHTN PA SMC [Ca2+]i. However, the effect of ryanodine was significantly greater in PHTN PA SMC. Previous work (23) suggests that oxygen causes a decrease in fetal PA SMC [Ca2+]i through the quantal, localized release of intracellular calcium from a ryanodine-sensitive store close to the cell membrane in the region of the calcium-sensitive K+ channel. These small bursts of calcium have been termed calcium sparks. Ca2+ sparks are localized events that do not contribute to the global [Ca2+]i and do not reach a high enough concentration to cause contraction. Ca2+ spark activation of the KCa channel hyperpolarizes the cell, closing voltage-dependent Ca2+ channels and causing a decrease in cytosolic Ca2+ and, ultimately, vasodilation (11, 21). The relatively greater response to ryanodine in PHTN compared with normotensive PA SMC [Ca2+]i suggests chronic intrauterine hypertension affects the ryanodine-sensitive Ca2+ stores such that the stores contain Ca2+ in greater amounts than in controls. We speculate that Ca2+ spark release may no longer be localized or in the region of the KCa channel, and Ca2+ release from the store may lead to an increase in PA SMC [Ca2+]i from PHTN animals. While the underlying reason for the relatively replete ryanodine-sensitive stores is unknown, this observation seems to preclude the possibility that diminished KCa channel activation results primarily from depletion of the ryanodine-sensitive store.
Consideration of these data in concert with the decrease in KCa channel activity noted in PA SMC from hypertensive animals leads to the conclusion that release of Ca2+ from ryanodine-sensitive stores does not result in KCa channel activation. It is possible that chronic intrauterine pulmonary hypertension changes the relationship of the ryanodine-sensitive intracellular stores and the KCa channel as has been described in cardiac myocytes derived from animals with congestive heart failure (13). The inability of Ca2+ release from the ryanodine-sensitive Ca2+ store to activate KCa channels, thereby causing membrane hyperpolarization and closure of voltage-operated Ca2+ channels, may underlie the incomplete response to pulmonary vasodilator stimuli that characterizes persistent pulmonary hypertension of the newborn. Further support for this notion derives from the observation that both NO (29) and O2 (24) cause pulmonary vasodilation through ryanodine-dependent activation of KCa channels. Thus, if calcium release from ryanodine-sensitive stores cannot activate KCa channels, then both oxygen and NO-induced vasodilation may be compromised.
Given the generalized decrease in responsiveness of hypertensive PA SMC, intrauterine exposure to hypertension may prompt PA SMC to change from a contractile to a synthetic phenotype (12). To address such a possibility, PA SMC from hypertensive animals were serum deprived for periods of time ranging between 24 h and 7 days before the study. Serum deprivation had no effect on the responsiveness of these cells to any of the agents included in the present manuscript. On the basis of these results, we conclude that diminished responsiveness of these cell types cannot be explained on the basis of a change in phenotype.
There was no difference in basal [Ca2+]i between HTN and control PA SMC. Given clear evidence that chronic intrauterine pulmonary hypertension increases fetal pulmonary vascular tone, relatively higher levels of SMC [Ca2+]i might be anticipated in PHTN compared with control SMC. The similar levels of [Ca2+]i in these two cell populations suggests that the contractile state of the pulmonary vasculature is not determined solely by [Ca2+]i. Putative mechanisms include enhanced sensitivity of the contractile proteins to Ca2+, upregulation of Rho kinase activity, or a decrease in phosphatase activity (15). Alternatively, changes in intracellular calcium buffering, such as augmented cADP-ribose activity, may modulate SMC contractility (18).
In summary, the present study provides data that chronic intrauterine pulmonary hypertension has direct effects on PA SMC. PA SMC from animals with chronic intrauterine pulmonary hypertension do not respond to an acute increase in PO2 with a decrease in [Ca2+]i. Moreover, the K+ channel that determines basal [Ca2+]i changes from a KCa to a Kv channel. The physiological changes are consistent with changes in gene expression as KCa mRNA and protein levels are decreased and Kv mRNA levels are not significantly changed in PA SMC derived from animals with chronic intrauterine pulmonary hypertension. The observation that ryanodine causes a greater response in PA SMC [Ca2+]i in hypertensive compared with normotensive animals provides further insight into the mechanism whereby chronic intrauterine pulmonary hypertension alters perinatal pulmonary vascular reactivity. Further study is necessary to determine whether Ca2+ spark physiology is altered by chronic intrauterine pulmonary hypertension.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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