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Am J Physiol Regul Integr Comp Physiol 293: R1650-R1656, 2007. First published July 25, 2007; doi:10.1152/ajpregu.00300.2007
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DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY

Calcium-dependent and calcium-sensitizing pathways in the mature and immature ductus arteriosus

Ronald I. Clyman,1 Nahid Waleh,2 Hiroki Kajino,1 Christine Roman,1 and Francoise Mauray1

1Cardiovascular Research Institute and Department of Pediatrics, University of California, San Francisco, and 2SRI International, Menlo Park, California

Submitted 27 April 2007 ; accepted in final form 21 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Studies performed in sheep and baboons have shown that after birth, the normoxic muscle media of ductus arteriosus (DA) becomes profoundly hypoxic as it constricts and undergoes anatomic remodeling. We used isolated fetal lamb DA (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine why the immature DA fails to remain tightly constricted during the hypoxic phase of remodeling. Under normoxic conditions, mature DA constricts to 70% of its maximal active tension (MAT). Half of its normoxic tension is due to Ca2+ entry through calcium L-channels and store-operated calcium (SOC) channels. The other half is independent of extracellular Ca2+ and is unaffected by inhibitors of sarcoplasmic reticulum (SR) Ca2+ release (ryanodine) or reuptake [cyclopiazonic acid (CPA)]. The mature DA relaxes slightly during hypoxia (to 60% MAT) due to decreases in calcium L-channel-mediated Ca2+ entry. Inhibitors of Rho kinase and tyrosine kinase inhibit both Ca2+-dependent and Ca2+-independent DA tension. Although Rho kinase activity may increase during gestation, immature DA develop lower tensions than mature DA, primarily because of differences in the way they process Ca2+. Calcium L-channel expression increases with advancing gestation. Under normoxic conditions, differences in calcium L-channel-mediated Ca2+ entry account for differences in tension between immature (60% MAT) and mature (70% MAT) DA. Under hypoxic conditions, differences in both calcium L-channel-dependent and calcium L-channel-independent Ca2+ entry, account for differences in tension between immature (33% MAT) and mature (60% MAT) DA. Stimulation of Ca2+ entry through reverse-mode Na+/Ca2+ exchange or CPA-induced SOC channel activity constrict the DA and eliminate differences between immature and mature DA during both hypoxia and normoxia.

normoxia; hypoxia; oxygen concentration; Y27632; tyrosine kinase; calcium channels; gestation; calcium L-channels; store-operated calcium channels; RhoA; RhoB; Rho kinase

IN THE FULL-TERM NEWBORN, the ductus arteriosus closes its lumen within 24 h of birth. Anatomic remodeling and permanent obliteration of the lumen follow the initial constriction. Studies performed in sheep and baboons have shown that the initial postnatal constriction produces profound ischemic hypoxia within the ductus wall. The ischemic hypoxia initiates the chain of events leading to anatomic remodeling and permanent obliteration of the lumen (8, 26). During the period of hypoxic remodeling, it is essential for the ductus to maintain a tight degree of constriction so that the newly formed neointima can obstruct and occlude the constricted lumen (8). In contrast with the full-term newborn, the preterm newborn frequently relaxes its constriction during the hypoxic phase of anatomic remodeling and reopens its lumen before it has been remodeled (31).

Regulation of ductus contractility depends on a balance between vasoconstricting and vasodilating influences. The ductus arteriosus of the preterm newborn has an increased sensitivity to locally produced vasodilators like prostaglandins and nitric oxide that prevent its closure after birth (9, 11, 27, 42). Current interventions, designed to close the preterm patent ductus arteriosus, have targeted vasodilating factors that block ductus constriction. However, even when inhibitors of prostaglandin and nitric oxide production have been used to close the preterm patent ductus arteriosus, a significant number will reopen and require additional therapies (27, 31).

New therapies targeting the factors that promote vasoconstriction might complement existing approaches. Smooth muscle constriction is determined by the degree of myosin light chain phosphorylation (35). Events that elevate intracellular Ca2+, activate myosin light chain kinase, which, in turn, phosphorylates myosin light chain (35). On the other hand, constriction is inhibited by myosin phosphatase, which dephosphorylates the phosphorylated myosin light chain. Events or drugs that interfere with myosin phosphatase activity increase the sensitivity of the contractile apparatus to Ca2+-induced activation and reduce the requirement for Ca2+ influx (34).

Many investigators have demonstrated that oxygen constricts the ductus after birth (28). Oxygen depolarizes ductus smooth muscle cells and increases intracellular calcium through a series of interactions involving potassium (K+) channels, cytochrome P-450, and endothelin (12, 13, 18, 30, 33, 40). In addition to the tone induced by oxygen, there is an intrinsic oxygen-independent tone in the ductus that keeps it closed during the hypoxic phase of ductus remodeling (25). To date, there is little information about the factors that contribute to this intrinsic oxygen-independent tone in the ductus and how these factors change over gestation. We designed the following studies to identify the factors that are responsible for both the oxygen-dependent and oxygen-independent tone in the ductus. We also examined the factors that are responsible for the difference in tension between the immature and mature ductus. We were particularly interested in the factors that might be responsible for the relaxation that occurs in preterm infants during the hypoxic phase of ductus remodeling. We used a pharmacologic approach to examine the roles of extracellular Ca2+ and altered Ca2+ sensitivity in maintaining ductus tensions.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Forty-seven late gestation sheep fetuses (mixed Western breed: 137 ± 5 day gestation, term = 145 days) and 52 immature fetuses (105 ± 3 days) were delivered by Cesarean section and anesthetized with ketamine HCl (30 mg/kg iv) before rapid exsanguination. These procedures were approved by the Committee on Animal Research at the University of California, San Francisco, CA.

In vitro isometric contractility studies. The ductus arteriosus was divided into 1-mm-thick rings (2–3 rings per animal), and isometric tension was measured in a Krebs-bicarbonate solution {[x10–3 M]: 118 NaCl, 4.7 KCl, 2.5 CaCl2, 0.9 MgSO4, 1 KH2PO4, 11.1 glucose, 23 NaHCO3 (pH 7.4)} containing indomethacin (5 x 10–6 M) and N{omega}-nitro-L-arginine methyl ester (10–4 M), to inhibit endogenous prostaglandin and nitric oxide production, respectively (26). An oxygen electrode (model 53 Biological Oxygen Monitor; YSI, Yellow Springs, OH) placed in the 10-ml organ bath measured the oxygen concentration. The bath solution was equilibrated with gas mixtures containing 5% CO2 and was changed every 20 min. Rings were stretched to initial lengths (preterm = 5.0 ± 0.4 mm; late gestation = 6.7 ± 0.6 mm) that produce maximal contractile responses when exposed to K+-Krebs solution (containing 0.1 M KCl substituted for an equimolar amount of NaCl) equilibrated with 95% O2 (10).

After the rings reached a steady-state tension in 30% O2 (~100–120 min), K+-Krebs solution (equilibrated with 95% O2) was used to measure the maximal contraction that could be developed by the ductus. After returning the rings to the initial modified Krebs solution, equilibrated with 15% O2, the rings were sequentially exposed to two different oxygen conditions: 2% and 15% O2. We chose bath O2 concentrations of 2% and 15% since they produce tissue oxygen concentrations similar to the physiologic extremes experienced by the ductus in vivo (8). The 15% O2 concentration was used to produce tissue O2 concentrations associated with the expected postnatal increase in arterial PO2 (Normoxia). The 2% O2 concentration was used for the hypoxic condition since it produces 0.2% tissue O2 concentration (the O2 concentration needed to induce remodeling in vivo) (26). The rings were then equilibrated with one of several experimental solutions before exposing them to 2% and 15% O2 again. The experimental solutions included: 10–5 M nifedipine (calcium L-channel antagonist), 10–6 M BAY K 8644 (calcium L-channel agonist), 25 x 10–6 M ryanodine (to deplete ryanodine-sensitive sarcoplasmic reticulum Ca2+ stores), 10–5 M cyclopiazonic acid (CPA) [sarcoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, to block SERCA-dependent uptake and deplete sarcoplasmic reticulum Ca2+ stores], 2.5 x 10–6 M LaCl3, 2.5 x 10–6 M NiCl2, 2.5 x 10–6 M CdCl2 (multivalent ion Ca2+ channel inhibitors), Ca2+-free Krebs solution (containing 0.5 x 10–3 M EGTA without Ca2+), low Na+-Krebs solution (containing 0.1 M choline chloride substituted for an equimolar amount of NaCl; to inhibit forward-mode Na+/Ca2+ exchange), 10 x 10–6 M Y27632 (Rho kinase inhibitor), 100 x 10–6 M genistein (tyrosine kinase inhibitor), and K+-Krebs solution. When rings were exposed to low Na+-Krebs solution, 10–6 M atropine was added to the bath solution to prevent any potential stimulation from the elevated choline chloride concentrations; atropine has no effect on ductus tension at 2% or 15% O2 (data not shown). In experiments involving LaCl3, NiCl2, or CdCl2, 10–2 M HEPES buffer was substituted for NaHCO3/CO2 buffer. We have previously shown that there is no difference in the contractile response of the ductus to hypoxia or oxygen when repeatedly exposed to hypoxia or oxygen in time-matched control experiments (26). In all experiments, we allowed the tension in the rings to reach a new steady-state plateau after the rings were exposed to a new experimental condition or drug. Sodium nitroprusside (10–3 M) was added to each ring at the end of the experiment to determine its minimal tension.

The difference in tensions between any measured steady-state tension and the minimal tension produced by sodium nitroprusside was considered the net active tension. The difference in tensions between the maximal tension (produced by K+-Krebs/95% O2) and the minimal tension (with sodium nitroprusside) was treated as the maximal active tension (MAT) capable of being developed by the rings.

Active tensions are expressed as a percentage of the MAT. MATs were 16.8 ± 3.6 g in immature fetuses and 21.7 ± 4.4 g in mature fetuses. After the experiment, the tissues were removed from the baths, were blotted dry, and wet weights were determined (30 ± 9 mg, immature fetuses; 34 ± 9, mature fetuses). Chemicals were from Sigma (St. Louis, MO).

Preparation of total RNA, reverse transcription, and quantitative PCR. Total RNA was isolated from the frozen ductus of seven immature and seven mature fetuses as described elsewhere (4). We used the TaqMan Universal PCR master mix of PE Applied Biosystems (Foster City, CA) to quantify the expression of the calcium L-channel subunits ({alpha}1c, beta2, and beta3), in addition to RhoA, RhoB, and Rho kinase 1. TaqMan probes were designed by using the Primer Express program and were labeled with fluorophores 6-caboxy-fluorescein and 6-carboxy-tetramethyl-rhodamine as reporter and quencher dyes, respectively. An ABI PRISM 7500 Sequence Detection System was used to determine the cycle threshold. Reactions were carried out in triplicates. Data were analyzed by using the Sequence Detector version 1.6.3 program. Malate dehydrogenase was used as an internal control to normalize the data (42).

Statistics. Statistical analyses of unpaired and paired data were performed by the appropriate t-test and by ANOVA. Schéffe's test was used for post hoc analysis. Values are expressed as means ± SD. Drug concentrations refer to their final molar concentration in the bath.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mature ductus under normoxic conditions. In the absence of prostaglandin and nitric oxide production, the mature ductus under normoxic conditions (15% O2) develops an active tension that is ~70% of its MAT (Fig. 1). Fifty percent of the normoxic active tension depends on the presence of extracellular Ca2+ (utilizing both calcium L-channel-dependent and calcium L-channel-independent pathways) (Fig. 1). Nifedipine, the calcium L-channel antagonist, decreased normoxic ductus tension by 20% of MAT (Fig. 1). Following the removal of extracellular Ca2+, nifedipine had no effect on ductus tension (data not shown).


Figure 1
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  		Fig. 1. Active tension in the mature ductus under normoxic and hypoxic conditions. Ductus rings from mature fetuses were studied in the presence or absence of either extracellular Ca2+, 10–5 M nifedipine (nifed), 10–6 M BAY K 8644, or K+-Krebs solution. Ca2+-free Krebs solution contained 0.5 x 10–3 M EGTA without Ca2+. n, Number of ductus rings from separate animals exposed to each condition. Height of column represents active tension (means ± SD) as %maximal active tension. *P < 0.05 normoxia compared with hypoxia (same experimental condition); +P < 0.05 compared with hypoxic control; #P < 0.05 compared with normoxic control; ns, not significant.

 
A portion of the ductus tension, that depends on extracellular Ca2+ was not affected by the calcium L-channel inhibitor nifedipine but was inhibited by La3+, Ni2+, and Cd2+. These multivalent cations inhibited normoxic tension even after the rings had been exposed to nifedipine [%MAT, n = 4: normoxic control = 72 ± 7, nifedipine (10–5 M) = 55 ± 6, nifedipine + La3+ (2.5 x 10–6 M) = 43 ± 6, nifedipine + Ni2+ (2.5 x 10–6 M) = 49 ± 5, and nifedipine + Cd2+ (2.5 x 10–6 M) = 42 ± 6].

The other 50% of normoxic active tension in the mature ductus is independent of extracellular Ca2+ (Fig. 1). Ca2+ release from the sarcoplasmic reticulum, through ryanodine-sensitive Ca2+ channels, does not appear to play a significant role in maintaining ductus tone; neither ryanodine (25 x 10–6 M, n = 5, data not shown) nor caffeine (7) had any effect on ductus tension under either normoxic or hypoxic conditions.

Mature ductus under hypoxic conditions. In the mature ductus, there is a small but significant decrease in active tension as the tissue becomes hypoxic (Fig. 1). The small difference in active tension between the hypoxic and normoxic ductus appears to be due to differences in the rate of extracellular Ca2+ entry through voltage-gated calcium L-channels. Removal of extracellular Ca2+ from the bath solution decreases ductus tension and eliminates the difference in tensions between the normoxic and hypoxic ductus (Fig. 1). The same phenomenon occurs when nifedipine is added to the organ bath. Conversely, stimuli that increase calcium L-channel opening (e.g., K+ depolarization and BAY K 8644) increase the active tension of the ductus and eliminate any difference in tension between the normoxic and hypoxic conditions (Fig. 1). The contractile effects of K+ depolarization and BAY K 8644 are due to Ca2+ entry through voltage-gated calcium L-channels; incubating the ductus in the absence of Ca2+ or in the presence of nifedipine eliminates their contractile effects (data not shown).

Immature ductus under normoxic conditions. Under normoxic conditions, the immature ductus also develops an active tension that is maintained by extracellular Ca2+-dependent and extracellular Ca2+-independent pathways (Fig. 2). In the absence of prostaglandin and nitric oxide production, there is a small but significant difference in the active tensions between the normoxic immature and normoxic mature ductus (~10% MAT) (Fig. 2). This difference appears to be due to differences in the rate of extracellular Ca2+ entry through voltage-gated calcium L-channels. Conditions that inhibit Ca2+ entry through calcium L-channels (e.g., nifedipine or the absence of extracellular Ca2+) eliminate the difference in tension between the two age groups, as do conditions that open calcium L-channels (e.g., BAY K 8644 or K+-induced depolarization) (Fig. 2). In addition, we found that with advancing gestation there was a significant increase in the expression of both the calcium L-channel {alpha}1c-subunit, which confers most of the functional properties to the calcium L-channel (36), and the calcium L-channel beta2-subunit, which modulates the channel's physiological responses (36) (Fig. 3).


Figure 2
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  		Fig. 2. Comparison of normoxic tensions in the immature and mature ductus. Ductus rings from immature and mature fetuses were studied in the presence or absence of either extracellular Ca2+, nifedipine, BAY K 8644, or K+-Krebs solution (see Fig. 1 for concentrations). n, Number of ductus rings from separate animals exposed to each condition. *P < 0.05 immature compared with mature (same experimental condition); +P < 0.05 compared with immature control; #P < 0.05 compared with mature control.

 

Figure 3
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  		Fig. 3. Real-time PCR measurements of calcium L-channel subunits ({alpha}1c, beta2, and beta3), RhoA, RhoB, and Rho kinase 1 (ROCK-1) in the mature and immature ductus and aorta. [{Delta}CT(MDH-gene)], where CT is cycle threshold and MDH is malate dehydrogenase, represents the difference in CT between the expression of the housekeeping gene MDH and the gene of interest. Each unit of {Delta}CT(MDH-gene) represents a 2-fold increase in the mRNA of a gene. The more negative the {Delta}CT(MDH-gene), the fewer the number of starting copies of a gene (mRNA). n, Number of separate animals used. *P < 0.01, mature compared with immature. In the ductus, the P value for the difference between the mature and immature groups was >0.05, but was <0.10 for measurements of the RhoB and ROCK-1. There were also significant (P < 0.01) differences between the ductus and aorta in the immature (CaL-{alpha}1c, CaL-beta3, RhoA, and RhoB) and the mature (CaL-{alpha}1c, CaL-beta2, CaL-beta3, RhoA, and RhoB) fetus.

 
Immature ductus under hypoxic conditions. In contrast with the mature ductus, the immature ductus loses a significant degree of its active tension as the tissue becomes hypoxic. The hypoxic tension in the immature ductus is <40% of MAT. In the hypoxic immature ductus, pathways that depend on extracellular Ca2+ no longer appear to play a role in maintaining ductus tension; the hypoxic ductus tension appears to be maintained only by pathways that are independent of extracellular Ca2+ (Fig. 4). Neither the absence of extracellular Ca2+ nor the presence of nifedipine affect hypoxic tone in the immature ductus (Fig. 4). Similarly, conditions that usually open calcium L-channels (e.g., BAY K 8644 and K+-induced depolarization), either have no effect (BAY K 8644) or a blunted effect (K+-induced depolarization) on hypoxic tone (Fig. 4). This contrasts with the mature ductus where a significant proportion of the hypoxic tension is maintained by pathways that depend on the presence of extracellular Ca2+ (Fig. 4). Differences in both calcium L-channel-dependent and calcium L-channel-independent Ca2+ entry appear to account for the difference in hypoxic tensions between the immature and mature ductus.


Figure 4
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  		Fig. 4. Comparison of hypoxic tensions in the immature and mature ductus. See Fig. 2 legend for details. *P < 0.05 immature compared with mature (same experimental condition); +P < 0.05 compared with immature control; #P < 0.05 compared with mature control.

 
Hypoxic immature ductus: effects of alterations in Na+/Ca2+ exchange and SERCA pump activity. Since the hypoxic immature ductus does not respond to changes in extracellular Ca2+ concentration or to drugs that perturb calcium L-channel opening, we investigated whether it was capable of responding to other mechanisms that might increase extracellular Ca2+ entry into the cell. The Na+/Ca2+ exchange system in the plasma membrane has a high capacity for Ca2+ exchange. The Na+/Ca2+ exchanger operates in a forward (Ca2+ exit) or reverse (Ca2+ entry) mode, depending on the Na+ and Ca2+ gradients and the transmembrane potential (3). Reducing extracellular Na+ (by replacing it with choline) inhibits forward-mode Ca2+ exit and stimulates reverse-mode Ca2+ entry into the cell (Fig. 5). Reducing extracellular Na+ increased normoxic and hypoxic tensions in both the mature and immature ductus. Reducing extracellular Na+ eliminated the difference in hypoxic tensions (as well as the difference in normoxic tensions) between the two age groups (Fig. 5).


Figure 5
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  		Fig. 5. Comparison of hypoxic and normoxic tensions in the immature and mature ductus. Ductus rings from immature and mature fetuses were studied in the presence or absence of either low extracellular Na+ solution (containing 0.1 M choline chloride substituted for an equimolar amount of NaCl), 10–5 M cyclopiazonic acid (CPA), or low extracellular Na+ solution + CPA. n, Number of ductus rings from separate animals exposed to each condition. *P < 0.05 immature compared with mature (same experimental condition); +P < 0.05 compared with immature control [low Na+(-)/CPA(-)]; #P < 0.05: compared with mature control [low Na+(-)/CPA(-)].

 
Extracellular Ca2+ entry into ductus smooth muscle can also be potentiated by inhibiting the SERCA pump. Selective inhibitors of SERCA pump activity (e.g., CPA) stimulate extracellular Ca2+ entry through two linked pathways: store-operated calcium (SOCs) channels and the Na+/Ca2+ exchanger (1, 5, 45). CPA increased normoxic and hypoxic tensions in both the mature and immature ductus and eliminated the difference in hypoxic tensions between the two age groups (Fig. 5).

The contractile responses induced by lowering extracellular Na+ and by inhibiting SERCA pump activity were not blocked by the presence of nifedipine (Fig. 6) but were completely inhibited by the removal of extracellular Ca2+ (Fig. 6). This suggests that alterations in Na+/Ca2+ exchange and SERCA pump activity affect ductus tone primarily by regulating extracellular Ca2+ entry through pathways that are not dependent on calcium L-channels. It also appears that increased extracellular Ca2+ entry resulting from manipulations of SERCA pump activity or Na+/Ca2+ exchange can eliminate the difference in tensions between the immature and mature ductus under both normoxic and hypoxic conditions. This is in marked contrast to the lack of effect that manipulations of calcium L-channels have on ductus tension in the immature hypoxic ductus (Fig. 4).


Figure 6
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  		Fig. 6. Dependence of CPA and low Na+ induced tensions on the presence of extracellular Ca2+. Ductus rings from immature fetuses were studied under normoxic conditions in the presence and absence of extracellular Ca2+. Rings were exposed to the following conditions sequentially: 1) absent Ca2+, 2) Ca2+ present, 3) absent Ca2+ + nifedipine, 4) Ca2+ present + nifedipine, 5) absent Ca2+ + nifedipine plus CPA, 6) Ca2+ present + nifedipine plus CPA, 7) absent Ca2+ + nifedipine + CPA + low Na+, and, finally, 8) Ca2+ present + nifedipine + CPA + low Na+. n, Number of ductus rings from separate animals exposed to each condition. #P < 0.05 compared with control [nifedipine(-)/CPA(-)/low Na+(-)].

 
Inhibition of protein tyrosine kinase and Rho kinase in mature and immature ductus. The ductus maintains an active tension even when its Ca2+ stores are depleted. The residual active tension following Ca2+ store depletion (by simultaneous removal of extracellular Ca2+ and CPA-induced blockade of sarcoplasmic reticulum Ca2+ uptake) is approximately one-third of the MAT of the ductus (Fig. 6). An important determinant of vascular tension is the sensitivity of its actin/myosin to cytosolic Ca2+ (34, 35). Prior studies have shown that Rho kinase and protein tyrosine kinase both play a significant role in actin/myosin Ca2+ sensitization (21, 34). Therefore, we examined their impact on ductus contractility.

Genistein, a selective inhibitor of protein tyrosine kinases (37), relaxed both the mature and immature ductus under hypoxic and normoxic conditions (Fig. 7). Genistein was less effective in the mature ductus than it was in the immature ductus (Fig. 7). However, when Ca2+ entry into the ductus was maximally stimulated by K+ induced depolarization, genistein had the same relaxant effect on the mature and immature ductus (Fig. 7).


Figure 7
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  		Fig. 7. Effects of Y27632 and genistein on the immature and mature ductus exposed to hypoxia, normoxia, and K+ depolarization. Ductus rings from immature and mature fetuses were studied in the presence or absence of either 10 x 10–6 M Y27632, or 100 x 10–6 M genistein. K+-Krebs solution (equilibrated with 95% O2) was used to produce K+ depolarization. n, Number of ductus rings from separate animals exposed to each condition. *P < 0.05 immature compared with mature (same experimental condition). Compared with control conditions, Y27632 and genistein inhibited tension (P < 0.01) in both the immature and mature ductus under all 3 experimental conditions (hypoxia, normoxia, and K+ depolarization).

 
Y27632, a selective inhibitor of Rho kinase (2, 23, 41), also relaxed the ductus, under hypoxic and normoxic conditions (Fig. 7). Y27632 was less effective in the mature ductus than it was in the immature ductus even when Ca2+ entry into the ductus was maximally stimulated by K+ depolarization (Fig. 7). Higher concentrations of Y27632 (50 x 10–6 M) were needed in the mature ductus to inhibit ductus tensions to the same degree as in the immature ductus (data not shown). We also found that there was a significant increase in the expression of RhoA with advancing gestation and a trend for increased expression of RhoB and Rho kinase 1 in the fetal ductus. These changes were not observed in the fetal aorta (Fig. 3).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
After birth, it is essential for the ductus arteriosus to close its lumen and to keep it closed during the period of hypoxic/ischemic remodeling (8). When endogenous prostaglandin and nitric oxide production have been inhibited, the mature sheep ductus generates a steady-state tension under normoxic conditions that is ~70% of its MAT (Fig. 1). Tension decreases as the mature ductus becomes hypoxic. However, the decrease in tension is relatively small. Therefore, the mature ductus is able to maintain a tension that is ~60% of its MAT even under conditions of profound hypoxia (Fig. 1).

The purpose of our study was to identify the pathways responsible for the difference in tension between the hypoxic and normoxic ductus and for the difference in tension between the immature and mature ductus under hypoxic and normoxic conditions.

Normoxic tension. We used a bath O2 concentration of 15% O2 to mimic the normoxic arterial PaO2 that the ductus is exposed to after birth. Approximately half of the normoxic active tension of the ductus depends on the presence of extracellular Ca2+. The other half is independent of extracellular Ca2+ (Fig. 1). Forty percent of the tension that depends on extracellular Ca2+ relies on Ca2+ entry through voltage-gated calcium L-channels (Fig. 1). Ca2+ entry through SOC channels may also contribute to the component of normoxic tension that depends on extracellular Ca2+. SOC channels have recently been shown to play a significant role in maintaining the normoxic tension of the rabbit ductus arteriosus (20). In the sheep ductus, multivalent cations, like La3+, Ni2+, and Cd2+, reduce normoxic tension even after calcium L-channels have been inhibited by nifedipine. Similarly, in the presence of nifedipine and CPA, which empties Ca2+ stores in the sarcoplasmic reticulum, an increase in extracellular Ca2+ concentration from 0 to 2.5 mM, increases ductus tension much more than a similar increase in Ca2+ concentration in the presence of nifedipine alone (Fig. 6). This pattern of response to multivalent cations and CPA has been attributed to Ca2+ entry through SOC channels in other blood vessels (6, 22, 32).

The remaining 50% of normoxic tension is resistant to both Ca2+ depletion and CPA inhibition of sarcoplasmic reticulum filling (Fig. 6). This may be due to increased Ca2+ sensitization, a process whereby constriction occurs independently of ongoing increases in cytosolic Ca2+ (34, 35). The ductus has been shown to be more sensitive to cytosolic Ca2+ than the aorta or pulmonary artery (15). Prior studies suggest that protein tyrosine kinase and Rho kinase (a serine threonine kinase) may affect Ca2+ sensitization (21) (34). Smooth muscles possess high levels of nonreceptor tyrosine kinase activity (17), and genistein, a selective inhibitor of protein tyrosine kinase, attenuates smooth muscle contraction (37). The Rho family of GTPases also has been shown to increase smooth muscle contractility by activating Rho kinase and inhibiting myosin phosphatase (34). Rho kinase activation has been shown to be an essential distal step in agonist-induced smooth muscle constriction (19, 41). Recently, Rho kinase has been implicated in ductus arteriosus constriction (14, 20, 24). We found that both Rho kinase and tyrosine kinase appear to play a significant role in maintaining active tension in the sheep ductus (Fig. 7).

Hypoxic vs. normoxic tensions in the mature ductus. We found that the difference in tensions between the hypoxic and normoxic mature sheep ductus was primarily due to differences in the rate of extracellular Ca2+ entry through calcium L-channels. Both calcium L-channel inhibitors and calcium L-channel activators eliminated the difference between hypoxic and normoxic tensions (Fig. 1). Our results are consistent with prior studies that found that oxygen increased Ca2+ entry through voltage-dependent calcium L-channels by inhibiting K+ currents and depolarizing ductus smooth muscle (29, 30, 33, 39, 40). Similarly, delayed ductus closure has been noted in vivo when Ca2+ entry was inhibited by Mg2+ (16) or verapamil (38).

Immature vs. mature ductus. The preterm ductus generates smaller tensions than the mature ductus under both normoxic and hypoxic conditions (26). This occurs even after endogenous prostaglandin and nitric oxide production have been inhibited (Figs. 2 and 4). The difference between the two age groups is most marked under hypoxic conditions. We hypothesize that the reduced ability of the immature ductus to maintain tension under hypoxic conditions is an important factor in the high incidence of ductus reopening (26, 31).

Recent studies in other species have suggested that alterations in Ca2+ sensitization may contribute to the increase in ductus tension that occurs with advancing gestation (14, 20, 24). Our findings suggest that tyrosine kinases and Rho kinases may play a role in this process (Fig. 7). Genistein, a tyrosine kinase inhibitor, significantly inhibited ductus tension. Genistein was less effective in the mature than in the immature ductus (under both hypoxic and normoxic conditions). Prior studies have shown that tyrosine kinases can influence smooth muscle constriction by activating calcium L-channels (43, 44). We found that when Ca2+ entry through calcium L-channels was maximized (by K+-induced membrane depolarization) there was no longer a difference in the ability of genistein to inhibit the mature and immature ductus (Fig. 7). This suggests that in the presence of similar degrees of Ca2+ entry, endogenous tyrosine kinase activity has similar effects in the mature and immature ductus.

The Rho kinase inhibitor Y27632 also was a potent inhibitor of ductus tension. Our findings are consistent with Rho kinase being present in greater amounts or having a greater activity in the mature ductus: 1) there was a significant increase in the expression of RhoA and a trend for increased expression of RhoB and Rho kinase 1 with advancing gestation (these changes were not observed in the fetal aorta) (Fig. 3); 2) Y27632 was less effective in the mature ductus even when Ca2+ entry was maximally stimulated by K+-induced depolarization (Fig. 7); 3) higher concentrations of Y27632 were needed to inhibit ductus tensions in the mature ductus to the same degree as in the immature ductus. Similar findings have been observed in other species (14, 20, 24).

Although there appears to be a difference in Rho kinase activity between the immature and mature ductus (Fig. 7) (14, 24), our studies suggest that the major factor responsible for the developmental difference in tensions is the way the immature ductus processes extracellular Ca2+. In the absence of extracellular Ca2+, the tension in the full-term ductus drops to the same level as the preterm ductus (33 ± 8% MAT) (Figs. 2 and 4).

We found that there was a significant increase in the expression of calcium L-channels with advancing gestation (Fig. 3). Under normoxic conditions, the difference in extracellular Ca2+ entry through the nifedipine-sensitive calcium L-channels can completely account for the difference in tension between the mature and immature ductus (Fig. 2).

In contrast with normoxia, the difference in tension between the mature and immature ductus under hypoxic conditions appears to be due to differences in both calcium L-channel-dependent and calcium L-channel-independent extracellular Ca2+ entry (Fig. 4). The immature ductus does not appear to use extracellular Ca2+ to maintain its active tension under hypoxic conditions. Neither eliminating extracellular Ca2+ from the bath solution nor adding drugs that open calcium L-channels, like BAY K 8644, had any effect on tension in the hypoxic immature ductus (Fig. 4). These findings suggest that, while manipulating calcium L-channels may be a reasonable approach to increase tension under normoxic conditions, it is unlikely to promote increased tension when the immature ductus develops profound hypoxia after birth (Fig. 4).

We examined other possible mechanisms for increasing extracellular Ca2+ entry into the immature ductus to see if these could increase ductus tension under hypoxic conditions. Both stimulation of reverse-mode Na+/Ca2+ exchange (by reducing extracellular Na+) and inhibition of SERCA pump activity (with CPA) increased hypoxic ductus tension and eliminated the difference in hypoxic tensions between the immature and mature ductus (Fig. 5). This contrasts with the lack of effect that manipulations of calcium L-channels have on ductus tension under hypoxic conditions. Future studies designed to measure intracellular Ca2+ fluxes will be necessary to identify the exact pathways that have been altered by our pharmacologic manipulations. We hypothesize that Ca2+ removal/sequestration mechanisms may be potential targets for new therapies aimed at closing the preterm patent ductus arteriosus.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-46691 and HL-56061 and by a gift from the Jamie and Bobby Gates Foundation.


   ACKNOWLEDGMENTS
 
Present address of H. Kajino: Department of Pediatrics, Asahikawa Medical College, Hokkaido, Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. Clyman, Univ. of California San Francisco, 513 Parnassus Ave., Rm. 1408 HSW, UCSF Box 0544, San Francisco, CA 94143-0544 (e-mail: clymanr{at}peds.ucsf.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.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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