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Chronic hypoxia modulates relations among calcium, myosin light chain phosphorylation, and force differently in fetal and adult ovine basilar arteries

¹Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts; ²Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California; and ³Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada

Submitted 7 October 2004 ; accepted in final form 5 March 2005

	ABSTRACT

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The present study tests the hypothesis that age-related differences in contractility of cerebral arteries from hypoxic animals involve changes in myofilament Ca²⁺ sensitivity. Basilar arteries from term fetal and nonpregnant adult sheep maintained 110 days at 3,820 m were used in measurements of cytosolic calcium concentration ([Ca²⁺]_i), myosin light chain phosphorylation, and contractile tensions induced by graded concentrations of K⁺ or serotonin (5-HT). Slopes relating [Ca²⁺]_i to tension were similar in fetal (0.83 ± 0.07) and adult (1.02 ± 0.08) arteries for K⁺ contractions but were significantly greater for fetal (3.77 ± 0.64) than adult (2.00 ± 0.13) arteries for 5-HT contractions. For both K⁺ and 5-HT contractions, these relations were left shifted in fetal compared with adult arteries, indicating greater Ca²⁺ sensitivity in fetal arteries. In contrast, slopes relating [Ca²⁺]_i and %myosin phosphorylation for K⁺ contractions were less in fetal (0.37 ± 0.08) than adult (0.81 ± 0.07) arteries, and the fetal curves were right shifted. For 5-HT contractions, the slope of the Ca²⁺-phosphorylation relation was similar in fetal (0.33 ± 0.09) and adult (0.33 ± 0.23) arteries, indicating that 5-HT depressed Ca²⁺-induced myosin phosphorylation in adult arteries. For slopes relating %myosin phosphorylation and tension, fetal values (K⁺: 1.52 ± 0.22, 5-HT: 7.66 ± 1.70) were less than adult values (K⁺: 2.13 ± 0.30, 5-HT: 8.29 ± 2.40) for both K⁺- and 5-HT-induced contractions, although again fetal curves were left shifted relative to the adult. Thus, in hypoxia-acclimatized basilar arteries, a downregulated ability of Ca²⁺ to promote myosin phosphorylation is offset by an upregulated ability of phosphorylated myosin to produce force yielding an increased fetal myofilament Ca²⁺ sensitivity. Postnatal maturation reprioritizes the mechanisms regulating hypoxic contractility through changes in the source of activator Ca²⁺, the pathways governing myosin light chain phosphorylation, and its interaction with actin.

cerebral arteries; myofilament calcium sensitivity; postnatal maturation; myosin phosphorylation; thin filament regulation

Another strong modulator of cerebrovascular reactivity is chronic hypoxia. In ovine cerebral arteries, acclimatization at altitude generally alters patterns of receptor expression, induces changes in artery composition, and depresses contractility to a variety of agonists, in addition to a variety of other significant effects (18, 31, 34). The range and magnitude of these effects are also highly age dependent. Whereas these patterns of effects of chronic hypoxia on cerebrovascular reactivity are well documented, the mechanisms responsible remain obscure. Given that cerebrovascular myofilament Ca²⁺ sensitivity is strongly regulated in an age-dependent manner, it is reasonable to propose that age-related differences in myofilament Ca²⁺ sensitivity persist in hypoxia-acclimatized animals. Thus the present study is focused on the hypothesis that age-dependent differences in cerebrovascular contractility documented in hypoxia-acclimatized animals involve corresponding differences in myofilament Ca²⁺ sensitivity. Because myofilament Ca²⁺ sensitivity, in turn, can be viewed as the composite sum of the relations between cytosolic Ca²⁺ and myosin phosphorylation and between myosin phosphorylation and contractile force (20, 21), we further propose that age-related differences in myofilament Ca²⁺ sensitivity involve underlying differences in these relations.

Because serotonin (5-HT) has been shown to potently enhance myofilament Ca²⁺ sensitivity in ovine cerebral arteries (1, 3, 5), the present studies were designed to explore the simultaneous effects of varying concentrations of 5-HT on cytosolic Ca²⁺, MLC phosphorylation, and contractile force in chronically hypoxic basilar arteries from both term fetal lambs and nonpregnant adult sheep. Because contractions induced by K⁺ do not involve myofilament Ca²⁺ sensitization at least in ovine basilar arteries (3, 22, 24), and thereby reflect basal Ca²⁺ sensitivity, parallel experiments were performed using K⁺ as a stimulant. Together, these experiments enable a unique assessment of the relative importance of changes in the relations among cytosolic Ca²⁺, myosin phosphorylation, and contractile force in corresponding changes in age-dependent myofilament Ca²⁺ sensitivity in basilar arteries from chronically hypoxic sheep.

	METHODS

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General methods.   All procedures and protocols governing the care and use of laboratory animals strictly followed federal regulations and were approved by the Institutional Animal Care and Use Committee of Loma Linda University. Basilar arteries were obtained from nonpregnant adult sheep (18–24 mo old) and near-term (140 days gestation) fetuses. Sheep were maintained at an altitude of 3,820 m for 110 days at White Mountain Research Institute, Bishop, CA, and then euthanized with pentobarbital sodium (60 mg/kg iv). After removal from the surface of the brain, basilar arteries were placed in sodium Krebs buffer containing (in mM) 122 NaCl, 25.6 NaHCO₃, 5.17 KCl, 2.49 MgSO₄, 1.60 CaCl₂, 5.56 dextrose, 0.0270 EGTA, and 0.114 ascorbic acid. All buffer solutions were continuously bubbled with 5% CO₂ in 95% O₂, which maintained buffer pH at 7.35.

Basilar artery segments were cut into 5-mm lengths after mechanical abrasion of the endothelium, and viability of the segments was determined by maximum contraction (designated as 100%) with 122 mM K⁺. Various concentrations of K⁺ (50, 75, and 122 mM) or 5-HT (10^–7, 10^–6, or 10^–5 M) were used to obtain graded contractile responses, changes in cytosolic Ca²⁺, and phosphorylation of MLC at various durations of contraction (0, 5, 10, 15, 20, 30, 60, and 120 s). To measure the dynamics of these parameters precisely and accurately, contractile force (Kent no. TRN011) and cytosolic Ca²⁺ (via fura 2 with a Jasco CAF-110 fluorometer) were measured in one set of segments, and in another set of equivalent segments contractile force and myosin phosphorylation (via Western blot) were measured.

Simultaneous measurement of contractile tension and MLC phosphorylation.   Basilar artery segments were equilibrated for at least 1 h at 38°C in 5-ml jacketed tissue baths at levels of optimal passive stretch previously determined for these preparations (23). Basilar arteries were then contracted with one of three concentrations of either K⁺ or 5-HT for a designated period of time (0 to 120 s). The tensions were recorded until the segments were immersed into freezing-cold (–70°C) acetone with 10% trichloroacetic acid, 10 mM dithiothreitol (DTT), and 5 mM sodium fluoride (NaF) on dry ice. Basilar artery segments were then washed with acetone solution containing 5 mM DTT and 5 mM NaF. To lyophilize the basilar artery segments, segments were centrifuged under vacuum overnight. Total MLC was extracted as previously reported (12). Segments were weighed and incubated overnight at 4°C in 30 µl of extraction buffer (pH 8.6) containing 8 M urea, 10% glycerol, 0.04% bromophenol blue, and (in mM) 20 Tris base, 23 glycine, 10 DTT, 10 EGTA, and 5 NaF.

Western blot analysis using glycerol-urea gels was performed to determine levels of MLC phosphorylation (27). Briefly, 30 µl of each extraction sample were loaded on 10% acrylamide native gels and run for 5–6 h at 5 mA. Proteins were transferred onto cellulose membranes at a constant 25 V for 30 min. Cellulose membranes were then blocked with 0.1 M Tris base containing 0.05% Tween 20 and 0.5% gelatin. Primary MLC antibody (MY21) was used at 1:200 dilution followed by goat anti-rabbit secondary antibody conjugated with alkaline phosphatase. Membranes were then scanned to determine the levels of both phosphorylated and nonphosphorylated MLC by using a ChemiImager 4400 from AlphaInnotech. Percent MLC phosphorylation was calculated on the basis of integrated optical density values of the lower phosphorylated band divided by the total of the phosphorylated and nonphosphorylated (upper) bands.

Simultaneous measurement of contractile tension and cytosolic Ca²⁺.   To relate corresponding levels of MLC phosphorylation and cytosolic Ca²⁺, we measured both contractile force and cytosolic Ca²⁺ at the same durations of contraction used in the segments frozen for the phosphorylation measurements. In these experiments, basilar artery segments were incubated with the Ca²⁺-sensitive fluorescent dye fura 2-AM (5 µM) premixed with 0.01% Pluronic F127 for 4 h at 25°C under protection from light, as previously described (4). After loading, the segments were washed and mounted in a Jasco CAF-110 fluorometer at optimal passive stretch (23). Basilar artery segments were excited with wavelengths of 340 and 380 nm. The fluorescence emitted from the preparation was recorded from a photomultiplier circuit through a 500-nm filter. Values of the F340/F380 fluorescence ratios were continuously calculated and recorded by online computer.

After loading, basilar arteries were contracted with 122 mM K⁺, and the responses of both tension and Ca²⁺ to this concentration of K⁺ were taken as the 100% (maximal) values for both variables. We have previously shown that contraction with 122 mM K⁺ produces the same amount force per unit of cross-sectional area in both fetal and adult cerebral arteries (26). Basilar arteries were then washed, relaxed, and subsequently exposed to either K⁺ (50, 75, or 122 mM) or 5-HT (10^–7, 10^–6, or 10^–5 M) for several predesignated durations. Cytosolic Ca²⁺, tension, and time of contraction were recorded. At the end of each experiment, basilar arteries were incubated in Ca²⁺-free buffer containing 140 mM K⁺, 2 mM EGTA, and 10 µM ionomycin at pH 6.5 to enable measurement of the minimum fluorescence ratio. Basilar arteries were then exposed to 10 mM Ca²⁺ to obtain the maximum signal ratio. All measurements of fluorescence were then corrected for autofluorescence. Cytosolic Ca²⁺ concentrations under basal conditions were calculated by use of the Grynkiewicz equation (14) with a fixed fura K_d of 161 nM, as previously described (4).

Materials.   Fura 2-AM and Pluronic F127 were obtained from Molecular Probes. All Western blot materials and reagents, except for the myosin MY21 antibody, were ordered from Pierce. Unless otherwise indicated, all other materials and reagents, including the MY21 antibody, were purchased from Sigma.

Data analysis.   For all comparisons, peak values of contractile force, cytosolic Ca²⁺, and percent phosphorylation were taken from each time course measurement and used for subsequent statistical analyses. To compare two independent values of one variable between two groups, Student's t-tests were performed. In addition, a series of two-way ANOVAs was performed for each of the measured variables (contractile force, cytosolic Ca²⁺, and percent myosin phosphorylation) with age as one factor and treatment (K⁺ or 5-HT) as the other. Within each ANOVA data set, Barlett's test was run to verify homogeneity of variance (homoscedasticity). Post hoc differences were detected by use of Fisher's probable least significant difference test. To compare slopes of the relations between Ca²⁺ and contractile force, between Ca²⁺ and percent phosphorylation, and between percent phosphorylation and force, we calculated the individual slopes and intercepts of each three-point dataset and then analyzed the resulting values of slope and intercept using two-way ANOVAs with age as one factor and treatment (K⁺ or 5-HT) as the other. To plot these data, however, we averaged the values of each variable at each treatment level and plotted these averages against one another and then connected the points. The resulting line segments in the figures reflect the slopes of the averages, whereas all statistics were performed on the averages of the slopes relating these values. For all comparisons, power analyses were performed and all comparisons reported had power 0.8. Throughout the text, a significant difference implies P < 0.05 unless stated otherwise. All values are given as means and standard errors throughout the text. The reported values of n refer to the number of animals contributing to the mean and not the number of segments.

	RESULTS

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A total of 480 basilar artery segments were obtained from 38 fetuses and 32 adult sheep. The values of the dry weights for respective fetal and adult basilar arteries averaged 101 ± 6 and 162 ± 6 µg/5 mm, respectively (P < 0.05). At optimal length, the basal cytosolic Ca²⁺ concentrations measured via fura 2 were significantly greater in fetal (83.7 ± 2.5 nM) than in adult (55.0 ± 2.2 nM) arteries.

Effects of maturation on myofilament Ca²⁺ sensitivity.   When myofilament Ca²⁺ sensitivity was analyzed by plotting peak Ca²⁺ concentration against the peak value of tension for corresponding concentrations of K⁺ or 5-HT (Fig. 1), both fetal and adult arteries exhibited significant correlations between cytosolic Ca²⁺ and tension. The slopes of the Ca²⁺-tension relations for K⁺- and 5-HT-induced tone averaged 0.83 ± 0.07 and 3.77 ± 0.64, respectively, in fetal arteries. Corresponding values in adult arteries averaged 1.02 ± 0.08 and 2.00 ± 0.13. When these slopes were compared, the Ca²⁺-tension relations for both K⁺-induced and 5-HT-induced tone were significantly left shifted in fetal relative to adult arteries. When the extent of agonist-induced myofilament Ca²⁺ sensitization was estimated by comparing slopes for K⁺-induced and 5-HT-induced tone, the magnitude of this effect was significantly greater in fetal (slope = 2.94) than adult (slope = 0.98) arteries.

View larger version (16K): [in this window] [in a new window]   Fig. 1. Effects of maturation on myofilament Ca2+ sensitivity. A: 3 different concentrations of K+ (50, 75, and 122 mM) were used to contract fetal and adult ovine basilar arteries. Changes in cytosolic Ca2+ and contractile force were recorded simultaneously using fura 2-loaded arteries in a Jasco fluorometer. B: agonist-induced contractions were induced with 10–7, 10–6, and 10–5 M serotonin (5-HT). Peak values of 5-HT-induced tension are plotted against corresponding peak values in cytosolic Ca2+. For all means shown, error bars indicate standard errors for 5–7 animals in each group. Maximum values of tension and cytosolic Ca2+ were defined as the peak values observed in response to 122 mM K+. For both K+-induced and 5-HT-induced contractions, the relations between Ca2+ and tension were left shifted in fetal compared with adult arteries. As detailed in RESULTS, the slopes of these relations were significantly greater for 5-HT-induced than for K+-induced contractions.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Effects of maturation on K+-induced myosin light chain (MLC) phosphorylation. A: extent of MLC phosphorylation was measured at 0, 5, 10, 15, 20, 30, 60, and 120 s of exposure to K+ via Western blotting (inset). The time course of MLC phosphorylation after contraction with K+ was closely similar in fetal and adult arteries. Shown is the response to 122 mM K+. Arrows indicate the locations of bands containing unphosphorylated (MLC) and phosphorylated (MLC-P) myosin, respectively. B: peak values of cytosolic Ca2+ and phosphorylation were measured at 3 different concentrations of K+, and the relations between cytosolic Ca2+ and percent phosphorylation for each of these concentrations are indicated. For all means shown, error bars indicate standard errors for 5–7 animals in each group. As detailed in RESULTS, the slopes of these relations were significantly greater in adult than in fetal arteries, and the fetal curve was significantly right shifted relative to the adult curve.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Effects of maturation on 5-HT-induced MLC phosphorylation. A: as for K+-induced contractions, the extent of MLC phosphorylation was measured at 0, 5, 10, 15, 20, 30, 60, and 120 s of exposure to 5-HT via Western blotting (inset). The time course of MLC phosphorylation after contraction with 5-HT, however, was markedly different in fetal and adult arteries. Shown is the response to 10–5 M 5-HT. B: peak values of cytosolic Ca2+ and phosphorylation were measured at 3 different concentrations of 5-HT, and the relations between cytosolic Ca2+ and percent phosphorylation for each of these contractions are indicated. For all means shown, error bars indicate standard errors for 5–7 animals in each group. As detailed in RESULTS, the slopes of these relations were not significantly different in fetal and adult arteries but were significantly less than observed for K+-induced tone, indicating that 5-HT may depress the ability of Ca2+ to promote myosin phosphorylation in hypoxic basilar arteries.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Effects of maturation on the dependence of K+-induced force on myosin phosphorylation. A: time courses of contractile responses to K+ were highly similar in fetal and adult arteries. Shown here are the average responses to 122 mM K+. B: to assess differences in the ability of phosphorylated myosin to produce contractile force, peak values of tension were plotted against corresponding values of percent phosphorylation for each of the concentrations of K+ used to contract the arteries. For all means shown, error bars indicate standard errors for 5–7 animals in each group. As detailed in RESULTS, the slopes of these relations were similar in fetal and adult arteries, although the fetal curve was significantly left shifted relative to the adult curve, as indicated by differences in the corresponding y-intercept values.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Effects of maturation on the dependence of 5-HT-induced force on myosin phosphorylation. A: time courses of contractile responses to 5-HT were not significantly different in fetal and adult arteries. Shown here are the average responses to 10–5 M 5-HT. B: when peak values of tension were plotted against corresponding values of percent phosphorylation for each of the concentrations of 5-HT used to contract the arteries, the slopes of the fetal and adult curves were similar but the fetal curve was significantly left shifted relative to that of the adult, as indicated by significant differences in the corresponding y-intercept values. Equally important, the slopes of these relations were significantly greater than observed for K+-induced tone. Please see RESULTS for details. For all means shown, error bars indicate standard errors for 5–7 animals in each group.

	DISCUSSION

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Ovine basilar arteries from adult sheep acclimatized to chronic hypoxia for 110 days at 3,820 m exhibit an increased content of base-soluble protein, an enhanced capacity for endothelium-dependent relaxation, and a concomitant decrease in K⁺-induced contractile tension (18). In contrast, basilar arteries from fetal sheep born of ewes similarly acclimatized to hypoxia exhibit increased protein content of base-soluble protein but with no change in endothelial vasodilator function or responsiveness to K⁺. Whereas the mechanisms responsible for these marked differences in contractility remain largely unexplained, it is tempting to speculate that age-related differences in myofilament Ca²⁺ sensitivity are involved. In normoxic sheep, basal myofilament Ca²⁺ sensitivity is greater in fetal than adult sheep, and this difference is amplified in a GTP-dependent manner by 5-HT (1). Despite the well-established presence of this effect in other species and artery types (2, 5, 9), its role in age-related differences in vasoreactivity among arteries from chronically hypoxic animals remains unexplored.

The present data demonstrate that age-related differences in myofilament Ca²⁺ sensitivity persist but are altered in cerebral arteries from animals acclimatized to high-altitude hypoxia (Fig. 1). For both K⁺-induced and 5-HT-induced contractions, the relations between cytosolic Ca²⁺ and contractile force were left shifted in fetal compared with adult arteries. Equally important, 5-HT left shifted the relations between Ca²⁺ and force in both fetal and adult arteries, and these shifts were associated with significant increases in slope, thus demonstrating agonist-induced Ca²⁺ sensitization in both age groups. Together, these data suggest that age-related differences in vasoreactivity observed in cerebral arteries from chronically hypoxic animals can be explained at least in part by corresponding differences in myofilament Ca²⁺ sensitivity.

To further understand the observed age-related differences in Ca²⁺ sensitivity, we adopted a simple model of Ca²⁺ sensitivity originally advanced by Murphy and colleagues (20, 21). As proposed by this model, myofilament Ca²⁺ sensitivity can be viewed as the integrated sum of the relation between cytosolic Ca²⁺ and myosin phosphorylation and the subsequent relation between myosin phosphorylation and contractile force (Fig. 6). From this perspective, the relation between cytosolic Ca²⁺ concentration and the extent of MLC phosphorylation reflects the composite effects of the many mechanisms that together determine the extent of Ca²⁺-dependent MLC phosphorylation. In addition to this first family of mechanisms, which focuses primarily on thick filament events culminating in myosin phosphorylation, there is a second family of mechanisms that govern actin's ability to interact with phosphorylated myosin to produce contractile force. This second family of mechanisms involving predominantly thin filament proteins is characterized by the relation between the extent of MLC phosphorylation and contractile force. The key to employing this model experimentally is thus the measurement of MLC phosphorylation, together with simultaneous measures of cytosolic Ca²⁺ and contractile force. Owing to the dynamic nature of each of these variables, together with the fact that each reaches its maximum value at a different time, it is essential to obtain at least semicontinuous measures of each variable to enable the analysis. However, because each measurement of phosphorylation requires homogenization of one artery sample, it is not possible to obtain time courses for all three essential variables in a single artery segment. To solve this problem, we prepared up to eight serial segments from a single basilar artery. These equivalent sections were then separately assigned for simultaneous measurements of contractile force and cytosolic Ca²⁺, or for serial measurements of contractile force and myosin phosphorylation. The resulting data were then integrated by matching equivalent levels of treatment, time of contraction, and percent maximum contractile force. Although this approach assumed that adjacent artery segments were highly similar in terms of their contractile properties, this is a reasonable approach that has been used extensively in vascular studies (6, 25, 33). Given this assumption, this experimental approach provided a unique opportunity to partition regulation of myofilament Ca²⁺ sensitivity into mechanisms involved with regulation of the relations between Ca²⁺ and myosin phosphorylation (thick filament mechanisms) and mechanisms involved with regulation of the relations between myosin phosphorylation and force development (thin filament mechanisms).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Myofilament Ca2+ sensitivity: a composite of interactions among Ca2+, myosin phosphorylation, and force production. The thick filament-based mechanisms that govern myosin activation can be characterized by the relation between cytosolic Ca2+ concentration and MLC phosphorylation. In turn, the ability of phosphorylated myosin to interact with actin to produce force can be characterized by the relation between MLC phosphorylation and contractile force. Thus the 2 independent functions describing thick filament-based and thin filament-based mechanisms can be summed algebraically to yield the relation between cytosolic Ca2+ concentration and contractile force, which is classically defined as myofilament Ca2+ sensitivity.

In contrast to the relations between cytosolic Ca²⁺ concentration and the extent of MLC phosphorylation, the relations between MLC phosphorylation and force development appeared to be enhanced in fetal compared with adult arteries during K⁺-induced contractions (Fig. 4). Upon stimulation with 5-HT, the curves relating percent phosphorylation to contractile force were left shifted and exhibited steeper slopes (Fig. 5) than observed for K⁺ contractions. This result suggests that 5-HT-induced increases in myofilament Ca²⁺ sensitivity are mediated largely through increases in the ability of thin filaments to interact with phosphorylated myosin. Because the magnitude of this effect was significantly greater in fetal than adult ovine basilar arteries acclimatized to altitude hypoxia, age-related differences in agonist-induced Ca²⁺ sensitization also appear attributable to receptor-mediated increases in thin filament reactivity. In many respects, this is a novel finding because it suggests a possible direct coupling between activation of a G protein receptor and increased reactivity of actin filaments with phosphorylated myosin. The ability of G protein receptor activation to increase overall myofilament Ca²⁺ sensitivity has long been attributed largely to inhibition of MLC phosphatase activity mediated through activation of rho-kinase (32). Direct connection of these receptors to changes in the ability of thin filaments to interact with myosin has not been widely discussed. On the other hand, it is well established that Rho GTPases interact extensively with the actin cytoskeleton and through these interactions can dramatically influence smooth muscle contractile behavior (16, 42). Another Rho family GTPase-dependent kinase, p21-activated protein kinase, also can modulate actin reactivity (37), but how this kinase may be influenced by G protein receptor activation remains unknown at present. Other proteins, including heat shock protein (HSP)20 (29) and HSP27 (8, 41), might also modulate actin reactivity through pathways potentially coupled to G protein receptor activation, but, again, the physiological significance of these mechanisms remains unclear. Despite these uncertainties, there is little doubt that multiple mechanisms exist that could potentially explain the coupling observed between 5-HT-receptor activation and increased actin reactivity. The exact identity of these mechanisms remains a promising topic for further investigation, particularly in relation to the apparent age dependence of this coupling in chronically hypoxic basilar arteries.

Overall, the present results reinforce the view that myofilament Ca²⁺ sensitivity is greater in immature than in mature cerebral arteries, even after acclimatization to high-altitude hypoxia. Relative to the contractile effects of K⁺, which presumably reflect basal Ca²⁺ sensitivity, age-related differences in Ca²⁺ sensitivity were enhanced during agonist-induced contractions, and these effects were attributable primarily to an increased ability of phosphorylated myosin to interact with thin filaments to produce contractile force. In contrast, the ability of Ca²⁺ to promote myosin phosphorylation was attenuated in immature compared with mature arteries, and this attenuation was amplified during agonist-induced contractions. Thus the effects of maturation on myofilament Ca²⁺ sensitivity appeared balanced between opposing effects on thick and thin filament mechanisms, such that postnatal maturation upregulated thick filament and downregulated thin filament contributions to overall myofilament Ca²⁺ sensitivity. This shift is particularly interesting given a greater contractile dependence on extracellular Ca²⁺ entry, and a lesser dependence on intracellular Ca²⁺ release, in immature compared with mature arteries (4). Together, these findings demonstrate that postnatal maturation dramatically reprioritizes the mechanisms regulating contractility and that this process involves not only the source of activator Ca²⁺ but also the pathways governing MLC phosphorylation and the ability of phosphorylated myosin to interact with thin filaments to produce force in hypoxia-acclimatized animals. From a clinical perspective, these findings also suggest that the pharmacological strategies for managing cerebrovascular homeostasis will be quite different in the hypoxic neonate and the hypoxic adult. For example, approaches designed to enhance myosin phosphorylation (e.g., via increased Ca²⁺ influx or decreased MLC phosphatase activity) will be more effective in hypoxic neonates than in adults owing to the downregulation of Ca²⁺-phosphorylation relations in neonates. The present data also argue against the use of phosphodiesterase inhibitors for treatment of the long-term neonatal hypoxia associated with persistent pulmonary hypertension (35), because these inhibitors raise cyclic nucleotide levels that can enhance MLC phosphatase activity (38) and further reduce levels of myosin phosphorylation in the hypoxic neonate. In view of the high-frequency and long-lasting morbidity associated with cerebrovascular dysfunction and pathophysiology in hypoxic neonates, particularly those born prematurely (7, 13), further exploration of the mechanisms governing myosin phosphorylation and thin filament interactions with phosphorylated myosin in immature arteries appears well justified and offers great potential for identification of new strategies of pharmacological management of these fragile patients.

	GRANTS

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The work reported here has been supported by National Heart, Lung, and Blood Institute Grants HL-54120 and HL-064867 to W. J. Pearce and a grant from the Polycystic Kidney Disease Foundation to S. M. Nauli.

	FOOTNOTES

 

Address for reprint requests and other correspondence: W. J. Pearce, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, CA 92350 (E-mail: wpearce{at}llu.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.

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