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Postnatal maturation modulates relationships among cytosolic Ca²⁺, myosin light chain phosphorylation, and contractile tone in ovine cerebral arteries

¹Department of Physiology and Pharmacology, Center for Perinatal Biology, Loma Linda University, School of Medicine, Loma Linda, California; and ²Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada

Submitted 5 June 2007 ; accepted in final form 26 July 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The present study tests the hypothesis that age-related changes in patterns of agonist-induced myofilament Ca²⁺ sensitization involve corresponding differences in the relative contributions of thick- and thin-filament regulation to overall myofilament Ca²⁺ sensitivity. Posterior communicating cerebral arteries from term fetal and nonpregnant adult sheep were used in measurements of cytosolic Ca²⁺, myosin light chain (MLC) phosphorylation, and contractile tensions induced by varying concentrations of K⁺ or serotonin [5-hydroxytryptamine (5-HT)]. The results were used to assess the relative contributions of the relationships between cytosolic Ca²⁺ and MLC phosphorylation (thick-filament reactivity), along with the relationships between MLC phosphorylation and contractile tension (thin-filament reactivity), to overall myofilament Ca²⁺ sensitivity. For K⁺-induced contractions, both fetal and adult arteries exhibited similar basal myofilament Ca²⁺ sensitivity. Despite this similarity, thick-filament reactivity was greater in fetal arteries, whereas thin-filament reactivity was greater in adult arteries. In contrast, 5-HT-induced contractions exhibited increased myofilament Ca²⁺ sensitivity compared with K⁺-induced contractions for both fetal and adult cerebral arteries, and the magnitude of this effect was greater in fetal compared with adult arteries. When interpreted together with our previous studies of 5-HT-induced myofilament Ca²⁺ sensitization, we attributed the present effects to agonist enhancement of thick-filament reactivity in fetal arteries mediated by G protein receptor activation of a PKC-independent but RhoA-dependent pathway. In adult arteries, agonist stimulation enhanced thin-filament reactivity was also probably mediated through G protein-coupled activation of RhoA-dependent and PKC-independent mechanisms. Overall, the present data demonstrate that agonist-enhanced myofilament Ca²⁺ sensitivity can be partitioned into separate thick- and thin-filament effects, the magnitudes of which are different between fetal and adult cerebral arteries.

myofilament Ca²⁺ sensitivity; myosin phosphorylation; thick- and thin-filament regulation; serotonin

Another important modulator of cerebrovascular Ca²⁺ sensitivity is the ability of agonists such as serotonin [5-hydroxytryptamine (5-HT)] to bind to cell-surface G protein-coupled receptors and to enhance myofilament sensitivity to Ca²⁺. Agonist-induced Ca²⁺ sensitization is significantly upregulated in fetal compared with adult arteries (5), but the exact mechanisms that enhance Ca²⁺ sensitivity remain unclear, particularly in the fetus. To help identify which mechanisms are involved in agonist-induced myofilament Ca²⁺ sensitization, the overall relation between cytosolic Ca²⁺ concentration and contraction can be divided into two interacting families of mechanisms. The first of these includes the mechanisms that govern the relationships between cytosolic Ca²⁺ concentration and the extent of myosin light chain (MLC) phosphorylation, which can be referred to collectively as thick-filament regulation. The second main family of mechanisms governs the relationships between the extent of MLC phosphorylation and the magnitude of contractile force and can be referred to collectively as thin-filament regulation (28). To date, most studies of myofilament Ca²⁺ sensitization in cerebral arteries have focused on thick-filament regulation (4, 5), and our previous studies have revealed that it is independent of PKC activity but requires Rho-kinase activity (5, 41). Conversely, the effects of G protein receptor activation on thin-filament regulation and its contribution to overall myofilament Ca²⁺ sensitization have not been well investigated. Evidence obtained in basilar arteries from chronically hypoxic sheep, however, suggests that the contributions of both thick and thin filament reactivity to overall myofilament Ca²⁺ sensitization may vary significantly with postnatal age (32). Given that cerebrovascular myofilament Ca²⁺ sensitivity also varies with postnatal age in arteries from normoxic animals, it seems likely that the independent mechanisms regulating thick- and thin-filament reactivity may also vary with postnatal age. Thus the present study is focused on the hypothesis that age-related changes in patterns of agonist-induced myofilament Ca²⁺ sensitization involve corresponding differences in the relative contributions of thick- and thin-filament regulation to overall myofilament Ca²⁺ sensitivity.

Because K⁺-induced contractions do not require the activation of cell-surface G protein-coupled receptors and subsequent enhancement of myofilament Ca²⁺ sensitivity (5, 31, 33), the experimental approach in the present study used K⁺-induced contractions to define baseline characteristics of both thick- and thin-filament reactivity in term fetal and adult ovine cerebral arteries. Because 5-HT is a potent agonist that activates G protein-coupled receptors (5-HT-2) and enhances myofilament Ca²⁺ sensitivity in ovine cerebral arteries (4, 5), the present experimental design also employed 5-HT-induced contractile responses to define shifts in thick- and thin-filament reactivity. To separate thick-filament and thin-filament reactivity, the protocols included independent measurements of cytosolic Ca²⁺ concentration, the extent of MLC phosphorylation, and contractile force in ovine posterior communicating cerebral arteries from both term fetal and nonpregnant adult sheep. Together, these experiments offer a unique view of age-related differences in agonist-induced changes in the relationships among cytosolic Ca²⁺, MLC phosphorylation, and contractile force and illuminate the relative contributions of thick- and thin-filament reactivity to basal and agonist-induced myofilament Ca²⁺ sensitivity.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
General preparation. All procedures and protocols were approved by the Institutional Animal Care and Use of Committee of Loma Linda University and strictly followed all federal rules and regulations governing the care and use of laboratory animals. Posterior communicating arteries were harvested from young nonpregnant adult sheep (18–24 mo old) and near-term (140 days of gestation) fetuses euthanized with an overdose of pentobarbital sodium (60 mg/kg iv). Following their removal from the surface of the brain, the arteries were placed in Na⁺-Krebs buffer solution that was continuously bubbled with 95% O₂-5% CO₂ and contained (in mM) 120 NaCl, 25.6 NaHCO₃, 5.17 KCl, 2.49 MgSO₄, 1.60 CaCl₂, 2.56 dextrose, 0.0270 EGTA, and 0.114 ascorbic acid. Extracellular and loose connective tissue was gently removed. The arteries were then cut into 4-mm segments and mounted on wires suspended between a force transducer and a micrometer used to adjust resting length and passive diameter of each individual tissue segment. The tissue diameter recordings were used to obtain the optimal stretch ratios necessary to obtain maximal isometric force in each segment, as previously described (11). To minimize endothelium-mediated effects, we removed the vascular endothelium by gently rotating each arterial segment around its mounting wire several times to scrape the entire luminal surface. All buffers also included 100 µM N^G-nitro-L-arginine methyl ester and 100 µM nitro-L-arginine to ensure complete inhibition of endothelial nitric oxide (NO) synthase and NO release.

Simultaneous determination of contractile tension and MLC phosphorylation. Tissue segments were equilibrated for 30 min in Na⁺-Krebs buffer bubbled with 95% O₂-5% CO₂ at 38°C (normal ovine core temperature). Unstressed artery diameter (slack length) was defined as the artery diameter observed at a passive tension of 0.03 g (D₀), and this measurement was made in each artery segment. Thereafter, artery stretch was calculated as a stretch ratio (D/D₀), which was the working diameter divided by the unstressed diameter. Each segment studied was stretched to an optimal length [D/D₀ of 1.9 (39)] and allowed to equilibrate until a stable resting tension was obtained (usually in about 1 h). The artery segments were then contracted with 120 mM K⁺-Krebs buffer solution to determine tissue viability and maximal contractile tension (designated as 100%). After reequilibration in Na⁺-Krebs buffer solution for 45 min, artery segments were contracted with one of three of K⁺ concentrations (50, 75, or 120 mM) or one of three 5-HT concentrations (10^–7, 10^–6, or 10^–5 M) to obtain graded contractile time-course responses for a standardized duration (0–120 s). Following this first contraction, the arteries were reequilibrated in Na⁺-Krebs buffer solution for 45 min and then contracted again with the same agonist concentration used for the first contraction. At predetermined times during this second contraction (K⁺: 0, 3, 6, 9, 12, 15, and 18 s; and 5-HT: 0, 4, 8, 12, 16, 20, and 24 s), the segments were immersed into freezing cold (–70°C) acetone containing 10% trichloroacetic acid, 10 mM dithiothreitol (DTT), and 5 mM sodium fluoride (NaF). Artery segments were then washed with acetone containing 10 mM DTT and 5 mM NaF and air dried. Once the dry weight of each segment was measured, the segments were extracted in buffer (pH 8.6) containing 8 M urea, 10% glycerol, and 0.04% bromophenol blue and (in mM) 20 Tris base, 23 glycine, 10 DTT, 10 EGTA, and 5 NaF. The tissue-to-buffer ratio was carefully controlled to be 1 mg/250 µl. The extractions were carried out for 90 min at room temperature, after which the extracts were assayed for total protein content using the Bradford BCA reagent (Bio-Rad, Hercules, CA) calibrated against known amounts of bovine serum albumin dissolved in extraction buffer.

To quantify the extent of MLC phosphorylation, an MLC standard pool was prepared from adult common carotid arteries. Aliquots of samples and MLC standards were always analyzed together via immunoblotting using 10% urea gels. The separation gel consisted of 30%/0.8% acrylamide, 1.5 M Tris at pH 8.6 in 40% glycerol, and 10% ammonium persulfate. The stacking gel consisted of 30%/1.6% acrylamide, 1.0 M Tris at pH 6.8 with 10% urea, and 10% ammonium persulfate. A 0.05 M Tris and 0.1 M glycine tank buffer was used. Identical amounts of total protein were loaded for each sample and run for 2.5 h at 200 V. Proteins were transferred onto nitrocellulose membranes at constant current (50 mA) for 3 h. The nitrocellulose membranes were then blocked with TBS buffer (pH 7.5) containing 5% milk for 1 h. After being blocked, the membranes were placed in TBS buffer with 5% milk and 0.1% Tween-20 and primary mouse monoclonal anti-MLC20 (clone MY-21, Sigma) at a titer of 1:300 for 3 h and then visualized with horseradish peroxidase-conjugated goat anti-mouse secondary antibody at a titer of 1:1,000 for 1 h. Membranes were scanned to determine both the levels of nonphosphorylated and phosphorylated MLC using an AlphaInnotech ChemiImager. Integrated optical density values (IDVs) for the MLC protein standards were plotted against the mass of protein loaded to give an IDV-mass curve. The IDVs for the unknown samples were read from the standard curve to give relative mass values for both the nonphosphorylated (upper band) and phosphorylated (lower band) MLC blots. %MLC phosphorylation was calculated as the phosphorylated mass divided by the total of the phosphorylated and nonphosphorylated masses.

Measurement of cytosolic Ca²⁺. To measure cytosolic Ca²⁺, arterial segments were mounted in a Jasco fluorescence photometer (Jasco CAF-110), and slack length was determined, after which the segments were stretched to a D/D₀ ratio of 1.3 and were then loaded with 5 µM fura-2 AM in the presence of 0.01% pluronic acid for 3 h at 25°C. The loading buffer also included (in mM) 122 NaCl, 25 HEPES, 5.16 KCl, 2.40 MgSO₄, 50 µM EDTA, 11.1 dextrose, and 1.60 CaCl₂. Following loading, the segments were washed in the same buffer without fura-2 AM and then gradually warmed to 38°C (normal ovine core temperature) and bubbled with 95% O₂-5% CO₂. After 30 min of equilibration to allow deesterification of fura-2 AM, the initial 340-to-380 ratio was recorded. Following this wash and equilibration, each segment was stretched to optimal length (D/D₀ = 1.9) and allowed to equilibrate for 1 h. Once responses had stabilized, the segments were contracted with 120 mM K⁺-HEPES buffer, containing (in mM) 120 KCl, 25 HEPES, 11.1 dextrose, 5.16 NaCl, 2.4 MgSO₄, 1.6 CaCl₂, and 0.05 EDTA, to obtain maximal responses for both tension and cytosolic Ca²⁺ that were taken as the 100% (maximum) values. After reequilibration with Na⁺-HEPES buffer, the arteries were contracted with one of the K⁺ or 5-HT concentrations used in the phosphorylation protocol, and both contractile tensions and cytosolic Ca²⁺ were recorded for 5 min. Artery segments were washed and reequilibrated with fresh buffer and then exposed to buffer containing 2 mM EGTA without Ca²⁺ in the presence of 10 µM ionomycin to enable determination of minimum fluorescence intensity values at 380 nm. After stable 340-to-380 ratio values were obtained, Ca²⁺ was added at a final concentration of 24 µM to obtain maximum fluorescence values at 380 nm. Autofluorescence was then determined via manganese quench as previously described (46). Intracellular Ca²⁺ concentrations were then determined using the Grynkiewicz equation (15).

Measurement of MLC kinase and MLC phosphatase abundance in fetal and adult cerebral arteries. Adult and fetal posterior communicating cerebral arteries were harvested to compare age-related differences in MLC kinase (MLCK) and MLC phosphatase (MLCP) abundances. Arteries were pulverized in liquid nitrogen and then glass-on-glass homogenized in extraction buffer containing 50 mM Tris·HCl (pH 7.5), 10 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.5 M NaCl, 0.5% protease inhibitor cocktail (P8340, Sigma), and 0.1% -mercaptoethanol. The tissue-to-buffer ratio used was 1 mg wet wt of tissue sample to 6 µl of extraction buffer (see Simultaneous determination of contractile tension and MLC phosphorylation) for both fetal and adult cerebral arteries. After homogenization, the sample extracts were centrifuged at 15,000 g for 15 min at 4°C, and the supernatants were analyzed for total protein content using Bio-Rad Bradford Protein Assay (No. 500-0006). Samples from both fetal and adult cerebral tissue extracts containing 15 µg of total protein were combined with equal volumes of sample buffer (containing 0.125 M Tris·HCl, 4% SDS, 20% glycerol, and 10% -mercaptoethanol, pH 6.8) and loaded onto a 15-lane, 8% SDS-PAGE gel. In one lane, molecular weight protein markers (No. 161-0374, Bio-Rad) were loaded, and in five other lanes, different amounts of a relative standard (made from ovine posterior communicating cerebral arteries) were loaded to enable construction of a standard curve. Proteins were separated at 15 mA per gel for 90 min using Bio-Rad Mini-Protean system and then wet transferred onto nitrocellulose (Protran, Schleicher and Schuell) at 270 mA for 2 h with the use of an ice bath to prevent overheating. The membrane was then blocked overnight at 4°C [5% milk in TBS (pH 7.5) and 0.1% Tween-20] and then probed with mouse anti-MLCK antibody (No. M7905 at 1:7,000 in blocking agent, Sigma) or mouse MYPT1 antibody (No. 612164 at 1:3,000 in blocking agent, Transduction Laboratories) for 2 h. After a 15-min wash in blocking agent, a horseradish peroxidase-conjugated goat anti-mouse secondary antiserum (No. 1858413, Pierce) was applied at 1:1,000 in blocking agent for 1 h, followed by two 15-min washes in blocking agent and a 5-min TBS wash. Pierce Super Signal West Femto Substrate was used, in conjunction with the AlphaInnotech camera and densitometry software, to visualize and quantify the chemiluminescent standard curve and the MLCK and MLCP bands. The MLCK and MLCP bands were subsequently normalized with the use of a standard curve prepared from pooled ovine cerebral arteries relating mass and optical density. This approach minimized nonlinearity and enabled highly consistent quantification of abundances. The relative abundances were calculated such that fetal values equaled unity.

Measurement of relative heat shock protein 27 and caldesmon abundance in fetal and adult cerebral arteries. Adult and fetal posterior communicating arteries were also analyzed to reveal age-related differences in heat shock protein 27 (HSP27) and caldesmon abundance. Artery segments from both age groups were frozen in liquid nitrogen, pulverized, and then glass-on-glass homogenized in an extraction buffer, containing (in mM) 190 NaCl, 50 Tris·HCl, 6 EDTA, 5 NaF, 0.25 PMSF, 2 leupeptin, and 1 Na₃VO₄ at pH 7.5, at a 1 mg/20 µl tissue-to-buffer ratio. Following centrifugation for 10 min at 10,000 g at 4°C, each sample was analyzed for total protein concentration using Bio-Rad Bradford Protein Assay (BCA No. 500-006), combined with an equal volume of sample buffer [containing 0.125 M Tris·HCl, 4% SDS, 20% glycerol, and 10% -mercaptoethanol (pH 6.8)] and loaded onto a 15-lane 12% SDS-PAGE gel. Different amounts of a relative standard (made from ovine arteries) were loaded onto each gel to enable construction of a standard curve. Proteins were separated at 30 mA for 90 min using Bio-Rad Mini-Protean system and then wet transferred onto nitrocellulose (Protran, Schleicher and Schuell) at 400 mA for 2 h with the use of an ice bath to prevent overheating. The membranes were blocked for 30 min at room temperature in a solution containing 5% milk, 0.15% Tween-20, 150 mM Tris·HCl, and 150 mM NaCl (pH 7.5) and then probed with either mouse anti-HSP27 monoclonal antibody (No. YSGSPA800 at 1:5,000 in blocking agent, Accurate Chemical and Scientific) or mouse anti-caldesmon monoclonal antibody (No. 07-135 at 1:4,000, in blocking agent, Upstate Biotechnology) for 2 h. After 15 min of washing in blocking agent, horseradish peroxidase-conjugated goat anti-mouse secondary antiserum (No. 1858413, Pierce) was applied at 1:1,000 in the blocking agent for 1 h followed by three 5-min washes in the blocking agent and 5 min in TBS. Pierce SuperSignal West Femto Maximum Sensitivity Substrate was used, in conjunction with an AlphaInnotech ChemiImager camera and densitometry software, to visualize and quantify the chemiluminescent signals from both the HSP27 and caldesmon bands. The bands were subsequently normalized with the use of a standard curve, prepared from pooled ovine cerebral arteries, relating mass and optical density.

Calculation, data analysis, and statistics. For all comparisons, peak values of contractile tension, cytosolic Ca²⁺, and %MLC phosphorylation were analyzed for each time-course measurement and used to determine the values used in statistical analyses. Reported values of contractile tensions induced by graded concentrations of both K⁺ and 5-HT were expressed as percentages relative to the maximum contractile capacity of each artery, as defined by the responses to Krebs buffer containing 120 mM K⁺. Measurements of cytosolic Ca²⁺ induced by graded concentrations of both K⁺ and 5-HT were expressed as percentages relative to maximum cytosolic Ca²⁺ produced by exposure to 120 mM K⁺. Myosin phosphorylation ratios were determined as the ratio between the mass of the phosphorylated band divided by the sum of the masses of the phosphorylated and unphosphorylated bands times 100. All statistical comparisons were performed using two-way ANOVA and Duncan's post hoc analysis. The significance of correlations between peak tension, peak cytosolic Ca²⁺, and peak %MLC phosphorylation were determined with Fisher r-to-z conversion at P < 0.05. Homogeneity of variance was verified for all ANOVA procedures using Cochran's analysis. Statistical power reached minimum value of 0.8 unless stated otherwise. All values are reported as means ± SE for the number of animals studied.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Effects of maturation on myofilament Ca²⁺ sensitivity. Basal and agonist-induced myofilament Ca²⁺ sensitivities were determined by plotting the relationships between peak cytosolic Ca²⁺ concentrations and peak values of contractile tensions produced in response to graded concentrations of either K⁺ or 5-HT. Peak cytosolic Ca²⁺ concentration was significantly correlated with peak contractile tensions produced in response to K⁺ (r = 0.931 for fetus, and r = 0.806 for adult) and 5-HT (r = 0.629 for fetus, and r = 0.634 for adult) in both age groups (Fig. 1). However, these relationships did not vary significantly with age when the arteries were contracted with K⁺ (Fig. 1, top). K⁺-induced contractions at 50, 75, and 120 mM yielded similar peaks in cytosolic Ca²⁺ concentration in fetal (48 ± 5.1%, 70.6 ± 6.6%, and 100 ± 0.0% of 120 mM K⁺ maximum, respectively) and adult (47.2 ± 6.3%, 64.2 ± 4.2%, 100 ± 0.0% of 120 mM K⁺ maximum, respectively) cerebral arteries. Peak contractile tensions did not differ significantly with age and averaged 29.2 ± 2.0%, 64.5 ± 2.7%, 87.7 ± 1.8% of 120 mM K⁺ maximum in fetal arteries and 37.9 ± 2.0%, 60.4 ± 5.6, and 88.1 ± 1.3% of 120 mM K⁺ maximum in adult arteries, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Effects of maturation on myofilament Ca2+ sensitivity. Top: 3 different concentrations of K+ (50, 75, and 120 mM) were used to contract fetal and adult ovine posterior communicating cerebral arteries. Bottom: agonist-induced contractions were induced with 10–7, 10–6, 10–5 M serotonin [5-hydroxytryptamine (5-HT)]. Changes in cytosolic Ca2+ and contractile tensions were measured simultaneously using fura-2-loaded arteries in Jasco fluorometer. Peak values for K+- and 5-HT-induced tensions were plotted against peak values of cytosolic Ca2+ and expressed as a percentage of the corresponding response to 120 mM K+ in each segment. There were no significant differences in peak cytosolic Ca2+ (*) or peak tension (#) between fetal and adult cerebral arteries for any concentration of K+ used. For 5-HT-induced contractions, peak tensions (#) were significantly greater in adult than in fetal cerebral arteries at 10–7 and 10–5 5-HT. Also, peak cytosolic Ca2+ (*) was significantly greater in adult than fetal cerebral arteries contracted with 10–6 and 10–5 5-HT. Error bars indicate standard errors for arteries from 6 adults and 6 fetuses. %Kmax, percent of maximum contractile response to 120 mM K+ in Krebs buffer. *Fetal values were significantly different than adult values of peak cytosolic Ca2+ following identical treatments with 5-HT. #Fetal values were significantly different than adult values of peak tension following identical treatments with 5-HT.

Effects of maturation on relationships between Ca²⁺ and MLC phosphorylation: thick-filament reactivity. The results of K⁺- and 5-HT-induced peaks in cytosolic Ca²⁺ concentration values from Fig. 1 were plotted against corresponding values of peak %MLC phosphorylation to determine the contribution of thick-filament reactivity to the overall myofilament Ca²⁺ sensitivity in both fetal and adult cerebral arteries (Fig. 2). In fetal arteries, contractions produced by graded K⁺ concentrations of 50, 75, and 120 mM generated peak myosin phosphorylation values that averaged 36.8 ± 6.0%, 51.9 ± 5.2%, and 53.8 ± 1.7%, respectively. In adult arteries, corresponding values averaged 38.4 ± 2.1%, 42.5 ± 4.2%, and 58.2 ± 3.0%, respectively (Fig. 2, top). For K⁺-induced contractions, peak cytosolic Ca²⁺ and myosin phosphorylation were significantly correlated in both fetal (r = 0.719) and adult (r = 0.696) arteries. However, this relationship was significantly left shifted only at 75 mM K⁺ in fetal relative to adult arteries, suggesting a modest age-related difference in thick-filament reactivity.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Effects of maturation on K+- and 5-HT-induced thick-filament reactivity. Top: peak values of cytosolic Ca2+ plotted against peak values of percent myosin light chain (%MLC) phosphorylation induced by 3 concentrations of K+ (50, 75, and 120 mM) to determine K+-induced thick-filament reactivity in both fetal and adult posterior communicating cerebral arteries. Bottom: peak values of cytosolic Ca2+ plotted against peak values of %MLC phosphorylation induced by 3 concentrations of 5-HT (10–7, 10–6, and 10–5 M) to determine agonist-induced thick-filament reactivity in both fetal and adult posterior communicating cerebral arteries. Peak cytosolic Ca2+ values are expressed as a percentage of the corresponding response to 120 mM K+. For K+-induced contractions, peak %MLC phosphorylation () was significantly greater in fetal than in adult cerebral arteries only at 75 mM K+. There was no significant difference in peak cytosolic Ca2+ (*) between fetal and adult cerebral arteries for any concentration of K+ used. For 5-HT-induced contractions, there was no significant difference in peak %MLC phosphorylation () between fetal and adult cerebral arteries at all concentrations of 5-HT used. However, peak cytosolic Ca2+ (*) was significantly greater in adult than in fetal cerebral arteries contracted with 10–6 and 10–5 5-HT. Error bars indicate standard errors for arteries from 6 adults and 6 fetuses. Fetal values were significantly different than adult values of peak %MLC phosphorylation following similar treatments with K+. *Fetal values were significantly different than adult values of peak cytosolic Ca2+ following similar treatments with 5-HT.

Effects of maturation on relationships between MLC phosphorylation and tension: thin-filament reactivity. The results of K⁺ and 5-HT-induced peak values of contractile tension from Fig. 1 were plotted against corresponding peak %MLC phosphorylation (Fig. 2) to determine the contribution of changes in thin-filament reactivity to changes in overall myofilament Ca²⁺ sensitivity in both fetal and adult cerebral arteries (Fig. 3). The relationships between MLC phosphorylation and contractile force were significantly correlated in both fetal and adult cerebral arteries contracted with either K⁺ (r = 0.794 for fetal, and r = 0.764 for adult arteries) or 5-HT (r = 0.800 for fetal, and r = 0.930 for adult arteries). For K⁺-induced contractions, the relationship of peak contractile tension to peak %MLC phosphorylation was significantly left shifted in adult compared with fetal cerebral arteries only at 75 mM K⁺ (Fig. 3, top). Another important feature of these results was that both fetal and adult arteries achieved similar maximal contractile forces during 120 mM K⁺ contractions (87.7 ± 1.8% and 88.1 ± 1.3% of 120 mM K⁺ maximum, respectively). In contrast, fetal arteries required a lower concentration of K⁺ (75 mM) to achieve maximal MLC phosphorylation (51.9 ± 2.1%) than did adult arteries (58.2 ± 3.0% at 120 mM).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effects of maturation on K+- and 5-HT-induced thin-filament reactivity. Top: peak values of %MLC phosphorylation values plotted against peak contractile tensions generated by 3 concentrations of K+ (50, 75, and 120 mM) to determine K+-induced thin-filament reactivity in both fetal and adult posterior communicating cerebral arteries. Bottom: peak values of %MLC phosphorylation values plotted against peak contractile tensions generated by 3 concentrations 5-HT (10–7, 10–6, and 10–5 M) to determine agonist-induced thick-filament reactivity in both fetal and adult posterior communicating cerebral arteries. Peak contractile tension values are expressed as a percentage of the corresponding response to 120 mM K+. For K+-induced contractions, peak %MLC phosphorylation () was significantly greater in fetal than in adult cerebral arteries only at 75 mM K+. There was no significant difference in peak tension between fetal and adult cerebral arteries for any concentration of K+ used. For 5-HT-induced contractions, there was no significant difference in peak %MLC phosphorylation between fetal and adult cerebral arteries at any concentration of 5-HT used. However, peak tensions (#) were significantly greater in adult than in fetal cerebral arteries contracted with 10–7 and 10–5 5-HT. Error bars indicate standard errors for arteries from 6 adults and 6 fetuses. Fetal values were significantly different than adult values of peak %MLC phosphorylation following similar treatments with K+. #Fetal values were significantly different than adult values of peak tension following identical treatments with 5-HT.

Contractile protein abundance in fetal and adult cerebral arteries. The relative abundances of the thick-filament regulatory proteins MLCK and MLCP, as measured via Western blot analysis, were significantly greater in adult than in fetal cerebral arteries (Fig. 4, A and B). When artery abundances were normalized relative to fetal values, the relative abundances averaged 1.0 ± 0.39 and 3.8 ± 0.74 for MLCK and 1.0 ± 0.09 and 1.8 ± 0.16 for MLCP in fetal and adult arteries, respectively. The relative abundances of both caldesmon and HSP27 were also significantly greater in adult than in fetal cerebral arteries (Fig. 4, C and D) and averaged 1.0 ± 0.14 and 3.2 ± 0.67 for caldesmon and 1.0 ± 0.04 and 2.0 ± 0.19 for HSP27 in fetal and adult arteries, respectively.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Contractile protein abundance in fetal and adult cerebral arteries. The relative abundances of MLC kinase (MLCK; A), MLC phosphatase (MLCP; B), caldesmon (C), and heat shock protein 27 (HSP27; D) were determined by Western blot analysis and normalized relative to an external standard prepared from pooled carotid or cerebral artery homogenates. The relative abundances for all four proteins were significantly greater (*) in adult than fetal cerebral arteries. Error bars indicate SE for arteries from 5 adults and 5 fetuses.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Overall, contractile force in vascular smooth muscle is governed by two main variables: cytosolic Ca²⁺ concentration and myofilament Ca²⁺ sensitivity. Myofilament Ca²⁺ sensitivity can be increased by agonist-induced activation of G protein-coupled receptors, and this effect is a well-established characteristic of vascular smooth muscle that is regulated by a broad and complex variety of physiological mechanisms. These mechanisms, in turn, can be divided into two main families. The first family regulates thick-filament reactivity and includes all mechanisms that influence the relations between cytosolic Ca²⁺ concentration and the extent of MLC phosphorylation. The second family regulates thin-filament reactivity and includes all mechanisms, including possible changes in cross-bridge kinetics and efficiency (13) that influence the relationships between the extent of MLC phosphorylation and contractile force. Within this conceptual framework, myofilament Ca²⁺ sensitivity can be viewed as the net sum of thick- and thin-filament reactivity (28). Although the physiological regulation of overall myofilament Ca²⁺ sensitivity has been well documented (41), the relative importance of changes in thick- and thin-filament reactivity in this regulation remains largely unexplored.

One situation strongly associated with physiological changes in Ca²⁺ sensitivity is postnatal maturation of the cerebral circulation (3–5). For example, in the ovine cerebral circulation, the ability of 5-HT to enhance myofilament sensitivity was significantly upregulated in fetal compared with adult arteries (5, 32). Similar effects have also been documented in other species and artery types (1, 2, 10), including chronically hypoxic ovine basilar arteries (32). Despite these well-established effects, the independent and age-related contributions of changes in thick- and thin-filament reactivity to agonist-induced myofilament Ca²⁺ sensitization remain largely unknown.

The present data further confirm that G protein agonist-induced increases in myofilament Ca²⁺ sensitivity are markedly upregulated in fetal compared with adult cerebral arteries as previously shown (3–5, 32). For K⁺-induced contractions in nonpermeabilized arteries, the strong correlations between peak cytosolic Ca²⁺ concentrations and peak contractile tensions were quite similar in fetal and adult cerebral arteries (Fig. 1). This absence of age-related differences in basal myofilament Ca²⁺ sensitivity suggests that regulation of basal Ca²⁺ sensitivity is not age related. In contrast, during 5-HT-induced contractions, correlations between peak cytosolic Ca²⁺ concentrations and peak contractile tensions were markedly left shifted in both age groups, and the magnitude of this shift was greater in fetal than in adult cerebral arteries. Together, these data reinforce the view that agonist-induced increases in myofilament Ca²⁺ sensitivity are highly age related.

To better understand the mechanisms involved in age-related differences in agonist-induced myofilament Ca²⁺ sensitization, the experimental approach employed in this study expanded the basic thick-/thin-filament model proposed by Murphy and Walker (28). The application of this model required simultaneous time-dependent measurements of cytosolic Ca²⁺, MLC phosphorylation, and contractile force. Because each measurement of MLC phosphorylation required homogenization of a separate artery segment, it was not possible to measure all three parameters in a single artery segment. Thus the experimental approach involved serial measurements in matched adjacent artery segments, all obtained from a single parent artery. Some segments provided simultaneous measurements of contractile force and MLC phosphorylation, whereas adjacent segments from the same artery enabled simultaneous measurements of contractile force and cytosolic Ca²⁺ concentration. Integration of these results across matching levels of treatment required normalization relative to maximum contractile tone and maximum Ca²⁺ concentration. This approach enabled assessments of thick-filament reactivity, as indicated by the relationships between Ca²⁺ concentration and MLC phosphorylation (Fig. 2). The same approach also provided assessments of thin-filament reactivity as indicated by the relationships between MLC phosphorylation and contractile force (Fig. 3). Most importantly, the exact same data sets used to assess thick- (Fig. 2) and thin-filament (Fig. 3) reactivity also yielded plots of Ca²⁺ versus contractile force that indicated overall myofilament Ca²⁺ sensitivity. Together, these results demonstrate that the Murphy model is both feasible and useful for partitioning changes in myofilament Ca²⁺ sensitivity into respective thick- and thin-filament components.

Assessments of thick-filament reactivity during K⁺-induced contractions revealed that the relationship between Ca²⁺ and MLC phosphorylation was significantly left shifted in fetal relative to adult arteries only at 75 mM K⁺ (Fig. 2, top). This observation suggests that the ability of K⁺-induced increases in Ca²⁺ to stimulate myosin phosphorylation is only slightly upregulated in fetal compared with adult arteries (Fig. 1, top). In marked contrast, the relationships between MLC phosphorylation and cytosolic Ca²⁺ were significantly left shifted during 5-HT-induced contractions at both 1 and 10 µM, and the magnitude of this shift was significantly greater in fetal than in adult arteries (Fig. 2, bottom). This finding suggests that 5-HT enhanced the ability of Ca²⁺ to stimulate myosin phosphorylation in fetal and adult cerebral arteries as previously suggested (3–5) and more so in fetal than in adult cerebral arteries.

Although the present data do not directly identify specific enzymatic mechanisms responsible for the enhancement of thick-filament reactivity observed during 5-HT-induced contractions, when combined with our previous studies of 5-HT-induced Ca²⁺ sensitization in ovine cerebral arteries, the data help indicate which mechanisms are most likely to be involved. In our previous studies of ovine cerebral arteries, concentrations of calphostin-A that blocked Ca²⁺ sensitization induced by indolactam had no effect on 5-HT-induced Ca²⁺ sensitization in either fetal or adult ovine cerebral arteries (5). This finding suggests that PKC probably does not play a major role in 5-HT-induced myofilament Ca²⁺ sensitization in ovine cerebral arteries. In the same study, however, our results demonstrated that exotoxin C3 completely inhibited 5-HT-induced myofilament sensitization in both fetal and adult cerebral arteries (5), suggesting that RhoA, which is the ribosylation target for exotoxin C3 (35), is a common mediator of agonist-induced Ca²⁺ sensitization in ovine cerebral arteries. Within this context, the enhancement of thick-filament reactivity caused by 5-HT (Fig. 2) could be caused by either one of two main categories of PKC-independent mechanisms: 1) those that involve the coupling between 5-HT receptor activation and activation of RhoA and 2) those that govern the influence of RhoA on MLC phosphorylation. Regarding the first category, the ability of 5-HT to stimulate RhoA activation could be influenced by changes in receptor density, receptor isoform, agonist affinity, and the efficiency of coupling between receptor activation and RhoA activation (41). Because our previous studies of 5-HT receptor types in ovine cerebral arteries have revealed that postnatal maturation involves little change in 5-HT receptor type (43), this possibility can be excluded. Similarly, age-related changes in agonist affinity for 5-HT are probably not involved based on our previous findings in fetal and adult ovine common carotid arteries (6), although detailed studies of this possibility in cerebral arteries are still needed. Together, the evidence is most consistent with the hypothesis that the increased ability of fetal arteries to enhance thick-filament reactivity is due to either an upregulated 5-HT-2a receptor density or an increased efficiency of coupling between receptor binding and Rho activation in fetal cerebral arteries.

Regarding the second family of mechanisms, which endeavors to explain increased thick-filament reactivity in fetal arteries through more efficient coupling between RhoA and MLC phosphorylation, the most obvious mechanism is increased inhibition of MLCP through the activation of Rho kinase (20, 22, 42). Because we have previously demonstrated in ovine cerebral arteries that inhibition of RhoA activation completely blocked 5-HT-induced Ca²⁺ sensitization (5), other Rho-independent mechanisms influencing MLCP activity are probably not involved in the age-related changes in the thick-filament reactivity that we observed. These include changes in CPI-17 (17), arachidonate (14), PKC (44), PKG (30), Zip-like kinase (23, 24), Zip kinase (34), and myotonic dystrophy protein kinase (27). What this leaves are possible age-related changes in Rho-kinase and/or MLCP abundance and/or activity. As shown in Fig. 4, the fetal arteries exhibited a significantly lower abundance of MLCP, which is consistent with upregulated thick-filament reactivity in the fetus. However, many other factors are possible, including altered MLCP enzymatic activity or an altered ratio of MLCK activity relative to MLCP activity. Because the abundance of MLCK was also significantly less in fetal than in adult arteries (Fig. 4), it is clear that simple differences in enzyme abundance cannot explain the observed age-related differences in thick-filament reactivity. Instead, age-related regulation of MLCK- and/or MLCP-specific activity must be involved.

Apart from the potential contributions of changes in thick-filament reactivity to agonist-induced myofilament Ca²⁺ sensitization, the results also suggest that changes in thin-filament reactivity may contribute to agonist-induced Ca²⁺ sensitization. Plots of phosphorylation values against corresponding values of contractile force revealed that under basal conditions, thin-filament reactivity was significantly right shifted at 75 mM K⁺ and therefore downregulated in fetal compared with adult arteries (Fig. 3, top). However, during stimulation with 5-HT, both fetal and adult relationships between MLC phosphorylation and contractile force were left shifted relative to the relationships obtained during K⁺ contractions (Fig. 3, bottom). Equally important, this upregulation was greater in magnitude in adult compared with fetal cerebral arteries for all concentrations of 5-HT used. This finding suggests that the 5-HT-induced increases in myofilament Ca²⁺ sensitivity shown in Fig. 1 also involved increases in the ability of thin filaments to interact with activated myosin cross bridges. This observation further suggests more efficient coupling between activation of G protein receptors and increased reactivity of actin filaments with myosin cross bridges in adult compared with fetal cerebral arteries.

Regarding the mechanisms possibly involved in upregulation of thin-filament reactivity, these must be RhoA dependent and PKC independent, as demonstrated by our previous studies of Ca²⁺ sensitization in ovine cerebral arteries (5). Because Rho GTPases can interact with the actin cytoskeleton to dramatically influence smooth muscle contractility (21, 49), these are obvious candidates as mediators of 5-HT-induced increases in thin-filament reactivity. In particular, p21-activated protein kinase, which belongs to the Rho family of GTPases that modulates actin reactivity (45), could be involved. In addition, other actin regulatory proteins such as HSP20 (26, 36–38), HSP27 (8, 9, 48), and caldesmon (12) might also participate in the regulation of actin reactivity by G protein receptor activation. Although differences in the abundance of thin-filament regulatory proteins might contribute to age-related differences in thin-filament reactivity, this appears not to be the case. As shown in Fig. 4, the abundances of both caldesmon and HSP27 were reduced in fetal compared with adult arteries, which might suggest reduced inhibition of thin-filament reactivity with myosin cross bridges. However, this latter relation was not observed, suggesting that other mechanisms must be involved. Together, this evidence is most consistent with the hypothesis that 5-HT-induced increases in thin-filament reactivity are enhanced more in adult than in fetal arteries through Rho-dependent mechanisms, which are more active in mature than in immature arteries.

Overall, the present data reinforce the view that myofilament Ca²⁺ sensitivity is upregulated in fetal compared with adult cerebral arteries as previously suggested (3–5, 32). With the use of the model proposed by Murphy and Walker (28), the present experiments enable an assessment of the relative contributions of thick- and thin-filament regulation to both basal and agonist-enhanced myofilament Ca²⁺ sensitivity. The data demonstrate that despite a general similarity in basal myofilament Ca²⁺ sensitivity between fetal and adult cerebral arteries, thick-filament reactivity was increased and thin-filament reactivity was decreased in fetal compared with adult cerebral arteries. In contrast, fetal and adult arteries contracted with 5-HT demonstrated enhanced myofilament Ca²⁺ sensitivity compared with K⁺ contractions, and the magnitude of this effect was greater in fetal than in adult cerebral arteries as previously shown (5). These age-related differences in agonist-induced sensitization were mediated predominantly by agonist-enhanced thick-filament reactivity in fetal arteries and agonist-enhanced thin-filament reactivity in adult cerebral arteries both mediated by PKC-independent but RhoA-dependent mechanisms (5).

In terms of the potential relevance to human health, the present results predict that cerebrovascular responses to contractile stimuli, including membrane depolarizing agents and G protein receptor agonists, will have a greater effect on the extent of MLC phosphorylation in fetal than in adult arteries. This, in turn, predicts that MLCP inhibitors, such as the Rho kinase inhibitors now being used in clinical trials (16), should have dramatically different effects in pediatric and adult patients, including those in intensive care units due to cerebrovascular complications. Similarly, other treatments that influence MLCP activity through changes in the NO/cGMP/PKG pathway (29), including common clinical therapies such as NO inhalation and phosphodiesterase inhibition (7), would all be expected to have greater effects in infants than in adults. On the thin-filament side, any agent that alters actin reactivity or cytoskeletal protein regulation, such as statins (19), would be expected to have a greater effect in adult than in infant arteries. Altogether, the present data emphasize that thick- and thin-filament reactivities are critical components of overall myofilament Ca²⁺ sensitivity and vascular reactivity that have important age-related consequences for cerebrovascular homeostasis.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The study was supported by National Institutes of Health Grants HL-54120, HD-31266, and HL-64867 and the Loma Linda University School of Medicine.

	FOOTNOTES

 

Address for reprint requests and other correspondence: W. J. Pearce, Dept. of Physiology and Pharmacology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92354 (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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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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