AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H548-H556, 2007. First published March 23, 2007; doi:10.1152/ajpheart.00134.2007
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/1/H548    most recent
00134.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sandoval, R. J.
Right arrow Articles by Pearce, W. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sandoval, R. J.
Right arrow Articles by Pearce, W. J.

Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries

Renan J. Sandoval, Elisha R. Injeti, James M. Williams, William T. Georthoffer, and William J. Pearce

Department of Physiology and Pharmacology, Center for Perinatal Biology, School of Medicine, Loma Linda University, Loma Linda, California

Submitted 1 February 2007 ; accepted in final form 22 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of cytosolic calcium and myofilament calcium sensitivity varies considerably with postnatal age in cerebral arteries. Because these mechanisms also govern myogenic tone, the present study used graded stretch to examine the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in term fetal compared with adult cerebral arteries. Term fetal and adult posterior communicating cerebral arteries exhibited similar myogenic responses, with peak tensions averaging 24 and 26% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K+ (Kmax) at optimum stretch ratios (working diameter/unstressed diameter) of 2.19 and 2.23, respectively. Graded stretch increased cytosolic Ca2+ concentration at stretch ratios >2.0 in adult arteries, but increased Ca2+ concentration only at stretch ratios >2.3 in fetal arteries. In permeabilized arteries, myogenic tone peaked at a stretch ratio of 2.1 in both fetal and adult arteries. The fetal %Kmax values at peak myogenic tone were not significantly different at either pCa 7.0 (23%) or pCa 5.5 (25%) but were significantly less at pCa 8.0 (8.4 ± 2.3%). Conversely, adult %Kmax values at peak myogenic tone were significantly less at both pCa 8.0 (10.4 ± 1.8%) and pCa 7.0 (16%) than at pCa 5.5 (27%). The maximal extents of stretch-induced increases in myosin light chain phosphorylation in intact fetal (20%) and adult (17%) arteries were similar. The data demonstrate that the cerebrovascular myogenic response is highly conserved during postnatal maturation but is mediated differently in fetal and adult cerebral arteries.

beta-escin; fura 2; length-tension relations; myosin light chain phosphorylation

THE TRANSITION FROM LATE FETAL to postnatal life involves numerous changes in vascular morphology and function that significantly alter contractility in brain arteries (2, 15, 23, 58). For excitable tissues, many of these functional differences are a consequence of the relatively small total body mass of calcium present in most mammalian fetuses at term (60). This reduced calcium mass has important effects on the sizes of intracellular calcium pools and the signaling pathways used for cell activation (49). Specifically, contractile tone is more dependent on calcium influx in immature than in mature arteries (1). Conversely, immature arteries exhibit an enhanced ability to increase myofilament calcium sensitivity in response to G protein-dependent contractile agonists (2, 3). In contrast to these well-established effects of postnatal development, however, the influences of maturation on myogenic reactivity have not been well studied, even though it is clear that myogenic tone is critically important for cardiovascular homeostasis, regardless of age (12). Because cerebral autoregulation is highly dependent on myogenic reactivity (33), appears to be regulated quite differently in mature and immature cerebral arteries (28), and is highly vulnerable to pathophysiological insults (61), a close examination of the mechanisms mediating myogenic reactivity in immature cerebral arteries is due.

In adult cerebral arteries, myogenic contractions depend on both an increase in cytosolic calcium concentration and stretch-induced increases in myofilament calcium sensitivity (35, 53). These increases in calcium sensitivity, in turn, can be partitioned between effects of stretch on the relations between cytosolic calcium concentration and the extent of myosin light chain (MLC) phosphorylation, and the parallel effects of stretch on the relations between MLC phosphorylation and contractile force. As originally proposed by Murphy and Walker (48), the relation between cytosolic calcium concentration and the extent of MLC phosphorylation can be considered to be the result of the family of mechanisms that together govern thick-filament regulation. Alternatively, relations between the extent of MLC phosphorylation and contractile force reflect the combined influence of those mechanisms that determine thin-filament reactivity. These mechanisms include the influences of thin-filament regulatory proteins, such as caldesmon, heat shock protein (HSP) 27, and HSP20 (8, 17, 42, 43), as well as integrin-linked interactions of the actin cytoskeleton with the extracellular matrix (40). The relative importance of these general classes of mechanisms during myogenic responses remains unstudied in cerebral arteries of any age. Similarly, the ability of stretch to activate or modulate any of these mechanisms remains completely unstudied, particularly in immature cerebral arteries.

Given the importance of myogenic tone for cerebrovascular regulation, and evidence that the calcium-dependent contractile mechanisms governing myogenic tone are quite different in mature and immature cerebral arteries, the present study explores the hypothesis that myogenic tone is less dependent on calcium influx and more dependent on myofilament calcium sensitization in fetal compared with adult cerebral arteries. To test this hypothesis, we examined myogenic responses in posterior communicating cerebral arteries taken from term fetal and nonpregnant adult sheep. Owing to the possible contribution of the vascular endothelium to myogenic responses in cerebral arteries (19), the endothelium was gently removed in all arteries studied. The protocols involved application of graded stretch, normalized as a ratio of working diameter divided by unstressed diameter, followed by recordings of the corresponding changes in cytosolic calcium via fura 2 photometry. These protocols also included rapid freezing of artery segments at varying durations of graded stretch to enable determination of the corresponding extent of MLC phosphorylation, as measured using Western blots of membranes transferred from urea gels. Parallel experiments in intact and beta-escin-permeabilized arteries helped identify the relative importance of changes in cytosolic calcium and myofilament calcium sensitization in myogenic responses to artery stretch. Together, these approaches provided a unique view of the effects of postnatal maturation on the roles of cytosolic calcium transients and myofilament calcium sensitivity in the myogenic reactivity of ovine cerebral arteries.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General preparation. All procedures and protocols strictly followed all federal rules and regulations governing the care and use of laboratory animals and were approved by the Institutional Animal Care and Use of Committee of Loma Linda University. 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). We chose to study the posterior communicating artery because this artery plays a critical regulatory role in shunting Circle of Willis blood flow between the anterior and posterior cerebral territories, and its length permits the study of up to eight artery segments per animal. Following their removal from the surface of the brain, the arteries were placed in a calcium-replete sodium Krebs buffer solution that was continuously bubbled with 95% O2–5% CO2 and contained the following (in mM): 122 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 for both fetal and adult arteries and then mounted on wires suspended between a force transducer that was attached to a micrometer used to adjust resting length and determine the passive diameter of each individual tissue segment. The tissue diameter recordings were utilized to obtain the optimal stretch ratios necessary to obtain maximal isometric force in each segment, as previously described (16). To minimize endotheliummediated effects, we removed the vascular endothelium by gently rotating each arterial segment around a mounting wire several times to scrape the entire luminal surface. All buffers also included 100 µM NG-nitro-L-arginine methyl ester and 100 µM nitro-L-arginine to ensure complete inhibition of endothelial nitric oxide synthase and nitric oxide release.

Determination of active myogenic tone in intact arteries. Tissue segments were equilibrated for 30 min in calcium-replete 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, 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. As shown previously (16, 50), this approach normalized the increments of stretch applied to the resting unstressed artery diameter and thereby minimized the effects of differences in initial artery diameter. For each segment studied, the D/D0 was initially set at 1.3, after which the artery was allowed to equilibrate until a stable total tension (recorded continuously via computer) was obtained (usually in ~10 min). The artery segment was then stretched to a D/D0 of 1.5 and allowed to reequilibrate. This cycle was repeated in D/D0 increments of 0.2 until a final D/D0 of 2.5 was obtained. Then the arteries were frozen in liquid nitrogen, after which they were incubated in a calcium-free Na+ Krebs buffer containing 1 mM EGTA. As shown previously (58), freezing the arteries before incubation in EGTA accelerates the rate of calcium chelation and produces optimally reproducible estimates of passive tension. Following this treatment, the levels of tension observed at each D/D0 ratio were again recorded to define the passive length-tension relation in each segment. Active myogenic tone was calculated as the difference between the initial total tension and the subsequent passive tension measured at each D/D0 ratio.

Stretch-induced changes in global cytosolic calcium. To enable measurement of global cytosolic calcium, arterial segments were mounted in a Jasco fluorescent photometer (Jasco CAF-110), and slack length was determined after which the segments were stretched to a D/D0 ratio of 1.3, then were 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 the following (in mM): 122.1 NaCl, 25 HEPES, 5.16 KCl, 2.4 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-nm ratio (340/380 ratio) was recorded, after which each segment was contracted with 120 mM potassium and then washed with normal buffer. Following this wash and equilibration, each segment was stretched to one and only one D/D0 ratio between 1.5 and 2.5 (in increments of 0.2). During and following this stretch, changes in the 340/380 ratio were recorded along with contractile force. Once responses were stabilized, the segments were washed with fresh buffer and then exposed to buffer containing 2 mM EGTA without calcium in the presence of 10 µM ionomycin to determine minimum tissue signal intensity recorded during exposure to near-zero concentrations of free calcium (Fmin) values. After stable 340-to-380 ratio values were obtained, calcium was added at a final concentration of 24 µM to obtain maximum tissue signal intensity recorded during exposure to saturating concentrations of free calcium (Fmax). Autofluorescence was then determined via manganese quench (71). Intracellular calcium values were then determined using the Grynkiewicz equation (25).

Determination of active myogenic tone in permeabilized arteries. Tissue segments were equilibrated for 30 min in calcium-replete Na+ Krebs buffer bubbled with 95% O2–5% CO2 at 25°C. Unstressed artery diameter (slack length) was determined as before, and artery stretch was calculated as a stretch ratio (D/D0). For each segment studied, the D/D0 was initially set at 1.3, after which each segment was permeabilized with HEPES buffer (in mM): 110 K(CH3COO), 6 Mg(CH3COO)2, 5 EGTA, 5 ATP, 5 DTT, 20 HEPES, 0.001 calmodulin, 0.01 leupeptin containing 150 µM beta-escin at pCa 6.0. After equilibration, each tissue segment was washed with HEPES buffer at pCa 8.0 and allowed to equilibrate. The segments were then incubated with HEPES buffer at either pCa 8, 7, or 5.5 and allowed to equilibrate. The concentration of pCa 8 represented a sub-baseline concentration that should eliminate most, if not all, calcium-dependent responses. The pCa 7 concentration represented the approximate average basal concentration of calcium in uncontracted term fetal and adult ovine cerebral arteries (1). Conversely, the concentration of pCa 5.5 represented a maximal concentration previously shown to yield maximal contractile responses in both term fetal and adult ovine cerebral arteries (2). The artery segment was then stretched to a D/D0 of 1.5, and the total tension (recorded continuously via computer) was obtained (usually in ~10 min). The artery segment was stretched to D/D0 1.7 and allowed to reequilibrate. This cycle was repeated in D/D0 increments of 0.2 until a final D/D0 of 2.5 was obtained. Then the arteries were frozen in liquid nitrogen, after which they were incubated in a calcium-free Na+-Krebs buffer containing 1 mM EGTA. Following this treatment, the levels of tension observed at each D/D0 ratio were again recorded to define the passive length-tension relation in each segment. Active myogenic tone was calculated as the difference between the initial total tension and the subsequent passive tension measured at each D/D0 ratio.

Determination of MLC phosphorylation in intact arteries. Multiple adjacent tissue segments from both fetal and adult posterior communicating arteries were mounted in 5-ml baths and were allowed to equilibrate for 30 min in calcium-replete Na+-Krebs buffer solution constantly 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, and this measurement was made in each artery segment. Thereafter, artery stretch was calculated as a stretch ratio (D/D0) (at slack length, D/D0 = 1). After equilibration, each tissue segment was quickly stretched to a desired stretch ratio and frozen at exactly 10 s after stretch. The freeze time of 10 s was used because preliminary experiments indicated that the time to peak phosphorylation following quick stretch in intact arteries did not vary significantly with either age or D/D0 (between 1.5 and 2.3) and averaged 9.8 ± 5.1 s (N = 56). Each frozen segment's dry weight was measured and extracted in 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 added at a ratio of 1 mg/250 µl for 90 min at room temperature. The samples were then stored in microcentrifuge tubes at –80°C.

Each arterial extract was first assayed for total protein content using the Bradford reagent (Bio-Rad, Hercules, CA) calibrated against known amounts of BSA protein dissolved in extraction buffer. An MLC standard pool was prepared from common carotid arteries with a tissue-to-buffer ratio of 1 mg/30 µl using overnight extraction at room temperature; the higher tissue-to-buffer ratio was used to increase the relative concentration of MLC in the extracts. Aliquots of samples and MLC standards were analyzed 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 on 10% acrylamide native gels 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 Tris-buffered saline (pH 7.5) containing 5% milk for 1 h. After blocking, the membranes were placed in Tris-buffered saline buffer with 5% milk and 0.1% Tween 20 with primary mouse monoclonal anti-MLC20 (20-kDa MLC; clone MY-21) 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 the levels of both nonphosphorylated and phosphorylated MLC using an AlphaInnotech ChemiImager. Integrated optical density values (IDV) for the MLC protein standards were plotted against the mass of protein loaded to give an IDV-mass curve. The IDV 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. Percent MLC phosphorylation was calculated as the phosphorylated mass divided by the total of the phosphorylated and nonphosphorylated masses.

Determination of MLC phosphorylation in permeabilized arteries. Multiple adjacent tissue segments from both fetal and adult posterior communicating arteries were mounted in 5-ml baths and allowed to equilibrate for 30 min in HEPES buffer solution constantly bubbled with 95% O2–5% CO2 at 25°C. Unstressed artery diameter (slack length) was defined as the artery diameter observed at a passive tension of 0.03 g, and this measurement was made in each artery segment. Thereafter, artery stretch was calculated as a stretch ratio (D/D0). For each segment studied, the D/D0 was initially set at 1.3, after which each segment was permeabilized with HEPES buffer containing 150 µM beta-escin at pCa 6.0. After equilibration, each tissue segment was washed with HEPES buffer at pCa 8.0 and allowed to equilibrate. The segments were then incubated with HEPES buffer containing either pCa 8, 7, or 5.5 and allowed to equilibrate. Each tissue segment was then stretched to a D/D0 of 1.9 and frozen at times of 0, 4, 8, and 12 s, or 0, 1, 2, and 3 min after initial stretch. Each frozen segment was then analyzed on urea gels to quantify MLC phosphorylation, as previously described. The percent MLC phosphorylation was then plotted against time to identify the effect of stretch on myosin phosphorylation at each pCa for each group.

Calculation, data analysis, and statistics. Myogenic tone was calculated as the difference between total contractile tone and passive contractile tone, which was determined following freezing in liquid nitrogen and subsequent incubation in 1 mM EGTA. Reported values of active myogenic tone are expressed relative to the maximum contractile capacity of each artery, as defined by the response to calcium-replete Krebs buffer containing 120 mM K+. Measurements of cytosolic calcium were initially obtained as fura 2 340/380 ratios, which were subsequently corrected for Fmax, Fmin, and autofluorescence and then reported as values of the Grynkiewicz ratio (25). These ratio values were not converted to cytosolic calcium concentrations due to uncertainties regarding the actual value of the fura 2 Kd values in our preparations. Instead, calcium concentration estimates were normalized relative to the maximal change in calcium produced in response to Krebs buffer containing 120 mM K+ as obtained in each individual artery preparation. Myosin phosphorylation ratios were reported as percent total phosphorylation, as determined by the ratio between the mass of the phosphorylated band divided by the sum of the phosphorylated and unphosphorylated bands. All statistical comparisons were performed using ANOVA routines with age as one factor and either stretch ratio or time as the other factor. Homogeneity of variance was verified for all ANOVA procedures using Cochran's analysis. All sample distributions were verified for normalcy via analysis of kurtosis and skewness values. When multiple measurements were taken from a single artery segment (see Figs. 1, Go3, and Go5), comparisons between groups were performed using a repeated-measurements ANOVA. When multiple segments were taken from a single animal and used in the same protocol, the results were averaged and counted as an N of 1. Statistical power reached minimum value of 0.8, unless stated otherwise. All values are reported as means ± SE for the number of animals studied.


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 1. Myogenic length-tension relations in intact fetal and adult cerebral arteries. Segments of fetal and adult posterior communicating arteries were stretched from a stretch ratio [working diameter/unstressed diameter (D/D0)] of 1.3 to a ratio of 2.7 before (total tension) and after (passive tension) freezing in liquid N2 and incubation in 1 mM EGTA. Myogenic tone was calculated as the difference between total and passive tensions at each stretch ratio, then expressed as a percentage of the corresponding response to 120 mM K+ in each segment. Error bars indicate SEs for arteries from 6 adults and 7 fetuses, respectively. There were no significant differences between fetal and adult values at any stretch ratio. Kmax, maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K+.

 

Figure 2
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 2. Relations between stretch and cytosolic calcium in fetal and adult cerebral arteries. Shown here are values of change above baseline in the Grynkiewicz ratio ({Delta}Rg) for fetal and adult cerebral arteries plotted as a function of artery stretch. Initial values averaged 0.221 ± 0.019 and 0.214 ± 0.027 in fetal (N = 33) and adult (N = 38) arteries, respectively. These ratio values were calculated as an index of cytosolic calcium and exhibited no significant changes in response to stretch ratios <2.0. At stretch ratios between 2.0 and 2.3, stretch increased cytosolic calcium, but only in adult arteries. Increases in fetal artery cytosolic calcium were observed only at a stretch ratio of 2.5. *Significantly greater than baseline (average {Delta}Rg values between stretch ratios of 1.3 and 1.7). Error bars indicate SE of the mean for N = 5 in both experimental groups.

 

Figure 3
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 3. Myogenic length-tension relations in permeabilized fetal and adult cerebral arteries. Fetal and adult arteries were permeabilized with beta-escin and then equilibrated at pCa 8.0, 7.0, or 5.5 and progressively stretched from a D/D0 of 1.3 to 2.5 in increments of 0.2, before (total tension) and after treatment with 1 mM EGTA (passive tension). Myogenic tone was calculated as the difference between total and passive tensions at each stretch ratio and then expressed as a percentage of the corresponding response to 120 mM K+ obtained before permeabilization in each segment. Error bars indicate SE of the mean for 50 fetal and 32 adult arteries taken from 9 fetuses and 11 adults; the N values for each pCa in each age group are indicated in the legends. *Significantly greater than baseline (D/D0 = 1.3) at each pCa. In adult arteries, the stretch-response curve obtained at pCa 5.5 was significantly different than that observed at either pCa 7.0 or 8.0; the latter two curves were not significantly different (ANOVA). For fetal arteries, the stretch-response curves at pCa 5.5 and 7.0 were both significantly different than that at pCa 8.0. The fetal curves at pCa 5.5 and 7.0 were not significantly different (ANOVA).

 

Figure 4
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 4. Relations between stretch and myosin light chain (MLC) phosphorylation in intact cerebral arteries. Both fetal and adult cerebral arteries were equilibrated at slack length (D/D0 = 1) and then quickly stretched to the desired D/D0 and frozen 10 s after stretch. Frozen arteries were analyzed for phospho-MLC content using urea gels, and the percent MLC phosphorylation was calculated as the phosphorylated mass divided by the total of the phosphorylated and nonphosphorylated masses. *Significantly greater than baseline (D/D0 = 1). Fetal and adult responses to stretch were not significantly different. The individual curves shown are lines of best fit calculated using least squares minimization. Error bars indicate SE of the mean for 6 fetal and 6 adult arteries.

 

Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 5. Relations between stretch and MLC phosphorylation in permeabilized fetal and adult cerebral arteries. Both fetal and adult cerebral arterial segments were equilibrated at a D/D0 of 1.3 at pCa values of 8.0, 7.0, or 5.5, then frozen at 0, 1, 2, or 3 min after stretch to a D/D0 of 1.9. Percent MLC phosphorylation was determined as described in Fig. 4 legend. Rapid stretch significantly increased MLC phosphorylation in both fetal and adult arteries. *Significantly greater than baseline (time 0) at each pCa. The phosphorylation time course was significantly different at pCa 5.5 than that at either pCa 7.0 or 8.0 in adult arteries. For fetal arteries, the phosphorylation time courses at both pCa 5.5 and 7.0 were significantly different than that at pCa 8.0. Error bars indicate SE of the mean for 64 fetal and 64 adult arteries taken from 16 fetuses and 16 adults. Individual N values for each pCa in each age group are indicated in the legends.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of graded stretch on myogenic tone in intact cerebral arteries. Fetal and adult cerebral arteries exhibited similar myogenic responses with peak tensions averaging 23.9 ± 6.8 and 26.0 ± 4.8% of maximum contractile force produced in any given tissue in response to an isotonic Krebs buffer containing 122 mM K+ (Kmax) values at D/D0 values of 2.19 ± 0.04 and 2.23 ± 0.02, respectively (Fig. 1). None of these values varied significantly with age.

Effects of graded stretch on cytosolic calcium concentrations in cerebral arteries. When equilibrated at a stretch ratio of 1.3, values of the Grynkiewicz ratio averaged 0.221 ± 0.019 and 0.214 ± 0.027 in fetal (N = 33) and adult (N = 38) arteries, respectively. Graded stretch produced no significant increases in these ratio values at D/D0 values <2.0 in either fetal or adult cerebral arteries (Fig. 2). There was, however, an increase in values of the Grynkiewicz ratio at D/D0 ratios >2.0 for adult cerebral arteries. No increases in ratio values were observed in fetal arteries until D/D0 ratios >2.3 were applied. Thus significantly more stretch was required to elicit increases in fetal compared with adult arteries.

Effects of graded stretch on myogenic tone in permeabilized cerebral arteries. Both fetal and adult cerebral arteries exhibited stretch-induced myogenic tone under constant calcium conditions in beta-escin permeabilized arteries. In fetal arteries, peak myogenic tone was observed at a D/D0 of 2.1 at all three pCa values (Fig. 3). The fetal %Kmax values at peak myogenic tone were not significantly different at either pCa 7.0 (22.7 ± 3.0%) or pCa 5.5 (24.8 ± 5.0%) but were significantly less at pCa 8.0 (8.4 ± 2.3%). Repeated-measures ANOVA revealed that the fetal responses observed at pCa 5.5 and 7.0 were not significantly different but that the responses at pCa 8.0 were significantly less than at either pCa 7.0 or 5.5. Conversely, peak myogenic tone in adult arteries was observed at a D/D0 of 2.1 for pCa 5.5, and at D/D0 of 2.3 for pCa 7.0 and 8.0. Adult %Kmax values at peak myogenic tone were significantly less at pCa 8.0 (10.4 ± 1.8%) and pCa 7.0 (15.7 ± 4.2%) than at pCa 5.5 (26.6 ± 3.0%). As indicated by a repeated-measures ANOVA, the adult responses were not significantly different at pCa 8.0 and 7.0.

Effects of stretch on the extent of MLC phosphorylation in intact cerebral arteries. As shown in Fig. 4, both fetal and adult cerebral arteries exhibited similar patterns of stretch-induced MLC phosphorylation in intact cerebral arteries. However, there was no significant difference in the maximal extent of MLC phosphorylation above baseline between fetal (19.9 ± 4.9%) and adult (17.1 ± 2.7%) cerebral arteries. There were also no significant differences in basal MLC phosphorylation between fetal (18.0 ± 2.3%) and adult (15.9 ± 2.8%) arteries. The stretch ratios that produced half-maximal (EC50) MLC phosphorylation were also highly similar in fetal (1.68 ± 0.10) and adult (1.64 ± 0.18) cerebral arteries, respectively.

Effects of graded stretch on the extent of MLC phosphorylation in permeabilized cerebral arteries. When the arteries were held at a pCa of 8.0, basal values of %MLC phosphorylation were not significantly different in fetal (21.0 ± 2.1%) and adult (22.0 ± 1.9%) arteries (Fig. 5). Rapid stretch from a D/D0 of 1.3 to a D/D0 of 1.9 had no time-dependent effects on phosphorylation at pCa 8.0 in either fetal or adult arteries over a time course of 3 min. In arteries held at a pCa of 7.0, baseline phosphorylation was significantly elevated in fetal (40.5 ± 1.4%) but not adult (23.0 ± 1.9%) arteries. Rapid stretch at pCa 7.0 from a D/D0 of 1.3 to a D/D0 of 1.9 produced no significant increase in the extent of MLC phosphorylation in either fetal or adult arteries over a time course of 2 min. After 3 min of stretch, the averaged fetal value of phosphorylation (44.8 ± 0.5%) at pCa 7.0 was slightly but significantly elevated over baseline; no such difference was observed in the adult arteries. In arteries held at pCa 5.5, baseline values of phosphorylation were not significantly different than observed at pCa 7.0 in either fetal (42.0 ± 1.4%) or adult (26.0 ± 1.2%) arteries. Rapid stretch from a D/D0 of 1.3 to a D/D0 of 1.9 at pCa 5.5 significantly enhanced %MLC phosphorylation above basal levels to 37.0 ± 5.5% and 46.1 ± 1.5% in adult and fetal arteries, respectively.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Virtually all of the mechanisms that determine vascular reactivity change progressively throughout fetal development and early postnatal life, particularly in the cerebral circulation (58). Immature cerebral arteries exhibit greater dependence on calcium influx, and less dependence on intracellular calcium release, than is typical of functionally mature adult cerebral arteries (1, 49). In parallel, immature cerebral arteries also demonstrate a greater ability to enhance myofilament calcium sensitivity when stimulated by G protein receptor agonists (2). Together, these maturational changes have a major impact on many aspects of overall cerebrovascular regulation, including endothelial function (72), responses to hypoxia (56), and the role of the perivascular adrenergic innervation (57). Aside from this broad variety of work, however, relatively little is known about how postnatal maturation influences myogenic reactivity in the cerebral circulation. Although many of the mechanisms that govern myogenic reactivity have been studied intensively (12), almost all of these studies have been performed using preparations of adult tissues and have revealed the importance of the myogenic response in cerebral blood flow regulation (26, 32, 45). Equally important, these studies have helped establish that the primary mechanisms mediating myogenic contraction include increased calcium influx and enhanced myofilament calcium sensitivity (12).

Although evidence of the myogenic response in fetal arteries was first reported many years ago (63), only recently has this response been reported in fetal cerebral arteries (19, 20). To examine the mechanistic basis for this response, the present study explored the main hypothesis that postnatal maturation modulates the relative importance of each of the main mechanisms governing the myogenic response, with emphasis on changes in the relations among myogenic stretch, cytosolic calcium concentration, extent of MLC phosphorylation, and contractile force (48). Our first objective was to compare the relations between artery stretch and myogenic tone as a function of postnatal age. As shown in Fig. 1, both fetal and adult cerebral arteries exhibited robust myogenic contractions whose magnitudes (fetal: 23.9 ± 6.8%; adult 26.0 ± 4.8% Kmax) were similar and agreed well with previously published results in both cerebral (19, 35, 54) and noncerebral (7, 55, 64, 65, 66) arteries. In contrast to most previous measurements of myogenic tone in either cerebral or fetal arteries, our measurements were not based on diameter responses to changes in artery pressure. Instead, our stretch stimulus was normalized relative to passive diameter to obtain stretch ratios that enabled direct estimates of optimal diameter and stretch, as previously described (16). These estimates of optimal stretch were very similar in fetal (D/D0 = 2.19 ± 0.04) and adult (D/D0 = 2.23 ± 0.02) arteries, indicating that the mechanisms determining the myogenic response are fully developed in fetal cerebral arteries. This result was unexpected, given that regulation of contractility differs greatly between fetal and adult cerebral arteries (2, 3). In turn, this result suggests that the mechanisms mediating the myogenic response may be different in fetal and adult arteries, despite the fact that the overall response was independent of age.

The isometric preparations used in this study offered several advantages that simplified comparisons among arteries of different size and also facilitated direct measurements of wall calcium by minimizing movement of the artery wall. This enabled repeated measurements of cytosolic calcium during graded stretch in both fetal and adult cerebral arteries (Fig. 2). Consistent with reports of similar experiments in other preparations (22, 29, 53, 62, 68, 69), gradual stretch did not produce a significant increase in calcium at any stretch ratio (D/D0) <1.9 in either fetal or adult arteries. The same range of gradual stretch, however, significantly increased myogenic tone in both age groups (Fig. 1). At stretch ratios >1.9, however, only adult arteries exhibited a significant rise in calcium, whose peak correlated closely with maximum myogenic tone. In contrast, stretch significantly increased calcium in fetal arteries only at a much greater stretch ratio of 2.5, and this may have been attributable to forced dilatation (53). Together, these results demonstrate that cytosolic calcium is more sensitive to graded stretch in adult than in fetal cerebral arteries. From a more general perspective, these results also reinforce the view that low levels of myogenic tone do not require large steady-state increases in cytosolic calcium. Given that highly localized calcium transients can play a major role in regulation of smooth muscle contraction (36, 74), it remains possible that stretch-induced calcium influx might also involve localized calcium transients, as has been implied in recent reviews of myogenic reactivity (12, 30). If so, such transients would constitute a calcium-mediated response to stretch that would be very difficult to observe using fura 2, which is subject to extensive spatial averaging (67).

To evaluate the possibility that stretch-induced myogenic contractions in our preparations were being mediated by highly regional changes in calcium, we examined the effects of graded stretch on myogenic tone in beta-escin permeabilized cerebral arteries. As shown by others, beta-escin permeabilization enables equilibration of the cytosol with the extracellular medium (59), eliminates gradients in calcium throughout the cell, and simultaneously stabilizes calcium at any desired concentration. Under these conditions, any changes in localized calcium should be buffered by regional EGTA, which is present at high concentrations throughout the cell. In arteries held at a low-calcium concentration of 10 nM (pCa 8), graded stretch still produced small but significant myogenic contractions in both fetal and adult preparations (Fig. 3). The peak magnitudes of these contractions were similar in fetal (8.4 ± 2.3% Kmax) and adult (10.4 ± 1.8% Kmax) arteries. However, when ambient calcium was held at 100 nM (pCa 7.0), myogenic responses to stretch were significantly greater in fetal (22.7 ± 3.0% Kmax) than adult (15.7 ± 4.2% Kmax) arteries. These results suggest that stretch enhanced myofilament calcium sensitivity, as previously suggested (41), and the magnitude of this effect was greater in fetal than adult cerebral arteries. These observations are consistent with previous reports that agonist-induced myofilament calcium sensitization is upregulated in fetal compared with adult cerebral arteries (2). In addition, the stretch ratios at which peak myogenic tone was observed were similar in the permeabilized and intact preparations in both age groups. When the arteries were incubated and held at high-calcium concentrations of 3 µM (pCa 5.5), age-related differences in myogenic tone disappeared; the relation between stretch and peak tone was significantly enhanced in adult (26.6 ± 3.0% Kmax) but not fetal (24.8 ± 5.0% Kmax) cerebral arteries. Again, the stretch ratios at which peak myogenic tone was observed were similar in the permeabilized and intact preparations in both age groups. Together, these results suggest that 100 nM calcium is sufficient to obtain a maximal response in fetal, but not adult, arteries. This helps explain why significant myogenic tone was observed (Fig. 1) in the absence of stretch-induced increases in cytosolic calcium in fetal arteries at stretch ratios <1.9 (Fig. 2). The development of myogenic tone in the absence of major changes in global calcium is consistent with previously reported observations (22, 29, 53, 62).

In light of the age-related differences observed in stretch-induced changes in global calcium (Fig. 2), we explored the possibility that the effects of gradual stretch on MLC phosphorylation might also vary with postnatal age. When artery segments were rapidly frozen after 10 s of stretch, MLC phosphorylation increased significantly, as previously reported (6), but the response was closely similar in fetal and adult cerebral arteries (Fig. 4). Because significant increases in MLC phosphorylation occurred in both fetal and adult arteries at a stretch ratio of 1.9 that was not associated with significant increases in calcium (Fig. 2), the data suggest a possible contribution from calcium-independent kinases in the response to stretch. Recent studies have identified several candidates capable of phosphorylating serine 19 of MLC (5, 14, 34, 37, 51, 52, 70, 73). Another possibility is that stretch inhibits phosphatases that reduce the level of MLC phosphorylation, and, here again, several possible mechanisms for this effect have been reported. These include calcium-independent roles for arachidonate (24), zipper-interacting protein kinase (34), zipper-interacting protein-like kinase (38), integrin-linked kinase (13, 31, 47), and protein kinase C (27).

To test the possibility that stretch-induced changes in MLC phosphorylation are mediated by highly regional changes in calcium, we also examined the effects of stretch on MLC phosphorylation in permeabilized arteries. When adult arteries were held at low-calcium concentrations of 10 or 100 nM, stretch induced no changes in phosphorylation over either very short or long durations (Fig. 5). In adult arteries held at 3 µM calcium, however, stretch stimulated a gradual increase in MLC phosphorylation that reached a peak in ~2 min. This response suggests that calcium is required for stretch-induced increases in MLC phosphorylation but that some mechanism other than changes in cytosolic calcium are also involved in the development of myogenic tone at calcium concentrations at or below 100 nM (Fig. 3). One possibility is that stretch inhibits MLC phosphatase activity (13, 24, 27, 34, 38, 39, 47), which would be evident only if a significant fraction of MLC were phosphorylated. Such a system would be calcium dependent but would not require calcium transients to exhibit stretch-induced changes in MLC phosphorylation. This arrangement could also involve possible stretch-induced changes in MLC kinase activity, as reported in other preparations (46). Clearly, in adult cerebral arteries, calcium is required for stretch to stimulate increased MLC phosphorylation and maximum myogenic contraction (Figs. 1 and 3), even though calcium transients are not.

In contrast to adult cerebral arteries, fetal cerebral arteries did not exhibit stretch-induced increases in MLC phosphorylation when cytosolic calcium concentrations were held constant (Fig. 5). At very low calcium concentrations of 10 nM, baseline MLC phosphorylation was similar to that observed in the adult. Increasing the calcium concentration to only 100 nM elevated MLC phosphorylation to near maximal levels: at both 100 nM and 3 µM calcium, the extent of MLC phosphorylation was near maximal and did not respond to stretch. Consistent with the results shown in Fig. 3 and previous findings that myofilament calcium sensitization is markedly upregulated in the fetus (2, 3), the data suggest that 100 nM calcium are sufficient to support near maximal MLC phosphorylation in fetal cerebral arteries at a stretch ratio of 1.3 or higher. In addition, these data also support the view that the effects of stretch-induced increases in calcium are near maximal at low levels of stretch and that calcium increases observed at high levels of stretch may reflect forced dilatation and not a physiological response (53). As for the adult arteries, this response could involve parallel effects of stretch on both calcium-dependent MLC phosphorylation, as well as coupling between stretch and inhibition of MLC phosphatase activity. However, some additional mechanism must also be involved. As shown in Fig. 3, stretch clearly enhanced myofilament calcium sensitivity in fetal cerebral arteries. For this response to occur in the absence of stretch-induced changes in MLC phosphorylation, stretch must also enhance thin-filament reactivity. Correspondingly, a broad variety of recent studies has suggested that such mechanisms may play a key role in myogenic reactivity (4, 9, 10, 17, 18, 21, 44). For example, rates of actin polymerization can be increased by stretch, which in turn can elevate the number of active binding sites for myosin cross bridges, resulting in greater force development (4, 10, 11, 21, 44). In addition, stretch can activate protein kinase C and rho kinase (35, 66), leading to increased phosphorylation and disinhibition of actin regulatory proteins such as caldesmon, HSP20, and HSP27 (8, 17, 18, 42). Aside from the mechanisms involved, the data strongly suggest that the relative importance of these individual mechanisms in determining the overall myogenic response is markedly different in fetal and adult cerebral arteries.

Overall, the present data support the view that the myogenic response is highly conserved in cerebral arteries, regardless of postnatal age. Aside from this consistency, the relations between stretch and cytosolic calcium, and between stretch and myofilament calcium sensitization, appear to be regulated very differently in fetal and adult cerebral arteries. These age-dependent differences appear to be a natural consequence of corresponding differences in the ability to store and release intracellular calcium and are consistent with the general view that myofilament calcium sensitivity is markedly upregulated in fetal compared with adult arteries. A key feature of the present data, however, is the suggestion that stretch-induced modulation of thin-filament reactivity may be more important for the myogenic response in fetal compared with adult cerebral arteries. This suggestion predicts that it may be valuable to further explore the role of thin-filament regulatory proteins in the myogenic response, particularly in relation to the postnatal development of cerebrovascular reactivity.


   ACKNOWLEDGMENTS
 
The work reported in this paper was supported by United States Public Health Service Grants HL54120, HD31266, and HL64867 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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Akopov SE, Zhang L, Pearce WJ. Maturation alters the contractile role of calcium in ovine basilar arteries. Pediatr Res 44: 154–160, 1998b.[ISI][Medline]
  2. Akopov SE, Zhang L, Pearce WJ. Physiological variations in ovine cerebrovascular calcium sensitivity. Am J Physiol Heart Circ Physiol 272: H2271–H2281, 1997.[Abstract/Free Full Text]
  3. Akopov SE, Zhang L, Pearce WJ. Regulation of Ca2+ sensitization by PKC and rho proteins in ovine cerebral arteries: effects of artery size and age. Am J Physiol Heart Circ Physiol 275: H930–H939, 1998.[Abstract/Free Full Text]
  4. Albinsson S, Nordstrom I, Hellstrand P. Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization. J Biol Chem 279: 34849–34855, 2004.[Abstract/Free Full Text]
  5. Amano M, Ito M, Kimura K, Fukata Y, Chihara K, Nakano T, Matsuura Y, Kaibuchi K. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem 271: 20246–20249, 1996.[Abstract/Free Full Text]
  6. Barany K, Ledvora RF, Mougios V, Barany M. Stretch-induced myosin light chain phosphorylation and stretch-release-induced tension development in arterial smooth muscle. J Biol Chem 260: 7126–7130, 1985.[Abstract/Free Full Text]
  7. Belik J. The myogenic response of arterial vessels is increased in fetal pulmonary hypertension. Pediatr Res 37: 196–201, 1995.[ISI][Medline]
  8. Bitar KN. HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction. Am J Physiol Gastrointest Liver Physiol 282: G894–G903, 2002.[Abstract/Free Full Text]
  9. Cipolla MJ, Gokina NI, Osol G. Pressure-induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior. FASEB J 16: 72–76, 2002.[Abstract/Free Full Text]
  10. Cipolla MJ, Osol G. Vascular smooth muscle actin cytoskeleton in cerebral artery forced dilatation. Stroke 29: 1223–1228, 1998.[Abstract/Free Full Text]
  11. Civelek M, Ainslie K, Garanich JS, Tarbell JM. Smooth muscle cells contract in response to fluid flow via a Ca2+-independent signaling mechanism. J Appl Physiol 93: 1907–1917, 2002.[Abstract/Free Full Text]
  12. Davis MJ, Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiol Rev 79: 387–423, 1999.[Abstract/Free Full Text]
  13. Deng JT, Sutherland C, Brautigan DL, Eto M, Walsh MP, Muranyi A, MacDonald JA, Wilson DP, Haystead TA, Erdodi F, Kiss E, Wu Y, Hartshorne DJ, Csortos C, Gergely P, Ito M. Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase. Biochem J 367: 517–524, 2002.[CrossRef][ISI][Medline]
  14. Deng JT, Van Lierop JE, Sutherland C, Walsh MP. Ca2+-independent smooth muscle contraction. A novel function for integrin-linked kinase. J Biol Chem 276: 16365–16373, 2001.[Abstract/Free Full Text]
  15. Docherty CC, Kalmar-Nagy J, Engelen M, Nathanielsz PW. Development of fetal vascular responses to endothelin-1 and acetylcholine in the sheep. Am J Physiol Regul Integr Comp Physiol 280: R554–R562, 2001.[Abstract/Free Full Text]
  16. Elliott SR, Pearce WJ. Effects of maturation on alpha-adrenergic receptor affinity and occupancy in small cerebral arteries. Am J Physiol Heart Circ Physiol 267: H757–H763, 1994.[Abstract/Free Full Text]
  17. Foster DB, Shen LH, Kelly J, Thibault P, Van Eyk JE, Mak AS. Phosphorylation of caldesmon by p21-activated kinase. Implications for the Ca(2+) sensitivity of smooth muscle contraction. J Biol Chem 275: 1959–1965, 2000.[Abstract/Free Full Text]
  18. Franklin MT, Wang CL, Adam LP. Stretch-dependent activation and desensitization of mitogen-activated protein kinase in carotid arteries. Am J Physiol Cell Physiol 273: C1819–C1827, 1997.[Abstract/Free Full Text]
  19. Geary GG, Buchholz JN, Pearce WJ. Maturation depresses mouse cerebrovascular tone through endothelium-dependent mechanisms. Am J Physiol Regul Integr Comp Physiol 284: R734–R741, 2003.[Abstract/Free Full Text]
  20. Geary GG, Osol GJ, Longo LD. Development affects in vitro vascular tone and calcium sensitivity in ovine cerebral arteries. J Physiol 558: 883–896, 2004.[Abstract/Free Full Text]
  21. Gokina NI, Osol G. Actin cytoskeletal modulation of pressure-induced depolarization and Ca2+ influx in cerebral arteries. Am J Physiol Heart Circ Physiol 282: H1410–H1420, 2002.[Abstract/Free Full Text]
  22. Gokina NI, Park KM, McElroy-Yaggy K, Osol G. Effects of Rho kinase inhibition on cerebral artery myogenic tone and reactivity. J Appl Physiol 98: 1940–1948, 2005.[Abstract/Free Full Text]
  23. Gollasch M, Wellman GC, Knot HJ, Jaggar JH, Damon DH, Bonev AD, Nelson MT. Ontogeny of local sarcoplasmic reticulum Ca2+ signals in cerebral arteries: Ca2+ sparks as elementary physiological events. Circ Res 83: 1104–1114, 1998.[Abstract/Free Full Text]
  24. Gong MC, Fuglsang A, Alessi D, Kobayashi S, Cohen P, Somlyo AV, Somlyo AP. Arachidonic acid inhibits myosin light chain phosphatase and sensitizes smooth muscle to calcium. J Biol Chem 267: 21492–21498, 1992.[Abstract/Free Full Text]
  25. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]
  26. Halpern W, Osol G. Influence of transmural pressure of myogenic responses of isolated cerebral arteries of the rat. Ann Biomed Eng 13: 287–293, 1985.[ISI][Medline]
  27. Hamaguchi T, Ito M, Feng J, Seko T, Koyama M, Machida H, Takase K, Amano M, Kaibuchi K, Hartshorne DJ, Nakano T. Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N. Biochem Biophys Res Commun 274: 825–830, 2000.[CrossRef][ISI][Medline]
  28. Hernandez MJ, Brennan RW, Bowman GS. Autoregulation of cerebral blood flow in the newborn dog. Brain Res 184: 199–202, 1980.[CrossRef][ISI][Medline]
  29. Hill MA, Zou H, Davis MJ, Potocnik SJ, Price S. Transient increases in diameter and [Ca2+]i are not obligatory for myogenic constriction. Am J Physiol Heart Circ Physiol 278: H345–H352, 2000.[Abstract/Free Full Text]
  30. Hill MA, Zou H, Potocnik SJ, Meininger GA, Davis MJ. Invited review: arteriolar smooth muscle mechanotransduction: Ca2+ signaling pathways underlying myogenic reactivity. J Appl Physiol 91: 973–983, 2001.[Abstract/Free Full Text]
  31. Huang J, Mahavadi S, Sriwai W, Hu W, Murthy KS. Gi-coupled receptors mediate phosphorylation of CPI-17 and MLC20 via preferential activation of the PI3K/ILK pathway. Biochem J 396: 193–200, 2006.[CrossRef][ISI][Medline]
  32. Johansson B. Myogenic tone and reactivity: definitions based on muscle physiology. J Hypertens Suppl 7: S5–S9, 1989.[Medline]
  33. Johnson PC. The myogenic response in the microcirculation and its interaction with other control systems. J Hypertens Suppl 7: S33–S40, 1989.[CrossRef][Medline]
  34. Kiss E, Muranyi A, Csortos C, Gergely P, Ito M, Hartshorne DJ, Erdodi F. Integrin-linked kinase phosphorylates the myosin phosphatase target subunit at the inhibitory site in platelet cytoskeleton. Biochem J 365: 79–87, 2002.[CrossRef][ISI][Medline]
  35. Lagaud G, Gaudreault N, Moore ED, Van Breemen C, Laher I. Pressure-dependent myogenic constriction of cerebral arteries occurs independently of voltage-dependent activation. Am J Physiol Heart Circ Physiol 283: H2187–H2195, 2002.[Abstract/Free Full Text]
  36. Lee CH, Poburko D, Kuo KH, Seow CY, van Breemen C. Ca2+ oscillations, gradients, and homeostasis in vascular smooth muscle. Am J Physiol Heart Circ Physiol 282: H1571–H1583, 2002.[Abstract/Free Full Text]
  37. Li F, Zhang Y, Wu C. Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats. J Cell Sci 112: 4589–4599, 1999.[Abstract]
  38. MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshorne DJ, Haystead TA. Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci USA 98: 2419–2424, 2001.[Abstract/Free Full Text]
  39. MacDonald JA, Eto M, Borman MA, Brautigan DL, Haystead TA. Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase. FEBS Lett 493: 91–94, 2001.[CrossRef][ISI][Medline]
  40. Martinez-Lemus LA, Wu X, Wilson E, Hill MA, Davis GE, Davis MJ, Meininger GA. Integrins as unique receptors for vascular control. J Vasc Res 40: 211–233, 2003.[CrossRef][ISI][Medline]
  41. McCarron JG, Crichton CA, Langton PD, MacKenzie A, Smith GL. Myogenic contraction by modulation of voltage-dependent calcium currents in isolated rat cerebral arteries. J Physiol 498: 371–379, 1997.[Abstract/Free Full Text]
  42. McLemore EC, Tessier DJ, Thresher J, Komalavilas P, Brophy CM. Role of the small heat shock proteins in regulating vascular smooth muscle tone. J Am Coll Surg 201: 30–36, 2005.[CrossRef][ISI][Medline]
  43. Meeks MK, Ripley ML, Jin Z, Rembold CM. Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery. Am J Physiol Cell Physiol 288: C633–C639, 2005.[Abstract/Free Full Text]
  44. Mehta D, Gunst SJ. Actin polymerization stimulated by contractile activation regulates force development in canine tracheal smooth muscle. J Physiol 519: 829–840, 1999.[Abstract/Free Full Text]
  45. Mellander S. Functional aspects of myogenic vascular control. J Hypertens Suppl 7: S21–S31, 1989.[Medline]
  46. Morrison DL, Sanghera JS, Stewart J, Sutherland C, Walsh MP, Pelech SL. Phosphorylation and activation of smooth muscle myosin light chain kinase by MAP kinase and cyclin-dependent kinase-1. Biochem Cell Biol 74: 549–557, 1996.[ISI][Medline]
  47. Muranyi A, MacDonald JA, Deng JT, Wilson DP, Haystead TA, Walsh MP, Erdodi F, Kiss E, Wu Y, Hartshorne DJ. Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. Biochem J 366: 211–216, 2002.[ISI][Medline]
  48. Murphy RA, Walker JS. Inhibitory mechanisms for cross-bridge cycling: the nitric oxide-cGMP signal transduction pathway in smooth muscle relaxation. Acta Physiol Scand 164: 373–380, 1998.[CrossRef][ISI][Medline]
  49. Nauli SM, Williams JM, Akopov SE, Zhang L, Pearce WJ. Developmental changes in ryanodine- and IP3-sensitive Ca2+ pools in ovine basilar artery. Am J Physiol Cell Physiol 281: C1785–C1796, 2001.[Abstract/Free Full Text]
  50. Nauli SM, Williams JM, Pearce WJ. Effects of maturation on mechanisms of cGMP-induced cerebral vasodilatation. Dev Neurosci 23: 224–233, 2001.[CrossRef][ISI][Medline]
  51. Niiro N, Ikebe M. Zipper-interacting protein kinase induces Ca(2+)-free smooth muscle contraction via myosin light chain phosphorylation. J Biol Chem 276: 29567–29574, 2001.[Abstract/Free Full Text]
  52. Obara K, Uchino M, Koide M, Yamanaka A, Nakayama K. Stretch-induced triphosphorylation of myosin light chain and myogenic tone in canine basilar artery. Eur J Pharmacol 534: 141–151, 2006.[CrossRef][ISI][Medline]
  53. Osol G, Brekke JF, McElroy-Yaggy K, Gokina NI. Myogenic tone, reactivity, and forced dilatation: a three-phase model of in vitro arterial myogenic behavior. Am J Physiol Heart Circ Physiol 283: H2260–H2267, 2002.[Abstract/Free Full Text]
  54. Osol G, Laher I, Cipolla M. Protein kinase C modulates basal myogenic tone in resistance arteries from the cerebral circulation. Circ Res 68: 359–367, 1991.[Abstract/Free Full Text]
  55. Parker TA, Grover TR, Kinsella JP, Falck JR, Abman SH. Inhibition of 20-HETE abolishes the myogenic response during NOS antagonism in the ovine fetal pulmonary circulation. Am J Physiol Lung Cell Mol Physiol 289: L261–L267, 2005.[Abstract/Free Full Text]
  56. Pearce W. Hypoxic regulation of the fetal cerebral circulation. J Appl Physiol 100: 731–738, 2006.[Abstract/Free Full Text]
  57. Pearce WJ, Duckles SP, Buchholz J. Effects of maturation on adrenergic neurotransmission in ovine cerebral arteries. Am J Physiol Regul Integr Comp Physiol 277: R931–R937, 1999.[Abstract/Free Full Text]
  58. Pearce WJ, Hull AD, Long DM, Longo LD. Developmental changes in ovine cerebral artery composition and reactivity. Am J Physiol Regul Integr Comp Physiol 261: R458–R465, 1991.[Abstract/Free Full Text]
  59. Pfitzer G. Invited review: regulation of myosin phosphorylation in smooth muscle. J Appl Physiol 91: 497–503, 2001.[Abstract/Free Full Text]
  60. Prentice A. Calcium in pregnancy and lactation. Annu Rev Nutr 20: 249–272, 2000.[CrossRef][ISI][Medline]
  61. Pryds A, Tonnesen J, Pryds O, Knudsen GM, Greisen G. Cerebral pressure autoregulation and vasoreactivity in the newborn rat. Pediatr Res 57: 294–298, 2005.[CrossRef][ISI][Medline]
  62. Shirasawa Y, Benoit JN. Stretch-induced calcium sensitization of rat lymphatic smooth muscle. Am J Physiol Heart Circ Physiol 285: H2573–H2577, 2003.[Abstract/Free Full Text]
  63. Smiesko V, Kriska M, Kovalcik V. Bayliss myogenic response in the isolated ductus arteriosus of guinea-pig and rabbit fetuses. Experientia 34: 745, 1978.[CrossRef][ISI][Medline]
  64. Storme L, Parker TA, Kinsella JP, Rairigh RL, Abman SH. Chronic hypertension impairs flow-induced vasodilation and augments the myogenic response in fetal lung. Am J Physiol Lung Cell Mol Physiol 282: L56–L66, 2002.[Abstract/Free Full Text]
  65. Storme L, Rairigh RL, Parker TA, Kinsella JP, Abman SH. In vivo evidence for a myogenic response in the fetal pulmonary circulation. Pediatr Res 45: 425–431, 1999.[ISI][Medline]
  66. Su BY, Reber KM, Nankervis CA, Nowicki PT. Development of the myogenic response in postnatal intestine: role of PKC. Am J Physiol Gastrointest Liver Physiol 284: G445–G452, 2003.[Abstract/Free Full Text]
  67. Takahashi A, Camacho P, Lechleiter JD, Herman B. Measurement of intracellular calcium. Physiol Rev 79: 1089–1125, 1999.[Abstract/Free Full Text]
  68. Tanaka Y, Nakayama K. Measurement of intracellular Ca2+ concentration changes by use of fura-2 in the generation of myogenic contraction of dog cerebral artery in response to quick stretch. Res Commun Mol Pathol Pharmacol 99: 169–186, 1998a.[ISI][Medline]
  69. Tanaka Y, Nakayama K, Shigenobu K. Changes in cytosolic Ca2+ measured by use of fura-2 and contraction produced by quick stretch and various stimulants in canine cerebral artery. Res Commun Mol Pathol Pharmacol 102: 79–92, 1998b.[ISI][Medline]
  70. Weber LP, Van Lierop JE, Walsh MP. Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments. J Physiol 516: 805–824, 1999.[Abstract/Free Full Text]
  71. White C, McGeown JG. Regulation of basal intracellular calcium concentration by the sarcoplasmic reticulum in myocytes from the rat gastric antrum. J Physiol 529: 395–404, 2000.[Abstract/Free Full Text]
  72. Williams JM, Hull AD, Pearce WJ. Maturational modulation of endothelium-dependent vasodilatation in ovine cerebral arteries. Am J Physiol Regul Integr Comp Physiol 288: R149–R157, 2005.[Abstract/Free Full Text]
  73. Wu C, Dedhar S. Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. J Cell Biol 155: 505–510, 2001.[Abstract/Free Full Text]
  74. Zou H, Lifshitz LM, Tuft RA, Fogarty KE, Singer JJ. Visualization of Ca2+ entry through single stretch-activated cation channels. Proc Natl Acad Sci USA 99: 6404–6409, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
R. J. Sandoval, E. R. Injeti, W. T. Gerthoffer, and W. J. Pearce
Postnatal maturation modulates relationships among cytosolic Ca2+, myosin light chain phosphorylation, and contractile tone in ovine cerebral arteries
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2183 - H2192.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
<