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REPORT
DEVELOPMENTAL PHYSIOLOGY AND PREGNANCY
Department of Physiology and Pharmacology, Center for Perinatal Biology, Loma Linda University, School of Medicine, Loma Linda, California
Submitted 13 December 2006 ; accepted in final form 30 May 2007
ABSTRACT
Although postnatal maturation potently modulates agonist-induced cerebrovascular contractility, its effects on the mechanisms mediating cerebrovascular myogenic tone remain poorly understood. Because the regulation of calcium influx and myofilament calcium sensitivity change markedly during early postnatal life, the present study tested the general hypothesis that early postnatal maturation increases the pressure sensitivity of cerebrovascular myogenic tone via age-dependent enhancement of pressure-induced calcium mobilization and myofilament calcium sensitivity. Pressure-induced myogenic tone and changes in artery wall intracellular calcium concentrations ([Ca2+]i) were measured simultaneously in endothelium-denuded, fura-2-loaded middle cerebral arteries (MCA) from pup [postnatal day 14 (P14)] and adult (6-mo-old) Sprague-Dawley rats. Increases in pressure from 20 to 80 mmHg enhanced myogenic tone in MCA from both pups and adults although the normalized magnitudes of these increases were significantly greater in pup than adult MCA. At each pressure step, vascular wall [Ca2+]i was also significantly greater in pup than in adult MCA. Nifedipine significantly attenuated pressure-evoked constrictions in pup MCA and essentially eliminated all responses to pressure in the adult MCA. Both pup and adult MCA exhibited pressure-dependent increases in calcium sensitivity, as estimated by changes in the ratio of pressure-induced myogenic tone to wall [Ca2+]i. However, there were no differences in the magnitudes of these increases between pup and adult MCA. The results support the view that regardless of postnatal age, changes in both calcium influx and myofilament calcium sensitivity contribute to the regulation of cerebral artery myogenic tone. The greater cerebral myogenic response in P14 compared with adult MCA appears to be due to greater pressure-induced increases in [Ca2+]i, rather than enhanced augmentation of myofilament calcium sensitivity.
rat cerebral arteries; myofilament calcium sensitivity
Based almost exclusively on studies performed using adult arterial preparations, myogenic reactivity is commonly attributed to alterations in intracellular calcium concentration ([Ca2+]i), mediated in large part via pressure-induced changes in plasmalemmal calcium influx through several possible types of calcium channels, the most important of which appears to be the voltage-gated L-type channels (9, 11, 25, 40, 46, 49). Myogenic responses also may involve pressure-induced calcium release from intracellular stores (22, 30), but again the relative importance of this mechanism in the overall contractile response to changes in transmural pressure remains a subject of continued study. Changes in transmural pressure may also modulate myofilament calcium sensitivity (17, 24), possibly through changes in the enzyme activities of myosin light chain phosphatase, protein kinase C, Rho-kinase, and other enzymes (5, 17, 24, 28, 33, 34, 46).
Given that most mechanisms proposed to mediate the myogenic response also appear to change significantly during postnatal maturation, it is reasonable to expect that the regulation of myogenic tone should also be quite different in immature and mature cerebral arteries. Consistent with this possibility, cerebral autoregulation operates over lower and narrower ranges of arterial pressures in neonates compared with adults (8), suggesting that myogenic sensitivity to pressure changes may be upregulated in neonatal, compared with adult, cerebral arteries. Similarly, at low pressures (10–60 mmHg), endothelium-intact pressurized middle cerebral arteries (MCA) from neonatal mice develop significantly greater tone than MCA from adult mice (15). However, endothelium removal eliminated the age-related differences at high, but not at low, pressures suggesting that the endothelium modulates myogenic responses in both an age-dependent and pressure-dependent manner, at least in mouse cerebral arteries. Altogether, the available evidence strongly suggests that regulation of myogenic tone varies considerably between immature and mature cerebral arteries, but the mechanistic basis for this variation remains poorly understood and largely unexplored.
Owing to the importance of myogenic tone for the regulation of cerebrovascular homeostasis, and the findings that the calcium-dependent contractile mechanisms governing myogenic tone are quite different in adult and neonatal cerebral arteries, the present study explores the hypothesis that the relations among transmural pressure, cytosolic calcium, myofilament calcium sensitivity, and myogenic tone are significantly greater in immature and mature cerebral arteries. To evaluate this general hypothesis, we examined and compared pressure-dependent myogenic tone and changes in wall [Ca2+]i in endothelium-denuded MCA from adult (6-mo) and pup [postnatal day 14 (P14)] Sprague-Dawley rats. Removal of the endothelium enabled the study of pressure-induced myogenic responses independently of the previously reported complications due to endothelial modulation. With this approach, we tested the specific hypotheses that in cerebral arteries: 1) postnatal maturation depresses the sensitivity of myogenic constriction to pressure, 2) myogenic constriction is more dependent on the influx of extracellular calcium in immature than in mature cerebral arteries, and 3) increased myofilament calcium sensitivity contributes more to the myogenic response in cerebral arteries from pups than in arteries from adults.
MATERIALS AND METHODS
General preparation. Loma Linda University's Institutional Animal Care and Use Committee approved all procedures used in this study. We compared two groups of male Sprague-Dawley rats: adult rats (6-mo-old) and pups (P14) representing mature adult and juvenile rats were housed under a 12:12-h light-dark cycle with food and water available ad libitum. In terms of vascular maturity and reactivity, the P14 rat can be considered an early juvenile that is prepubertal but exhibits vascular characteristics midway between those of a newborn and those of an adult (43). As described previously (14, 15), main-branch MCA without side branches were dissected and cut to lengths of 5 mm and mounted on cannulas in an organ chamber positioned on the stage of an inverted microscope. (Living Systems, Burlington, VT). The chamber contained physiological salt solution (PSS) with (in mM): 130 NaCl, 10.0 HEPES, 6.0 glucose, 4.0 KCl, 4.0 NaHCO3, 1.8 CaCl2, 1.18 KH2PO4, 1.2 MgSO4, and 0.025 EDTA, pH 7.4. The proximal cannula was connected to a pressure transducer, a reservoir of PSS, and a servocontrolled pump system used to set transmural pressure. The distal cannula was connected to a luer-lock valve that was open to gently flush the lumen during the initial cannulation. After cannulation, the distal valve was closed, and all measurements were conducted under no-flow conditions. Arterial diameters were recorded with the SoftEdge Acquisition Subsystem (Ion-Optix, Milton, MA).
Endothelium removal. Vascular endothelium can significantly influence responses to stretch and pressure in cerebral arteries (15). Thus, all arteries used in this study were denuded of endothelium by perfusing 1 ml of air through the artery lumen. Removal of endothelium was verified by the lack of a vasodilatory response to 10 µM ACh in arteries equilibrated at 60 mmHg.
Measurement of smooth-muscle Ca2+ in pressurized arteries. Cannulated arteries were loaded for 20 min at room temperature with fura-2 AM (Molecular Probes, Eugene, OR) at a concentration of 1 µM as previously described (13). The fura-2-loaded arteries were then washed with PSS, and bath temperature was increased to 37°C; background-corrected fluorescence emission was measured at 510 nm at a sampling rate of 3 Hz using an IonOptix photomultiplier system (14).
We estimated arterial wall [Ca2+]i using both in vitro and in vivo calibration methods as previously described (41, 50). In vitro values for the fura-2 dissociation constant (Kd) were determined using a commercial calibration kit (Molecular Probes) with known calcium concentrations ranging from 0 to 39 µM. Each calcium standard was loaded with 4 µM of fura-2 pentapotassium salt drawn into a microcapillary tube and placed on the microscope stage where the fluorescent intensities during excitation at 340 and 380 nm were measured. The ratios of the intensities at 340 and 380 nm were then plotted against the known calcium concentrations to determine the fura-2 Kd values. For the in vivo method, fura-2 loaded arteries were incubated in 1 mM EGTA to reduce extracellular [Ca2+] to near zero values, after which fluorescence intensities at 380 nm (Fmin), and the 340/380 ratio (Rmin) were recorded for at least 1 min. The extracellular medium was then replaced with PSS containing 10 mM calcium, 120 mM K+, and ionomyocin (1 µM). Once stabilized, fluorescence intensity values were recorded to obtain fluorescence values at 380 nm (Fmax) and the 340/380 ratio (Rmax) when fura-2 was saturated with calcium. Overall, the values obtained for Fmin, Rmin, Fmax, and Rmax were similar for the in vitro and in vivo methods. In addition, the Kd values obtained for fura-2 agreed well with values reported in other studies (32). Given this pattern of agreement, the Kd values obtained from the in vitro calibration were used to convert the experimental fluorescent intensity ratios (R) to [Ca2+]i over the physiological range by iterative fit to the Grynkiewicz equation: [Ca2+]i = Kd[(R – Rmin)/(Rmax – R)]Sf (19). In our calculations using this equation, we used the following averaged values: Sf (20.5), Rmin (0.3), Rmax (6.3), and Kd (251 nM).
Protocols. In all experiments, artery viability and minimum possible diameter for each artery was first assessed by measuring the changes in diameter and wall [Ca2+]i produced in response to calcium-replete PSS containing 120 mM K+, as indicated in Fig. 1. Next, the arteries were equilibrated in normal PSS after which artery diameters and wall calcium concentrations were recorded at transmural pressures of 20, 40, 60, and 80 mmHg. We used this range of pressures because: 1) it spanned the range of arterial pressures typical of young rats between 5 and 14 days of age (23, 42, 53), and 2) previous studies in the rat have suggested that pressure-induced increases in calcium influx are most evident at these lower pressures (35). Each pressure was applied for at least 5 min until values for both diameter and Ca2+ were obtained. Next, the arteries were equilibrated in PSS containing no calcium and 3 mM EGTA after which diameter measurements were again obtained at transmural pressures of 20, 40, 60, and 80 mmHg to define the maximum possible diameter for each artery.
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Calculations, data analysis, and statistics. For each calculation of myogenic tone, artery diameters measured under the various experimental conditions were subtracted from the maximum diameters observed in each artery in 0 Ca2+ PSS at the corresponding transmural pressures. These diameter differences were then normalized relative to the maximum active change in diameter defined in each artery as the difference in maximum diameter observed in 0 calcium PSS minus the minimum diameter measured in calcium-replete PSS containing 120 mM K+, obtained at a pressure of 60 mmHg. Values of myogenic tone, artery diameter, and wall calcium were analyzed via two-way ANOVA with repeated measures using age (pup vs. adult) and pressure as factors. Post hoc comparisons were performed using the Fischer paired least significant difference analysis. The slopes of the relations between myogenic tone and transmural pressure were calculated by using linear regression and then were compared between pups and adults using t-statistics. Throughout the text, all values are given as means ± SE, and statistical significance implies P < 0.05 unless stated otherwise.
RESULTS
Validation of fura-2 AM loading and deesterification. To verify that comparable amounts of fura-2 AM were taken up and deesterified by both pup and adult arteries, the intensity of the fluorescence signal at 510 nm was measured during illumination at 380 nm to obtain a signal (F380) proportional to the amount of loaded active dye (31). The F380 values obtained averaged 3,735 ± 191 and 3,578 ± 133 for adult and pup arteries, respectively. These data demonstrate that the amount of fura-2 AM taken up and deesterified was not significantly different in the two age groups and thus are consistent with our previous results from superior cervical ganglia suggesting that the F380 does not change with advancing age from adult to senescence (41, 50). These results also suggest that overall esterase activity is sufficient to fully convert fura-2 AM into its free salt in both pup and adult MCA.
Effects of K+ and a 50-mmHg step increase in pressure on diameter and wall [Ca2±]i.
Exposure of adult artery segments to PSS containing 120 mM K+ produced an abrupt decrease in artery diameter, and this contraction was reversed over an interval of 
200 s following K+ washout (Fig. 1A). Exposure of adult artery segments to 120 mM K+ also produced a parallel rapid increase in [Ca2+]i, and this increase was also reversed over an interval of 200 s following K+ washout (Fig. 1B). In arteries from pups, exposure to 120 mM K+ produced changes in diameter (Fig. 1C) and [Ca2+]i (Fig. 1D) that were qualitatively similar to the changes observed in adult arteries but developed more slowly, were smaller in magnitude, and recovered more quickly.
When transmural pressure was quickly changed from 10 to 60 mmHg, adult artery diameters increased abruptly but did not reach maximum passive diameter, which was defined as the diameter attained in 0 calcium PSS with 3 mM EGTA (Fig. 2A). Following this step change in pressure, arteries in calcium-replete PSS gradually contracted over a period of 200 s, whereas arteries in calcium-free PSS maintained a near-constant diameter. The step change in pressure also initiated a rapid increase in [Ca2+]i, followed by a gradual increase over a period of 200 s (Fig. 2B); the dynamics of the diameter and [Ca2+]i responses were qualitatively quite similar following the step change in pressure. Similarly, in pup arteries with a single pressure step from 10 to 60 mmHg, diameters increased abruptly but did not reach maximum passive diameter (Fig. 2C). Following this step change in pressure, pup arteries in calcium-replete PSS contracted over a period of 200 s, whereas arteries in calcium-free PSS maintained a near-constant diameter. In addition, the constriction caused by a single pressure step is greater in pup compared with adult arteries. The step change in pressure also initiated a more gradual increase in [Ca2+]i, over a period of 200 s (Fig. 2D); the dynamics of the diameter and [Ca2+]i responses were qualitatively quite similar following the step change in pressure. However, in pup arteries, the calcium levels are greater compared with adults.
Effects of pressure and nifedipine on myogenic tone in pup and adult arteries. In calcium-free PSS containing 3 mM EGTA, progressive increases in transmural pressure from 20 to 80 mmHg produced corresponding increases in diameter in both adult (Fig. 3A) and pup (Fig. 3B) arteries. Maximum passive diameters at 20 mmHg were significantly larger in adult (212 ± 5.6 µm) than in pup (192 ± 3.1 µm) arteries, and similarly at 80 mmHg were significantly larger in adult (254 ± 6 µm) than in pup (227 ± 4 µm) arteries (P < 0.02, ANOVA). In the presence of normal PSS containing 1.8 mM Ca2+, the diameters associated with each transmural pressure were significantly less than observed in calcium-free PSS, indicating the development of myogenic tone in both adult (Fig. 3C) and pup (Fig. 3D) arteries. Most importantly, the magnitude of the pressure-induced increase in myogenic tone was significantly greater in pup than in adult arteries.
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To directly compare myogenic reactivity in adult and pup arteries, we plotted the pressure-induced changes in myogenic tone against the corresponding transmural pressures (Fig. 4). Regression of these values of tone against pressure yielded slopes of 0.50 ± 0.10 and 0.90 ± 0.20 in adult and pup arteries, respectively. These results suggest that myogenic reactivity to increases in pressure was significantly less in adult than in pup arteries.
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Cerebrovascular contractility depends on the coordinated integration of multiple mechanisms, most of which change steadily throughout fetal development and early postnatal maturation (24, 25, 37–39, 47). Moreover, during maturation there is an increase in systemic blood pressure, which increases hemodynamic stress on blood vessels. Thus, the contractile mechanisms that govern myogenic responses in cerebral blood vessels must adapt to control cerebral blood flow and minimize the risk of blood vessel rupture (52). In addition, the overall regulation of cerebrovascular resistance involves a coordinated integration of both large artery and small artery responses; up to 50% of total cerebrovascular resistance can be accounted for in arteries proximal to the pial circulation (7, 36). These findings illustrate the importance of larger cerebral arteries, such as the MCA, in overall regulation of the immature cerebral circulation.
Although the consequences of fetal development and postnatal maturation have been established for a variety of contractile mechanisms in smooth muscle, their effects on cerebrovascular myogenic reactivity remain largely unexplored. This deficit offered a novel opportunity for the application of recently developed, in vitro, small vessel perfusion techniques with simultaneous measurements of lumen diameter and wall [Ca2+]i (10, 12, 27, 45, 47) to the study of myogenic reactivity in immature arteries. To that end, an initial goal of the present study was to establish that fura-2 AM uptake and overall esterase activity were sufficient in cerebral arteries from P14 rats to enable comparable measurements of [Ca2+]i in arteries from both pups and adults. The values we obtained for maximum fluorescence at 380 nm (F380) agreed within 4% in pup and adult arteries, and thus validated the use of the fura-2 methodology in our preparations.
To further validate the preparations used in our experiments, we also measured the magnitudes of changes in [Ca2+]i and artery diameter observed following a step change in pressure. These were comparable to previously reported results obtained using similar methods in other preparations (27). As shown in Fig. 1, we found that exposure to 120 mM K+ produced significant changes in both diameter and wall calcium. The dynamics of the changes in diameter and wall calcium were similar in adult and pup arteries, although the changes developed more slowly, were smaller in magnitude, and recovered more quickly in pup than in adult arteries. In parallel, the experiments in adult and pup arteries further revealed that a 50-mmHg step change in transmural pressure produced an initial rapid increase in both diameter and wall [Ca2+]i followed by a gradual vasoconstriction and a slow continued rise in wall [Ca2+]i. This pattern of response was similar to that reported by Meininger et al. (27), although in their study the myogenic constriction returned diameter completely back to baseline with a corresponding sustained increase in wall [Ca2+]i of only 10–15%. In contrast, in the present study, the pressure step produced a sustained increase in diameter that was not completely reversed by myogenic constriction despite an increase in wall [Ca2+]i of 30% and 15% in adult and pup arteries, respectively (Fig. 2, A and C). Regardless, the two sets of results agree that arteries from very different vascular beds (cremaster muscle vs. cerebral circulation) were stimulated with quite different pressure stimuli (step increase from 90 to 130 cmH2O vs. 10 to 60 mmHg). This agreement strongly suggests that the methods used to study myogenic responses in our preparations were valid, accurate, and reliable.
The main goal of the present study was to test the general hypothesis that the relations among transmural pressure, cytosolic calcium, myofilament calcium sensitivity, and myogenic tone are significantly different in immature and mature cerebral arteries. In the first series of experiments designed to explore this hypothesis, stepwise increases in transmural pressure from 20 to 80 mmHg produced changes in diameter, which, when compared with corresponding changes observed in calcium-free PSS, enabled calculations of myogenic tone at each pressure (Fig. 3). These calculations revealed that both adult and pup MCA significantly increase myogenic tone in response to increased transmural pressure. As indicated by the slopes of the relations between pressure and myogenic tone (Fig. 4), the data also indicated that myogenic reactivity was up to 30% greater in arteries from pups than in arteries from adults. This finding is consistent with previous studies of cerebral arteries (14–16) and further supports the concept that greater myogenic reactivity in neonatal arteries may be an advantageous adaptation to the lower hydraulic pressures that are typical of the immature cerebrovascular circulation (6, 8, 18).
To understand more completely why myogenic reactivity might be greater in immature than mature cerebral arteries, we also examined myogenic reactivity to changes in transmural pressure in the presence of an optimally effective concentration of nifedipine, a dihydropyridine blocker of L-type calcium channels (48). Nifedipine dramatically attenuated the development of pressure-induced myogenic tone in both pup and adult arteries (Fig. 3). This finding supports the view that myogenic reactivity is largely dependent upon the entry of extracellular calcium through plasmalemmal calcium channels (1, 9, 11, 20, 47). Although a significant fraction of myogenic tone persisted in the presence of nifedipine, the reactivity of this component to changes in transmural pressure was greatly attenuated in pup arteries and completely eliminated in adult arteries. This suggests that the specialized coupling between mechanotransduction and voltage-dependent calcium channels proposed to explain myogenic reactivity in other vascular beds (21) also may be important in both mature and immature rat cerebral arteries. In addition, our finding that the degree of attenuation of myogenic reactivity by nifedipine was greater in adult than in pup arteries is consistent with, and may be a consequence of, the tendency for calcium influx to be a more important source of activator calcium in pup compared with adult cerebral arteries (3). Alternatively, it could also be explained by the presence of a small, nifedipine-resistant component of pressure-sensitive calcium influx or release in pup but not adult arteries.
To examine more closely the age-dependence of the role of calcium in myogenic reactivity, an additional set of measurements focused on the relations between transmural pressure and cytosolic calcium concentration as measured via fura-2. Consistent with the results shown in Fig. 3, increases in transmural pressure also produced significant increases in cytosolic calcium, and the magnitudes of these increases were greater in pup than in adult arteries (Fig. 5). In addition, nifedipine virtually eliminated the pressure-induced calcium increases in the adult arteries and significantly attenuated but did not eliminate these increases in the pup arteries. The small pressure-dependent and nifedipine-resistant increases in cytosolic calcium observed in pup, but not adult, arteries suggest the possible presence either of an additional nifedipine-resistant pathway for calcium influx in the pup arteries, or of a mechanism mediating pressure-sensitive calcium release. This latter possibility is consistent with the observation that pressure increased cytosolic calcium even in pup arteries equilibrated in calcium-free PSS. In either case, the fura-2 data are quite consistent with the nifedipine data, and strongly suggest that the increased myogenic reactivity observed in pup compared with adult arteries is attributable, at least in part, to an enhanced ability of pressure to stimulate increases in cytosolic calcium.
In light of convincing evidence that increased transmural pressure can lead to increased myofilament calcium sensitivity (17, 24, 52), we calculated the ratio of myogenic tone to cytosolic calcium concentration to obtain estimates of calcium sensitivity. Consistent with the reports from Gokina et al. (17) and Lagaud et al. (24), our estimates of calcium sensitivity rose in direct proportion to transmural pressure in both adult and pup arteries (Fig. 6). This indicates that pressure-induced increases in myofilament calcium sensitivity make a significant contribution to overall myogenic reactivity in rat cerebral arteries. However, the magnitudes of these estimates did not vary significantly between adult and pup arteries at any pressure. This finding suggests that age-related differences in overall myogenic reactivity probably do not involve corresponding differences in the ability of increased transmural pressure to enhance myofilament calcium sensitivity in pup and adult arteries.
In conclusion, the present data demonstrate that pressure-induced myogenic reactivity is greater in MCA from P14 rats than from adult rats. This myogenic reactivity appears dependent on pressure-induced increases in calcium-influx through nifedipine-sensitive calcium channels but also may involve a small contribution from pressure-dependent or nifedipine-resistant increases in cytosolic calcium in pup (not adult) cerebral arteries. Increases in transmural pressure also appear to enhance myofilament calcium sensitivity significantly in rat cerebral arteries, but the magnitude of this effect is quite similar in cerebral arteries from P14 and adult rats. Overall, the observed age-related differences in myogenic reactivity are best explained by an enhanced ability of increased transmural pressure to elevate cytosolic calcium, largely via increased calcium influx through nifedipine-sensitive calcium channels in pup compared with adult cerebral arteries. The mechanisms responsible for the increased ability of transmural pressure to enhance calcium influx remain unclear but could involve numerous possible mechanisms related to age-related differences in artery structure, G protein expression, calcium channel density, and intracellular calcium pool size (21, 30).
GRANTS
This work was supported, in part, by National Heart, Lung, and Blood Institute Grants R01-HL-69078 and R01-HL-54210 and National Institute of Child Health and Human Development Grant P01-31226.
ACKNOWLEDGMENTS
The authors thank Conwin Vanterpool and Charles Hewitt for their technical assistance with the experiments conducted for this study. The authors also appreciate the many excellent suggestions given by Dr. Michael A. Hill during preparation of this manuscript.
FOOTNOTES
Address for reprint requests and other correspondence: J. N. Buchholz, Dept. of Physiology and Pharmacology, Loma Linda Univ., School of Medicine, Loma Linda, CA, 92350 (e-mail: jbuchholz{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
1-adrenergic receptors, IP3, responses, and NE-induced contraction in cerebral arteries. Am J Physiol Heart Circ Physiol 271: H2313–H2319, 1996.This article has been cited by other articles:
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  J. B. Samora, J. C. Frisbee, and M. A. Boegehold Increased myogenic responsiveness of skeletal muscle arterioles with juvenile growth Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2344 - H2351. [Abstract] [Full Text] [PDF]
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