INTRODUCTION

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IN CEREBROVASCULAR SMOOTH muscle, agonist-induced elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) occurs through G protein-coupled mechanisms involving Ca²⁺ release from the sarcoplasmic reticulum (SR) (24). Stimulation of ₁-adrenergic receptors (₁-AR) by norepinephrine (NE) results in production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], which stimulates SR Ca²⁺ release via Ins(1,4,5)P₃-receptors (6, 27, 28). In most smooth muscle, NE also induces Ca²⁺ influx through both voltage-gated and receptor-operated channels (19, 29), and electrophysiological studies have demonstrated the importance of membrane potential in the regulation of cerebral vascular tone (21, 29). Nonetheless, the extent to which L-type voltage-gated and/or receptor-operated Ca²⁺ channels are important in NE-induced contraction of cerebral arteries is not established. Neither is the role of these Ca²⁺ channels in cerebral arteries of the fetus established, nor how their function changes with development.

We and others have demonstrated that adrenergic-mediated cerebrovascular reactivity changes dramatically with development from fetus to newborn to adult (25, 27, 31, 41). Changes in membrane ₁-AR density (27), Ins(1,4,5)P₃ synthesis (27), and Ins(1,4,5)P₃ receptor density (43) appear to be involved in developmental differences in NE-induced contractility. However, changes in these factors alone cannot explain fully the changes in vascular contractility (26, 27). Thus differences in related intracellular signaling mechanisms in addition to membrane receptor binding events appear to play an important role in the genesis of age-related differences in cerebrovascular tone.

The present studies test the hypotheses that Ca²⁺ flux via L-type plasma membrane Ca²⁺ channels and/or receptor-operated Ca²⁺ channels plays a key role in NE-induced contraction of cerebral arteries and that the role of these channels is different in the fetus from the adult.

	METHODS

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Experimental animals and tissues. For these studies, we used middle cerebral arteries (MCA) from normoxic control near-term fetuses (~140 days) and nonpregnant adult sheep (2 yr) obtained from Nebeker Ranch (Lancaster, CA) that had been maintained near sea level (~300 m). The ewes were anesthetized and killed with 100 mg/kg intravenous pentobarbital sodium, following which we obtained isolated cerebral artery segments. We have shown that this method of death has no significant effect on vessel reactivity compared with use of other anesthetic agents (31). All studies were performed in isolated main branch MCA (adult ~450 µm; fetus ~350 µm) cleaned of adipose and connective tissue, as previously described (27, 28). To avoid the complication of endothelial-mediated effects, we removed the endothelium by carefully inserting a small wire three times (27, 28). To confirm endothelium removal, we contracted the vessel with 10⁵ M 5-hydroxytryptamine and at the plateau added 10⁶ M ADP. Vessels that relaxed >20% after this treatment were rejected for further study. Cerebral arteries were used immediately for simultaneous measurements of [Ca²⁺]_i and tensions (36). Unless otherwise noted, all chemical compounds were purchased from Sigma (St. Louis, MO).

Contractility measurements. We cut the MCA into rings of 2 mm in length, mounted them on two tungsten wires (0.13 mm diameter; A-M Systems, Carlsborg, WA), attaching one wire to an isometric force transducer (Kent Scientific, Litchfield, CT) and the other to a post attached to a micrometer used to vary resting tension in a 5-ml tissue bath mounted on a Jasco CAF-110 intracellular Ca²⁺ analyzer (Jasco, Easton, MD). We equilibrated the arteries at 38°C for 30 min in a bicarbonate Krebs solution (pH 7.4) containing (in mM) 115.2 NaCl, 22.14 NaHCO₃, 7.88 D-glucose, 4.7 KCl, 1.18 KH₂PO₄, 1.16 MgSO₄, 1.80 CaCl₂, 0.114 ascorbic acid, and 0.027 Na₂ EDTA continuously bubbled with 95% O₂-5% CO₂. We obtained micrometer readings and thereby inside diameter measurements for each arterial segment under unstressed conditions (<0.1 g tension) and similarly at optimum resting tension. To establish optimal resting tension for the studies, we performed K⁺-induced contractility experiments with resting tension ranging from 0.2 to 0.5 g (n = 4 each). In both fetal and adult arteries, optimal results for both tension and fluorescence ratio were obtained at 0.3 g, the value chosen for the present studies. The vessel inside diameter measurements, in combination with measurements of vessel wall thickness, length, and potassium-induced force, enabled calculation of force per unit cross-sectional area, as previously described (30, 31).

Intracellular calcium measurement in smooth muscle. MCA rings were equilibrated under 0.3 g tension at 25°C for 40 min before loading with the acetoxymethyl ester of fura 2 (fura 2/AM; Molecular Probes, Eugene, OR), a fluorescent Ca²⁺ indicator (15). To sequester the dye in the cytosol, loading of the smooth muscle with the fura 2 was performed by incubating it for 4 h at 25°C in Krebs buffer containing 5 µM fura 2, 0.17% DMSO, 0.02% Cremophore EL, and 0.1% BSA. After loading, the vessel ring was washed with Krebs buffer five times in 30 min to allow for complete hydrolysis of the fura 2 ester groups by endogenous esterase. Fura 2 fluorescence and force were measured simultaneously at 38°C. Vessels were illuminated alternatively (125 Hz) at excitation wavelengths of 340 and 380 nm by means of two monochromators in the light path of a 75 W xenon lamp. Tissue fluorescence emission was measured at 510 nm by a photomultiplier. The fluorescence intensity at each excitation wavelength (F₃₄₀ and F₃₈₀) and the ratio of these fluorescence values (R_340/380) were recorded with a time constant of 250 ms and stored with the force signal.

When fura 2 is present only as free acid there is an inverse change in fluorescence at 340 and 380 nm excitation. At the end of the study we determined the maximum R_340/380 (R_max) by stimulation with 10⁴ M NE and 10⁵ M ionomycin in a phosphate-free, bicarbonate-free 120 mM K⁺, 5 mM Ca²⁺ Krebs buffer. The minimum R_340/380 (R_min) was elicited by chelating Ca²⁺ with 50 mM EGTA. R_max and R_min were used to normalize changes in fluorescence ratios obtained in different preparations. Because of uncertainty about the dissociation constant for [Ca²⁺]_i under intracellular conditions, we used R_340/380 as an indicator of relative changes in [Ca²⁺]_i, rather than calculating actual [Ca²⁺]_i. Although some investigators may prefer the transformation of fluorescence to [Ca²⁺]_i, in tissues such as cerebral arteries the presentation of the ratio is less ambiguous.

We first contracted the arterial segment by adding 120 mM isotonic potassium chloride to the Krebs buffer. After peak tension was reached, we washed the artery with normal sodium Krebs solution and allowed it to return to baseline tension for 15 min. Subsequent contractions were then induced with NE at half log doses (10⁸-10⁴ M). In earlier studies in ovine cerebral arteries, we have shown this to produce maximal stable contractions (31). During all contractility experiments, we continuously digitized, normalized, and recorded contractile tensions and the fluorescence ratio (R_340/380) using an online computer. For all vessels we evaluated the contractile response for tension and fluorescence ratio by measuring the maximum peak height, and expressing it as percent K_max (a measure of "efficacy") and calculated pD₂ (the negative logarithm of the EC₅₀, or half-maximal concentration, for NE and an index of tissue "sensitivity" or "potency").

Role of Ca²⁺ channel blockers. To determine the role of L-type Ca²⁺ channels in MCA arteries (n = 4 each group), we quantified NE-induced tension and [Ca²⁺]_i after blockade by each of three groups of agents as follows: dihydropyridines (e.g., nifedipine, 10⁹-10⁵ M), phenylalkylamines (e.g., verapamil, 10⁹-10⁵ M), and benzothiazepines (e.g., diltiazem, 10⁹-10⁵ M). For each of these agents we first quantified the [Ca²⁺]_i and tension in response to 10⁵ M NE. After a 40-min washout and reequilibration, we repeated the experiment after administration of 10⁵ M NE to increase [Ca²⁺]_i and tension. Then, at the plateau of the response, we added increasing concentrations of the Ca²⁺ channel blocker to examine the inhibition of the [Ca²⁺]_i signal and tension. For each compound we determined threshold concentration, EC₅₀ or pIC₅₀ (negative logarithm of the half-maximal inhibitory concentration), and maximum response. We also quantified these parameters after the addition of the Ca²⁺ channel antagonist lanthanum ion (10³ M LaCl₃) for 2 min (34, 42). Because La³⁺ forms an La₂ (CO₃)₃ precipitate in Krebs buffer, those studies of La³⁺ effects were conducted in HEPES-buffered salt solution containing (in mM) 120 NaCl, 6.0 KCl, 1.8 CaCl₂, 1.2 MgCl₂, 11.4 D-glucose, and 5.0 HEPES. pH was adjusted to 7.4 by titration with NaOH, and this solution was gassed with 100% O₂. To examine the role of receptor-operated or nonselective cation channels, we used the blocker 2-nitro-4 carboxyphenyl-N,N-diphenyl carbamate (NCDC) (34).

Role of extracellular Ca²⁺. To determine the role of extracellular Ca²⁺ in traversing calcium channels, we measured [Ca²⁺]_i and tension after NE stimulation at the value of half-maximal contraction (pD₂), after vessels had been exposed to calcium-free (<10⁸ M of Ca²⁺ with EGTA 5 × 10⁴ M to chelate Ca²⁺) Krebs buffer for 2 min (n = 4 each). Again, we evaluated the contractile response for tension and fluorescence ratio by measuring the maximum peak height and expressing it as percent K_max. We quantified the duration of contractile response by measuring the peak width (s) at 50% of peak height, arbitrarily defining this at t_1/2.

Statistical analysis. All values were calculated as means ± SE. In all cases, n refers to the number of vessel segments (which corresponds to the number of animals) studied. Because of the nature of these studies, several statistical tests were used to test for significant differences. For testing differences between two groups, we used a simple unpaired Student's t-test. For multiple comparisons, one- and two-way ANOVA (vessel, age) coupled with Duncan's multiple range test was used. Where appropriate, we used ANOVA with repeated measures. The correlation between tension and fluorescence ratio was determined by linear regression (8). A P value of <0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Contractile and [Ca²⁺]_i responses to potassium and norepinephrine. Figure 1A shows the NE-induced contractile tension (g) changes of main branch MCA in response to increasing NE concentration under control conditions for fetus and adult. Fetal and adult maximal K⁺-induced tensions were 0.79 ± 0.06 (n = 10) and 1.43 ± 0.08 g (n = 12), respectively. The corresponding maximal NE-induced tensions (g) were 0.91 ± 0.12 and 1.61 ± 0.13, respectively (P < 0.01), whereas the corresponding stress values (10⁶ dynes/cm²) were 0.36 ± 0.05 and 0.41 ± 0.05, respectively. The corresponding pD₂ values were 6.0 ± 0.1 for each. Figure 1B shows the NE-induced tension as percent of maximal response. The NE maximum values as %K_max for fetus and adult MCA were 107 ± 16 and 119 ± 7, respectively (Fig. 1, inset). The Hill coefficients of the fetal and adult NE dose-response curves were 1.03 ± 0.12 and 1.02 ± 0.14, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Norepinephrine (NE) dose-response relationships for fetal and adult main branch middle cerebral arteries (MCA) under control conditions. Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, we induced subsequent contractions using cumulative doses of NE added in half-log increments (see METHODS for details). A: vascular tensions in g for adult (n = 12; , solid line) and fetus (n = 10; , dashed line). Maximum NE-induced tensions were 1.61 ± 0.13 and 0.91 ± 0.12 g, respectively. Points shown are means and SE. B: vascular tensions expressed as %maximal NE responses for fetal () and adult () MCA. PD2 values were each 6.0 ± 0.1. Inset, NE dose response as %K+ maximum for fetal (open bar) and adult (shaded bar) MCA, 107 ± 16 and 119 ± 7, respectively. C: fluorescence ratio for fetal () and adult () MCA expressed as %maximal response. Fetal and adult pD2 values were 6.2 ± 0.1 and 6.4 ± 0.1, respectively. Inset, dose response as %K+ maximum for fetus (open bar) and adult (shaded bar), 81 ± 9 and 103 ± 5, respectively.

Figure 1C shows the fura 2 fluorescence ratios (R_340/380), a measure of free Ca²⁺ concentration in response to NE, for fetal and adult MCA. For adult MCA, NE produced dose-dependent increases of [Ca²⁺]_i with a pD₂ value of 6.4 ± 0.1 and %K_max of 103 ± 15 (Fig. 1, inset). The NE-induced pD₂ value for the fluorescence ratio for fetal MCA was 6.2 ± 0.1, whereas the %K_max was 81 ± 9. Neither the pD₂ values nor the maximal K⁺-induced tensions (%K_max) were significantly different between fetus and adult. The relation of NE-induced tension (%K_max) to fluorescence ratio (%K_max) for fetal and adult MCA were plotted, the slopes being 1.66 ± 0.1 (r² = 0.90) and 1.40 ± 0.1 (r² = 0.78), respectively (P < 0.05).

Role of "L-type" Ca²⁺ channels. To determine the role of L-type Ca²⁺ channels in fetal and adult MCA contractile responses, we quantified NE-induced tensions and [Ca²⁺]_i after administration of three L-type Ca²⁺ channel blockers. As shown in Fig. 2A, in fetal and adult MCA (n = 4 each), nifedipine administered in half-log doses from 10⁹ to 10⁵ M produced dose-dependent inhibition of contraction to 10⁵ M NE, with pIC₅₀ values of 7.3 ± 0.1 and 6.6 ± 0.1, respectively (P < 0.01). As shown in Fig. 2B, in fetal and adult MCA, the pIC₅₀ values for the inhibition of NE-induced [Ca²⁺]_i response to nifedipine were 7.2 ± 0.1 and 6.9 ± 0.1, respectively (P < 0.05) (see Table 1).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   A: percent inhibition of NE-induced tensions in fetal () and adult () MCA in response to L-type Ca2+ channel blocker nifedipine (n = 4 each). Vessels were first contracted with 105 M NE, then at plateau of contraction blocker was given in half log increments. For vascular tension, pIC50 values for fetal and adult MCA were 7.3 ± 0.1 and 6.6 ± 0.1, respectively (P < 0.01). Points shown are mean and SE of measurements. B: percent inhibition of NE-induced intracellular Ca2+ concentration ([Ca2+]i) response, i.e., fluorescence ratio, in fetal () and adult () MCA (pIC50 values 7.2 ± 0.1 and 6.9 ± 0.1, respectively; P < 0.05).

In response to verapamil, NE-induced vascular tension in fetal and adult MCA (n = 4 each) was also inhibited, with pIC₅₀ values of 6.7 ± 0.1 and 6.1 ± 0.1, respectively (P < 0.01) (Table 1). The pIC₅₀ values for verapamil-induced inhibition of the NE-induced [Ca²⁺]_i in fetal and adult MCA were each 5.9 ± 0.1 (Table 1). Diltiazem also inhibited NE-induced vascular tension in fetal and adult MCA (n = 4 each), with pIC₅₀ values of 6.2 ± 0.1 and 5.2 ± 0.1, respectively (P < 0.01). For diltiazem, the pIC₅₀ values for [Ca²⁺]_i in fetal and adult MCA were 5.8 ± 0.1 and 5.4 ± 0.1, respectively (P < 0.05) (Table 1).

Role of receptor-operated Ca²⁺ channels. To evaluate the role of receptor-operated Ca²⁺ channels in fetal and adult NE-induced responses, we quantified tension and [Ca²⁺]_i responses to NE after blockade by the receptor-operated Ca²⁺ channel blocker NCDC. When we administered the blocker at the plateau of the NE response, NCDC failed to inhibit significantly the NE-induced tension or [Ca²⁺]_i responses in either age group until a relatively high concentration was reached. With administration of NCDC in fetal and adult MCA (n = 4 each), the pIC₅₀ values for tension were 4.3 ± 0.1 and 4.8 ± 0.1, respectively (P < 0.05). The comparable values for fetal and adult MCA [Ca²⁺]_i were 4.9 ± 0.01 and 4.2 ± 0.1, respectively (P < 0.01) (Table 1).

Role of extracellular [Ca²⁺]. To determine the role of extracellular Ca²⁺ in NE-induced contraction, we exposed both fetal and adult MCA to zero extracellular Ca²⁺ for 2 min before contraction with 10⁵ M NE. As shown in Fig. 3, A and B, in response to 10⁵ M NE in the presence of 1.8 mM extracellular [Ca²⁺] ([Ca²⁺]_o), adult MCA showed a typical rise in vascular tension with a sustained plateau phase and increase in [Ca²⁺]_i. In the absence of [Ca²⁺]_o, however (n = 4), tension (Fig. 3C) showed an initial rise to only 39 ± 7% of that peak tension in the presence of Ca²⁺. This was followed by a rapid decline to baseline values, rather than maintaining a plateau. The t_1/2 for the contraction was 60 ± 12 s. The peak fluorescence ratio under these conditions (Fig. 3D) was 73 ± 8% of the peak ratio in the presence of Ca²⁺ (Fig. 3B); however, the t_1/2 was 23 ± 8.0 s. As predicted and as seen in Fig. 3D, after the change to Ca²⁺-free buffer, baseline fluorescence ratio decreased significantly (0.10 ± 0.03) over the 2-min period. In contrast, baseline tension remained relatively constant. When the adult artery was exposed to zero [Ca²⁺]_o for 10 rather than 2 min (n = 3), there was no detectable NE-induced response of either tension or [Ca²⁺]_i.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   A and B: adult MCA contractile and [Ca2+]i responses to 105 M NE in presence of normal Krebs buffer (1.8 mm Ca2+). C and D: adult MCA contractile response to 105 M NE after exposure to zero extracellular [Ca2+] for 2 min. Note attenuated responses to NE of both tension and fluorescence ratio, as well as decrease in [Ca2+]i, but not in vascular tension, during exposure to zero extracellular [Ca2+].

Figure 4, A and B, shows the 10⁵ M NE-induced increase in tension and [Ca²⁺]_i in fetal MCA in the presence of 1.8 mM [Ca²⁺]_o. Tension and [Ca²⁺]_i plateaued in a manner similar to that of the adult. However, in contrast to the adult, in the absence of extracellular Ca²⁺ (n = 4; Fig. 4C), after NE stimulation both the initial rise in tension was much less (~6 ± 2% that of control) and the plateau was markedly attenuated. [Ca²⁺]_i (Fig. 4D) showed a similar attenuated response (12 ± 3% of control). Again, after the change to Ca²⁺-free buffer, the baseline fluorescence ratio decreased significantly (0.12 ± 0.02).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   A and B: fetal MCA contractile and [Ca2+]i responses to 105 M NE in presence of normal Krebs buffer (1.8 mm Ca2+). C and D: fetal MCA contractile response to 105 M NE in presence of zero extracellular [Ca2+]. Note markedly attenuated responses of both tension and fluorescence ratio, as well as marked decrease in [Ca2+]i during 2-min exposure to zero extracellular [Ca2+].

After exposure to Ca²⁺-free buffer, the vessel segments were allowed to reequilibrate in Ca²⁺ Krebs buffer for 40 min and then were replaced in Ca²⁺-free buffer for 2 min and NE exposure was repeated. Under these conditions, the contractile and fluorescence ratio responses to the second agonist exposure were somewhat less than that of the first. For fetal and adult MCA, NE-induced tensions (%K_max) were 5 ± 1 and 22 ± 8, respectively. Fluorescence values (%K_max) were 8 ± 1 and 14 ± 4, respectively. For adult MCA, the t_1/2 for these responses were only half of that of the first NE exposure, e.g., 26 ± 3 and 13 ± 1 for tension and fluorescence ratio, respectively. For fetal MCA, the t_1/2 values were 29 ± 7 and 20 ± 2, respectively.

To explore further the role of extracellular Ca²⁺ channels in contraction of fetal and adult cerebral arteries, before giving 10⁵ M NE we administered lanthanum ion (10³ M LaCl₃) for 2 min to irreversibly block the several types of plasmalemmal Ca²⁺ channels (n = 4 each). As shown in Fig. 5, A and B, in adult MCA after La³⁺ pretreatment, the contractile and [Ca²⁺]_i responses to NE stimulation were significantly attenuated (43 ± 7% for each peak response). For fetal MCA in the presence of La³⁺ (Fig. 5, C and D), there was essentially no NE-induced response of either tension or [Ca²⁺]_i.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   A and B: adult MCA tension and [Ca2+]i responses to 105 M NE after exposure to 103 M LaCl3 in HEPES buffer for 2 min (see METHODS). C and D: fetal MCA tension and [Ca2+]i responses to 105 M NE in presence of 103 M LaCl3 for 2 min.

	DISCUSSION

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The present studies are the first to present simultaneous measurements of [Ca²⁺]_i and tensions of cerebral arteries of fetus and adult and their responses to NE. As such they offer several important observations. First is the significant role of extracellular Ca²⁺ in NE-induced contractility in fetal and adult MCA, with the extracellular Ca²⁺ dependence for the fetus being greater than that for the adult. For NE-induced tension and fluorescence ratios in fetal and adult MCA, neither the pD₂ values nor the maximum tension and fluorescence ratios expressed as %K_max were significantly different from one another (Fig. 1). Nonetheless, in terms of %K_max tension as a function of %K_max fluorescence ratio (i.e., [Ca²⁺]_i), the fetal MCA showed significantly increased sensitivity compared with that of the adult. Second, fetal MCA showed a dramatically greater sensitivity to voltage-gated Ca²⁺ channel blockade compared with that of the adult (Fig. 2 and Table 1). This was especially evident for nifedipine (Fig. 2), but was also true for verapamil and diltiazem (Table 1). By comparison, both fetal and adult MCA were relatively insensitive to NCDC blockade of receptor-operated channels. In agreement with this, MCA of both fetus and adult showed great dependence on extracellular Ca²⁺ for contraction, with that for the fetus being greater than that of the adult (Figs. 3-5). In the absence of extracellular Ca²⁺, the fetal MCA peak contractile responses and fluorescence ratios were more markedly attenuated and the plateaus were not sustained in comparison with those of the adult. Determination of the [Ca²⁺]_i and tension in the presence of Ca²⁺ channel blockers and zero [Ca²⁺]_o demonstrates the important role of Ca²⁺ channels and other non-Ins(1,4,5)P₃ mechanisms in NE-induced contraction in both fetal and adult cerebral arteries.

These differences between fetal and adult calcium handling mechanisms fit with previous studies from our group and others on developmental differences in cerebral artery contractility. Fetal arteries develop less tone but have greater aminergic activity than those of adult (31), newborn MCA require more transmembrane calcium uptake than the adult (44), fetal arteries show greater calcium sensitivity (1, 3), and fetal arteries rely less on Ins(1,4,5)P₃-mediated contractile mechanisms (27, 43).

Vascular tension and [Ca²⁺]_i. [Ca²⁺]_i is a key determinant of vascular contractility. Ca²⁺ flux across the plasma membrane from the extracellular space occurs via a resting Ca²⁺ leak and the several types of Ca²⁺ channels: the voltage dependent or L-type, which is the most common, receptor-operated channels, stretch-activated channels, and others.

Both fetal and adult MCA exhibited a highly reproducible K⁺- and NE-induced dose-dependent increase in tension and [Ca²⁺]_i. In a previous study, we reported the pD₂ for ovine fetal and adult MCA as 6.3 ± 0.1 and 6.2 ± 0.1, respectively (27). Also, in a study of NE responsiveness of second- and fourth-order MCA of newborn lambs vs. adult sheep, Elliott and Pearce (13) demonstrated markedly increased tissue sensitivity (e.g., pD₂) in fourth-order MCA of younger animals, but not the second-order branches. The pD₂ values for fourth-order newborn and adult MCA were 6.2 ± 0.2 and 5.2 ± 0.2, respectively, whereas in second-order branches the values were 5.9 ± 0.2 and 5.5 ± 0.2, respectively (13). These values compare to the present values of 6.0 ± 0.1 in each vessel. The previous studies used larger vessel segments (~5 mm length) compared with the present study (2 mm), as well as somewhat different vessel bath ionic concentrations. In addition, Akopov and coworkers (1) reported increased Ca²⁺ sensitivity of common carotid and basilar arteries in 9-day-old rabbits compared with adults. These changes appeared to be associated with a G protein-dependent mechanism (1). The present results of greater NE sensitivity of fetal, compared with adult, MCA are in agreement with these studies. Of interest, and in contrast to findings of studies of some workers (7, 40), Elliott and Pearce (13) showed no change in NE sensitivity as a function of cerebral artery diameter in a given age group. This was also the case in the baboon (16).

Because of the prolonged duration of this study (3 h after loading with fura 2), to examine the possibility of desensitization to NE and to determine the reproducibility of the NE-induced contraction results over time, we repeated the NE dose response three times (with 5 min for dose response followed by 40 min washout and reequilibration in Krebs buffer) over a total period of ~180 min (n = 5). At 10⁶ M (approximately EC₅₀) NE-induced tension and fluorescence ratio each decreased (~20% by the second and third contractions).

Some may argue that the [Ca²⁺]_i data should be presented in absolute values rather than as the fluorescence ratio. As noted above, because of uncertainty about K_D for [Ca²⁺]_i, use of the ratio is less ambiguous. Nonetheless, regardless of the method used, the conclusions would be identical.

Plasma membrane calcium channels and contractility. In several tissues, calcium handling has been shown to vary with developmental age. For instance, neonatal myocardium is much more dependent on transmembrane Ca²⁺ flux and less dependent on Ca²⁺ release from intracellular storage sites than adult myocardium (11, 20). This agrees with the present study. In addition, several lines of evidence indicate that the volume of sarcoplasmic reticulum (i.e., Ca²⁺ storage site) in vascular smooth muscle is less in immature tissues (38) as well as in the smaller arteries (4, 37).

In addition to releasing compartmentalized Ca²⁺ from intracellular stores, vascular smooth muscle can increase [Ca²⁺]_i by evoking Ca²⁺ influx from the extracellular space via several types of Ca²⁺ channels: voltage-dependent L-type, receptor operated, stretch activated, and/or others (17, 19, 29). Other investigators (1, 4, 21, 35) have noted that cerebral arteries are more dependent on Ca²⁺ flux through voltage-gated plasma membrane channels than arteries from other vascular beds. This is especially true for smaller cerebral arteries (2). Results of the present study also agree with findings that cerebral arteries of the newborn sheep are more dependent on extracellular Ca²⁺ than those of the adult (44). In rat tail artery (18) and rat skeletal muscle (32) membrane electrical properties change with age. This suggests that the voltage-current relations for voltage-sensitive Ca²⁺ channels may also differ in fetal vs. adult cerebral arteries, and this needs to be explored.

L-type calcium channel blockers. Vascular L-type Ca²⁺ channels can be blocked by several classes of compounds: dihydropyridines, phenylalkyamines, and benzothiazepines (10, 14, 33). As demonstrated in the present study, the dihydropyridine nifedipine was much more effective in blocking fetal MCA (pIC₅₀ for tension = 7.3 ± 0.1), compared with that of the adult (pIC₅₀= 6.6 ± 0.1). Nonetheless, both the phenylalkamine verapamil (fetal and adult pIC₅₀ = 6.7 ± 0.1 and 6.1 ± 0.1, respectively) and the benzothiazepine diltiazem (fetal and adult pIC₅₀ = 6.2 ± 0.1 and 5.2 ± 0.1, respectively, see Table 1) were markedly effective in this regard. These findings further support the idea that fetal cerebral arteries are more dependent on extracellular Ca²⁺ than are those of the adult. In addition, the data on inhibition of contractility suggest that in the presence of the Ca²⁺ channel blockers the Ca²⁺ sensitivity of fetal MCA is greater than that of the adult, e.g., greater inhibition of tone for a given [Ca²⁺]_i (see Fig. 2).

We caution that the age-related differences in sensitivity of cerebral arteries to blockers of L-type voltage-operated Ca²⁺ channels, may not necessarily reflect differences in the relative roles of these channels in transmembrane Ca²⁺ flux or the development of tension. A number of related processes may relate to the latter. In addition, the density of these channels and their molecular structure may differ considerably between fetus and adult. Thus these several factors may contribute to an apparent developmental change in sensitivity to Ca²⁺ channel blockers. We currently are exploring these possibilities.

Receptor-operated and other calcium channel blockers. Several studies have demonstrated a class of Ca²⁺-permeable receptor-activated, nonselective cation channels in vascular smooth muscle, which are insensitive to the classical Ca²⁺ channel blockers (5, 23, 34). To date, a pharmacological classification of these channels has not been possible because of the lack of specific and potent pharmacological blockers. Perhaps surprisingly, in the present studies in both adult and fetal MCA, except at high concentrations, the receptor-operated Ca²⁺ channel blocker NCDC (34) displayed little effect on NE-induced contractility and thus presumably on transmembrane Ca²⁺ flux.

As noted above, the blockade of essentially all plasma membrane Ca²⁺ channels by lanthanum ions, with resultant diminution of NE-induced increases in tension and [Ca²⁺]_i (Fig. 5), confirms the key role of plasmalemmal Ca²⁺ channels in cerebral artery contractile responses. This is compatible with findings in human uterine artery (22), rabbit renal artery (42), rat aorta (12), and dog coronary artery (39). In addition, it further emphasizes the dependence of cerebral arteries on extracellular Ca²⁺ because of small intracellular stores (9), this being even more true for the fetus than the adult.

Perspectives

The present studies are the first to quantify simultaneously [Ca²⁺]_i and tension in fetal and adult cerebral arteries and to demonstrate in these vessels that plasma membrane L-type (but not receptor operated) Ca²⁺ channels play a key role in the NE-induced contractile response. Fetal cerebral arteries demonstrate almost total dependence on extracellular Ca²⁺. This dependence on extracellular Ca²⁺ is associated with fetal cerebral arteries also having increased Ca²⁺ sensitivity compared with those of the adult. For both fetus and adult, plasma membrane Ca²⁺ channels are probably an important regulating element of cerebral artery contraction in vivo as well as in vitro. Nonetheless, the role of changes with development in the many other elements of the signal transduction cascade that modulate NE-induced Ca²⁺ flux and contraction needs to be explored. These include the influence of ₁-AR subtypes, intracellular sarcoplasmic reticulum Ca²⁺ stores, protein kinase C and protein kinase A, enzyme phosphorylation, the several K⁺ channels, and so forth. These topics are the subject of current studies in the elucidation of the mechanisms of adrenergic-mediated cerebral vascular contraction and their change with development.