INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

CONTRACTION OF VASCULAR SMOOTH muscle is dependent on an increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) as a result of rapid Ca²⁺ release from intracellular stores, chiefly sarcoplasmic reticulum (SR), and from Ca²⁺ flux via plasma membrane Ca²⁺ channels. Recently we reported that fetal ovine cerebral arteries, in contrast to those in the adult, are exquisitely sensitive to extracellular Ca²⁺ concentration ([Ca²⁺]_o), with both [Ca²⁺]_i and tension being markedly attenuated by exposure to either zero [Ca²⁺]_o or pharmacological blockade of plasma membrane L-type Ca²⁺ channels (19). The question thus arises as to the role of SR Ca²⁺ stores in vascular contraction of fetal vs. adult cerebral arteries.

In smooth muscle cells, including those of the vasculature, the SR serves as the principal Ca²⁺ store and contributes significantly to Ca²⁺ release and intracellular signaling (3, 16, 27). Functionally, the vascular smooth muscle SR Ca²⁺ store may be divided into two compartments (41). One, the inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-releasable store, is activated by Ins(1,4,5)P₃, the receptor of which is a major channel mediating receptor-operated signal transduction (37). The second, or ryanodine (RYN)-sensitive Ca²⁺ store, releases Ca²⁺ in response to RYN and to caffeine (Caf) and appears to be involved in Ca²⁺-induced Ca²⁺ release (CICR) (12, 18). The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) is believed to fill both of these stores and is sensitive to the inhibitors thapsigargin (TSG) and cyclopiazonic acid (CPA) (5, 33).

The role of intracellular Ca²⁺ stores and their sensitivity to various agents may differ greatly as a function of vessel type, species, and developmental age. The marked dependence of fetal cerebral arteries on [Ca²⁺]_o (19) fits with previous studies from our group and others on developmental differences in cerebral artery contractility. Fetal cerebral arteries develop less tone, but have greater aminergic activity, than those of the adult (28); the newborn middle cerebral artery (MCA) requires more transmembrane calcium uptake than the adult (45); fetal arteries show greater calcium sensitivity (1, 19); and fetal arteries rely less on Ins(1,4,5)P₃-mediated contractile mechanisms (21, 44). In addition, the presence of relatively small intracellular stores in immature vessels (7, 38) further emphasizes the dependence of fetal cerebral arteries on extracellular Ca²⁺ compared with the adult.

The present studies tested the hypothesis that Ca²⁺ flux from SR, although playing a key role in norepinephrine (NE)-induced contraction of adult cerebral arteries, plays a less significant role in the fetal vessels. Specifically, the studies attempted to examine the role of SERCA-dependent Ca²⁺ stores, as well as the relative roles of SR Ca²⁺ and extracellular Ca²⁺ stores in the two age groups.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Experimental animals and tissues. For these studies, we used MCA from near-term fetal (~140 days) and nonpregnant adult sheep (2 yr) obtained from Nebeker Ranch (Lancaster, CA), as we have previously described (19, 20, 21). The ewes were anesthetized and killed with 100 mg/kg iv pentobarbital sodium, after 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 (28). To avoid the complication of endothelium-mediated effects, we removed the endothelium by carefully inserting a small wire three times (20). To confirm endothelium removal, we contracted the vessel with 10⁵ M NE 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 the [Ca²⁺]_i and tensions (19). Unless otherwise noted, all chemical compounds were purchased from Sigma (St. Louis, MO).

Contractility and intracellular calcium measurements. We cut the MCA into rings of 2 mm in length and mounted them on two tungsten wires (0.13 mm diameter; A-M Systems, Carlsborg, WA). We attached 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 Jasco CAF-110 intracellular Ca²⁺ analyzer (Jasco, Easton, MD). The tension value, along with vessel inside diameter measurements, wall thickness, length, and potassium-induced force, enabled calculation of force per unit cross-sectional area, as previously described (28). 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. Mean cytoplasmic [Ca²⁺]_i depends on where fura 2 is located (11). Fura 2 fluorescence and force were measured simultaneously at 38°C, as previously described (19). As we have noted, 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 (19). During all contractility experiments, we continuously digitized, normalized, and recorded contractile tensions and the fluorescence ratio (F_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 half-maximal concentration for NE and an index of tissue "sensitivity" or "potency") (19). In the presence of fura 2, neither K⁺- nor NE-induced tensions were significantly different from those contractions in the absence of the dye (data not shown).

Role of SR SERCA-dependent Ca²⁺ stores. In an attempt to determine the role of the SERCA-dependent Ca²⁺ stores in fetal and adult MCA, we measured [Ca²⁺]_i and tension after administration of TSG (10⁹ to 10⁵ M in half-log increments), an irreversible inhibitor of Ca²⁺-ATPase, for 15 min in Krebs buffer. We also measured 10⁵ NE-induced [Ca²⁺]_i and tension 15 min after TSG administration and every 2 min to examine the time course of Ca²⁺ depletion of the SERCA-sensitive pool. In addition, we examined the effect of CPA (10⁹ to 10⁵ M), a reversible inhibitor of Ca²⁺-ATPase, in blocking the SR Ca²⁺ uptake. Again, after 15 min, we repeated the response of [Ca²⁺]_i and tension to 10⁵ NE at 2-min intervals. We also repeated these studies in Ca²⁺-free media.

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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. The n values for the different experiments are given in Table 1. 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. A P value of <0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Role of SERCA-dependent Ca²⁺ stores in [Ca²⁺]_i and tension. To determine the role of SERCA-dependent Ca²⁺ stores in adult MCA NE-induced [Ca²⁺]_i and contraction responses, we first exposed the vessels to 10⁵ M NE and quantified vascular tension (Fig. 1A) and the fluorescence ratio (F_340/380, an index of [Ca²⁺]_i) (Fig. 1B) in normal Krebs buffer (1.8 mM Ca²⁺). As shown in Fig. 1, A and B, in response to 10⁵ M NE, adult MCA showed typical increases in vascular tension and [Ca²⁺]_i with sustained plateaus. The mean maximal NE-induced increases from baseline tension and [Ca²⁺]_i were 1.7 ± 0.1 g and 0.15 ± 0.01 units, respectively (n = 20; Table 1). To examine the role of TSG per se in Ca²⁺ release and contractility, after a 30-min recovery period, we administered TSG in increasing doses of half-log increments (10⁹ to 10⁵ M). At 10⁵ M TSG, fluorescence ratio slowly increased 0.04 ± 0.01 units, with a minimal change in tension (n = 4; Table 1). In other experiments, we administered 10⁶ M TSG, then after 15 min gave 10⁵ M NE. As shown in Fig. 1C, after TSG the NE-induced contractile response increased to only 37 ± 5% (P < 0.01) of the peak control tension with a maintenance of the plateau. The peak fluorescence ratio under these conditions (Fig. 1D) was 47 ± 7% of the peak ratio in the absence of TSG (n = 4; Table 1). As also shown in Figs. 1, C and D, when 10⁶ M nifedipine (a blocker of L-type Ca²⁺ channels) was given on the NE-induced plateau, both tension and [Ca²⁺]_i returned to baseline values in a manner similar to the response in the absence of TSG (19). In a related study, 13 min after administration of TSG, the media was changed to Ca²⁺-free Krebs buffer. Then after 2 min 10⁵ M NE was given. As shown in Fig. 1, E and F, the NE-induced increase in peak tension and [Ca²⁺]_i under these circumstances was about half of that seen in the presence of external Ca²⁺, e.g., 32 ± 5 and 13 ± 3%, respectively (P < 0.01 for each), and there was no plateau of either tension or [Ca²⁺]_i (n = 3; Table 1). As an aside, in several experiments, we added 10⁶ M nifedipine 13 min after TSG and then in 2 min gave 10⁵ M NE. Under these conditions, the initial rise in tension and fluorescence ratio was similar to that seen in Fig. 1, C and D. However, in a manner similar to that seen in Ca²⁺-free buffer (Fig. 1, E and F), the plateau was not maintained, presumably as Ca²⁺ flux through the L-type Ca²⁺ channels was blocked. Finally, in another study after administration of TSG, 3 × 10³ M Caf was given after 10 min. Then after 5 min, 10⁵ NE was given, after which there was essentially no increase in tension or [Ca²⁺]_i (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Typical adult middle cerebral artery (MCA) contractile and intracellular Ca2+ concentration ([Ca2+]i) responses. Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, cumulative doses of ryanodine (RYN) were added in half-log increments (see METHODS for details). A and B: control responses of tension (g) and fluorescence ratio (F340/380) to 105 M norepinephrine (NE) in normal Krebs buffer (1.8 mM Ca2+). C and D: MCA contractile and [Ca2+]I responses to 105 M NE after exposure to 106 M thapsigargin (TSG) for 15 min. Note the attenuated responses of both tension and fluorescence ratio to NE. In addition, these responses were essentially eliminated by exposure to 106 M nifedipine (Nif). E and F: attenuated MCA contractile and fluorescence ratios in response to 105 M NE after exposure to 106 M TSG for 15 min and Ca2+-free Krebs buffer for 2 min (see METHODS for details).

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View larger version (17K): [in this window] [in a new window]   Fig. 2.   Typical fetal MCA contractile and [Ca2+]i responses. A and B: control responses of tension (g) and fluorescence ratio (F340/380) to 105 M NE in the presence of normal Krebs buffer (1.8 mM Ca2+). C and D: fetal MCA contractile and [Ca2+]i responses to 105 M NE after exposure to 106 M TSG for 15 min. Note the responses of both tension and fluorescence ratio to NE, which were only slightly less than those in the absence of TSG. In addition, the responses of both tension and fluorescence ratio were essentially eliminated by exposure to 106 M nifedipine. E and F: markedly attenuated MCA contractile and fluorescence ratios in response to 105 M NE after exposure to 106 M TSG for 15 min and Ca2+-free Krebs buffer for 2 min.

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View larger version (19K): [in this window] [in a new window]   Fig. 3.   Typical responses of adult and fetal main branch MCA to 105 M NE 15 min after administration of 106 M cyclopiazonic acid (CPA). Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, CPA was added (see METHODS for details). A: vascular tensions (g) for adult MCA, with significant increase after 105 M NE to a maximum tension of 1.6 ± 0.1 g. B: fluorescence ratio for adult MCA, with a significant increase after 105 M NE. Note in both A and B the return to baseline values after administration of 106 M nifedipine. C: vascular tensions (g) for fetal MCA in response to 105 M NE 15 min after administration of 106 M CPA. D: fluorescence ratio for fetal MCA showing a significant increase in [Ca2+]i after 105 M NE in the presence of CPA. Again, in C and D, note the return to baseline of tension (C) and [Ca2+]i after 106 M nifedipine.

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Role of RYN-sensitive Ca²⁺ stores in [Ca²⁺]_i and tension. To explore the role of RYN-sensitive Ca²⁺ stores in contraction of adult cerebral arteries, we administered RYN in increasing half log doses (10⁹ to 10⁴ M) to deplete these stores before giving 10⁵ M NE. As shown in Fig. 4, A and B, in adult MCA 10⁵ M RYN produced significant increases in both tension and [Ca²⁺]_i, which peaked at 3 × 10⁵ M RYN and which approximated those increases seen in response to 10⁵ M NE (n = 4; Table 1). Of interest, 10⁶ M nifedipine given on the plateau of the response resulted in rapid and complete reversal of the RYN-induced increase in [Ca²⁺]_i and tension. In a related experiment, 15 min after 10⁷ M RYN, administration of 10⁵ M NE resulted in increases from baseline tension and [Ca²⁺]_i of 53 ± 7 and 33 ± 5%, respectively, of control NE (P < 0.01 for each; Table 1). When the study was repeated in the absence of extracellular Ca²⁺, the responses were barely detectable (Table 1).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   RYN (RYA) dose-response relationships for adult and fetal main branch MCA under control conditions. Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, cumulative doses of RYN were added in half-log increments (see METHODS for details). A: vascular tensions (g) for adult MCA, with significant increase after 105 M RYN to a maximum tension of 2.7 ± 0.1 g. B: fluorescence ratio for adult MCA, with a significant increase after 105 M RYN. C: vascular tensions for fetal MCA, showing a significant increase in tension after 3 × 105 M RYN. D: fluorescence ratio for fetal MCA in response to RYN. Again, note the return to baseline of tension and [Ca2+]i in both adult and fetal MCA when 106 M nifedipine was given on the plateau of the NE-induced contraction.

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View larger version (17K): [in this window] [in a new window]   Fig. 5.   RYN dose-response relationships for fetal and adult main branch MCA under control conditions. Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, cumulative doses of RYN were added in half-log increments (see METHODS for details). A: vascular tensions (g) for adult (n = 4; , solid line) and fetal MCA (n = 4; , dashed line). Maximum RYN-induced tensions were 1.6 ± 0.1 and 1.4 ± 0.1 g, respectively. Points shown are mean and standard errors. B: fluorescence ratios (F340/380) for fetal and adult MCA. Maximum ratios for fetus and adult were 0.18 ± 0.05 and 0.07 ± 0.02, respectively (P < 0.05).

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View larger version (22K): [in this window] [in a new window]   Fig. 6.   Adult and fetal MCA responses to 3 × 103 M caffeine. A and B: adult MCA contractile and [Ca2+]i responses to 3 × 103 M caffeine (Caf), followed in 15 min by 105 M NE. Note the large rise in tension and [Ca2+]i immediately after the administration of Caf. Also note the markedly attenuated response to 105 M NE after 15-min exposure to caffeine and the return to baseline of these responses after administration of 106 M nifedipine. C and D: fetal MCA contractile and [Ca2+]i responses to 3 × 103 M Caf. Note the rise in tension and [Ca2+]i immediately after the administration of Caf that is much less than in the adult. In addition, as with that of the adult, the responses to 105 M NE after exposure to Caf for 15 min were markedly attenuated and eliminated in response to 106 M nifedipine.

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View larger version (15K): [in this window] [in a new window]   Fig. 7.   Caffeine dose-response relationships for adult and fetal main branch MCA under control conditions. Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, cumulative doses of Caf were added in half-log increments (see METHODS for details). A: vascular tensions (g) for adult (n = 12; , solid line) and fetus (n = 10; , dashed line). B: fluorescence ratio (F340/380) for adult and fetal MCA as a function of Caf dose.

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View larger version (20K): [in this window] [in a new window]   Fig. 8.   Typical responses of adult and fetal main branch MCA to 105 M ryanodine 15 min after administration of 106 M TSG. Arterial segments were first contracted with 120 mM K+ to obtain peak tension. After washing and reequilibration to baseline tension, TSG was added, followed in 15 min by RYN (see METHODS for details). A: vascular tension (g) for adult MCA, with significant increase after 105 M RYN to a maximum tension of 1.61 ± 0.44. B: fluorescence ratio for adult MCA, with a significant increase to 0.09 ± 0.02 units after 105 M RYN. Note in both A and B that the addition of 105 M NE failed to increase tension of fluorescence ratio significantly above the peak values after RYN. C: vascular tensions (g) for fetal MCA in response to 105 M ryanodine 15 min after administration of 106 M TSG, with increase to 0.09 ± 0.05 g. D: fluorescence ratio for fetal MCA showing a significant increase in [Ca2+]i to 0.04 ± 0.02 units after 105 M RYN in the presence of TSG. In contrast to the adult, in C and D note the further increase of tension to 0.27 ± 0.20 g (C) and [Ca2+]i to 0.07 ± 0.04 units after 105 M NE.

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	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present studies offer several important observations. The first is that although SR Ca²⁺ stores play a significant role in NE-induced contractility in adult MCA, for the fetal cerebral vessels that role is much less (Figs. 1-9). As a corollary of this, although both adult and fetal cerebral arteries showed considerable dependence on extracellular Ca²⁺ for sustained contraction, for the fetus that dependence was essentially complete. Second, in the adult MCA, SR Ca²⁺-ATPase blockade by TSG had a major effect on inhibiting the NE response. In contrast, such inhibition was negligible in fetal MCA (see Table 1 and Figs. 1, 2, and 9). For fetal MCA, the responses to TSG and CPA were similar. However, adult MCA showed much greater sensitivity (less NE-induced tension) to TSG than to CPA (Figs. 1-3, Table 1). The much greater dependence of the fetal arteries on extracellular Ca²⁺ became evident when [Ca²⁺]_o equalled zero for both TSG and CPA. Third, in response to RYN stimulation of Ca²⁺ release, fetal cerebral arteries were more sensitive by one order of magnitude than the adult vessels (Figs. 4 and 5). This increased RYN sensitivity and the elimination of the RYN response by nifedipine-mediated L-type Ca²⁺ channel blockade or zero extracellular Ca²⁺ also fits with the greater dependence of the fetal arteries on extracellular Ca²⁺. Fourth, in view of the lack of response to RYN in Ca²⁺-free media or in the presence of a blocker of L-type Ca²⁺ channels, it would appear that in both adult and fetal cerebral arteries, the RYN receptor is closely linked with the plasma membrane L-type Ca²⁺ channel (Figs. 4 and 8). In addition, the lack of response to RYN in the presence of the K_Ca channel blocker iberiotoxin, suggests coupling of the RYN receptor to the K_Ca channel, which, in turn, is linked to the L-type Ca²⁺ channel. Fifth, adult cerebral arteries showed a robust response of tension and [Ca²⁺]_i to Caf release of SR Ca²⁺ stores; however, this was much less evident in the fetal arteries, which were totally dependent on extracellular [Ca²⁺]_i (Fig. 6). In addition, although fetal cerebral arteries were more sensitive than adult vessels to RYN (Fig. 4 and 5), the reverse was the case for Caf (Fig. 7). These differences in response to RYN and Caf may suggest different Ca²⁺ pools sensitive to these agents. Alternatively, this may suggest that Caf acted as an inhibitor of phosphodiesterase to a greater extent in fetal cerebral arteries than in adult vessels (see below). Overall, for both adult and fetal cerebral arteries, nifedipine blockade of Ca²⁺ channels or zero extracellular Ca²⁺ each markedly attenuated the NE-induced peak contractile responses and fluorescence ratios. Thus determination of the [Ca²⁺]_i and tension in the presence of SR Ca²⁺ store blockers, blockade of plasma membrane L-type Ca²⁺ channels, or zero extracellular [Ca²⁺]_o are further demonstrations of the critical role of extracellular Ca²⁺, as opposed to SR Ca²⁺ stores, in NE-induced contraction in fetal cerebral arteries compared with those of the adult. In addition, the idea that in cerebral arteries the RYN receptor is coupled to the plasma membrane L-type Ca²⁺ channel may be important.

View larger version (46K): [in this window] [in a new window]   Fig. 9.   Summary of fetal and adult MCA contractile (A) and fluorescence ratio (B) responses to several agents, as % of response to 105 M NE. [Ca2+]o = 0, zero extracellular [Ca2+]. Stippled bar, fetus; hatched bar, adult.

The SR and its function. The SR of vascular smooth muscle is a membranous tubular system with components closely approximating the plasma membrane, as well as deeper portions contiguous with rough endoplasmic reticulum and the nuclear membrane (36). In addition to actively transporting Ca²⁺ from the cytoplasm into its lumen via its Ca²⁺-ATPase (thereby enhancing relaxation) on smooth muscle stimulation by neurotransmitters and autocoids, the SR rapidly releases its luminal Ca²⁺ into the cytosol. SR also buffers Ca²⁺ entry from the extracellular space into the cell (15, 27). As noted above, SR contains at least two types of Ca²⁺-release channels (41). Both the Ins(1,4,5)P₃-receptor/Ca²⁺-release mechanism and the RYN receptor/Ca²-induced Ca²⁺ release mechanism are major channels that mediate pharmacomechanical coupling in smooth muscle (37) and have been localized to both junctional and central SR (18, 26). Several lines of evidence suggest that Ca²⁺ compartmental heterogeneity allows smooth muscle (and other) cells to generate spatially and temporally distinct Ca²⁺ signals to regulate specific Ca²⁺-dependent processes (3, 10). Nonetheless, little information on SR structure-function relationships is available for fetal and/or newborn arteries compared with those of the adult.

Role of SERCA-dependent Ca²⁺ stores. The SERCA, believed to fill both Ins(1,4,5)P₃- and RYN-sensitive Ca²⁺ stores, is irreversibly inhibited by TSG (5, 22, 25, 40). Because of its slow depletion of the SR Ca²⁺ store, application of TSG may result in gradual elevation of cytosolic free Ca²⁺, as seen in the present study (Figs. 1D, 2D, and 8, B and D). To what extent TSG may differently inhibit SERCA in fetal and adult cerebral vessels is unknown, although it is reported to inhibit each of the several isoforms with equal potency (24). As an aside, in the rat aorta and portal vein, the Ins(1,4,5,)P₃-receptor has been reported to switch from type 3 isoform with low Ins(1,4,5)P₃ affinity in the neonate to type 1 isoform with high affinity in the adult (39). Should such a developmental isoform switch occur in cerebral arteries, it would certainly fit with the present results.

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Role of RYN-sensitive Ca²⁺ stores. Considerable evidence suggests that SR Ca²⁺ release through RYN receptor channels plays a key role in elevating [Ca²⁺]_i during agonist stimulation of smooth muscles (42). In vascular smooth muscle, RYN receptors are present in both peripheral and central SR (18). Again, although there are at least three isoforms of this receptor, and in skeletal muscle these may change with developmental age (4), we know of no data as to how these might vary in fetal vs. adult cerebral arteries. RYN at high concentrations appears to deplete the SR Ca²⁺ store, thereby attenuating agonist-induced contraction dependent on Ca²⁺ release. In single neurons and some other cells, at low concentrations (10⁷ to 10⁶ M), RYN stimulates Ca²⁺ release, whereas at high concentrations (10⁵ to 10⁴ M) it may act to inhibit Ca²⁺ release (9, 34).

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Perspectives

By simultaneously measuring [Ca²⁺]_i and tension, the present studies are the first to demonstrate in fetal and adult cerebral arteries the role of the SR in the NE-induced contractile response. As we showed in a previous study, for their contraction, fetal cerebral arteries demonstrate considerable dependence on extracellular Ca²⁺ flux via L-type Ca²⁺ channels. These vessels also have increased Ca²⁺ sensitivity compared with adult arteries (19). The present studies provide additional evidence that SR Ca²⁺ stores play a key role in adult cerebral artery contractility. In comparison, in fetal vessels these stores are negligible and secondary in importance to extracellular Ca²⁺ availability via plasmalemmal Ca²⁺ channels for NE (or probably other agonist)-induced contraction. In addition, the present studies suggest that although SERCA fills both Ins(1,4,5)P₃- and RYN-sensitive Ca²⁺ pools, these two stores are more completely separated in the adult than in the fetal MCA. Finally, the studies support the novel idea of coupling between SR RYN receptors and plasma membrane K_Ca and/or L-type Ca²⁺ channels in ovine cerebral arteries.

Of course, an obvious question is at what time during the course of development, from fetus to newborn to adult, the SR increases in density and activity as a Ca²⁺ store and the cerebral vessels become less dependent on extracellular [Ca²⁺] and the molecular mechanisms thereof? Additional questions relate to the role of developmental changes in the density, affinity, and isoforms of the several receptors and enzymes relevant to SR function [SERCA, the Ins(1,4,5)P₃ receptor, RYN receptor, etc.] that modulate pharmacomechanical coupling and the mechanisms by which the RYN receptor is coupled to the plasma membrane K_Ca and L-type Ca²⁺ channels. These areas are the subject of current studies in the elucidation of the mechanisms of cerebral vascular contraction and their change with development.