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Am J Physiol Regul Integr Comp Physiol 279: R860-R873, 2000;
0363-6119/00 $5.00
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Vol. 279, Issue 3, R860-R873, September 2000

Cerebral artery sarcoplasmic reticulum Ca2+ stores and contractility: changes with development

Wen Long, Lubo Zhang, and Lawrence D. Longo

Center for Perinatal Biology, Departments of Physiology/Pharmacology and Obstetrics and Gynecology, School of Medicine, Loma Linda University, Loma Linda, California 92350


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To test the hypothesis that sarcoplasmic reticulum (SR) Ca2+ stores play a key role in norepinephrine (NE)-induced contraction of fetal and adult cerebral arteries and that Ca2+ stores change with development, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal (~140 days) and nonpregnant adult sheep, we measured NE-induced contraction and intracellular Ca2+ concentration ([Ca2+]i) in the absence and presence of different blockers. In adult MCA, after thapsigargin (10-6 M), the NE-induced responses of tension and [Ca2+]i were 37 ± 5 and 47 ± 7%, respectively, of control values (P < 0.01 for each). In the fetal artery, in contrast, this treatment resulted in no significant changes from control. When this was repeated in the absence of extracellular Ca2+, adult MCA increases in tension and [Ca2+]i were 32 ± 5 and 13 ± 3%, respectively, of control. Fetal cerebral arteries, however, showed essentially no response. Ryanodine (RYN, 3 × 10-6 to 10-5 M) resulted in increases in tension and [Ca2+]i in both fetal and adult MCA similar to that seen with NE. For both adult and fetal MCA, the increased tension and [Ca2+]i responses to RYN were essentially eliminated in the presence of zero extracellular Ca2+. These findings provide evidence that in fetal MCA, in contrast to those in the adult, SR Ca2+ stores are of less importance in NE-induced contraction, with such contraction being almost wholly dependent on Ca2+ flux via plasma membrane L-type Ca2+ channels. In addition, they suggest that in both adult and fetal MCA, the RYN receptor is coupled to the plasma membrane Ca2+-activated K+ channel and/or L-type Ca2+ channel.

cerebrovascular circulation; vascular smooth muscle; sympathetic nervous system; norepinephrine; intracellular calcium; thapsigargin; cyclopiazonic acid; ryanodine; L-type calcium channel; fetus; adult


    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 10-5 M NE and at the plateau added 10-6 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 (F340/380) 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 Kmax (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 (10-9 to 10-5 M in half-log increments), an irreversible inhibitor of Ca2+-ATPase, for 15 min in Krebs buffer. We also measured 10-5 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 (10-9 to 10-5 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 10-5 NE at 2-min intervals. We also repeated these studies in Ca2+-free media.

To determine the role of the SR RYN-sensitive Ca2+ pool in MCA of fetus and adult, we measured tension and [Ca2+]i after administration of 10-9 to 10-4 M RYN in half-log doses. In addition, we quantified tension and [Ca2+]i in response to 10-5 M NE stimulation given 15 min after 10-7 M RYN. We also measured [Ca2+]i and tension in response to depletion of this Ca2+ pool by 3 × 10-3 M Caf (1,3,7-trimethylxanthine). Then we administered 10-5 M NE every 2 min to examine the time course of tension and [Ca2+]i after Ca2+ depletion of the Caf-sensitive pool. We then allowed the vessel to restore its Ca2+ stores for 30 min and replaced the Krebs media with Ca2+-free media for 10 min and repeated the Caf dose (n = 5 each). In a related study, after stimulation by low dose (10-8 or 10-6 M) RYN, we administered 3 × 10-3 M Caf to deplete Ca2+ stores. Then we gave 10-5 M NE and examined the response of tension and [Ca2+]i. We repeated this study with a higher dose of RYN (10-4 M). We also repeated the studies in Ca2+-free buffer and in separate experiments in the presence of the Ca2+-activated K+ channel blocker iberiotoxin (10-6 M).

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.

                              
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  		Table 1.   Peak responses of vascular tension and fluorescence ratio in absolute values and as percent of NE


    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 10-5 M NE and quantified vascular tension (Fig. 1A) and the fluorescence ratio (F340/380, 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 10-5 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 (10-9 to 10-5 M). At 10-5 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 10-6 M TSG, then after 15 min gave 10-5 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 10-6 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 10-5 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 10-6 M nifedipine 13 min after TSG and then in 2 min gave 10-5 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 × 10-3 M Caf was given after 10 min. Then after 5 min, 10-5 NE was given, after which there was essentially no increase in tension or [Ca2+]i (data not shown).


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  		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 10-5 M norepinephrine (NE) in normal Krebs buffer (1.8 mM Ca2+). C and D: MCA contractile and [Ca2+]I responses to 10-5 M NE after exposure to 10-6 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 10-6 M nifedipine (Nif). E and F: attenuated MCA contractile and fluorescence ratios in response to 10-5 M NE after exposure to 10-6 M TSG for 15 min and Ca2+-free Krebs buffer for 2 min (see METHODS for details).

Figure 2, A and B, show the 10-5 M NE-induced increase from baseline tension and [Ca2+]i in fetal MCA under control conditions in normal Krebs buffer (1.8 mM Ca2+), with both tension and [Ca2+]i plateauing in a manner similar to that of the adult. The mean increases in maximal NE-induced tension and [Ca2+]i from baseline were 1.40 ± 0.10 g and 0.25 ± 0.02 units, respectively (n = 18; Table 1). Also similar to the adult, after the administration of TSG in increasing half-log doses (10-9 to 10-5 M), there was no significant change in tension, despite a small increase in [Ca2+]i (0.02 ± 0.01 units; Table 1). When 10-6 M TSG was given, followed in 15 min by 10-5 M NE, the responses of tension (Fig. 2C) and [Ca2+]i (Fig. 2D) were 81 ± 10 and 84 ± 9% of control values, respectively (n = 4; Table 1). Also note the modest (0.03 ± 0.01) increase in baseline fluorescence ratio by 15 min after TSG (Fig. 2D). Again, as shown in Fig. 2, C and D, the administration of 10-6 M nifedipine on the plateau of the NE-induced contraction resulted in both tension and [Ca2+]i rapidly returning to baseline values in a manner similar to that seen in the absence of TSG (n = 4) (19). In a related study, 13 min after administration of TSG, the media was changed to Ca2+-free Krebs buffer and 10-5 M NE was given after 2 min. As shown in Fig. 2, E and F, under this circumstance the NE-induced responses of tension and [Ca2+]i were essentially nil (n = 3; Table 1). Again, in another experiment, we added the 10-6 M nifedipine 13 min after the TSG, then in 2 min administered 10-5 M NE. In this instance, there was no detectable increase in either tension or [Ca2+]i, presumably because of blockade of transmembrane Ca2+ flux.


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  		Fig. 2.   Typical fetal MCA contractile and [Ca2+]i responses. A and B: control responses of tension (g) and fluorescence ratio (F340/380) to 10-5 M NE in the presence of normal Krebs buffer (1.8 mM Ca2+). C and D: fetal MCA contractile and [Ca2+]i responses to 10-5 M NE after exposure to 10-6 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 10-6 M nifedipine. E and F: markedly attenuated MCA contractile and fluorescence ratios in response to 10-5 M NE after exposure to 10-6 M TSG for 15 min and Ca2+-free Krebs buffer for 2 min.

In adult MCA, in a manner similar to the minimal response of [Ca2+]i and tension to TSG, when CPA was administered in increasing half-log doses at 10-5 M CPA, the vessel showed no significant increase in tension despite a small increase in [Ca2+]i (Table 1). As shown in Fig. 3, A and B, in a related study, 15 min after 10-6 M CPA, 10-5 M NE resulted in robust increases from baseline of tension and [Ca2+]i (78 ± 9 and 100 ± 11%, respectively, of control NE), not unlike that seen with NE alone (n = 4). Nonetheless, when 10-6 M nifedipine was given on the plateau of the response, both tension and [Ca2+]i rapidly returned to baseline (n = 3). In a manner similar to that seen with TSG, when CPA was given for 13 min and then continued for 2 min in the presence of Ca2+-free Krebs buffer, adult MCA NE-induced increases in tension and [Ca2+]i were markedly attenuated (11 ± 3 and 7 ± 1%, respectively, Table 1).


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  		Fig. 3.   Typical responses of adult and fetal main branch MCA to 10-5 M NE 15 min after administration of 10-6 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 10-5 M NE to a maximum tension of 1.6 ± 0.1 g. B: fluorescence ratio for adult MCA, with a significant increase after 10-5 M NE. Note in both A and B the return to baseline values after administration of 10-6 M nifedipine. C: vascular tensions (g) for fetal MCA in response to 10-5 M NE 15 min after administration of 10-6 M CPA. D: fluorescence ratio for fetal MCA showing a significant increase in [Ca2+]i after 10-5 M NE in the presence of CPA. Again, in C and D, note the return to baseline of tension (C) and [Ca2+]i after 10-6 M nifedipine.

Similarly, in fetal MCA, administration of CPA alone resulted in a small elevation of [Ca2+]i with essentially no change in tension. Fifteen minutes after 10-6 M CPA, administration of 10-5 M NE resulted in essentially normal increases of tension and [Ca2+]i (Fig. 3, C and D, respectively) (41 ± 7 and 64 ± 8%, respectively, of control; Table 1). As also shown in Fig. 3, C and D, 10-6 M nifedipine given on the plateau of the NE response resulted in both tension and [Ca2+]i returning to baseline. As with TSG, when CPA was given for 13 min and then continued for 2 min in the presence of Ca2+-free Krebs solution, the fetal MCA showed essentially no response to 10-5 M NE (Table 1).

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 (10-9 to 10-4 M) to deplete these stores before giving 10-5 M NE. As shown in Fig. 4, A and B, in adult MCA 10-5 M RYN produced significant increases in both tension and [Ca2+]i, which peaked at 3 × 10-5 M RYN and which approximated those increases seen in response to 10-5 M NE (n = 4; Table 1). Of interest, 10-6 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 10-7 M RYN, administration of 10-5 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).


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  		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 10-5 M RYN to a maximum tension of 2.7 ± 0.1 g. B: fluorescence ratio for adult MCA, with a significant increase after 10-5 M RYN. C: vascular tensions for fetal MCA, showing a significant increase in tension after 3 × 10-5 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 10-6 M nifedipine was given on the plateau of the NE-induced contraction.

In a similar manner, in fetal MCA, both tension and [Ca2+]i (Fig. 4, C and D) increased strikingly in response to 3 × 10-6 M RYN (Table 1). After administration of 10-6 M nifedipine, both returned to near baseline. (As an aside, administration of 10-3 M Caf after the return to baseline failed to increase either [Ca2+]i or tension.) In a related experiment, 15 min after 10-7 M RYN, administration of 10-5 M NE resulted in tension and [Ca2+]i increases of 19 ± 4 and 24 ± 4%, respectively, of NE control (P < 0.01 for each; Table 1). Again, when the study was repeated in the absence of extracellular Ca2+, the increases were negligible (Table 1). In contrast to the responses of tension and [Ca2+]i to RYN in the presence of 1.8 mM extracellular Ca2+ (Fig. 4), in the presence of Ca2+-free media, neither adult nor fetal MCA showed any significant responses to 10-5 to 10-4 M RYN (n = 3 each; data not shown). Also in a separate experiment, we first administered 10-6 M iberiotoxin [Ca2+-activated K+ channel (KCa) blocker] and then after 15 min examined the response to 10-5 M RYN. Under these conditions, neither adult nor fetal MCA showed a significant response to RYN.

For fetal and adult MCA, Fig. 5 shows the RYN dose-response curves of tension and fluorescence ratio (Fig. 5, A and B, respectively). The pD2 values for the increases in tension for fetal and adult vessels were 5.9 ± 0.1 and 5.3 ± 0.1, respectively (P < 0.05), whereas those for fluorescence ratio were 5.9 ± 0.1 and 5.4 ± 0.1, respectively (P < 0.05). The increases in tension and fluorescence ratio were similar when plotted as %Kmax.


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  		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; black-triangle, 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).

To explore the role of Caf-sensitive Ca2+ stores on fetal and adult cerebral artery contractility, we exposed MCA to 3 × 10-3 M Caf treatment. As seen in Fig. 6, A and B, in adult MCA both tension and [Ca2+]i showed a dramatic increase from baseline after Caf administration, with an initial peak similar to that seen with 10-5 M NE (107 ± 10 and 107 ± 11%, respectively, of NE control; Table 1). However, after Caf-induced depletion of SR Ca2+ stores, tension decreased rapidly rather than being maintained on a plateau (Fig. 6A). In turn, after 15 min, the response of tension and [Ca2+]i to 10-5 M NE were only 6 ± 1 and 13 ± 3%, respectively, of controls (Fig. 6, A and B; n = 4; Table 1). Furthermore, with administration of 10-6 M nifedipine on the plateau of the post-Caf NE-induced response, both tension and [Ca2+]i rapidly returned to baseline values (Fig. 6, A and B). When the study was repeated in Ca2-free buffer, the tension and [Ca2+]i responses to 10-5 M NE were negligible (Table 1).


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  		Fig. 6.   Adult and fetal MCA responses to 3 × 10-3 M caffeine. A and B: adult MCA contractile and [Ca2+]i responses to 3 × 10-3 M caffeine (Caf), followed in 15 min by 10-5 M NE. Note the large rise in tension and [Ca2+]i immediately after the administration of Caf. Also note the markedly attenuated response to 10-5 M NE after 15-min exposure to caffeine and the return to baseline of these responses after administration of 10-6 M nifedipine. C and D: fetal MCA contractile and [Ca2+]i responses to 3 × 10-3 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 10-5 M NE after exposure to Caf for 15 min were markedly attenuated and eliminated in response to 10-6 M nifedipine.

In contrast to the responses in the adult, in fetal cerebral arteries, 3 × 10-3 M Caf resulted in increases of tension of only 27 ± 4% (Fig. 6C) and [Ca2+]i of 64 ± 12% (Fig. 6D) compared with the control 10-5 M NE response (n = 4). As with the responses in the adult, the fetal tension response to Caf was only briefly maintained. In addition, in fetal MCA after 10-5 M NE, both tension and [Ca2+]i increased significantly (Fig. 6, C and D), but these NE-induced responses were almost totally ablated by 10-6 M nifedipine (n = 4). Finally, when the study was repeated in Ca2+-free buffer, the tension and [Ca2+]i responses to 10-5 M NE were negligible (Table 1).

Figure 7 shows the Caf dose-response curves for adult and fetal MCA in normal Krebs buffer, with increases in tension and fluorescence ratio (Fig. 7, A and B, respectively). In response to Caf, adult MCA showed a slight increase in tension (~15% Kmax, Fig. 7A) and larger increase in fluorescence ratio (~50% Kmax, Fig. 7B). In contrast, fetal MCA showed no significant increase in tension and only a modest increase in fluorescence ratio (Fig. 7, A and B, respectively). In both adult and fetal vessels, the attenuated responses to increasing Caf dose were less than the single dose response (for instance, 3 × 10-3 M as in Fig. 6; see DISCUSSION). The pD2 value for adult tension was 4.2 ± 0.02, whereas the values for fetal and adult fluorescence ratio were 2.8 ± 0.2 and 3.3 ± 0.2, respectively.


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  		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; black-triangle, dashed line). B: fluorescence ratio (F340/380) for adult and fetal MCA as a function of Caf dose.

To examine the extent to which the RYN-sensitive Ca2+ store was similar to, or differed from, the SERCA-inhibited Ca2+ pool, we performed the following study. After equilibration in normal Krebs buffer, we exposed the MCA to 10-6 M TSG for 15 min. We then administered 10-5 M RYN followed in 10 min by 10-5 M NE. As shown in Fig. 8, A and B, after TSG, adult MCA demonstrated minimal increase in tension (0.10 ± 0.03 g) and fluorescence ratio (0.04 ± 0.04 units). However, in response to RYN, both tension and fluorescence ratio increased significantly from baseline (1.61 ± 0.34 g and 0.09 ± 0.02 units). Later, addition of 10-5 M NE after the peak values had declined resulted in further significant increases in tension and fluorescence ratio, but not to values higher than those after RYN per se (n = 3). Again, in Ca2+-free buffer with this protocol after RYN administration, adult MCA failed to show an increase in either tension or fluorescence ratio (n = 2; data not shown).


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  		Fig. 8.   Typical responses of adult and fetal main branch MCA to 10-5 M ryanodine 15 min after administration of 10-6 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 10-5 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 10-5 M RYN. Note in both A and B that the addition of 10-5 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 10-5 M ryanodine 15 min after administration of 10-6 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 10-5 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 10-5 M NE.

Figure 8, C and D, show the pattern of response in fetal MCA. Again after TSG, tension and fluorescence ratio (Fig. 8, C and D, respectively) showed minimal responses (0.06 ± 0.01 g and 0.01 ± 0.01 units). Then after administration of 10-5 M RYN, in contrast to the adult, tension increased slowly (0.31 ± 0.07 g), although fluorescence ratio increased 0.04 ± 0.02 units from baseline. Subsequently, administration of 10-5 M NE resulted in further increase of tension and ratio (to 0.87 ± 0.05 g and 0.07 ± 0.04 units, respectively) above that seen in response to RYN alone (Fig. 8, C and D; Table 1) (n = 3). As with the adult, in Ca2+-free buffer fetal MCA showed no increase in either tension or fluorescence ratio in response to RYN preceded by TSG (n = 2; data not shown).


    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 KCa channel blocker iberiotoxin, suggests coupling of the RYN receptor to the KCa 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.


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  		Fig. 9.   Summary of fetal and adult MCA contractile (A) and fluorescence ratio (B) responses to several agents, as % of response to 10-5 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.

Several isoforms of the SR Ca2+-ATPase exist; however, we know of no data as to how expression of these might differ in fetal as opposed to adult cerebral arteries. In the rat (both WKY and SHR) aorta, the level of SERCA 2a mRNA increased with development from 5 to 17 wk of age, whereas that for the 2b isoform remained constant (17). A possible increase in SERCA in cerebral arteries with development certainly fits with the present results. Several studies have examined SR function in aging. For instance in rat cardiac myocytes, Ca2+ buffering decreases with age due to decreased SERCA activity (43). Although SERCA levels as determined with Western immunoblotting did not change, SR Ca2+ cycling proteins and their phosphorylation were markedly decreased by aging from 6-8 mo to 26-28 mo (43). To our knowledge, however, no studies have examined vascular smooth muscle SERCA activity or Ca2+ handling in relation to early development.

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.

Shown in Fig. 1, C and D, in adult MCA after administration of 10-6 M TSG, 10-5 M NE resulted in markedly attenuated contractile and [Ca2+]i responses (Table 1). For fetal MCA (Fig. 2, C and D), TSG administration resulted in the NE-induced responses being near normal (Table 1). A reversible SERCA inhibitor, which is structurally different than TSG and has differences in selectivity and efficacy, is the mycotoxin CPA (23, 25, 33). As shown in Fig. 3, administration of 10-6 M CPA followed by 10-5 M NE resulted in near normal increases in tension and fluorescence ratio in the adult MCA (Fig. 3, A and B, and Table 1) but somewhat attenuated responses in fetal cerebral arteries (Fig. 3, C and D, and Table 1). Again, this suggests the relative independence of the fetal arteries on intracellular Ca2+ stores compared with the adult. The greater effect of TSG compared with CPA in inhibiting adult MCA responses to NE, but less effect in fetal arteries (see Table 1), may relate to TSG being an irreversible, as opposed to a reversible, SERCA inhibitor. Alternatively, the difference in action of the drugs (particularly in the adult) may relate to somewhat different mechanisms of action. Nonetheless, the contractile and [Ca2+]i responses to both SERCA inhibitors were essentially eliminated when L-type Ca2+ channels were blocked by nifedipine or when [Ca2+]o = 0. This is in line with our previous studies on the role of L-type Ca2+ channels in contractile responses (19).

Figure 9 presents in summary form the relative responses (as %10-5 M NE) of tension and fluorescence ratio for fetal and adult MCA in the presence of TSG. Evident is the sustained response of fetal MCA to NE after the administration of TSG, whereas that of the adult vessel is markedly attenuated. In addition, although adult MCA showed a modest response of tension and [Ca2+]i in the presence of zero [Ca2+]o, that was not the case for the fetus.

A related consideration from the data in Fig. 9 and Table 1 is to estimate the fraction of the total change in [Ca2+]i that originates from SR Ca2+ stores vs. that amount deriving from Ca2+ flux across the plasma membrane. Because we did not quantify Ca2+ flux per se (as with 45Ca2+), this can be only an approximation. As seen in Table 1, for adult MCA, the peak [Ca2+]i increases in response to NE after TSG or CPA were 50 to 100%, respectively, compared with that of NE alone. In contrast, in Ca2+-free buffer these responses were <15%, suggesting that in the adult vessel, although SR stores make a definite contribution to the NE-induced [Ca2+]i increase, Ca2+ flux across the plasma membrane plays the major role in this regard. For fetal MCA, the results are even more striking. The NE-induced [Ca2+]i increases after TSG and CPA were 60-80% of the control NE response; however, in Ca2+-free buffer these responses were negligible. This again emphasizes the essentially total dependence of the fetal vessels on extracellular Ca2+ compared with that of the adult.

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 (10-7 to 10-6 M), RYN stimulates Ca2+ release, whereas at high concentrations (10-5 to 10-4 M) it may act to inhibit Ca2+ release (9, 34).

In vascular smooth muscle, the RYN receptor, which may allow Ca2+ to be released as "sparks," appears to be functionally coupled to plasma membrane KCa, so that by activating KCa the Ca2+ sparks hyperpolarize the cell, thereby acting in negative feedback regulation of [Ca2+]i (15, 29). (Of course, Ca2+ sparks would not be expected to be observed by use of the present techniques.) Contrary to expectations, in both adult and fetal cerebral arteries, low RYN concentrations (10-7 to 10-6 M) failed to increase significantly either [Ca2+]i or tension (Fig. 4). This may have resulted because the present studies were conducted in tissue (segments of MCA) rather than in single cells. Under such circumstances, the intracellular drug concentration may be much lower than that which would be present in isolated cells. Nonetheless, at higher concentrations (3 × 10-5 M for adult and 3 × 10-6 M for fetal MCA), RYN application resulted in marked increase in both [Ca2+]i and tension (Fig. 4 and Table 1). In addition, the increased sensitivity of fetal MCA to RYN, compared with the adult, fits with the fetal vessel being more dependent on extracellular Ca2+ for contraction. For both adult and fetal MCA, the L-type Ca2+ channel blocker nifedipine (10-6 M) eliminated the RYN responses (Fig. 4). Similarly, neither adult nor fetal MCA responded to RYN in Ca2+-free buffer. In addition, as noted in Fig. 8 for both adult and fetal MCA after TSG administration, 10-5 M RYN resulted in significant increases in tension and fluorescence ratio. These findings suggest that in adult MCA the TSG- and RYN-sensitive Ca2+ pools are to a certain extent separate (Fig. 8, A and B). In contrast, in fetal MCA the pools may overlap to a greater extent (Fig. 8, C and D). Thus developmental maturation may be associated with these pools becoming more distinct. In addition, in the presence of either Ca2+-free buffer or the KCa channel blocker iberiotoxin, responses to RYN in both adult and fetal MCA were eliminated. This strongly suggests coupling between the RYN receptor and plasmalemmal KCa channel and the L-type Ca2+ channel (14, 29) and may be analogous to the coupling between the Ins(1,4,5)P3 receptor and plasma membrane transient receptor potential family of plasma membrane channel proteins (6, 8, 30, 31).

As noted above, Fig. 9 attempts to put some of these observations in perspective. In a manner similar to TSG, for adult MCA, application of 10-5 M NE after 10-7 M RYN resulted in relatively robust increases in tension and [Ca2+]i (53 ± 7 and 33 ± 5%, respectively). In contrast, in the fetal artery, the NE responses in the presence of RYN were more modest (19 ± 4 and 24 ± 4%, respectively). The difference in fetal MCA response to RYN vs. TSG suggests several possibilities. Perhaps the most likely is that the relative depletion of Ca2+ stores by these two compounds differs. Alternatively, RYN and TSG may have very different effects on plasma membrane Ca2+ and/or K+ channels that are responsible for mediating the Ca2+ fluxes (as suggested in Figs. 4 and 8). Also, as noted above, the RYN responses in both fetal and adult arteries were eliminated in the absence of extracellular Ca2+, again emphasizing the extracellular Ca2+ dependence of the cerebral arteries for contraction. Again, whereas in adult MCA after TSG administration application of RYN resulted in robust increases in tension and [Ca2+]i, that was not the case in the fetal vessel.

Caf has been shown to activate the RYN-sensitive, TSG-resistant Ca2+ store (32), which in mesenteric artery smooth muscle comprises ~80% of the total Ca2+ stores (2). In myocardial (35) and cecal smooth muscle (13) cells, Caf increases Ca2+ sensitivity of the SR-Ca2+ release channels involved in CICR. Thus it serves as a convenient tool to investigate some characteristics of the RYN-sensitive store. As shown in Fig. 6, A and B, and Fig. 7, in the adult MCA application of 3 × 10-3 M Caf resulted in a marked increase in both [Ca2+]i and tension, which was essentially the response to 10-5 M NE (Table 1). Although tension returned to baseline value within ~15 min (Fig. 6A), [Ca2+]i decreased more slowly (Fig. 6B). In contrast, in fetal arteries, the Caf-induced peak increases in tension and [Ca2+]i were 27 ± 4 and 64 ± 12%, respectively, of the NE-induced responses, and the increase in tension was much more transient (Fig. 6, C and D). In adult MCA, subsequent administration of 10-5 M NE resulted in negligible responses of tension and [Ca2+]i (Fig. 6, A and B; Table 1). By comparison, after Caf store depletion in response to 10-5 M NE, fetal MCA showed a modest response of [Ca2+]i (20 ± 3%), whereas the increase in tension was only 9 ± 2% (Fig. 6, C and D). Nonetheless, in both adult and fetal MCA, 10-6 M nifedipine eliminated these NE-induced responses, again suggesting that the [Ca2+]i was derived from extracellular Ca2+ rather than the SR Ca2+ store. As an aside, in both adult and fetal vessels, with increasing half-log Caf doses (Fig. 7) the tension and fluorescence ratio responses were less than that after a single dose (Fig. 6). These attenuated responses may have been a result of desensitization. Alternatively, the multiple doses may have resulted in a more prolonged and gradual depletion of Ca2+ stores compared with the rapid release of such stores after a single dose. In addition, Caf can inhibit phosphodiesterase (PDE) activity, thereby resulting in increases in cAMP and protein kinase A (PKA) activity, with decreased tone. Thus an alternative explanation for the failure of the increase in tone to be sustained after Caf administration (Fig. 6, A and C) is that fetal cerebral vessels (and perhaps other smooth muscle) may be more dependent on changes in Ca2+ sensitivity than on changes in [Ca2+]i. This dependence may be associated with reliance on cAMP-dependent phosphorylation events to modify the tone of smooth muscle in the adult. The apparent difference in fetal MCA sensitivity is evident when RYN-induced changes in tension and [Ca2+]i are compared with those of Caf. For example, as seen in Fig. 4, C and D, and Fig. 6, C and D, RYN resulted in an increase of 0.19 ± 0.04 fluorescent ratio units compared with 0.16 ± 0.04 ratio units for Caf (Table 1). These increases are roughly equivalent. In contrast, vascular tension increases were dramatically different in the two groups (1.23 ± 0.17 vs. 0.38 ± 0.01 g). An additional factor to consider is that the PDE isoform and/or activity in fetal cerebral arteries may differ from that of the adult; however, we are unaware of information about differences in PDE sensitivity to Caf in the two age groups.

Perspectives

NE-induced vascular tension is an index of alpha -adrenergic receptor-mediated smooth muscle activation. In vitro, this represents the integrated output of several complex signal transduction cascades that converge on the myofilaments and contractile apparatus. Previously, we demonstrated major differences between fetal and adult cerebral artery alpha 1-AR density and NE-induced Ins(1,4,5)P3 responses (21), Ins(1,4,5)P3-receptor density (44), and other elements of this cascade (28). However, none of the differences can fully account for the developmental differences observed in NE-induced contractility.

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 KCa 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 KCa 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.


    ACKNOWLEDGEMENTS

We thank James W. Putney, Jr., for reviewing the manuscript and providing several useful insights and Brenda Kreutzer for preparing the manuscript.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HD/HL-03807 and PO1-HD-31226 to L. D. Longo.

Address for reprint requests and other correspondence: L. D. Longo, Center for Perinatal Biology, Loma Linda Univ., School of Medicine, Loma Linda, CA 92350 (E-mail: llongo{at}som.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. §1734 solely to indicate this fact.

Received 14 June 1999; accepted in final form 31 March 2000.


    REFERENCES
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ABSTRACT
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METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 279(3):R860-R873
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