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
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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.
 |
RESULTS |
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).
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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.
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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.
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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.
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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; , 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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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.
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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; , 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 |
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.
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|
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
-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
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.
 |
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