1 Department of Pharmacology, University of Vermont, Burlington, Vermont 05405; and 2 Institute of Neurobiology, University of Puerto Rico, San Juan, Puerto Rico 00901
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ABSTRACT |
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Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) Ca2+-ATPase, and this inhibition is relieved by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. The role of PLB in regulating Ca2+ release through ryanodine-sensitive Ca2+ release channels, measured as Ca2+ sparks, was examined using smooth muscle cells of cerebral arteries from PLB-deficient ("knockout") mice (PLB-KO). Ca2+ sparks were monitored optically using the fluorescent Ca2+ indicator fluo 3 or electrically by measuring transient large-conductance Ca2+-activated K+ (BK) channel currents activated by Ca2+ sparks. Basal Ca2+ spark and transient BK current frequency were elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, an activator of adenylyl cyclase, increased the frequency of Ca2+ sparks and transient BK currents in cerebral arteries from control mice. However, forskolin had little effect on the frequency of Ca2+ sparks and transient BK currents from PLB-KO cerebral arteries. Forskolin or PLB-KO increased SR Ca2+ load, as measured by caffeine-induced Ca2+ transients. This study provides the first evidence that PLB is critical for frequency modulation of Ca2+ sparks and associated BK currents by PKA in smooth muscle.
cerebral arteries; smooth muscle; ryanodine receptors; cAMP-dependent protein kinase; ion channels; calcium; potassium; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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CALCIUM
RELEASE through ryanodine-sensitive Ca2+ channels
(ryanodine receptors or RyRs) in the sarcoplasmic reticulum (SR) can influence the contractile state of smooth muscle by regulating, directly or indirectly, average ("global") intracellular
Ca2+ (1, 6, 15, 28). Rapid activation of RyRs,
for example, by the Ca2+ current or by a pharmacological
agent such as caffeine, can lead to a global Ca2+ transient
(7, 9, 27) and a transient contraction (18). Localized Ca2+ release events from RyRs
("Ca2+ sparks") have also been detected in smooth
muscle cells (15, 28). Ca2+ sparks appear to
be caused by the activation of a small number of clustered RyRs, which
leads to a transient elevation of Ca2+ in ~1% of the
cell volume. Ca2+ sparks activate nearby large-conductance
Ca2+-activated K+ (BK) channels in the plasma
membrane to cause a transient membrane potential hyperpolarization,
which closes voltage-dependent Ca2+ channels, and thus can
lower global Ca2+ (15, 28). In some smooth
muscle types, Ca2+ sparks also activate
Ca2+-sensitive Cl channels, which can cause
membrane potential depolarization (20, 38).
RyRs are defined by their activation to increased cytoplasmic Ca2+ (15). It is also now known that the level of Ca2+ within the SR can modulate RyR channel open probability (40). An elevation of luminal SR Ca2+ increases RyR open probability, which can manifest itself as a rise in Ca2+ spark frequency (5, 34, 39). The open probability of RyRs is also increased by protein kinases including cGMP-dependent protein kinase and cAMP-dependent protein kinase (PKA) (8, 40). In arterial myocytes, agents that activate PKA elevate Ca2+ spark frequency as well as the frequencies and amplitudes of the associated transient BK currents (31), which may contribute to the vasodilatory action of these agents.
The molecular basis for the cAMP-induced elevation in Ca2+ spark frequency is not known. Two logical targets of PKA in smooth muscle are the RyR itself (8, 24, 36) and phospholamban (PLB), which inhibits the SR Ca2+-ATPase. Phosphorylation of PLB by PKA leads to dissociation of its pentameric structure and a disinhibition of the SR Ca2+-ATPase (35). Thus phosphorylation of PLB leads to an increase in SR Ca2+-ATPase activity and elevation of SR Ca2+ load, which could potentially increase Ca2+ spark frequency and amplitude.
The goal of this study was to determine the role of PLB in the regulation of Ca2+ spark frequency in cerebral arteries. The effects of the adenylyl cyclase activator forskolin on Ca2+ sparks and associated BK channel currents were examined in smooth muscle cells in cerebral arteries of control and PLB-deficient knockout (PLB-KO) mice. Our results indicate that PLB has a key role in the frequency modulation of Ca2+ sparks and BK currents and in the regulation of these events by PKA.
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METHODS |
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Adult PLB-KO mice were purchased from the University of Cincinnati. Ablation of the gene for PLB and the absence of this protein in vascular smooth muscle of these commercially available animals has previously been demonstrated (21, 23, 40). Control mice used in this study (SVJ-129 strain) were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were euthanized under deep pentobarbital anesthesia (intraperitoneal; 150 mg/kg body wt). After decapitation, the brain was removed and quickly transferred to cold (4°C), oxygenated (95% O2-5% CO2) physiological saline solution (PSS) of the following composition (in mM): 119 NaCl, 4.7 KCl, 24 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, 0.023 EDTA, and 11 glucose. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23) and followed protocols approved by the Institutional Animal Use and Care Committee of the University of Vermont.
Ca2+ spark measurements. To examine Ca2+ sparks in intact artery segments, cerebral arteries (100-150 µm in diameter) from control and PLB-KO mice were slipped over glass cannulas as previously described (9, 14, 16). Cannulated arteries were placed into HEPES-PSS containing 10 µM fluo 3-AM (Molecular Probes) and 0.05% Pluronic acid (Molecular Probes) and were incubated at 22°C for 60 min. Arteries were washed for 30 min at 22°C to allow deesterification of the intracellular dye. The HEPES-PSS had the following composition (in mM): 135 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH).
A Noran Oz laser scanning confocal system coupled to an inverted Nikon TMD microscope equipped with a ×60 water-immersion lens (N.A. 1.2) was used to image intact artery segments. Fluo 3 was excited with the 488-nm line of a krypton/argon laser. The light emitted (520 nm) by fluo 3 was separated from the excitation light and collected; images of the vessel wall (56.3 µm × 52.8 µm) were acquired at a frequency of 60 Hz (every 16.7 ms), with each area scanned for a period of 10 s. All data were recorded at room temperature (20-22°C). Ca2+ sparks were detected and analyzed using custom software (IDL 5.0.2; Research Systems, Boulder, CO). Baseline fluorescence (Fo) was determined by averaging 10 images without Ca2+ spark activity. Fractional fluorescence (F/Fo) increases were determined in areas (1.54 µm × 1.54 µm) where Ca2+ sparks were detected. Ca2+ sparks were defined as local fractional fluorescence increases >1.3.K+ current recordings.
Smooth muscle cells from cerebral arteries were enzymatically isolated
as previously described (30, 37). Whole cell
K+ currents were measured in these freshly isolated
cerebral artery myocytes from control and PLB-KO mice with the
perforated-patch configuration (13) of the patch-clamp
technique (11). The external solution contained (in mM)
134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose,
and 10 HEPES (pH 7.4). Patch pipettes (r = 3-5
M) were filled with a solution that contained (in mM) 110 KAsp, 30 KCl, 10 NaCl, 1 MgCl2, 10 HEPES, and 0.05 EGTA (pH 7.2). Amphotericin B was dissolved in dimethyl sulfoxide (DMSO) and diluted
into the pipette solution to give a final concentration of 200 µg/ml.
Experiments were performed at room temperature (20-22°C).
Arterial wall [Ca2+].
Measurements of arterial wall [Ca2+] were obtained in
cerebral artery segments that were prepared in a manner similar to
those used for Ca2+ spark measurements (i.e., placed on
glass cannulas in HEPES-PSS). Artery segments were loaded with the
ratiometric Ca2+-sensitive fluorescent dye fura 2-AM (2 µM) for 45 min as previously described (17). Ratio
images were obtained from background corrected images of the 510-nm
emission from the arteries alternately excited at 340 and 380 nm
with software developed by IonOptix (Milton, MA). Arterial
wall [Ca2+] was calculated using the following equation
(10): [Ca2+] = Kd × × (R
Rmin)/(Rmax
R). At the end of every
experiment, Rmin and Rmax, the ratios of
emission signals under Ca2+-free and
Ca2+-saturated conditions, respectively, were
measured from ionomycin-treated arteries, and
was determined. An
apparent dissociation constant (Kd) of 282 nM of
fura 2 for Ca2+ was used (17).
Materials. Fura 2-AM, fluo 3-AM, and Pluronic acid were purchased from Molecular Probes (Eugene, OR). Stock solutions (1 mM) of fluo 3-AM and fura 2-AM were made using DMSO as the solvent. Caffeine was from Serva (Hauppauge, NY). Ryanodine and the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) were obtained from Calbiochem (La Jolla, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).
All values are means ± SE. Differences were considered statistically significant at P < 0.05 using either the unpaired Student's t-test or Student-Newman-Keuls test when appropriate. ![]() |
RESULTS |
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Ca2+ spark and BK channel current
frequency are elevated in smooth muscle cells of cerebral arteries from
PLB-KO mice.
Ca2+ sparks were measured in intact cerebral arteries from
control and PLB-KO mice (Fig.
1). Ca2+ spark
frequency was significantly higher in cerebral arteries from PLB-KO
mice (2.1 ± 0.3 Hz, n = 14 areas from 5 animals)
compared with Ca2+ spark frequency in similar tissue from
control animals (1.4 ± 0.2 Hz, n = 16 areas from
4 animals; Figs. 1, A and B, and 2). While the
frequency of Ca2+ sparks was elevated in cerebral artery
segments from PLB-KO mice, the amplitude of these intracellular
Ca2+ release events, measured as F/Fo (see
METHODS), was not different in control and PLB-KO arteries
(control: 1.57 ± 0.02, n = 109 sparks, vs.
PLB-KO: 1.57 ± 0.02, n = 162 sparks,
P = 0.9).
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Forskolin elevates Ca2+ spark and BK channel current frequency to a greater extent in smooth muscle cells of control compared with PLB-KO mice. Activators of PKA (e.g., forskolin and cAMP) have been shown to increase Ca2+ spark and associated BK current frequency in freshly isolated rat cerebral artery myocytes (31). However, the molecular identity of the protein(s) involved in this PKA-signaling pathway has not been established. The PLB-KO model provides a unique approach to dissect, at the molecular level, the PKA-signaling pathway in arterial smooth muscle. Forskolin increased the frequency of Ca2+ sparks by more than twofold in intact cerebral artery segments from control mice (Figs. 1, A and C, and 2) and increased transient BK current frequency by a similar degree in myocytes isolated from these control animals (Fig. 3). The inhibitor of PKA, Rp-cAMPS, reversed this stimulatory effect of forskolin on BK current frequency (Fig. 3). Forskolin also increased the amplitude of the transient BK currents in control myocytes, an effect reversed by Rp-cAMPS (Fig. 3). Consistent with previous reports (31), we found that forskolin did not alter Ca2+ spark amplitude in cerebral arteries from control mice (F/Fo = 1.57 ± 0.02, n = 109, vs. 1.61 ± 0.02, n = 204, in the absence and presence of forskolin, respectively). These data suggest that elevation of cAMP levels and PKA activation can lead to an increase in the frequency of Ca2+ sparks and associated BK currents in cerebral artery myocytes from control mice. To test the hypothesis that phosphorylation of PLB is involved in the PKA-induced increase in Ca2+ spark frequency, the actions of forskolin were also examined in cerebral arteries from PLB-KO mice. In cerebral arteries from PLB-KO mice, forskolin did not significantly increase Ca2+ spark (P = 0.136, Fig. 2) or BK current frequency (Fig. 3). Ca2+ sparks were abolished in all groups by ryanodine (10 µM), an inhibitor of ryanodine-sensitive Ca2+ release channels (n = 4-6, Fig. 2). These results indicate that the presence of PLB leads to a decrease in basal Ca2+ spark frequency. Furthermore, our data demonstrate that PLB plays a significant role in the frequency modulation of these events by PKA.
Caffeine increases BK current frequency in the presence of
forskolin.
One possible explanation for the reduced effect of forskolin on
arterial myocytes from PLB-KO animals is that Ca2+ spark
activity may already be near maximal levels. To examine this
possibility, the effect of caffeine, a pharmacological activator of
RyRs, was tested. In the presence of forskolin, a low concentration of
caffeine (10 µM) increased transient BK activity in cerebral artery
myocytes isolated from both control (1.6 ± 0.2-fold,
n = 7) and PLB-KO (1.4 ± 0.2-fold,
n = 6) mice (Fig. 4).
These data suggest that the lack of effect of forskolin on myocytes
from PLB-KO mice is not due to a maximal level of transient BK channel activity or underlying Ca2+ sparks under these conditions.
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Forskolin and PLB do not influence the kinetics of
Ca2+ sparks or transient BK currents in
mouse cerebral artery myocytes.
The data described above suggest that forskolin, acting via PLB,
increases the frequency of localized intracellular Ca2+
release events (i.e., Ca2+ sparks). Because phosphorylation
of PLB can increase SR Ca2+-ATPase activity, we next
investigated the effects of forskolin and PLB on the kinetics of
Ca2+ sparks and associated BK currents. The time required
for local fractional fluorescence changes to decline 50% from peak
values (half-time of decay) was used to study Ca2+ spark
decay in myocytes from intact cerebral artery segments. Half-time of
decay was not significantly different in myocytes from control and
PLB-KO mice either in the absence (control: 42.1 ± 1.5 ms,
n = 88; PLB-KO: 42.2 ± 1.6 ms, n = 102) or presence of forskolin (control + forskolin: 42.4 ± 1.5 ms, n = 102; PLB-KO + forskolin: 44.3 ± 2.0 ms, n = 91). In addition, no difference was
observed in the half-time of decay for transient BK currents in
cerebral artery myocytes isolated from control or PLB-KO mice in the
presence or absence of forskolin (Fig.
5). These data suggest that
Ca2+ uptake by the SR Ca2+-ATPase is not a
major contributor to the decay of Ca2+ sparks in smooth
muscle.
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Forskolin and ablation of the gene for PLB lead to an elevation of SR Ca2+ load. In cardiac and smooth muscle, elevation of SR Ca2+ levels leads to an increase in Ca2+ spark frequency (4, 34, 39). Phosphorylation of PLB by PKA or targeted disruption of the PLB gene should lead to an elevation of SR Ca2+ load through activation of the SR Ca2+-ATPase (22, 31). We therefore sought to examine the effects of forskolin and PLB deficiency on SR Ca2+ content in cerebral artery myocytes. Rapid application of high concentrations of caffeine (10 mM) leads to a significant release of Ca2+ from the SR through activation of RyRs. The relative amplitude of these caffeine-induced Ca2+ transients has routinely been used as an index of SR Ca2+ content (18, 31, 34).
Arterial wall Ca2+ was measured in unpressurized cerebral artery segments (9, 14, 16) with the Ca2+ indicator dye fura 2 (17) (Fig. 6). Note that these arterial wall Ca2+ measurements represent the spatially average Ca2+ signal throughout the vessel wall, which includes the total contribution of global cytosolic Ca2+ levels and Ca2+ sparks as well as Ca2+ waves. Under these experimental conditions, arterial wall Ca2+ was 85.0 ± 9.9 nM (n = 17) and unaffected by forskolin (89.0 ± 17.1 nM, n = 11). Arterial wall Ca2+ levels were also similar in cerebral arteries isolated from PLB-KO mice in the absence (85.4 ± 9.5 nM, n = 9) and presence (85.7 ± 12.0 nM, n = 7) of forskolin. Consistent with PKA activation increasing SR Ca2+ load in cerebral arteries from control animals, forskolin increased the magnitude of caffeine-induced Ca2+ transients from 218.1 ± 32.6 nM (n = 15) to 360 ± 49.6 nM (n = 11). In the absence of forskolin, caffeine-induced Ca2+ transients were significantly higher in cerebral arteries from PLB-KO mice (363.6 ± 55.6 nM, n = 9). This influence of PLB on SR Ca2+ load parallels changes in Ca2+ spark frequency observed in this tissue. Interestingly, in the presence of forskolin, caffeine-induced Ca2+ transients appeared to decrease in arteries from PLB-KO mice (207 ± 32.4 nM, n = 7, P = 0.07) compared with caffeine transients in PLB-KO mice in the absence of forskolin. These data suggest that the SR Ca2+-ATPase regulatory protein PLB exerts a tonic inhibitory influence on SR Ca2+ load that is removed by activation of PKA. The apparent decrease in SR Ca2+ content caused by forskolin in PLB-KO animals may represent an additional direct effect of PKA to increase Ca2+ efflux from the SR through RyRs.
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DISCUSSION |
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In this study, we provide the first evidence suggesting that PLB
is a critical component of the frequency modulation of Ca2+
sparks and their associated transient BK currents by PKA in smooth muscle. We propose that PKA phosphorylates PLB to augment SR
Ca2+-ATPase activity, which, in turn, leads to an increase
in SR Ca2+ load. This increase in SR Ca2+ load
leads to enhanced Ca2+ spark frequency and BK channel
activity (Fig. 7).
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Ca2+ spark and BK channel current frequency are elevated in cerebral arteries from PLB-deficient mice. PLB regulates SR Ca2+ load indirectly by inhibiting the SR Ca2+-ATPase (19, 35). We found that SR Ca2+ load and Ca2+ spark frequency were elevated in cerebral arteries from PLB-KO mice (Figs. 1 and 6), consistent with the idea that PLB exerts a tonic inhibitory effect on the SR Ca2+-ATPase. The frequency of transient BK currents was also elevated in PLB-KO mice (Fig. 3), consistent with the causal relationship between Ca2+ sparks and BK channel activity in arterial smooth muscle (3, 30). Heart muscle cells from PLB-KO mice also exhibited an elevated basal Ca2+ spark frequency (34). The observed increase in Ca2+ spark frequency is likely a result of elevated SR Ca2+ load, which has been shown to increase the open probability of RyR (5) and Ca2+ spark frequency (39). Consistent with a causal role for RyR activation in the generation of Ca2+ sparks, ryanodine (10 µM) abolished Ca2+ sparks under all experimental conditions observed in the present study. Although the activation of D-myo-inositol 1,4,5-trisphosphate (IP3) receptors does not directly contribute to Ca2+ spark genesis, one could argue that PKA phosphorylation of IP3 receptors could indirectly modulate Ca2+ spark frequency (18). However, in the present study, IP3 receptor activity would be expected to be low (i.e., in the absence of agonists to stimulate IP3 production), and thus these channels would be expected to have a minimal contribution to the frequency modulation of Ca2+ sparks.
An elevation of SR Ca2+ load might be expected to increase Ca2+ spark amplitude, as evidenced in cardiac myocytes (33) or nonvascular smooth muscle (39). However, we did not observe an increase in Ca2+ spark amplitude, measured as F/Fo, in smooth muscle cells from PLB-KO cerebral arteries. The amplitude of Ca2+ spark-activated BK current transients also was not elevated in myocytes from the PLB-KO animals, consistent with the Ca2+ spark data. Ca2+ spark amplitude depends on the flux of calcium ions through open RyR channels. A recent study, using purified cardiac RyRs incorporated into planar lipid bilayers, has demonstrated that the relationship between luminal Ca2+ concentration and the unitary Ca2+ current through RyRs is hyperbolic (26). One possible explanation for the lack of effect of elevating SR Ca2+ load on Ca2+ spark amplitude is that the Ca2+ flux through the RyR channel may be near saturation with respect to luminal Ca2+.PLB plays an important role in the modulation of Ca2+ sparks by cAMP. In a previous study (31), we found that activators of PKA elevate Ca2+ spark frequency as well as the frequency and amplitude of transient BK currents in smooth muscle cells from rat cerebral arteries. PKA could exert its effects on Ca2+ sparks through phosphorylation of PLB and/or the RyR. In either case, this could lead to elevation of RyR channel open probability, i.e., increase Ca2+ spark frequency. To explore the molecular basis of cAMP modulation of Ca2+ sparks, we examined the effects of forskolin on Ca2+ spark and BK current frequency in smooth muscle cells of cerebral arteries from control and PLB-KO mice. Forskolin clearly elevated Ca2+ spark and BK current frequency more than twofold in smooth muscle cells from control mice cerebral arteries (Figs. 2 and 3). However, in cerebral arteries from PLB-KO mice, forskolin had no significant effect on Ca2+ spark and BK current frequency. This diminished effect of forskolin on Ca2+ spark and BK current frequency was not due to saturation of Ca2+ spark frequency because caffeine, a pharmacological activator of RyRs, could further increase transient BK current frequency in the presence of forskolin in cells from both control and PLB-KO mice (Fig. 6). These results support a significant role of PLB in the frequency modulation of Ca2+ sparks by cAMP. However, the reduction of caffeine-induced Ca2+ transients in the presence of forskolin in PLB-KO myocytes suggests the possibility that PKA may also directly activate RyRs, which could lead to a decrease in SR Ca2+ content.
Physiological implications of PLB modulation of Ca2+ sparks and BK channel currents. Small diameter arteries in many vascular beds (e.g., cerebral and coronary circulations) autoregulate by constricting to increased intravascular pressure (2, 12, 25). Recent evidence suggests that the activation of BK channels by Ca2+ sparks plays a negative feedback role to counteract pressure-induced depolarization and constriction (18, 28, 30). A likely mechanism responsible for a pressure-dependent increase in BK channel activity is an increase in the frequency of Ca2+ sparks arising from elevated cytoplasmic and SR Ca2+. Our data demonstrating an elevation of basal Ca2+ spark frequency in cerebral arteries from PLB-deficient animals reveal the crucial role of this protein in regulating Ca2+ uptake into the SR and thus Ca2+ spark activity.
Activators of PKA cause vasodilation, which is partially reversed by the BK channel inhibitor iberiotoxin in a number of vascular beds, including the cerebral circulation (29, 31, 32). Our data demonstrating that PLB greatly contributes to forskolin-induced increases in Ca2+ spark and transient BK channel activity suggest that this protein plays a role in the iberiotoxin-sensitive component of vasodilations mediated via the cAMP pathway. In conclusion, PLB appears to be an important regulatory protein in the control of Ca2+ spark activity within cerebral arterial smooth muscle. Thus it is likely that PLB, via its inhibitory effects on the SR Ca2+-ATPase, exerts significant control within the cerebral circulation by influencing SR Ca2+ load and Ca2+ sparks. Ultimately, this pathway through BK channel activity regulates membrane potential and voltage-dependent Ca2+ channel activity that influences smooth muscle contractile state and cerebral artery diameter. ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Dr. Litsa Kranias, University of Cincinnati, for assistance in providing the phospholamban knock-out mice. We also thank Dr. Litsa Kranias, Dr. Maria Gomez, and Christine Saundry for helpful comments in the preparation of this manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HL-44455 and HL-63722 (to M. T. Nelson), F32-HL-09920 (to G. C. Wellman), NS-39405 (to M. T. Nelson and L. F. Santana), and P40 PR-12358 (to E. G. Kranias), American Heart Association Grant 003029N (to G. C. Wellman), and the Totman Trust for Human Cerebrovascular Research.
Address for reprint requests and other correspondence: M. T. Nelson, Dept. of Pharmacology, Univ. of Vermont, Burlington, Vermont 05405 (E-mail: nelson{at}salus.med.uvm.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 March 2001; accepted in final form 30 April 2001.
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