Laboratory for Research in Neonatal Physiology, Cardiovascular Renal Center, Department of Physiology and Biophysics, The University of Tennessee, Memphis, Tennessee 38163
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ABSTRACT |
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Endothelin-1 (ET-1) is the most potent vasoconstrictor agent known. ET-1 is elevated in the cerebrospinal fluid following hemorrhage and brain injury and can compromise cerebral microvascular homeostasis. The modulation of ET-1 production by cerebral microvascular endothelial cells and the mechanism by which such changes take place are very important in our understanding of the pathological roles of ET-1. In the present study, we investigated the effects of vasoconstrictor agents that can be released from hemolyzed blood, cAMP-dependent dilators, and the role of protein kinase C (PKC) in the regulation of ET-1 production by piglet cerebral microvascular endothelial cells in culture. ET-1 was measured by RIA. 1) Cerebral microvascular endothelial cells synthesize and release ET-1 into the media; 2) 5-hydroxytryptamine (5-HT), lysophosphatidic acid (LPA), thromboxane analog U-46619, fetal bovine serum (20%), and phorbol 12-myristate 13-acetate significantly increase ET-1 production; 3) basal and vasoconstrictor agent-induced increases in ET-1 production by endothelial cells may be mediated via PKC; 4) cAMP-dependent vasodilators attenuate the basal production of ET-1 by cerebral microvessels; and 5) pretreatment of endothelial cells with a higher concentration of LPA, U-46619, or 5-HT counterbalances the cAMP-dependent dilator agent-induced reduction in basal ET-1 production. Therefore, by-products of hemolyzed blood can stimulate the production of ET-1 by a PKC-mediated mechanism. cAMP-dependent dilators can attenuate the vasoconstrictor agent-induced elevation in ET-1 production. These results suggest that cerebral microvascular homeostasis could be compromised by effects of interactions among vasoactive agents released during conditions injurious to the brain and they may further the understanding of potential contributions of hemolyzed blood clots to subarachnoid hemorrhage-induced vasospasm.
adenosine 3',5'-cyclic monophosphate; peptide; radioimmunoassay; dilators; constrictors; endothelin-1; protein kinase C
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INTRODUCTION |
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ONE DIRECT CONSEQUENCE of cerebral hemorrhage is the exposure of cerebral tissues to blood constituents. Numerous substances are released in response to the blood on and in the cerebral cortex. In addition, by-products of hemolyzed blood, such as oxyhemoglobin, leukotrienes, bilirubins, 5-hydroxytryptamine (5-HT), lysophosphatidic acid (LPA), and thromboxane (6, 11), are released onto the cerebral cortex as a consequence of brain trauma. Most of the substances so released are vasoactive and may act individually or collectively on cerebral microvascular cells, causing modification of cerebral microcirculation (6, 11, 24, 25). Their activities may involve effects on pial arteriolar reactivity, through modifications of receptors and/or alterations in second messenger pathways. Such by-products may also cause the release of other vasoactive agents, which by themselves or in conjunction with others could adversely affect cerebral metabolism. The effects of these vasoactive agents together could play a significant role in the modulation of vascular reactivity and circulation reported following cerebral hemorrhage (6, 11, 24, 25).
Elevated endothelin-1 (ET-1) has been observed in the cerebrospinal fluid (CSF) following subarachnoid hematoma (13, 25). The elevated ET-1 has been shown to be involved in the alteration of cerebral microcirculation and changes in pial arteriolar reactivity (13, 25). Treatment with agents that inhibit ET-1 production and/or block ET-1A receptor activation attenuates the aforementioned atleration and changes (2, 5, 8, 14, 15). Previous studies have shown increased production of ET-1 and prostanoids by human cerebromicrovascular endothelial cells following treatments with other vasoconstrictive peptides (19). Spatz et al. (19) detected an interrelationship between the production of ET-1 and prostanoids and suggested that they may play a role in the regulation of cerebral microcirculation. In addition, ET-1 has been implicated in the pathogenesis of stroke, hypertension, cerebral hemorrhage, and atherosclerosis (13, 18, 22). However, the mechanism(s) behind increased ET-1 production under such pathophysiological conditions have not been fully investigated. Also, the effects of dilator agents on ET-1 production have not been well defined. Specifically, the roles of constrictor agents released by hemolyzed blood and the influence of dilator agents that act by generating cAMP on increased production of ET-1 from cerebral microvascular endothelial cells are unknown. The interactions between these two classes of vasoactive agents help to maintain the microvascular tone. It will be important to understand their influence on ET-1 production. In the present study, we have investigated the hypotheses that vasoconstrictor agents released from hemolyzed blood following cerebral hemorrhage stimulate ET-1 production from cerebral microvascular endothelial cells, that cAMP-dependent dilator agents antagonize the basal production of ET-1, and that the increase in ET-1 production is mediated by the activation of protein kinase C (PKC).
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METHODS |
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Primary culture of cerebral microvascular endothelial cells. Primary cultures of cerebral microvascular endothelial cells from newborn pig brain were established as previously described (9). Briefly, cerebral cortical microvessels (60-300 µm) were isolated by differential filtration of cerebral cortex homogenate, first through 300-µm and then through 60-µm nylon mesh screens. The isolated microvessels were incubated in collagenase-dispase solution (1 mg/ml) for 2 h at 37°C. At the end of the incubation, the dispersed microvascular endothelial cells were separated using Percoll density gradient centrifugation. Endothelial cells were resuspended in culture medium consisting of 20% fetal bovine serum (FBS), 2 mg/ml sodium bicarbonate, 1 U/ml heparin, 30 mg/ml endothelial cell growth supplement, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2.5 mg/ml amphotericin B. Endothelial cells were plated on 12-well Costar plates coated with Matrigel. Endothelial cell cultures were maintained in a 5% CO2-95% air incubator at 37°C. The culture medium was changed frequently until the cells attained confluence. Confluent cells (after 5-7 days of cultivation) were used for the experiments.
Determination of the effects of vasoconstrictor agents
on ET-1 production by endothelial cells. To investigate
the effects of vasoconstrictor agents, the PKC activator phorbol
12-myristate 13-acetate (PMA), and 20% FBS on ET-1 production by
endothelial cells, confluent endothelial cells were treated with 20%
FBS, PMA (1 µM), U-46619 (0.1 µM), LPA (1 µM), or 5-HT (1 µM)
and incubated for 4 h in a 5%
CO2-95% air incubator at
37°C. The media were collected and stored at 70°C for
the determination of ET-1 produced.
Determination of the role of PKC. The
role of PKC in vasoconstrictor agent-induced ET-1 production was
investigated by pretreatment of confluent endothelial cells with PKC
inhibitors (1 µM calphostin C, 1 µM staurosporine, or 10 µM
Gö-6976) for 15 min. After the pretreatment, cells were exposed
to 20% FBS, PMA (1 µM), U-46619 (0.1 µM), LPA (1 µM), or 5-HT (1 µM) and coincubated for 4 h in a 5%
CO2-95% air incubator at
37°C. The media were collected and stored at 70°C for
the determination of ET-1 produced.
Effects of cAMP-dependent vasodilators on basal
production of ET-1. Confluent endothelial cells were
treated with the cAMP analog 8-bromo-cAMP (8-BrcAMP; 0.1 mM), adenylase
cyclase activator forskolin (0.1 mM), and cAMP-dependent dilator agents
[histamine (10 µM), dilator prostanoids (10 µM
PGE2), or iloprost (1 µM)] and incubated for 1 h in a 5%
CO2-95% air incubator at
37°C. At the end of the incubation, the media were collected and
stored at 70°C for the determination of ET-1 content.
Effects of higher concentrations of the
vasoconstrictor agents on cAMP-dependent vasodilator-induced reduction
in basal production of ET-1. Confluent microvascular
endothelial cells were pretreated for 15 min with U-46619 (0.1 mM), LPA
(1 mM), or 5-HT (1 mM) before addition of 8-BrcAMP (0.1 mM), forskolin
(0.1 mM), histamine (10 µM),
PGE2 (10 µM), or iloprost (1 µM). Cells so treated were then incubated with such cotreatment for 1 h in a 5% CO2-95% air incubator at 37°C. The media were collected and stored at 70°C
until the ET-1 level was determined.
Determination of ET-1 in the cells.
After incubation of the cells with vasoactive agents and removal of the
media, cells were treated with 0.1 N HCl for 30 min. At the end of the
treatment, cells were scraped off the plates. ET-1 was extracted from
the cells by sonication using a high-intensity cell disrupter. The cell
homogenates were centrifuged, and the cell extract and original media
were stored at 70°C until assayed.
ET-1 assay. ET-1 levels were determined in the collected media and cell extracts using RIA (ET-1,2-specific RIA kit; Amersham Life Sciences, Arlington Heights, IL), according to the manufacturer's instructions. Cell extracts were neutralized with 1 N NaOH before assay. The system utilizes a high specific activity of 125I-labeled ET-1 synthetic tracer, together with a highly specific and sensitive antiserum. Separation of the bound antibody from the free fraction was achieved with the addition of an Amerlex-M second antibody preparation to the reaction mixture (unknown, antibody, and 125I-ET-1), which was incubated for 16-24 h at 2-8°C (overnight delay addition protocol). The mixture was centrifuged at 3,000 rpm at 16°C for 15 min. The supernatant was removed by vacuum suction, and radioactivity in the pellet was determined. The concentration of the unlabeled ET-1 in the sample was determined by interpolation from the standard curve (0.25-32 fmol/tube). The sensitivity, as determined by a response at 50% displacement of tracer, was 4.5 fmol.
Statistics. All the experiments were conducted in duplicate and the means used as data points. There was minimal variation in protein concentration among wells (48 ± 5 mg/well). Therefore, the results are expressed as femtomoles per well. All values are presented as means ± SE. The results were subjected to two-way ANOVA for repeated measures with Fisher protected least significant difference to isolate differences among groups. A level of P < 0.05 was considered significant.
Drugs. Iloprost (a stable analog of prostacyclin) was a gift from Schering Pharmaceutical Research (Berlin, Germany); histamine, PGE2, 8-BrcAMP, PMA, and forskolin were purchased from Sigma Chemical (St. Louis, MO); LPA was purchased from Avanti Polar Lipids (Alabaster, AL); and calphostin C, Gö-6976, and staurosporine were purchased from Calbiochem (San Diego, CA). ET-1 RIA kits were purchased from Amersham Life Sciences.
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RESULTS |
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ET-1 formation and release by cerebral microvascular endothelial cells. Under basal conditions, cerebral microvascular endothelial cells produce ET-1 and release it into the media. At the end of incubation, the ET-1 released from the cells into the media was 133 ± 17 fmol/well, whereas that retained in the cells was 33 ± 2 fmol/well. This indicates that the endothelial cells synthesize ET-1 and release it into the media. Because the ET-1 of interest is extravascular, extracellular, and adjacent to the vascular smooth muscle, we determined the levels of ET-1 in the media.
Effects of vasoconstrictor agents on ET-1 produced by
cerebral microvascular endothelial cells. To
investigate the effects of these vasoactive agents on ET-1 production
from the endothelial cells, confluent cells were treated with 20% FBS,
PMA (1 µM), the thromboxane analog U-46619 (0.1 µM), LPA (1 µM),
and 5-HT (1 µM). These treatments increased ET-1 production
significantly compared with controls (Fig.
1). Concentrations of
naturally occurring agents or their stable analogs were selected to
simulate a condition comparable with hematoma or brain injury, in which
a high concentration of these agents might be released or produced by
hemolyzed blood. Concentrations so selected are also consistent with
those used by others in similar studies and observed in the CSF
following hemorrhage (6, 11, 19).
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Role of PKC in ET-1 production by endothelial
cells. To investigate the mechanism by which the above
agents increase ET-1 production, confluent cerebral microvascular
endothelial cells were pretreated with the PKC inhibitors calphostin C
(1 µM), Gö-6976 (10 µM), and staurosporine (1 µM).
Pretreatment of cultured cells with the PKC inhibitors for 15 min
before the addition of the vasoactive agents attenuated the production
of ET-1 induced by 20% FBS, PMA (1 µM), U-46619 (0.1 µM), LPA (1 µM), and 5-HT (1 µM) (Fig. 2).
Calphostin C and Gö-6976 also inhibited the basal production of
ET-1 by the endothelial cells (Fig. 2,
A and
B). However, staurosporine did not
have any significant effect on the basal production of ET-1 by the
endothelial cells except in the presence of U-46619 and LPA (Fig.
2C).
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Effects of cAMP-dependent vasodilators on ET-1
production. The roles of cAMP-dependent vasodilator
agents in the regulation of ET-1 production were investigated.
Confluent microvascular endothelial cells were treated with 8-BrcAMP
(0.1 mM), forskolin (0.1 mM), or the cAMP-dependent vasodilator agents
histamine (10 µM), PGE2 (10 µM), or iloprost (1 µM). The basal production of ET-1 by the
microvascular endothelial cells in culture was reduced by the cAMP
analog, the adenylase cyclase activator, and cAMP-dependent vasodilators. Treatments with histamine and
PGE2 resulted in the highest
reduction in basal ET-1 production by the microvascular endothelial
cells (Fig. 3).
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Effects of cotreatment of endothelial cells with
vasoconstrictor and vasodilator agents on the attenuation of ET-1
production. To determine whether higher concentrations
of the vasoconstrictor agents prevent the attenuation of ET-1
production from the endothelial cells, confluent endothelial cells were
cotreated with higher concentrations of 5-HT (1 mM), LPA (1 mM), and
U-46619 (0.1 mM) in the presence of cAMP-dependent vasodilators. The
higher concentrations counterbalanced the attenuation of basal
production of ET-1 by the cAMP-dependent vasodilators (Fig.
4). The lower concentrations of 5-HT, LPA,
and U-46619 did not have any significant effects on the attenuation of
ET-1 production by the dilator agents.
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Effects of cAMP-dependent vasodilators on ET-1 release
from the cell cytoplasm. ET-1 levels in the media as
well as within the cell cytoplasm were determined following treatment
with cAMP-dependent dilators. ET-1 levels in the cell cytoplasm were
not affected following treatment with cAMP-dependent dilator agents
(Table 1). This indicates that
the attenuation of ET-1 production by cAMP-dependent vasodilator agents
does not involve the inhibition of ET-1 secretion into the media from
the cell cytoplasm.
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DISCUSSION |
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Interactions between the vasoconstrictor and dilator agents play a significant role in the maintenance of cerebral microvascular tone during both normal and pathophysiological conditions. During pathophysiological conditions, the balance in the cerebral microvascular tone may be altered in favor of vasoconstriction due to increased production of vasoconstrictor agents, resulting in reduced cerebral perfusion. ET-1 is the most potent naturally produced vasoconstrictor agent known today and has been implicated in several vascular diseases (13, 18, 22). The present study not only investigated the roles of selected vasoactive agents in ET-1 production but also investigated the potential contribution of PKC to the mechanism of vasoactive agent-induced ET-1 production. The results presented indicate that 1) cerebral microvascular endothelial cells synthesize ET-1 and release it into the media; 2) 5-HT, LPA, the thromboxane analog U-46619, 20% FBS, and PMA significantly increase ET-1 production; 3) basal and vasoconstrictor agent-stimulated ET-1 production by endothelial cells may be mediated via PKC activation; 4) cAMP-dependent vasodilators attenuate the basal production of ET-1 by cerebral microvessels; and 5) pretreatment of endothelial cells with higher concentrations of LPA, U-46619, and 5-HT counterbalance the cAMP-dependent dilator agent-induced reduction of basal production of ET-1 by microvascular endothelial cells.
By-products of hemolyzed blood have been suspected as potential candidates for the cause of the pial arteriolar narrowing observed following subarachnoid hemorrhage. Their role in the pathogenesis of microvascular diseases has been the subject of several reviews (6, 11). These reports have concluded that, alone, none of these elements released from the blood clot is capable of causing the modification of cerebral microvascular circulation observed following hemorrhage. This conclusion was based on 1) the potency of the individual agents, 2) inadequate concentration in the vicinity of the affected pial arterioles, and 3) the time course of release and the manifestation of alteration in microvascular reactivity (1, 7). However, persistent increases in CSF ET-1 following cerebral hemorrhage have been reported in both clinical and experimental studies (10, 21, 25). In the present study, we have shown that agents that can be released by blood clots and hemolyzed blood, 5-HT, thromboxane, and LPA stimulate increased ET-1 production from endothelial cells in culture. Similarly, serum and phorbol ester significantly increased ET-1 production by the cerebral endothelial cells in culture compared with the basal production. These agents per se are not known to be potent vasoconstrictors (4, 6, 11, 24), but they increased ET-1 production and secretion by cerebral microvascular endothelial cells. The increased ET-1 levels could contribute to the changes in cerebral microvascular circulation and vascular remodeling observed following hemorrhage. In addition, these vasoactive agents, at the low levels that may be present in the CSF, could synergize with ET-1 to affect pial arterioles, resulting in the modification of cerebral microvascular circulation. In the present study, we did not study the effects of interactions of these vasoactive agents on vascular reactivity. However, Yang et al. (28) have shown potentiation of vasoconstrictor effects of ET-1 following treatment with 5-HT. These data suggest that the hemolyzed blood by-products, besides stimulating the increased production of ET-1, could also interact with one another to potentiate vasoconstrictor effects on microvascular vessels.
The elevated ET-1 production caused by potential by-products of hemorrhaged blood was attenuated by calphostin C, Gö-6976, and staurosporine, indicating that the production of ET-1 by these agents is mediated via the activation of PKC. Calphostin C, Gö-6976, and staurosporine are known inhibitors of PKC (12, 23). The PKC inhibitors not only attenuated the vasoconstrictor agent-induced increases in ET-1 but also inhibited basal production of ET-1 by endothelial cells. Furthermore, the PKC activator PMA enhanced ET-1 production. These data indicate that stimulated production of ET-1 by the vasoconstrictor agents may be mediated through PKC activation and that the basal production of ET-1 is PKC-dependent. Gö-6976 and calphostin C, but not staurosporine, selectively attenuated basal production of ET-1. In the presence of LPA and U-46619, but not the other vasoactive agents, staurosporine attenuated the basal production of ET-1 from the cerebral microvascular endothelial cells. The reasons behind the differences in the actions of staurosporine and the other PKC inhibitors used are not known. The differences observed, however, could be attributed to differential sensitivity of the different PKC isoforms involved in ET-1 production.
Eleven PKC isozymes (,
1,
2,
,
,
,
, µ,
,
, and
) are known to exist in mammalian cells (3, 16, 17).
These isoforms of PKC can be divided into three categories.
"Classical" PKC isoforms are calcium dependent, "novel" PKC
isoforms are calcium independent, and the "atypical" PKC isoforms
are phosphatidylserine dependent (3, 16). The
-subclass of PKC is
found only in the brain, whereas
- and
-subclasses have been
found in many tissues and cell lines (16). The differences in the
sensitivity of PKC isoforms to calcium ions might be reflected in the
varied effects of PKC inhibitors observed in this study. In the present study, however, we did not investigate the role of calcium ions or
phosphatidylserine in the vasoactive agent-induced regulation of ET-1
production by cerebral microvascular endothelial cells. Vasoconstrictor
agent-induced stimulation of ET-1 production was equally attenuated by
calphostin C, Gö-6976, and staurosporine. Basal production of
ET-1 was attenuated by calphostin C and Gö-6976 but not
staurosporine. These observations suggest that stimulated and basal
production of ET-1 could require activation of different PKC isozymes.
At this moment, we are not aware of any report indicating that
different and specific PKC isozymes are required to be activated for basal or stimulated production of ET-1.
The mechanism responsible for the apparent synergistic effects of LPA and U-46619 on the staurosporine-induced attenuated basal production of ET-1 is not known. However, calphostin C and Gö-6976 are selective inhibitors for PKC with inhibitor constant (Ki) values in the nanomolar range, whereas staurosporine, apart from showing a high affinity for PKC, also shows equal affinity for PKG, PKA, myosin light chain kinase (MLCK), and calmodulin kinase (3, 16, 17). It is therefore possible that the effects of staurosporine observed in the present study may be due to inactivation of other kinases apart from PKC. Also, the isozymes of PKC that may be involved in the basal production of ET-1 may not be sensitive to staurosporine. Similarly, the concentrations of U-46619 and LPA employed in this study might have differential effects on other kinases apart from PKC, which happen to be sensitive to staurosporine too. Such effects could account for the differential effects observed. To fully understand the mechanisms behind these differences, studies involving the use of PKC isoform-selective agents and the use of PKC coenzymes need to be performed.
In addition, the concentrations of PKC inhibitors employed in these studies are high, especially that of the Gö-6976. There is therefore the possibility that this high dose employed may act on other kinases, contributing to the regulation of ET-1 production by the cerebral microvascular endothelial cells. The IC50/Ki of Gö-6976 for PKC is 8 nM, whereas for PKG it is 6 µM and for MLCK is ~5.8 µM; for PKA it is >100 µM (3, 16, 17). Possible inactivation of MLCK and PKG may contribute to the effects of Gö-6976 observed in the regulation of ET-1 production. However, in comparing the affinity of Gö-6976 for other kinases, it has a relatively higher affinity for PKC with a magnitude of 1,000-fold. Hence the effects observed on ET-1 production are most likely mediated via inhibition of PKC rather than nonspecific or toxic actions on other kinases.
We observed a reduction in the basal production of ET-1 by cerebral microvascular endothelial cells following pretreatment with histamine, iloprost, PGE2, 8-BrcAMP, and forskolin. In the presence of cAMP-dependent dilators, the elevated production of ET-1 induced by vasoconstrictor agents was attenuated. During normal physiological conditions, the endothelium produces dilator and constrictor agents to maintain cerebral microvascular tone. This balance could be affected by pathology. Here we report that ET-1 production is affected by the presence of cAMP-dependent vasodilators. As such, elevation in the level of cAMP immediately following hemorrhage or conditions that could lead to increased ET-1 production may temper ET-1 production. However, treatment of the endothelial cells with pathological concentrations of vasoconstrictor agents counterbalances the cAMP-dependent dilator agent-induced attenuation of basal production of ET-1. This indicates that, following conditions that increase the level of vasoconstrictor agents, the cerebral microvascular tone may be altered in favor of reduction in cerebral blood flow. The pathological concentrations of these vasoactive agents are hard to define, but the concentrations employed in the present study could be in excess of even those produced with severe injuries.
The mechanism(s) behind the cAMP-dependent vasodilator agent-induced attenuation of ET-1 production is not known. The possibility exists that the mechanism(s) may involve interference with the ET-1 synthetic pathway. In the present studies, we have shown that attenuated ET-1 production, induced by the cAMP-dependent dilators, from the cerebral microvascular endothelial cells does not involve the inhibition of ET-1 release from the cell cytoplasm. The precise mechanism by which these agents regulate ET-1 production needs to be further investigated.
The present study indicates that the by-products of blood elements released following hemorrhage or brain injury can stimulate an increased production of ET-1. Mechanisms stimulating ET-1 production can involve PKC. Basal and stimulated productions of ET-1 from endothelial cells are attenuated by agents that stimulate increases in cAMP. In addition, the basal and stimulated production of ET-1 by cerebral microvascular endothelial cells might involve activation of different PKC isozymes, which might be differentially sensitive to PKC inhibitors.
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ACKNOWLEDGEMENTS |
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We thank Jin Emerson-Cobb for editorial assistance and Laura Malinick and Danny Morse for the illustrations.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grants HL-42851 and HL-34059 and by the American Heart Association, Tennessee Affiliate.
Address for reprint requests: M. A. Yakubu, The Univ. of Tennessee, Memphis, Dept. of Physiology and Biophysics, 894 Union Ave., Memphis, TN 38163.
Received 10 September 1997; accepted in final form 29 September 1998.
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