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1 Department of Veterinary Biomedical Sciences, University of Missouri, Columbia, Missouri 65211; 2 Department of Medical Physiology, Texas A & M University College of Medicine, College Station, Texas 77843; and 3 Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Increases in intraluminal shear stress are thought to cause vasodilation of coronary arterioles by activation of Ca2+-dependent endothelial nitric oxide synthase followed by release of nitric oxide. We tested the hypothesis that endothelium-dependent vasodilation of isolated coronary arterioles to shear stress and agonists is necessarily preceded by an increase in endothelial cell Ca2+ concentration ([Ca2+]i). After selective loading of endothelium in isolated rabbit coronary arterioles with fura 2, simultaneous changes in diameter and [Ca2+]i were recorded. Vasodilations recorded in response to ACh, substance P, or shear stress were accompanied by significant increases in endothelial cell [Ca2+]i. Vasodilations to shear stress were accompanied by smaller changes in endothelial cell [Ca2+]i than equivalent dilations evoked by substance P or ACh. To test the role for Ca2+ as an activator of endothelial nitric oxide synthase, the endothelium was treated with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid eliminated significant changes in endothelial cell [Ca2+]i and inhibited dilations to ACh and substance P but did not significantly affect shear stress-induced vasodilation. The data indicate that endothelium-dependent vasodilation of coronary arterioles in response to agonists and shear stress is mediated in part through a rise in endothelial cell [Ca2+]i but that a substantial component of the shear stress-induced response occurs through a Ca2+-insensitive pathway.
calcium; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; nitric oxide; nitric oxide synthase; isolated arterioles
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IN THE CORONARY CIRCULATION, fluid shear stress acts as an important, moment-to-moment regulator of vascular resistance. Coronary arterioles display profound vasodilation in response to luminal shear stress (19-21), a response shown to be mediated by endothelium-dependent release of nitric oxide (NO) (19, 20). In cultured cells, increases in fluid shear stress elicit increases in endothelial cell Ca2+ concentration ([Ca2+]i) (1, 2, 4, 10, 13, 15, 30-32) and release of NO (3, 4, 12, 17, 18).
Although it is generally believed that acute NO production is controlled and preceded by changes in endothelial cell [Ca2+]i, evidence to support this idea is inconclusive, both in cultured cell systems (28) and in intact vessel preparations (3). For example, Korenaga et al. (17) found that removal of extracellular Ca2+ blocked shear stress-dependent production of NO in fetal bovine aortic endothelial cells, but others reached different conclusions (1, 10, 13, 31, 32). Kuchan and Frangos (18) reported that an initial increase in laminar flow stimulated release of NO that was Ca2+-calmodulin dependent but that sustained increases in flow were associated with Ca2+-independent NO production. Recent data indicate that a Ca2+-independent pathway involving serine/threonine and tyrosine kinases may be activated by shear stress in cultured endothelial cells (3, 4, 8, 14, 34).
A close correlation also exists between the rise in endothelial cell [Ca2+]i produced by endothelium-dependent vasodilators and the subsequent relaxation of vascular smooth muscle (16). Agonist-induced vasodilation that is mediated by NO can generally be blocked by removal of extracellular Ca2+ (12, 22). Because the constitutive form of NO synthase (NOS) expressed in endothelial cells (ecNOS) is thought to be a Ca2+-calmodulin-regulated enzyme (9), these findings suggest that increases in endothelial cell [Ca2+]i are necessary for agonist-dependent production of NO. However, this idea has recently been challenged by several studies using cultured cell systems (3, 4, 8, 34).
Because endothelial cells have been shown to undergo changes in signaling pathways and ion channel expression in culture (26, 33) and significant differences exist between large and small vessels, it is important to investigate these mechanisms in intact resistance vessels. Using isolated rat cremaster arterioles, Falcone et al. (7) found that intraluminal flow produced vasodilation and a slight but significant increase in endothelial cell [Ca2+]i. However, flow-induced dilation in the cremaster preparation was later shown not to be completely dependent on NO (19). Hecker et al. (12) found that removal of extracellular Ca2+ significantly decreased NO production in response to ACh but had no effect on shear stress-induced release of NO in rabbit femoral artery. In coronary arterioles, shear stress-induced vasodilation can be blocked completely by treatment with arginine analogs, indicating that the response is mediated entirely by release of NO (19). Because ecNOS is dependent on Ca2+-calmodulin for activation (9), we speculated that Ca2+ would be of cardinal importance in the signaling cascade initiated by shear stress. The purpose of this study was threefold: 1) to determine whether shear stress-induced vasodilation of isolated coronary arterioles is accompanied by an increase in endothelial cell [Ca2+]i, 2) to determine whether alterations in endothelial cell [Ca2+]i produced in response to shear stress differ from the changes in endothelial cell [Ca2+]i produced by endothelium-dependent, nitroxidergic agonists, and 3) to determine whether agonist- and shear stress-induced vasodilation can be eliminated by blocking changes in endothelial cell [Ca2+]i.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolation and cannulation of coronary arterioles. New Zealand White rabbits (n = 42) of either gender weighing 1-2 kg were anesthetized with xylazine (Rompun, 10 mg/kg im) and ketamine (35 mg/kg im) and given heparin (1,000 U/kg iv). A midline thoracotomy was performed, and the heart was excised and placed in cold (4°C) saline.
Coronary arterioles were dissected and cannulated according to the method of Kuo et al. (19). Physiological saline solution (PSS) containing 3% gelatin and india ink was infused into the left anterior descending artery, the circumflex artery, and the right coronary artery to visualize coronary arterioles. Arterioles (65-110 µm intraluminal diameter) were dissected free from the myocardium under a dissection microscope and then transferred to a Lucite chamber for cannulation with micropipettes (50-60 µm diameter) of matched tip resistance.Evaluation of vasodilatory responses. The microvessel chamber was transferred to the stage of an inverted microscope (Zeiss Axiovert 100) equipped with a videocamera and video caliper system for determination of intraluminal diameter. Arterioles were pressurized at 50 cmH2O with two independent hydrostatic pressure reservoirs. Leaks were detected by pressurizing the vessel, then closing the valves to the reservoirs and verifying that intraluminal pressure remained constant; arterioles exhibiting leaks were discarded. Arterioles were bathed in PSS containing (in mM) 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.22 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 MOPS buffer, along with 1 g/100 ml BSA. The PSS was adjusted to pH 7.4 and warmed to 36-37°C. During an initial equilibration period the arterioles were allowed to develop spontaneous tone. Only vessels that developed spontaneous tone were studied.
Vasodilatory responses to shear stress were determined by the method described previously in this laboratory (19, 20). Altering the heights of two independent pressure reservoirs in equal and opposite directions generated pressure differences (
P = 4, 10, 20, 40, and 60 cmH2O) between the cannulating
pipettes without changing mean intraluminal pressure. Previous work
demonstrated that intraluminal pressure was not altered by this method
of controlling intraluminal flow (19). Actual flow rates were computed
from measurements of vessel radius and the velocity of red blood cells injected into the vessel lumen. The relationship between P and flow
rate was determined for each pair of micropipettes used for a given
size of vessel. Shear stress (
) was calculated from the following
equation: = 4
/
r3,
where is viscosity (0.8 cP), is volumetric
flow rate, and r is the steady-state
vessel radius (21).
On displaying a steady level of spontaneous tone, arterioles were
exposed to graded increases in shear stress or increasing concentrations of endothelium-dependent vasodilators. Vasodilatory responses to substance P (SP, 1 × 10
12 to 1 × 108 M) or ACh (1 × 1010 to 1 × 105 M) were determined at
constant intraluminal pressure in the absence of shear stress. In some
experiments the arginine analog
NG-monomethyl-L-arginine
(L-NMMA, 1 × 105 M for 30 min) was used
to inhibit endothelial cell NO production. In all vessels, sodium
nitroprusside (SNP, 105 M)
was applied near the end of the experiment to determine the maximal
passive diameter with intraluminal pressure set to 50 cmH2O. ACh, SP, and SNP were
applied extraluminally.
Determination of endothelial cell
[Ca2+]i.
To facilitate selective loading of the endothelial cell layer (7), the
Ca2+-sensitive dye fura 2-AM was
applied intraluminally for a short time period (5 min). In some
experiments the Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM was applied intraluminally in combination with fura
2-AM. Fura 2-AM was prepared fresh daily by initial dissolution in DMSO
and dilution to 5 µM final concentration in PSS containing 0.5%
DMSO. BAPTA-AM was prepared as a 5 mM stock solution in DMSO and
diluted to 40 µM in PSS. PSS containing fura 2 and/or BAPTA was
introduced into the lumen by backfilling one pipette and then initiating flow into the vessel lumen by creating a pressure gradient of 20 cmH2O across the vessel for
5 min. A 5-min loading period in the absence of flow was followed by 15 min of backflushing (P = 20 cmH2O) to remove any traces of
fura 2 or BAPTA from the lumen and pipettes. This procedure produced
consistent and specific loading of the endothelium in rabbit coronary arterioles.
Chemicals. Fura 2 pentapotassium salt, fura 2-AM, and BAPTA-AM were purchased from Molecular Probes (Eugene, OR). Albumin was purchased from Amersham Life Science (Chicago, IL). All other chemicals were purchased from Sigma Chemical (St. Louis, MO).
Statistical analysis. Diameter changes were expressed as a percentage of the maximal dilation measured in response to 100 µM SNP. For Ca2+ responses, peak endothelial cell [Ca2+]i was measured after each intervention, and the responses were averaged. Differences within and between groups (with and without BAPTA) were determined using ANOVA for repeated measures. Post hoc analyses were performed when appropriate.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Component of dilation due to NO.
An initial series of experiments was performed to determine what
fraction of the response to ACh, SP, and shear stress was NO dependent
in isolated rabbit coronary arterioles. Figure
1 summarizes the results of these
experiments. Arterioles developed spontaneous tone (an average
constriction to 78 ± 3 µm from a maximal passive diameter of 104 ± 3 µm) and dilated in response to abluminal application of ACh
or SP. The responses shown in Fig. 1 are expressed as a percentage of
the dilation from the baseline diameter (with spontaneous tone) to the
maximal passive diameter. In PSS, increasing concentrations of ACh
caused a dose-dependent vasodilation (Fig.
1A), with a threshold response
occurring at 1 × 109
M and the SNP response at 1 × 106 M ACh (to 90% of the
maximal dilation). After treatment with the NO inhibitor
L-NMMA (1 × 105 M) for 30 min, this
response was eliminated; rather than dilation, the two highest
concentrations of ACh produced constriction in the presence of the
inhibitor. In PSS, increasing concentrations of SP also caused a
dose-dependent dilation, with a threshold response occurring at 1 × 1012 M and the
maximal dilation at 1 × 108 M SP. In contrast to
ACh, the SP response was only partially blocked by
L-NMMA treatment, so ~25%
dilation still occurred at 1 × 108 M SP (Fig.
1B). The response to shear stress
was intermediate between the responses to ACh and SP (Fig.
1C); increasing shear stress
produced graded dilation, with a threshold occurring at <1
dyn/cm2 and the maximal response
at just above 2 dyn/cm2.
L-NMMA completely inhibited the
dilation at all levels of shear stress, suggesting that the effects of
shear stress in this tissue are mediated entirely by NO.
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Time course of [Ca2+]i and diameter responses. If a rise in endothelial cell [Ca2+]i is required for NO release, then increases in endothelial cell [Ca2+]i would be predicted to precede arteriolar dilation. To monitor the time course of changes in [Ca2+]i and diameter, the arteriolar preparation was viewed simultaneously under fluorescence and near-infrared illumination. [Ca2+]i measurements were determined from fura 2 fluorescence when the microscope objective was focused on the endothelial cell layer (at the bottom surface of the vessel lumen); diameter measurements were determined after refocusing on the vessel midpoint during the time when the image acquisition system was storing fura 2 data sets.
In almost all vessels studied, vasodilatory responses to ACh or SP were preceded by significant increases in endothelial cell [Ca2+]i. However, in response to shear stress, the differences in the time courses of endothelial cell [Ca2+]i and diameter were not as clear. Those results are summarized in Fig. 2. We analyzed data only from the first two pressure steps (P = 10 and 20 cmH2O), because the greatest
changes in diameter occurred during these steps (Fig.
1C). For the step at P = 10 cmH2O, the first significant
increase in endothelial cell
[Ca2+]i
occurred 21 s after the initiation of flow, whereas the first significant increase in diameter occurred 20 s after the initiation of
flow (Fig. 2A). For the step at P = 20 cmH2O, the first significant increase in endothelial cell
[Ca2+]i
occurred 21 s after the initiation of flow, whereas the first significant increase in diameter occurred 35 s after the initiation of
flow (Fig. 2B). These findings are
not inconsistent with the idea that the
[Ca2+]i
rise precedes the dilation (although increases in these two parameters
are essentially indistinguishable for P = 10 cmH2O), but they do not, of
course, demonstrate a cause-and-effect relationship.
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Steady-state responses to ACh before and after BAPTA. Figure 3 summarizes the steady-state [Ca2+]i and diameter responses after administration of ACh. Figure 3A shows the average [Ca2+]i, and Fig. 3B shows the average diameters at each dose of ACh. Under control conditions, where the endothelium was loaded only with fura 2, significant dilations occurred at the three higher ACh doses, and each of these was accompanied by significant increases in [Ca2+]i above the baseline level. Although the relative time courses of the [Ca2+]i and diameter changes are not apparent in Fig. 3, the [Ca2+]i increases preceded the dilations in all 11 experiments (data not shown).
To test whether the dilations to ACh were dependent on increases in endothelial cell [Ca2+]i, the endothelium was preloaded with BAPTA (40 µM) along with fura 2. After this treatment, endothelial cell [Ca2+]i remained at <100 nM at all doses of ACh, indicating that no change from the average [Ca2+]i before agonist application occurred. After BAPTA loading, vasodilatory responses to ACh were completely blocked, and the diameter-dose curve (Fig. 3B) appeared to be quite similar to that recorded in the presence of L-NMMA (Fig. 1A). Under these conditions, the highest concentration of ACh produced vasoconstriction (Fig. 3B).Steady-state responses to SP before and after BAPTA.
Figure 4 summarizes steady-state
[Ca2+]i
and diameter responses after administration of SP. A response pattern
slightly different from that with ACh was produced. Each dose of SP
between 1 × 1012 and
1 × 108 M produced
significant dilation, but only the highest dose was associated with a
significant increase in endothelial cell
[Ca2+]i
(Fig. 4A, filled squares).
[Ca2+]i
changes associated with maximal SP-induced dilation were very modest
(to <100 nM; Fig. 4A) compared
with those observed at the ACh dose producing maximal dilation (235 nM;
Fig. 3A).
8 M
SP was not statistically significant (Fig.
4A, open squares). Vasodilation to SP
was only partially attenuated by BAPTA (Fig. 4B, open circles). Thus the BAPTA
effect on the SP response was much less pronounced than that for ACh.
Steady-state responses to shear stress before and after BAPTA. In fura 2-loaded vessels, elevations in shear stress produced significant arteriolar vasodilation (Fig. 5B) at all shear stress levels >1 dyn/cm2. Under control conditions, these dilations were associated with small but significant increases in steady-state endothelial cell [Ca2+]i (Fig. 5A). For example, the shear stress produced by a pressure difference of 40 cmH2O (4.3 ± 0.4 dyn/cm2) caused arterioles to dilate by 56 ± 7%, and this response was associated with a 46 ± 14% increase in endothelial cell [Ca2+]i. The highest shear stress (6.2 ± 0.6 dyn/cm2) increased endothelial cell [Ca2+]i by 49 ± 17% and produced 69 ± 6% dilation.
To test whether the dilations to shear stress were dependent on increases in endothelial cell [Ca2+]i, the endothelium was preloaded with BAPTA (40 µM) along with fura 2. In the presence of BAPTA, the patterns of steady-state [Ca2+]i and diameter responses to shear stress elevation were different from those for ACh or SP. After BAPTA, endothelial cell [Ca2+]i increased by <10 nM over the entire range of shear stress applied, indicating that effective Ca2+ buffering occurred. In marked contrast to its effect on ACh- or SP-induced dilation, BAPTA treatment had no significant effect on the vasodilation to shear stress (Fig. 5B), with the maximal shear stress-induced dilation remaining at 67 ± 8%.| |
DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We have made the first direct measurements of [Ca2+]i in the endothelial cell layer of coronary arterioles during measurement of the physiological end point of NO production, namely, vasodilation. The results of our study indicate that the role of endothelial cell [Ca2+]i in eliciting endothelium-dependent, nitroxidergic vasodilation differs among pharmacological agonists and levels of shear stress. By selective treatment of the endothelium with a Ca2+ chelator, we directly tested the idea that a rise in endothelial cell [Ca2+]i is required for short-term NO release. The endothelium-dependent agonists ACh and SP produced increases in endothelial cell [Ca2+]i that preceded vasodilations, whereas increases in endothelial cell [Ca2+]i after shear stress coincided very closely with the dilation. Chelation of endothelial cell [Ca2+]i using BAPTA eliminated the vasodilation to ACh, supporting the conclusion that, in isolated rabbit coronary arterioles, this agonist produces NO-mediated vasodilation through a Ca2+-dependent pathway. The mechanism of SP-induced dilation in this tissue appeared to be partially Ca2+ independent and to involve at least one endothelium-derived factor in addition to NO. The most remarkable finding in our study was that shear stress caused a significant increase in endothelial cell [Ca2+]i that was apparently unrelated to NO release. These data provide direct evidence in intact vessels that short-term regulation of NO production by shear stress can occur through endothelial cell signaling pathways that are independent of Ca2+.
Methodological considerations. A number of technical issues must be considered when these data are interpreted. Our experiments were performed on intact coronary arterioles, a multicellular preparation consisting of endothelium and smooth muscle. To measure vasodilatory responses to agonists or shear stress, the smooth muscle layer must be actively contractile, a process that may require changes in smooth muscle cell [Ca2+]i, and in most cases such changes would be predicted to proceed in a direction opposite to those occurring in the endothelium (6). However, we were also investigating the changes in endothelial cell [Ca2+]i that occurred in response to vasodilators and shear stress. Through application of fura 2 from the luminal side of the vessel and use of a very short loading protocol (5 min), we were able to achieve selective loading of the endothelial cell layer and, therefore, monitor changes in endothelial cell [Ca2+]i without substantial interference from changes in smooth muscle [Ca2+]i. This procedure worked well for rabbit coronary arterioles, but, for reasons we do not fully understand, similar protocols were ineffective for loading the coronary endothelium of pig arterioles. In rabbit vessels we established that the fura 2 fluorescence signal was derived from the endothelium by employing several tests: 1) mechanical denudation of fura 2-loaded arterioles showed that 90% of the fluorescence was eliminated after removal of the endothelium (n = 3); and 2) the direct smooth muscle vasodilator SNP produced significant dilation without a change in the fura 2 fluorescence signal. Similar observations were reported by Falcone et al. (7) in isolated rat cremaster arterioles. Thus changes in the fura 2 ratio were not caused by changes in vessel diameter, thickness of the light path, or smooth muscle [Ca2+]i.
Intraluminal perfusion also permitted selective loading of the endothelium with BAPTA, and we were thereby able to test vasodilatory responses with endothelial cell [Ca2+]i clamped. Fura 2 measurements confirmed the degree of Ca2+ buffering during the various interventions. We found that selective loading of the endothelium was dependent on the loading time and the BAPTA concentration. It was difficult to achieve adequate BAPTA loading of the endothelium without altering smooth muscle cell [Ca2+]i, as indicated by a loss of active tone with prolonged incubation in solutions containing high concentrations of BAPTA. In ~50% of the vessels that regained spontaneous tone after loading with 40 µM BAPTA, a nearly complete clamp of endothelial cell [Ca2+]i was obtained (i.e., no significant [Ca2+]i increase from control). There was a slight effect on baseline diameter in these vessels, as shown by the absolute diameters listed in Table 1. In BAPTA-loaded vessels, even the highest concentration of agonist did not produce a significant increase in endothelial cell [Ca2+]i (Figs. 3A and 4A). Thus the vasodilatory responses measured after BAPTA loading were taken from arterioles where endothelial cell [Ca2+]i did not change significantly over the entire range of agonist concentration. In those vessels we were confident that diameter responses to agonists or shear stress occurred independent of significant global changes in endothelial cell [Ca2+]i.
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Ca2+-independent NO production after shear stress. Evidence from a number of studies has suggested that an increase in endothelial cell [Ca2+]i occurs when the endothelium is exposed to shear stress (1-4, 7, 10, 13, 15, 30-32). Prasad et al. (29) and Nollert et al. (27) showed that flow triggers an increase in endothelial cell inositol trisphosphate levels; they suggested that an increase in inositol trisphosphate levels may be part of an intracellular signaling process leading to release of intracellular Ca2+. However, the majority of studies of this nature have been performed using cultured cells and, consequently, do not address the functional significance of the [Ca2+]i increase as it relates to production of endothelial cell NO and vasodilation. The only study to address this issue in isolated arterioles is that of Falcone et al. (7), who showed that flow provokes a concurrent increase in endothelial cell [Ca2+]i and vasodilation in cremaster arterioles. However, the vasodilatory response to shear stress in cremaster arterioles is now known to be mediated in part by prostacyclin, so it is not the best model for studying NO signaling mechanisms. In contrast, we have demonstrated that vasodilation to shear stress in porcine coronary arterioles is mediated entirely by release of NO (19), and the data in Fig. 1 suggest that this is also the case for rabbit coronary arterioles. Our measurements of [Ca2+]i and diameter during shear stress elevation, coupled with the BAPTA protocol we employed, allowed us to make additional conclusions about the mechanism of NO production in response to shear stress in intact arterioles. Thus, although an increase in endothelial cell [Ca2+]i may precede shear stress-induced dilation in coronary arterioles, the NO-mediated vasodilation does not appear to be substantially Ca2+ dependent.
In cultured cell studies the existing body of evidence supports the idea that shear stress may initiate Ca2+-sensitive and Ca2+-insensitive intracellular signaling pathways leading to NO production. Ayajiki et al. (3) found that shear stress stimulated biphasic NO release from rabbit iliac artery consisting of a transient peak followed by sustained but reduced NO production; the initial, transient increase in NO production was eliminated by removal of extracellular Ca2+, whereas the sustained release was unaffected. Kuchan and Frangos (18) found that antagonists of calmodulin inhibited NO production by human umbilical vein endothelial cells at the onset of shear stress but did not alter the sustained release of NO on continued exposure to flow. In bovine aortic endothelial cells, shear stress-induced release of NO persisted after removal of Ca2+ from the media and after chelation of intracellular Ca2+ with BAPTA (28). Collectively, results from studies of cultured cells support the contention that a Ca2+-insensitive mechanism for activation or modulation of NOS exists in endothelium. Such a pathway may not be the only one involved in control of NO release (e.g., in the case of agonists such as ACh), but it may be the major pathway controlling shear stress-induced NO production under the conditions of our experiments. A number of shear stress-induced endothelial cell signaling mechanisms have now been investigated that appear to occur independently of changes in [Ca2+]i. In cultured bovine aortic endothelial cells, shear stress activated 42- and 44-kDa isoforms of mitogen-activated protein kinase. Although an increase in endothelial cell [Ca2+]i occurred on exposure to shear stress, the mitogen-activated protein kinase activation was not affected by chelation of Ca2+ with BAPTA (34). Corson et al. (4) reported that phosphorylation of ecNOS occurs in cultured endothelial cells on exposure to shear stress. They demonstrated that endothelial cell [Ca2+]i increased on initial exposure to flow, but step increases in flow augmented phosphorylation of ecNOS and increased NO production in the absence of a change in endothelial cell [Ca2+]i. In intact rabbit iliac artery, shear stress induced an initial spike in NO production that was eliminated by removal of extracellular Ca2+, yet the sustained phase of shear stress-induced NO production was unaffected by removal of Ca2+ from the media (3). The conclusion that sustained NO production was Ca2+ independent is also supported by subsequent observations that the tyrosine kinase inhibitor erbstatin A inhibits both phases of NO release (3) whereas tyrosine phosphatase inhibition induces NO production in a Ca2+-independent manner (8). We have also shown that flow-induced vasodilation of coronary arterioles involves activation of one or more tyrosine kinases (25). Collectively, these results suggest that shear stress activates a signaling pathway involving tyrosine kinases to modulate NO production independent of changes in endothelial cell [Ca2+]i. This may possibly occur through an association of ecNOS with one or more signaling proteins or protein kinases that modify its activation. Evidence to support the latter idea has recently been provided by Fleming and colleagues (8).Ca2+ dependence of agonist-mediated dilation. Our data agree with several reports in the literature that the Ca2+ dependence of agonist-induced NO release may differ from that for shear stress-induced NO release. For example, NO production in response to bradykinin (16, 23), thimerosal (23), ATP (16), and ACh (3, 12, 22) is inhibited by removal of extracellular Ca2+. In rabbit femoral artery, Ca2+-free bathing solutions inhibited ACh-stimulated release of NO but did not alter shear stress-dependent release of NO (12). Ca2+-free solutions also eliminated ACh-induced NO release from rabbit thoracic aorta (3). Similarly, in human umbilical vein endothelial cells (18), treatment with the Ca2+-ATPase inhibitor thapsigargin inhibited ACh-induced release of NO from rabbit femoral artery but did not alter shear stress-induced NO production (24).
Our present results make an important contribution to the literature by showing that a Ca2+-independent mechanism for NO production is 1) operative in the coronary circulation, 2) important in intact arterioles, and 3) involved in the arteriolar response to relevant levels of an important physiological stimulus, shear stress. Taken together, the existing literature and our data suggest that agonist-induced, endothelium-dependent production of NO (and subsequent vasodilation) is dependent on extracellular Ca2+, whereas shear stress-induced NO production is largely insensitive to removal of extracellular Ca2+. Similar to agonists, shear stress may stimulate release of Ca2+ from intracellular stores, but sustained NO production appears to occur independent of intracellular Ca2+ release. The shear stress-induced increase in endothelial cell [Ca2+]i may well be required for one of the other short- or long-term signaling events elicited by shear stress (5).| |
ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the technical contributions made by Judy A. Davidson.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-32788 and HL-51748 (to W. M. Chilian), HL-46502 (to M. J. Davis), HL-48179 (to L. Kuo), and F32 HL-80975-02 (to J. M. Muller) and an American Heart Association Established Investigator Award to M. J. Davis.
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.
Address for reprint requests and other correspondence: M. J. Davis, Dept. of Medical Physiology, Texas A & M University Health Science Center, College Station, TX 77843-1114 (E-mail: mjd{at}tamu.edu).
Received 1 October 1998; accepted in final form 21 January 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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, Control measurements in
physiological saline solution (PSS); 
, measurements after 30 min of
incubation in 1 × 10
Significant differences
(P < 0.05) between control and
L-NMMA groups. For ACh and SP,
significance was determined using a repeated-measures ANOVA (with
repeated measures on both factors), and post hoc comparisons were made
using Bonferroni tests. For shear stress, significance was determined
using a repeated-measures ANOVA (within-factor only) with Bonferroni's
post hoc comparisons.
