pressure from. 20 to 120 mm. Hg produce graded depolarization of the vascular smooth muscle cells in the wall of isolated perfused renal arcuate arteries from.
Cellular and Ionic Signal Transduction Mechanisms for the Mechanical Activation of Renal Arterial Vascular Smooth Muscle1 Richard
J. Roman
and
David
R. Harder2
renal R.J.
Poman,
Medical
(J. Am.
DR.
College
Soc.
Harder,
Nephrol.
pressure increase from most vascular
organs. arteries
of vascular
Physiology, WI
E
4:986-996)
active beds,
response has been shown in the regulation of blood
kidney and other isolated perfused larization
of
Milwaukee,
1993;
ABSTRACT Elevations in transmural cular tone in arteries this myogenic important role
Department
of Wisconsin,
cle
to play an flow in the
The myogenic is associated
smooth
muscle
vasand
response in with depocells
and
a rise
in intracellular calcium concentration, which is dependent on calcium influx through voltage-sensitive calcium channels. Recent studies have indicated that the myogenic response in renal arteries is associated with the activation of phospholipase C and that arachidonic acid potentiates, whereas inhibitors of cytochrome
P-450
and
protein
kinase
C attenuate,
this response. Renal arteries produce 20-hydroxyeicosatetraenoic acid (20-HETE) via the cytochrome P450 pathway when incubated with arachidonic acid. 20-HETE is a potent constrictor of canine and rat renal arterioles. It Inhibits K channel activity, depolarizes renal vascular smooth muscle cells, and produces a sustained tration.
increase in intracellular calcium In this regard, the vasoconstrictor
to 20-HETE mimics the arteries after elevations studies C and
suggest
that
concenresponse
myogenic activation of renal in transmural pressure. These
the
activation
of phospholipase
subsequent increases in the intracellular levels of diacylglycerol, 1,4,5 inositoi triphosphate, and cytochrome P-450 metabolites of arachidonic acid may participate in the myogenic response of renal arteries and in the regulation of renal vascular tone.
Key
Words:
Kidney,
glomerulus,
‘Rceived January 2Coespondenc.
16, 1993. to Dr. DR.
leg.
8701
of Wisconsin.
renal
hemodynamics,
Accepted May 10. 1993. Harder. Department of Physiology.
Watertown
Plank
Road.
Milwaukee,
1046-6673/0404-0986$03.00/0 Journal of the American Society of Nephroiogy Copyright © 1993 by the American society of Nephrology
986
kidney
Medical
WI 53226.
Col-
vasculature,
P-450,
eicosanoids,
vascular
smooth
mus-
levations in transmural pressure increase active tone in arteries from most vascular beds, and this response plays a major role in the local regulation of blood flow (1 -3). This myogenic response sets the basal level of vascular tone upon which metabolic, humoral, and neurogenic influences adjust vascular resistance. Despite the fact that the myogenic response was first described by Bayliss (4) over 90 yr ago, the intracellular signal transduction pathways coupling changes in transmural pressure to stretch and contraction of vascular smooth muscle cells remain relatively unknown. Studies with isolated perfused renal arterioles (5,6) and the in vitro perfused rat juxtamedullary microvascular preparation (7-9) have established that renal arcuate, interlobular, and afferent arterioles contract in response to elevations in transmural pressure. Even large renal arteries, which only maintain constant diameter when transmural pressure is elevated, still exhibit increases in active wall tension when transmural pressure is elevated (10,1 1). There is now general agreement that the autoregulation of RBF is mediated by a myogenic response In preglomerular arteries acting in series with tubuloglomerular feedback, which regulates the diameter of the terminal portion of the afferent arteriole (1216). Indeed, recent studies, in which the autoregulation of glomerular capillary pressure has been measured before and after the interruption of tubuloglomerular feedback, have indicated that myogenic and tubuloglomerular feedback mechanisms contribute about equally to the autoregulation of single-nephron GFR and RBF (1 2, 1 6, 1 7). Thus, myogenic tone is an important determinant of renal vascular resistance, glomerular capillary pressure, and GFR. There is also considerable evidence suggesting that alterations In the myogenic response is associated with the renal vascular abnormalities that accompany the development of hypertension, diabetes, cyclosporine nephropathy, and glomerulosclerosis (1 5). Thus, a better understanding of the cellular mechanisms underlying the myogenic response is required to develop more effective therapies for the treatment of these diseases. This review will focus on what is currently known about the cellular, molecular, and ionic mech-
Volume
4’
Number
4’
1993
Roman
anisms renal
mediating arteries.
the
PRESSURE-INDUCED ARTERIAL MUSCLE
generation
of myogenic
DEPOLARIZATION
OF
tone
in
RENAL
Elevations in transmural pressure from 20 to 120 mm Hg produce graded depolarization of the vascular smooth muscle cells in the wall of isolated perfused renal arcuate arteries from -57 ± 2 to -38 ± 2 mV (Figure 1 A). Even smaller elevations in pressure, within the physiologic range from 80 to 1 20 mm Hg, results in significant membrane depolarization from -45 ± 2 to -38 ± 2 mV. Pressure-induced depolarization of renal arterial vascular smooth muscle is often accompanied by the generation of Ca2”-dependent “spikelike” action potentials as is seen in Figure 1 D. Pressure-induced depolarization of vascular smooth muscle cells has also been observed in isolated arteries from other vascular beds (18,19). In cerebral arteries, the slope of the curve relating membrane potential and transmural pressure, as well as the amplitude of the action potential, is dependent on the extracellular Ca2’ concentration (18,19). The dependency of stretch depolarization of vascular smooth muscle cells on extracellular Ca2 concentration is consistent with previous observations that the mechanical response of arteries to elevations in transmural pressure is associated with a calcium influx from the extracellular fluid (18-22) that is
A
-30
*
40
E
.
‘U -60
TRANSMURAL
PRESSURE
(mmHg)
B 20 mmHg
100
C mmHg
100
D mmHg
I-I
4 sec.
Figure 1 . Effect of transmural pressure tial (Em) in vascular smooth muscle
potenwail of isolated perfused canine renal arcuate arteries. (A) Relationship between transmural pressure and membrane potential. (B) Original recording showing membrane potential at a transmural pressure of 20 mm Hg. (C) Original recording demonstrating membrane depolarization at a transmural pressure of 100 mm Hg. (D) Original recording demonstrating the generation of spontaneous action potentials at a transmurai pressure of 100 mm Hg in a canine renal arcuate artery. Reprinted from reference 5 with permission.
Journal
of the
American
Society
on membrane cells in the
of Nephrology
and
Harder
blocked by calcium channel antagonists (3.23-25). An important consequence of the pressure-induced depolarization is that the resting membrane potential of arterial vascular smooth muscle is much closer to the threshold potentials required for the opening of voltage-sensitive calcium channels than is generally recognized. As a result, vascular sensitivity to a number of vasoconstrictor agonists is potentiated, and the dose response curves relating vascular diameter to agonist concentration are shifted to the left (26,27). For example, the dose response curve relatIng vascular diameter versus norepinephrine concentration in isolated perfused canine renal arcuate arteries (Figure 2) is shifted to the left by about 3 orders of magnitude when transmural pressure is increased from 60 to 140 mm Hg (27). Vice versa, a and a2 adrenoceptor activation have been reported to greatly enhance the myogenic response of arterioles to elevations in intravascular pressure (28,29).
POTENTIAL
MECHANISM
RESPONSE
OF
Stretch-Activated
RENAL Ion
OF
THE MYOGENIC
ARTERIES Channels
Several hypotheses have been proposed to explain the myogenic activation of arterial vascular smooth muscle. The first is based on the observation that certain ion channels can be activated by the physical stretching of cell membranes. Accordingly, membrane stretch activates ion channels, allowing the influx of extracellular calcium, which increases vascular tone and depolarizes these cells. The existence of stretch-activated ion channels has been documented in a variety of cells by the use of singlechannel patch-clamp recording techniques (30-32). These channels exhibit very high unitary conductances and, therefore, have a large effect on membrane potential and calcium influx (30). However, all of the stretch-activated channels that have been described to date conduct ions in a relatively nonspecific manner and are only selective for cations or anions (30). Mechanosensitive channels specific for Ca2 or K have not been identified, nor can these channels be blocked with pharmacologic agents known to block other well-characterized ion channels (30). The activation of stretch-activated cation channels would promote calcium influx refractory to voltage-sensitive calcium channel blockers, and the rise in the potassium conductance of the membrane would hyperpolarize the cell. However, the pressureinduced contraction of vascular smooth muscle is associated with membrane depolarization, and the myogenic response can be blocked by calcium channel blockers and can be up- or down-regulated by potassium channel agonists and antagonists, respectively. These findings argue against a role for stretchactivated channels in the myogenic response. More-
987
Myogenic
Response
in Renal
Arteries
100
#{163} 60mm
60
-
u) ‘
0
Control
(n=6)
Denuded
200
-
(n=6)
1.
100
80
Pressure Figure
3. Effect of the mechanical
arcuate mechanical
difference endothelium
attenuated
Pressure
diameter
of the endothelium
relationships
for six vessels
indicate arteries release
of Phospholipase
that
the myogenic response not dependent on the of a constrictor factor from is
C
Another hypothesis for the generation of myogenic tone in renal arteries is based on recent observations that the stretching of cells can activate phospholipases (41-44) and recent reports that inhibitors of protein kinase C (PKC) antagonize (45,46), whereas phorbol esters enhance, the response to arteries to elevations in transmural pressure (3). According to this hypothesis, the stretching of arterial vascular smooth muscle cells activates phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-biphosphate into 1 ,2-diacylglycerol (1 ,2-DG) and inositol 1,4,5triphosphate (1 ,4,5-1P3). 1 ,4,5-1P3 elevates intracellular Ca2 through an action on Ca2 channels in the sarcoplasmic reticulum (47). 1 ,2-DG activates PKC,
Journal
160
‘
These findings In renal arcuate pressure-induced the endothelium.
Role
140
(mmHg)
on the myogenic response of isolated perfused canine renal are presented under control conditions and after the removal of the endothelium by rubbing the lumen of the vessel with a glass micropipette. indicates significant in vascular diameter from control measured at 80 mm Hg. The inset illustrates that the mechanical removal of the had no effect on the vasoconstrictor response to norepinephrine (NE; 3 x i0 M) but that it significantly the response to acetylchoiine (ACH; I x 10 M).
arteries.
removal
120
of the
American
Society
of Nephrology
which phosphorylates a number of important intracellular regulatory proteins (47). In this regard. isoforms of PKC have been found to subserve many cellular formations, including regulation of intracellular Mg2 concentration, Ca2-ATPase, and phosphorylation of membrane channels, including voltage-activated Ca2 channels (48). One of the problems with this hypothesis is the lack of direct experimental evidence indicating that PLC activity in arterial vascular muscle is increased by elevations in transmural pressure. We have recently found that elevations of transmural pressure from 60 to 120 mm Hg increase 1 ,4,5-1P3 concentration in isolated canine renal arteries from 15.5 ± 3.4 to 23.2 ± 6 pmol/mg of protein and increase 1 ,2-DG concentration from 230 ± 45 to 336 ± 69 pmol/mg of protein (49). The rise in IP3 levels in renal vessels exposed to increases in transmural pressure was not blocked by the mechanical removal of the endothehum or by the voltage-sensitive calcium channel antagonist nifedipine (1 /2m). These observations sug-
989
Myogenic
Response
in Renal
Arteries
gest that stretch activates PLC in arterial smooth muscle cells and that this response is not dependent on extracehlular calcium influx through voltage-sensitive calcium channels. Although there are several pathways that could contribute to the elevation of 1 .2-DG levels (i.e. via phosphohipase D or C), the only metabolic pathway that could account for the rise in 1 ,4,5-1P3 levels is through the metabolism of phosphatidyhinositol 4,5biphosphate by PLC (41 ,47). These results, therefore, provide direct evidence that PLC activity in renal arteries increases after elevations in transmural pressure. Our results are consistent with previous findings that mechanical flexing of a variety of cell types increases IP3 levels at least in vitro (43,44,50). At present, one can only speculate on the mechanisms by which changes in transmural pressure activate PLC activity in renal arteries. It may be that mechanical deformation of the cell membrane allows cytoplasmic PLC access to phosphohipids along the inner surface of the cell membrane. It could also involve stimulation of a G protein, or PLC activity may increase through a Ca2-dependent mechanism secondary to the opening of stretch-activated cation channels.
o-
#{176}cofltrol + Indo #{149}-U AA + Indo
2000
+ P450
inhibitor
1500
,
Role of Metabolites Myogenic Response
of Arachidonic
Acid
in the
Recent studies have indicated that endogenous P450 metabohites of arachidonic acid (AA) can modulate the myogenic response of isolated canine renal arcuate arteries (10). In these studies, elevations in transmural pressure from 80 to 1 60 mm Hg reduced vascular diameter and produced a threefold increase in active wall tension (Figure 4). The administration of AA (50 M) to the bath constricted these arteries and potentiated the myogenic response to elevations in transmural pressure ( 1 0). This response was further enhanced by the blockade of cyclooxygenase activity and was reversed by structurally and mechanistically different inhibitors of cytochrome P450. These findings support the view that an endogenous P-450 metabolite of AA modulates the myogenic response in renal arteries ( 1 0). In other studies, microsomes prepared from canine renal arcuate arteries produced a P-450 metabolite that coeluted with 20-hydroxyeicosatetraenoic acid (20-HETE) when incubated with AA (10). Because 20-HETE is a potent constrictor of rat aortic vascular rings (51), this substance could potentially serve as an endogenous P-450 metabolite of AA that modulates the myogenic response. This possibility was investigated by characterizing the effects of 20-HETE on the vascular diameter and membrane potential of renal arteries (52). The addi-
990
1000 0
500
.
- -
a)
80
-
120
Pressure
160
(mmHg)
Figure
4. Effect of AA (50 m) and P-450 inhibitors (ketoconSKF525; 100 ,m) on the relationship between transmural pressure active and active wail tension in isolated perfused canine renal arcuate arteries (N = 7). Indomethacm (Indo) (IBM) was included in the bath to block cyciooxygenase. indicates significant difference from the control value measured at 80 mm Hg within a group. indicates a significant difference from control values measured at the same level of transmurai pressure. Reprinted from reference 10 with permission.
azole,
‘
tion of 20-HETE to the bath or lumen of isolated perfused canine renal arcuate arteries depolarized vascular smooth muscle cells from -43 ± 1 to -34 1 mV and reduced vascular diameter in a concentration-dependent manner (Figure 5). The threshold
±
-1 0 E
-20 E
-30
b ‘I
-40 -50
-D-
Vehicle
-0--
20-HETE,
I
enzymatic,
I
-8.0
-7.5
lumen
I
-7.0 Log
I
I
-6.5
-6.0
[MI
Figure 5. Cumulative concentration-response curves for the effects of 20-HETE on the internal diameter of isolated perfused canine renal arteries. The basal diameter of arteries that received vehicle or 20-HETE averaged 266 ± 24 (N = 10) and 289 ± 15 m (N = 12), respectively. indicates significant change in diameter from control. indicates significant difference from corresponding value observed in vehicle control group. Reprinted from reference 52 with permission. ‘
Volume
4
‘
Number
4
‘
1993
Roman
concentration of 20-HETE that constricted these arteries was 0. 1 zM (52). At higher concentrations, 20HETE (1 zM) reduced the diameter of these vessels by about 25% of the response produced by an equivalent concentration of norepinephrine (52). Previous studies have indicated that the vasoconstrictor response to 20-HETE in the rat aorta was dependent on an intact endothehium and was mediated by the formation of a vasoconstrictor cyclooxygenase metabolite (51). However, the vasoconstrictor response to 20-HETE in canine renal arteries is not blocked by either indomethacin or the endoperoxide/thromboxane receptor antagonist. SQ29548 or by a combined blockade of the cyclooxygenase, hipoxygenase, and P-450 pathways (52). These findings suggest that the vasoconstrictor response to 20HETE in renal arteries is not mediated by a metabolite formed in the endothehium but is probably due to
and
Harder
a direct effect of this compound on vascular smooth muscle cells. In these same studies, we did find that the mechanical removal of the endothehium attenuated the vasoconstrictor response to 20-HETE (52). Additional work, however, suggested that this may be due to the loss of the vasodilatory influences of nitric oxide on the basal tone of the artery and/or to the stimulation of the endogenous production of 20HETE in the vessel secondary to the removal of the endothehium, rather than being the result of evidence that 20-HETE is metabolized to a constrictor factor in the endothehium (52). Patch-clamp and intracellular calcium studies that have examined the ionic basis of the vasoconstrictor response to 20-HETE further support our view that 20-HETE has direct actions on canine renal vascular smooth muscle cells. As can be seen in Figure 6, the application of 1 zM 20-HETE to vascular smooth
CONTROL -20
mV
C .rLIll’
VVI
c1J .1
1 jM
-20
-
20-HETE
mV
C
C
0
40 ms Figure 6. Effects of 20-HETE on single potassium channel currents in vascular smooth muscle cells isolated from canine renal arcuate arteries. Representative tracings of single potassium channel currents recorded in the cell-attached mode before (top panel) and after (bottom panel) the addition of 20-HETE (1 m) to the bath. c denotes closed state of the channel. Reprinted from reference 52 with permission.
Journal
of the
American
Society
of Nephrology
991
Myogenic
muscle teries in the
Response
in Renal
Arteries
cells isolated from canine renal arcuate arinhibited single K channel activity recorded cell attached mode (52). The response to 20HETE is concentration dependent, and the threshold concentration that produces significant inhibition of the open-state probability of the potassium channels in renal vascular smooth muscle cells is 0.01 M (52). This is lower than the threshold concentration required to constrict intact renal arteries, but this apparent discrepancy might be because the endogenous production of 20-HETE in isolated pressurized arteries may be much greater than that seen in isolated cells not exposed to membrane stretch: therefore, a much higher concentration of 20-HETE is needed to elicit a response in pressurized vessels. In dog and rat renal microvascular smooth muscle cells, we found that the predominant potassium channel type is voltage sensitive and exhibits a large conductance (1 86 p5) when recorded by the insideout detached patch mode (53). The open state probabihity of this channel is increased by raising Ca2 concentration on the cytoplasmic surface from 0. 1 to 0.5 M and is blocked by externally applied tetraethylammonium (0. 1 mM) or charybdotoxin (50 nM). These characteristics are consistent with those of the large-conductance Ca-activated K channel that has been previously described in smooth muscle cells isolated from arteries obtained from a variety of vascular beds. It remains to be determined whether 20HETE also inhibits the activity of other K’ channels, such as the ATP-sensitive K channel or the delayed rectifier. The ability of 20-HETE to block the Ca2activated K channel is consistent with the depolarization of renal vascular smooth muscle cells and the contraction of renal arteries seen after the administration of this compound. 20-HETE also increases intracellular calcium (Figure 7) in renal vascular smooth muscle cells (52). This effect may be secondary to the depolarization of these cells and the opening of voltage-sensitive Ca2 channels. Overall, the response to 20-HETE in canine renal arcuate arteries mimics the myogenic activation of these vessels, which is characterized by an increase in vascular tone, a depolarization of vascular smooth muscle cells, and an increase in intracellular calcium concentration secondary to calcium influx through voltage-sensitive channels. These observations are also consistent with previous findings indicating that inhibitors of the metabolism of AA by P-450 can block the myogenic response of isolated canine renal arteries (10). Nevertheless, the available data remain insufficient to conclude that 20-HETE is a mediator rather than a modulator of the myogenic response in renal arteries. Such a conclusion will require additional evidence that the endogenous production of 20-HETE is stimulated by elevations in transmural pressure to levels sufficient to constrict these vessels.
992
B
40
w
I-
600
w
30 500400-
.
A I’
-a
*
‘‘-.4,
0,
20 50 Sec
Cu
2.
300
-
200
10
10::
Control
20-HETE
Figure 7. Effects of 20-HETE (I ,M) on intracellular calcium concentration measured with Fura2 in vascular smooth muscle cells isolated from canine renal arcuate arteries. The inset presents the time course of changes in intracellular calcium in a representative experiment. The bar graph presents the mean response from eight experiments. indicates a significant difference from control. Reprinted from reference 52 with permission. ‘
Previous studies have indicated that the vascular responses to P-450 metabolites of AA may be species dependent (54). To address this issue, we have reexamined the effects of P-450 inhibitors on the myogenic response and the effects of 20-HETE in the renal circulation of the rat (55,56). The effects of several mechanistically different P-450 inhibitors on the response of afferent arterioles to changes in perfusion pressure were studied with the in vitro perfused rat juxtamedullary nephron microvascular preparation of Casellas and Navar (57). Under control conditions, the diameter of the proximal and distal afferent arterioles decreased by 7% when perfusion pressure was increased from 80 to 1 60 mm Hg (55,56). The addition of the P-450 inhibitors 1 7-octadecynoic acid (17-ODYA) (20 SM), 7-ER (10 SM), miconazole (20 SM), or ketoconazole (100 M) to the bath and perfusate eliminated this autoregulatory response (55,56). After the addition of the P-450 inhibitors, the diameters of the afferent arterioles increased by 8% in response to the same elevation in perfusion pressure and the autoregulation of glomerular capillary pressure was markedly impaired. The ability of chemically and mechanistically dissimilar P-450 inhibitors to block the contraction of the proximal portion of the afferent arteriole in response to an elevation in perfusion pressure supports our previous findings suggesting a permissive role for P-450 metabolites of AA in the myogenic response of canine renal arteries (10). The finding that P-450 inhibitors also block the vasoconstrictor response of the terminal portion of
Volume
4’
Number
4’
1993
Roman
the afferent arteriole in the juxtamedullary nephron microvascular preparation to elevations in perfusion pressure suggests a potential role for P-450 metabohites in the tubuloglomerular feedback because this response is dependent on the chloride concentration of the fluid delivered to the macula densa and is blocked by furosemide (8). To examine the role of P450 metabolites of AA in tubuloglomerular feedback in rats in vivo, Imig et at. (55) have examined the effects of P-450 inhibitors on this response. In these experiments, basal stop flow pressure averaged 4 1 ± 2 mm Hg and fell by 1 5 ± 2 mm Hg as the loop of Henle perfusion rate was increased from 0 to 50 nL/ mm. The addition of several different P-450 inhibitors, 1 7-ODYA (20 SM), 7-ER (1 0 zM) or clotrimazole (20 SM), to the tubular perfusate significantly reduced the stop flow pressure responses to changes in perfusion rate by >73 ± 3%. These findings suggest that endogenous P-450 metabolites of AA may also play a role in the tubuloglomerular feedback response. In other experiments, Imig et at. (58) reported that afferent and interlobular arteries isolated from the rat kidney produce 20-HETE when incubated with AA and that 20-HETE is an extremely potent constrictor of rat afferent arterioles. Significant reductions of afferent arteriolar diameter could be detected after the addition of 1 nM 20-HETE (58). Similar to the results obtained with canine renal arteriolar smooth muscle cells, 20-HETE inhibits K channel activity at nanomolar concentrations and increases intracellular calcium concentration in vascular smooth muscle cells isolated from rat afferent arte-
and
Harder
rioles dicate
(unpublished observations). These studies inthat 20-HETE is also a potent endogenous constrictor in the renal microcirculation of the rat. Because P-450 metabolites of AA appear to play a role in both myogenic and tubuloglomerular feedback responses in the renal circulation of the rat, the functional significance of this system In the autoregulation of RBF in the rat in viva has recently been examined. The autoregulation of RBF was studied with an electromagnetic flowprobe placed on the renal artery of rats as renal perfusion pressure was lowered from 1 50 to 80 mm Hg with an adjustable clamp on the aorta above the renal arteries (55). Control RBF averaged 8.9 ± 0.6 mL/min (N = 10) and was well autoregulated as pressure was lowered from 1 50 to 1 00 mm Hg. The RBF autoregulatory index averaged 0.23 ± 0.05 over this range of pressures. The infusion of the mechanism-based P-450 inhibitor 1 7-ODYA (33 nmol/min) directly into the renal artery impaired the autoregulation of RBF. After the intrarenal infusion of 1 7-ODYA, the RBF autoregulatory index rose to 0.97 ± 0. 1 0 as pressure was varied over the same range (55). These studies provide experimental evidence that an endogenous P450 metabohite of AA participates in the autoreguhation of RBF in vivo. These findings may be relevant to the possible contribution of P450 metabohites of AA in elevating renal vascular tone (56,59) and resetting the pressure-natriuretic relationship in spontaneously hypertensive rats (60). In this regard, recent studies have indicated that the expression of the P4504A2 gene (61) and the production of 20-HETE is elevated in the kidney of spontaneously hyperten-
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ACTIVATION PIP2
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