(Stroke. 2001;32:249.)
© 2001 American Heart Association, Inc.
Original Contributions |
Ethanol-Induced Contractions in Cerebral Arteries
Role of Tyrosine and Mitogen-Activated Protein Kinases
From the Departments of Physiology and Pharmacology (Z-w.Y., T.Z., B.T.A., B.M.A.), Anesthesiology (J.W.), and Medicine (B.M.A.), and the Center for Cardiovascular and Muscle Research (B.T.A., B.M.A.), State University of New York, Health Science Center at Brooklyn.
Correspondence to Dr B.M. Altura, Box 31, SUNY Health Science Center at Brooklyn, 450 Clarkson Ave, Brooklyn, NY 11203.
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Abstract |
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Background and Purpose—The relationship between alcohol consumption and stroke appears complex; moderate ingestion is associated with reduced stroke risk, while heavy intake is associated with increased stroke risk. Ethanol has been shown both experimentally and epidemiologically to induce hemorrhagic and ischemic strokes, which are associated with cerebral vasoconstriction. Ethanol is known to induce contraction in isolated cerebral arteries and intact microvessels from diverse mammalian animals. The relationships between ethanol-induced contractions in cerebral arteries, intracellular free Ca2+ ([Ca2+]i), tyrosine kinases (including the src family), and mitogen-activated protein kinases (MAPK) were investigated in the present study.
Methods—Cerebral arterial muscle tension and [Ca2+]i were quantified by an isometric contraction technique and direct visualization of Ca2+ in single cells.
Results—Ethanol induces concentration-dependent contractions in intact canine basilar arteries, which are attenuated significantly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein); an src homology 2 (SH2) domain inhibitor peptide; a highly specific antagonist of p38 MAPK (SB-203580); a potent, selective antagonist of MEK1/MEK2 (U0126); and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059). IC50 levels obtained for these 5 antagonists are consistent with reported Ki values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol induces transient and sustained increases in [Ca2+]i in primary single smooth muscle cells from canine basilar arteries, which are markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, and PD-98059. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the ethanol-induced contractions or the elevation of [Ca2+]i.
Conclusions—The present study suggests that activation of protein tyrosine kinases (including the src family) and MAPK appear to play important roles in the ethanol-induced contractions and the elevation of [Ca2+]i in smooth muscle cells from canine basilar arteries. The results could be used to suggest that selective antagonists of protein tyrosine kinases and MAPK may be useful both prophylactically and therapeutically in alcohol-induced strokes.
Key Words: alcohol • basilar artery • calcium • dogs • protein kinases • tyrosine kinases
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Introduction |
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Although it is believed that moderate alcohol intake,1 especially moderate wine intake,2 may exert cardioprotective action, numerous studies over the past 2 decades have affirmed that the risk for developing cerebral vascular diseases, such as stroke, clearly increases with increasing alcohol consumption.3 4 5 Recent observations of the in situ cerebral microcirculation and a variety of isolated mammalian cerebral arteries indicate that acute treatment with ethanol produces prolonged constrictions in cerebral blood vessels,5 6 7 8 suggesting that such vasoconstrictive actions of ethanol are involved in the hypoxic, ischemic, and hemorrhagic actions of alcohol in the brain. Support for the latter concept derives from recent studies of ethanol-induced stroke in intact animals using 31P nuclear magnetic resonance and reflectance spectroscopy to follow in vivo changes in brain bioenergetics, blood flow, and blood volume.9 10 Except for Mg2+ or selective antioxidants, nothing else is known to either ameliorate or prevent experimentally induced alcohol-induced strokes.11 12 13
Multiple signaling pathways may participate in mechanisms of peripheral vasoconstriction. Protein tyrosine kinases have been suggested to be important signal transduction pathways in the regulation of tone and intracellular free Ca2+ ([Ca2+]i) in vascular smooth muscle.14 Activated and autophosphorylated receptor tyrosine kinases recruit src homology 2 (SH2) domain-containing adaptor proteins and play a role in agonist-induced activation of Ras.15 The mitogen-activated protein kinase kinase (MAPKK), or MEK (a cytosolic nonreceptor protein kinase), is in the family of tyrosine kinases.16 Mitogen-activated protein kinases (MAPK), substrates of MAPKK, known as extracellular signal-regulated kinases, serve to relay, amplify, and integrate diverse signals, thus allowing a cell to coordinate a physiological response. Several observations raise the possibility that activation of tyrosine kinase might be involved in ethanol-induced constriction in gastric smooth muscle,17 and ethanol might induce a stimulation of MAPK in aortic smooth muscle.18 However, there is no direct evidence that tyrosine kinases or MAPK are associated with ethanol-induced cerebral vasoconstriction.
The mechanisms underlying ethanol-induced contraction of cerebral vascular smooth muscle and stroke remain less well understood. With these points in mind, we designed our present study to test the hypothesis that the contractile effects of ethanol on cerebral arteries are due, in large measure, to activation of protein tyrosine kinases, recruitment of SH2 domain adaptor proteins, and activation of MAPK and that recruitment of these enzyme pathways collectively results in modulation of [Ca2+]i.
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Materials and Methods |
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General Procedures
Rings of canine basilar arteries were obtained from male mongrel dogs (weight, 18 to 22 kg), after administration of pentobarbital sodium anesthesia (40 mg/kg IV), as described previously,8 and placed in normal Krebs-Ringer bicarbonate solution at pH 7.4 containing the following (in mmol/L): NaCl 118, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 2.5, dextrose 10, and NaHCO3 25.19 The rings were 3 to 4 mm in length. The endothelium was denuded as described previously,8 and the denuded endothelium rings precontracted with prostaglandin F2
(PGF2) failed to relax >10% to maximal
concentrations of substance
P.8 The segments were
mounted on stainless steel pins under 2 g resting tension in
isolated organ baths, attached to force transducers (Grass model FT
03), and connected to Grass model 7 polygraphs. The organ baths,
containing normal Krebs-Ringer bicarbonate solution, were gassed
continuously with 95% O2 and 5%
CO2 and warmed to 37°C (pH 7.4). Tissues were
allowed to equilibrate for at least 90 minutes before data collection.
At the beginning of an experiment, rings were exposed for 30 to 45
minutes to 80 mmol/L KCl, and this was repeated every 30 to 45
minutes, until responses were stable (2 to 3 times). When tissues were
pretreated by various drugs, the drug was applied for at least 15
minutes before the concentration-response curves were obtained. All of
the animal experimental procedures were approved by our institutional
animal care and use committee.
Intracellular
Ca2+ Measurement
Primary smooth muscle cells from canine basilar
arteries for image analysis experiments were seeded on glass
coverslips (12 mm diameter; approximately
1x104 cells per coverslip) and used 2 to 3
days after seeding, as described in the
literature.20
Monolayers of the smooth muscle cells, grown on the coverslips, were
loaded with 2.0 µmol/L fura 2-AM and 0.12% pluronic acid F-127 (60
minutes, 37°C), and the experimental procedures for
[Ca2+]i
measurements were performed as described previously using fura
2-AM.20 The
resulting images were then used to calculate
[Ca2+]i in smooth
muscle cells.
[Ca2+]i was
calculated according to the following
equation21 :
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Drugs
The following pharmacological agents were purchased
from Sigma Chemical Co: daidzein, EGTA, genistein, naloxone HCl,
and propranolol HCl. Atropine sulfate was bought from MANN
Research Laboratory Inc. U0126 was purchased from Promega Co. SB-203580
was bought from Tocris Cookson Inc. Cimetidine HCl and diphenhydramine
HCl were received from Smith Kline & French Laboratories Ltd. Dimethyl
sulfoxide, PD-98059, and an SH2 domain inhibitor peptide
were purchased from CALBIOCHEM Corporation. Phentolamine
methanesulfonate was purchased from CIBA Pharmaceutical Company.
Methysergide maleate was received from Sandoz Pharmaceuticals.
Indomethacin was received as a gift from Merck Inc. All
other organic and inorganic chemicals were obtained from Fisher
Scientific and were of the highest purity.
Calculations and Statistical
Analysis
The contractile response (g), percentage of maximal
KCl-induced contraction, and
[Ca2+]i were
expressed as mean±SEM. Statistical evaluation of the results was
performed by analysis by the Newman-Keuls test and ANOVA with
Scheffé’s contrast test. The results were considered significant at
a value of
P<0.05.
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Results |
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Ethanol-Induced Vasoconstrictions Are Inhibited by a Tyrosine Kinase Antagonist and an SH2 Domain Inhibitor
To our knowledge, 20 mmol/L ethanol should be considered moderate ethanol intake, because it can be found in the blood of most humans after oral ingestion of only 1 to 2 oz of ethanol,6 22 23 and 90 to 200 mmol/L ethanol should be considered heavy to very heavy ethanol intake; 88 mmol/L ethanol is known to be found in the blood of humans with ethanol-induced strokelike episodes.23 Therefore, we used a concentration range of 20 to 200 mmol/L ethanol in the present study. Although not shown, intact versus endothelium-denuded cerebral arterial rings failed to manifest any significant differences in contractile concentration-response curves to ethanol (n=36; P>0.05). Only intact vessel rings were used in the present study. As shown in Figure 1A
, ethanol produces a rapid contractile
response (rapid component) that is followed by a prolonged, slightly
diminished stable increase in vessel tension (stable component) in
intact canine basilar arterial rings. Pretreatment of
intact canine basilar arteries with genistein (an
antagonist of protein tyrosine kinases) or an SH2 domain
inhibitor peptide, but not daidzein, an inactive homologue
of genistein,24 for
15 minutes significantly attenuates ethanol-induced contractions (both
rapid and stable components) in a concentration-dependent manner
(Figure 1A and 1B). The IC50 values
for genistein and an SH2 domain inhibitor for such
inhibition of the contractions are
5.6±0.23x10-5 and
0.73±0.06x10-6 mol/L
(Figure 1B), respectively. Mean values for vasoconstrictions
induced by varying concentrations of ethanol, in the absence (control)
and presence of genistein or an SH2 domain inhibitor, are
shown in
Figure 1C.
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Tyrosine Kinase Antagonist and SH2
Domain Inhibitor Attenuate Ethanol-Induced Elevations
in
[Ca2+]i
Figure 2A demonstrates that ethanol produces a rapid
[Ca2+]i peak,
followed by a steady state
[Ca2+]i plateau in
primary cultured single smooth muscle cells obtained from canine
basilar arteries. Preincubation of primary cultured smooth muscle cells
from intact canine basilar arteries with genistein or an SH2 domain
inhibitor, but not daidzein, for 15 minutes effectively
prevents both the transient elevation in
[Ca2+]i and the
additional sustained rise of
[Ca2+]I induced by
ethanol
(Figure 2A). Lower steady states and a loss of the rapid,
peak increment in
[Ca2+]i are now
seen. Such inhibitory effects of these 2
antagonists display concentration-dependent effects
(Figure 2B). The concentrations producing 50% of the maximal
inhibitory effects (IC50 values) for
genistein and an SH2 domain inhibitor, for such attenuation
of the increases in
[Ca2+]i, are
5.2±0.16x10-5 and
0.68±0.04x10-6 mol/L, respectively
(Figure 2B), which is consistent with the reduced
vasoconstriction induced by ethanol in the presence of these
antagonists under the same conditions. Mean peak
[Ca2+]i values
obtained under different concentrations of ethanol, in the absence and
presence of these antagonists, are shown in
Figure 2C.
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Ethanol-Induced Contractions Are Inhibited by
MAPK and MAPKK Antagonists
Figure 3A and 3B illustrates that the presence of PD-98059
(a selective antagonist of
MAPKK25 ), SB-203580
(a highly specific antagonist of p38
MAPK26 ), or U0126 (a
potent, selective antagonist of
MEK1/MEK227 )
attenuates contractile responses (both rapid and stable components) of
intact canine basilar arteries to ethanol in a concentration-dependent
manner. The calculated IC50 values for PD-98059,
SB-203580, and U0126 for such inhibition of the contractions are
8.1±0.2x10-6,
1.62±0.2x10-6, and
0.58±0.09x10-6 mol/L, respectively
(Figure 3B). Mean values for varying concentrations of
ethanol-induced contractions, in the absence (control) and presence of
PD-98059, SB-203580, or U0126, are shown in
Figure 3C.
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MAPK and MAPKK Antagonists
Attenuate Ethanol-Induced Elevations in
[Ca2+]i
Figure 4A demonstrates that preincubation of the cells with
PD-98059, SB-203580, or U0126 for 15 minutes effectively inhibits both
the transient
[Ca2+]i peak and
the sustained plateau of
[Ca2+]i induced by
ethanol (to lower steady states) in basilar arterial smooth
muscle cells. The inhibitory effects of these 3
antagonists show clear concentration-dependent effects
(Figure 4B). The calculated IC50
values for PD-98059, SB-203580, and U0126 for such attenuation of the
increases in
[Ca2+]i are
7.8±0.3x10-6,
1.53±0.09x10-6, and
0.46±0.08x10-6 mol/L, respectively,
which is in close agreement with the IC50 values
found for the reduced vasoconstriction produced by ethanol in the
presence of these antagonists under the same conditions.
Mean values for varying concentrations of ethanol-induced elevation in
[Ca2+]i, in the
absence (control) and presence of PD-98059, SB-203580, or U0126, are
shown in
Figure 4C.
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Effects of Tyrosine Kinase and MAPK
Antagonists and a SH2 Domain Inhibitor on
Ethanol- and PGF2-Precontracted Canine
Basilar Arterial Segments
After achieving full contractile responses of isolated
intact canine basilar arterial rings to 200 mmol/L
ethanol, we noted that administration of
6.0x10-6 mol/L genistein,
10-6 mol/L SH2 domain
inhibitor, 10-5 mol/L
PD-98059, 2.0x10-6 mol/L SB-203580,
10-6 mol/L U0126, but not
6.0x10-6 mol/L daidzein, led to a
reduction of the ethanol contractions to 60%~70% of the initial
level
(Figures 5A and 6A). The administration of the same
concentrations of genistein, an SH2 domain inhibitor,
PD-98059, SB-203580, and U0126, but not daidzein, also brought about
significant relaxation in PGF2-precontracted
isolated cerebral arteries
(Figures 5B and 6B). However, the effects of these
antagonists on ethanol-precontracted cerebral
arterial segments
(Figures 5A and 6A) are much stronger than those of equipotent
PGF2-precontracted segments
(P<0.05,
Figures 5B and 6B).
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Failure of Several Specific Pharmacological
Antagonists to Attenuate or Interfere With Ethanol-Induced
Vasoconstrictions
Incubation of canine basilar arterial rings
with a variety of pharmacological antagonists (ie,
diphenhydramine [10-6 mol/L], cimetidine
[10-5 mol/L], phentolamine
[10-6 mol/L], methysergide
[10-6 mol/L], propranolol
[10-5 mol/L], atropine
[10-6 mol/L], naloxone
[10-5 mol/L], and
indomethacin [10-5
mol/L]) for 15 minutes, before stimulation with ethanol, failed to
either inhibit or attenuate cerebrovasospasms induced by the alcohol
(n=6 each; data not shown). Likewise, these antagonists
failed to attenuate the rises in
[Ca2+]i produced by
ethanol (data not shown).
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Discussion |
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The present investigation aimed to determine whether activation of tyrosine kinase (including the src family) and MAPK is associated with contractile responses of cerebral arteries to ethanol, thereby giving us some insight into the potential contribution of these 2 cellular signaling pathways to ethanol-induced cerebral vasoconstriction.
Previous studies have demonstrated that tyrosine kinase (including the src family) activation is important in several vasoconstrictor- and epidermal growth factor–induced vascular contractions.14 28 29 30 These studies provide the foundation that, in addition to their mitogenic activities, tyrosine kinase(s) and src itself may play some important roles in agonist-induced smooth muscle contraction. However, little knowledge is currently available concerning the actions of tyrosine kinases in ethanol-induced cerebral vasoconstriction. As the present study has demonstrated, the ability of genistein (an antagonist of protein tyrosine kinase) and an SH2 domain inhibitor peptide to impair ethanol-induced contractions in canine basilar arterial segments implicates the involvement of tyrosine kinase activation (phosphorylation, including src) in the cerebrovascular contractile responses of cerebral smooth muscle to ethanol. We used daidzein, a structurally similar but inactive form of genistein, in the present study as a control agent to test the selectivity of tyrosine kinase antagonists, especially genistein. The calculated IC50 values reported herein for genistein and the SH2 domain inhibitor peptide are in a range similar to the reported Ki values of genistein for protein tyrosine kinase24 and SH2-SH3/phosphoprotein interaction.31 Our present findings are well supported by a recent study in which the tyrosine kinase antagonists, genistein and tyrphostin-47, were reported to inhibit the contractile action of ethanol on guinea pig gastric smooth muscle, although these data were not obtained from cerebral blood vessels.17
The involvement of tyrosine kinase, including the src family, is reinforced by the present findings that genistein and an SH2 domain inhibitor peptide suppress both the ethanol-induced transient and sustained increments in [Ca2+]i in single canine basilar smooth muscle cells at calculated IC50 values, which are in good agreement with those of ethanol-induced arterial contractions under the same conditions and are consistent with previously published Ki values for these 2 antagonists.24 31 These data suggest that contraction of the cerebral arteries to ethanol may be mediated, at least partially, by an elevation in [Ca2+]i in canine basilar arterial smooth muscle cells modulated by activation of tyrosine kinase, including src. This conclusion gains strong support from several lines of recent experimental findings: (1) platelet-derived growth factor BB elicits Ca2+ influx in human cultured vascular smooth muscle cells via a tyrosine kinase–dependent mechanism32 ; (2) genistein can inhibit the activity of L-type Ca2+ channels in vascular smooth muscle cells from rat portal vein33 ; (3) serotonin-evoked Ca2+ release from the sarcoplasmic reticulum in vascular smooth muscle cells is blocked by genistein34 ; and (4) tyrosine phosphorylation by both nonreceptor and receptor tyrosine kinases could be an important mechanism by which voltage-operated channels are regulated in vascular muscle.35 36
Several recent studies indicate that MAPK plays important roles in diverse vasoconstrictor-induced contraction in rat cerebral arteries, rat aorta, and guinea pig gastric longitudinal smooth muscle.28 30 37 These previous findings implicate the MAPK pathway in modulation of vascular smooth muscle contractility and, as a tyrosine kinase, MAPKK might be a logical candidate to be activated by ethanol stimulation. An important observation presented herein is that PD-98059 (a specific MAPKK antagonist25 ), SB-203580 (a highly specific antagonist of p38 MAPK26 ), and U0126 (a potent and selective antagonist of MEK1/MEK227 ) produce significant concentration-dependent attenuation of ethanol-induced contractions in intact canine basilar arteries. The calculated IC50 values for PD-98059, SB-203580, and U1026 are consistent with the reported Ki values for these 3 antagonists.25 26 27 These results suggest that activations of both MAPKK and MAPK pathways in cerebral arterial smooth muscle cells play important roles in these ethanol-induced contractile responses. This concept derives strong support from a very recent study that demonstrates that concentrations of ethanol (17 to 170 mmol/L) similar to those used in the present study could induce a dose-dependent stimulation of p44/p42 MAPKs in rat aortic smooth muscle cells.18
It has been shown previously that MAPK is involved in angiotensin and low [Mg2+]0-induced contraction and elevation of [Ca2+]0-stimulated [Ca2+]i increases in rat cerebral and aortic smooth muscle cells.16 37 38 39 This may be a major pathway by which ethanol leads to increases of [Ca2+]i in smooth muscle cells from canine basilar arteries and activates MAPKK and MAPK in the smooth muscle cells. In this context, our present findings indicate that PD-98059, SB-203580, and U0126 significantly inhibit the ethanol-induced concomitant rise in [Ca2+]i in single cells from canine basilar smooth muscle at calculated IC50 values, which are in close agreement with those of the ethanol-induced arterial contractions under the same conditions and are consistent with previously reported Ki values for these 3 antagonists.25 26 27 These results could thus be used to support the aforementioned contention that MAPKK and MAPK are indeed important in ethanol-evoked contractile responses.
It was noted that the antagonists of tyrosine
kinase and MAPK used herein produced relaxant effects (albeit much
smaller effects compared with those against ethanol) on
PGF2-precontracted isolated canine cerebral
arteries. In our opinion, this does not indicate that the signaling
pathways activated by ethanol are not specific.
PGF2 may contract, in part, the basilar
arterial segments by activating tyrosine kinases and MAPK.
Similar results have been reported by other investigators for
PGF2 and other vasoactive substances in other
type of smooth
muscle.30 40 41
A variety of specific pharmacological antagonists of known endogenously formed vasoconstrictors did not inhibit or attenuate the ethanol contractions and elevation of [Ca2+]i, which suggests no involvement or release of endogenous vasoconstrictors (ie, histamine, catecholamines, serotonin, acetylcholine, opiates, or prostanoids) in such ethanol-induced contractile actions.
In summary, several points are noteworthy regarding the potential physiological and clinical significance of our present study. First, the ethanol-induced contractions in canine cerebral arterial segments can be significantly attenuated by tyrosine kinase and MAPK antagonists. Second, concomitantly, the increase in [Ca2+]i in single cells from canine cerebral arterial smooth muscle induced by ethanol can be suppressed by the aforementioned antagonists, also in concentrations associated with their specific inhibitory actions on their target enzymes. Overall, these results support the importance of tyrosine kinases, including the src family and MAPK pathways, to ethanol-associated cerebral vascular contraction. The present studies thus may help to shed new light on the etiologies of cerebral ischemia, cerebral vasoconstriction, and diverse cerebrovascular/stroke disease states associated with ethanol. Although ethanol-induced cerebral vasoconstriction is clearly a complicated event, the approach taken in this study may prove useful in development of prophylactic and therapeutic tools for prevention and amelioration of alcohol-induced stroke and hypertension.
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Acknowledgments |
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The authors are grateful to the National Institutes of Health (grant AA-08674 to Dr Burton M. Altura), whose support was helpful in performing these studies.
Received July 3, 2000; revision received August 30, 2000; accepted August 30, 2000.
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Editorial Comment
Role of Tyrosine and Mitogen-Activated Protein Kinases
Department of Anesthesiology/Critical Care Medicine, Johns Hopkins Medical Institutions, Johns Hopkins Hospital, Baltimore, Maryland
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Alcohol drinking and abstinence are both part of our social culture. The epidemiological relationships between alcohol drinking and stroke appear to be "U-shaped" and are a frequent source of controversy in both popular press and medical/scientific literature. Light-to-moderate alcohol consumption was recently reported to reduce overall stroke risk and risk of ischemic stroke in male physicians,R1 stimulating ongoing interest and cautionsR2 about alcohol’s potential health benefits. Notably, moderate alcohol consumption (frequently defined as 2 drinks per day) decreases ischemic stroke risk in elderly, multiethnic subjects of both sexes after adjustment for cardiac disease, hypertension, smoking, and diabetes.R3 Ethanol’s dose-restrictive, antioxidant activity may be important in these results (for review, see HillbomR4 ). Whether ethanol ingestion increases or decreases overall stroke or stroke subtype risk appears to be dependent not only on oral dose and formulation but also on drinking pattern (regular versus binge drinking) and the presence of alcohol-induced hypertension and proembolic changes, including thromboxane-mediated platelet activation.R5 Nevertheless, numerous studies indicate that heavy recent alcohol drinking is an independent risk factor for all major subtypes of stroke. In animals, the majority of work agrees that acute alcohol induces cerebral arterial constriction. It is to these observations that the present study of Yang et al offers pathophysiological insight and new information about the signaling pathways of ethanol in intact canine basilar artery and primary vascular smooth muscle cells. These data carefully characterize dose-response relationships within ethanol concentrations that would be expected to be pathological, not protective, in human stroke. Ethanol-induced vasoconstrictor mechanisms are not limited to tyrosine kinase and mitogen-activated protein kinase in large cerebral vessels (for review, see Altura and AlturaR6 ). Future work will be tasked with determining cross-talk among controllers of calcium cycling in the cerebrovascular contractile apparatus.
Received July 3, 2000; revision received August 30, 2000; accepted August 30, 2000.
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1. Berger K, Ajani UA, Kase CS, Gaziano M, Buring JE, Glynn RJ, Hennekens CH. Light-to-moderate alcohol consumption and the risk of stroke among U.S. male physicians. N Engl J Med.. 1999;341:1557-1564.
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