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Hypertension. 2004;43:1086-1091
Published online before print March 29, 2004, doi: 10.1161/01.HYP.0000125995.85427.fd
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(Hypertension. 2004;43:1086.)
© 2004 American Heart Association, Inc.

Scientific Contributions

p38 Mitogen-Activated Protein Kinase Contributes to the Diminished Aortic Contraction by Endothelin-1 in DOCA-Salt Hypertensive Rats

Bokyung Kim; Junghwan Kim; Young M. Bae; Sung I. Cho; Seong C. Kwon; Jin Y Jung; Jung -C. Park; Hee Y. Ahn

From the Department of Physiology (B.K., J.K., Y.M.B., S.I.C.), College of Medicine, Konkuk University, Choongju, Korea; Department of Physiology (S.C.K.), College of Medicine, Kwandong University, Kangneung, Korea; Department of Pharmacology (J. Y. J., J.C.P., H.Y.A.), College of Medicine, Chungbuk National University, Cheongju, Korea.

Correspondence to Dr Hee Y. Ahn, Department of Pharmacology, College of Medicine, Chungbuk National University, Cheongju 361-763, Korea. E-mail hyahn{at}chungbuk.ac.kr


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
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We investigated whether the diminished contractile responsiveness to endothelin-1 (ET-1) is associated with the altered activation of mitogen-activated protein kinase (MAPK) in aortic smooth muscles from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. ET-1 dose-dependently increased contractions in aortic smooth muscle strips, and the contractions were significantly attenuated in tissues from DOCA-salt hypertensive rats compared with those from sham-operated rats. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was elevated by ET-1, with the magnitude and time-course being similar between strips. Although ET-1 also increased the phosphorylation of p38 MAPK in both strips, the increment was markedly lower in the strips from DOCA-salt hypertensive rats compared with sham-operated controls. 5-Hydroxytryptamine (5-HT) increased vascular contraction and phosphorylation of both MAPK isoforms; these were greater in DOCA-salt hypertensive rats than in sham-operated rats. ET-1 also increased the phosphorylation of caldesmon, an actin-binding protein, in sham-operated and DOCA-salt hypertensive rats. However, the increment was markedly lower in the strips from DOCA-salt hypertensive rats compared with sham-operated controls. The phosphorylation of MAPK isoforms and caldesmon elevated by ET-1 was inhibited by PD098059, an inhibitor of ERK1/2 kinase, and SB203580, an inhibitor of p38 MAPK, respectively. These results suggest that ET-1 and 5-HT induce contraction by activating the MAPK pathway in rat aortic smooth muscle and that the diminished responsiveness to ET-1 in the DOCA-salt hypertensive rat may be, in part, mediated by the decrease of caldesmon phosphorylation after the decreased activation of p38 MAPK.


Key Words: endothelin • hypertension • vasoconstriction • deoxycorticosterone • protein kinases


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
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Endothelin-1 (ET-1) is an important vasoconstrictor that contributes to vascular disorders, including hypertension, vascular remodeling, and coronary artery disease.1,2 ET-1 induces vascular smooth muscle contraction. It is widely accepted that smooth muscle contraction is triggered by intracellular Ca2+ ([Ca2+]i) released from intracellular Ca2+ stores and from the extracellular space. The increased [Ca2+]i can phosphorylate the 20-kDa myosin light chain (MLC) by activating MLC kinase, and this initiates smooth muscle contraction.3,4 Previous reports have shown that ET-1 induces a sustained contraction, which results from the increase of [Ca2+]i in isolated vascular smooth muscle.5,6 In addition to the [Ca2+]i-MLC kinase pathway, a number of intracellular signal molecules, including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), phosphatidylinositol 3 kinase (PI3K), and Rho kinase, play important roles in the regulation of smooth muscle contraction.4,7–10 ET-1 can also stimulate these kinases.

MAPK is a family of serine/threonine-specific protein kinase, consisting of three isoforms: extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK).11,12 MAPK plays a central role in intracellular signal transduction initiated by extracellular stimuli, including growth factors, neurotransmitters, and hormones.13,14 ERK1/2 is activated by receptor agonists, including angiotensin II and phenylephrine, which induce smooth muscle contraction.15,16 ET-1 also increases the activity of ERK1/2 in vascular smooth muscle.17–19 There is accumulating evidence that the MAPK pathway is closely linked to modulating the intensity of contraction in vascular smooth muscle.15,20–23 Moreover, the inhibition of p38 MAPK diminishes contractility in smooth muscle cells, indicating that p38 MAPK, as well as ERK1/2, can contribute to the elevation of contraction. In DOCA-salt hypertensive rats, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure.24 The contractile responses of vascular smooth muscle to vasoconstrictors, including 5-hydroxytryptamine (5-HT) and norepinephrine, are significantly increased in hypertensive animals.16,24–26 In contrast, the contractile response to ET-1 is significantly diminished in the DOCA-salt hypertensive rat compared with the normotensive rat.27,28 Furthermore, the lower density of ET-A receptor on vascular smooth muscle has been reported in DOCA-salt hypertensive rats.27 In addition, the increment of [Ca2+]i induced by ET-1 also decreased in vascular smooth muscle from DOCA-salt hypertensive rats.28,29 Although the changes in the receptor number and intracellular Ca2+ levels have been reported, the clear reason for this diminution in response to ET-1 in DOCA-salt hypertensive rats has not been elucidated.

In the present study, because the MAPK pathway is an important mediator of vascular contraction, we hypothesized that this pathway regulates the diminished contractile response to ET-1 during DOCA-salt hypertension. To test this hypothesis, we examined the roles of MAPK isoforms and the decreased responsiveness to ET-1 in contraction between sham-operated and DOCA-salt hypertensive rats.


*    Methods
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 up arrowIntroduction
 *Methods
down arrowResults
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Animals
All experiments were performed in accordance with the institutional guidelines of Konkuk University, Korea. Male Sprague-Dawley rats (190 to 200 g; Daehan Biolink, Korea) were uninephrectomized, and, after 1 week, received a silicon rubber implant impregnated with DOCA (200 mg kg–1) subcutaneously under intramuscular anesthesia (35 mg kg–1 ketamine/5 mg kg–1 xylazine). DOCA-salt hypertensive rats received 0.9% NaCl plus 0.2% KCl drinking water solution. A control group (sham-operated rat) was also uninephrectomized and operated without receiving an implant. Sham-operated rats received normal tap water. All animals were fed standard laboratory rat chow and had ad libitum access to both food and water. Systolic blood pressure was directly determined using a pressure transducer (Statham P23XL; Viggo Spectramed) at the common carotid artery under ketamine/xylazine anesthesia. At 4 weeks after the silicon rubber implantation, the blood pressure was significantly higher in DOCA-salt hypertensive rats (187±7 mm Hg, n=25) than in sham-operated rats (119±2 mm Hg, n=27).

Measurement of Isometric Contraction
Rats were euthanized by stunning and bled; the thoracic aorta was removed, cut into strips (3x8 mm), and the endothelium was removed. Isometric muscle contraction was recorded, as described previously.30

Measurement of Protein Phosphorylation
Aortic strips were isolated and snap-frozen in liquid N2 after treatment with various stimulants for different times. The samples were then homogenized in sample buffer containing 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L EGTA, 20 mmol/L ß-glycerophosphate, 1 mmol/L NaF, 2 mmol/L Na3VO4, 5 µg/mL aprotinin, 5 µmol/L leupeptin, 1% Triton-X 100, 10% glycerol, 300 µmol/L phenylmethylsulfonyl fluoride, 5 mmol/L dithiothreitol, and 150 mmol/L NaCl. The homogenate was centrifuged at 14 000g for 10 minutes at 4°C, and the supernatant was collected.15,30–32 Proteins separated with SDS-PAGE were transferred to polyvinylidene fluoride membranes (Millipore). These were incubated with phosphate-buffered saline containing 0.1% Tween 20 and 5% nonfat dried milk for 60 minutes, and then incubated with individual rabbit antiphosphorylated MAPK and antiphosphorylated caldesmon antibodies diluted 1:1000 to 1:5000 overnight at 4°C. In some experiments, the membrane was stripped and then reprobed with nonphosphorylated antibodies for detection of total protein expression. After incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1:1000) for 60 minutes, the blots were developed using an enhanced chemiluminescence detection system (Amersham). Antibody-specific bands were quantified using an image analyzer (BioRad).

Materials
Polyclonal antiphosphorylated and nonphosphorylated ERK1/2 antibodies, Triton-X 100, and dithiothreitol were purchased from Promega. Polyclonal antiphosphorylated and nonphosphorylated p38 MAPK antibodies were purchased from Cell Signaling. Polyclonal antiphosphorylated and nonphosphorylated caldesmon was purchased from Upstate. DOCA, ET-1, 5-HT, NaF, ß-glycerophosphate, Na3VO4, phenylmethylsulfonyl fluoride, leupeptin, and aprotinin were purchased from Sigma. PD098059 and SB203580 were purchased from Tocris.

Data Analysis
The results of experiments are expressed as means±SEMs. Unpaired Student t tests were used to compare the data, and P<0.05 was considered significantly different.


*    Results
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 *Results
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ET-1 induced sustained contractions in dose-dependent manners in both sham-operated and DOCA-salt hypertensive rats (Figure 1A). The response to ET-1 was significantly decreased in the experimental rats compared with sham-operated controls. 5-HT dose-dependently increased muscle contraction in both strips, and the contractions were significantly greater in DOCA-salt hypertensive rats than in sham-operated rats (Figure 1B). The results from mechanical study are consistent with earlier studies.24,27,28



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  		Figure 1. Contractile responses to ET-1 and 5-HT in aortic smooth muscle strips from sham-operated and DOCA-salt hypertensive rats. The muscle strips from sham-operated and DOCA-salt hypertensive rats were stimulated repeatedly with 70 mmol/L K+. After the response to high K+ was determined, ET-1 (0.1 to 100 nmol/L) or 5-HT (0.1 nmol/L to 100 µmol/L) was applied cumulatively. Dose–response curves of the contraction induced by ET-1 (A) and 5-HT (B). The contractile levels of 70 mmol/L K+ before the treatment of vasoconstrictors were defined as 100%. Each point represents the mean±SEM of 6 to 14 experiments. *Significant difference from the results of strips from sham-operated rat (P<0.05).

To determine whether MAPK influences receptor agonist-induced responses, the activity of MAPK was measured using phosphorylated MAPK antibodies in aortic smooth muscles from sham-operated and DOCA-salt hypertensive rats. In the quiescent state without any stimulant, the phosphorylation of ERK1/2 was significantly greater in DOCA-salt hypertensive rat (182.0±19.3% of sham-operated rats, n=5). ET-1 (30 nmol/L) increased the phosphorylation of ERK1/2 in a time-dependent manner in both strips, and reached a maximum at 15 minutes (Figure 2A and B). The magnitudes and time-courses of phosphorylation of ERK1/2 elevated by ET-1 did not differ between strips. The treatment of muscle strips with 5-HT (10 µmol/L) increased the phosphorylation of ERK1/2 in both strips (Figure 2C). The increase in ERK1/2 phosphorylation by 5-HT was significantly greater in DOCA-salt hypertensive rats than in sham-operated rats (Figure 2D). In the Western blot analysis using a nonphosphorylated ERK1/2 antibody, the total expression of the kinase was not changed between both strips (Figure 2A and C). In the quiescent state, the phosphorylation of p38 MAPK was significantly greater in DOCA-salt hypertensive rats (171.0%±25.5% of sham-operated rats, n=5). ET-1 (30 nmol/L) also increased the phosphorylation of p38 MAPK in both strips, and maximal phosphorylation of the kinase were recorded at 15 minutes (Figure 3A and B). The response to ET-1 was significantly attenuated in strips from DOCA-salt hypertensive rats compared with sham-operated controls. The treatment of muscle strips with 5-HT (10 µmol/L) increased the phosphorylation of p38 MAPK in aortic strips, which is significantly increased in DOCA-salt hypertensive rats than in sham-operated rats (Figure 3C and D). The total expression of p38 MAPK, determined using a nonphosphorylated p38 MAPK antibody, was not different between strips (Figure 3A and C). These results suggest that ET-1 and 5-HT induce contraction resulting from the activation of p38 MAPK, as well as ERK1/2, and the diminished responsiveness to ET-1 in DOCA-salt hypertensive rat may result from the lowered activation caused by p38 MAPK, but not by ERK1/2.



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  		Figure 2. Effects of ET-1 and 5-HT on ERK1/2 phosphorylation in aortic smooth muscles from sham-operated and DOCA-salt hypertensive rats. The strips treated with ET-1 (30 nmol/L) or 5-HT (10 µmol/L) were prepared as described in Methods. A and C, The total expressions of ERK1/2 in the quiescent and vasoconstrictor-stimulated strips were measured using a nonphosphorylated ERK1/2 antibody (upper panels). The phosphorylation of ERK1/2 was demonstrated using a phosphorylated ERK1/2 antibody in both strips (lower panels). B and D, Statistical results of levels of phosphorylated ERK1/2 in both strips were obtained from 4 to 5 independent experiments. The level of phosphorylated ERK1/2 in the quiescent state was defined as 100%. *Significant difference from the results of strips from sham-operated rat (P<0.05).



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  		Figure 3. Effects of ET-1 and 5-HT on p38 MAPK phosphorylation in aortic smooth muscle from sham-operated and DOCA-salt hypertensive rats. The strips treated with ET-1 (30 nmol/L) or 5-HT (10 µmol/L) were prepared as described in Methods. A and C, The total expressions of p38 MAPK in the quiescent and vasoconstrictor-stimulated strips were measured using a nonphosphorylated p38 MAPK antibody (upper panels). The phosphorylation of p38 MAPK was demonstrated using a phosphorylated p38 MAPK antibody in both strips (lower panels). B and D, Statistical results of levels of phosphorylated p38 MAPK in both strips were obtained from 4 to 5 independent experiments. The level of phosphorylated p38 MAPK in the quiescent state was defined as 100%. *Significant difference from results of muscles from sham-operated rats (P<0.05).

PD098059 (50 µmol/L), an inhibitor of ERK1/2 kinase, partially attenuated the sustained contraction induced by ET-1 (30 nmol/L) in sham-operated (64.0%±1.2% of ET-1-induced contraction, n=4) and DOCA-salt hypertensive rats (33.6%±11.6% of ET-1-induced contraction, n=4: Figure 4A). SB203580 (50 µmol/L), an inhibitor of p38 MAPK, strongly attenuated ET-1–induced contraction to 13.7%±4.8% (n=5) and 5%±1.3% (n=5) in sham-operated and DOCA-salt hypertensive rats, respectively (Figure 4B).



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  		Figure 4. Effects of inhibitors of MAPK on contractile response and MAPK phosphorylation elevated by ET-1 in aortic smooth muscles from sham-operated and DOCA-salt hypertensive rats. The strips were stimulated with ET-1 30 nmol/L for 15 minutes and then treated with PD098059 and SB203580 for 15 minutes, respectively. The phosphorylation of ERK1/2 (C) and p38 MAPK (D) were detected using phosphorylated MAPK antibodies as described in Figure 2 and 3Up. The levels of contractility (A and B) and MAPK phosphorylations (C and D) before treatment with inhibitors were defined as 100%. Each result represents the mean±SEM of 4 to 6 experiments. *Significant difference from the ET-1–stimulated strips (P<0.05).

ET-1 (30 nmol/L) elicited ERK1/2 phosphorylation in strips from sham-operated (343.1%±26.2% of the resting state, n=6) and DOCA-salt hypertensive rats (282.0%± 29.3% of resting state, n=6), and these responses were abolished by 10 µmol/L PD098059 in sham-operated (115.2%±19.7% of resting states, n=6) and DOCA-salt hypertensive rats (110.3%±21.8% of resting states, n=6: Figure 4C). ET-1 (30 nmol/L) also increased p38 MAPK phosphorylation in strips from sham-operated (415.6%±52.1% of resting state, n=5) and DOCA-salt hypertensive rats (207.0%±28.7% of resting state, n=5). These responses were also abolished by 10 µmol/L SB203580 in sham-operated (123.6%±14.3% of resting states, n=5) and DOCA-salt hypertensive rats (103.7%±11.8% of resting states, n=5: Figure 4D).

To evaluate whether the activation of the MAPK pathway regulates caldesmon, an actin-binding protein, we measured the phosphorylation of caldesmon using a Ser789 phosphorylated h-caldesmon antibody. In the quiescent state without any stimulant, the phosphorylation of caldesmon did not differ between sham-operated and DOCA-salt hypertensive rats (120.2%±14.4% of sham-operated control, n=6). ET-1 (30 nmol/L) increased the phosphorylation of caldesmon, which was significantly greater in sham-operated controls (235.8%±24.4% of resting state, n=6) than in DOCA-salt hypertensive rats (167.5%±14% of resting state, n=6). The increment of caldesmon phosphorylation was inhibited by 60-minute pretreatment of 10 µmol/L PD098059 in sham-operated (152.7%±17.3%, n=6) and DOCA-salt hypertensive rats (98.8%±8.8%, n=6, Figure 5). The 60-minute pretreatment with 10 µmol/L SB203580 also decreased the level of caldesmon phosphorylation by 30 nmol/L ET-1 in sham-operated (170.8%±13.8%, n=6) and DOCA-salt hypertensive rats (89.3%±7.8%, n=6, Figure 5).



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  		Figure 5. Effects of ET-1 on the phosphorylation of caldesmon in aortic smooth muscles from sham-operated and DOCA-salt hypertensive rats. The strips were pretreated with PD098059 (10 µmol/L) and SB203580 (10 µmol/L), respectively, for 60 minutes, and then treated with ET-1 30 nmol/L for 30 minutes. The total expressions of caldesmon were measured using a nonphosphorylated caldesmon antibody in both strips. The phosphorylation of caldesmon was detected using a Ser789 phosphorylated h-caldesmon antibody in both strips. The levels of caldesmon phosphorylation in resting states were defined as 100%. Each result represents the mean±SEM of 6 experiments. *Significant difference from the ET-1-stimulated strips (P<0.05).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we found that ET-1 activates MAPK pathways, ERK1/2 and p38 MAPK, resulting in increased contraction in aortic smooth muscles. The contractile responses and p38 MAPK phosphorylation by ET-1 were significantly attenuated in strips from DOCA-salt hypertensive rats compared with sham-operated controls. Moreover, treatment of aortic strips with inhibitors of MAPK abolished the contraction and the phosphorylation of MAPK, as reported elsewhere.16,18,19,33,34 Although there was no difference in the expression and the kinetic of ERK1/2 between both strips, p38 MAPK phosphorylation by ET-1 was significantly attenuated in strips from DOCA-salt hypertensive rats. In contrast to the results with ET-1, the previous report24 and the present study showed that contractile responses and MAPK, both ERK1/2 and p38 MAPK, phosphorylation induced by 5-HT were significantly increased in strips from DOCA-salt hypertensive rats compared with those from sham-operated rats. These results indicate that ET-1 and 5-HT evoke a sustained contraction resulting from the activation of p38 MAPK, as well as ERK1/2, in rat aortic smooth muscle. Furthermore, the diminution of ET-1–induced contraction in aortic strips from DOCA-salt hypertensive rats may be mediated, at least in part, by the reduced activation of p38 MAPK, but not that of ERK1/2. The p38 MAPK inhibitor, SB203580, completely inhibited both p38 MAPK phosphorylation and contraction by ET-1 in sham-operated and DOCA-salt hypertensive rats. Although PD098059, an inhibitor of ERK1/2 kinase, abolished ERK phosphorylation by ET-1, the contraction in response to ET-1 was not completely abolished by PD098059 in sham-operated and DOCA-salt hypertensive rats. These findings show that p38 MAPK activation is more important than that of ERK1/2 in ET-1–induced contraction of sham-operated and DOCA-salt hypertensive rats. Although previous reports showed that the ET-1–increased MAPK activity is slightly elevated in DOCA-salt hypertensive rats,18,35 the role of p38 MAPK has not been demonstrated in vascular smooth muscle. To our knowledge, this is the first study to show that altered activation of the p38 MAPK pathway mediates the diminished ET-1–induced contraction in muscle from DOCA-salt hypertensive rats.

DOCA-salt hypertensive rats show decreased density of ET-A receptors.27,28 The increment of [Ca2+]i to ET-1 was reduced in DOCA-salt hypertensive rats.28 In addition, ERK activity elevated by ET-1 was partially inhibited in the absence of extracellular Ca2+.36,37 Moreover, the activity of p38 MAPK, as well as ERK1/2, can be regulated by both Ca2+-dependent and Ca2+-independent mechanisms in vascular smooth muscle.17,38,39 Therefore, it can be assumed that the lowered activity of p38 MAPK cannot be associated with the decrease of [Ca2+]i, which may be mediated by decreased density of ET-A receptor, in DOCA-salt hypertensive rats. Furthermore, in the present study, despite alteration of the phosphorylation of p38 MAPK in DOCA-salt hypertensive rats, that of ERK1/2 by ET-1 did not differ between both strips, implying that the possibility of coupling between changed receptor properties and MAPK activity during DOCA-salt hypertension can be excluded. These results show that reduced p38 MAPK activity, as well as impaired [Ca2+]i,28 may be an alternative factor, which contributes to the lowered contractility to ET-1 in DOCA-salt hypertensive rats.

In the present study, the levels of MAPK in the quiescent state were significantly increased in the muscles of DOCA-salt hypertensive rats, which is consistent with previous reports on aortic smooth muscle from spontaneous and DOCA-salt hypertensive rats.18 Several vasoconstrictors, including 5-HT and norepinephrine, increased the contractility and the activity of MAPK in vascular smooth muscle, and the magnitude was greater in DOCA-salt hypertensive rats compared with normotensive rats.16,17,24–26 Furthermore, the application of MAPK inhibitor attenuated the systolic blood pressure in hypertensive rats.40 From these results, it can be assumed that the increased activity of MAPK may contribute to the elevation of blood pressure in DOCA-salt hypertensive rats. In contrast, the diminished responsiveness to ET-1 may act as a compensatory mechanism to the increased vascular resistance and blood pressure produced during DOCA-salt hypertension.

Previous studies have demonstrated that the activation of ERK1/2 participates in smooth muscle contraction by inhibiting the hindrance of caldesmon, an actin-binding protein, onto crossbridges.15,31 Although the total expression of h-caldesmon did not differ between sham-operated and DOCA-salt hypertensive rats, ET-1 increased the phosphorylation of h-caldesmon in both strips. The inhibitors of ERK1/2 and p38 MAPK inhibited the phosphorylation of caldesmon in response to ET-1, respectively. Moreover, the increment of h-caldesmon phosphorylation by ET-1 was significantly attenuated in strips from DOCA-salt hypertensive rats compared with sham-operated controls. These results indicate that p38 MAPK, as well as ERK1/2, increases the phosphorylation of caldesmon,41 and that the reduced activation of p38 MAPK to ET-1 contributes to the diminished caldesmon phosphorylation, which causes the diminution of the contraction in DOCA-salt hypertensive rats. Our current findings strongly support the hypothesis that part of the diminished responsiveness to ET-1 in DOCA-salt hypertensive rats may be caused by the decreased activation of p38 MAPK, but not caused by ERK1/2.

In summary, findings of the present study demonstrate that the MAPK pathway plays a central role in the contractile signaling initiated by ET-1 and 5-HT in vascular smooth muscle. The diminution of p38 MAPK activity contributes to the decreased contraction to ET-1 in the DOCA-salt hypertensive rats, and this downregulation may act a compensatory mechanism to the increased vascular resistance and blood pressure in DOCA-salt hypertensive rats. These studies suggest the roles of MAPK on the mineralocorticoid hypertension.


*    Acknowledgments
 
This work was supported by grant 1999-1-214-001-3 from the Korea Science & Engineering Foundation, and by grants from the Bio-Food & Drug Research Center at Konkuk University and the Research Centres for Bioresource & Health at Chungbuk National University, Korea.


*    Footnotes
 
The first 2 authors contributed equally to this work.

Received October 2, 2003; first decision October 31, 2003; accepted March 4, 2003.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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