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Nonspecific Inhibition of Myogenic Tone by PD98059, a MEK1 Inhibitor, in Rat Middle Cerebral Arteries
Biochemical and Biophysical Research Communications 257, 523–527 (1999) Article ID bbrc.1999.0350, available online at http://www.idealibrary.com on
Nonspecific Inhibition of Myogenic Tone by PD98059, a MEK1 Inhibitor, in Rat Middle Cerebral Arteries Guy. J. L. Lagaud, Eugene Lam, Amy Lui, Cornelis van Breemen, and Ismail Laher 1 Vancouver Vascular Biology Research Centre (VVBRC), and Department of Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada
Received February 1, 1999
Activation of MAP kinase kinase, also called ERK kinase (MEK), may lead to desinhibition of thin filament regulatory proteins and we therefore investigated the acute effects of the potent MEK inhibitor, PD98059 on the contractile properties of pressurized rat middle cerebral arteries. Cerebral arteries (diameter 100-150 mm) were mounted on a pressure myograph and PD98059 (10 mM, 40 mM) significantly inhibited (15% and 64%) myogenic tone (P < 0.001). At these concentrations, PD98059 also significantly reduced the vasopressin (0.1 mM)- and KCl (60 mM)-induced tone. Cumulative addition of exogenous Ca 21 (0.4-1.6 mM) increased myogenic tone to ;50% of constriction at 80 mmHg. This effect was inhibited by PD98059 (P < 0.001). These results demonstrate that pressure-induced myogenic tone is inhibited by PD98059 at the concentrations that have been reported to be selective for inhibition of MEK and the MAP kinase cascade. However, our results also demonstrate that PD98059 may have nonspecific effects on voltage-sensitive Ca 21 entry in vascular smooth muscle. © 1999 Academic Press
Arteries from the cerebral, coronary and renal circulations constrict to increases, and dilate to decreases, in transmural pressure either in situ or in vitro. A major component of this mechanism, which facilitates blood flow autoregulation, appears to be myogenic in nature, by definition, a direct response of vascular smooth muscle to pressure or stretch (1). However, the precise cellular signaling cascade that controls vascular myogenic tone has not been fully elucidated. Extra1
Author for correspondence: Dr. Ismail Laher, Department of Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, B.C. V6T 1Z3 Canada. Fax: (604) 822-2281 E-mail: [email protected]. Abbreviations: AVP, arginine vasopressin; Ca 21, calcium; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase, MEK, MAPK or ERK kinase; PD98059, [2-(29-amino-39methoxyphenyl)-oxanaphthalen-4-one]; PKC, protein kinase C.
cellular Ca 21 is obligatory for basal tone in pressurized resistance arteries (2). The increase in intracellular calcium concentration [Ca 21] i activates myosin light chain kinase to phosphorylate myosin light chain and in addition protein kinase C (PKC) to enhance myofilament Ca 21 sensitivity (3, 4). Furthermore, a role for mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) has recently been suggested in mediating signal transduction from stretch to phosphorylation of caldesmon, a thin filament-associated protein, in conduit arteries (5, 6, 7). To our knowledge, there is no published report on the involvement of the MAP kinase in the development of myogenic tone. Since activation of MAP kinase kinase also called ERK kinase (MEK) may lead to disinhibition of thin filament regulatory proteins (e.g. caldesmon), we have investigated the effect of MEK inhibition of the contractile properties of pressurized rat middle cerebral arteries. To this end we used PD98059, a putative selective inhibitor of MEK and the MAP kinase cascade that has recently been characterised (8, 9, 10, 11). METHODS Vessel isolation and cannulation. Male Sprague-Dawley rats (200-300 g) were anesthetized with sodium pentobarbital (30 mg.kg 21, i.p.) containing heparin (500 U/kg), and then killed by decapitation. The brain was removed and placed in physiological salt solution (PSS) at 4°C. Second order middle cerebral arteries (inner diameter 125 6 5.03 mm, range 100-150 mm; n 5 19) were dissected and transferred to an arteriograph filled with oxygenated PSS at 37°C. Vessels were tied onto microcannulae (tip diameter 60-80 mm). Under no flow conditions, the intraluminal pressure was increased to 60 mm Hg using a pressure servo system (Living Systems, Burlington, VT), and the vessel was equilibrated for 60 min whereupon the vessel spontaneously and reliably developed myogenic tone (reduced luminal diameter). A detailed description of the method is given elsewhere (4). Experimental procedure. After equilibration at 80 mmHg, PD98059 (10 and 40 mM) was added to the PSS in order to determine its effect on myogenic tone (n 5 6). In other experiments, transmural pressure was lowered to 20 mmHg, a maneuver that places the vessel below the lower limit of the pressure range for myogenic tone.
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0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 257, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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FIG. 3. (A) Representative traces of KCl (60 mM)-induced contractions of rat middle cerebral arteries in the absence and presence of PD98059 (40 mM). (B) The mean 6 S.E.M (vertical bars) of 6 experiments of KCl-induced tone in the absence and presence of PD98059 (10 mM, 40 mM). *P , 0.05; ***P , 0.001 significantly different as compared to KCl response in the absence of PD98059. ###P , 0.001 significantly different from KCl response in the presence of PD98059 (10 mM).
Luminal diameter was allowed to stabilize for 15-20 min before arginine vasopressin (AVP, 0.1 mM) or potassium (K 1, 60 mM) were added, either in the absence or presence of PD98059. For studies of Ca 21-induced myogenic tone, cerebral arteries were equilibrated at 80 mmHg and the bathing PSS was replaced with Ca 21 free medium for 20 min. Cumulative additions of Ca 21 (0.4-1.6 mM; n 5 5) were made in the absence and presence of PD98059. Expression of the results and statistical analysis. Myogenic tone at each pressure was expressed as a percent constriction 5 100% x [(D Ca-free 2 D PSS)/D Ca-free], where D is the diameter in calcium free or PSS. All results are expressed as mean 6 S.E.M of n animals. Statistical evaluation was made using ANOVA, and means were considered significantly different when P , 0.05.
Solutions and chemicals. The composition of the PSS was (in mM): NaCl 119, KCl 4.7, KH 2PO 4 1.18, NaHCO 3 24, MgSO 4-7H 2O 1.17, CaCl 2 1.6, glucose 5.5 and EDTA 0.026. Ca 21 free solution was a PSS solution containing no CaCl 2. 60 mM K 1 solution was a PSS solution in which Na 1 was substituted for an equimolar concentration of KCl. PD98059 was purchased from BIOMOL (PA, USA); AVP was obtained from Sigma Chemical Company (St Louis, MO).
RESULTS Myogenic tone developed during equilibration at 80 mmHg, reducing diameter by an average of 52 6
FIG. 1. (A) Representative traces of the pressure (80 mmHg) induced myogenic tone in rat middle cerebral arteries in the absence and presence of PD98059 (40 mM). (B) The mean 6 S.E.M of 6 experiments of pressure-induced tone at 80 mmHg in the absence and presence of PD98059 (10 mM, 40 mM). *P , 0.05; ***P , 0.001 significantly different as compared to the control. ###P , 0.001 significantly different from the myogenic tone in the presence of PD98059 (10 mM). FIG. 2. (A) Representative traces of the contractile response of rat middle cerebral arteries to AVP (0.1 mM) in the absence and presence of PD98059 (40 mM). (B) The mean 6 S.E.M of 6 experiments of AVP-induced tone in the absence and presence of PD98059 (10 mM, 40 mM). **P , 0.01 significantly different as compared to AVP response in the absence of PD98059. 525
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FIG. 4. (A) Representative traces of the effect of PD98059 (40 mM) on the CaCl 2 (0.4-1.6 mM)-induced myogenic tone of rat middle cerebral arteries in the absence and presence of PD98059 (40 mM). (B) The mean 6 S.E.M (vertical bars) of 5 experiments of the CaCl 2 concentration-response curve (0.4-1.6 mM) in the absence and presence of PD98059 (40 mM). ***P , 0.001 significantly different as compared CaCl 2 concentration-response curve in the absence of PD98059.
3% (n 5 6) (Figs. 1A, 1B). Beyond this, further increases in intravascular pressure (up to 120 mmHg, n 5 6) did not change the diameter significantly. PD98056 was used at 10 and 40 mM to investigate the role of MAP kinase on the myogenic tone of pressurized rat middle cerebral arteries. Myogenic tone was inhibited by 15% in the presence of 10 mM (from 52 6 3% to 44 6 5%; n 5 6), and by 64% in the presence of 40 mM of PD98059 (from 52 6 3% to 18 6 3%; n 5 6, Fig. 1B). A second series of experiments was conducted to verify the specificity of PD98059 as an inhibitor of MAP kinase. Tone was induced by 1) AVP (0.1 mM), an agent that produces contraction of vascular smooth muscle by increasing intracellular Ca 21 (12, 13) and activating MAP kinase (14), or 2) by 60 mM KCl (which exclusively stimulates influx of extracellular Ca 21 through voltage-dependent Ca 21 channels). This served
as positive and negative controls for the specificity of PD98059 as an inhibitor of MEK respectively. AVPinduced contraction was inhibited by 30% (from 53 6 5% to 38 6 7%; n 5 6) and 38% (from 53 6 5% to 33 6 5%; n 5 6) after addition of 10 and 40 mM PD98059 respectively (Figs. 2A, 2B). PD98059 also inhibited the contraction observed following exposure to 60 mM KCl (Fig. 3A). Here, 10 mM PD98059 inhibited the contraction by 13% (from 73 6 4% to 63 6 6%; n 5 6) and 40 mM PD98059 further reduced the contraction by 60% (from 73 6 4.1% to 30 6 8%; n 5 6) (Fig. 3B). We further investigated the effect of PD98059 on the Ca 21-induced myogenic tone. As Fig. 4A shows, the concentration-response curve of Ca 21 (0.4-1.6 mM) was significantly inhibited by PD98059. For example, the response to 1.6 mM CaCl 2 was inhibited by 65% (from 51.8 6 2.83 to 18.30 6 2.1%; n 5 6).
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DISCUSSION
ACKNOWLEDGMENTS
Our study aimed to elucidate the effects of MEK inhibition on myogenic tone and evoked contractility of the middle cerebral artery of the rat. We report two main findings of this study: (1) pressure-induced myogenic tone was inhibited by PD98059 at concentrations which had previously been reported to be selective for inhibition of MEK and the MAP kinase cascade. (2) PD98059 mediated inhibition of myogenic tone may have nonspecific effects on voltage-sensitive Ca 21 entry in vascular smooth muscle. PD98059 inhibits both the activation and phosphorylation of MEK with half-maximal inhibition values of 5-10 mM and maximal effects occurring at 10-100 mM (8, 9, 10). We found that 40 mM PD98059 inhibited AVP-induced constriction, which may be partly mediated by the MAP kinase cascade (40% inhibition). However, the same concentration of PD98059 also inhibited pressure and high K 1-induced tone by 60%. We and others have previously shown that PKC modulates myogenic tone (3, 4). Consistent with the fact that PKC phosphorylates MAPK, studies made in isolated cells (15, 16) and conduit arteries (5, 6, 7) indicate a role for MAPK in myogenic activation. Of interest are the studies from Adam and colleagues (7) who could demonstrate that prolonged (90 min) mechanical stretch of porcine carotid arteries activated MAPK, as did 110 mM KCl. The authors suggest that a dual activation of MAPK during vascular distension through changes in wall tension as well as by depolarization induced Ca 21 entry. Our results provide the first demonstration that inhibition of MAPK reduces vascular tone, although they do not distinguish between inhibition of kinase activity and non-specific blockade of voltage gated Ca entry, since all forms of tone were inhibited at near equivalent concentrations of PD98059. Thus this data suggests caution in the use of PD98059 as a selective inhibitor of MAPK.
Supported by funds from the Canadian Heart and Stroke Foundation and Canada Foundation for Innovation. The authors express gratitude to Dr. Ruehlmann for carefully reading the manuscript.
REFERENCES 1. Meninger, G. A., and Davis, M. J. (1992) Am. J. Physiol. 263, H647–H659. 2. Knot, H. J., Standen, N. B., and Nelson, M. T. (1998) J. Physiol. 508, 211–221. 3. Laher, I., and Bevan, J. A. (1987) J. Pharmacol. Exp. Ther. 242, 666 – 672. 4. Osol, G., Laher, I., and Kelley, M. (1993) Am. J. Physiol. 265, H415–H420. 5. Adam, L. P., Gapinski, C. J., and Hathaway, D. R. (1992) FEBS Lett. 302, 223–226. 6. Adam, L. P., Franklin, M. T., Raff, G. J., and Hathaway, D. R. (1995) Circ. Res. 76, 183–190. 7. Franklin, M. T., Wang, C. L., and Adam, L. P. (1997) Am. J. Physiol. 273, C1819 –C1827. 8. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686 –7689. 9. Pang, L., Sawada, T., Decker, S. J., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 13585–13588. 10. Alessi, D. R., Cuanda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489 –27494. 11. Lazar, D. F., Brady, J., Wiese, R. J., Mastick, C. C., Waters, S. B., Yamauchi, K., Pressin, J. E., Cuatrecasas, P., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 20801–20807. 12. Caramalo, C., Okada, K., Tsai, P., Linas, S. L., and Schrier, R. W. (1990) Kidney Int. 38, 47–54. 13. Thibonnier, M., Bayer, A. L., Simonson, M. S., and Kester, M. (1991) Endocrinology 129, 2845–2856. 14. Kribben, A., Wieder, E. D., Li, X., Van Putten, V., Granot, Y., Schrier, R. W., and Nemenoff, R. A. (1993) Am. J. Physiol. 265, C939 –C945. 15. Sadoshima, J., and Izumo, S. (1993) EMBO J. 12, 1681–1692. 16. Khalil, R. A., and Morgan, K. G. (1993) Am. J. Physiol. 265, C406 –C411.
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Nonspecific Inhibition of Myogenic Tone by PD98059, a MEK1 Inhibitor, in Rat Middle Cerebral Arteries Guy. J. L. Lagaud, Eugene Lam, Amy Lui, Cornelis van Breemen, and Ismail Laher 1 Vancouver Vascular Biology Research Centre (VVBRC), and Department of Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada
Received February 1, 1999
Activation of MAP kinase kinase, also called ERK kinase (MEK), may lead to desinhibition of thin filament regulatory proteins and we therefore investigated the acute effects of the potent MEK inhibitor, PD98059 on the contractile properties of pressurized rat middle cerebral arteries. Cerebral arteries (diameter 100-150 mm) were mounted on a pressure myograph and PD98059 (10 mM, 40 mM) significantly inhibited (15% and 64%) myogenic tone (P < 0.001). At these concentrations, PD98059 also significantly reduced the vasopressin (0.1 mM)- and KCl (60 mM)-induced tone. Cumulative addition of exogenous Ca 21 (0.4-1.6 mM) increased myogenic tone to ;50% of constriction at 80 mmHg. This effect was inhibited by PD98059 (P < 0.001). These results demonstrate that pressure-induced myogenic tone is inhibited by PD98059 at the concentrations that have been reported to be selective for inhibition of MEK and the MAP kinase cascade. However, our results also demonstrate that PD98059 may have nonspecific effects on voltage-sensitive Ca 21 entry in vascular smooth muscle. © 1999 Academic Press
Arteries from the cerebral, coronary and renal circulations constrict to increases, and dilate to decreases, in transmural pressure either in situ or in vitro. A major component of this mechanism, which facilitates blood flow autoregulation, appears to be myogenic in nature, by definition, a direct response of vascular smooth muscle to pressure or stretch (1). However, the precise cellular signaling cascade that controls vascular myogenic tone has not been fully elucidated. Extra1
Author for correspondence: Dr. Ismail Laher, Department of Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, B.C. V6T 1Z3 Canada. Fax: (604) 822-2281 E-mail: [email protected]. Abbreviations: AVP, arginine vasopressin; Ca 21, calcium; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase, MEK, MAPK or ERK kinase; PD98059, [2-(29-amino-39methoxyphenyl)-oxanaphthalen-4-one]; PKC, protein kinase C.
cellular Ca 21 is obligatory for basal tone in pressurized resistance arteries (2). The increase in intracellular calcium concentration [Ca 21] i activates myosin light chain kinase to phosphorylate myosin light chain and in addition protein kinase C (PKC) to enhance myofilament Ca 21 sensitivity (3, 4). Furthermore, a role for mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) has recently been suggested in mediating signal transduction from stretch to phosphorylation of caldesmon, a thin filament-associated protein, in conduit arteries (5, 6, 7). To our knowledge, there is no published report on the involvement of the MAP kinase in the development of myogenic tone. Since activation of MAP kinase kinase also called ERK kinase (MEK) may lead to disinhibition of thin filament regulatory proteins (e.g. caldesmon), we have investigated the effect of MEK inhibition of the contractile properties of pressurized rat middle cerebral arteries. To this end we used PD98059, a putative selective inhibitor of MEK and the MAP kinase cascade that has recently been characterised (8, 9, 10, 11). METHODS Vessel isolation and cannulation. Male Sprague-Dawley rats (200-300 g) were anesthetized with sodium pentobarbital (30 mg.kg 21, i.p.) containing heparin (500 U/kg), and then killed by decapitation. The brain was removed and placed in physiological salt solution (PSS) at 4°C. Second order middle cerebral arteries (inner diameter 125 6 5.03 mm, range 100-150 mm; n 5 19) were dissected and transferred to an arteriograph filled with oxygenated PSS at 37°C. Vessels were tied onto microcannulae (tip diameter 60-80 mm). Under no flow conditions, the intraluminal pressure was increased to 60 mm Hg using a pressure servo system (Living Systems, Burlington, VT), and the vessel was equilibrated for 60 min whereupon the vessel spontaneously and reliably developed myogenic tone (reduced luminal diameter). A detailed description of the method is given elsewhere (4). Experimental procedure. After equilibration at 80 mmHg, PD98059 (10 and 40 mM) was added to the PSS in order to determine its effect on myogenic tone (n 5 6). In other experiments, transmural pressure was lowered to 20 mmHg, a maneuver that places the vessel below the lower limit of the pressure range for myogenic tone.
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0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
Vol. 257, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
524
Vol. 257, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. (A) Representative traces of KCl (60 mM)-induced contractions of rat middle cerebral arteries in the absence and presence of PD98059 (40 mM). (B) The mean 6 S.E.M (vertical bars) of 6 experiments of KCl-induced tone in the absence and presence of PD98059 (10 mM, 40 mM). *P , 0.05; ***P , 0.001 significantly different as compared to KCl response in the absence of PD98059. ###P , 0.001 significantly different from KCl response in the presence of PD98059 (10 mM).
Luminal diameter was allowed to stabilize for 15-20 min before arginine vasopressin (AVP, 0.1 mM) or potassium (K 1, 60 mM) were added, either in the absence or presence of PD98059. For studies of Ca 21-induced myogenic tone, cerebral arteries were equilibrated at 80 mmHg and the bathing PSS was replaced with Ca 21 free medium for 20 min. Cumulative additions of Ca 21 (0.4-1.6 mM; n 5 5) were made in the absence and presence of PD98059. Expression of the results and statistical analysis. Myogenic tone at each pressure was expressed as a percent constriction 5 100% x [(D Ca-free 2 D PSS)/D Ca-free], where D is the diameter in calcium free or PSS. All results are expressed as mean 6 S.E.M of n animals. Statistical evaluation was made using ANOVA, and means were considered significantly different when P , 0.05.
Solutions and chemicals. The composition of the PSS was (in mM): NaCl 119, KCl 4.7, KH 2PO 4 1.18, NaHCO 3 24, MgSO 4-7H 2O 1.17, CaCl 2 1.6, glucose 5.5 and EDTA 0.026. Ca 21 free solution was a PSS solution containing no CaCl 2. 60 mM K 1 solution was a PSS solution in which Na 1 was substituted for an equimolar concentration of KCl. PD98059 was purchased from BIOMOL (PA, USA); AVP was obtained from Sigma Chemical Company (St Louis, MO).
RESULTS Myogenic tone developed during equilibration at 80 mmHg, reducing diameter by an average of 52 6
FIG. 1. (A) Representative traces of the pressure (80 mmHg) induced myogenic tone in rat middle cerebral arteries in the absence and presence of PD98059 (40 mM). (B) The mean 6 S.E.M of 6 experiments of pressure-induced tone at 80 mmHg in the absence and presence of PD98059 (10 mM, 40 mM). *P , 0.05; ***P , 0.001 significantly different as compared to the control. ###P , 0.001 significantly different from the myogenic tone in the presence of PD98059 (10 mM). FIG. 2. (A) Representative traces of the contractile response of rat middle cerebral arteries to AVP (0.1 mM) in the absence and presence of PD98059 (40 mM). (B) The mean 6 S.E.M of 6 experiments of AVP-induced tone in the absence and presence of PD98059 (10 mM, 40 mM). **P , 0.01 significantly different as compared to AVP response in the absence of PD98059. 525
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FIG. 4. (A) Representative traces of the effect of PD98059 (40 mM) on the CaCl 2 (0.4-1.6 mM)-induced myogenic tone of rat middle cerebral arteries in the absence and presence of PD98059 (40 mM). (B) The mean 6 S.E.M (vertical bars) of 5 experiments of the CaCl 2 concentration-response curve (0.4-1.6 mM) in the absence and presence of PD98059 (40 mM). ***P , 0.001 significantly different as compared CaCl 2 concentration-response curve in the absence of PD98059.
3% (n 5 6) (Figs. 1A, 1B). Beyond this, further increases in intravascular pressure (up to 120 mmHg, n 5 6) did not change the diameter significantly. PD98056 was used at 10 and 40 mM to investigate the role of MAP kinase on the myogenic tone of pressurized rat middle cerebral arteries. Myogenic tone was inhibited by 15% in the presence of 10 mM (from 52 6 3% to 44 6 5%; n 5 6), and by 64% in the presence of 40 mM of PD98059 (from 52 6 3% to 18 6 3%; n 5 6, Fig. 1B). A second series of experiments was conducted to verify the specificity of PD98059 as an inhibitor of MAP kinase. Tone was induced by 1) AVP (0.1 mM), an agent that produces contraction of vascular smooth muscle by increasing intracellular Ca 21 (12, 13) and activating MAP kinase (14), or 2) by 60 mM KCl (which exclusively stimulates influx of extracellular Ca 21 through voltage-dependent Ca 21 channels). This served
as positive and negative controls for the specificity of PD98059 as an inhibitor of MEK respectively. AVPinduced contraction was inhibited by 30% (from 53 6 5% to 38 6 7%; n 5 6) and 38% (from 53 6 5% to 33 6 5%; n 5 6) after addition of 10 and 40 mM PD98059 respectively (Figs. 2A, 2B). PD98059 also inhibited the contraction observed following exposure to 60 mM KCl (Fig. 3A). Here, 10 mM PD98059 inhibited the contraction by 13% (from 73 6 4% to 63 6 6%; n 5 6) and 40 mM PD98059 further reduced the contraction by 60% (from 73 6 4.1% to 30 6 8%; n 5 6) (Fig. 3B). We further investigated the effect of PD98059 on the Ca 21-induced myogenic tone. As Fig. 4A shows, the concentration-response curve of Ca 21 (0.4-1.6 mM) was significantly inhibited by PD98059. For example, the response to 1.6 mM CaCl 2 was inhibited by 65% (from 51.8 6 2.83 to 18.30 6 2.1%; n 5 6).
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Vol. 257, No. 2, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION
ACKNOWLEDGMENTS
Our study aimed to elucidate the effects of MEK inhibition on myogenic tone and evoked contractility of the middle cerebral artery of the rat. We report two main findings of this study: (1) pressure-induced myogenic tone was inhibited by PD98059 at concentrations which had previously been reported to be selective for inhibition of MEK and the MAP kinase cascade. (2) PD98059 mediated inhibition of myogenic tone may have nonspecific effects on voltage-sensitive Ca 21 entry in vascular smooth muscle. PD98059 inhibits both the activation and phosphorylation of MEK with half-maximal inhibition values of 5-10 mM and maximal effects occurring at 10-100 mM (8, 9, 10). We found that 40 mM PD98059 inhibited AVP-induced constriction, which may be partly mediated by the MAP kinase cascade (40% inhibition). However, the same concentration of PD98059 also inhibited pressure and high K 1-induced tone by 60%. We and others have previously shown that PKC modulates myogenic tone (3, 4). Consistent with the fact that PKC phosphorylates MAPK, studies made in isolated cells (15, 16) and conduit arteries (5, 6, 7) indicate a role for MAPK in myogenic activation. Of interest are the studies from Adam and colleagues (7) who could demonstrate that prolonged (90 min) mechanical stretch of porcine carotid arteries activated MAPK, as did 110 mM KCl. The authors suggest that a dual activation of MAPK during vascular distension through changes in wall tension as well as by depolarization induced Ca 21 entry. Our results provide the first demonstration that inhibition of MAPK reduces vascular tone, although they do not distinguish between inhibition of kinase activity and non-specific blockade of voltage gated Ca entry, since all forms of tone were inhibited at near equivalent concentrations of PD98059. Thus this data suggests caution in the use of PD98059 as a selective inhibitor of MAPK.
Supported by funds from the Canadian Heart and Stroke Foundation and Canada Foundation for Innovation. The authors express gratitude to Dr. Ruehlmann for carefully reading the manuscript.
REFERENCES 1. Meninger, G. A., and Davis, M. J. (1992) Am. J. Physiol. 263, H647–H659. 2. Knot, H. J., Standen, N. B., and Nelson, M. T. (1998) J. Physiol. 508, 211–221. 3. Laher, I., and Bevan, J. A. (1987) J. Pharmacol. Exp. Ther. 242, 666 – 672. 4. Osol, G., Laher, I., and Kelley, M. (1993) Am. J. Physiol. 265, H415–H420. 5. Adam, L. P., Gapinski, C. J., and Hathaway, D. R. (1992) FEBS Lett. 302, 223–226. 6. Adam, L. P., Franklin, M. T., Raff, G. J., and Hathaway, D. R. (1995) Circ. Res. 76, 183–190. 7. Franklin, M. T., Wang, C. L., and Adam, L. P. (1997) Am. J. Physiol. 273, C1819 –C1827. 8. Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686 –7689. 9. Pang, L., Sawada, T., Decker, S. J., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 13585–13588. 10. Alessi, D. R., Cuanda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489 –27494. 11. Lazar, D. F., Brady, J., Wiese, R. J., Mastick, C. C., Waters, S. B., Yamauchi, K., Pressin, J. E., Cuatrecasas, P., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 20801–20807. 12. Caramalo, C., Okada, K., Tsai, P., Linas, S. L., and Schrier, R. W. (1990) Kidney Int. 38, 47–54. 13. Thibonnier, M., Bayer, A. L., Simonson, M. S., and Kester, M. (1991) Endocrinology 129, 2845–2856. 14. Kribben, A., Wieder, E. D., Li, X., Van Putten, V., Granot, Y., Schrier, R. W., and Nemenoff, R. A. (1993) Am. J. Physiol. 265, C939 –C945. 15. Sadoshima, J., and Izumo, S. (1993) EMBO J. 12, 1681–1692. 16. Khalil, R. A., and Morgan, K. G. (1993) Am. J. Physiol. 265, C406 –C411.
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