INTRODUCTION

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PERIPHERAL RESISTANCE depends on the tone of small arteries and arterioles, which is regulated by neural, humoral factors [e.g., angiotensin II (ANG), norepinephrine (NE)], and local mechanisms intrinsic to the vessel wall (17, 18). Among the intrinsic mechanisms, the pressure-sensitive arteriolar myogenic response plays an important role in the local regulation of resistance, hence tissue blood flow (6, 8, 22, 33, 37, 38). It is thought that by reducing vessel diameter the pressure-induced response represents a negative feedback mechanism that helps to maintain wall tension according to the law of Laplace and Frank (17, 18). However, the pathways in arteriolar smooth muscle that contribute to the mechanotransduction of pressure-wall tension have not been clearly elucidated (8, 15, 30).

It has been shown that increases in pressure-wall tension are associated with increases in smooth muscle intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to the influx of extracellular Ca²⁺ and constriction (42, 50). Recent studies also raised the possibility that pressure-wall tension activates Ca²⁺ independent pathways and/or pathways that increase the sensitivity of the contractile machinery to Ca²⁺ (8, 15, 27, 30, 49). These cascades were shown to involve PKC (1, 11, 49); however, the roles of mitogen-activated protein (MAP) kinases, which may also affect smooth muscle contractility (24), in arteriolar myogenic constriction have not been fully explored.

From the 12 identified MAP kinases, the p42/44 (ERK1/2) and p38 MAP kinase pathways were proposed as likely modulators of vascular smooth muscle contractions (8, 24, 28, 29, 46, 48). Studies on large vessels and isolated smooth muscle cells suggested that activation of MAP kinases contribute to vascular smooth muscle contraction induced by agonists (4), such as NE and ANG II (29, 39). Interestingly, in conduit vessels that do not develop spontaneous tone in response to increases in intraluminal pressure, MAP kinases were also shown to be phosphorylated and activated in vitro by mechanical stretch (2, 26, 32, 36), whereas pharmacological inhibition of p42/44 MAP kinase activation was reported to abolish the tone developed by middle cerebral arteries to 80 mmHg intraluminal pressure (24).

Because of the aforementioned findings, we hypothesized that the roles of PKC, p42/44, and/or p38 MAP kinases are different in agonist- and pressure-induced responses of skeletal muscle arterioles and aimed to elucidate whether specific relationships exist among these pathways. Thus changes in diameter of isolated rat gracilis muscle arterioles were obtained as a function of changes in intraluminal pressure-wall tension and in response to NE and ANG II before and after inhibition of the PKC, p42/44 MAP kinase, or p38 MAP kinase pathways.

	METHODS

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Isolation of arterioles: videomicroscopy. Skeletal muscle arterioles were prepared as previously described in detail (37, 43). Briefly, male Wistar rats (387 ± 3 g) were housed in an American Association for Accreditation of Laboratory Animal Care-accredited animal care facility at the New York Medical College, and all protocols were approved by the Institutional Animal Care and Use Committee. Animals were anesthetized with pentobarbital sodium (50 mg/kg ip), and the gracilis muscle was removed and placed in a dissecting dish containing cold (4°C) physiological saline solution (PSS) containing (in mmol/l): 118.3 NaCl, 24 NaHCO₃, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 1.9 CaCl₂, and 11.1 glucose (pH 7.4). Arterioles were isolated and mounted onto two glass micropipettes in a vessel chamber (15 ml) and slowly pressurized to 80 mmHg by using a pressure-servo syringe reservoir system (Living Systems Instruments, www.livingsys.com). PSS flow through the system was set at 40 ml/min. Inner arteriolar diameter was measured with a video micrometer system and continuously recorded using a computerized data acquisition system (Biopac MP100; Goleta, CA). All vessels were allowed to stabilize for 60 min in oxygenated (10% O₂-5% CO₂-85% N₂) PSS warmed to 37°C.

Experimental protocols. After an equlibration period, changes in arteriolar diameter in response to ANG II (10⁹-10⁶ mol/l) and NE (10⁹-10⁶ mol/l) were obtained at an intraluminal pressure of 80 mmHg before and after incubation with the benzophenanthridine alkaloid chelerythrine, an inhibitor of total PKC activity (10 µmol/l for 30 min) (1, 13, 21) or PD-98059 (10 µmol/l for 30 min) (24, 26, 46), an inhibitor of p42/44 (ERK1/2) MAP kinase activation by inhibition of its phosphorylation by MAP kinase kinase (7), or SB-203580 (5 µM for 30 min) (29), a pyridinyl imidazole inhibitor of the p38 MAP kinase (7). Concentration-response curves for NE were obtained in a cumulative manner, whereas for ANG II, vessels were washed for 15 min after administration of each dose to obtain the maximal response to each concentration of ANG II. Arterioles were washed with PSS for 15 min after the completion of each concentration-response curve.

Western blotting. Phosphorylation of PKC, p42/44, and p38 MAP kinases was detected by Western blotting according to the modified protocols of Wesselman et al. (47), Matrougui et al. (28), and Dessy et al. (9). In brief, arterioles (n = 9) were cannulated in a dual vessel chamber (model CH/1/QT, Living Systems), pressurized to 80 mmHg (for 30 min, at 37°C), and then snap frozen in liquid nitrogen. Pressurized arterioles treated with PD-98059 and nonpressurized vessels served as controls. Protein samples were prepared, as previously described (5). Na₃VO₄ (1 mmol/l) and phosphatase inhibitor cocktail I and II (10 µl, Sigma; St. Louis, MO) were added to the homogenization buffer to inhibit phosphatases. Equal amounts of protein (8 µg) were electrophoresed on 10% SDS-PAGE gel, transferred to a membrane with a semidry blotting system, and labeled with the following antibodies: a phosphorylation-specific primary antibody for p42/44 MAP kinase, p38 MAP kinase, and PKC- (Cell Signaling, www.cellsignal.com) in 1:1,000 dilution for 1 h at room temperature. The membranes were washed with PBS and incubated for 1 h with sheep anti-rabbit IgG-horseradish peroxidase or donkey anti-mouse IgG-horseradish peroxidase (Amersham, www.amershambiosciences.com) at the final titer of 1:4,000. The membranes were developed with ECL-Plus (Amersham). Prestained rainbow markers (Amersham) were used as molecular mass standards.

Measurement of smooth muscle [Ca²+]_i. In separate experiments arteriolar smooth muscle was loaded with fura-2 (2 µmol/l fura 2-AM in the superfusate; 30 min at 25°C) as previously described (42). Changes in background-corrected, normalized fura 2 fluorescence ratios, the estimates of smooth muscle [Ca²⁺]_i, were measured by the ratiometric fluorescence method by using the Ionoptix Microfluorimeter System (Ionoptix, Milton, MA) (3, 44). After the loading period, changes in arteriolar [Ca²⁺]_i to chelerythrine (10 µmol/l), PD-98059 (10 µmol/l), SB-203580 (5 µmol/l), or nimodipine (10⁶ mol/l; an L-type Ca²⁺ channel antagonist) were obtained (at 80 mmHg intraluminal pressure).

Drugs and solutions. PD-98059, SB-203580, chelerythrine, and fura 2 AM were initially dissolved in DMSO (0.01% of total volume) and then diluted with PSS. Final concentrations and incubation time for PD-98059, SB-203580 (7, 24, 26, 28, 29, 46), and chelerythrine (1, 21) were based on previously published data. All other drugs were dissolved in distilled water. The vehicle had no effect on arteriolar responses. PD-98059 was purchased from Cal-Biochem (San Diego, CA); all other chemicals were purchased from Sigma Chemical.

Data analysis. The slope of the pressure-diameter relationship between 40 and 100 mmHg for each curve was determined by linear regression analysis. Myogenic tone was calculated by dividing the active diameter by the passive diameter (expressed as percent) at each pressure step. Circumferential wall tension was calculated as: T_w = P_i × r_i, where T_w is the circumferential wall tension, P_i is intraluminal pressure, and r_i is the vessel radius (22, 50). The 50% effective value of T_w (T₅₀) was calculated from the linear part of the wall tension-myogenic tone curves. The amplitudes of agonist- and pressure-induced constrictions were normalized (%) to the passive diameter at 80 mmHg. Constrictor responses before and after treatment were compared by using ANOVA for repeated measures followed by a modified Student's t-test with the Bonferroni correction for multiple comparisons. Pre- and posttreatment comparisons were made by using paired Student's t-tests when appropriate. Statistical significance was set at P < 0.05. Data are expressed as means ± SE.

	RESULTS

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Arteriolar constrictions to NE and ANG II. NE elicited concentration-dependent constrictions of isolated rat gracilis muscle arterioles that were abolished by 10 µmol/l chelerythrine (Fig. 1A). Inhibition of the p42/44 MAP kinase pathway with 10 µmol/l PD-98059 or inhibition of the p38 MAP kinase with 5 µmol/l SB-203580 did not significantly alter the maximal NE-induced constriction (Fig. 1A).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Arteriolar constrictions to norepinephrine (A) and angiotensin II (ANG) (B) under control conditions and after treatment with PD-98059 (10 µmol/l), an inhibitor of the p42/44 mitogen-activated protein (MAP) kinase pathway, or SB-203580 (5 µmol/l), an inhibitor of the p38 MAP kinase pathway, or chelerythrine (10 µmol/l), a PKC inhibitor. Data are means ± SE (n = 5-11). * P < 0.05.

Pressure-induced arteriolar constriction. In the absence of Ca²⁺ in the bath solution, step increases in pressure elicited continuous increases in arteriolar diameter (Fig. 2A; passive diameter at 80 mmHg: 132 ± 4 µm). In the presence of Ca²⁺, arterioles developed active myogenic tone in response to step increases in intraluminal pressure (20-140 mmHg) without the use of any vasoactive agent (Fig. 2A). Initially, the diameter of the vessels increased from ~85 µm to ~100 µm in response to an increase in intraluminal pressure from 20 to 40 mmHg. Beyond this point, further increases in pressure resulted in constrictions of arterioles. Inhibition of both PKC (10 µmol/l chelerythrine) and p38 MAP kinases (5 µmol/l SB-203580, Fig. 2A) altered the shape of the pressure-diameter curve as indicated by the slopes of the regression line fitted to the pressure-diameter relationship between 40 and 80 mmHg (control: 0.76 ± 0.18, chelerythrine: 0.02 ± 0.05, SB-203580: 0.08 ± 0.15; P < 0.05). Inhibition of the p42/44 MAP kinase pathway with 10 µmol/l PD-98059 also resulted in significantly increased arteriolar diameter at each pressure step (Fig. 2A). However, in the presence of PD-98059 the shape of the myogenic curve was similar to control (slope: 0.73 ± 0.06, not significant).

View larger version (41K): [in this window] [in a new window]   Fig. 2.   A: normalized changes in arteriolar diameter as a function of intraluminal pressure under control conditions and after treatment with PD-98059 (10 µmol/l), an inhibitor of the p42/44 MAP kinase pathway, SB-203580 (5 µmol/l), an inhibitor of the p38 MAP kinase pathway, or chelerythrine (10 µmol/l), a PKC inhibitor, or after removal of extracellular Ca2+. B: myogenic tone as a function of wall tension before and after treatment with chelerythrine, PD-98059, or SB 203580. * P < 0.05 vs. control. C: values of wall tension that are associated with the development 50% of maximal myogenic tone (T50) in the absence and presence of PD-98059 or SB-203580. Data are means ± SE (n = 7-12). * P < 0.05 vs. control. D: representative Western blots showing pressure (80 mmHg)-induced PKC, p42/44, and p38 MAP kinase phosporylation in isolated arterioles in various conditions. E: changes in smooth muscle intracellular Ca2+ concentration ([Ca2+]i) in response to chelerythrine, PD-98059, SB-203580, or the Ca2+ channel antagonist nimodipine (106 mol/l). * P < 0.05. F: proposed scheme for the differential role of PKC and MAP kinase-dependent pathways in eliciting constrictions of skeletal muscle arterioles. Agonists and intraluminal pressure-wall tension elicit increases in smooth muscle [Ca2+]i and at the same time increase the Ca2+ sensitivity of the contractile apparatus by activating PKC. Activation of PD-98059-sensitive p42/44 MAP kinase and SB-203580-sensitive p38 MAP kinase pathways regulates the wall tension sensitivity of the myogenic machinery by modulating Ca2+ sensitivity. Role of MAP kinases in receptor-mediated arteriolar constriction is agonist specific.

Wall tension-myogenic tone relationship. To further analyze the effect of inhibition of the PKC and MAP kinase pathways on the pressure-sensitive myogenic machinery of arterioles, from our data we calculated myogenic tone (percentage of corresponding passive diameter) and plotted these values against the calculated circumferential wall tension (Fig. 2B). In control conditions the wall tension-myogenic tone relationship was linear in the range of 0.2-0.5 N/m (slope: 1.3). Inhibition of the p42/44 MAP kinase pathway with PD-98059 resulted in a parallel rightward shift in the wall tension-myogenic tone relationship, indicating decreased tension sensitivity of the myogenic mechanism, without any significant change in the slope (1.3). In contrast, inhibition of both the p38 MAP kinase pathway with SB-230580 and PKC with chelerythrine resulted in a significant decrease in the slope of the wall tension-myogenic tone relationship (slope: 0.3 and 0.1, respectively) and an increased T₅₀ (Fig. 2C).

Pressure-induced activation of PKC, p42/44, and p38 MAP kinases. In arterioles submitted to pressure (80 mmHg), phosphorylation of PKC was increased (by ~170%) compared with nonpressurized vessels (Fig. 2D). Pressure also increased phosphorylation of p42/44 MAP kinase (by ~114%), which was prevented by the same concentration of PD-98059 that was used in the functional studies (Fig. 2D). Phosphorylation of p38 MAP kinase was also increased by pressure (by ~42%, Fig. 2D).

Measurement of smooth muscle [Ca²+]_i. Incubation of arterioles (pressurized to 80 mmHg) with chelerythrine (10 µmol/l), PD-98059 (10 µmol/l), or SB-230580 (5 µmol/l) did not significantly alter smooth muscle [Ca²⁺]_i (Fig. 2D). In contrast, administration of the Ca²⁺ channel inhibitor nimodipine elicited a significant decrease in smooth muscle [Ca²⁺]_i (Fig. 2D).

	DISCUSSION

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The new findings of the present study are that in skeletal muscle arterioles 1) constrictions to ANG II are inhibited by the PKC inhibitor chelerythrine (10 µmol/l) and PD-98059 (10 µmol/l), an inhibitor of the p42/44 MAP kinase pathway, but not by SB-203580 (5 µmol/l), an inhibitor of the p38 MAP kinase pathway; 2) constrictions to NE are inhibited by chelerythrine, whereas they are not affected by PD-98059 and SB-203580; and 3) myogenic constriction is inhibited completely by chelerythrine and partially by PD-98059 and SB-203580.

Mechanical forces, such as pressure-wall tension and pharmacological stimuli, may activate multiple signaling pathways in arteriolar smooth muscle that determine microvascular tone development. Previous studies demonstrated that in addition to increases in [Ca²⁺]_i, activation of various kinase cascades regulating Ca²⁺ sensitivity of the contractile apparatus may modulate the level of arteriolar constriction (8, 15, 30). In the present study, we conducted experiments on isolated skeletal muscle arterioles to compare the role of the PKC, p42/p44 MAP kinase, and p38 MAP kinase pathways in the modulation of constrictions to agonists and pressure-wall tension by using pharmacological inhibitors. We found that ANG II- and NE-induced constrictions of skeletal muscle arterioles involve activation of PKC (Fig. 1A), extending previous findings on vessels from other vascular beds (25). Moreover, it is likely that p42/44 MAP kinases are also involved in the signal transduction of ANG II, because ANG II-induced arteriolar constrictions were significantly inhibited by PD-98059 (Fig. 1A) adding previous findings in vascular smooth muscle cells (39, 40). Also, PD-98059 was shown to significantly decrease ANG II responses of rat mesenteric and human subcutaneous arterioles (28, 39, 40). Moreover, PD-98059 was shown to lower blood pressure in ANG II-induced hypertension in rats (31). It has been noted, however, that there are important tissue- and/or stimulus-specific differences in the effects of various agonists on the p42/44 MAP kinase pathway. For example, Watts (46) reported that PD-98059 reduced serotonin- but not ANG II-induced contraction of rat aortic rings. In contrast, at the level of arterioles, p42/44 MAP kinases, unlike PKC, play only a minor role, if any, in NE-induced vascular responses, because in the present study arteriolar constrictions to NE were essentially unaffected by PD-98059 (Fig. 1B). Similarly, PD-98059, while preventing -adrenergic receptor-dependent activation of p42/44 MAP kinase, did not affect NE-induced contractions of rat aortic rings under control conditions, but it did so in the absence of extracellular Ca²⁺ (9). Arteriolar responses to NE were also insensitive to inhibition of p38 MAP kinases (Fig. 1B). A recent study reported that in the rat aorta, ANG II-induced contractions can be blocked by SB-203580 (29). In contrast, in the present study in skeletal muscle arterioles, SB-203580 did not alter ANG II-induced constrictions (Fig. 1A). Thus it seems that the p38 MAP kinase pathway, unlike p42/44 MAP kinases, does not play a substantial role in agonist-induced arteriolar constrictions.

Myogenic constriction of skeletal muscle arterioles depends on both the influx of extracellular Ca²⁺ and the activity of the PKC pathway (Fig. 2, A and D), as shown in the present and previous studies (1, 10, 14, 19, 25, 34, 49). Because PKC inhibitors abolish both agonist- and pressure-induced constrictions (1), it is likely that PKC represents a common pathway leading to arteriolar constriction shared by several stimuli. In contrast, PD-98059 elicited a significant rightward shift in the wall tension-myogenic tone relationship without altering its slope (Fig. 2, B and C), suggesting that the p42/p44 MAP kinase pathway modulates the sensitivity of the myogenic mechanism to wall tension, although it may not be essential for the development of pressure-induced constriction (36). The findings that the p38 MAPK inhibitor SB-203580 significantly attenuated both the maximal myogenic tone and the sensitivity of the myogenic mechanism to wall tension (Fig. 2, A-C) suggest that in gracilis muscle arterioles activation of the p38 MAP kinase pathway is involved in the mechanotransduction of wall tension. From previous studies, it can be hypothesized that changes in wall tension are sensed by extracellular matrix-coupled integrins that activate tyrosine phosphorylation events (8), which lead to activation of MAP kinases (8). The idea that MAP kinase pathways are sensitive to pressure-induced increases in wall tension is supported by the findings that p42/p44 and/or p38 MAP kinase phosphorylation increased in rat skeletal muscle arterioles exposed to pressure (Fig. 2D) (36). These results provide physiological importance to previous findings on pressure or stretch-dependent PD-98059-sensitive increase in p42/p44 MAP kinase activity in rabbit aorta (2) and facial vein (26) and in cultured smooth muscle cells (32).

The findings that chelerythrine, PD-98059, and SB-203580 did not elicit significant decreases in smooth muscle [Ca²⁺]_i (Fig. 2E), yet inhibited arteriolar myogenic tone, suggest that PKC- (1, 10, 35) and MAP kinase-dependent pathways modulate primarily the Ca²⁺ sensitivity of the myogenic mechanism (3). It is likely that Ca²⁺ sensitivity of the myogenic mechanism and development of myogenic constriction is regulated by a complex interaction of multiple signaling pathways, involving activation of RhoA/Rho kinase (3, 32, 45, 49), myosin light chain kinase, and Ca²⁺-calmodulin-dependent protein kinase II (20).

Because the effects of PD-98059 and SB-203580 on agonist-induced and myogenic arteriolar constrictions are different, one can speculate that p42/44 and p38 MAP kinases interfere with the smooth muscle contractile apparatus and/or the myogenic mechanism at different steps of the signal transduction pathways (Fig. 2F). The importance of our findings lies in the fact that in pathophysiological conditions, alterations in PKC- and MAP kinase-dependent signal transduction may result in specific changes in arteriolar contractility (39, 43) and thus impaired regulation of peripheral resistance. Several studies have demonstrated that in hypertension an increased vascular MAP kinase activity (12, 23, 39) is associated with an upregulated arteriolar myogenic mechanism due to an increased Ca²⁺ sensitivity of the smooth muscle contractile apparatus (42) and that increases in pressure-wall tension elicit enhanced constriction of skeletal muscle arterioles (16, 42). In theory, wall tension-sensitive MAP kinase pathways are potential candidates for the upregulation of myogenic arteriolar constriction that would provide the means to normalize increased wall tension during elevations of blood pressure (22, 42). In addition, chronic increases in MAP kinase and/or PKC activity in hypertension (41) by inducing smooth muscle hypertrophy may also serve to reduce wall tension.

Collectively, the results of this study suggest that in skeletal muscle arterioles, by modulating the Ca²⁺ sensitivity of the contractile machinery, PKC is involved in both myogenic- and agonist-induced constrictions, PD-98058-sensitive p42/44 MAP kinases modulate both wall tension-dependent and ANG II-induced constrictions, whereas the SB-203580-sensitive p38 MAP kinase pathway seems to be specifically involved in the mechanotransduction of wall tension.