Attenuation of pressure-induced myogenic contraction and tyrosine phosphorylation by fasudil, a cerebral vasodilator, in rat cerebral artery

Abstract

The mechanism by which fasudil inhibits pressure-induced myogenic contraction was studied with regard to tyrosine phosphorylation in rat cerebral artery. Intracellular Ca(2+) concentration ([Ca(2+)](i)) and vessel diameter were simultaneously measured. Total tyrosine phosphorylation level and phosphorylation of tyrosine 419 on pp60(src) required for its full catalytic activity were immunocytochemically detected in situ. Fasudil (1 - 100 microM) partially suppressed the increase in [Ca(2+)](i), and totally attenuated contraction elicited by pressurization from 10 to 60 mmHg. Furthermore, fasudil (100 microM) significantly attenuated tyrosine phosphorylation and the activity of pp60(src) augmented in situ by pressure. Herbimycin A (1 - 100 nM) and genistein (3 - 30 microM), tyrosine kinase inhibitors, effectively attenuated the pressure-induced increase in [Ca(2+)](i), contraction, tyrosine phosphorylation, and activation of pp60(src). Both fasudil and herbimycin A directly inhibited the pp60(src) activity in a cell free system. Orthovanadate (100 microM), a tyrosine phosphatase inhibitor, significantly potentiated the pressure-induced increase in [Ca(2+)](i) and contraction. Nicardipine (100 nM), a Ca(2+) antagonist, completely inhibited pressure-induced increase in [Ca(2+)](i) and contraction, but affected neither tyrosine phosphorylation nor activity of pp60(src) in the pressurized arteries. Arginine-glycine-aspartic acid-serine peptide (1 - 100 microM) concentration-dependently reduced the pressure-induced contraction. In addition to the hitherto reported vasodilatory actions of fasudil, the present results suggest the inhibition by fasudil of pressure-induced tyrosine phosphorylation and pp60(src) activation. The wide spectrum of inhibitory actions of fasudil may contribute to the effective attenuation of the pressure-induced contraction in the cerebral artery.

Figure 1

Pressure-induced increase in [Ca²⁺]_i and myogenic contraction of rat cerebral artery loaded with indo-1. (a) Time course of [Ca²⁺]_i and arterial diameter in response to a pressure stimulus from 10 to 60 mmHg. [Ca²⁺]_i (open circle; upper part of panel A) is expressed as the ratio of fluorescence intensity at 405 to that at 485 nm (R_405/485) in the upper panel. Arterial diameter (closed circle; lower part of panel A) at 60 mmHg in the presence of 100 μM papaverine (197.6±13.5 μm, n=5) was taken as 100% in the lower panel, and that at 10 mmHg was 143.5±12.0 μm (per cent diameter=72.4±1.7). The [Ca²⁺]_i and contractile response lasted stable for at least 60 to 90 min (outer diameter=146.8±10.8 μm, per cent diameter=74.2±1.4). (b) The relationship between [Ca²⁺]_i and the change in diameter (μm) in response to pressurization from 10 to 20, 40 or 60 mmHg. Each point represents the mean±s.e.mean of five preparations.

Figure 2

Effects of fasudil and nicardipine on the pressure-induced changes in [Ca²⁺]_i and diameter of rat cerebral artery. (a) Simultaneous measurements of [Ca²⁺]_i and diameter changed by a pressure stimulus from 10 to 60 mmHg and the effect of fasudil. (b) Summarized data representing the concentration-response relation as to the effects of fasudil (open circle; upper part of panel a) and nicardipine (closed circle; lower part of panel a) on the [Ca²⁺]_i and arterial diameter. Each point represents the mean±s.e. mean of four preparations. Corresponding control values in the absence of fasudil are not significantly different from those in the absence of nicardipine.

Figure 3

Effects of tyrosine kinase inhibitors on the [Ca²⁺]_i and arterial diameter of rat cerebral arteries in response to a pressure stimulus. (a) Herbimycin A (100 nM) significantly inhibited the [Ca²⁺]_i (open circle; upper part of panel a) and the resultant contraction (closed circle; lower part of panel a) increased by a pressure stimulus from 10 to 60 mmHg. (b) Summarized data representing the concentration-response relation as to the effects of herbimycin A (open circle) and genistein (closed circle) on the [Ca²⁺]_i and arterial diameter. Each point represents the mean±s.e. mean of four to five preparations. Corresponding control values in the absence of herbimycin A are not significantly different from those in the absence of genistein.

Figure 4

Effect of fasudil on pressure-induced tyrosine phosphorylation. Staining of phosphorylated tyrosine residues in rat cerebral arteries under control conditions (a) and following exposure to pressurization from 10 to 60 mmHg for 5 min in the absence (b) or the presence of fasudil (100 μM) (c). The image stained by anti-phosphotyrosine antibody (b) was identical to that stained by anti-α-smooth muscle actin antibody (d), indicating that tyrosine phosphorylation occurred in the medial smooth muscle cells of the same artery.

Figure 5

Effects of fasudil, nicardipine, herbimycin A, and genistein on tyrosine phosphorylation of rat cerebral artery. (a) Pressurization from 10 to 60 mmHg significantly increased the fluorescence intensity. Note that fasudil (Fas) (100 μM), herbimycin A (Her) (100 nM), and genistein (Gen) (30 μM) except nicardipine (Nic) (100 nM) treatments significantly reduced the fluorescence intensity. (b) U46619 (100 nM) significantly potentiated fluorescence intensity. Note that fasudil (100 μM) and genistein (30 μM) except nicardipine (100 nM) and herbimycin A (100 nM) treatments significantly reduced the fluorescence intensity. Each column represents the mean±s.e. mean of five preparations. Cont, control; Pres, pressure; U, U46619. ^** indicates P<0.01 vs corresponding control values. # indicates P<0.05 vs corresponding values stimulated with either pressure (a) or U46619 (b).

Figure 6

Effects of fasudil and herbimycin A on pressure-induced phosphorylation of tyrosine 419 on pp60^src required for its full catalytic activity. Typical images stained by anti-src [pY⁴¹⁸] phosphospecific antibody in rat cerebral arteries under control conditions (a) and following exposure to pressurization from 10 to 60 mmHg for 5 min in the absence (b), or the presence of fasudil (100 μM) (c) were shown. Image of an pressurized artery with herbimycin A (100 nM) was also shown (d). Note that the stained individual smooth muscle cells were oriented perpendicular to the longitudinal axis of the artery.

Figure 7

Effects of fasudil, nicardipine, herbimycin A, and genistein on phosphorylation of tyrosine 419 on pp60^src in rat cerebral artery. Pressurization from 10 to 60 mmHg significantly increased the fluorescence intensity. Note that fasudil (Fas) (100 μM), herbimycin A (Her) (100 nM), and genistein (Gen) (30 μM) except nicardipine (Nic) (100 nM) treatments significantly reduced the fluorescence intensity. Each column represents the mean±s.e.mean of five preparations. Cont, control: Pres, pressure. ^** indicates P<0.01 vs corresponding control values. ## indicates P<0.01 vs corresponding values stimulated with pressure.

Figure 8

Effects of fasudil and herbimycin A on pp60^src activity in cell free system. Note that fasudil significantly inhibited the pp60^src activity in a concentration-dependent manner (IC₅₀ value = 32.5±2.3 μM). Herbimycin A also significantly reduced the enzyme activity (IC₅₀ value=8.0±0.5 nM). The absorbance in each well was measured by use of a spectrophotometric plate reader at a wavelength of 450 nm. Each column represents the mean±s.e.mean of five determinations. ^* and ^** indicate P<0.05 and P<0.01, respectively, vs corresponding control values.