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Am J Physiol Heart Circ Physiol 281: H1047-H1056, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 3, H1047-H1056, September 2001

Tyrosine phosphorylation following alterations in arteriolar intraluminal pressure and wall tension

Timothy V. Murphy, Brian E. Spurrell, and Michael A. Hill

Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria 3083, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Arterioles respond to increased transmural pressure with myogenic constriction. The present study investigated the role of tyrosine phosphorylation in myogenic activity. Cannulated segments of a rat cremaster arteriole were fixed under pressure, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-phosphotyrosine. Smooth muscle cell fluorescence intensity was measured with the use of confocal laser-scanning microscopy. Anti-phosphotyrosine fluorescence intensity in muscle cells of arterioles maintained at 100 mmHg was reduced by the tyrosine kinase inhibitor tyrphostin A47 (30 µM) and increased by the tyrosine phosphatase inhibitor pervanadate (100 µM). In time-course experiments, anti-phosphotyrosine fluorescence increased slowly (over 5 min) after an acute increase in intraluminal pressure, and was dissociated from myogenic contraction (within 1 min). In contrast, angiotensin II (0.1 µM) caused rapid constriction and increased tyrosine phosphorylation. Anti-phosphotyrosine fluorescence was also pressure dependent (10-100 mmHg). Abolition of myogenic activity, either through removal of extracellular Ca2+, or exposure to verapamil (5 µM) or forskolin (0.1 µM) caused a further increase in anti-phosphotyrosine fluorescence. We conclude that transmural pressure and/or wall tension in arterioles causes increased tyrosine phosphorylation; however, this is not involved in the acute phase of myogenic constriction but may be involved in later responses, such as sustained myogenic tone or mechanisms possibly related to growth.

myogenic constriction; rat cremaster arteriole; pervanadate; tyrphostin; confocal microscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

INCREASED INTRALUMINAL PRESSURE within arteries and arterioles initiates a variety of responses both acutely and over a longer time period. In arterioles, an acute increase in pressure typically elicits a rapid constriction after which a sustained increase in smooth muscle tone persists for the duration of the pressure stimulus. These myogenic responses are a property of the vascular smooth muscle and are not initiated by neurohumoral or endothelial substances (6, 18). Prolonged increases of intravascular pressure have been suggested (14, 24, 25) to lead to cytoskeletal reorganization within smooth muscle cells and remodeling of the vessel wall, resulting in a thickened medial layer and ultimately smooth muscle hyperplasia and hypertrophy. Such pressure-induced responses, in addition to involvement in the moment-to-moment regulation of the microcirculation, have been implicated in longer-term adaptive responses and the etiology of pathophysiological states including hypertension (3, 11).

The precise cellular mechanisms underlying the effects of increased intraluminal pressure are not fully understood. Acute myogenic constriction is dependent on an initial increase in smooth muscle intracellular Ca2+ concentration ([Ca2+]i) via Ca2+ entry (5, 16, 32), followed by phosphorylation of the 20-kDa regulatory light-chain of myosin and contraction (34). In addition, a number of other signaling molecules have been implicated; i.e., protein kinase C, metabolites of arachidonic acid, and small molecular weight G proteins (13, 15, 19). Other components of myogenic contraction are poorly understood, in particular, the membrane events coupling an increase in pressure to contraction and the maintenance of myogenic tone (6). There is also uncertainty as to the mechanisms coupling increased transmural pressure to remodeling and smooth muscle cell growth.

Because vascular smooth muscle cells contain high levels of tyrosine kinase activity (7-9), the possible role of such signaling pathways in myogenic constriction has recently been considered (27). Tyrosine phosphorylation-dependent events provide possible mechanisms by which intraluminal pressure (via changes in vessel wall tension) could be transduced across smooth muscle cell membranes. Increased wall tension in porcine carotid artery (12) and ovine trachealis (28) resulted in increased tyrosine phosphorylation of integrin-associated proteins (focal adhesion kinase and paxillin) and the intracellular signal mitogen-activated protein kinase. In support of a role for integrins in myogenic activity, the disruption of integrin-matrix binding with Arg-Gly-Asp (RGD) peptide attenuated pressure-induced myogenic constriction (5, 22). Furthermore, tyrosine phosphorylation has also been implicated in the regulation of voltage-sensitive Ca2+ channels in smooth muscle (30, 31), which would be expected to modulate both myogenic and agonist-induced reactivity.

In studies (27) of isolated skeletal muscle arterioles, we recently demonstrated that tyrosine kinase inhibitors genistein and tryphostin A47 caused concentration-dependent dilation of vessels with spontaneous myogenic tone, whereas the phosphatase inhibitor pervanadate enhanced the level of tone. However, despite the presence of the tyrosine kinase inhibitors, acute increases in intraluminal pressure continued to stimulate myogenic constriction, and furthermore, the inhibitors did not prevent the rapid increase in [Ca2+]i after the pressure increase. Taken together, these observations suggested a role for tyrosine kinase activity in the maintenance of tone rather than the myogenic constriction itself, and further suggested that regulation of smooth muscle Ca2+ entry was not a primary target of tyrosine kinases in this preparation (27). These studies could not, however, exclude the possibility that tyrosine kinase was principally involved in pathways other than those relating to contraction. For example, the tyrosine kinase inhibitor genistein has been shown to attenuate the pressure-induced increase in protooncogenes (c-myc and c-fos) in isolated resistance vessels (21).

On the basis of the above, the present study had two aims: 1) to demonstrate levels and time course of tyrosine phosphorylation after alterations in intraluminal pressure in isolated arterioles, and 2) to determine whether the level of phosphorylation was related to specific mechanical events such as changes in wall tension or contractile activity. Tyrosine phosphorylation was measured by rapid fixation of vessels, followed by incubation with a fluorescent antibody to phosphotyrosine and subsequent quantitation of fluorescence intensity by using confocal laser-scanning microscopy. Changes in wall tension were dissociated from myogenic contraction by examining pressure-induced changes in tyrosine phosphorylation in the presence of dilator stimuli (verapamil, forskolin, and 0 mM extracellular Ca2+).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Animals used in this study where housed in a dedicated facility with a 12:12-h light-dark cycle. Rats were provided with standard rat chow and drinking water ad libitum. The protocols and procedures were approved by the RMIT Animal Ethics and Experimentation Committee.

Isolated Arteriole Preparation

Male Sprague-Dawley rats, age 6-9 wk and weight 200-350 g, were anesthetized with Pentothal Sodium (100 mg/kg ip) and the cremaster muscles were excised and placed in a cooled (4°C) chamber composed of dissection buffer [3 mM of 3-N-morpholino propanesulfonic acid (MOPS)] and (in mM) 145 NaCl, 5 KCl, 2.5 CaCl2, 1 MgSO4, 1 NaH2PO4, 0.02 EDTA, 2 pyruvate, 5 glucose, and 1% albumin. Segments of the main intramuscular arteriole were dissected as described previously (20). Vessel segments were cannulated with glass micropipettes, secured, and mounted in a tissue superfusion chamber. The cannulated arterioles were continuously superfused (4 ml/min) with a physiological salt solution (PSS) composed of (in mM) 111 NaCl, 25.7 NaHCO3, 4.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 11.5 glucose, and 10 HEPES. Vessel segments were gradually pressurized to 70 mmHg and were warmed to 34°C during a 60-min equilibration period. During this period, the vessels were checked for pressure leaks and were allowed to develop spontaneous tone. Vessels without spontaneous tone were discarded.

The vessel preparation was positioned on the stage of an inverted microscope. Measurement of internal vessel diameter was accomplished by using an electronic video caliper.

In Situ Demonstration of Smooth Muscle Tyrosine Phosphorylation

Pressurized arteriole segments were fixed in an ice-cold mixture of 50% (vol/vol) ethanol and 4% polyethylene glycol (Kryofix) for 20 min and stored at 4°C. The fixation procedure halted biochemical processes and abolished all myogenic tone, rendering the vessels passive and thereby avoiding potential complications with differing distribution of fluorescence in arterioles contracted to differing extents. Vessels fixed at 10 mmHg tended to have a smaller diameter than those fixed at higher pressures due to the elastic nature of the vessel wall and the relative lack of distending pressure at 10 mmHg. Therefore, the pressure in vessels initially fixed at 10 mmHg was raised to 100 mmHg for the final 30 s of the fixation period to ensure these vessels were distended and to minimize possible optical artifacts. In preparation for antibody labeling, vessel segments were washed with phosphate-buffered saline (PBS) composed of (in mM) 2.7 KCl, 1.5 KH2PO4, 137 NaCl, 8 Na2HPO4, and 2% paraformaldehyde (2 g/100 ml) and incubated with 0.1% Triton X-100 in PBS for 10 min at room temperature. Vessels were washed with PBS and then incubated in a solution containing 0.9% Na-citrate, 2% filtered goat serum, 1% bovine serum albumin, 0.05% Triton X-100, 0.025% NaN3 and fluorescein isothiocyanate (FITC)- or tetramethyl rhodamine isothiocyanate (TRITC)-labeled phosphotyrosine monoclonal antibody (1:50) for 45 min at 21°C. TRITC-labeled antibody was used for the endothelium-denuded and angiotensin II (ANG II)-treated samples. After incubation, the vessel segments were washed with 0.02% Triton X-100/0.9% Na-citrate in PBS. The vessel segments were then mounted on glass slides in Fluomount (Dako Industries) and were examined by laser confocal microscopy.

Confocal Microscopy and Image Analysis

Most of the images were obtained with a confocal scanning laser microscope (model MRC-500, Bio-Rad Laboratories), in conjunction with a Nikon inverted-stage microscope. The argon-ion laser with primary excitation peak at 488 nm was used with maximum power setting. For the studies examining ANG II time course and comparison of deendothelialized and intact preparations, an Optiscan Confocal Laser Scanning Microscope System (Optiscan; Melbourne, Australia) was used. The same laser wavelength and power setting was used as for the Bio-Rad instrument. A Nikon ×60 oil-immersion objective (numerical aperture 1.3) was used with each microscope. Acquired 8-bit images (Bio-Rad) or 16-bit images (Optiscan) were 12-frame averages; 0.5 s/frame, exposure being limited due to the potential for rapid bleaching of FITC. Image thickness was set at 0.36 µm. Quantification of the pixel intensity in confocal images of vascular smooth muscle cells was performed with the use of the histogram/area functions of the MRC/Comos software or Scion Image (ANG II-treated and deendothelialized preparations). The regions examined were outlined manually to include only smooth muscle cells in sections where other structures were visible, and the calculated mean fluorescence intensity was obtained for the selected region. Corrections were made for background fluorescence by using a region of the slide adjacent to the fixed vessel. Pixel intensity was assigned on a grayscale of 0-256 (8-bit or 16-bit images of 512 × 512 pixels). Data are presented as the mean fluorescence intensity of the selected regions. In preliminary experiments, specificity of labeling was confirmed by preabsorbing the antibody with 1 mM of phosphotyrosine before incubation with the vessels.

Experimental Protocols

In situ demonstration of tyrosine phosphorylation during pressure stimulation. Vessels were allowed to develop spontaneous myogenic tone while maintained at 70 mmHg and were then treated with 10 µM of acetylcholine. The diameter achieved in the presence of acetylcholine was recorded as an estimate of maximal diameter. The vessel was then washed with the PSS and spontaneous tone was allowed to redevelop to the preacetylcholine level after which the intraluminal pressure was lowered to 30 mmHg for 20 min. In studies where extracellular Ca2+ was reduced, vessels were superfused with a nominally Ca2+-free PSS during this 20-min period (no added CaCl2 and 2 mM of EGTA) and for the remainder of the experiment. The Ca2+-channel blocking agent verapamil, where present, was also added to the PSS during this period. The addition of other drugs (tyrphostin A47 and forskolin) followed this same protocol. After this equilibration period, intraluminal pressure was instantaneously altered, or "stepped," to 10, 70, or 100 mmHg. Vessels were maintained at a particular test pressure for 15 min. In most of the experiments, pervanadate (100 µM) was added to the PBS for the final 5 min of this period to cause rapid and full inhibition of endogenous tyrosine phosphatase activity, as described previously (23). Experiments where this procedure was not carried out are indicated in the text. At the conclusion of the test period, vessels were fixed in the vessel chamber as described above.

In four experiments, the effect of endothelium removal on pressure-induced phosphotyrosine formation was examined. After vessel maximum diameter in response to acetylcholine was obtained (see Isolated Arteriole Preparation) and the vessel was allowed to regain spontaneous tone, an air bubble was passed through the vessel lumen until the response to acetylcholine was abolished. Experiments were then performed as described above. Paired control experiments were performed as a different confocal microscope system and image analysis package was used for these experiments.

Time-course studies. Experiments were performed to examine the time course of changes in anti-phosphotyrosine fluorescence after a pressure step or, for comparison, ANG II treatment. ANG II was used as a positive control because it is a known stimulator of tyrosine kinase activity. The vessel was either maintained at 30 mmHg for 20 min and stepped to 100 mmHg as described above or maintained at 70 mmHg throughout and treated with ANG II (0.1 µM). The vessel was rapidly fixed with the use of ice-cold Kryofix at various time points after the pressure step ranging from 15 s to 60 min. These experiments were performed in the presence of 1 µM of pervanadate, throughout the entire experiment to inhibit tyrosine phosphatase. This variation in protocol from the studies mentioned above was to prevent the large constriction of the vessel caused by 100 µM of pervanadate, which prevents measurement of the myogenic contraction.

Drugs and Chemicals

Tyrphostin A47 (RG 50864; Sigma) and forskolin (Sigma) were dissolved in dimethyl sulfoxide and were diluted further in PSS (final concentration of dimethyl sulfoxide ~0.1%). [Sar1,Ile8]ANG II (Sigma) was dissolved in deionized water and was diluted in Krebs solution. Verapamil (Sigma) was initially dissolved in ethanol and diluted further in PSS (final concentration ethanol <0.1%). Sodium pervanadate was prepared as a 50 mM stock solution by using the following procedure. Sodium orthovanadate (2 ml of 100 mM; ICN; Aurora, OH) solution was activated by mixing with 2 ml of a 100 mM hydrogen peroxide solution (30% vol/vol; Ajax Chemicals; Auburn, NSW, Australia) and the reaction allowed to proceed for 15 min at room temperature. The reaction was terminated by the addition of 400 U/ml of catalase (15 min), after which the pervanadate solution was ready for use. Monoclonal FITC-conjugated anti-phosphotyrosine antibody was obtained from Sigma.

Statistical Analysis

Data are expressed as means ± SE. Simple comparisons of means ± SE were performed with the use of Student's t-test. Multiple comparisons were determined by using analysis of variance with the paired least-squares difference post hoc test. Values of P < 0.05 were considered to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Specificity of FITC-Conjugated Anti-Phosphotyrosine for Labeling Phosphotyrosine Residues

Isolated, unpressurized cremaster muscle arterioles were incubated with 10 µM of phenylephrine in the presence of the tyrosine phosphatase inhibitor pervanadate (100 µM) for 10 min at room temperature, and then fixed and incubated with FITC-conjugated anti-phosphotyrosine. The mean fluorescence intensity of these arterioles was 122.6 ± 14.6 units (n = 3). In contrast, when the fluorescent antibody was preabsorbed with excess phosphotyrosine (1 mM) for 60 min before incubation with the blood vessel, the fluorescence intensity after phenylephrine and pervanadate treatment was 32.8 ± 0.3 units (n = 3), a significant reduction of ~75% (P < 0.05, Student's t-test).

In further experiments, pressurized arterioles were maintained at 30 mmHg for 20 min before being subjected to an acute increase in intraluminal pressure to 100 mmHg, which was maintained for 15 min. Vessels were then fixed and incubated with FITC-conjugated anti-phosphotyrosine as described above. The mean fluorescence intensity of these vessels was 22.4 ± 2.4 units (Fig. 1). The addition of pervanadate (100 µM) to the superfusate for the final 5 min of the pressure step caused a large increase in pressure-induced anti-phosphotyrosine fluorescence (Fig. 1). The tyrosine kinase inhibitor tyrphostin A47 (30 µM) reduced the pressure-induced increase in anti-phosphotyrosine fluorescence both in the absence and presence of pervanadate (Fig. 1).


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  		Fig. 1.   Effect of pervanadate or tyrphostin A47 on fluorescence intensity of rat cremaster arterioles incubated with fluoroscein isothiocyanate (FITC)-conjugated antibody to phosphotyrosine after a pressure step from 30 to 100 mmHg. Vessels were maintained at 100 mmHg for 15 min before being fixed and incubated with the antibody. Fluorescence intensity was subsequently measured with the use of confocal laser scanning microscopy. Bars are means ± SE. *P < 0.05, significant difference from control (Student's t-test). **P < 0.05, significant interaction between pervanadate and tyrphostin [analysis of variance (ANOVA), followed by Student's t-test].

Time Course of Phosphotyrosine Accumulation After Pressure Stimulus or ANG II Treatment

Isolated segments of arteriole were maintained at a transmural pressure of 30 mmHg for 20 min in the presence of 1 µM of pervanadate to inhibit endogenous tyrosine phosphatase activity. A lower concentration of pervanadate was used in this protocol compared with that described above, to allow measurement of the myogenic response. In the continued presence of pervanadate, the transmural pressure was raised to 100 mmHg in an acute step. The vessels were fixed for 15 s, 30 s, or 1, 2, 5, 10, 20, or 60 min after the step and the vessel diameter recorded at the time. Phosphotyrosine levels were measured with the use of FITC-conjugated antiphosphotyrosine and measurement of fluorescence intensity as described in MATERIALS AND METHODS. Figure 2 shows the vessel diameter after the pressure step in relation to the intensity of anti-phosphotyrosine fluorescence at each time point. After the initial distension caused by the pressure step, the vessels exhibited a myogenic contraction that was largely completed within 1 min of the step. However, the rate of phosphotyrosine generation was markedly slower. Anti-phosphotyrosine fluorescence was not significantly elevated above that of control vessels (maintained at 30 mmHg) during the contractile phase; however, there was a significant increase in phosphotyrosine after the myogenic contraction was completed. After 30 s at 100 mmHg, anti-phosphotyrosine fluorescence intensity was 109.0 ± 8.9% of control (P > 0.05); after 10 min at 100 mmHg, this intensity had risen to 203.0 ± 9.9% (P < 0.05); by 60 min, anti-phosphotyrosine fluorescence intensity was 396.3 ± 23.0%. Vessel diameter did not change significantly between the 1- and 20-min time points after the pressure step; however, further constriction was evident after 60 min (Fig. 2). In vessels maintained at 30 mmHg for 60 min, in the presence of 1 µM of pervanadate, neither diameter nor the level of anti-phosphotyrosine fluorescence intensity were altered over the period of the experiment (Fig. 2).


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  		Fig. 2.   Time course of changes in fluorescence pixel intensity and vessel diameter of rat cremaster arterioles incubated with FITC-conjugated antibody to phosphotyrosine after a pressure step from 30 to 100 mmHg (time 0). Experiments were performed in the presence of pervanadate (1 µM). Vessels were maintained at 100 mmHg for the period indicated before being fixed and incubated with the antibody. Also shown are the diameter and pixel intensity of vessels maintained at 30 mmHg for the duration of the experiment. Fluorescence intensity was subsequently measured by using confocal laser scanning microscopy. Points represent the means ± SE. *P < 0.05, significant difference from time 0 (ANOVA, followed by t-test).

For comparison, some vessels were treated with ANG II (0.1 µM) for 30 s, 15 or 30 min in the absence of pervanadate. In contrast to the pressure step, ANG II evoked a rapid increase in anti-phosphotyrosine fluorescence, which was maintained over 30 min. Vessels also demonstrated a rapid constriction in response to ANG II but showed marked tachyphylaxis over the time course (Fig. 3).


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  		Fig. 3.   Time course of changes in fluorescence intensity and vessel diameter of rat cremaster arterioles incubated with FITC-conjugated antibody to phosphotyrosine after application of angiotensin II (0.1 µM; time 0). Vessels were maintained at 70 mmHg for the duration of the experiment before being fixed at the time points indicated and incubated with the antibody. Fluorescence intensity was subsequently measured by using confocal laser scanning microscopy. Points represent means ± SE. *P < 0.05, significant difference from time 0 (ANOVA, followed by t-test).

Effect of Verapamil on Time Course of Phosphotyrosine Accumulation After Pressure Stimulus

A similar series of experiments were performed in the presence of the voltage-sensitive Ca2+-channel blocker verapamil (5 µM; Fig. 4). Verapamil abolished the myogenic response after the pressure step from 30 to 100 mmHg. After 30 s, the amount of phosphotyrosine formed was not statistically different from vessels in the absence of verapamil, but 20 min after the step, verapamil increased the amount of phosphotyrosine formed, compared with vessels without verapamil (P < 0.05; Fig. 4). At the 60-min time point, the anti-phosphotyrosine fluorescence intensity was no different to vessels in the absence of verapamil. Verapamil increased the formation of phosphotyrosine in vessels maintained at 30 mmHg over a 60-min period (Fig. 4).


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  		Fig. 4.   Effect of verapamil (5 µM) on time course of changes in fluorescence intensity of rat cremaster arterioles incubated with FITC-conjugated antibody to phosphotyrosine after a pressure step from 30 to 100 mmHg. Also shown are control experiments, where vessels were maintained at 30 mmHg for 0.5 or 60 min. All experiments were performed in the presence on pervanadate (1 µM). Vessels were maintained at 30 or 100 mmHg for the period indicated before being fixed and incubated with the antibody. Fluorescence intensity was subsequently measured using confocal laser scanning microscopy. Bars represent means ± SE. *P < 0.05, significant effect of verapamil on fluorescence intensity compared with control (100 mmHg) after 20 min; **P < 0.05, significant effect of verapamil on fluorescence intensity compared with control (30 mmHg) after 60 min (ANOVA, followed by t-test).

Pressure-Dependent Increases in Phosphotyrosine Formation and Effect of Endothelium Removal

In this series of experiments, vessels were maintained at 30 mmHg for 20 min, and pressure was then acutely stepped to 10, 70, or 100 mmHg. After 15 min at the test pressure, vessels were fixed and phosphotyrosine formation measured. Initial studies were performed in the absence of pervanadate. Arterioles stepped to 10 mmHg showed a reduced diameter as this was below the pressure range in which myogenic responses occur in this vessel (30-160 mmHg); the elastic elements in the vessel wall result in the arteriole partially collapsing in the absence of sufficient distending pressure. Vessels stepped to 70 or 100 mmHg also reduced their diameter as a result of myogenic constriction (Fig. 5A). There was a low level of phosphotyrosine in vessels maintained at 10 mmHg and there was no significant increase, compared with these vessels, in arterioles maintained at 70 mmHg (Fig. 6). Vessels stepped to 100 mmHg did show a small, but significant increase in anti-phosphotyrosine fluorescence intensity compared with vessels at 10 or 70 mmHg (P < 0.05; Fig. 6).


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  		Fig. 5.   Time course of changes in diameter of rat cremaster arterioles after a pressure step from 30 mmHg to 10, 70, or 100 mmHg. A: diameter in vessels maintained at the test pressure for 15 min. B: pervanadate (100 µM) was added for the final 5 min after the pressure step. Diameter was measured with the use of video calipers on a video image of the vessel. Shown is vessel diameter immediately before the pressure step (at 30 mmHg) and diameter 10 and 15 min after the step at the three test pressures. Points represent the means ± SE. *P < 0.05, significant difference from diameter prestep (30 mmHg); #P < 0.05, significant effect of pervanadate on diameter from test pressure for 10 min (Student's t-test).



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  		Fig. 6.   Fluorescence intensity of rat cremaster arterioles 15 min after a pressure step from 30 to 10, 70 or 100 mmHg. At the conclusion of this period, vessels were fixed and incubated with FITC-conjugated anti-phosphotyrosine, and fluorescence intensity was subsequently measured by using confocal laser scanning microscopy. Fluorescent pixel intensity is shown in the absence and presence of pervanadate (100 µM) for the final 5 min of the pressure step. Points represent means ± SE. *P < 0.05, significant increase in fluorescence intensity above vessels at 10 mmHg (Student's t-test). **P < 0.05, significant increase in fluorescence intensity above vessels at 10 and 70 mmHg (Student's t-test).

These experiments were repeated with pervanadate (100 µM) added to the bathing solution for the final 5 min of the pressure step to prevent metabolism of phosphotyrosine residues by endogenous phosphatases. This concentration of pervanadate constricted the vessels to a similar degree regardless of the preexisting pressure (Fig. 5B). At each test pressure, the level of anti-phosphotyrosine fluorescence intensity was significantly greater than in the absence of pervanadate. In the presence of pervanadate, there was a significant increase in phosphotyrosine formation with increasing pressure, from 10 to 70 to 100 mmHg (Fig. 6). Examples of confocal images of pervanadate-treated vessels fixed at the test pressures and incubated with FITC-conjugated anti-phosphotyrosine are shown in Fig. 7.


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  		Fig. 7.   Top: confocal laser microscope images of vessels maintained at 10 (left), 70 (middle), or 100 mmHg (right) for 15 min, with 100 µM of pervanadate present for the final 5 min, followed by fixation and incubation with FITC- (top) or tetramethyl rhodamine isothiocyanate-conjugated anti-phosphotyrosine (see MATERIALS AND METHODS). Bottom: images from vessels with intact endothelium (left) or endothelium denuded (right) at 100 mmHg, in the presence of 100 µM of pervanadate.

In four experiments, the endothelium was removed from the vessel and the 30- to 100-mmHg pressure step performed as previously described. Removal of the endothelium was confirmed by abolition of the dilatory response to acetylcholine (10 µM). There was no significant difference between deendothelialized arterioles and those with an intact endothelium in the amount of anti-phosphotyrosine fluorescence present following the 30- to 100-mmHg pressure step (with endothelium, 47.2 ± 1.2 units; no endothelium, 42.0 ± 7.5 units, n = 4 for both; P > 0.05, t-test). Images of cells from vessels with or without endothelium are shown in Fig. 7. In addition, removal of the endothelium did not significantly alter the myogenic contraction resulting from the pressure step (data not shown).

Effect of Removal of Extracellular Ca2+, Ca2+ Channel Blockade, and Forskolin on Phosphotyrosine Formation

Removal of Ca2+ from the superfusate abolished the myogenic contraction caused by a pressure step from 30 to 70 or 100 mmHg. Under these conditions, the pressure step caused the arterioles to dilate passively (Fig. 8A). The additional contraction caused by pervanadate was also abolished in the absence of extracellular Ca2+. Vessels subjected to a decrease in pressure to 10 mmHg continued to collapse to a narrower diameter in the absence of extracellular Ca2+, emphasizing the passive nature of this response (Fig. 8). Despite the absence of extracellular Ca2+ and the absence of an active mechanical response, anti-phosphotyrosine fluorescence was increased at 10 and 70 mmHg, but not at 100 mmHg compared with vessels in Ca2+-replete Krebs (Fig. 9).


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  		Fig. 8.   Time course of changes in diameter of rat cremaster arterioles after a pressure step from 30 mmHg to 10, 70, or 100 mmHg, in Ca2+-free physiological saline solution (PSS) (A) or in the presence of 5 µM of verapamil (B). Vessels were maintained at the test pressure for 15 min and pervanadate (100 µM) was added for the final 5 min. Diameter was measured using video calipers on a video image of the vessel. Shown is vessel diameter immediately before the pressure step (at 30 mmHg) and diameter 10 and 15 min after the step at the 3 test pressures. Points represent means ± SE. *P < 0.05, significant difference from diameter at prestep (30 mmHg); #P < 0.05, significant effect of pervanadate on diameter from test pressure for 10 min (Student's t-test).



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  		Fig. 9.   Fluorescence intensity of rat cremaster arterioles 15 min after a pressure step from 30 to 10, 70, or 100 mmHg, in normal PSS, Ca2+-free PSS, or PSS containing 5 µM of verapamil. In all vessels, pervanadate (100 µM) was added to the PSS for the final 5 min of the pressure step. At the conclusion of this period vessels were fixed and incubated with FITC-conjugated anti-phosphotyrosine, fluorescence intensity was subsequently measured by using confocal laser scanning microscopy. *P < 0.05, significant increase in fluorescence intensity above the pressure-matched control (Student's t-test).

Verapamil (5 µM) abolished the myogenic constriction associated with pressure steps from 30 to 70 or 100 mmHg (Fig. 8). The constrictor effect of 100 µM pervanadate was unaffected by verapamil (Fig. 8). Consistent with the data obtained after removal of extracellular Ca2+, verapamil increased the amount of anti-phosphotyrosine fluorescence at 70 and 100 mmHg, compared with vessels in the absence of verapamil (Fig. 9). However, the relationship between pressure and fluorescence intensity was maintained; in effect, verapamil caused a leftward shift in the pressure-fluorescence intensity curve.

A similar series of experiments were performed using forskolin (0.1 µM), a vasodilator that activates adenylate cyclase. In these experiments the effect of forskolin on diameter and vessel fluorescence intensity was examined on a single pressure step from 30 to 100 mmHg. Forskolin dilated the vessels to 94.1 ± 1.7% (n = 4) of maximum (passive) diameter at 30 mmHg and prevented myogenic constriction. Prevention of the myogenic response with forskolin also increased anti-phosphotyrosine fluorescence at 100 mmHg. Control fluorescence intensity, at 100 mmHg in the presence of pervanadate, was 85.4 ± 10.4 units; in the presence of forskolin, this became 127.9 ± 15.2 units (P < 0.05, n = 4 for each). The contraction induced by pervanadate was not altered by forskolin (51.9 ± 9.9% of passive, 36.4 ± 5.8% of control, and n = 4 for both).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The study examined tyrosine phosphorylation in skeletal muscle arterioles exhibiting spontaneous tone and undergoing myogenic constriction in response to acute increases in intra-luminal pressure. Detection of phosphorylation was accomplished by using a technique in which phosphotyrosine residues were labeled with a fluorophore-conjugated antibody and the fluorescence intensity of the vessels subsequently measured by using confocal scanning laser microscopy. This technique has been used previously (23) to demonstrate changes in tyrosine kinase activity in vascular endothelial cells in situ. The results suggest that increased intraluminal pressure in skeletal muscle arterioles causes an increase in tyrosine kinase activity in vascular smooth muscle cells. However, the slow accumulation of pressure-induced tyrosine phosphorylation suggests this enzyme activity is not involved in acute myogenic constriction, which follows an increase in intravascular pressure. Interventions that prevented the myogenic constriction, such as the removal of extracellular Ca2+ and inhibition of Ca2+ entry into vascular smooth muscle cells, caused a greater increase in pressure-induced tyrosine phosphorylation, suggesting that perhaps mechanical factors, such as vessel wall tension or cell stretch, are key regulators of tyrosine kinase activity. Conceivably, the increased tyrosine phosphorylation represents events involved in longer-term responses, such as maintenance of myogenic tone or possibly adaptive smooth muscle growth responses.

Preliminary studies tested the reliability of using FITC-conjugated anti-phosphotyrosine to measure tyrosine phosphorylation in arterioles. Preabsorption of the antibody with excess phosphotyrosine prevented detection of the increase in arteriolar fluorescence caused by phenylephrine. Furthermore, the increase in fluorescence caused by stepping intraluminal pressure from 30 to 100 mmHg was greatly attenuated by the tyrosine kinase inhibitor tyrphostin A47. This suggests that the increase in fluorescence was caused by increased tyrosine kinase activity, rather than solely by inhibition of tyrosine phosphatase caused by pervanadate. However, the large increase in anti-phosphotyrosine fluorescence caused by pervanadate suggests that there was considerable endogenous tyrosine phosphatase activity under basal conditions. Studies (10) in other tissues have also shown a high level of tyrosine phosphatase activity under basal or unstimulated conditions.

Tyrosine kinase activity in the cremaster muscle arterioles increased slowly, relative to the time course of myogenic contraction, after an acute increase in intraluminal pressure. The level of tyrosine phosphorylation was not significantly altered during the acute contractile phase, which was largely complete at ~1 min. In contrast, ANG II, a known activator of tyrosine kinases (29), induced a rapid accumulation of phosphotyrosine, suggesting that the slow time course of accumulation stimulated by increased pressure was not an artifact of the technique. The observations suggest tyrosine phosphorylation is not critically involved in the acute myogenic contraction, at least not in first-order arterioles from rat cremaster muscle. Indeed, these observations complement our earlier study (27), in which tyrosine kinase inhibitors did not alter immediate myogenic contraction or the increase in arteriolar [Ca2+] in cremaster muscle arterioles after an acute pressure step. In the present study, levels of tyrosine phosphorylation did not increase significantly above the prestep level until ~5 min after the step, and continued to increase over the following hour, suggesting that increased tyrosine kinase activity may be involved in longer-term responses of the arterioles to increased intraluminal pressure. In our previous study (27), we reported that tyrosine kinase inhibition, while not preventing acute myogenic contraction, did cause dilation of vessels with an established level of myogenic tone. Together with our current observations regarding the relatively slow increase in tyrosine phosphorylation, these studies suggested that tyrosine kinases may be involved in the prolonged maintenance of arteriolar smooth muscle tone after an increase in vessel pressure. Alternatively, only a proportion of the delayed increase in tyrosine phosphorylation observed in the current study may be related to smooth muscle tone, with other tyrosine-phosphorylated proteins possibly being involved in alternative responses to an increase in vessel pressure, such as protooncogene expression (1) or cellular hypertrophy (4). In contrast with these current findings, studies (34, 35) in the same tissue preparation found phosphorylation of myosin light chain was increased within 30 s after a pressure step and inhibition of myosin light-chain kinase attenuated myogenic contraction.

Tyrosine phosphorylation in the vessels increased with vessel transmural pressure, a relationship which was apparent in the absence of pervanadate (between 70 and 100 mmHg) but significantly enhanced in the presence of the tyrosine phosphatase inhibitor. A further increase in tyrosine phosphorylation was observed at 10 or 70 mmHg, when myogenic constrictions and tone were abolished by removal of extracellular Ca2+ from the superfusate or blockade of voltage-sensitive Ca2+ channels (5, 16, 32). The vasodilator forskolin, which also abolished myogenic contraction, increased tyrosine phosphorylation at 100 mmHg. Taken together, these observations suggest that vessel wall tension, rather than pressure alone, may be an important regulator of tyrosine kinase activity within smooth muscle cells. Wall tension is regarded as a likely candidate for a physiological regulator of myogenic activity in arterioles (6, 18, 34). Myogenic constriction of arterioles assists in these vessels attenuating the pressure-induced increase in the level of wall tension. Another possibility is that the degree of smooth muscle cell stretch may activate tyrosine kinase because abolition of the myogenic response would have increased smooth muscle cell stretch in response to pressure and increases in wall tension of nonmyogenically active smooth muscle, such as sheep trachealis (28) and rat carotid artery (12) have also been found to stimulate tyrosine kinase activity. However, simple cellular stretch cannot fully explain the increase in tyrosine kinase activity in the present study because anti-phosphotyrosine fluorescence increased with pressure when the myogenic response was functional. Removal of extracellular Ca2+ caused a particularly large increase in tyrosine phosphorylation in vessels maintained at 10 mmHg, which possessed the least active wall tension under the study conditions. It is possible that exposure to Ca2+-free extracellular solution containing EGTA leads to cellular effects other than maximal relaxation of the contractile proteins.

A potential criticism of the techniques used in the present study is the inability to identify individual proteins undergoing tyrosine phosphorylation. For example, whereas our observations suggested that tyrosine kinase activity increases slowly after raised arteriolar wall tension, a previous study (28) in sheep trachealis muscle showed increased tyrosine phosphorylation of the integrin-associated proteins focal adhesion kinase (pp125FAK) and paxillin within 30 s after increased muscle tension. Similarly, in porcine carotid artery, tyrosine phosphorylation of mitogen-activated protein kinase increased within 30 s of cell stretch (12). Integrins are transmembrane proteins that are activated by binding with extracellular matrix elements and are regarded as prime candidates for the "tension sensor" in vascular smooth muscle cells, which help convert an increase in cell wall tension to a contractile, myogenic response. Disruption of integrin-matrix binding in cremaster arterioles with RGD peptides reduced myogenic tone and inhibited the rise in [Ca2+]i after a pressure step (5, 22, 33). Because there are several proteins that undergo tyrosine phosphorylation as a result of integrin activation, including focal adhesion kinase (itself a tyrosine kinase), paxillin, CD45, and talin (17, 26), it seems likely that these proteins would undergo rapid tyrosine phosphorylation after an increase in vessel wall tension.

Whereas further studies are required to identify individual phosphoproteins, there are several possible explanations for the apparently "slow" increase in total tyrosine phosphorylation observed in the present study. As mentioned earlier, the tissue preparations used by Tang et al. (28) and Franklin et al. (12) do not demonstrate myogenic activity in response to increases in tension, in contrast to skeletal muscle arterioles. Acute myogenic constriction lowers wall tension and reduces the degree of cell stretch; therefore, it could be reasonably expected to attenuate the onset of a tension or stretch-induced intracellular event. Indeed, the abolition of the myogenic response increased the rate of phosphotyrosine formation and the amount of phosphotyrosine formed at higher pressures. This suggests that an increase in vessel wall tension, without a compensating myogenic constriction, accelerates the increase in tyrosine kinase activity and may explain the differences in our observations and those made in nonmyogenic tissues. Allen et al. (2) also demonstrated that myogenic constriction inhibited the pressure-induced increase in expression of c-myc and c-fos in cannulated rat mesenteric arterioles. As mentioned previously, a slow onset of tyrosine kinase activity supports our earlier finding with tyrosine kinase inhibitors, which failed to alter the acute phase of myogenic constriction in the cremaster arterioles but dilated vessels with spontaneous myogenic tone. Therefore, the slow increase in anti-phosphotyrosine fluorescence in the cremaster arterioles after a pressure step may accurately reflect the rate of tyrosine phosphorylation in pressurized, myogenic vascular smooth muscle. Alternatively, some proteins, such as focal adhesion kinase, may be rapidly phosphorylated but represent too small a fraction of the total population of tyrosine-phosphorylated proteins to impact the mass phosphotyrosine fluorescence assay used in the present studies.

In summary, elevated wall tension in pressurized skeletal muscle arterioles leads to increased tyrosine phosphorylation, most likely due to increased tyrosine kinase activity. The slow rate of increase in tyrosine phosphorylation suggests the enzymes are not involved in the acute effects of increased intravascular pressure, such as myogenic constriction. In fact, myogenic activity may inhibit tyrosine kinase activity because constriction lowers wall tension, and abolition of myogenic constriction increased tyrosine kinase activity in response to raised intraluminal pressure. Thus tyrosine kinase may play a role in longer-term responses to raised pressure, such as maintenance of myogenic tone or possibly pressure-stimulated growth of smooth muscle cells.


    ACKNOWLEDGEMENTS

We thank Dr. Sharmini Rajanayagam for critically reviewing this paper before submission.


    FOOTNOTES

This study was supported by a grant from the National Health and Medical Research Council of Australia.

Address for reprint requests and other correspondence: T. Murphy, Microvascular Biology Group, School of Medical Sciences, RMIT Univ., Plenty Rd., Bundoora, Victoria 3083, Australia (E-mail: t.murphy{at}rmit.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 July 2000; accepted in final form 23 April 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 281(3):H1047-H1056
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


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