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Hypotonic solutions cause vasoconstriction in rat tail arteries, due largely to activation of L-type calcium channels (CaV1.2). We studied possible roles of tyrosine kinases, particularly src family kinases (SFK) and extracellular signal-related kinases (ERK1/2), in this response.
Rat tail arteries were mounted on a myograph for measurement of isometric force. Arteries were bathed in isosmotic physiological saline solution (300 mOsm/l) containing 50 mmol/l mannitol and were stimulated by a hyposmotic solution containing 0 mmol/l mannitol (PSS-M). Activation of tyrosine kinases and ERK1/2 by hyposmotic solution was examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting on rat tail artery lysates with specific phospho-antibodies.
Western blotting showed SFK src and yes present in rat tail artery. PSS-M increased tyrosine phosphorylation of several proteins, including SFK and ERK1/2. Genistein blocked phosphorylation of SFK and ERK1/2 by PSS-M. In isolated arteries PSS-M caused a contraction inhibited by the tyrosine kinase inhibitor, genistein, and three structurally different selective SFK inhibitors, herbimycin-A, PP1 and SU6656. Mitogen-activated protein kinase kinase inhibitor PD98059 or selective inhibitors of platelet-derived growth factor receptor (AG1296) and epidermal growth factor receptor (AG1478) had no effect on contraction induced by a hypotonic solution.
Hyposmotic conditions activate SFK, src and yes, and contract rat tail artery by a SFK-dependent mechanism. ERK1/2 are activated by the hypotonic solution, but do not play a role in the contractile response. SFK modulation of CaV1.2 may be an important mechanism mediating vasoconstriction to mechanical stimuli in vascular smooth muscle.
Mechanical forces, causing stretching or swelling of cells, induce contraction in vascular smooth muscle [1,2]. This response underlies myogenic tone  and autoregulation of blood flow , and contributes to the regulation of normal and elevated blood pressure.
Hyposmotic stimuli cause swelling of cells and result in contraction of blood vessels. The mechanism by which hypotonic solutions cause contraction is similar to that of myogenic tone, in that contractions are preceded by membrane depolarization and increased intracellular calcium . L-type calcium channels (CaV1.2) play an essential role in vasoconstriction in response to both hyposmotic stimuli [5,6] and increased transmural pressure . We recently showed that rat isolated tail arteries contracted on exposure to a hyposmotic solution, and that this was largely dependent on Cav1.2 . The signalling mechanisms contributing to activation of CaV1.2 following such stimuli, however, are not clear.
Tyrosine kinases are important regulators of cell function . They are classified into two groups: receptor-linked tyrosine kinases [e.g. epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor (PDGFR)], and nonreceptor tyrosine kinases, such as the src family kinases (SFK). SFK consist of src, fyn, yes, lck, blk, hck, fgr, yrk and lyn. Of these, src, fyn and yes are ubiquitously expressed, whereas the other kinases show a distribution restricted to the central nervous system or the immune system . Interestingly, nonreceptor tyrosine kinase activity is very high in smooth muscle [11,12], and this is in large part attributable to SFK [12,13].
Tyrosine kinases have been well established to play a role in vascular contraction. Receptor-linked tyrosine kinases such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) cause contraction in some blood vessels [14,15]. Tyrosine kinases including SFK also mediate contractions induced by classical vasoconstrictor agonists including angiotensin II  and 5-hydroxytryptamine , although tyrosine kinases are not involved in all agonist-induced vasoconstrictions . In many cell types, mechanical forces including increased pressure  and osmotic stimuli  cause activation of SFK, and it has been suggested that SFK are also involved in pressure-induced contraction . Since SFK increase CaV1.2 opening [22-24], they may link mechanical stimuli to vasoconstriction. The present study was therefore undertaken to investigate whether activation of SFK mediates vasoconstriction induced by hypotonic solutions.
All animal work conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Male Wistar rats (175-250 g) were killed by cervical dislocation and small (~2 mm) segments of the rat main tail artery (internal diameter 400 - 600 μm) were dissected and mounted as rings in an isometric myograph . Arteries were bathed in 10 ml modified Kreb’s physiological saline solution (PSS+M) comprising: NaCl, 100 mmol/l; KCl, 5 mmol/l; CaCl2 6H2O, 1.6 mmol/l; MgCl2, 1 mmol/l; NaHCO3, 20 mmol/l; HEPES, 5 mmol/l; glucose, 5 mmol/l; and mannitol, 50 mmol/l (pH 7.4). The osmolarity of PSS+M was 300 mOsm/l. Hypotonic solution (PSS-M) was identical except for the omission of mannitol. Removal of mannitol reduced the osmolarity of PSS-M to 250 mOsm/l. The osmolarity of all solutions was checked with an osmometer (Osmomat 030; Gonotec, Berlin, Germany) prior to use. Arteries were maintained at 37°C and bubbled with 95% O2 and 5% CO2.
Arteries were allowed to equilibrate for 1 h and then set at a ‘normalized’ internal circumference (0.9 L100), estimated to be 0.9 times the circumference they would maintain if relaxed and exposed to 100 mmHg transmural pressure. This normalization was calculated for each individual vessel on the basis of the passive length-tension characteristics of the artery and the Laplace relationship . All experiments were started by repetitively stimulating vessels for 2 min with a high-potassium solution (KPSS), comprising PSS+M with equimolar substitution of NaCl (118 mmol/l) by KCl, until reproducible contractions were elicited. Contraction to hyposmotic solution was examined by changing the PSS+M to a mannitol-free solution (PSS-M). Contractions under isosmotic or hyposmotic conditions were conducted on a background of a small (~10% maximum) tone induced by the thromboxane mimetic, U46619 (100-200 nmol/l) to prevent tachyphylaxis . Peak contraction was measured following exposure to PSS-M. Typically this occurred between 2 and 6 min following exposure to PSS-M. Prazosin (20 nmol/l) was present throughout all experiments to prevent neural activation of α1-adrenoceptors - the major subtype present in rat tail artery : this concentration of prazosin was sufficient to abolish responses to 10 μmol/l norepinephrine (data not shown). For inhibitor studies, two arteries were studied in parallel, with one artery acting as control. Care was taken to ensure similar levels of background tone were achieved in test and control vessel.
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were used to detect SFK and tyrosine phosphorylation of cellular proteins. Segments of rat tail arteries were exposed to PSS+M or PSS-M in the presence or absence of inhibitors. Arteries were frozen in liquid nitrogen and stored at −80°C. To prepare lysates, frozen arterial segments were re-suspended in ice-cold lysis buffer (200 μl) containing: Tris, 50 mmol/l; NaCl, 150 mmol/l; EGTA, 1 mmol/l; NP-40, 1% v/v; sodium deoxycholate, 19% w/v; aprotinin, leupeptin and pepstatin, all 1 μg/ml; and PMSF, 200 μmol/l); the suspensions were then homogenized with a glass-on-glass homogenizer. Homogenates were centrifuged at 4°C at 15 000 rpm for 15 min and the supernatant collected. A protein assay was performed on the supernatant (BCA assay kit, Pierce, Illinois, USA) and the sample diluted appropriately. Samples were heated at 95°C for 5 min with SDS sample buffer (Tris–HCl, pH 6.8, 0.3 mmol/l, β-mercaptoethanol 25% v/v, SDS 10% v/v, glycerol 50% v/v, bromophenol blue 0.01% w/w). The samples were stored at −20°C until used for gel electrophoresis.
SDS-PAGE was carried out using a Bio Rad Protean II system (Bio-Rad Laboratories, Hercules, California, USA). A known amount of protein samples and molecular weight markers were loaded into wells and the gel was run in SDS running buffer (Tris 25 mmol/l, glycine 192 mmol/l and SDS 1% w/v, pH 8.3) at 85 V for 2 h. The separated proteins were transferred onto supported nitrocellulose membrane using a Bio-Rad transfer cell using transfer buffer (Tris 25 mmol/l, glycine 192 mmol/l, methanol 20% v/v) at 40 V overnight. In all western blots, equal loading of samples was confirmed by Ponceau S staining. The membrane was blocked for 1-2 h with 5% bovine serum albumin in Tris-buffered saline containing 0.001% v/v Tween 20.
To estimate the size of the bands, standard molecular weight markers (Novagen; Merck Chemicals Ltd, Nottingham, UK) were run along with the samples in all SDS-PAGE. To detect SFK, the blots were probed with specific monoclonal antibodies to src, yes, fyn and lck. A specific antiphosphotyrosine antibody (PY99) was used to detect tyrosine phosphorylation. To detect phosphorylated, active forms of SFK and extracellular signal-related kinases (ERK1/2), antibodies recognizing SFK phosphorylated on Tyr 416 (mouse monoclonal anti-src Tyr416) or recognizing antiphospho-ERK1/2, respectively, were used. Horseradish peroxidase-conjugated rabbit antimouse polyclonal antibody (1/1000 dilution), prepared in 10% milk in Tris-buffered saline and containing 0.001% v/v Tween 20 was used as a secondary antibody. The antigen-antibody complex was visualized using enhanced chemiluminescence and X-ray film. Films were scanned using an imaging densitometer (Bio-Rad Model GS-700; Bio-Rad Laboratories) and the optical density was measured using commercial software (Molecular Analyst version 1.5; Bio-Rad Laboratories) and all optical density results were normalized with respect to isoosmotic control conditions.
Purified active src and yes were used to assess the inhibitory effects of SU6656, PP1 and herbimycin on SFK. Enzyme activity was measured using a nonradioactive tyrosine kinase assay kit (Upstate, Lake Placid, New York, USA) according to the manufacturer’s instructions. Enzyme (100 ng/ml) was allowed to preequilibrate with SU6656 (10 μmol/l), PP1 (1 μmol/l) or herbimycin (500 nmol/l) for 30 min, prior to initiation of the kinase reaction by addition of the substrate (poly-glu-tyr) with ATP (1 mmol/l) and orthovanadate (1 mmol/l) for 30 min. Horseradish peroxidase-conjugated antiphosphotyrosine antibody was added followed by a peroxidase substrate Absorbance was measured using an enzyme-linked immunosorbent assay plate reader at 450 nm. A standard curve was constructed for every assay by using a range of known concentrations of poly-glutyr-phosphate provided with the assay kit. Each experiment was carried out in triplicate and the mean value was calculated.
PY99, anti-src, anti-fyn, anti-lck and anti-yes antibodies were purchased from Santa Cruz Biotechnology, Inc. (www.scbt.com). Antiphospho-src-Y416 antibody and antiphospho ERK1/2 were obtained from Upstate. All other reagents were obtained from Sigma-Aldrich (www.sigmaaldrich.com). Stock solutions of genistein, daidzein AG1296, AG1478 and PD98059 were made up in dry dimethyl sulphoxide, and final concentrations containing less than 0.01% dimethyl sulphoxide were made up in physiological saline. Stock solutions of 2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide (SU6656), (4-amino-5(4-methylphenyl)-7-(4 butyl)pyrazolo[3,4,d] pyrimidine (PP1) and herbimycin-A were made up in ethanol, and final concentrations (containing < 0.1% ethanol) were made up in physiological saline.
All data are presented as the mean ± SEM of n observations. Statistical comparisons of data were made using Student’s t-test for single paired comparisons or one-way repeated-measures analysis of variance with Dunnett’s multiple comparisons test using GraphPad InStat 3.05 (www.graphpad.com) as appropriate. P values less than 0.05 were considered statistically significant.
The presence of src and yes were demonstrated by SDS-PAGE and western blotting using specific anti-src and anti-yes antibodies in rat tail artery lysates (Fig. 1a). The bands corresponded to approximately 60 kDa. fyn and lck were not detected by western blotting.
Western blotting with an antiphosphotyrosine antibody PY99 showed that there are a number of tyrosine phosphorylated proteins in rat tail artery, as shown in the representative blot (Fig. 1b). PSS-M caused an increase in tyrosine phosphorylation of several of these proteins, including bands of approximate molecular weights 60 kDa and 42-44 kDa (Fig. 2a,b). On the basis of their molecular weight, and since they are known to be activated by mechanical stimuli in vascular smooth muscle, these proteins are likely to represent SFK and ERK1/2, respectively. Further studies were therefore undertaken with specific antibodies to confirm that SFK and ERK1/2 were activated by the hyposmotic solution. Western blotting with an antiphospho-ERK antibody showed that PSS-M increased tyrosine phosphorylation of ERK1/2 in rat tail arteries significantly (Fig. 3a). Activation of ERK1/2 was inhibited by 10 μmol/l genistein (Figs (Figs2b2b and and3a)3a) and 10 μmol/l SU6656 (Fig. 3b). Similarly, increased tyrosine phosphorylation of SFK was confirmed using a specific antiphospho-src antibody. Activation of SFK was inhibited by genistein (Fig. 4a). Expression of total immunodetectable src, yes or ERK1/2 was unaffected by the hypotonic solution (Fig. 4b).
Exposure to PSS-M caused a stable submaximal contraction in all arteries studied (Fig. 5) that was equivalent to 58 ± 6% of the contraction in response to KPSS.
Genistein (10 μmol/l) inhibited the contractile response to PSS-M significantly (2.3 ± 2% KPSS, n = 7, P < 0.001) (Fig. 5) - but daidzein (10 μmol/l), an inactive analogue of genistein, had no significant effect (55 ± 4% KPSS, n = 4, P = 0.8).
To further characterize the tyrosine kinases involved in the effect of PSS-M on contraction, we examined the effects of three structurally different inhibitors of SFK (SU6656, 10 μmol/l; PP1, 1 μmol/l; and herbimycin, 500 nmol/l). All three inhibitors of SFK reduced responses to PSS-M (19.1 ± 8% KPSS, 27 ± 7% KPSS and 24 ± 7% KPSS respectively; n = 5-6, P < 0.005 in all cases) (Fig. 5).
Using an in-vitro kinase assay, we also demonstrated that at the above concentrations SU6656, PP1 and herbimycin inhibited purified src and yes to similar extents (src: 35 ± 4%, 50 ± 13% and 55 ± 11%, respectively; yes: 47 ± 8%, 58 ± 11% and 43 ± 5%, respectively; n = 3-4).
In view of the activation of ERK1/2 by PSS-M in the present study, we also examined the effect of mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 and selective inhibitors of PDGFR (AG1296) or EGFR (AG1478) since PDGF and EGF have been shown to cause contraction in some arteries . AG1296 (30 μmol/l), AG1478 (30 μmol/l) and PD98059 (50 μmol/l) had no significant effects (Fig. 5) on the contraction induced by hyposmotic solution. Further, PDGF (100 ng/ml) and EGF (100 ng/ml) failed to contract rat tail artery (n = 3 for both).
We have shown that vasoconstriction in response to a hyposmotic solution is associated with increased tyrosine phosphorylation of several cellular proteins in rat tail artery, including SFK and ERK1/2. Further, we have demonstrated that the contractile response to a hypotonic solution is inhibited by a nonselective tyrosine kinase inhibitor, genistein, and by three structurally unrelated selective inhibitors of SFK, SU6656, PP1 and herbimycin-A. Previous studies have shown that these agents selectively inhibit SFK at the concentrations used in this study [24,27-30], and the efficacy of these agents as inhibitors of src and yes was confirmed in our study. In contrast, inhibitors of MEK or PDGFR or EGFR had no effect on hypotonic vasoconstriction. Endothelial factors do not play an important role in mediating contraction induced by hypotonic solution as we have shown previously under identical conditions that removal of the endothelium, or incubation with NG-monomethyl-l-arginine, oxyhaemoglobin and indomethacin did not influence contractile responses to hyposmotic solutions .
Increasing evidence suggests that tyrosine kinases mediate responses to mechanical stimuli in vascular smooth muscle. Increased transmural pressure causes an increase in tyrosine phosphorylation [31,32]. In addition, osmotic stress has been reported to induce HB-EGF gene expression and JNK activation in cultured rat aortic smooth muscle cells via activation of SFK , and there is extensive evidence in other cell types that hyposmotic stimuli result in SFK activation [20,34]. In support of a role for SFK in mechanically induced contraction, it has been shown that contraction induced by elevated transmural pressure was attenuated by SFK inhibition by herbimycin-A  and that activation of SFK was an early signal in response to elevated pressure . In our studies with rat tail artery, SFK was activated at 5 min by the hypotonic solution, which corresponded to the time of peak contraction following exposure to the hyposmotic solution.
SFK inhibitors attenuated the contractile response to the hypotonic solution. src and/or yes are likely candidates to mediate the contractile response induced by hyposmotic solution as both enzymes are present in this tissue, but due to the lack of selective inhibitors or phospho-antibodies for individual SFK, we were unable at present to further characterize the individual SFK responsible.
EGF and PDGF are known to cause vascular contraction in a number of vascular beds [15,35,36]. In addition, EGFR activation has been shown to have a role in pressure-induced contraction  and in hyposmolarity-induced  contraction. It is also known that PDGFR is activated by mechanical stimuli  and stretch has been reported to increase the expression of PDGF-β receptors in pulmonary artery . We therefore examined whether EGFR and PDGFR have a role in hypotonicity-induced contraction, but selective inhibitors of PDGFR and EGFR did not affect contraction induced by a hypotonic solution in rat tail artery and the growth factors PDGF or EGF did not cause a contraction. These data therefore do not support a role for PDGFR or EGFR in hyposmotic contraction in rat tail artery either via activation of SFK or by another mechanism. Although EGF and PDGF are known to stimulate src and ERK1/2 in some smooth muscle [41-43], there is currently no evidence that EGF or PDGF stimulate SFK or ERK in rat tail artery.
Mechanical stimulation has been reported to activate ERK1/2. Elevation of intravascular pressure in rabbit aorta activated ERK1/2 by a src-dependent mechanism . In rat cerebral arteries the MEK inhibitor PD980509 was shown to inhibit the myogenic response , but this effect was attributed to a nonspecific action of PD98059 on calcium entry. In cremasteric arterioles, increased intra-luminal pressure was shown to activate ERK1/2 at around 15 min, but this was temporally dissociated from the onset of myogenic contraction, which occurred within 5 min . In the present study, phosphorylation of ERK1/2 and the peak contraction induced by hypotonic solution both occurred at approximately 5 min, but inhibition of MEK by PD980509 did not inhibit the hypotonicity-induced contraction. Hyposmolarity-induced phosphorylation of ERK1/2 was attenuated by genistein and the SFK inhibitor SU6656. Although a small degree of inhibition of ERK by SU6656 under resting (isosmotic) conditions was observed in some experiments, this was not statistically significant and may be explained by inhibition by SU6656 of basally active SFK. These results suggest that hyposmotic stimulation in rat tail artery causes ERK1/2 activation as a result of SFK activation, but activation of the ERK pathway does not contribute to contraction. It is possible that ERK activation may play role in growth-related responses to mechanical stimulation such as c-fos and c-jun expression and protein synthesis .
We previously reported that exposure of rat tail artery to hypotonic solutions resulted in vasoconstriction that was largely dependent on CaV1.2 . Similar observations regarding the pivotal role of CaV1.2 in the response to hypotonic solution have been made in rat portal vein , rat aorta , guinea-pig aorta , rat cerebral artery  and canine basilar artery [6,50] suggesting that this is a common mechanism of Ca2+ entry in response to hyposmotic stimuli in a variety of arteries. Previous studies have shown that SFK activate Cav1.2 in vascular smooth muscle [22,23]. Taken together with our previously published data in rat tail artery  showing that, under identical conditions, hypotonicity-induced contraction was inhibited by more than 95% by Cav1.2 antagonists, we suggest that src and/or yes mediate hyposmotic contraction by increasing opening of Cav1.2. This mechanism is consistent with results of a study by Kimura et al. , who reported that cell swelling by hyposmotic solution was associated with an increase in CaV1.2 currents and this effect was blocked by herbimycin. Herbimycin and related benzaquinone ansamycins are generally considered to act by increasing expression of heat shock proteins  and promoting degradation of oncogenic tyrosine kinases . Our data show that herbimycin also acts as a direct inhibitor of SFK, and this action is therefore likely to account for its ability to inhibit responses to hyposmotic stimuli.
A possible common mechanism that could link mechanical stimuli such as cell swelling or increased pressure to activation of SFK and opening of Cav1.2 is integrin activation. Integrins are cell surface receptors that bind extracellular matrix proteins and so provide a mechanical link between the extracellular matrix and the cytoskeleton. Integrin activation recruits and activates SFK. Inhibition of αVβ3 and α5β1 integrins attenuates myogenic responses in skeletal muscle arterioles , and activation of α4β1 integrin increases CaV1.2 in isolated smooth muscle cells via SFK . Activation of SFK by integrins in response to cell swelling could therefore account for activation of CaV1.2 and contraction in response to cell swelling.
SFK inhibitors do not inhibit the contractile response to hyposmotic solution completely, suggesting that other mechanisms may contribute to the contraction to hypotonic stimulation. Ding et al.  recently showed that hyposmotic swelling stimulated L-type calcium channels through activation of protein kinase C in vascular smooth muscle cells; therefore it is possible that protein kinase C or other signalling pathways may also contribute to hypotonicity-induced contraction.
In summary, the SFK src and yes are present in rat tail artery and undergo tyrosine phosphorylation following exposure to a hypotonic solution. A hypotonic solution also induces a contraction that is sensitive to selective inhibitors of SFK. We propose that activation of SFK by a hypotonic solution causes opening of CaV1.2 resulting in vasoconstriction, and that this mechanism may contribute to vasoconstriction in response to a variety of mechanical stimuli, including elevated pressure.
All our studies utilized rat tail artery. This isolated tissue has been widely used in studies of hypertension [56,57], ionic transport  and vascular neuro-effector coupling , as it provides a plentiful supply of smooth muscle for biochemical studies and it is densely innervated. Owing to its involvement in thermoregulation , rat tail artery shows some differences from other arteries in terms of receptor population and intracellular signalling pathways. Nevertheless, given the ubiquity of both SFK and hypotonic contraction in blood vessels, we propose that activation of SFK may represent a common mechanism linking mechanical stimulation to calcium entry through L-type calcium channels and contraction. Further studies using other blood vessels would provide valuable confirmatory evidence on this point.
Mechanical stimuli, such as cell swelling and stretch are important regulators of arterial function. Abnormalities of myogenic tone have been reported to play a role in the aetiology [61,62] and pathological consequences  of hypertension. Our results provide novel insights into the molecular mechanisms by which mechanical forces modulate calcium channel opening through activation of SFK, and suggest the possibility that selective modulation of SFK could be a novel therapeutic strategy in hypertension and cardiovascular disease.
The study was supported by the British Heart Foundation and the Foundation for Circulatory Health.