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Am J Physiol Heart Circ Physiol 288: H1218-H1223, 2005; doi:10.1152/ajpheart.00512.2004
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Impaired flow-induced dilation in mesenteric resistance arteries from receptor protein tyrosine phosphatase-µ-deficient mice

Elianne A. Koop,1 Martijn F. B. G. Gebbink,1,2 Terrence E. Sweeney,4,5 Marie-Jeanne Mathy,3 Harry F. G. Heijnen,2 Jos A. E. Spaan,4 Emile E. Voest,1 Ed VanBavel,4 and Stephan L. M. Peters3

1Department of Medical Oncology and 2Department of Haematology, University Medical Centre Utrecht, Utrecht; 3Department of Pharmacology and Pharmacotherapy and 4Department of Medical Physics, Academic Medical Center Amsterdam, Amsterdam, The Netherlands; and 5Department of Biology, University of Scranton, Scranton, Pennsylvania

Submitted 28 May 2004 ; accepted in final form 5 November 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The transmembrane receptor-like protein tyrosine phosphatase-µ (RPTPµ) is thought to play an important role in cell-cell adhesion-mediated processes. We recently showed that RPTPµ is predominantly expressed in the endothelium of arteries and not in veins. Its involvement in the regulation of endothelial adherens junctions and its specific arterial expression suggest that RPTPµ plays a role in controlling arterial endothelial cell function and vascular tone. To test this hypothesis, we analyzed myogenic responsiveness, flow-induced dilation, and functional integrity of mesenteric resistance arteries from RPTPµ-deficient (RPTPµ–/–) mice and from wild-type littermates. Here, we show that cannulated mesenteric arteries from RPTPµ–/– mice display significantly decreased flow-induced dilation. In contrast, mechanical properties, myogenic responsiveness, responsiveness to the vasoconstrictors phenylephrine or U-46619, and responsiveness to the endothelium-dependent vasodilators methacholine or bradykinin were similar in both groups. Our results imply that RPTPµ is involved in the mechanotransduction or accessory signaling pathways that control shear stress responses in mesenteric resistance arteries.

endothelium; shear stress; protein phosphatase; transgenic mice; mechanotransduction

VASCULAR ENDOTHELIAL CELLS are known to be sensitive to a variety of local physical and chemical stimuli, including shear stress exerted by blood flow. Local shear stress induces a series of endothelial responses that are thought to play an important role in the development of atherosclerosis (28). The responses of endothelial cells to shear stress include changes in cell shape, protein phosphorylation, and gene expression. Moreover, in response to shear stress, endothelial cells produce vasoactive factors, including nitric oxide (NO), that mediate flow-induced dilation (10). Despite considerable research, the mechanisms by which endothelial cells sense shear stress remain poorly understood. Recently, Langille (19) suggested that drag forces on the luminal surface are transmitted to endothelial cell-cell contacts via the cytoskeleton. Considerable reorganization of the adherens junctions in particular leads to the alignment and reorientation of endothelial cells in response to mechanical loads.

We previously identified receptor-like protein tyrosine phosphatase-µ (RPTPµ) and showed that it can mediate homotypic cell-cell adhesion (12, 14, 39). RPTPµ belongs to the subfamily of the MAM (meprin, A5, RPTPµ) domain (2)-containing RPTPs (3). It has been suggested that RPTPµ as well as two other MAM domain-containing RPTPs, RPTP{kappa} and RPTP{lambda}, play a role in the regulation of cell adherens junctions via interactions with cadherins or with {beta}- or {gamma}-catenin (5, 7, 11). RPTPµ associates with catenin p120ctn and decreases phosphorylation of p120ctn upon increasing cell density (29, 40). By regulating p120ctn phosphorylation, RPTPµ may control reorganization of the actin cytoskeleton and/or the clustering of membrane proteins at sites of cell-cell contact.

We generated RPTPµ-deficient (RPTPµ–/–) mice that express the {beta}-galactosidase (LacZ) reporter gene under the control of the RPTPµ promoter (18). Both heterozygous RPTPµ-LacZ as well as RPTPµ–/– mice were viable and fertile and showed no aberrant phenotype under normal circumstances. Analysis of {beta}-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPµ expression in the vasculature, in the cardiac musculature, in neuronal tissues, and in a selected number of other cell types. Further analysis of the vasculature showed RPTPµ expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. The expression pattern of RPTPµ in arterial but not venous endothelial cells and the involvement of this phosphatase in the regulation of endothelial cell adherens junctions suggest that RPTPµ may play a role in controlling arterial endothelial cell function and in the regulation of vascular tone, notably in responses to shear stress.

In this study, we tested the hypothesis that RPTPµ plays a role in endothelial cell-dependent responses in small resistance arteries. We measured the responses to pressure, shear stress, and vasoactive compounds in mesenteric resistance arteries isolated from RPTPµ–/– mice compared with wild-type littermates.


   METHODS
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 METHODS
RESULTS
 DISCUSSION
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Mice. The generation of RPTPµ-null mice has been previously described (18). All experiments were performed using RPTPµ-LacZ+/– or RPTPµ-LacZ–/– (hereafter referred to as RPTPµ–/–) mice backcrossed 10 times in the FVB background. Mice were genotyped using Southern blot analysis (18). Wild-type and RPTPµ–/– mice used for all experiments here described were comparable in age, 20 ± 4 vs. 20 ± 4 wk, respectively, and weight, 31 ± 2 vs. 29 ± 2 g, respectively. Mice were kept in a controlled dark-light cycle and had access to pellet food and water ad libitum. Animal welfare was in accordance with institutional guidelines.

{beta}-Galactosidase staining in adult mice. Four-month-old RPTPµ-LacZ+/– mice were killed by cervical dislocation, and the mesentery was isolated. The mesentery was fixed in 1% paraformaldehyde and 0.2% glutaraldehyde for 1 h at 4°C and stained for 2 h at 30°C with 1 mg/ml X-gal [in 5 mmol/l K3Fe(CN)6, 5 mmol/l K4Fe(CN)6, and 2 mmol/l MgCl2]. The mesentery was postfixed with 2% paraformaldehyde, and whole mount pictures were made using a digital camera.

Immunoelectron microscopy. Human tissues were used for immunoelectron microscopy because the monoclonal antibody specifically recognizes human RPTPµ protein (13). Small mesenteric arteries were excised from human tumor-containing colon tissue immediately after colon resection. The small arteries were excised from the healthy tissue near the resection area. Arteries were fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1 mol/l sodium phosphate buffer (pH 7.4) for 2 h at room temperature. After being washed in 0.1 mol/l sodium phosphate buffer, samples were embedded in 10% gelatin, cooled on ice, and cut into 1-mm3 blocks in a cold room. The blocks were infused with 2.3 mol/l sucrose at 4°C for 24 h, frozen in liquid nitrogen, and stored until cryoultramicrotomy. Fifty-nm-thick cryosections were cut at –120°C using an Ultracut S ultramicrotome (Leica). The sections were collected on formvar-coated grids using a mixture of 1.8% methylcellulose and 2.3 mol/l sucrose (20) and incubated with primary monoclonal antibody 3D7 against RPTPµ (13) and protein A gold (34). Rabbit anti-mouse antibody was used as a bridging antibody. After being labeled, the sections were fixed with 1% glutaraldehyde, counterstained with uranyl acetate, and embedded in methyl cellulose-uranyl acetate. The sections were viewed in a JEOL 1200EX electron microscope.

Blood pressure and heart rate measurements. Blood pressure was measured as described previously (41). In brief, female mice were anesthetized with an induction dose of 0.075 ml/10 g ip containing 12.6 mg/100 g body wt ketamine, 20 µg/100 g body wt medetomidine, and 50 µg/100 g body wt atropine. Anesthesia was maintained by continuous infusion at a rate of 0.1 ml·10 g–1·h–1 containing 3.6 mg/100 g body wt ketamine, 4 µg/100 g body wt medetomidine, and 7.5 µg/100 g body wt atropine. After anesthesia was induced, a tracheotomy was performed, and mechanical ventilation was started at a rate of 100 breaths/min. The carotid artery was cannulated with a heparinized saline-filled catheter, which was connected to a blood pressure transducer. Mouse body temperature was maintained at 37.5°C with the use of rectal temperature monitoring, a temperature-controlled heating pad, and an infrared lamp.

Wire myograph experiments. The vasoconstrictor and relaxant effects of phenylephrine, U-46619, methacholine, and bradykinin on small resistance mesenteric arteries of knockout and wild-type mice were investigated using an isometric wire myograph. Both male and female mice were anesthetized with a mixture of ketamine (125 mg/kg ip) and xylazine (7.5 mg/kg ip). The mesenteric vascular bed was isolated, dissected free from its connective tissue, and transferred to physiological Tyrode solution [composition (in mmol/l) of 118.5 NaCl, 4.7 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 KH2PO4, 25 NaHCO3, 5.5 glucose, and 0.024 EDTA; oxygenated with 95% O2-5% CO2] at room temperature. Small segments of the first-branch mesenteric artery were prepared and mounted in an isometric wire myograph (Danish Myo Technology; Aarhus, Denmark). The preparations were equilibrated for 15 min, and, subsequently, the diameter was determined by a normalization procedure, according to Mulvany and Halpern (24). In this procedure, passive tension of the relaxed vessel is measured as a function of internal circumference, as set by the distance between the two wires. From these data and using the Laplace relationship, the equivalent diameter at a distending pressure of 100 mmHg is determined. The passive tension was set to 5 mN/mm (4). After an additional 15-min equilibration period, the preparations were exposed three times to a depolarizing Tyrode solution (equimolar substitution of 120 mmol/l KCl for NaCl) for 5 min with a 20-min interval. Hereafter, cumulative concentration response curves were constructed for the {alpha}1-adrenoceptor agonist L-phenylephrine and the thromboxane A2 receptor agonist 9,11-dideoxy-11{alpha},9{alpha}-epoxymethanoprostaglandin F2{alpha} (U-46619). Endothelium-dependent and -independent relaxations were studied by adding 10 µmol/l methacholine, 1 µmol/l bradykinin, or 10 µmol/l sodium nitroprusside (SNP), respectively, after precontraction with a submaximal concentration phenylephrine (1 µmol/l). With the use of GraphPad Prism (Graphpad; San Diego, CA), concentration-response curves for the different agonists were fitted to log concentration-response data of individual experiments.

Shear stress experiments. Male mice were anaesthetized with ketamine (125 mg/kg ip) and xylazine (7.5 mg/kg ip). The mesentery was isolated and immediately placed in cold (4°C) HEPES-buffered physiological salt solution [containing (in mmol/l) 10 HEPES, 142.0 NaCl, 4.7 KCl, 1.2 MgSO4, 2.0 CaCl2, 1.2 NaH2PO4, 5.0 glucose, and 2.0 pyruvate; pH 7.35 ± 0.02]. First-branch arteries were isolated and cannulated. Perfusion fluid contained 1% bovine serum albumin. Pressure in both cannula reservoirs was computer controlled. Internal diameter of the vessels was continuously measured using a video technique. The temperature was controlled and maintained constant at 38.5–38.8°C, the in vivo rectal temperature of mice (31). The intraluminal flow medium contained the HEPES-buffered physiological salt solution with 1% albumin. All agonists used during the experiments were added to the superfusion medium (HEPES-buffered physiological salt solution without albumin). We used a HEPES buffer in these experiments because carbonate-buffered systems would require carbogenation, which is difficult to apply to the luminal compartment of cannulated resistance vessels.

Internal diameter of the vessels was between 260 and 330 µm when vessels were fully dilated and pressurized to 75-mmHg mean pressure. Unlike vessels below 100 µm, such vessels do not always develop a substantial level of basal tone. To have a consistent level of tone in all protocols, we preconstricted all vessels with 20 nmol/l U-46619 at 75 mmHg. Arteries suitable for the experiments were selected on their endothelial cell response as measured by their dilation to 0.1 µmol/l bradykinin.

Passive pressure-diameter relations and myogenic responsiveness were studied by increasing the intravascular pressure by steps from 20 to 100 mmHg in the absence of flow. Pressure steps were maintained for 3 min. Passive pressure-diameter relations were determined at the end of each experiment. For this, the arteries were superfused with Ca2+-free HEPES buffer containing 10 mmol/l adenosine.

All flow profiles were applied at a constant intraluminal pressure of 75 mmHg. To calculate the cannula pressures for the desired shear stress, we first determine the required flow:

(1)
where Q is flow, {tau} is the required shear stress, d is the internal diameter of the vessel after tone development, and {eta} is the viscosity of 0.001 N·s·m–2. The left and right pressures (Pl and Pr, respectively) needed for these values of flow at a constant intraluminal pressure of 75 mmHg were then determined from

(2)

where Rl and Rr are the left and right cannula resistances, as determined before the start of the experiment. We used pipettes with matched resistances. On the basis of geometry, the resistance of the cannulated vessel was negligible compared with that of the cannulas. Steady shear stresses between 5 and 40 dyn/cm2 were achieved by a step change in cannula pressures calculated according to the above formulas. Cannula resistances were between 240 and 400 mmHg·ml–1·min. The different shear stress values were randomized. The average pressure gradient that was applied to get 5 dyn/cm2 of shear stress was 10 mmHg, to get 10 dyn/cm2 was 21 mmHg, to get 20 dyn/cm2 was 49 mmHg, and to get 40 dyn/cm2 was 91 mmHg. Shear stress was maintained for 3 min; shear stress steps were separated by 3-min periods of zero flow.

Drugs. All chemicals were of analytic grade and purchased from Sigma (St. Louis, MO). All stock solutions were prepared in distilled water.

Analysis. In wire-mounted vessels, vascular wall tension changes to different concentrations of phenylephrine and U-46619 are presented as the percentage of the final 120 mmol/l KCl response. Active tension changes in wire-mounted vessels to SNP, bradykinin, and methacholine are presented as the percentage response of the maximum possible relaxation after preconstriction with 10–6 mol/l phenylephrine. In cannulated vessels, diameters of the passive and active vessels are presented as percentages of the passive diameter at 100 mmHg, as determined at the end of the experiment. Vascular dilations in cannulated vessels to bradykinin and shear stress are represented as percentages of the maximum possible dilation. For each 3-min intervention, the diameter was obtained after the development of a stable response. Data are presented as means ± SE. Data were analyzed by Student's t-test or by two-way ANOVA. Significance was defined as P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To investigate the gene expression of RPTPµ in detail, we previously created knockin mice with a LacZ gene under direct control of the RPTPµ promoter (RPTPµ-LacZ mice) (18). Analysis of whole mount stained mesentery from the RPTPµ-LacZ+/– adult mice showed clear heterogeneity in expression between arteries and veins (Fig. 1, A and B), which has previously been shown for other organs (18). Using immunoelectron microscopy on fixed cryosections of human mesenteric arteries, we found that expression of RPTPµ is selectively localized at endothelial cell contact sites, particularly on luminally oriented lamellipodia (Fig. 1C).



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  		Fig. 1. Heterogeneous {beta}-galactosidase (LacZ) activity between arteries and veins in adult receptor-like protein tyrosine phosphatase-µ (RPTPµ)-LacZ+/– mice. A: whole mount staining of the adult mice mesentery showed heterogeneity in {beta}-galactosidase expression between arteries (positive) and veins (negative). Bar = 1 mm. B: histological section of the mesenteric artery shows specific expression in endothelial cells (ec) and not in smooth muscle cells (smc). Bar = 20 µm. C: immunoelectron microscopy image after immunogold labeling for RPTPµ on fixed cryosections of human mesenteric arteries. ecs I–IV are pointed out to depict the vessel lumen (L). RPTPµ (arrowheads) is exclusively localized at endothelial cell (ecs I–III) cell-cell contacts, to the luminal side of the junction. Bar = 100 nm.

 
We investigated whether RPTPµ deficiency would lead to changes in systemic blood pressure regulation. However, heart rate and mean arterial pressure in RPTPµ–/– and wild-type littermates did not significantly differ (Table 1).


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  		Table 1. Heart rate and MAP of wild-type vs. RPTPµ–/– mice

 
To test whether RPTPµ plays a role in contractile vascular responses in small mesenteric resistance arteries, we performed a wire myograph study. No significant differences were measured in the response to KCl between RPTPµ–/– versus wild-type mice (Table 2). Subsequent cumulative concentration-response curves for phenylephrine and the thromboxane analog U-46619 showed no significant differences between RPTPµ–/– versus wild-type mice in both pD2 (–log EC50) and the maximal effect of the drug (Table 2). Next, the relaxation to endothelium-independent and endothelium-dependent dilators was tested. We exposed the vessels on a wire myograph to a submaximal concentration phenylephrine followed by methacholine, bradykinin, or the NO donor SNP. No significant differences were found concerning the preconstriction with phenylephrine (Table 2). Furthermore, we found no significant difference in dilation to all three dilators between the RPTPµ–/– versus wild-type arteries (Table 2).


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  		Table 2. Constriction to KCl, PE, and U-46619 and dilation to SNP, methacholine, and bradykinin of mesenteric resistance arteries isolated from RPTPµ–/– and wild-type mice

 
In cannulated arteries, we examined whether the passive pressure-diameter relation and active diameter responses of RPTPµ–/– mesenteric arteries to pressure steps were altered. The passive arterial pressure-diameter relation did not significantly differ between the RPTPµ–/– and wild-type mice (Fig. 2A), indicating similar elastic properties of the wall. Accordingly, the wall cross-sectional area was not significantly different between both groups: 18,680 ± 758 µm2 in wild-type arteries (n = 6) versus 17,250 ± 1,239 µm2 in RPTPµ–/– arteries (n = 7). Stepwise increases in pressure induced distension followed by myogenic contraction. The steady-state active pressure-diameter relation had a negative slope, representative of strong myogenic responsiveness, in the range between 40 and 100 mmHg. These relations were not significantly different between both groups (Fig. 2B).



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  		Fig. 2. A: passive diameter in mesenteric resistance arteries isolated from RPTPµ–/– (n = 5) and wild-type (n = 5) mice. Data are normalized to the passive diameter at 100 mmHg and are expressed as means ± SE. B: active diameter response to pressure in mesenteric resistance arteries isolated from RPTPµ–/– (n = 7) and wild-type (n = 6) mice preconstricted with 20 nmol/l U-46619. Data are normalized to the passive diameter at 100 mmHg and are expressed as means ± SE. C: shear stress-induced dilation in 20 nmol/l U-46619-preconstricted mesenteric resistance arteries isolated from RPTPµ–/– (n = 7) and wild-type (n = 6) mice. Data are normalized to the maximum possible dilation and are expressed as means ± SE. Two-way ANOVA showed no significant difference in passive and active response between the groups (P > 0.05) and a significant difference in shear stress-induced dilation between the two groups (P < 0.01).

 
In the vessels to be subjected to shear stress, 20 nmol/l U-46619 at 75 mmHg in the absence of shear stress induced similar preconstriction in both groups: 65.2 ± 2.2% of the passive diameter in wild-type arteries (n = 6) versus 68.5 ± 1.9% in RPTPµ–/– arteries (n = 7). This tone development was comparable with the U-44619-induced tone as observed at 80 mmHg during the recording of the active pressure-diameter relation (Fig. 2B). Before each shear stress protocol on the cannulated arteries, we checked the endothelial cell viability by the addition of 10–7 mol/l bradykinin to the superfusion fluid. The dilatory response to bradykinin was similar in both groups (Table 2). Flow in either direction induced a significant dilation in mesenteric resistance arteries in both groups. Shear stress-induced dilation was significantly reduced in mesenteric resistance arteries isolated from RPTPµ–/– mice compared with arteries from wild-type mice (Fig. 2C). Thus RPTPµ–/– mice have a decreased flow-induced dilation, whereas mechanical properties, myogenic responsiveness, and responsiveness to vasoconstrictors and endothelium-dependent vasodilators are similar.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we show that RPTPµ is specifically expressed at arterial endothelial cell-to-cell contacts in vivo and plays a key role in flow-dependent dilation. The absence of RPTPµ in this knockout model resulted in impaired dilation to flow, whereas mechanical properties and sensitivity to pressure, vasoconstrictors, and vasodilators were unaffected.

In vivo, arteries are exposed to tensile and shear loads; the former are due primarily to blood pressure and the latter are due primarily to blood flow. Changes in tensile loads are transduced at least partially through the matrix attachments and medial smooth muscle cells, whereas shear-induced stresses are imposed directly on the apical surface of endothelial cells. Here, we show that shear stress-induced dilation is significantly decreased in mesenteric resistance arteries isolated from RPTPµ–/– mice, whereas wall cross-sectional area and passive and active diameter responses to pressure changes are not affected. We also found normal constriction to KCl, phenylephrine, and the thromboxane analog U-46619 in these vessels. In contrast with the altered shear stress-induced dilation, these arteries had a normal endothelium-dependent dilation to bradykinin and methacholine. This shows that generalized endothelial dysfunction or defects in smooth muscle contractility or vasorelaxant properties are not involved in the reduced dilation to shear stress found in arteries from RPTPµ–/– mice. We therefore conclude that RPTPµ is involved in either shear stress sensing or in the cellular signaling cascade that regulates shear stress-induced responses.

Although RPTPµ deficiency led to a reduced flow-induced dilation, blood pressure in the knockout animals was preserved. We cannot exclude that other regulatory mechanisms in vivo compensate for the RPTPµ deletion, leading to a normal mean arterial pressure in the RPTPµ–/– mice. However, the unchanged mean arterial pressure in these mice might also strengthen the hypothesis that flow-induced dilation has a key role in the control of local blood flow but is not necessarily directly related to the basal level of systemic blood pressure (15, 22). The further consequences of impaired flow-dependent dilation in the RPTPµ–/– mice at the level of the intact organism remains to be investigated.

The endothelium is the primary transducer of shear stress, and several proteins and protein structures have been found to be involved in shear stress sensing and adaptation. Shear forces on the luminal surface of the endothelium are transmitted to endothelial cell-to-cell contacts via the cytoskeleton (19). The considerable reorganization of especially the adherens junctions leads to the alignment and reorientation of endothelial cells in response to mechanical loads. The role of tyrosine phosphorylation of many proteins in the organization of cell-to-cell contacts is well appreciated. Although kinases have been studied to some extent, cellular homeostasis also requires that phosphatases are active. Recent evidence has suggested a core function for p120ctn in regulating cadherin stability and turnover (6, 9, 23, 38). Phosphorylation of these cadherin/catenin complexes regulates both their stability and their participation in junctional complexes (30) and thereby the role of these complexes in shear stress responses (1, 26, 32, 37). RPTPµ is a transmembrane protein, which, like adherens and tight junction components (33), localizes at cell contact sites in endothelial cell-to-cell contacts. RPTPµ is thought to interact directly with catenin p120ctn (40) and cadherins (5) at the site of endothelial cell-cell adherens junctions. Disturbed regulation of these cadherins-catenin complexes in RPTPµ–/– mice might be a possible reason for the observed impaired flow-induced dilation.

Putative sensors of shear stress are associated with the cytoskeleton and transmembrane structures that undergo dramatic changes when shear is changed. Vimentin and dystrophin are both cytoskeletal structure proteins that are sensitive to shear stress (8, 36). Mice lacking one of these genes have a similar phenotype as RPTPµ–/– mice in mesenteric resistance arteries, with decreased flow-induced dilation, whereas pressure-induced tone is unaffected (15, 21, 22). Whether all three proteins can be linked in the same mechanotransduction pathway remains to be investigated.

Our reason for studying the role of RPTPµ in flow-dependent dilation was based on its involvement in the regulation of endothelial adherens junctions, which are believed to form a site for sensing of shear stress. It was not our purpose to analyze the further downstream events, which could include NO, prostaglandins, and EDHFs, because we had little reason to suspect differences in these downstream pathways. Activation of NO-dependent and -independent mechanisms of dilation by other stimuli was not affected in the RPTPµ–/– mice, as shown by the metacholine responsiveness in the wire-mounted vessels and bradykinin reactivity in the cannulated vessels. In pilot experiments, we found no apparent differences in relative inhibition of dilation to either N-nitro-L-arginine or indomethacin between RPTPµ–/– mice and wild-type controls (data not shown).

There are no specific inhibitors of RPTPµ available. Therefore, to investigate the role of this phosphatase in flow-dependent dilation, we relied on a knockout model. On the basis of the present findings, one would expect that more general phosphatase inhibitors also suppress flow-dependent dilation. Indeed, the broad-spectrum phosphatase inhibitor vanadate attenuated flow-induced vasodilation. However, vanadate also influenced basal tone significantly, and we were therefore reluctant to derive any conlusions on specific mechanisms of action from these data (data not shown).

An alternative explanation for the impaired shear stress response in RPTPµ–/– mice might be disturbed K+ channel function. The activation of K+ membrane currents is one of the fastest known responses to fluid mechanical shear stress in endothelium and may play a role in the sensing and transduction of shear forces (17, 25, 27). Interestingly, it has been demonstrated that RPTPµ can regulate mRNA expression of the potassium channel gene Kv1.5 in cardiac myocytes (16). Thus it remains interesting to investigate potassium channel functions in this knockout model in the future.

In conclusion, we found that RPTPµ is involved in the regulation of mechanotransduction of shear stress by the vascular endothelium in resistance arteries. The present findings support the concept that cell-cell junctional elements connected to the cytoskeleton are important in transducing the signal from shear stress to the dilator response elements in vascular endothelial cells.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
T. E. Sweeney was supported by the University of Scranton, The Netherlands Organization for Scientific Research Grant B 94-188, and National Heart, Lung, and Blood Institute Grant HL-72548-01. M. F. B. G. Gebbink was supported by Dutch Cancer Society Grant 1999-2114.


   ACKNOWLEDGMENTS
 
We thank Dr. Coert Zuurbier and Anneke Koeman, Department of Anaesthesiology, Academic Medical Center Amsterdam, for assistance with the blood pressure and heart rate measurements.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. B. G. Gebbink, Dept. of Haematology, Univ. Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands (E-mail: m.gebbink{at}azu.nl)

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.


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