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Circulation Research. 2002;91:360-369
Published online before print July 25, 2002, doi: 10.1161/01.RES.0000030861.13850.F1
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(Circulation Research. 2002;91:360.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Phosphoinositide 3-Kinase Mediates Enhanced Spontaneous and Agonist-Induced Contraction in Aorta of Deoxycorticosterone Acetate-Salt Hypertensive Rats

Carrie A. Northcott, Matthew N. Poy, Sonia M. Najjar, Stephanie W. Watts

From the Department of Pharmacology and Toxicology (C.A.N., S.W.W.), Michigan State University, East Lansing, Mich; and Medical College of Ohio (M.N.P., S.M.N.), Toledo, Ohio.

Correspondence to Carrie A. Northcott, MS, Dept of Pharmacology and Toxicology, B445 Life Science Building, Michigan State University, East Lansing, MI 48824-1317. E-mail taetscar@ msu.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Arteries from deoxycorticosterone acetate (DOCA)-salt and N{omega}-nitro-L-arginine (L-NNA) hypertensive but not normotensive rats develop spontaneous tone. LY294002 and wortmannin, phosphoinositide 3-kinase (PI3-kinase) inhibitors, eliminate spontaneous tone. We hypothesized that PI3-kinase protein and/or activity was increased in hypertension and contributed to the observed enhanced contractility. PI3-kinase activity assays revealed 2-fold higher activity in thoracic aorta from DOCA-salt [systolic blood pressure (SBP)=184±5 mm Hg] compared with sham rats (SBP=111±2 mm Hg). Western analyses of aortic homogenates revealed the presence of p85{alpha}, p110{alpha}, p110ß, and p110{delta} but not p110{gamma} PI3-kinase subunits; p110{delta} protein was elevated in aorta of hypertensive rats as compared with sham. Aortic homogenates from L-NNA rats also had elevated p110ß protein density, but neither L-NNA nor DOCA-salt had differences in p85{alpha} and p110{alpha}. Total Akt density was unaltered, but pAkt was significantly lower in homogenates from DOCA-salt rats. LY294002 (20 µmol/L) and nifedipine (50 nmol/L) abolished Ca2+-induced spontaneous tone in aorta from DOCA-salt rats. However, LY294002 did not alter BayK8644-induced contraction, indicating that LY294002 does not inhibit L-type Ca2+ channels directly. PTEN (phosphatase and tensin homolog) and pPTEN were expressed but not different in aorta from DOCA-salt and sham rats. LY294002 corrected the enhanced contraction to KCl and norepinephrine in aorta from DOCA-salt rats. These data support an increase in PI3-kinase activity and p110{delta} density in aorta from L-NNA and DOCA-salt rats. Importantly, this increase contributes to the enhanced contractility observed in two models of hypertension.


Key Words: phosphoinositide 3-kinase • artery • deoxycorticosterone acetate-salt hypertension • phosphatase and tensin homolog • N{omega}-nitro-L-arginine hypertension


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Phosphoinositide 3-kinase (PI3-kinase) is a multifaceted enzyme, possessing both lipid and protein kinase activity. It participates in multiple signaling pathways, receiving input from a variety of receptors ultimately leading to changes in gene expression, metabolism, cell division, apoptosis, cellular trafficking, and contractility.1–6 In general, PI3-kinase transfers a phosphate group to the D3 position of phospholipids. Cloning of the catalytic subunits has led to organizing the multigene family into 3 groups based on substrate specificity.1–6 Importantly, PI3-kinase has been implicated in the modulation of vascular and nonvascular smooth muscle contractility.7–11

Hypertension is a disease characterized by enhanced agonist-stimulated contraction, reduced agonist-stimulated relaxation, smooth muscle cell growth, and spontaneous arterial tone. Because PI3-kinase plays a role in modulating contraction and growth, it is logical to question the involvement of PI3-kinase in vascular changes associated with hypertension. Of specific interest to this work is spontaneous tone, where arteries contract with no exogenous stimulus and which can be associated with oscillatory contractions. Spontaneous tone occurs in arteries of hypertensive rats and is due, at least in part, to altered Ca2+ sensitivity and/or handling.12–16 All the Class I PI3-kinase subunits associate with L-type Ca2+ channels and increase current directly17 or via protein kinase C (PKC).18 The end result is an increase in intracellular Ca2+. Thus, we investigated the profile of PI3-kinase proteins present in arteries and whether this profile is altered in arteries from 2 models of hypertension. We also assessed PI3-kinase Ca2+-dependence with respect to mediating spontaneous tone in the aorta.

A potential mechanism of altered PI3-kinase activity is a change in phosphatase and tensin homolog (PTEN) activity. PTEN and several phosphatases are regulators of PI3-kinase activity. PTEN is a unique tumor suppressor gene that encodes a dual-specificity phosphatase and functions by removing the 3-phosphate from inositol moieties and proteins phosphorylated by PI3-kinase.19–22 PTEN exists primarily in the inactive phosphorylated state (pPTEN) and the phosphorylation suppresses PTENs activity by preventing its recruitment to the PTEN-associated complex (PAC).22–24 Thus, we determined if PTEN and pPTEN were present and whether they were altered between deoxycorticosterone acetate (DOCA)- salt and sham rat aorta.

Arterial responsiveness to NE is increased in hypertension and NE, as well as a host of other vasoactive compounds, and activates PI3-kinase in vascular smooth muscle and small arteries in the rat.25–31 Thus, enhanced arterial contraction to NE may be PI3-kinase mediated, and we have examined this idea. Overall, we tested the hypothesis that there is an increase in PI3-kinase protein and/or activity in hypertension that is ultimately responsible for physiologically relevant changes in contractility.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Surgical and Blood Pressure Protocol
DOCA-Salt Hypertension
Male Sprague-Dawley rats (250 to 300 g; Charles River Laboratories, Inc, Portage, Mich) underwent uninephrectomy and implantation of DOCA (200 mg kg-1) under isoflurane anesthesia as described previously.32 Animals remained on the regimen for 4 weeks. Systolic blood pressures were measured using standard tail cuff methods. All animal procedures complied with The Guide for the Care and Use of Laboratory Animals (DHEW publication No. NIH 78-23, revision 1996).

L-NNA Hypertension
Male Sprague-Dawley rats (250 to 300 g; Harlan Laboratories, Indianapolis, Ind) were given L-NNA (N{omega}-nitro-L-arginine) (0.5 g/L) in their drinking water for 2 weeks. Systolic blood pressures were measured using standard tail cuff methods.

Isolated Tissue Bath Protocol
Endothelial cell-denuded or intact strips of thoracic aorta removed from pentobarbital (60 mg kg-1, IP) anesthetized rats were pair-mounted (Sham/DOCA; Sham/L-NNA) in isolated tissue baths for measurement of isometric force.32 Tissues were challenged with a maximal concentration of {alpha}-adrenergic agonist, phenylephrine (PE) (10-5 mol/L). Inhibitors (LY294002, 20 µ mol/L; LY303511, 20 µmol/L; nifedipine, 50 nmol/L) or vehicle were added to the bath for 1 hour before measurements of spontaneous tone. NE (1x 10-9 to 3x10- 5 mol/L), BayK 8644 (1x10- 10 to 3x10-6 mol/L) and KCl (6x10-3 to 1x 10-1 mol/L) were added in a cumulative fashion after a 1-hour incubation with LY294002 (20 µmol/L) or vehicle (0.1% to 0.2% DMSO). Concentration response curves to LY294002 and wortmannin were generated by adding increasing concentrations of vehicle, LY294002 (1x 10-7 to 3x10- 4 mol/L) or wortmannin (1x10- 8 to 3x10-4 mol/L) every 30 minutes and measuring spontaneous tone. For Ca2+ experiments, arteries were first incubated in normal Ca2+ (1.6 mmol/L) and challenged with PE (10- 5 mol/L). Tissues were washed and then incubated for 30 minutes in Ca2+-free buffer supplemented with 1 mmol/L EGTA. Tissues were switched to a Ca2+-free EDTA buffer, equilibrated for 10 minutes, and LY294002 (20 µmol/L) or vehicle added. Ca2+ was then added back to the bath in a cumulative fashion (1x10-6 to 3x 10-3 mol/L) with additions every 5 minutes.

Western Protocol
Protein Isolation
Aorta were cleaned, pulverized in liquid nitrogen and solubilized in lysis buffer (0.5 mol/L Tris HCl [pH 6.8], 10% SDS, 10% glycerol) with protease inhibitors (0.5 mmol/L PMSF, 10 µg/mL aprotinin and 10 µg/mL leupeptin). Homogenates were centrifuged (11 000 g for 10 minutes, 4°C), and supernatant total protein measured. Vascular smooth muscle cells were derived from aorta of male Sprague-Dawley rats in an explant method described previously.33

Western Blotting
Equivalent amounts of aortic protein from sham, DOCA-salt, and L-NNA rats were separated on 7% SDS-polyacrylamide gels and transferred to Immobilon-P membrane for standard Western analyses using p85{alpha} (1:100; Upstate Biotechnology), p110{alpha} (1:250; BD Transduction Laboratories), p110ß, p110{delta}, p110{gamma} (1:1000; Santa Cruz Biotechnology, Inc), PTEN, pPTEN, Akt, and pAkt (1:1000; Cell Signaling) antibodies. Positive controls for p110{alpha} were Jurkat cells (BD Transduction Laboratories); p110ß were K-562 cells; p110{delta} were U-87 MG cells; p110{gamma} were U-937 cells; PTEN were PTEN(FL) epitopes (Santa Cruz Biotechnology, Inc); and pPTEN positive control were EGF-stimulated A431 cells (Upstate Biotechnology). Smooth muscle {alpha}-actin (1:400; Oncogene) was used as a marker to ensure equal loading of protein.

Immunoprecipitation and PI3-Kinase Activity Assay
Aorta were cleaned, pulverized in liquid nitrogen, and solubilized in PI3-kinase lysis buffer. p85{alpha} antibody (5 µL) and protein A agarose beads (70 µL) were added to equal amounts of total protein and the samples rocked (4°C) for 2 hours. The PI3-kinase assay was performed as previously described.34,35 Briefly, the p85{alpha} aortic homogenate immunoprecipitants from DOCA-salt and sham rats were incubated with phosphatidylinositol (PI) in the presence of [32P] adenosine triphosphate (ATP). Reactions were terminated with 15 µL 4N HCL and phospholipids extracted with 130 µL CHCl3/methanol (1:1). The radioactive product of the reaction (PI3-monophosphate) was detected using thin layer chromatography (TLC) and quantified with Bio-Rad software.

Materials
Materials included acetylcholine hydrochloride, DOCA, BayK8644, L-NNA, nifedipine, NE, PE, KCl, wortmannin (Sigma Chemical Co), EGF (Gibco Life Technologies), LY294002 (Biomol), and LY303511 (gift from Dr Chris Vlahos, Eli Lilly, Indianapolis, Ind).

Data Analyses
Data are presented as mean±SEM for the number of animals (n) stated. Contraction is reported as tension (milligrams) or as a percentage of response to maximum contraction to PE, NE, or KCl. EC50 values were determined using GraphPad Prism and reported as the mean of the negative logarithm (-log) of the EC50 value. Band density quantitation was performed using NIH Image (version 1.61). When comparing 2 groups, the appropriate Student’s t test were used. An ANOVA followed by Student-Newman-Keuls post hoc tests were performed when comparing 3 or more groups. A value of P<=0.05 was considered statistically significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Spontaneous Tone and PI3-Kinase
The systolic blood pressures of DOCA-salt and sham rats were 184±5 and 111±2 mm Hg, respectively. Spontaneous tone developed in aorta isolated from DOCA-salt (Figure 1A, 2nd tracing) but not sham rats (Figure 1A, 1st tracing). LY294002 (20 µmol/L), a specific and reversible PI3-kinase inhibitor, significantly reduced spontaneous tone (Figures 1A and 1B). Similarly, LY294002 (20 mol/L) inhibited spontaneous tone in aortic strips with the endothelium-intact from DOCA-salt rats (vehicle, +1.6±1.5% versus LY294002, -13.9±5.6% initial PE (10-5 mol/L) contraction). Systolic blood pressures of the L-NNA and sham rats were 200±8 and 121±3 mm Hg, respectively. Similar to the DOCA-salt model, spontaneous arterial tone developed (Figures 1C, 4th tracing) in aortic strips from the L-NNA model and not in sham. LY294002 (20 µmol/L) reduced the spontaneous tone that developed (Figures 1C and 1D). Increasing concentrations of LY294002 (10-7 to 3x 10-4 mol/L) were added to tissues from DOCA-salt rats in the absence of agonist and spontaneous tone monitored to ensure that the concentration utilized in the experiments (20 µmol/L) was appropriate. LY294002 reduced spontaneous tone in a concentration-dependent manner (Figures 1E and 1G). The effect of LY294002 was reversible in all experiments, as spontaneous tone was restored on washing out of LY294002. Similarly wortmannin, another pharmacological inhibitor of PI3-kinase, also reduced spontaneous tone in a concentration-dependent manner (Figures 1F and 1H) in the aortic strips from the DOCA-salt rats.



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  		Figure 1. A, Representative tracing of spontaneous arterial tone in endothelium-denuded aorta from DOCA-salt and sham rats. Tissues were under passive tension for optimal force production; vehicle (0.1% DMSO) or LY294002 (20 µmol/L) was added and allowed to equilibrate for 1 hour. B, Effect of PI3-kinase inhibitor LY294002 on spontaneous tone in endothelium-denuded rat aorta from DOCA-salt and sham rats. C, Representative tracing of spontaneous arterial tone in endothelium-denuded aorta from L-NNA and sham rats. Tissues were under passive tension for optimal force production; vehicle (0.1% DMSO) or LY294002 (20 µmol/L) was added and allowed to equilibrate for 1 hour. D, Effect of PI3-kinase antagonist LY294002 on spontaneous tone in endothelium-denuded rat aorta from L-NNA and sham rats. Bars represent the mean spontaneous tone development ±SEM. Data are presented as a percentage of initial phenylephrine (PE) (10-5 mol/L) contraction. There was no difference in this initial contraction between aortic strips from DOCA-salt and sham rats, as well as no difference between L-NNA and their respective sham rats (1475.8±67.5 vs 1394.2±39.3 mg and 917.3±90.2 vs 1015.9±35.0 mg, respectively). Representative tracings of vehicle, (E) LY294002 (10-7 to 3x10-4 mol/L), and (F) wortmannin (10-8 to 3x10- 4 mol/L) concentration response curves in endothelium-denuded aorta from DOCA-salt and sham rats. Effect of cumulative additions of vehicle, (G) LY294002, and (H) wortmannin in endothelium-denuded rat aorta from DOCA-salt and sham rats on spontaneous tone. Data are presented as a percentage of the initial phenylephrine (PE) (10-5 mol/L) contraction. Points represent mean±SEM. *P< 0.05.

To ensure that LY294002 was acting selectively, we utilized LY303511, an inactive analog of LY294002. In contrast to LY294002, LY303511 did not inhibit epidermal growth factor (EGF)-induced activation of pAkt, a substrate of PI3-kinase, in cultured aortic smooth muscle cells (Figure 2A); Akt protein density was similar in all samples. LY303511 (20 µmol/L) failed to inhibit spontaneous tone (Figure 2B), but elicited a contraction in the aorta from DOCA-salt rats without altering tone of sham aorta.



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  		Figure 2. A, Western blot of pAkt, reflective of PI3-kinase activity, of rat aortic smooth muscle cells exposed to vehicle, LY294002 (20 µmol/L), and LY303511 (20 µmol/L) with or without EGF (10 nmol/L). B, Representative tracing of spontaneous arterial tone in endothelium-denuded aorta from DOCA-salt hypertensive and sham normotensive rats. Tissues were under a tension for optimal force production; vehicle (0.1% DMSO) or LY303511 (20 µmol/L) were added and allowed to equilibrate for 1 hour.

PI3-Kinase Biochemistry
Having established a functional change in PI3-kinase in aorta from DOCA-salt rats, we proceeded to measure PI3-kinase activity. As Figure 3A reveals, p85{alpha}-associated PI3-kinase activity was significantly greater in aorta from DOCA-salt rats as compared with sham (5.7±0.9 versus 2.8±0.9 adjusted volume optical densityxmm2, respectively). Equal amounts of p85{alpha} protein were present in the immunoprecipitates from DOCA-salt and sham rats (Figure 3B), and this was confirmed with standard Western analyses (Figure 3C). To examine how this alteration in activity may affect proteins downstream of PI3-kinase, we measured Akt and pAkt protein in aortic lysates from DOCA-salt and sham rats. Similar concentrations of Akt were found, but there was significantly lower pAkt protein density in aortic lysate from DOCA-salt rats as compared with sham (Figure 3D). These data suggested the enhanced PI3-kinase activity was being funneled to an alternative effector.



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  		Figure 3. A, p85{alpha}-associated PI3-kinase activity in aorta from hypertensive DOCA-salt and normotensive sham rats. PI(3)P was detected using thin layer chromatography and quantified with Bio-Rad software. Bars represent mean adjusted volume optical densityxmm2±SEM. B, Immunoprecipitation of p85{alpha} from aorta from hypertensive DOCA-salt and normotensive sham rats to confirm equal amounts of protein loaded for PI3-kinase assay. Western analysis of (C) p85{alpha}, (D) Akt, and pAkt protein in aorta from hypertensive DOCA-salt and normotensive sham rats. Bars represent mean arbitrary densitometry units±SEM. *P< 0.05.

A profile of p110 subunits was next performed using Western analyses. The Class IA catalytic subunits p110{alpha}, p110ß, and p110{delta}, but not p110{gamma}, were present in the aorta of both DOCA-salt and sham rats, with p110{delta} being significantly higher in the aorta from DOCA-salt as compared with sham rats (P<0.05) (Figure 4). Confirmation of the specificity of the antibodies was determined by examining a positive control for each antibody. These data suggest that the increase in p85{alpha}-associated PI3-kinase activity may be due to the increase in the p110{delta} subunit, because it is the only PI3-kinase subunit that exhibited greater density in the aorta from DOCA-salt as compared with sham rats.



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  		Figure 4. Western analyses of protein isolated from aorta from DOCA-salt and sham rats with antibodies for (A) p110{delta} (control=U-87 MG cells), (B) p110{alpha} (control=Jurkat cells), (C) p110ß (control=K-562 cells), and (D) p110{gamma} (control=U937 cells). Bars represent mean arbitrary densitometry units±SEM. *P< 0.05.

We also examined aortic lysates from hypertensive L-NNA and normotensive sham rats. p85{alpha}, p110{alpha}, p110ß, and p110{delta}, but not p110{gamma} PI3-kinase subunits, were detected (Figure 5). There were significantly higher p110ß and p110{delta} protein densities in the L-NNA compared with sham (Figures 5C). There were also no significant changes in pAkt (Figure 5F). These data demonstrate that both models have alterations in arterial PI3-kinase, suggesting this is common to hypertension.



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  		Figure 5. Western analyses of protein isolated from aorta from L-NNA and sham rats with antibodies for (A) p85{alpha}, (B) p110{delta}, (C) p110ß, (D) p110{alpha}, (E) p110{gamma}, control=U937 cells, (F) Akt, and pAkt. Bars represent mean arbitrary densitometry units±SEM. *P< 0.05.

Ca2+, PI3-Kinase, and Spontaneous Tone
The role of Ca2+ in mediating arterial spontaneous tone is established. Nifedipine, an L-type Ca2+ blocker, inhibited spontaneous tone in a manner similar to LY294002 (compare Figures 1A, 1C, and 6A). This concentration of nifedipine was the minimum concentration that maximally inhibited KCl-induced contraction in aorta.33 To ensure that LY294002 did not directly inhibit L-type Ca2+ channels, we examined the effect of LY294002 (20 µmol/L) on BayK8644, a direct L-type channel agonist. In agreement with others,36 we observed an enhanced contraction to BayK8644 in aorta from the DOCA-salt rat. LY294002 did not alter BayK8644-induced contraction (Figure 6B), indicating that LY294002 was not acting directly to inhibit L-type Ca2+ channels. To link Ca2+ and PI3-kinase, all Ca2+ was removed from the tissue bath and added back in increasing concentrations in the presence of LY294002 or vehicle and spontaneous tone development measured. LY294002 completely inhibited the development of spontaneous tone in aorta from DOCA-salt rats, whereas vehicle-incubated aorta developed calcium-dependent spontaneous tone (Figure 6C). Aorta from sham rats did not develop spontaneous tone. Additionally, KCl-induced arterial contraction depends on Ca2+ channel activation but may necessitate the activation of other signaling pathways. LY294002 did not shift KCl-induced contraction in arteries from sham rats. However, LY294002 significantly shifted KCl-induced contraction in arteries from DOCA-salt rats, back to that similar in sham (Figure 6D).



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  		Figure 6. A, Representative tracing of spontaneous arterial tone in endothelium-denuded aorta from DOCA-salt hypertensive and sham normotensive rats. Tissues were under a tension for optimal force production; vehicle (0.1% DMSO) or nifedipine (50 nmol/L) was added and allowed to equilibrate for 1 hour. B, Effect of L-type voltage gated Ca2+ channel agonist BayK8644 and PI3-kinase antagonist LY294002 (20 µmol/L) in endothelium-denuded rat aorta from DOCA-salt and sham rats. LY294002 or vehicle was added to the tissues 1 hour before cumulative addition of BayK8644. C, Effect of Ca2+ chloride and PI3-kinase antagonist LY294002 in endothelium-denuded rat aorta from DOCA-salt and sham rats on spontaneous tone. LY294002 (20 µmol/L) or vehicle (0.1% DMSO) was added to the tissues 1 hour before cumulative addition of Ca2+ chloride. Data are presented as a percentage of the initial phenylephrine (PE) (10- 5 mol/L) contraction. D, Effect of KCl and PI3-kinase antagonist LY294002 (20 µmol/L) in endothelium-denuded rat aorta from DOCA-salt and sham rats. LY294002 or vehicle was added to the tissues 1 hour before cumulative addition of KCl. Points represent mean±SEM. *P< 0.05.

PTEN
We next examined aortic homogenates for the presence of PTEN and its inactive form, pPTEN. We found both PTEN and pPTEN in the thoracic aorta of both DOCA-salt and sham rats (Figure 7), but density of these proteins was not different between the aorta of DOCA-salt and sham rats. Appropriate positive controls were used to confirm antibody specificity. To our knowledge, this is the first time that PTEN and pPTEN have been localized to the arterial tissue.



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  		Figure 7. Western analyses of PTEN and pPTEN in aorta from DOCA-salt hypertensive and sham normotensive rats. Bars represent mean arbitrary densitometry units± SEM.

Norepinephrine and PI3-Kinase
We last examined the effect of LY294002 on NE-induced contraction, a contraction that is enhanced in DOCA-salt hypertension (Figure 8). LY294002 shifted the NE-induced aortic contraction of sham and the DOCA-salt rats compared with vehicle treated control tissues in both endothelium-denuded (-E) (representative tracing, Figure 8A) and endothelium-intact (+E) tissues, supporting a role for PI3-kinase in NE-induced contraction. Interestingly, the potency of NE in the presence of LY294002, in aorta from DOCA-salt and sham rats incubated with LY294002, was not significantly different (-log EC50 [M] sham-E=7.2±0.1; DOCA-salt-E=7.2±0.1; sham+E=6.6±0.1; DOCA-salt+E=6.4±0.1) (Figures 8B and 8C). Thus, an alteration in PI3-kinase may partially explain the enhanced arterial contractility to NE observed in DOCA-salt hypertension.



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  		Figure 8. A, Representative tracing of the effect of PI3-kinase antagonist, LY294002 (20 µmol/L), on NE-induced contraction in endothelium-denuded aorta from DOCA-salt and sham rats. B and C, Effect of NE and PI3-kinase antagonist LY294002 (20 µmol/L) in endothelium-denuded (B) and endothelium-intact (C) aorta from DOCA-salt and sham rats. LY294002 (20 µmol/L) or vehicle (0.1% DMSO) was added to the tissues 1 hour before cumulative addition of NE. Data are presented as a percentage of the maximal NE contraction. Points represent mean±SEM. Values are the -log EC50 of the NE-induced contractions in the presence of LY294002. *P< 0.05.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Vascular growth and hyperresponsiveness in hypertension has been observed for decades. PI3-kinase plays a key role in cellular growth and apoptosis and has been implicated in modulating vascular contraction.1– 6 LY294002 given acutely in vivo to conscious hypertensive DOCA-salt rats lowered blood pressure (data not shown), but also lowered heart rate, making it difficult to determine the role PI3-kinase in hypertension in the vasculature of conscious animals. With this in mind, we examined alterations in PI3-kinase activity and protein in DOCA-salt and L-NNA hypertension and how this alteration affects vascular contraction in aorta isolated from these models.

Our main hypothesis stemmed from the initial finding that LY294002 eliminated spontaneous tone in aorta from a DOCA-salt and L-NNA rats and had no effect on sham basal tone. Wortmannin, another PI3-kinase inhibitor, also inhibited spontaneous tone in aorta from a DOCA-salt rat and not sham. Renal arterial spontaneous tone has been observed in hypertensive patients, with resultant phasic changes in renal perfusion that can be reduced by calcium channel blockade.37,38 These findings suggested an important role for PI3-kinase in spontaneous tone development, which may have serious physiological implications.

To examine the specificity of LY294002, we used LY303511. LY303511 is a single atom substitution of LY294002 that has no known actions on PI3-kinase and thus provides an excellent structural control for evaluating possible nonspecific effects of LY294002.39 LY303511 did not inhibit spontaneous tone, but it did elicit a contraction in the DOCA-salt tissue and had no effect on the sham. The reason for this contraction is unclear, but LY303511 did not inhibit PI3-kinase activity as it did not reduce EGF-induced phosphorylation of Akt. Interestingly, the contraction occurred only in the aorta from DOCA-salt and not sham rats.

PI3-Kinase Biochemistry
To assay for PI3-kinase activity, we used an antibody against p85{alpha}. We chose to use this antibody versus a phosphotyrosine antibody because of the specificity for PI3-kinase association provided by p85{alpha}. There were no significant differences in the p85{alpha} protein levels, but there was significantly elevated PI3-kinase activity in aorta from DOCA-salt rats as compared with sham.

This led us to inquire if the increase in activity was associated with an increase in p110 protein density. We detected p110{alpha}, p110ß, and p110{delta} in the thoracic aorta of DOCA-salt, L-NNA, and sham rats with a significantly greater amount of p110{delta} in the aorta of DOCA-salt and L-NNA compared with the sham rats, as well as a significantly greater p110ß in the L-NNA model of hypertension. This is in contrast to the findings of Macrez et al17 in which p110{alpha}, p110ß, and p110{gamma} were found, but not p110{delta}, in rat portal vein myocytes. The localization of p110{delta} to the aorta was unexpected because p110{delta} had been reported to be restricted to hematopoietic cells.40 The upregulation of p110 subunit density may constitute a potential mechanism of the enhanced PI3-kinase activity observed. It is unlikely changes in PTEN function contribute to this increase in activity as PTEN density and state of activation was not different in aorta between DOCA-salt and sham rats. This is the first time, to the best of our knowledge, that PTEN and pPTEN have been localized in the aorta. Thus, other factors are implicated in the regulation of PI3-kinase activity in aorta of DOCA-salt rats, and this warrants further investigation.

PI3-Kinase and Ca2+
In experimental and clinical hypertension, investigators have identified defects in arterial Ca2+ handling resulting in inappropriately high basal Ca2+ levels.41 This is reflected in development of spontaneous, nonagonist induced arterial tone. We suggest that this increase in intracellular Ca2+ level is due, at least in part, to an alteration in PI3-kinase. Altered membrane depolarization, a main stimulus for Ca2+ current through voltage-gated Ca2+ channels, is a critical parameter which may be also affected by PI3-kinase.

Our findings of dramatically enhanced contraction to BayK8644 in aorta from DOCA-salt rats supported previous observations in coarctation-hypertensive rats, DOCA-salt hypertensive rats, L-NNA, and spontaneously hypertensive rats (SHR) compared with their respective normotensive controls.36,42,43 These data suggest an upregulation of L-type calcium channel activity in DOCA-salt rat hypertension. Recently, Molero et al44 found an increase in L-type Ca2+ channel expression in membrane protein from DOCA-salt compared with sham rats in small mesenteric arteries. Viard et al18 demonstrated that Gß{gamma} dimers have the ability to stimulate vascular L-type Ca2+ channels through PI3-kinase, which adds support to the notion that PI3-kinase and L-type Ca2+ channels are linked and G protein-coupled receptors have the potential to tap into this interaction. Macrez et al17 demonstrated that p110 subunits can directly associate with L-type Ca2+ channels. Thus, if there is an alteration in PI3-kinase activity, this may further activate Ca2+ channels. The regulation of Ca2+ flux by PI3-kinase was strengthened by the finding that LY294002 inhibited all Ca2+-induced spontaneous tone as well as corrected the enhanced KCl-induced contraction in aorta from DOCA-salt rats. We realize that KCl and NE are mechanistically different in how they induce vascular contraction, but LY294002 corrected the enhanced contraction observed in aorta from DOCA-salt rats in both types of contraction. Thus, this supports a role for PI3-kinase in some aspect of contraction for both compounds. At the present time, it is unclear whether a change in Ca2+ channel, Ca2+ flux, PI3-kinase, or a combination of these is sufficient to enable the development of arterial spontaneous tone and hyperresponsiveness observed in hypertension.

However, PI3-kinase may be connected to Ca2+ channels, their interaction has implications for the hyperresponsiveness to contractile agonists that is characteristic in hypertension. LY294002 normalized NE-induced contraction in sham and DOCA-salt tissues, indicating that it is the increased activity of PI3-kinase that is responsible for enhanced contraction to NE. Because PI3-kinase is integral to signaling of hormones involved in contraction and growth, an increase in PI3-kinase activity has significant implications for arterial function.

Limitations
Several limitations of these studies should be acknowledged. In the studies we performed, we used only the thoracic aorta. We chose to do so because the aorta is well characterized and easier to use for protein isolation. We are cognizant of the fact that this is not a resistance artery, and thus, we cannot state that our observations apply to resistance arteries. We also recognize that there are limitations with the PI3-kinase inhibitors. LY294002 has the ability to inhibit casein kinase 2 (CK2).45 Because of the lack of selective CK2 inhibitors, it was not possible to control for this limitation. Wortmannin, another PI3-kinase inhibitor, inhibits myosin light chain kinase.45 We chose to focus our studies using the inhibitor LY294002 after finding that both inhibitors inhibited spontaneous tone, as well as the fact that we had a control for LY294002, LY303511. Finally, we have studied alterations in PI3-kinase only after the development of hypertension, making it difficult to draw any conclusions when these changes in the vasculature occur.

Summary
In conclusion, we have demonstrated a role for PI3-kinase in spontaneous tone development and hyperreactivity in thoracic aorta from DOCA-salt and L-NNA rats. This may, in part, be due to an increase in PI3-kinase activity via increased p110{delta} and p110ß protein density observed in aorta of hypertensive rats. We are the first, to our knowledge, to perform a complete profile of Class I p110 PI3-kinase subunits in the aorta. Alteration in activity/protein may be linked to calcium modulation, perhaps one mechanism is via L-type calcium channels. This is also the first demonstration of the presence of PTEN and pPTEN in arterial tissue. Increased PI3-kinase activity has functional significance in that when PI3-kinase is inhibited spontaneous tone, KCl and NE-induced hyperreactivity is decreased. Collectively, these data provide an argument for the importance of PI3-kinase in the vascular smooth muscle and in hypertension.


*    Acknowledgments
 
This work was supported by AHA grant 024033 to S.W. Watts and NIH research grants DK54254 and DK57497 to S.M. Najjar. S.W. Watts is an Established Investigator of the American Heart Association (AHA). C.A. Northcott is a predoctoral fellow of the Midwest affiliate of the AHA (grant No. 0110207Z). M.N. Poy is a predoctoral fellow of the Institutional National Research Service Award (grant T32-CA79450).

Received February 1, 2002; revision received July 12, 2002; accepted July 15, 2002.


*    References
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