PI3-Kinase–Induced Hyperreactivity in DOCA-Salt Hypertension Is Independent of GSK-3 Activity
Phosphatidylinositol 3-kinase (PI3K) activity is increased in aortae from deoxycorticosterone (DOCA)-salt rats and enhanced PI3K activity contributes to the arterial hyperreactivity in these animals. Because PI3K activity is increased in DOCA-salt hypertension, we postulated that phosphorylation of Akt and glycogen synthase kinase 3 (GSK-3), serine threonine kinases that are downstream of PI3K, would be increased in DOCA-salt hypertension. In this study, we focused on GSK-3. Because GSK-3 activity is reduced by phosphorylation, we expected that its activity would be reduced in DOCA-salt hypertensive arteries and that reduced GSK-3 activity could contribute to enhanced adrenergic signaling and vascular smooth muscle hypertrophy that augment the heightened contractile response in DOCA-salt hypertension. Surprisingly, we observed a decrease in phosphorylation of GSK-3, indicating an increase in GSK-3 activity. To determine whether increased GSK-3 activity contributes to altered arterial reactivity in DOCA-salt animals, we measured isometric contraction to norepinephrine (NE) in the presence and absence of PI3K or GSK-3 inhibition. Addition of LY294002 (20 μmol/L), a PI3K inhibitor, resulted in a rightward shift in response to NE and normalized the NE-induced contractions in the DOCA hypertensive vessels. SB415286, a GSK-3 inhibitor, resulted in a slight rightward shift in response to NE in the DOCA-salt vessels. Thus, enhanced GSK-3 activity modestly augments the effects of PI3K but does not appear to contribute greatly to the altered arterial reactivity in DOCA-salt hypertension.
Arteries isolated from hypertensive animals consistently demonstrate potentiated contractions to a variety of agonists, and recent evidence has underlined the importance of phosphatidylinositol 3-kinase (PI3K) activity in this potentiation.1–5 For example, Northcott et al5 have demonstrated that PI3K activity is elevated in aortae from rats with DOCA-salt hypertension and that the increased activity contributes to enhanced arterial reactivity.
To investigate the mechanisms involved in the PI3K-mediated contractile response seen in arteries from DOCA-salt hypertensive animals, we have focused on the downstream serine-threonine kinase, glycogen synthase kinase 3 (GSK-3). GSK-3 is a ubiquitously expressed kinase that was first characterized as a major regulator of glycogen synthase activity.6 GSK-3 is a direct substrate of PKB/Akt7 and functions in several signaling pathways, including the PI3K pathway, to affect protein synthesis, carbohydrate metabolism, gene transcription, and apoptosis.8 Of the 2 known GSK-3 isoforms, the β isoform is best characterized and seems to be predominant in adult tissues.9 In contrast to most serine-threonine kinases, activity of GSK-3 is reduced by phosphorylation and increased by dephosphorylation. Hence, PI3K and resultant Akt activation cause GSK-3 inactivation.
GSK-3 activity has been shown to be regulated by both α and β adrenergic receptor activation.10,11 Activation of α adrenergic receptors induces phosphorylation of GSK-3β, the major GSK-3 isoform, thus inhibiting its activity.11 In addition, phosphorylation and inhibition of GSK-3β leads to cardiac hypertrophy, which can be prevented by overexpression of active GSK-3β.12 Because PI3K activity is increased in DOCA-salt hypertension, we postulated that GSK-3β activity would be reduced in this model and that persistent inhibition of GSK-3β would in turn contribute to the augmented adrenergic signaling and/or to vascular smooth muscle hypertrophy that might augment the contractile response. However, in contrast to this hypothesis, we have observed a decrease in GSK-3 phosphorylation and an increase in GSK-3 activity. In addition, we have confirmed a decrease in Akt phosphorylation in arteries from DOCA-salt hypertensive animals. These results are unexpected because phosphorylation of both enzymes is usually increased when PI3K activity is enhanced. We have also tested the role of GSK-3 activity on the enhanced reactivity observed in arteries from DOCA-salt hypertensive animals and have found that it has only a minor effect on vascular reactivity in this model.
Polyclonal antibodies for phosphoGSK-3α/βSer9/21, total Akt, phosphoAktThr308, and phosphoPDK1Ser241 were obtained from Cell Signaling Technology. The antibody for total GSK-3α/β was from Upstate Biotechnology. Antibodies for total PKB kinase/PDK1-BD were from Transduction Laboratories. SB216763 and SB415286 were obtained from GlaxoSmithKline. All other compounds were from Sigma-Aldrich Corporation.
Male Sprague Dawley rats (250 to 300 g; Charles River Laboratories Inc, Portage, Mich) were uninephrectomized and impregnated with a subcutaneous silastic implant containing DOCA (200 mg/kg) under isoflurane anesthesia as described previously.13 Animals were maintained on water containing 1.0% NaCl and 0.2% KCl for 4 weeks. Sham animals were uninephrectomized but did not receive a DOCA implant and were maintained on normal drinking water. Systolic blood pressures were measured using standard tail-cuff procedures.
Endothelial cell-denuded or intact strips of thoracic aortae and carotid arteries were removed from pentobarbital (60 mg kg−1, IP) anesthetized rats and were mounted in isolated tissue baths for measurement of isometric force.13 Tissues were preincubated in the presence of SB415286 (1 μmol/L and 10 μmol/L) for 1 hour before measurements of contractility. Norepinephrine (NE, 1×10−9 to 3×10−5 mol/L) was added in a cumulative fashion after preincubation with inhibitors.
Aortae and carotid arteries were cleaned and pulverized in liquid nitrogen and solubilized in lysis buffer and prepared as previously reported.14 Whole cell lysates were separated by SDS-PAGE and immunoblotting was performed as previously reported.5,14
GSK-3 Activity Assay
Tissue homogenates from aortae and carotid arteries were prepared as described above using a homogenization buffer comprised of HEPES pH 7.5 (50 mmol/L), EGTA (1 mmol/L), EDTA (1 mmol/L), β-glycerophosphate (10 mmol/L), sodium pyrophosphate (5 mmol/L), potassium chloride (100 mmol/L), Triton X-100 (0.5%), benzamidine (1 mmol/L), PMSF (0.1 mmol/L), aprotonin (10 μg/mL), vanadate (0.5 mmol/L), and dithiothreitol (1 mmol/L). In some samples, the GSK-3 inhibitor, SB415286, was added to the homogenization buffer before sample preparation for control experiments. Homogenates were sonicated for 10 seconds at 40% duty cycle (Sonicator Vibracell, Sonics and Materials Inc) and were centrifuged at 13 000 rpm for 20 minutes at 4°C. The supernatants were transferred to fresh microfuge tubes and protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Pierce). Samples (10 μg) were added to 5 μL of 5× reaction buffer (HEPES [250 mmol/L], MgCl2 [50 mmol/L], and 2.5 mg/mL phosphoglycogen synthase peptide-2 substrate [Upstate Biotechnology]) and preincubated for 2 minutes at 30°C. The reaction was initiated by the addition of 250 μmol/L ATP and 2 μCi/10 μL 32P-γ-ATP (Amersham) and incubated at 30°C for 15 minutes. Addition of 200 mmol/L EDTA+5 mmol/L ATP terminated the reaction and samples were transferred to Whatman P81 filter paper and washed 3 times in 100 mmol/L phosphoric acid followed by a final rinse in 95% ethanol. Filter paper was allowed to air dry and radioactivity was assessed by scintillation counting.
Data are presented as mean±standard error of the mean. Contraction 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 National Institutes of Health (NIH) Image (v.1.61). When comparing 2 groups, a Student t test was used. For multiple comparisons, an ANOVA followed by Student-Newman-Keuls or Bonferroni post hoc tests was performed. Probability values ≤0.05 were considered statistically significant.
Arteries from DOCA-salt hypertensive rats and normotensive controls (systolic blood pressure: DOCA 180.5±4.17; sham 115.63±2.39 mm Hg) were isolated and vascular reactivity was assessed. As previously reported,5 inhibition of PI3K with LY294002 attenuated the enhanced norepinephrine-induced reactivity seen in arteries from DOCA-salt rats, (−log EC50 values [mol/L]: DOCA −7.85±0.15 versus DOCALY294002 −6.80±0.14) (Figure 1), confirming the functional effect of enhanced PI3K activity in hypertensive vessels. Incubation with LY294002 more modestly reduced reactivity in normotensive aortae (−log EC50 values [mol/L]: sham −6.97±0.2 and shamLY294002 −6.48±0.14). Similar results were found in carotid arteries from both hypertensive and normotensive animals (−log EC50 values [mol/L]: DOCA −8.36±0.19 versus DOCALY294002 −7.33±0.07 and sham −8.26±0.32 versus shamLY294002 −7.42±0.06).
To determine whether enhanced PI3K activity resulted in increased phosphorylation of the downstream kinase, GSK-3β, we evaluated phosphoGSK-3 levels in arteries from DOCA-salt and normotensive rats. Surprisingly, there was a decrease in GSK-3β phosphorylation in vessels from hypertensive rats (relative GSK-3β phosphorylation: aortae, sham 0.767±0.153 versus DOCA 0.253±0.031; carotid arteries, sham 1.06±0.334 versus DOCA 0.125±0.081) (Figure 2).
To confirm that the decreased GSK-3β phosphorylation correlated with increased GSK-3 activity,15 we assessed the level of GSK-3 activity in arterial homogenates from DOCA-salt hypertensive and control rats. As expected, GSK3 activity was approximately twice as high in tissue homogenates from carotid arteries of DOCA-salt hypertensive animals when compared with control homogenates (sham 3532±438 versus DOCA 6543±566 cpm/μg protein/5 minutes) (Figure 3A). The GSK-3 inhibitor, SB415286, reduced GSK-3 related kinase activity in arterial homogenates at 1 μmol/L and, especially, at 10 μmol/L, confirming the specificity of this inhibitor in arterial tissue (Figure 3B).
To further characterize the apparent dissociation of PI3K and GSK-3 activities in DOCA-salt vessels, we assessed the activity of signaling intermediates between PI3K and GSK-3. We previously reported that Akt Ser473 phosphorylation was decreased in vascular smooth muscle of hypertensive animals.5 Because activity of Akt is dependent on the phosphorylation state of both Ser473 and Thr308, we assessed the phosphorylation state of Akt Thr308 and found a 43.5±1.2% decrease in its phosphorylation in aortae from hypertensive animals (Figure 4A). In addition, we examined the phosphorylation of PDK1, which mediates PI3K-induced phosphorylation of Akt. There was no difference in PDK1 phosphorylation in DOCA-salt and sham vessels (relative PDK1 phosphorylation: sham 1.53± 0.23, DOCA 152±0.15) (Figure 4B).
To determine whether increased GSK-3 activity contributes to enhanced reactivity of the hypertensive vessels, we assessed the effect of 2 GSK-3 specific inhibitors, SB415286 (1 μmol/L and 10 μmol/L) and SB216763 (1 μmol/L) (data not shown) on aortae and carotid arteries from normotensive and hypertensive animals. SB415286 had no effect on reactivity in the aortae at 1 μmol/L (−log EC50 values [mol/L]: DOCA −8.07±0.10 versus DOCASB415286 −7.87±0.07 and sham −7.70±0.10 versus shamSB415286 −7.53±0.06) but modestly attenuated reactivity at 10 μmol/L (−log EC50 values [mol/L]: DOCA −8.06±0.18 versus DOCASB415286 −7.72±0.16 and sham −7.91±0.14 versus shamSB415286 −797±0.21) of the hypertensive vessels at (Figure 5). Essentially no effects were seen in the carotid arteries incubated with SB415286 at 1 μmol/L (−log EC50 values [mol/L]: DOCA −8.06±0.16 versus DOCASB415286 −8.04±0.13 and sham −7.98±0.22 versus shamSB415286 −7.98±0.13) and 10 μmol/L (−log EC50 values [mol/L]: DOCA −7.84±0.11 versus DOCASB415286 −7.79±0.10 and sham −7.84±0.11 versus shamSB415286 −8.26±0.09) (Figure 6). These data suggest that increased GSK-3 activity may contribute modestly to the enhanced reactivity in DOCA-salt aortae but plays no role in the carotid artery reactivity.
Recent results have shown that the enhanced contractile response to norepinephrine in DOCA-salt hypertensive arteries is due to enhanced PI3K activity.5 The current study addresses whether downstream kinases known to be regulated by PI3K, specifically PDK1, Akt, and GSK-3, are involved in this enhanced contractile response. Contrary to expectations, phosphorylation of 2 of these downstream kinases, Akt and GSK-3, was reduced, not enhanced, in DOCA-salt vessels. Besides suggesting a complex regulation of Akt and GSK-3 in vascular smooth muscle cells, these data support the hypothesis that PI3K enhances contractility through signaling pathways other than the Akt/GSK-3 pathway.
GSK-3β has been shown to be an important signaling molecule in regulating glucose transport and glycolytic metabolism in a variety of tissues including vascular smooth muscle.15,16 Previous reports have demonstrated a significant decrease in both glucose transporter expression and glucose transport in vascular smooth muscle cells from aortae and carotid arteries of DOCA-salt hypertensive rats and that this impaired glycolytic metabolism contributes to the altered contractility profile in hypertension.14 Thus, we determined whether the surprising decrease in GSK-3 phosphorylation and the concomitant increase in activity contributed to the enhanced norepinephrine-induced contractions seen in the arteries from hypertensive animals.
Whereas the use of the GSK-3 inhibitors, SB415286 and SB216762, at a 1 μmol/L had no effect on the norepinephrine-induced contraction, SB415286 at 10 μmol/L demonstrated a relatively modest attenuation of the norepinephrine-induced reactivity in aortae from the hypertensive animals. Although these compounds have not been used previously to study the effects of GSK-3β activity on arterial contractility, the 10 μmol/L dose appears to be the effective dose in in vivo preparations.17 While further experiments will be required to definitively assess the functional effects of increased GSK-3β activity in hypertensive vessels, any effects of increased GSK-3 activity on contractility in hypertensive vessels would likely be independent of, and additive to, the well-documented effects of enhanced PI3K activity on arterial contractility.5
The surprising reduction in Akt and GSK-3β phosphorylation in hypertensive aortae despite increased PI3K activity suggests a number of potential future lines of investigation. First, the dissociation between PI3K and Akt and GSK-3β signaling found in DOCA-salt hypertensive arteries is quite unusual. The cellular mechanisms behind this dissociation have yet to be elucidated but may involve other vascular signaling pathways that are induced by DOCA-salt hypertension. Second, these data make very clear that the enhanced PI3K-associated arterial reactivity found in this model is not due to changes in Akt/GSK-3β signaling. Finally, although enhanced GSK-3β activity appears to play little direct role in vascular reactivity, it may participate in long-term modulation of hypertrophy in hypertensive vascular smooth muscle cells as it does in cardiac myocytes. Enhanced activity may protect against hypertrophy12 and may be an important counterbalance to hypertrophic stimuli in hypertension. Further augmentation of GSK-3 activity in vascular smooth muscle cells might prevent or reduce such changes in hypertension.
This work was supported by National Institutes of Health (NIH) grants RO1 HL 60156 and HL 65567 to F.C.B. and an institutional NIH predoctoral National Research Service Award (T32 GM08322) to R.D.L.
- Received December 6, 2002.
- Revision received December 31, 2002.
- Accepted February 5, 2003.
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