Mislocalization of eNOS and Upregulation of Cerebral Vascular Ca2+ Channel Activity in Angiotensin-Hypertension
We tested the hypothesis that endothelial dysfunction induced by angiotensin II (Ang-hypertension) would impair regulatory control of vascular smooth muscle L-type Ca2+ channels by endothelial nitric oxide synthase (eNOS). We studied cerebral lenticulostriate arterioles (LSAs) from control rats, from rats infused with Ang (240 μg · kg−1 · h−1 SQ ×4 days), which were normotensive, and from Ang-hypertensive rats (AHR; 240 μg · kg−1 · h−1 ×28 days). Patch-clamp measurements on isolated LSA smooth muscle cells (SMCs) showed a significant increase in Ca2+ channel availability with 4- and 28-day infusions versus controls (0.47±0.03 and 0.66±0.05 vs 0.36±0.03 pS/pF, respectively; P<0.01), with Western blots showing no change in channel protein expression, consistent with altered channel regulation. In LSAs from 28-day AHR, 4,5-diaminofluorescein diacetate imaging showed diminished NO production in response to acetylcholine stimulation in vivo, and inhibition of eNOS with NG-nitro-l-arginine methyl ester failed to increase Ca2+ channel availability in isolated SMCs, indicating an abnormality with the eNOS/NO-signaling pathway regulating the channel. Immunofluorescence imaging showed that in 1 of 53, 33 of 109, and 53 of 62 LSAs from controls and from rats with 4- and 28-day infusions, respectively, eNOS was absent from its normal location at the abluminal border and was mislocalized to perinuclear Golgi. Ca2+ channel availability in LSA SMCs from controls and from rats with 4- and 28-day infusions was proportional to the fraction of LSAs showing eNOS mislocalization, but not blood pressure. These data provide the first evidence linking Ang-induced eNOS mislocalization, eNOS dysfunction, and Ca2+ channel upregulation, and they provide novel mechanistic insights into pathological changes in LSAs associated with stroke.
- calcium channels
- angiotensin II
- nitric oxide synthase
- hypertension, experimental
- muscle, vascular, smooth
Pathophysiological processes associated with hypertension that damage cerebral blood vessels and lead to stroke are poorly understood. Endothelial dysfunction, which is well documented in hypertension, is regarded as an important early contributor1 because it is a leading factor predisposing to altered contractility and structural remodeling of the medial layer. However, specific mechanisms by which endothelial dysfunction leads to changes in underlying vascular smooth muscle cells (SMCs) are not well understood.
Abnormalities of the renin-angiotensin system are encountered in many forms of hypertension. Furthermore, the level of circulating angiotensin II (Ang) is an important determinant of the incidence and severity of ischemic injury in experimental stroke.2 Animal models of hypertension associated with increased levels of circulating Ang, including spontaneously hypertensive rats (SHR), stroke-prone SHR (spSHR), and angiotensinogen-overexpressing mice, are associated with increased incidence of stroke, and blockade of this pathway with either angiotensin-converting enzyme inhibitors or angiotensin receptor blockers reduces stroke risk and infarct volume, both in animal models3–6 and in humans.7
Ca2+ is involved in multiple functions in smooth muscle, including contraction, proliferation, and gene regulation,8,9 and the L-type Ca2+ channels in SMCs are the most important and highly regulated route of Ca2+ entry into these cells. Given that nitric oxide (NO) generated by endothelial NO synthase (eNOS) normally downregulates the L-type Ca2+ channel in SMCs,10 we hypothesized that in Ang-related hypertension, endothelial dysfunction induced by Ang11,12 would result in an increase in availability of L-type Ca2+ channels. Larger Ca2+-channel currents have been observed in SMCs of cerebral vessels from Ang-related hypertensive animals, including spSHR and Goldblatt 2-kidney, 1-clip rats.13,14 However, direct evidence linking Ang-induced endothelial dysfunction and increased availability of Ca2+ channels in SMCs has not been reported.
In the present study, we examined the effects of subpressor doses of Ang,15 infused for 4 or 28 days, on lenticulostriate arterioles (LSAs), the small cerebral vessels that typically undergo pathological deterioration leading to stroke. We found that Ca2+-channel availability in isolated SMCs was significantly increased in LSA SMCs with Ang infusion and that this abnormality was correlated with abnormal eNOS activation and abnormal eNOS localization, both of which are novel findings in vivo. Moreover, eNOS mislocalization, which appeared after 4 days of Ang infusion, preceded development of hypertension and was directly proportional to Ca2+-channel availability, consistent with Ang-induced endothelial dysfunction, rather than hypertension per se, being responsible for abnormal channel regulation. These findings provide direct evidence linking Ang-induced endothelial dysfunction and dysregulation of smooth muscle Ca2+ channels, and they demonstrate an important mechanism, ie, an increase in Ca2+ channel availability, by which pathophysiological changes in the medial layers of cerebral arterioles associated with stroke may be initiated.
Animal Models and Cell Isolation
Ang-hypertensive rats (AHR) were prepared by implanting female Wistar-Kyoto (WKY) rats, 220 to 300 g, with osmotic minipumps (Alzet 2002, Alza Corp) that delivered 0.9% NaCl with Ang (Sigma; 240 μg · kg−1 · h−1) for 28 days.16 Controls were age-matched, female WKY rats with no implanted pump. We also studied animals, with implanted pumps that delivered Ang at the same rate as in AHR, that were euthanized at 4 days, before the development of hypertension. Systolic blood pressure (mean±SEM), measured in awake animals by tail-cuff plethysmography (Harvard Instruments), was 137±13, 135±15, and 205±10 mm Hg in control rats and in rats with 4-day and 28-day infusions of Ang, respectively.
After death (sodium pentobarbital, 100 mg/kg IP), animals underwent transcardiac perfusion with perfusion solution containing (in mmol/L) NaCl 116.3, KCl 5.4, NaH2PO4 10.4, MgSO4 0.83, glucose 5.5, and NaHCO3 26.2, together with papaverine · HCl 6 μg/mL, equilibrated with 95% O2/5% CO2. For typical experiments, the brain was subsequently removed, but for other experiments, an additional perfusion step was included before the brain was removed.
For experiments that blocked NOS activity, a second perfusion was performed with perfusion solution (100 mL) supplemented with 1 mmol/L NG-nitro-l-arginine methyl ester (l-NAME, Calbiochem-Novabiochem Corp). For these experiments, all subsequent solutions used for cell isolation were also supplemented with 1 mmol/L l-NAME. LSAs were dissected from posterior and middle cerebral arteries and were processed for isolating single cells by enzymatic digestion.17
Methods used for perforated patch recording in this laboratory have been described.10,17 For macroscopic Ca2+-channel recordings, the bath solution contained (in mmol/L) tetraethylammonium (TEA) · Cl 130, MgCl2 1, BaCl2 10, HEPES 10, glucose 12.5, and 4-aminopyridine 2, pH 7.2, with TEA · OH, and the pipette solution contained (in mmol/L) CsCl 130, MgCl2 8,and HEPES 10, pH 7.35, with CsOH plus nystatin. For cell-attached patch recordings, the pipette contained (in mmol/L) TEA · Cl 100, MgCl2 1, BaCl2 40, HEPES 10, glucose 12.5, and 4-aminopyridine 2, pH 7.2, with TEA · OH, and the bath contained (in mmol/L) KCl 145, MgCl2 2, HEPES 10, and glucose 12.5, pH 7.4, with NaOH.
In Situ NO Production
Imaging for in situ NO production was carried out with 4,5-diaminofluorescein diacetate (DAF-2DA) by using a modification of protocols previously described.18 An animal was anesthetized with ketamine (60 mg/kg) plus xylazine (7.5 mg/kg), and a femoral venous catheter was placed. DAF-2DA was given as 2 separate bolus injections (70 μL of a 5 mmol/L stock solution in dimethyl sulfoxide) 20 minutes apart. Ten minutes after the second bolus, a bolus of acetylcholine (6 μL of 100 mmol/L stock solution in saline) was given to stimulate eNOS. Animals were then perfusion-fixed with 2% glutaraldehyde, brains were removed and quickly frozen in 2-methylbutane, and 10-μm frozen sections were cut and examined with a Nikon E1000 epifluorescence microscope.
LSAs isolated from 3 to 5 rats per batch were lysed in liquid nitrogen, thawed, and homogenized in lysis buffer (50 mmol/L Tris-Cl, pH 7.5, 250 mmol/L sucrose, 1 mmol/L EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate [SDC], 10 mmol/L phenylmethylsulfonyl fluoride, 1% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail). After normalizing for protein concentrations in the supernatant of lysates, protein was denatured and separated by 10% SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes. Immunoblotting was performed with antibodies directed against the α1C subunit of the L-type Ca2+ channel (1:200, Alomone), eNOS (1:200, Santa Cruz Biotechnology), and phosphotyrosine (1:1000, Upstate Biotechnology). Blots were visualized with an enhanced chemiluminescence system (Amersham). Levels of immunoreactive proteins were determined by densitometry using Scion Image software (Scion Corp).
Protein isolates obtained as described in the preceding section were pretreated with 20 μL/mL rabbit serum and 50 μL/mL protein A–Sepharose beads for 2 hours at 4°C. Supernatants obtained after centrifugation were incubated with 50 μL/mL protein A–Sepharose beads and eNOS antibody overnight at 4°C. Immunocomplexes were washed 3 times in buffer (50 mmol/L Tris-Cl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% NP-40, 0.1% SDS, and 0.5% SDC) at 4°C and resuspended in 50 μL of SDS-PAGE loading buffer. Antigen was eluted by heating the tubes to 70°C for 10 minutes and was analyzed by Western blotting.
Animals were perfusion-fixed with 10% formaldehyde in phosphate-buffered saline, and brains were removed and processed for paraffin sectioning (4 μm). Sections were permeabilized with 0.5% Triton X-100; nonspecific binding was blocked with 1% bovine serum albumin in 0.5% Triton X-100 for 60 minutes at room temperature and then exposed to primary antibodies at 37°C for 1 hour. The primary antibodies used are listed in the preceding section. Immunolabeled sections were examined with a Nikon Eclipse E1000 microscope. Images were captured and processed with a SenSys digital camera (Photometrics) and a personal computer with IP Laboratory software (v 3.06, Scanalytics).
Ca2+-channel availability was quantified and normalized as previously described.17 Statistical comparisons were evaluated with either ANOVA with Student-Newman-Keuls comparisons, Student t test, or χ2 analysis. Data are given as mean±SEM.
We compared macroscopic Ca2+-channel currents in LSA SMCs from control rats and from AHR (28-day infusion of Ang). In both groups, the kinetics of activation, inactivation, and deactivation (Figures 1A and 1B), as well as the voltage dependence (Figures 1C and 1D) of the currents were typical for L-type Ca2+ channels. However, the current density in LSA SMC from AHR was noticeably larger than from controls (Figures 1C and 1D). For control rats versus AHR, maximum current densities at +10 to +20 mV were −14.2±1.2 versus −21.0±2.0 pA/pF, respectively, which were significantly different (by t test, P<0.01).
Possible Mechanisms for Increased Current Density
We examined several variables that might account for larger currents in AHR. Cell sizes, estimated by cell capacitance, were 9.1±0.7 and 7.8±0.4 pF for control versus AHR, respectively, which were not different (by t test, P>0.05). Also, currents in AHR were highly sensitive to the blocking dihydropyridine nifedipine (Figure 2A), suggesting that no new Ba2+-permeable cation channel was present. Single-channel recordings obtained with the activating dihydropyridine, Bay K8644 showed an underlying single-channel conductance of 22 to 24 pS in AHR (Figures 2B and 2C), as found in controls,17 indicating no change in single-channel conductance.
Western blotting of lysates from LSAs was performed to examine the L-type Ca2+ channel α1C subunit (Figure 2D). In controls and AHR, bands were labeled at ≈200 kDa, the appropriate molecular weight, as indicated by the control tissue from brain. Densitometric analysis of 5 different batches of LSA tissues revealed a small decrease in channel protein in AHR compared with controls, but this was not statistically significant (by t test, P>0.05; Figure 2E). Given that L-type Ca2+ channels are present in SMCs but not in endothelium, these data indicated that the large increase in Ca2+-channel current density observed in SMCs from AHR was not due to an increase in channel expression but was more likely to be due to altered channel regulation.
Experiments were performed to determine whether the increase in current density could be attributed to an acute, direct effect of Ang on SMCs, which was being chronically infused in AHR, or of peroxynitrite, which might be generated in the vessel wall in response to Ang.1 Ang (100 nmol/L) or peroxynitrite (100 to 200 μmol/L) was added to the perfusion solution while patch-clamping LSA SMCs from normotensive control animals. Except for a brief, spontaneously reversing diminution in current immediately after application of Ang, neither agent caused a change in Ca2+-channel current (Figure 2F), suggesting that the increase in current density in AHR was unlikely to have been mediated by an acute, direct effect on ion channels.
eNOS Regulation of Ca2+ Channels
Given that the eNOS/NO-signaling pathway downregulates smooth muscle Ca2+ channels,10 we considered that the increase in current density in AHR might be due to endothelial dysfunction, with an associated decrease in bioavailability of NO. We tested the functional integrity of this pathway in LSAs by examining the effect of an eNOS inhibitor on the currents. In a vessel with normal, ongoing eNOS activity, pretreatment of the vessel with l-NAME before isolation of the SMCs results in an increase in Ca2+-channel currents in SMCs isolated from that vessel. Conversely, with dysfunction of eNOS, l-NAME pretreatment of the vessel has no effect on Ca2+-channel currents in SMCs isolated from that vessel.10
We first tested the effect of NOS inhibition in LSAs from control rats. Pretreatment of vessels with l-NAME before SMC isolation yielded cells that exhibited a significant increase (by ANOVA, P<0.01) in Ca2+-channel currents (Figures 3A and 3B) when compared with cells isolated from LSAs without l-NAME pretreatment (Figures 1A and 1C). Moreover, values of current density were similar to those observed in LSA SMCs from AHR (Figure 1D). Notably, l-NAME pretreatment had no effect on either the macroscopic (Figures 3A and 3B) or the single-channel (not shown) properties of the currents. Separate control experiments with LSA SMCs from untreated control rats showed that acute exposure to l-NAME during the patch-clamp experiment had no effect on Ca2+-channel properties or on the magnitude of the current.10 These findings confirmed that in LSAs from control rats, l-NAME–sensitive NOS was actively producing endogenous NO and that this NO was normally downregulating Ca2+ channels.
We repeated the NOS inhibition experiment with LSAs from AHR. Pretreatment of these vessels with l-NAME before SMC isolation had no effect, yielding cells that exhibited the same current density (Figures 3C and 3D) as did cells from AHR not pretreated with l-NAME (Figures 1B and 1D). Computation of Ca2+-channel availability from the 4 sets of data (control and AHR, with and without l-NAME) reaffirmed that l-NAME pretreatment had a significant effect in cells from controls but not from AHR (Figure 3E). Together, these findings that the increase in current density in AHR could be mimicked by eNOS inhibition in controls and that l-NAME pretreatment in AHR had no effect were consistent with the hypothesis that the increase in current density in AHR was due to an abnormality in the eNOS/NO-signaling pathway that regulates the channel.
Abnormal eNOS Activation
Our electrophysiological data suggested that the increase in current density in AHR could be due to eNOS dysfunction. To further assess this, we measured NO production in arterioles in vivo by using the NO-sensitive probe, DAF-2DA.18 We administered DAF-2DA intravenously to anesthetized animals, stimulated eNOS activity by administering an intravenous bolus of acetylcholine, and then stopped eNOS activity by perfusion-fixation. Fluorescence examination of LSAs from control rats showed strong DAF-2DA signal within the vessel wall (Figures 4A and 4B). In contrast, in LSAs from AHR, this protocol resulted in noticeably less signal being evident in the vessel wall (Figures 4C and 4D). Quantitative evaluation of the signals from 8 control LSAs and 6 AHR LSAs indicated that the DAF-2DA signal was significantly weaker (by t test, P<0.05) in LSAs from AHR (Figure 4E), giving direct evidence that eNOS activation in vivo was reduced.
We quantified eNOS protein on Western blots of lysates from LSAs (Figure 4F). Densitometric analysis indicated no significant difference in protein in vessels from AHR compared with controls (Figure 4F), suggesting that abnormal eNOS activation was not attributable to altered protein expression. Abnormal eNOS function could be due to tyrosine phosphorylation.19 To examine this, we immunoisolated eNOS from cerebral tissues and immunoblotted it for phosphotyrosine. These experiments showed no significant changes in AHR compared with controls (Figure 4G).
The finding of eNOS dysfunction prompted us to perform imaging experiments to examine eNOS localization in LSAs. In controls, immunofluorescence labeling for eNOS showed strong signal in the endothelial layer of all LSAs, with signal being prominent on the side of the endothelium near the basal lamina (Figures 5A and 5B). In LSAs from AHR, the signal for eNOS appeared to be as strong as in controls, but it was often located predominantly in perinuclear regions, presumably in the Golgi complex, away from the abluminal surface, indicating an abnormality with eNOS localization (Figures 5C and 5D). Defining an arteriole with eNOS mislocalization as one in which 2 or more endothelial cells showed perinuclear clumping of eNOS, we identified mislocalization in 53 of 62 arterioles from 3 AHR, compared with 1 of 53 arterioles from 3 control rats.
Four-Day Infusion of Ang
Finally, we considered that the abnormalities of eNOS mislocalization and increased Ca2+-channel availability observed in AHR could have been due to either hypertension per se or simply to infusion of Ang. To examine this, we studied rats infused with Ang at the same rate as in AHR, but for only 4 days instead of 28 days. After 4 days, systolic blood pressure was not elevated (Figure 6A). However, compared with controls, both the Ca2+-channel availability in LSA SMCs (0.47±0.03 nS/pF, 13 cells) and the index of eNOS mislocalization (33 of 109 arterioles in 3 rats) were significantly increased (by ANOVA, P<0.01, and by χ2 analysis, P<0.001, respectively).
Analysis of data from control animals and from animals infused with Ang for 4 or 28 days indicated that neither the index of eNOS mislocalization (Figure 6B) nor the magnitude of Ca2+-channel availability (Figure 6C) was related to systolic blood pressure, but both were significantly related to each other (r=0.999, P<0.01; Figure 6D).
Here we report 3 novel findings in LSAs from Ang-hypertensive rats: (1) that eNOS was dysfunctional, as shown by reduced production of NO in response to acetylcholine stimulation and by failure of upregulation of Ca2+-channel activity after exposure of vessels to l-NAME; (2) that eNOS was mislocalized in endothelium; and (3) that L-type Ca2+-channel activity in SMCs was upregulated. Our data indicated that these abnormalities were due to prolonged infusion of Ang, rather than an increase in blood pressure. Moreover, we found that eNOS mislocalization and Ca2+-channel availability were proportionally related to each other.
It is generally accepted that L-type Ca2+ channels play an important role in hypertension, based on reports of an increase in [Ca2+]i,20,21 and on the ability of Ca2+-channel blockers to reduce Ca2+ entry, restore endothelial function, reduce blood pressure, and normalize pathological changes in vessel walls.22 Despite an abundance of indirect evidence of this type, specific molecular mechanisms involving the channel have not been well characterized.23 Larger Ca2+-channel currents have been reported in Goldblatt 2-kidney, 1-clip hypertension14 as well as in genetic forms of hypertension. In SHR, this has been attributed to increased channel expression,24 but whether this mechanism is common to other forms of hypertension or whether existing channels are abnormally regulated had not previously been determined. Similarly, studies of NOS-III knockout models as well as of NOS inhibitors and NO scavengers have shown the importance of mechanisms involving abnormal eNOS function in the development of excessive vessel tone,25,26 but it had not previously been determined whether augmented Ca2+-channel availability was present in these conditions or whether other targets of cGMP-dependent protein kinase were involved.27 Here we showed that in AHR, Ca2+-channel activity was upregulated, with no change in channel protein expression, and that upregulation was due to eNOS dysfunction.
We showed dysfunction of eNOS by using 2 entirely different methods. First, we showed that the NOS inhibitor l-NAME failed to augment Ca2+-channel activity in AHR, a response normally observed with functional eNOS in an intact vessel.10 Second, we showed that acetylcholine failed to stimulate NO production, also a normal response expected with functional eNOS. For the latter experiments, we used the novel technique of administering DAF-2DA in vivo, stimulating the animals with acetylcholine, and then stopping eNOS activity by perfusion-fixation. This protocol allowed us to assess relative NO production in vivo without the confounding effects of dissection and harvesting that produce shear forces that affect NO production in isolated vessels.28 The DAF-2DA signal that we measured in AHR was significantly reduced compared with controls, but it may have been artifactually increased due to a possible elevation in [Ca2+]i, which may augment DAF-2DA signals.29 Thus, the magnitude of the reduction in NO levels that we measured in AHR might have been underestimated. Nevertheless, these experiments, coupled with the patch-clamp experiments with l-NAME, gave direct evidence that eNOS activation in vivo was reduced; ie, that eNOS was dysfunctional.
Reduced activity of the eNOS/NO-signaling pathway has previously been shown in several pathophysiological condition,30,31 with the most commonly observed mechanism being a decrease in eNOS protein expression.32,33 However, we found no alteration in eNOS protein levels in AHR, indicating that this mechanism is not common to all forms of hypertension.
Recent in vitro studies have shown that mislocalization of eNOS is associated with reduced activity of the eNOS/NO-signaling pathway. Optimal formation of NO depends on proper association with regulatory proteins, subcellular trafficking, and correct localization of the enzyme.34 Mislocalization of eNOS away from plasma membrane caveolae and its accumulation in the Golgi complex, which is invariably associated with reduced NO production, has been reported in cultured endothelial cells after brief exposure to oxidizing agents or to oxidized LDL (ox-LDL).35–38 Oxidative stress is a key mechanism of injury to endothelium with Ang, making it likely that this is the cause of the eNOS mislocalization that we observed.15 Ang causes upregulation of expression of the endothelial receptor for ox-LDL, LOX-1, causes a concentration-dependent increase in uptake of ox-LDL by endothelial cells, and enhances ox-LDL–mediated cell injury.39 Thus, the mislocalization and associated dysfunction of eNOS that we observed here might be due to oxidant stress brought on by elevated levels of Ang, although additional work would be required to confirm this.
Our data indicate that a characteristic feature of AHR is an increase in availability of Ca2+ channels in LSA SMCs. An increase in Ca2+-channel availability would be expected to result in an increase in vessel tone, which would compromise collateral flow in the penumbra of an infarct and might thereby account for worsened stroke associated with Ang-related hypertension.2 In addition, the increase in Ca2+-channel availability would be expected to activate cellular processes and upregulate expression of genes associated with degeneration of SMCs. Thus, the consequences of mislocalization and associated dysfunction of eNOS due to Ang are likely to be far-reaching in their implications for stroke.
Only one previous study has demonstrated a molecular mechanism responsible for increased L-type Ca2+-channel availability in hypertension.24 In that study, which involved SHR, it was found that Ca2+ channel α1C subunit mRNA and protein were increased. By contrast, here we show in Ang-related hypertension that increased channel availability is due not to an increase in channel protein expression but to channel dysregulation attributable to eNOS dysfunction. Our data showing mislocalization and associated dysfunction of eNOS in AHR provide the first evidence showing the importance of eNOS mislocalization with chronic exposure to Ang and the relevance of this mechanism in an in vivo pathological condition. The observed increase in Ca2+-channel availability in AHR as well as in SHR24 provides the best explanation for the success of Ca2+-channel blocker therapy in reversing vessel wall changes in hypertension, thereby pointing specifically to abnormal regulation of L-type Ca2+ channels as a key factor in initiating degenerative processes in SMCs.
This work was supported by grants (to J.M.S.) from the National Heart, Lung, and Blood Institute (HL51932), the National Institute for Neurological Diseases and Stroke (NS39956), and a Bugher award from the American Heart Association. We thank Lioudmila Melnitchenko and Jia Bi Yang for expert assistance.
- Received September 30, 2002.
- Revision received October 23, 2002.
- Accepted March 3, 2003.
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