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(Hypertension. 2002;39:704.)
© 2002 American Heart Association, Inc.

Scientific Contributions

Functional Significance of Activation of Calcium/Calmodulin–Dependent Protein Kinase II in Angiotensin II–Induced Vascular Hyperplasia and Hypertension

Mubarack M. Muthalif; Nour A. Karzoun; Ibrahim F. Benter; Lillian Gaber; Farid Ljuca; Mohammed R. Uddin; Zinat Khandekar; Anne Estes; Kafait U. Malik

From the Department of Pharmacology and Vascular Biology, Center of Excellence, College of Medicine (M.M.M., N.A.K., I.F.B., M.R.U., Z.K., A.E., K.U.M.), and the Department of Pathology (L.G., F.L.), The University of Tennessee, Baptist Memorial Hospital, Memphis, Tenn.

Correspondence to Kafait U. Malik, PhD, DSc, Professor of Pharmacology, Department of Pharmacology, College of Medicine, The University of Tennessee, Memphis, TN 38163. E-mail kmalik{at}utmem.edu

	Abstract

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We have reported that norepinephrine (NE) and angiotensin II (Ang II) increase CaM kinase II activity, which, in turn, activates cytosolic phospholipase A₂ (PLA₂) and releases arachidonic acid. The products of arachidonic acid generated via cytochrome P-450 and lipoxygenase contribute to the development of hypertension and vascular smooth muscle cell (VSMC) hyperplasia. The purpose of this study was to investigate whether CaM kinase II contributes to VSMC proliferation elicited by NE and Ang II and to hypertension induced by Ang II. NE (1 µmol/L) and Ang II (1 µmol/L) increased proliferation of rabbit aortic VSMC as measured by increased [³H]-thymidine incorporation; this effect of NE and Ang II was attenuated 88±10% and 64±11% by the CaM kinase II inhibitor KN-93, respectively. Infusion of Ang II with miniosmotic pumps (350 ng/min for 6 days) in rats elevated mean arterial pressure (MABP), which was reduced by simultaneous infusion of KN-93 (578 ng/min, for 6 days) (Ang II alone: MABP =174±3 mm Hg, n=12 versus Ang II + KN-93: MABP 123±5 mm Hg, n=4, P<0.05). Administration of KN-93 as a single bolus injection (16 mg/Kg), but not its vehicle, in Ang II–infused hypertensive animals also decreased MABP from 179±9 mm Hg to 109±6 mm Hg (n=5, P<0.05). CaM kinase II activity was increased in the kidney of Ang II–infused hypertensive animals compared with normotensive controls. Treatment with KN-93 reduced CaM kinase II activity and ameliorated the intravascular injury in the kidneys of Ang II–infused hypertensive rats. Our data indicate that CaM kinase activation represents an important component of the mechanism(s) initiating VSMC proliferation and the development and maintenance of Ang II–induced hypertension in rat.

Key Words: kinase • angiotensin II • hypertension, essential • muscle, smooth, vascular

	Introduction

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Many cellular stimuli produce oscillations in the intracellular concentration of calcium (Ca²⁺) ions. Ca²⁺/calmodulin (CaM) dependent kinase (CaM kinase II, CaMKII), a multi functional protein kinase family, can decode the frequency of Ca²⁺ spikes and regulates the activity of a range of cellular targets involved in many physiological processes including control of cell cycle, apoptosis, gene expression, neurotransmission, long term potentiation, memory, synaptic plasticity and early after depolarization and contractile responses of aorta.^1–7 CaM kinase II contains an N-terminal catalytic domain, an autoinhibitory domain with an overlapping Ca²⁺/CaM binding domain and a C terminal subunit association domain. CaM kinase II is maintained in an inactive state by an autoinhibitory regulatory domain and binding of Ca²⁺/CaM relieves the autoinhibition and increases autophosphorylation of kinase at Thr²⁸⁶ and subsequent phosphorylation of substrates.^7,8 Autophosphorylation promotes CaM trapping and decoding of the frequency of Ca²⁺ oscillations.^3,8 We have previously shown that the adrenergic transmitter norepinephrine (NE) and angiotensin II (Ang II) increases CaM kinase II activity in vascular smooth muscle cells (VSMC);^9,10 CaM kinase II directly phosphorylates cytosolic PLA₂ on Ser515 and liberates arachidonic acid by hydrolyzing arachidonyl phospholipids.¹¹ Arachidonic acid is metabolized by cyclooxygenase, lipoxygenase and cytochrome P-450 monooxygenase into various products with marked cardiovascular actions.¹² A cytochrome P-450 metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) constricts blood vessels, promotes proliferation of VSMC and has been shown to contribute to the regulation of myogenic tone and development of high blood pressure in some models of hypertension.^13–16 These observations and the evidence that CaM kinase II regulates smooth muscle contraction^5,6 raises the possibility that CaM kinase II might play a key role in the regulation of cardiovascular function and alterations in its activity may lead to vascular disease. To test this hypothesis, we have studied the effect of CaM kinase II inhibition on rabbit VSMC proliferation caused by NE and Ang II, and on blood pressure and organ damage in Ang II-induced hypertension in rats.

	Methods

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Materials
Ang II was obtained from Bachem Biosciences. Norepinephrine (NE) angiotensin II (Ang II), sodium pentobarbital, leupeptin, aprotinin, penicillin, streptomycin, and amphotericin were obtained from Sigma; KN-92 and KN-93 were purchased from Seikagaku; polyclonal phospho-CaM kinase II antibody from Promega; and M-199 medium from Celgro; [³H] thymidine (20 Ci/mmol) from New England Nuclear.

Preparation of VSMC
The aorta was removed from male New Zealand white rabbits (1 kg) and the VSMC were isolated as previously described.¹⁷ Cells between 4 to 8 passages were plated in 48 wells. Cells were maintained below 5% CO₂ in M-199 medium with penicillin, streptomycin, and 10% fetal bovine serum. We used rabbit VSMC because we have performed several of our previous studies in this species and we have substantial background information on the signaling mechanisms involved in the action of NE and Ang II, including CaMKII activation.

Measurement of [³H]-Thymidine Incorporation in VSMC
VSMC isolated from the rabbit aorta were cultured, and subconfluent cells were incubated with 0.05% fetal bovine serum containing M-199 for 48 hours to induce mitogenic quiescence. To investigate the contribution of CaM kinase II to NE and Ang II–induced DNA synthesis, quiescent cells were preincubated with CaM kinase II inhibitor KN-93 (1 µmol/L) or its inactive analogue KN-92 (1 µmol/L) for 2 hours and then NE (1 µmol/L) or Ang II (1 µmol/L) was added for 48 hours. [³H] thymidine (0.5 µCi/mL) was added to the cultures in each well during the last 24 hours of incubation period. The cells were washed twice with ice-cold PBS, 5% trichloroacetic acid, and 95% ethanol. The cells were then lysed with 0.5 N NaOH and radioactivity was measured in a scintillation counter. [³H] thymidine incorporation was measured as counts per minute per well and expressed as a percentage increase above basal.

Flow Cytometry
Cells were arrested for 48 hours in M-199 medium (0.05% serum) and then treated with NE (1 µmol/L) in the presence and absence of KN-93 (1 µmol/L) for 16 hours. Cells were trypsinized in 1 mL of trypsin/EDTA for at least 5 minutes, and then the reaction was stopped by the addition of 1 mL of serum-containing M-199. The samples were centrifuged at 1000 rpm for 5 minutes, washed in ice-cold PBS containing 1% bovine serum albumin, and then fixed in 70% ethanol and washed in BSA buffer. The washed pellet was then resuspended in 1 mL of the BSA buffer to which 100 µg/mL of RNAse A was added to remove interfering double stranded RNA, and 5 µg/mL propidium iodide was added to stain DNA. Cells were incubated at 37°C for 10 to 15 minutes in the dark. The cells were analyzed for DNA content using an Epics Profiler II (Coulter Electronics) with an Argon laser emitting at 488 nm. The emission maxima for analysis of PI fluorescence were 610 to 630 nm. The percentage of cells in various stages of the cell cycle was determined using the "multi-cycle" program (P. Rabinovitch, Phoenix Flow Systems).

Measurement of Blood Pressure
Male Sprague-Dawley rats (Charles River Laboratories) weighing 300 to 350 g were fed standard chow (Ralston Purina Co, Wilmington, MA). All animal protocols were approved by the institutional guidelines for animal research. Rats with Ang II–induced hypertension were prepared as previously described.^15,18 Ang II was infused at a rate of 350 ng/min for 6 days. To determine the effect of CaM kinase II on development of hypertension, KN-93 or its inactive analog KN-92 (578 ng/min for 6 days) was infused, together with Ang II, in osmotic minipumps. To study the acute effect of KN-93 on established hypertension, the animals were injected subcutaneously with KN-93 solution (16 mg/kg) or its vehicle (saline) on the sixth day of infusion of Ang II. The animals were killed, and the kidneys were processed for measurement of CaM kinase II activity and histological examination. Mean arterial blood pressure (MABP) was measured via a catheter inserted in the femoral artery.

Measurement of Phospho CaM Kinase II by Western Blotting
Kidneys were isolated and washed in ice-cold PBS and frozen immediately in liquid nitrogen. The kidney (200 mg) was ground into fine powder with a pestle and mortar and homogenized in a buffer (20 mmol/L Tris HCl, pH 7.5; sucrose 300 mmol/L; 0.5 mmol/L EDTA; 0.5 mmol/L EGTA; 10 mmol/L ß-mercaptoethanol; 10 µg/mL leupeptin; and 10 µg/mL aprotinin) using a Dounce homogenizer. Lysates were centrifuged to remove debris, 100 µg of proteins were processed by Western blotting, and signals were detected using enzyme-linked chemiluminescence (ECL) (Amersham).

Histological Examination
The kidneys removed from the Ang II–induced hypertensive animals, with or without treatment with KN-93, were processed, and semiquantitative assessment of the severity and extent of the arterial lesions was performed as described.¹⁵

Analysis of Data
The basal incorporation of [³H]-thymidine in rabbit VSMC ranged between 2851 to 7591 cpm per well in different batches of cells. Although the basal values of [³H]-thymidine incorporation and the agonist-induced stimulation were variable in different batches of cells, the effect of agonists and inhibitors on [³H]-thymidine incorporation was consistent within each batch of cells. Therefore, the increase in [³H]-thymidine incorporation produced by Ang II and NE was calculated as a percentage above basal (range 45 to 149) and was taken as 100%. The results are expressed as mean±SEM. The data were analyzed with one-way analysis of variance. The Newman-Keuls multiple range test was applied to determine the difference among multiple groups. Differences were considered significant at P<0.05.

	Results

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NE- and Ang II–Stimulated [³H]-Thymidine Incorporation in VSMC and Regulation of Cell Cycle Is Mediated by CaM Kinase II
NE and Ang II are known to increase [³H]-thymidine incorporation in VSMC.^19–21 To determine whether CaM kinase II is involved in NE- and Ang II–stimulated [³H]-thymidine uptake, the effect of CaM kinase II inhibitor KN-93 was examined. NE- and Ang II–induced [³H]-thymidine uptake was inhibited by KN-93 but not by its inactive analog KN-92 (Figure 1).

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of CaM kinase II inhibitor (KN-93) on norepinephrine and Ang II–induced [3H]thymidine incorporation in rabbit VSMC. Cells were arrested in M-199 medium containing 0.05% serum for 48 hours and treated with norepinephrine (1 µmol/L) for 48 hours. In the last 24 hours, [3H]thymidine (0.5 µCi/mL) was added, and incorporation was determined as described in Methods. Changes in [3H]-thymidine incorporation is presented as percent increase obtained with NE or Ang II (taken as 100%). Data are shown as means±SE (n=6 to 30 wells obtained with 2 or 3 different batches of cells). *Value significantly different from that obtained with vehicle.

Changes in intracellular Ca²⁺ concentration occur at the awakening of cells from quiescence, at the G₁/S transition, during S-phase, and at the exit from mitosis.^22,23 There are reports indicating that CaM kinase II plays a role in the G₁/S, G₂/M, and metaphase/anaphase transitions.^24,25 Consistent with its antiproliferative effects on VSMC growth, KN-93 produced a block primarily in the G₁ phase (Table). The percentage of cells in G₁ phase increased from 59% to 83% after treatment of cells with KN-93, with a corresponding decrease in S phase (from 40% to 8% with KN-93 treatment). KN-93 treatment resulted in G₁- and G₂-phase cell-cycle arrest in NIH3T3 and HeLa cells, respectively.^26,27 Our data are in agreement with the finding that the activation of CaM kinase II pathway is required for cells to pass the G₁ and G₂ restriction points.

View this table: [in this window] [in a new window]   Table 1. Effect of Norepinephrine on VSMC Cycle in the Presence and Absence of KN-93

CaM Kinase II in Hypertension
Arachidonic acid metabolites have been implicated in both the pro- and antihypertensive mechanisms in various forms of hypertension.^28–31 The products of arachidonic acid generated via lipoxygenase (12-HETE) and cytochrome P-450 (20-HETE) in blood vessels have been shown to contribute to VSMC hyperplasia and Ang II–dependent models of hypertension.^16,19,31 The ability of CaM kinase II to phosphorylate and activate cPLA₂, which results in arachidonic acid release in response to NE and Ang II, raises the possibility that CaM kinase II activation might be an important component in the development and maintenance of hypertension and organ damage. Moreover, CaM kinase II has been reported to play a role in vascular contraction.^5,6 Therefore, we studied the effect of CaM kinase II inhibitor KN-93 on the blood pressure of animals made hypertensive with Ang II infusion. KN-93 was infused (578 ng/min) for 6 days in combination with Ang II (350 ng/min) or its vehicle. Chronic infusion of KN-93 in hypertensive animals significantly reduced MABP as shown in Figure 2 (top) (Ang II alone: MAP=174±3 versus Ang II and KN-93: MAP=123±5, n=4 to 12, P<0.05). KN-92 infusion failed to reduce blood pressure in animals made hypertensive with the administration of Ang II (n=2, data not shown). Infusion of KN-93 in normotensive rats did not alter MABP (data not shown). Administration of KN-93 subcutaneously as a single bolus injection (16 mg/kg) to animals infused with Ang II for 6 days decreased MABP from 179±9 mm Hg to 109±6 mm Hg (n=5) (P<0.05). Reduction of MABP by KN-93 lasted about 20 to 40 minutes before MABP returned to control level. Administration of KN-93 as a bolus injection did not alter arterial blood pressure in normotensive rats (n=1, data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 2. Top, Effect of CaM kinase II inhibitor KN-93 on Ang II–induced elevation of blood pressure. Rats that were made hypertensive with Ang II (350 ng/min) were infused with KN-93 (578 ng/min) for a period of 6 days together with Ang II. Mean arterial pressure was measured through a catheter inserted into the left femoral artery. Data are presented as mean±SEM (n=4 to 12). * Indicates value significantly different from that obtained with vehicle; , value significantly different from that obtained with Ang II alone (P<0.05). Bottom, Effect of KN-93 on activation of CaM kinase II in kidney. One hundred micrograms of proteins obtained from kidney were resolved by SDS-PAGE and transferred onto nitrocellulose membrane. Control (lane 1), Ang II–infused animals treated with vehicle (lane 2), and KN-93 (lane 3). The blots were probed with phosphospecific CaM kinase II antibody. The experiments shown were performed 2 or 3 times, and a representative blot of these experiments is presented.

CaM Kinase II Phosphorylation in Hypertensive Animals
To determine whether CaM kinase II activity is altered during the development of hypertension and whether treatment with KN-93 inhibits its activation, we studied the phosphorylation of CaM kinase II in kidneys from hypertensive animals treated with either KN-93 or vehicle. Phosphorylation of CaM kinase II was markedly elevated in animals infused with Ang II compared with normotensive controls. Consistent with the reduction in MABP, phosphorylation of CaM kinase II was significantly attenuated in the kidneys of KN-93–treated hypertensive animals (Figure 2, bottom).

CaM Kinase II Contribution in End-Organ Damage
We examined kidneys from 4 groups of animals, including normotensive controls, normotensive animals that received Ang II, and animals that received Ang II and KN-93 or KN-92 or their vehicle. Kidneys from animals with vehicle alone were normal. Microscopic examination of kidneys from Ang II–stimulated hypertensive animals demonstrated severe vascular pathology as previously reported.^32,33 Vascular lesions in these animals consisted of hemorrhage in the media, commonly identified in the large-sized arteries and fibrinoid necrosis, which was observed in the smaller arteries (Figure 3). In addition, there was widespread enlargement of the myocytes, with evidence of single-cell necrosis. Lesions characteristic of hypertensive arteriosclerosis, such as concentric hyperplasia of the media, thickening of the wall, lumen narrowing, and frequent onion-skin pattern, were identified in the untreated hypertensive animals (Figure 3). On the other hand, the vascular pathology in the hypertensive animals treated with KN-93, but not KN-92 (data not shown), was ameliorated. KN-93–treated animals had minimal necrosis of the myocytes, in the form of single-cell necrosis, without florid damage in the vessel wall. Fibrinoid necrosis was detected in only a few small arteries (Figure 3).

View larger version (132K): [in this window] [in a new window]   Figure 3. Morphological examination of transverse sections of kidney of hypertensive rats. Photomicrography of a kidney from rats with Ang II–induced hypertension infused with KN-93 (top) or its vehicle (bottom). Top, In KN-93–treated rat, the interlobular artery (arrow) shows mild hypertrophy of myocytes and necrosis of the arterial wall. Bottom, In the vehicle of the KN-93–treated rat, the small arteries (arrow) display transmural hyalinization of the wall. Focal hyaline deposits were also present in the intima of large arteries (round pointer).

	Discussion

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Activation of cytosolic PLA₂ and release of arachidonic acid in response to some humoral and growth factors has been shown to be mediated by MAP kinase.^34,35 We have shown that CaM kinase II promotes arachidonic acid release by directly phosphorylating cytosolic PLA₂ on Ser-515.^9,11,36 The metabolites of arachidonic acid generated via lipoxygenase (12-HETE) and cytochrome P-450 (20-HETE) contribute to the action of NE in VSMC proliferation and to Ang II–dependent models of hypertension.^16,19 The cytochrome P-450 metabolite, 20-HETE, has also been shown to contribute to the development of hypertension in spontaneously hypertensive rats and in the deoxycorticosterone acetate (DOCA)/salt model of hypertension.^13–15 These observations and the ability of CaM kinase II to activate cytosolic PLA₂ raised the possibility that activation of CaM kinase II might represent a central component responsible for initiating VSMC proliferation and hypertension. The fact that activation of the CaM kinase II pathway may contribute to VSMC proliferation was suggested by our finding that CaM inhibitors W-7 and E-6 berbamine (data not shown) and CaM kinase II (KN-93), but not inactive analogue KN-92, inhibited [³H] thymidine incorporation in the rabbit VSMC. This observation, together with our earlier report that the cytosolic PLA₂ inhibitor methyl arachidonylfluorophosphate (MAFP) blocked NE-induced VSMC proliferation, supports the view that VSMC proliferation elicited by norepinephrine is mediated by activation of cytosolic PLA₂ by CaM kinase II.¹⁹ The inhibition of VSMC proliferation with KN-93 was associated with a block in the G₁ phase of the cell cycle as indicated by an increase in the number of cells in G₁ phase and a decrease in S phase. CaM kinase II has been reported to be a positive regulator of G₁/S, G₂/M, and metaphase/anaphase transitions of cell cycle.^26,27,37 Therefore, activation of CaM kinase II by NE and Ang II appears to mediate its effect on VSMC proliferation by promoting G₁ transition of the cell cycle.

Ang II has been shown to promote hypertrophy and hypertension.^28–33 Ang II also increased CaM kinase II activity, which, in turn, by activating cytosolic PLA₂, promotes release of arachidonic acid in VSMC.^10,36 The products of arachidonic acid generated through lipoxygenase (12-HETE) and cytochrome P-450 (20-HETE) contribute to Ang II–dependent models of hypertension.^16,28–31 Therefore, it is possible that activation of CaM kinase II by neurohumoral factors, including Ang II, might cause vascular hyperplasia and hypertension. Our demonstration that (1) the infusion of Ang II caused a rise in blood pressure in the rat that was associated with an increase in CaM kinase II phosphorylation in the kidneys of these animals, (2) the infusion of CaM kinase II inhibitor KN-93 attenuated CaM kinase II activity in the kidney, and (3) the infusion of KN-93 reduced the increase in blood pressure or the development of a rise in blood pressure by Ang II, suggests that activation of CaM kinase II might represent a central component of the mechanisms by which the products of cytosolic PLA₂-derived arachidonic acid metabolites, formed via lipoxygenase and cytochrome P-450, contribute to the development and maintenance of Ang II–dependent hypertension.

Ang II–induced hypertension is known to be associated with vascular hypertrophy and hyperplasia and fibrinoid necrosis.^32,33,37 In the present study, treatment with KN-93 in rats made hypertensive with Ang II infusion, the pathological changes were markedly diminished. There were minimal necrosis of myocytes, florid damage in the vessel wall, and fibrinoid necrosis. Therefore, it appears that activation of CaM kinase II by Ang II, NE, and probably other pressor agents by increasing cytosolic PLA₂ activity results in increased production of prohypertensive eicosanoids. These in turn, by increasing vascular tone and promoting vascular hypertrophy and VSMC proliferation, lead to the development and maintenance of hypertension and, consequently, vascular damage and necrosis.

KN-93 has been extensively used to investigate the contribution of CaM kinase II in cellular and animal models because it selectively inhibits CaM kinase II activity. It inhibits by binding directly and selectively to the CaM binding site of CaM kinase II and preventing the association of CaM and CaM kinase II. It inhibits autophosphorylation of both and ß subunits of CaM kinase II.³⁸ We have reported that KN-93 inhibits the activation of CaM kinase II in VSMC caused by norepinephrine and Ang II.^9,10 It has no significant effects on the activity of cyclic adenosine monophosphate (cAMP)-dependent protein kinase, Ca²⁺/phospholipid-dependent protein kinase, myosin light chain kinase, and Ca²⁺/phosphodiesterase.³⁸ However, we cannot exclude any possible nonspecific effects of KN-93 on other signaling molecules involved in vascular smooth muscle function. Moreover, we cannot draw any conclusion that the renal vascular protective action of KN-93 in our study is independent of a decrease in blood pressure. Further studies with other antihypertensive agents are needed to address this issue.

In conclusion, the present study demonstrates that activation of CaM kinase II mediates NE- and Ang II–induced VSMC proliferation and contributes to Ang II–induced hypertension and associated vascular pathology, including hypertrophy.

	Acknowledgments

 
This work was supported by NIH grant 19134-26 (to K.U.M.), and American Heart Association Grant-In-Aid Awards (to M.M.M. and I.F.B.).

Received October 1, 2001; first decision October 26, 2001; accepted November 21, 2001.

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