Molecular and Cellular Biochemistry 280: 107–117, 2005. c�Springer 2005

Role of MMP-2 in PKCδ-mediated inhibitionof Na+ dependent Ca2+ uptake in microsomesof pulmonary smooth muscle: Involvement ofa pertussis toxin sensitive protein

Sajal Chakraborti, Amritlal Mandal, Sudip Das and Tapati ChakrabortiDepartment of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India

Received 30 March 2005; accepted 1 June 2005

Abstract

Treatment of bovine pulmonary artery smooth muscle with the O•−2 generating system hypoxanthine plus xanthine oxidase

stimulated MMP-2 activity and PKC activity; and inhibited Na+ dependent Ca2+ uptake in the microsomes. Pretreatment ofthe smooth muscle with SOD (the O•−

2 scavenger) and TIMP-2 (MMP-2 inhibitor) prevented the increase in MMP-2 activityand PKC activity, and reversed the inhibition of Na+ dependent Ca2+ uptake in the microsomes. Pretreatment with calphostinC (a general PKC inhibitor) and rottlerin (a PKCδ inhibitor) prevented the increase in PKC activity and reversed O•−

2 causedinhibition of Na+ dependent Ca2+ uptake without causing any change in MMP-2 activity in the microsomes of the smoothmuscle. Treatment of the smooth muscle with the O•−

2 generating system revealed, respectively, 36 kDa RACK-1 and 78 kDaPKCδ immunoreactive protein profile along with an additional 38 kDa immunoreactive fragment in the microsomes. The 38 kDaband appeared to be the proteolytic fragment of the 78 kDa PKCδ since pretreatment with TIMP-2 abolished the increase inthe 38 kDa immunoreactive fragment. Co-immunoprecipitation of PKCδ and RACK-1 demonstrated O•−

2 dependent increasein PKCδ-RACK-1 interaction in the microsomes. Immunoblot assay elicited an immunoreactive band of 41 kDa Giα in themicrosomes. Treatment of the smooth muscle tissue with the O•−

2 generating system causes phosphorylation of Giα in themicrosomes and pretreatment with TIMP-2 and rottlerin prevented the phosphorylation. Pretreatment of the smooth muscletissue with pertussis toxin reversed O•−

2 caused inhibition of Na+ dependent Ca2+ uptake without affecting the protease activityand PKC activity in the microsomes. We suggest the existence of a pertussis toxin sensitive G protein mediated mechanismfor inhibition of Na+ dependent Ca2+ uptake in microsomes of bovine pulmonary artery smooth muscle under O•−

2 triggeredcondition, which is regulated by PKCδ dependent phosphorylation and sensitive to TIMP-2 for its inhibition. (Mol Cell Biochem280: 107–117, 2005)

Key words: endoplasmic reticulum, Giα, pertussis toxin, matrix metalloprotease, microsomes, Na+ dependent Ca2+ uptake,oxidant, superoxide, protein kinase C delta, pulmonary artery smooth muscle

Abbreviation: O•−2 , superoxide; SOD, superoxide dismutase; ER, endoplasmic reticulum; MMP-2, matrix metalloprotease-2;

TIMP-2, tissue inhibitor of metalloprotease-2; PKC, protein kinase C; RACK-1, receptor for activated C kinase-1; E-64d, trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; Giα, α-subunit of the inhibitory G protein; HBPS, Hank’s bufferedphysiological saline; IAP, islet activating protein (pertussis toxin)

Address for offprints: S. Chakraborti, Department of Biochemistry and Biophysics, University of Kalyani, Kalyani 741235, West Bengal, India(E-mail: saj chakra@rediffmail.com)

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Introduction

Ca2+ plays a role as a second messenger in many biochemicaland physiological events [1–3]. An increase in Ca2+ levelsin situ caused by a variety of agonists is due to an influxof extracellular Ca2+ and/or release of Ca2+ from its sub-organelle stores [1–4]. Oxidants have been shown to causeCa2+ overload in cells and tissues [5–7].

Infusion of oxidants in isolated lung has been demon-strated to cause pulmonary hypertension and edema withthe involvement of an increase in Ca2+ level in situ [8, 9].Under sustained pulmonary hypertension, excessive collagendegradation occurs within the wall of the vessel [5]. Matrixmetalloprotease-2 (MMP-2) with its unique ability to degradenative fibrillar collagen has been implicated in several patho-logical states, for example, vascular diseases [5]. We havepreviously demonstrated that MMP-2 regulates Na+ depen-dent Ca2+ uptake in microsomes of bovine pulmonary arterysmooth muscle under oxidant-triggered condition [10]. Acti-vation of MMP-2 by oxidants has been shown to inactivate itsambient protease inhibitor, TIMP-2, resulting in an alterationof the MMP-2-TIMP-2 balance in favour of MMP-2 [10].

Protein kinase C (PKC) represents a family of phospho-lipid dependent serine/threonine kinase. PKC was detectedin almost all types of cells and tissues in the body. PKC ac-tivation by different agonists mediates many signaling path-ways in cells and tissues [11, 12]. PKC has multiple isoforms.PKC-mediated cellular processes are tissue and isoform spe-cific [11, 12]. Protein kinase C (PKC) isozymes play pivotalroles in major serine/threonine kinases in signal transduc-tion cascades involved in agonist-induced responses of vari-ous cells and tissues [11, 12]. Several isozymes of PKC arepresent within a cell, each mediating unique cellular func-tions. Studies revealed a complex and specific localization ofPKC isozymes in their inactive as well as their active states[13–15]. Most isozymes are localized predominantly to thecytosol prior to cell stimulation, and translocate upon activa-tion to new distinct intracellular sites. Localization of PKCisozymes is determined by their capacity to bind to specificanchoring molecules termed RACKs (receptor for activated Ckinase) [13–15]. Two types of RACKs: RACK-1 and RACK-2 have been identified and characterized [13–15].

Signal processing systems mediate physiologic responsesof a variety of stimulants in different kinds of cells and tissues.Three systems have been extensively studied. These are thereceptor-adenylate cyclase complex, the phosphatidyl inosi-tol pathway; and protein kinase(s)-G protein direct couplingpathway [16, 17]. It has been suggested that agonists stimu-lated PKC alter the function of G proteins in some systemsleading to attenuation or augmentation of specific responses[18, 19].

Proteolytic processes play important roles in experimen-tally induced or physiologically occurring changes in cells

and tissues [20, 21]. Previous reports have indicated that en-dogenous proteases, for example, trypsin like proteases ac-tivate PKC [22, 23]. The trypsin like proteases and Ca2+

activated proteases have been shown to produce catalyticallyactive fragments of PKC in a variety of systems [22–24].Oxidants have been shown to increase PKC activity in pul-monary smooth muscle membrane [25]. Activation of PKChas been shown to be an important mechanism by whichbronchoconstrictors and vasoconstrictors act [26]. Oxidant-mediated pulmonary vasoconstriction has been shown to oc-cur through the involvement of an increase in Ca2+ level insitu [8, 9]. Microsomes play a pivotal role in producing Ca2+

overload in cells and tissues under oxidant stimulation [1, 2].We have previously demonstrated that inhibition of Na+ de-pendent Ca2+ uptake in microsomes is one of the mechanismsfor intracellular Ca2+ overload in pulmonary smooth muscleunder oxidant-triggered condition [10]. In view of this andto gain an insight into the biochemical mechanisms associ-ated with the inhibition of Na+ dependent Ca2+ uptake inmicrosomes of bovine pulmonary artery smooth muscle un-der O•−

2 triggered condition, we tested the role of MMP-2in activating PKC and also the involvement of a pertussistoxin sensitive protein in this scenario. We have attempted toaddress the following questions: (i) Does O•−

2 -mediated in-hibition of Na+ dependent Ca2+ uptake in the microsomes ofthe smooth muscle occur through the involvement of PKC?(ii) Does MMP-2 play a role in activating PKC and sub-sequently inhibiting the Na+ dependent Ca2+ uptake in themicrosomes? (iii) Which isoform(s) of PKC is responsiblefor O•−

2 -mediated inhibition of Na+ dependent Ca2+ uptakein the microsomes of the smooth muscle? (iv) Does the PKCanchoring proteins, RACK-1 or RACK-2 trigger transloca-tion of the PKC isoform(s) from cytosol to the microsomes?And, since pertussis toxin prevents platelet activating factorinduced pulmonary hypertension [27], we also attempted toascertain (v) whether pertussis toxin is able to reverse O•−

2caused inhibition of Na+ dependent Ca2+ uptake in the mi-crosomes? Our results suggest that treatment of bovine pul-monary artery smooth muscle with the O•−

2 generating sys-tem (HPX + XO) stimulates MMP-2 activity, which activatesPKCδ; PKCδ then translocated to the microsomes in associa-tion with RACK-1 resulting in phosphorylation of the 41 kDaα-subunit of Gi (Giα), which subsequently causes inhibitionof Na+ dependent Ca2+ uptake in the microsomes.

Materials and methods

Materials

Hypoxanthine, xanthine oxidase, superoxide dismutase, le-upeptin, antipain, phenylmethyl-sulfonyl fluoride, molec-ular wt markers, 4-chloro-1-naphthol, histone type IIIs,

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phosphatidyl-serine, diolein and pertussis toxin were pur-chased from Sigma Chemical Co., St. Louis, MO, USA.Calphostin C and rottlerin were procured from CalBiochem,San Diego, CA, USA. [γ -32P]ATP, [14C]-gelatin and [32P]-orthophosphoric acid were the products of New EnglandNuclear, Wilmington, DE, USA. Antigen and antibodies ofMMP-2, TIMP-2, α, β, δ, ε and ξ PKC isoforms and Giα

were the products of Chemicon International Inc, Temecula,CA, USA, ABCAM, Cambridge, UK and Invitrogen LifeTechnologies, Carlsbad, CA, USA. Mouse IgM anti-RACK1monoclonal antibody and horseradish peroxidase-conjugatedrabbit anti-(mouse IgM) antibody were the products of Trans-duction Laboratories (Lexington, KY, USA), and horseradishperoxidase-conjugated goat anti-rabbit secondary antibodywere the products of StressGen Biotechnologies Corporation(Victoria, British Columbia, Canada). Protein A/G PLUS-agarose beads was the product of Santa Cruz Biotechnology,Santa Cruz, CA, USA. BCA protein assay reagent was ob-tained from Pierce Biotechnology Inc, Rockford, USA. Allother chemicals used were of analytical grade.

Methods

Isolation of bovine pulmonary artery smooth muscle tissueBovine pulmonary artery collected from a slaughterhousewas washed several times with Hank’s buffered physiolog-ical saline (HBPS) (pH 7.4). The washed pulmonary arterywas used for further processing within 2 h of collection. Theintimal and external portions were removed and the tunicamedia i.e., the smooth muscle tissue was collected, charac-terized histologically [28] and used for the present studies.

Preparation of bovine pulmonary artery smooth muscletissue microsomesMicrosomes from the smooth muscle tissue were preparedby following the procedure described by Chakraborti et al.[28]. All experiments were carried out with freshly preparedmicrosomes.

Characterization of microsomesMicrosomes isolated from the smooth muscle tissue werecharacterized by electron microscopic study [29] and alsoby assaying marker enzymes of the subcellular fractions i.e.,mitochondria, lysosomes, endoplasmic reticulum and plasmamembrane fractions as previously described [29].

Assay of protease activity

Degradation of synthetic peptideMMP-2 activity was assayed with the dinitrophenyl labeledsynthetic peptide substrate Dnp-Pro-Gln-Gly-Ile-Ala-Gly-

Gln-D-Arg-OH, which we have synthesized using BIOLYNX4175 peptide synthesizer (LKB Biochrom Ltd, Cambridge,England). DNP peptide was dissolved in Tris-NaCl-CaCl2(50 mM tris-HCl buffer, pH 7.5 containing 150 mM NaCl,10 mM CaCl2 and 0.05% Brij 35) buffer containing 0.02%bovine serum albumin to make a concentration of 500 µM.The peptide (0.1 ml) was mixed with an equal volume of thesmooth muscle microsomal suspension (∼50 µg protein) andincubated for a period of 30 min at 37 ◦C. The enzymatic re-action was stopped by adding 0.5 ml of 1 M HCl, the DNPpeptide fragments released was extracted by vigorous shak-ing with 1 ml of ethyl acetate followed by centrifugation at5000 × g at room temperature for 10 min to separate the twolayers completely. The degree of hydrolysis was determinedby measuring the absorbance of the organic layer at 365 nm[30].

To measure O•−2 mediated increase in MMP-2 activity,

the smooth muscle tissue was exposed to the O•−2 gener-

ating system for 30 min. The microsomes were isolated andthe synthetic peptide degradation by the microsomal suspen-sion was determined. TIMP-2 (50 µg/ml), E-64d (10 µg/ml),calphostin C (1 µM), rottlerin (5 µM) and pertussis toxin(1 µg/ml) were added to the smooth muscle tissue for 30 minfollowed by treatment with the O•−

2 generating system for30 min. The microsomal fraction was isolated and the syn-thetic peptide degradation was determined.

Zymogram of protease activityPolyacrylamide gel (12%; 0.75 mm thick) was cast contain-ing 0.1% gelatin. Gelatin solution was made up as a 2%stock solution in distilled water and dissolved by heating.Bovine pulmonary artery smooth muscle microsomal sus-pension (∼20 µg of protein) was applied to the gel in stan-dard SDS loading buffer containing 0.1% SDS but lacking2-mercaptoethanol; it was not boiled before loading. The gelswere run at 200 V for 1 h in a Bio-Rad Mini Protean II ap-paratus and then soaked in 200 ml of 2% Triton X-100 indistilled water in a shaker for 1 h at 20 ◦C. Next the gelswere soaked in reaction buffer (100 mM Tris HCl, pH 8.0)for 12 h at 37 ◦C and then stained with Coomassie BrilliantBlue followed by washing with distilled water for 1 min. Theclear zone against the dark Coomassie background indicatesMMP-2 activity. The marker lane was separated from the geland destained with destaining solution containing methanol:acetic acid: water (4:1:5) [31].

To determine the effect of O•−2 on MMP-2 activity, the

smooth muscle was treated with the O•−2 generating system

for 30 min at 37 ◦C; then microsomes were isolated and theprotease activity in gelatin zymogram was determined.

To determine the effect of SOD on O•−2 induced MMP-

2 activity, SOD (80 µg/ml) was added to smooth muscle for30 min followed by treatment with the O•−

2 generating systemfor 30 min at 37 ◦C; the microsomes were then isolated and

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the protease activity in the microsomes was determined ingelatin zymogram.

To determine whether TIMP-2 can inhibit O•−2 induced

MMP-2 activity, the smooth muscle was treated with the O•−2

generating system for 30 min at 37 ◦C; then microsomes wereisolated and electrophoresis was carried out. After appropri-ate washing with triton X-100, the gel slice was incubatedwith TIMP-2 (50 µg/ml) for 12 h at 37 ◦C in reaction buffer.A control with the smooth muscle microsomal suspensionwithout any treatment was carried out simultaneously andthe zymogram was performed as mentioned.

Measurement of Na+ dependent Ca2+ uptakeNa+ dependent Ca2+ uptake measurements were carried outby following the procedure previously described [32]. Briefly,bovine pulmonary artery smooth muscle tissue was prein-cubated at 37 ◦C for 30 min with SOD (80 µg/ml), TIMP-2(50 µg/ml) and pertussis toxin (1 µg/ml) followed by treat-ment with the O•−

2 generating system for 30 min, and thenthe microsomal fraction was isolated. The microsomal sus-pension (∼50 µg protein) was added to 5 ml of a mediumcontaining 150 mM KCl, 20 mM MOPS-Tris buffer (pH 7.4)plus 10 µCi of [45Ca2+] for 30 min. Ca2+ uptake was termi-nated by adding 5 ml of ice-cold stopping solution contain-ing 140 mM KCl, 1 mM LaCl3, 20 mM MOPS-Tris buffer(pH 7.4), 250 µl aliquots were filtered through Millipore fil-ters (pore size: 0.45 µm) and radioactivity in the filters weredetermined.

Measurement of protein kinase C activityProtein content in the microsomal suspension was adjustedto ∼1 mg/ml with the homogenizing medium supplementedwith 0.1% Triton X-100. Protein kinase C activity was deter-mined by following the method of Kitano et al. [33].

To determine the effect of O•−2 on membrane PKC activity,

the smooth muscle tissue was treated with the O•−2 generating

system for 30 min. The microsomal fraction was isolated andPKC activity was then determined.

SOD (80 µg/ml), TIMP-2 (50 µg/ml), E-64d (10 µg/ml),calphostin C (1 µM), rottlerin (5 µM) and pertussis toxin(1 µg/ml) were added to the smooth muscle tissue for 30 minfollowed by addition of the O•−

2 generating system for 30 min.The microsomal fraction was isolated and then PKC activitywas determined.

Immunoblot assay of PKC subspecies in the microsomalfractionBovine pulmonary artery smooth muscle cytosol and micro-somal fractions were immunoblotted with α, β, δ, ε and ξ

monoclonal antipeptide antibodies of PKC. The smooth mus-cle tissue was treated with the O•−

2 generating system for30 min, then microsomal fraction was isolated. The cytosolicand the microsomal suspension were immunoblotted using

monoclonal α, β, δ, ε and ξ antipeptide antibodies by follow-ing the method of Towbin et al. [34]. E-64d (10 µg/ml) andTIMP-2 (50 µg/ml) were added to the smooth muscle tissuefor 30 min followed by treatment with the O•−

2 generatingsystem for 30 min. The microsomal fraction was isolated andthen immunoblotted using PKCδ antibody.

Immunoblot assay of RACK-1Cytosol and microsomal fractions isolated from bovine pul-monary artery smooth muscle tissue were immunoblottedwith monoclonal anti-RACK-1 antibody in combination withhorseradish peroxidase-conjugated rabbit anti-(mouse IgM)antibody by following the method of Towbin [34].

Co-immunoprecipitation of PKCδ and RACK-ITo carry out immunoprecipitations, 5 µg of anti-PKCδ an-tibodies were incubated with 50 µl of protein A/G-agarosebeads for 40 min at 4 ◦C as described previously [13]. Theanti-PKCδ antibodies were substituted with IgG in controls.The protein A/G agarose-anti-PKCδ complex was washedthree times with phosphate buffered saline containing 0.1%triton X-100. This was then incubated overnight at 4 ◦C withthe microsomal fraction (∼1 mg protein) isolated from bothcontrol and O•−

2 treated smooth muscle tissue. The beads werethen washed three times with PBS containing 0.1% triton X-100. The immunoprecipitates were subsequently subjected toWestern immunoblotting using anti-RACK1 antibody (to as-sess its co-immunoprecipitation with PKCδ), or anti- PKCδ

antibody.

Phosphorylation and localization of Giα

Phosphorylation of the microsomes isolated from the smoothmuscle tissue were carried out by following the methodof Neyses et al. [35] with some modifications. Briefly, thesmooth muscle tissues were exposed to the O•−

2 generatingsystem for 30 min. The microsomal fraction was isolated andthen [32P]Pi (5 mCi) was added and incubated for 30 minat 37 ◦C. In separate experiments, the smooth muscle tis-sues were pretreated with TIMP-2 (50 µg/ml) and rottlerin(5 µM) for 30 min followed by addition of the O•−

2 gener-ating system for 30 min. The microsomal fraction was iso-lated and protein phosphorylation was performed. The result-ing mixture was resuspended in lysis buffer that contained150 mM NaCl, 50 mM sodium phosphate (pH 7.2), 2 mMEDTA, 1 mM dithiothreitol, 0.5% SDS, 1% deoxycholate,1% triton X-100, 2 mM Na3PO4 and 2 mM Na3VO4. 20 µgof protein of the microsomal suspension was added to 30 µlof SDS-PAGE sample buffer and boiled for 4 min. Phospho-rylation of standard Giα was carried out by following themethod of Crouch and Lapetina [36] with some modifica-tions. Briefly, 5 µg of standard Giα was added to 100 µlof a reaction mixture containing 25 mM HEPES/NaOH (pH7.4), 5 µg of PKCδ, 2 µM dithiothreitol, 0.5 mM ATP, 10 µCi

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[γ -32P]ATP, 0.1 mM Na3VO4, 1.5 mM CaCl2, 25 µg/mlphosphatidyl serine and 0.5 µg/ml diolein. Incubation wasperformed at 37 ◦C for 30 min. The reaction was terminatedwith the addition of 100 µl of ice-cold stopping solution con-taining 20% TCA and 1 mM ATP. After centrifugation at 800× g for 20 min, the supernatant was discarded and the pel-let was suspended in 30 µl of SDS-PAGE sample buffer andboiled for 4 min. Samples were then separated using SDS-PAGE, blotted to nitrocellulose paper and then incubated withpolyclonal antibody of Giα for 2 h at room temperature with1 in 200 dilution with constant shaking. The blot was thenwashed three times (20 min each) with TBS, incubated for2 h with horseradish peroxidase conjugated goat anti(rabbitIgG) antibody followed by washing with TBS for three times(20 ml each). The nitrocellulose membrane was then devel-oped with 4-chloro-1-naphthol (0.2 mM). The nitrocelluloseblot was then exposed to X-ray film.

Viability of the smooth muscle tissueViability of the smooth muscle tissue was determined bymeasuring oxygen uptake polarographically using a Clark-type oxygen electrode fitted with a 2 ml water-jacketed closedchamber at 37 ◦C [37].

Estimation of proteinProteins were estimated by BCA protein assay reagent usingserum albumin as the standard [38].

Statistical analysisData were analyzed by unpaired t-test and analysis of vari-ance, followed by the test of least significant differences [39]for comparisons within and between the groups. p < 0.05was considered as significant.

Results and discussion

The smooth muscle microsomes were characterized by assay-ing marker enzymes of mitochondria, lysosomes, endoplas-mic reticulum and plasma membrane, and also by electronmicrosopic studies as outlined by Mandal et al. [29] (datanot shown). To generate O•−

2 , we used mixtures of hypoxan-thine (2 mM HPX) and xanthine oxidase (0.03 U ml−1 XO).When added to 1 ml of HBPS (30 min at 37 ◦C), the super-oxide generating system produced 16.12 ± 0.92 µM (mean± SE; n = 4) O•−

2 as measured by lucigenin chemiluminis-cence [40]. This value is in accordance with that previouslydetermined by Rajagopalan et al. [40]. None of the treat-ments used in the present study affected the smooth muscletissue viability as assessed by oxygen uptake studies (data notshown).

The present study suggest that stimulation of PKC activityis important for the inhibition of Na+ dependent Ca2+ uptake

in the microsomes isolated from bovine pulmonary arterysmooth muscle tissue under O•−

2 triggered condition. Threelines of evidence suggest that O•−

2 stimulation of PKC activityis an important mechanism for inhibition of Na+ dependentCa2+ uptake in the microsomes. First, treatment of the smoothmuscle tissue with the O•−

2 generating system stimulates PKCactivity and subsequently inhibits Na+ dependent Ca2+ up-take in the microsomes; and pretreatment with calphostin C,a general PKC inhibitor, prevents the increase in PKC ac-tivity and reverses Na+ dependent Ca2+ uptake (Table 1).Second, immunoblot study revealed an apparent increase inPKCδ immunoreactive profile in microsomes of the smoothmuscle tissue under O•−

2 triggered condition (Figs. 1 and 2).And, third, pretreatment of the smooth muscle tissue with thePKCδ inhibitor rottlerin prevents the increase in PKC activityand reversed the inhibition of Na+ dependent Ca2+ uptake in

Table 1. Effect of SOD, TIMP-2, E-64d, Calphostin C, rottlerin and IAP onMMP-2 activity, protein kinase C activity and Na+ dependent Ca2+ uptakein microsomes isolated from bovine pulmonary artery smooth muscle tissueunder O•−

2 triggered condition

MMP-2 PKC Na+ dependentTreatment activitya activityb Ca2+ uptakec

Control (none) 18.25 ± 2.02 84 ± 7 121.3 ± 12

O•−2 generating 90.75 ± 7.89d 754 ± 35d 36.4 ± 4d

system

SOD (80 µg/ml) 17.44 ± 1.68 76 ± 8 122.7 ± 11

SOD (80 µg/ml) + O•−2 18.51 ± 2.14f 88 ± 7f 120.1 ± 9f

generating system

TIMP-2 (50 µg/ml) 11 ± 1.32e 71 ± 8 126.6 ± 12

TIMP-2 (50 µg/ml) + O•−2 17.65 ± 2.42f 81 ± 9f 121.9 ± 11f

generating system

E-64d (10 µg/ml) 17.72 ± 1.76 82 ± 8 121.6 ± 9

E-64d (10 µg/ml) + O•−2 91.22 ± 8.16d,g 764 ± 32d,g 35.9 ± 4d,g

generating system

Calphostin C (1 µM) 17.45 ± 1.72 78 ± 7 122.4 ± 9

Calphostin C (1 µM) + O•−2 90.25 ± 8.22d,g 85 ± 7f 120.9 ± 12f

generating system

Rottlerin (5 µM) 17.78 ± 1.56 83 ± 6 121.5 ± 11

Rottlerin (5 µM) + O•−2 89.74 ± 6.98d,g 79 ± 6f 122.1 ± 12f

generating system

IAP (1 µg/ml) 17.48 ± 1.52 79 ± 7 122.3 ± 9

IAP (1 µg/ml) + O•−2 91.52 ± 8.16d,g 768 ± 38d,g 120.2 ± 11f

generating system

Note. Results are mean ± S.E. (n = 4).aMMP-2 activity was assessed by measuring degradation of the syn-thetic peptide (Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH) is expressedas mU/mg protein (1 U gelatinase catalyses the hydrolysis of 1 µmol dini-trophenyl labeled peptide/30 min at 37 ◦C); bPKC activity is expressed aspmol/mg protein/min; cNa+ dependent Ca2+ uptake is expressed as nmol45Ca2+/30 min/mg protein; d p < 0.001 compared with basal condition;e p < 0.05 compared with basal condition; f p < 0.001 compared with O•−

2treatment condition; g p < 0.001 compared with respective control.

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Fig. 1. Upper panel A: Immunoreactive PKCδ protein profile in cytosol andmicrosomal fraction of bovine pulmonary artery smooth muscle under nor-mal (C) and O•−

2 triggered (T) condition. Lane a, cytosol; lane b, microsome;lane c, standard PKCδ. Lower panel B: Histogram representing PKCδ in cy-tosol and microsomal fraction of the smooth muscle under normal (C) andO•−

2 triggered (T) condition. a, cytosol; b, microsome; c, standard PKCδ.

Fig. 2. Effect of different treatments on immunoreactive PKCδ profile inmicrosomes isolated from bovine pulmonary artery smooth muscle tissueafter treatment with the O•−

2 generating system. Lane a, control; b, treatmentwith the O•−

2 generating system; c, TIMP-2 (50 µg/ml) treatment; d, TIMP-2 (50 µg/ml) treatment followed by addition of the O•−

2 generating systemfor 30 min; e, E-64d (10 µg/ml) treatment; f, E-64d (10 µg/ml) treatmentfollowed by addition of the O•−

2 generating system for 30 min; g, standardPKCδ.

the microsomes isolated from the smooth muscle tissue upontreatment with the O•−

2 generating system (Table 1).It is known that PKC is involved in myocardial pressure

overload hypertrophy and it appears that various isoformsmay have different roles in the process [15]. There is dif-ferential expression of PKC isoforms among cell and tissuetypes with each type characteristically expressing multiplePKC isoforms [41, 42]. The mechanism by which a specific

function is conferred in a specific type of cell and tissue is notwell elucidated. It has been hypothesized that specific com-partmentalization of a PKC isoform within the cell or tissuedirects its unique biological functions. PKCα was shown tobe associated with nuclear membrane in InIICa cells [43];PKCε is found to be associated with the Golgi bodies inNIH 3T3 cells [43]; and PKCβ has been shown to be asso-ciated with melanosomes in melanocytes [43]. Our presentstudy suggests that the cytosol fraction isolated from bovinepulmonary artery smooth muscle elicited α, β, δ, ε and ξ

isoforms of PKC (Fig. 1: only PKCδ data shown).Recent studies indicate that RACK-1 is a scaffolding pro-

tein that is involved in the recruitment assembly and reg-ulation of a number of different signaling molecules intodifferent cellular compartments [13–15]. Upon stimulation,PKC undergoes a conformational change and then binds withRACK-1, which leads to its translocation from cytosol tosubcellular fractions [13–15, 44]. Conformational change inPKC from an inactive to an active state results in exposure ofdomains required for PKC anchoring to the cellular and sub-cellular compartments and in increased sensitivity of the PKCisoform(s) to protease(s) [44]. The inactive state has been sug-gested to exist in a closed conformation with the proteolyticsites protected, whereas the active state is an open conforma-tion with exposed proteolytic sites [44]. Structural alterationsfrom closed to open states involve alteration of intramolecu-lar interactions within the enzyme [44], conceivably, due toan alteration of thiol-disulfide linkage in the enzyme.

An increase in PKCδ has been related to the process ofcardiac hypertrophy [43, 45]. PKCδ has been shown to bemarkedly up regulated in microsomes of different systemsunder a variety of stimulation [46]. A distinctive feature ofmice with cardiac hypertrophy is that they exhibit substantialincrease in RACK expression [13–15, 44, 45]. Although mo-bilization of PKC isoform(s) and also RACK-1 or RACK-2were known to some extent in some systems under a variety ofstimulation [13–15, 44, 45], yet to the best of our knowledgeno such information is available based on any study in pul-monary vascular smooth muscle. Our present study demon-strates that RACK-1, a 36 kDa protein is present in bovinepulmonary artery smooth muscle cytosol fraction (Fig. 3).However, under O•−

2 stimulation of the tissue, RACK-1 isfound along with PKC-δ in the microsomes (Fig. 4). Thisapparently appears to be due to the fact that RACK-1 allowsPKCδ to translocate from cytosol to the microsomes underO•−

2 triggered condition in the smooth muscle due possiblyto structural alteration from closed to the open states involv-ing a change in intramolecular interaction within the enzyme[44].

The biochemical mechanisms associated with the activa-tion of PKC in cells has long been a subject of great interest.PKC is widely distributed in cells of mammals and other or-ganisms. It consists of a peptide chain (∼80 kDa) which is

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Fig. 3. Localization of RACK-1 in cytosol and microsomal fractions ofbovine pulmonary artery smooth muscle tissue. Lane a, cytosol; b, micro-somes isolated from bovine pulmonary artery smooth muscle tissue; c, stan-dard RACK-1 (Jurkat cell lysates was used as a positive control for identi-fication of RACK-1 (Pass JM et al. Am J Physiol Heart Circ Physiol 281:H2500–H2510, 2001)).

Fig. 4. Co-immunoprecipitation of RACK-1 and PKCδ. PKCδ-RACK1 in-teractions were assessed by immunoprecipitating (IP) PKCδ and subsequentimmunoblotting (IB) of precipitates with anti-RACK1 antibody as describedin methods section. Upper panel A: Lane a, standard RACK-1 (Jurkat celllysates was used as a positive control for identification of RACK-1 [PassJM et al. Am J Physiol Heart Circ Physiol 281: H2500–H2510, 2001]); b,microsomes obtained from the control smooth muscle tissue; c, microsomesobtained from the smooth muscle tissue after treatment with the O•−

2 gener-ating system; d, IgG control. Lower panel B: Lane a, microsomes obtainedfrom the control smooth muscle tissue; b, microsomes obtained from thesmooth muscle tissue after treatment with the O•−

2 generating system; c,standard PKCδ.

composed of two functionally different domains and that canbe separated by protease(s) [47, 48]. In platelets, neutrophils,rat liver plasma membrane and pulmonary endothelial cellmembrane, PKC has been demonstrated to be proteolyticallyactivated by a variety of proteases such as calcium dependentproteases, trypsin-like serine proteases and aprotinin sensi-tive protease [22–24, 49, 50]. The proteolytically activatedPKC showed an ability to phosphorylate H1 histone in cal-cium phospholipid dependent manner [51]. Hashimoto andYamamura [22] reported that a trypsin-like protease prote-olytically activates rat liver plasma membrane PKC in thepresence of Ca2+ and phospholipid. Previous research alsodemonstrated that stimulation of human platelets and humanfibroblasts causes an increase in the Ca2+ activated proteaseactivity, which irreversibly cleaves PKCδ at specific sites inthe hinge region between the catalytic and regulatory domainof the kinase [52, 53].

Herein, we have reported that MMP-2 plays an impor-tant role in activating PKCδ with subsequent inhibitionof Na+ dependent Ca2+ uptake in microsomes of bovine

Fig. 5. Effect of SOD and TIMP-2 on O•−2 induced MMP-2 (72 kDa gelati-

nase) activity in microsomes isolated from bovine pulmonary artery smoothmuscle tissue in gelatin zymogram. Lane a, control; b, treated with the O•−

2generating system for 30 min; c, treated with SOD (80 µg/ml) for 30 min; d,treated with SOD (80 µg/ml) for 30 min followed by addition of the O•−

2 gen-erating system for 30 min; e, control gel incubated with TIMP-2 (50 µg/ml)for 30 min; f, treated with O•−

2 generating system for 30 min and the resul-tant gel incubated with TIMP-2 (50 µg/ml) for 30 min; g, standard mol. wt.markers.

pulmonary artery smooth muscle under O•−2 triggered con-

dition (Table 1). The following evidence supports this con-cept: (i) the smooth muscle microsomes exhibit MMP-2 ac-tivity (Fig. 5); (ii) treatment of the smooth muscle tissuewith the O•−

2 generating system augments MMP-2 activity(tested with the synthetic substrate: Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH) and inhibits Na+ dependent Ca2+

uptake in the microsomes (Table 1); (iii) TIMP-2, the ambi-ent MMP-2 inhibitor [29] prevents the increase in MMP-2activity (Table 1 and Fig. 5), PKC activity, and also reversedO•−

2 caused inhibition of Na+ dependent Ca2+ uptake in themicrosomes of the smooth muscle (Table 1); (iv) treatmentof the smooth muscle tissue with the O•−

2 generating sys-tem causes an apparent increase in the 78 kDa PKCδ proteinprofile in the microsomes as revealed by immunoblot studies(Figs. 1 and 2). Under this condition, a low molecular wt.band (∼38 kDa) along with the 78 kDa immunoreactive pro-file was also observed (Figs. 1 and 2). In some types of cells,for example, COS-1 cells, proteolytic activation of PKCδ wasdemonstrated [54]. Thus, the 38 kDa proteolytic fragment de-tected in the immunoblot appears to be the active fragmentof PKCδ; (v) pretreatment with TIMP-2 not only reversesO•−

2 caused inhibition of Na+ dependent Ca2+ uptake in themicrosomes (Table 1) but also abolishes the 38 kDa prote-olytic fragment detected in the immunoblot (Fig. 2). In viewof the previous report that calpain causes proteolysis of PKC[48] and that calpain has been implicated in rat myocardialinjury after ischemia and reperfusion [41], we seek to de-termine whether pretreatment of the smooth muscle with E-64d, a calpain inhibitor [42] could inhibit O•−

2 triggered PKC

114

activity and formation of the 38 kDa proteolytic fragment inthe immunoblot. Our results suggest that pretreatment withE-64d did not prevent the 38 kDa protein band caused by theO•−

2 generating system in the immunoblot (Fig. 2). Addition-ally, E-64d has been found to be ineffective to prevent O•−

2caused increase in PKC activity in the microsomes (Table 1).Taken together, our present study suggest that MMP-2, butnot calpain, plays a crucial role in inhibiting Na+ dependentCa2+ uptake by proteolytic activation of PKCδ (Table 1 andFig. 2). These five pieces of evidence strongly support thatproteolytic activation of PKCδ by MMP-2 plays a pivotal rolein inhibiting Na+ dependent Ca2+ uptake in the microsomesisolated from the smooth muscle upon treatment with the O•−

2generating system.

Signal processing mechanisms mediate the physiologicalresponse to a variety of stimulants in different systems [16–19]. It has been suggested that PKC upon stimulation altersthe function of G proteins leading to attenuation or augmen-tation of specific responses [18, 19]. Immunoblot assay of themicrosomes isolated from the smooth muscle with polyclonalGiα antibody revealed a 41 kDa immunoreactive band (Fig.6A). Additionally, [32P] incorporation study in the micro-somes isolated from the smooth muscle tissue after treatmentwith the O•−

2 generating system phosphorylates a protein hav-ing molecular mass of 41 kDa (Fig. 6B). Our present study

(A)

(B)

Fig. 6. (A) Immunoblot study of the presence of Gi α in microsomes. Lanea, control microsomes; b, microsomes obtained from O•−

2 treated smoothmuscle tissue; c, microsomes obtained from the tissue after treatment withrottlerin (5 µM) for 30 min; d, microsomes obtained from the tissue aftertreatment with rottlerin (5 µM) for 30 min followed by addition of the O•−

2generating system for 30 min; e, microsomes obtained from the tissue aftertreatment with TIMP-2 (50 µg/ml); f, microsomes obtained from the tis-sue after treatment with TIMP-2 (50 µg/ml) followed by treatment with theO•−

2 generating system for 30 min; g, standard Gi α. (B) Immunoprecipitateobtained from [32P]Pi -labeled microsomes isolated from bovine pulmonaryartery smooth muscle tissue with Gi α antibody. Lane a, control microsomes;b, microsomes obtained from O•−

2 treated smooth muscle tissue; c, micro-somes obtained from the tissue after treatment with rottlerin (5 µM) for30 min; d, microsomes obtained from the tissue after treatment with rott-lerin (5 µM) for 30 min followed by addition of the O•−

2 generating systemfor 30 min; e, microsomes obtained from the tissue after treatment withTIMP-2 (50 µg/ml); f, microsomes obtained from the tissue after treatmentwith TIMP-2 (50 µg/ml) followed by treatment with the O•−

2 generatingsystem for 30 min; g, standard Gi α.

also revealed that pretreatment of the smooth muscle tissuewith pertussis toxin reversed the inhibition of Na+ dependentCa2+ uptake in the microsomes caused by the O•−

2 generatingsystem (Table 1).

Pertussis toxin action has been shown to bypass receptorsin some cells [55]. It is clear from the present study thatpertussis toxin treatment does not affect basal Na+ depen-dent Ca2+ uptake in microsomes of the smooth muscle tissue(Table 1). In contrast, pertussis toxin pretreatment reversesO•−

2 caused inhibition of Na+ dependent Ca2+ uptake in thesmooth muscle microsomes (Table 1). To ascertain clearlywhether Gi is present in the membrane, we performed im-munoblot study, which confirmed that Giα is present in themicrosomes (Fig. 6A). [32P]phosphorylation of the micro-somes isolated from the smooth muscle tissue after treatmentwith the O•−

2 generating system and subsequent immunoblotwith Giα antibody showed a 41 kDa band which confirm thatGiα has been phosphorylated. Importantly, pretreatment withTIMP-2 and rottlerin prevent phosphorylation of Giα (Fig.6B). Overall, it appears from the present study that activa-tion of PKCδ by MMP-2 is not sufficient for inhibition ofNa+ dependent Ca2+ uptake in microsomes of the smoothmuscle under O•−

2 triggered condition. It seems conceivablethat phosphorylation of a pertussis toxin sensitive G proteinof the Giα class by functionally active PKCδ (i.e., 38 kDaproteolytic fragment of the 78 kDa PKCδ) is required for in-hibition of Na+ dependent Ca2+ uptake in microsomes of thesmooth muscle under O•−

2 triggered condition. In some cellsPKC has been suggested to phosphorylate the α-subunit ofGi and alter its function [18, 49]. It is established that themost prominent chemical modification of Gi , which leads toextensive modification of its function as a signal transducer,is ADP ribosylation of its α-subunit, catalyzed by pertussistoxin (IAP) [49]. Phosphorylation by PKC has been proposedto be an additional modification of Gi . In fact, the α-subunitof Gi (Giα) purified from rabbit liver plasma membrane canbe phosphorylated by PKC in a cell free system. Giα in in-tact systems can also be phosphorylated by the kinase, ashas been observed with platelets, neutrophils and hepatocytes[56]. The data presented herein suggest a regulatory link ofNa+ dependent Ca2+ uptake by PKCδ-Gi signaling systemin microsomes of bovine pulmonary artery smooth muscle.Thus, under oxidant stress, an inhibitory guanine nucleotidebinding regulatory component (Gi ), which is inactivated afterADP ribosylation by pertussis toxin or activated after phos-phorylation by PKCδ with the involvement of MMP-2, maysomehow be associated with the regulation of Na+ dependentCa2+ uptake in the microsomes. Identification of the couplingcomponent(s), if any, of the phosphorylated Giα for Na+ de-pendent Ca2+ uptake occurs in vivo is an important area forfuture research.

In conclusion, our present study suggest that (i) treatmentof bovine pulmonary artery smooth muscle with the O•−

2

115

Fig. 7. Schematic representation for the underlying mechanism of superoxide (O•−2 ) triggered inhibition of Na+ dependent Ca2+ uptake in bovine pulmonary

smooth muscle microsomes. (A), anion channel; (B), diffusion; MMP-2, matrix metalloprotease-2; TIMP-2, tissue inhibitor of metalloprotease-2; PKCδ, proteinkinase C delta; RACK-1, receptor for activated C kinase-1; Giα, inhibitory G protein α subunit; GiαP, phosphorylated Giα; C, Na+-K+-ATPase/Na+-H+exchanger?

generating system causes an increase in MMP-2 activity inthe microsomes; (ii) under O•−

2 triggered condition, RACK-1 binds with PKCδ in the cytosol leading to translocation ofPKCδ to the microsomes of the smooth muscle; (iii) prote-olytic activation of PKCδ appears to be an important mecha-nism for the inhibition of Na+ dependent Ca2+ uptake in themicrosomes isolated from the smooth muscle upon treatmentwith the O•−

2 generating system; (iv) O•−2 induced MMP-2

stimulates PKCδ activity which phosphorylates Giα leadingto the inhibition of Na+ dependent Ca2+ uptake in the mi-crosomes. In some systems, for example, rat arterial smoothmuscle cells, oxidants have been shown to stimulate phos-phorylation of extracellular signal regulated kinase (ERK),and p38MAPK [57, 58]. In some systems, MAP kinases areactivated by PKC under stimulated conditions [59]. It remainsto be determined whether the PKC-mediated response in thesmooth muscle microsomes under O•−

2 triggered conditionoccurs through the involvement of mitogen activated proteinkinases. How the phosphorylated Giα affects Na+ dependentCa2+ uptake in microsomes is currently unknown. The phos-phorylated Giα may act directly on the Na+/Ca2+ exchanger

or influence the activity of the exchanger in the microsomesvia factors such as Na+/K+ATPase and Na+/H+ exchanger[2, 60, 61]. A schematic representation for Na+ dependentCa2+ uptake in the smooth muscle microsomes under O•−

2triggered condition is depicted in Fig. 7.

Activation of protein kinase C is an important mechanismby which bronchoconstrictors and vasoconstrictors act [26,62, 63]. In isolated rabbit lung, oxidant-mediated pulmonaryvasoconstriction has been shown to occur with the involve-ment of an increase in Ca2+ level in situ [8, 9]. Pulmonaryhypertension produces right ventricular atropy and failure [8,9, 62, 63] and causes a substantial increase in morbidity andmortality. Herein, we have demonstrated that treatment of thesmooth muscle with the O•−

2 generating system inhibits Na+

dependent Ca2+ uptake in the microsomes and that appears tobe a potential mechanism for increase in Ca2+ overload un-der oxidant triggered condition. A similar response on PKCactivation, for example, by platelet activating factor-inducedvasoconstriction has been demonstrated in pigs and that hasbeen shown to be mediated by a pertussis toxin sensitive Gprotein [62]. Thus, the role of MMP-2 on Na+ dependent

116

Ca2+ uptake via PKCδ in association with RACK-1 and theinvolvement of a pertussis toxin sensitive G protein in micro-somes isolated from bovine pulmonary artery smooth muscleupon treatment with the O•−

2 generating system appears to bephysiologically important.

Acknowledgments

Thanks are due to the Department of Biotechnology (Gov-ernment of India) and the Life Science Research Board (Min-istry of Defence, Government of India) for partly financingthe work. Thanks are also due to the Department of Science &Technology (DST), Government of India for supporting ourDepartment by DST-FIST program. The authors are gratefulto Prof. Gerry Shaw (Department of Neuroscience, Univer-sity of Florida, Gainesville, Fl, USA) and Late Prof GailH Gurtner (Departments of Physiology and Medicine, NewYork Medical College, Valhalla, NY, USA) for their help andsupport in this work.

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