Europcatr Journal of Plmrtnacology 0

1992 Elsevier ScieFx

EJPMOL

-

Mokculur

Pharmacology

Scc~io~t, 227

(1992)343-348

343

Publishers B.V. All rights reserved 0922-4106/92/$05.00

X0093

Short communication

Protein kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats Eulalia Bazan Depnrrmcnt

of Pharmacology

‘, Anita K. Campbell

and Robert

M. Rapoport

and Cell Biophysics and Vetcraw Affairs Medical Center. Uniwrsity Cincinrrati, OH 45267-0575.

of Cincinnati,

College of Medicine,

USA

Received 18 August 1992, accepted 25 August 1992

This study investigates the effects of phorbol dibutyratc (PDB) on protein kinase C (PKC) activation, as assessed by the translocation of PKC activity from the cytosolic to the particulate fraction, in aortas and mesenteric arteries from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The basal distribution of PKC activity between the cytosolic and particulate fractions of SHR and WKY aortas, and mesenteric arteries, was not significantly diffcrcnt. PDB induced a concentration-dependent decrease in cytosolic PKC activity in SHR and WKY aortas. PDB (0.01 ,uMJ decreased cytosolic PKC activity to a greater magnitude in SHR aorta as compared to WKY aorta, while 1.0 HIM PDB decreased cytosolic PKC activities to similar magnitudes in SHR and WKY aortas, and mcsentcric arteries. These results suggest that the increased sensitivity of SHR vessels to contraction by phorbol esters may be due. at least in part, to the greater ssnsitivity of PKC in these vessels to phorboi r;Ztr;i actkr;tion. Smooth muscle (vascular); Ca’+; Protein kinasc C activity (cytosolic, particulate);

1. Introduction

Although it is known that vascular contractility 3s increased in hypertension, the mechanism(s) that underlies the increase is not clear. One potential mechanism is that the increased contractility may be due to enhanced activation of protein kinase C (PKCS. In support of this hypothesis are the observations that numerous vessels, including the aorta, from the spontaneously hypertensive rat (SHR) and the stroke-prone SHR were more sensitive to contraction and/or were contracted to a greater magnitude by phorbol esters, which directly activate PKC, than corresponding Wistar-Kyoto rat (WKY) vessels (MacKay and Cheung, 1987; Turla and Webb, 1987; Bruschi et al., 1988; Silver et al., 1988, 1992). Furthermore, the purported selective PKC inhibitor, H7, was a more potent inhibitor of porepinephrine-, angiotensin II-, and phorbol myristate acetate-induced contractions of SHR

Correspondence to: Robert M. Rapoport, Department of Pharmacology and CelC Biophysics, University of Cincmnati College of Medicine. 231 Bethesda Ave., Cincinnati, OH 45267-0575, USA. Tel. 5 13-W-23%: Fax 5 IS-SW I 169. ’ Prcscnt address: Depastmcnto de Invcstiyacion. Plonta- I D, I-lohpiIUI Rzmon y Cujal. Carreter;~ dc Colmenar Km. 9.1, 2N034-Mndrid, Spain.

Phorbol esters

aorta than WKY aorta (Shibata et al., 1990). More recently, it was demonstrated that the Ca’+-dependent contractions elicited in detergent skinned aorta from SHR were more sensitive to inhibition by the PKC inhibitor, polymyxin B, and by H7, than contractions elicited in skinned WKY aorta (Soloviev and Bershtein, 1992). The relationship between PKC activity and vascular reactivity in SHR and WKY vessels, however, remains obscure. There have been no studies, to our knowledge, that assess the ability of phorbol esters to activate PKC in vessels or cultured vascular smooth muscle cells from SHR and WKY, Furthermore, studies on the basal distribution of PKC between the cytosolic and/or particulate fractions of SHR vessels and corresponding WKY vessels have yielded conflicting results. Murakawa et al. (1988) reported that the PKC-specific activity in the cytosolic fraction of SHR aorta correlated significantly with systolic blood pressure (particulate PKC activity was not reported), although wide variations in PKC activity were observed. In contrast, Silver et al. (1988) reported that the PKC specific activity of the cytosolic, as well as particulate, fractions of SHR and WKY aorta were not significantly differcnt. The PKC-specific activity of the cytosolic fraction of the SHR renal artcry was, however, greater than that of the WKY renal artcry (Silver cl al., 1988). In

cultured smoo~l muscle cells dcrivcd from SHR and WKY 30rta. the total amounts of PKC and the distrib~t~~~~of PKC between the cytosoiic and particulate frdctions. as asscsscd by photbol ester binding and immunoblotting. were also not significantly different (F&sink et al.. 1989L The rc~ationship between PKC activity and vascular reactivity also remains unclear because an experimental artifact may have greatly influenced the above resorted values for the distribution of PKC between the c~tosolic and particu4atc fictions of SHR and WKY aorta (Silver et al,, 198X). and cultured smooth muscle cells (Resink et al., 1989). The experimental artifact that may have occurred is that PKC was translocated from the cytosolic to the particutatc fraction during preparation of tissue or toll fractions, most tikcly due to the prescncc of free Ca” (Wolf et al., f985). In support of the suggestion that the inadvertant translocation of PKC from the cytosolic to the particulate fraction had indeed occurred in the PKC measurements reported by Silver ct al. (1988) and Resink et al. (1989) is their data demonstrating greater amounts of PKC in the particulate fraction as compared to the cytosolic fraction in SHR and WKY smooth muscle. Thiq qtudv. therefore. investigates whcthcl the greater reactivity of SHR than WKY vcsscls to phorbol esters may possibly be due to greater relative amounts in SHR vessels. as compared to WKY vessels. of ( 1) PKC in the particulate as compared to the cytosolis fraction; (2) phorbol ester-induced t~nstocati~)n of PKC from the cytosolic to particulate fraction. and/or (3) total (cytosolic + particulate) PKC.

2. Materials and methods

2.1. l.~olation

antitreatment ofrmel.s

Sprague-Dawley rats (male, 2.0-3.5 months old; 2611-360 g). or SHR and WKY rats (male, 7-Y months old; 409 it 6 ( 15) and 411 t 8 ( 15) g, respectively (means or S.E. (n)) were used. The systolic blood prcssures. detcrmincd using a tail-cuff method, of SHR and WKY ratswcre 203+3(1S)and 122~33lS5mm Hg (means & S.E. (nf), respectively. The :.>stolic prcssure of each rat was determined by averaging three separate pressure measurements. Rats were sacrificed by CO, asphyxiation and the thoracic aorta and mesenteric arterial bed removed and cleaned of extraneous fatty tissue. The endothelium was removed by gently rubbing the intimal surface of the vcsscls. Vessels were allowed to cquilibratc for at least 1.5 h in 37°C Krcbs-Ringer bicarbonate (KRB) solution prior to drug addition. The KRB solution was gassed with 9% Q,-Sn/, CO, and had the following composition ImM): NaCt, 1IX.5; KCE, 4.74; M&O,,

1.18; KHzPO,. i.IK; CaC’I,, 2.5; NaHCO,, 24.5); glucost. 10; Na?EDTA, 0.03. Experiments with SHR aad WKY vesscis were designed as follows, Each aorta, or mesenteric arterial bed, was divided, on the basis of visual assessment, into two portions of approximately equivalent mass. The portions were exposed to 1 PM phentolamine for 30 min. and one portion was then exposed to phorbol dibutyrate (PDBJ for 60 min, while the other portion was exposed to vehicle (dimethyl sulfoxide, DMSO). Following wash of vessels for 60 min in 4°C Ca”-free KRB solution containing 2 mM EGTA, vessels were frozen between clamps precooked in liquid nitrogen (Chuprun et al., 1991). Experiments with Sprague-Dawley aorta were designed as follows. Aortas were washed in Ca*“-free KRB soilution for various timeq (as indicated) or remained unwashed prior to freezing as described above. 2.2. Preparatim of frmrimis SHR and WKY aorta (i vessel) or mesenteric arterial bed (i bedf were homogenized f 1t): i (volume : wet weight); glass-on-glass at 4”) in 30 mM Tris-HCI (pH 7.4) containing 10 pg/ml Ieupeptin, 1 mM phenylmeihy~suif~ii~~ fiiiorids, 0.f mM EDT44 and 5 m.M EGTA. Sprague-Dawlcy rat aorta were homogeni~cd in the above solution cxccpt that in some experiments, as indicated, the concentration of EGTA was increased from 5 to 21) mM. Homogenates were then centrifuged (jUO,(~OOx g, h0 min), the resulting supernatant (cytosolic fraction) saved, and the pellet resuspended in 0.5 ml homogeni~tjon buffer containing 5 mM EGTA supp~emcntcd with 0.2% Triton X-i00 (TX-1001. After 60 min, the suspension was ccntrifugcd (100,i)OO X ,yq 20 min), the resulting supcrnatant (SP, 1 saved, and the pellet reextracted, centrifuged, and an addit~onat supernata~t (SP,) obtained. The two extractions of the pellet yielded greater than 95% of the total solubilizcd particulate PKC activity from control and PDB-treated tissues.

PKC activity was determined as previously described ichuprun et al., 1991). Briefly, PKC activity was measured in reaction mixtures (0.1 ml) containing 30 mM ‘Tris-HCI (pH 7.41, 10 mM MgSU,. 500 fig/ml histone (type Ifi-Sf, 40 rug/ml phosphatidylser~~e, 200 PM ATP (containing 10 pCi/ml [y-“PJATP), 0.04% TX100, 1.1 mM Ca”+, and I mM EGTA in the presence or absence of 1 PM phorbol my&ate acetate. The free Cal* concentration was &f mM and this concentration elicited maximal activation. Activity in IO pg cytosolic or 2-5 ~11 solubilizcd particu!atc protein was measured.

2 3. St9risticul

Reactions were initiated by the addition of cytosolic or solubilized particulate protein and were terminated after 5 mir by..the addition of 0.01 ml ice-cold 75 mM phosphoric acid. The samples were quickly placed on ice. aliquots (0.02 ml) spotted on filter paper, the filter paper washed 3 times with 400 ml of 75 mM phosphoric acid and for a fourth time with 400 ml of 90% ethanol (10 rnin washes), dried at WC, and then counted. PKC activity was calculated as the difference between the cpm in the presence of Ca’+, and in the presence of Ca’+ + phosphatidylserine + phorbol myristate acetate. PKC activity in the cytosolic and particulate fractions was calculated as a specific activity (pm01 P,/mg protein per min), total activity (pm01 P,/min) and as a percent of the total activity.

.E E “m P

S

Differences between multltz!e means were analyzed ucing analysis of variance followed by the NeumanKculs test for multiple means unless otherwise indicated. Differences between two means were analyzed using Student’s unpaired t-test. Significance was accepted at the 0.05 level of probability. 2.5. Materials Leupeptin, pherylmethylsulfonyl fluoride, phorboi myristate acetate (phorbol 1Zmyristate 13-acetate), phorbol dibutyrate (PDB; phorbol 12.13-dibutyrate), and phosphatidylseriae were obtained from Sigma, his-

A. 5 mM EGTA homogenization CYT.0601

malysis

B. 20mM EGTA homogenization TOTAL

SP2

SPl

CYTOSOL

E”,

TOT&L

SPP

b

CR*+-free

0 Ca2+ -free wash

_

SPl

1

Ca

_

+

SP2

I

_

+

_

TOTAL

I

+

1

2+4ree wash: Time (min) Tomp (QC)

-

5 4

45 4

60 37

-

5 4

45 4

60 37

-

5 4

45 4

60 37

-

5

-

4

45 4

60 37

-

+

tone III-S from Worthington. P-80 filter paper from atman. [ y-“-‘P]ATP (25 Ci/mmol) from ICN, and H;I~O from Harlan. Phcntolaminc was a gift from CibaG&y.

3.2. Basal PKC nctirity

b E

PL9B (0.01 p Mi significantly decreased cytosolic PKC activity in aortas from SHR and WKY when

a.Aorta

500 400

1

z E

fkc-

Qtosofic PKC activity (pm01 P,/min), expressed as a percent of the total activity kytov&c -I-particulate pmol P,/min) was not significantly different in SHR and WKY aorta (figs. 2A and B), and in mesenteric ar!crkl bed (fig. 2C1. The PKC-specific activity (pm01 PJmg protein per min) in the cytosolic, and particulate, fractions of Ca’ c4rce solution-exposed aorta and mcscnteric arterial bed of SHR and WKY rats was also not significantly different (fig. ?,A-C!. In the expelimernts depicted in fig. 2B, the tot,4 specific activity (cytosolic -+ particulate) of SHR aorta was greater than that of WKY aorta. However, a greater tctsl specific rrctivity of SHR aorta as compared to WKY aorta was not qbserved in all experiments, as was the case with the experiments depicted in fig. 2A. The tutal specific activity of the SHR and WKY mcsenteric arterial bed was not significantly different Ifig. 20.

Homogenization of aorta irr buffer containing 5 mM EGTA resulted in cytosolic PKC activity of approximately 25% of the total PKC activity (fig. 1Ak Blot or‘ tissues prior to homogenization, or increasing the EGTA concentration in the homogenization buffer to 30 mM. did not alter the distribution of PKC activity (fig. IB: data with blotted tissues not shownf. Cytosolic PKC activity incrcascd, and par!iculate PKC activity decreased. upon exposure to Ca?+-free KRB solution containing 2 mM EGTA (fig. IA-C). Exposure to Ca”-free KRB solution at 4’C for 5 and 45 min. or at 37°C for 60 min. resulted in cyto*;okic PKC activities of approximately 6X. 80 ar;d 7X%, rcspcctively. of the total PKC activity (fig. ic’).

E

in cytosolic and particulate

tims in SHR and WKY ressels

100

WKY

SHR

(15)

($4)

1

(15)

300

WKY

b

: 5

2SS

a.E 100 0

I

WKY (Jt

I



SHR f3f

WKY (3)

SHR (3)

Fig. 2. Effects of PDB on PKC activity in the cytosolic and pnrticulate fractions of SHR and WKY vessels. SHR ;md WKY aort:~ (A :rnd Rl+ rmd mrsab& urkrd bed (0. were exposed to O.(lI pM or I WM PDB for 60 min, or remamed unrxposrd, followed by wash in 4°C Cd+-free KRB SduliOn for fdj miu a$ descrdA in Mateds iand merhods. Cytosdic (open columns), sduhilizcd purticuline (darkly shaded columnsi, and total &&lfy k&d eO1urnfts) PKCspecific activities and cy~osolicactivily expressed as a percent of ~atsl aclivity (ri~ht-h~ln~ open c(~illrnns~ore shown (mans :t 5.E. fflf). (A) ” Sisni~~ntly less than values of tissues unexposed 10 pDB. ” Si~ni~c~lntly less th;m ail other values. fB) I’ Significantly b*

than o’tdif

fractkms of

lk~~es

uncxposrd to PDB. ” Significnntly grc;ller than p;lrticnl;ltc fr;lclions of tissues unexposed 10 PDB.

’ Sknificanlly less than Iold activity of Iissues unexposed10 PDB. d Significnhtly grculcr

Illill olhcr kIlnI iiclivilics. (Cl “ Significiinlly less thnn cyiosolic fractions of !&ties unexposed IO PDf?.

activity was expressed as a percent of the total activity (fig. 2Al. The magnitude of decrease in cytosolic PKC activity due to 0.01 PM PDB, expressed as a percent of the total activity. was significantly greater in SHR aorta as compared ro WKY aorta (fig. 2k). The Cl.01 PM PDB-induced decrease in cytosolic, and increase in particulate, PXC activity were not significantly different when activity was expressed as specific activity and analyzed using the Neuman-Kculs test for multiple means (fig. 2A). The changes in specific activity due to 0.01 PM PDB were significantly different, however, using the Fisher PLSD test for multiple means. PDB (U.01 PM) did not decrease the total specific activity in SHR and WKY aorta (fig. 2A). PDB (I hM) decreased cytosolic PKC specific activity to a similar magnitude in aorta, and mesenteric arteriai bed, from SHR and WKY rats (fig. 28 and 0. PDB (1 PM) also increased particulate PKC-specific activity in the aorta and mesenteric arterial bed of SHR and WKY rats (fig, 28 and Cl. However, the magnitude of increase in particulate PKC activity was not always significant, and could not account for the magnitude of decrease in cytosolis PKC activity. The greater magnitude of the 1 PM PDB-induced decrease in cytosolic PKC-specific activity as compared to the magnitude of increase in particulate PKC-specific activity was presumably due to down-regulation of PKC by 1 PM PDB. In support of this suggestion are the observations that I PM PDB decreased total PKCspecific activity (fig. 2B1, although the decrease was not always statistically significant using the Neuman-Keuls test for multiple means (fig. 20. Due to the I PM PDB-induced decrease in total PKC-specific activity in the experiments depicted in fig. 2B and C, cytosoiic PKC activity was not calculated as a percent of the total PKC activity.

4. Dhwsion The present results support the hypothesis that the increased sensitivity of SHR vessels to phorboi esters (IMacKay and Cheung, 1987; Turla and Webb, 1987; Bruschi ct al,, 1988; Silver et al., 1988, 1992; Soloviev and Bershtein, 19921 is due, in part, to an increased sensitivity of PKC to phorbol ester activation. The mechanism for the increased sensitivity is not known, but several possibilities should be considered. Different amounts of PKC isozymes may be present in SHR and WKY vessels, and the isozymes present in larger amounts in SHR vessels may be more sensitive to phorboi ester activation. Another possibility is that the greater concentration ofCa2+ in the SHR aorta (Jelicks and Gupta, 19901 results in increased sensitivity of the PKC to activation by phorbol ester, Determinations of the PKC isozymes present in SHR and WKY aortas,

a.nd the ability of phorbol esters to activate pKC in vessels largely depleted of Ca’+, will test these pos?;ibilities. The present results also demonstrate that the total PKC-specific activity of SHR vessels was not consistently elevated as compared to corresponding WKY vessels. Furthermore, the basal distribution of PKC activity between the cytosoiic and particulate fractions, expressed either as specific activity or as a percent of the total activity, was not significantly different in corresponding vessels from SHR and WKY rats. Silver et ai. (1988) also demonstrated that the total PKCspecific activity in SHR and WKY aortas, and the distribution of PKC-specific activity between the cytosolic and particulate fractions of SHR and WKY aortas, were not significantly different. It is important to note, however, that the values of distribution of PKC-specific activity between the cgtosolic and particulate fractions reported by Silver et al. (1988) may be difficult to interpret. This difficulty in interpretation arises from the likelihood that the values of PKC distribution were derived from experiments performed under conditions allowing the Ca’+-induced translocation of PKC during vessel homogenization, i.e., the vessels were not washed in Ca’+-free solution prior to homogenization. Thus, while Silver et al. (19881 reported an aortic cytosolic: particulate PKC-specific activity ratio of 0.35, we presently report a ratio of 2.5 in aorta washed in Cal+-free solution prior to homogcnization. Furthermore, the ratio of cytosolic to particulate PKC activity decreased markedly in the absence of wash of tissues in Ca’+-free so!ution prior to homogenization (present results), and addition of Ca’+ to aortic homogenates caused the translocation of PKC from the cytosolic to the particulate fraction (unpublished obscrvationl. The observation that wash of tissues in Ca”-free KRB solution increased cytosolic and decreased particulate PKC activity may account, in large part, for the wide variation in reported subcellular distributions of PKC activity measured using intact vascular smooth muscle. For example, the ratios of cytosolic to particulate PKC-specific activities reported for intact rat renal artery and bovine mesenteric artery were substantially less than I : I (Ahlner et al., 1988; Sauro et al., 1989). and these tissues wcrc not washed in Ca’+-free SOILItion prior to homogenization. Although these tissues were homogenized in buffer containing reasonably high concentrations of EDTA and/or ECTA, we presently demonstrate that EGTA was unable to complctclY prevent the translocation of PKC due to Ca’+ during homogenization. The present results

suggest, therefore, that it is technically quite difficult, or even currently not fcasiblc, to dcterminc accurate mcasurcments of the rcsting distribution of PKC in subcellular fractions following

cell disruption.

since the time

of wash

in Ca’+-free

buffer may decrease intracellula: Ca’*-levels and. thus. ~~~ernbr~~~e-b~~undCa’ +_ PKC distribution can be dett-r-

ntincd. h~~wcvc‘r.in tissues exposed tr) Ca”-free tion. Thus. one is limited

to measurements

dependent

in the

bound

PKC

SOIU-

of Ca’“-in-

particulate

fraction,

which is ~~doub~ed~y lower than in the resting state. In contrast, phorbol

ester-induced

bution can he accurately

changes in PKC distri-

determirxd

&+-free

KRB

solution.

memb~ne

in response to phorboi

lowing removal of Ca’+ Of potential greater SHR

basal

since

(Bazzi

following

PKC

bound

wash in to the

esters is stable fol-

and Nelsestuen.

198929).

relevance to the present study is that a concentration was found in the Ca”

than the WKY

aorta (Jclicks

and Gupta,

19901.

This observation, along with the present demonstration associated with the tissue greatly inthat the

Protein kinase C activity in blood vessels from normotensive and spontaneously hypertensive rats.

This study investigates the effects of phorbol dibutyrate (PDB) on protein kinase C (PKC) activation, as assessed by the translocation of PKC activity...
792KB Sizes 0 Downloads 0 Views