Ca2+-transport ATPases of vascular smooth muscle

JA Eggermont, M Vrolix, L Raeymaekers, F Wuytack and R CasteelsCa2+-transport ATPases of vascular smooth muscle

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266

Ca2+-Transport ATPases of VascularSmooth Muscle

Jan A. Eggermont, Matty Vrolix, Luc Raeymaekers, Frank Wuytack, and Rik Casteels

To characterize the Ca2+-transport properties of the plasma membrane and of the endopiasmicreticuium of bovine pulmonary artery, membrane vesicles are subfractionated by a procedure ofdensity-gradient centrifugation that takes advantage of the selective effect of digitonin on the densityof plasma-membrane vesicles. The obtained endoplasmic-reticulum fraction contains hardly anyplasma-membrane vesicles, whereas the plasma-membrane fraction is still contaminated by asubstantial amount of endoplasmic-reticulum vesicles. An adenosine 5'-triphosphate (ATP) energizedCa2+-transport system and a Ca2+-stimulated ATPase activity are present in both subcellular fractions.The Ca2+ transport by the plasma membrane is catalyzed by a (Ca2+,Mg2+)-ATPase of M, 130,000.It binds calmodulin and it has a low steady-state phosphoprotein intermediate level. The endoplasmic-reticulum vesicles contain a Ca2+-transport ATPase of Mr 100,000 that is characterized by a highsteady-state phosphointermediate level. It is antigenicaJly related to the Ca2+-pump protein of cardiacsarcoplasmic reticuium. Phospholamban, the regulatory protein of the Ca2+-transport enzyme ofcardiac sarcoplasmic reticuium, is also present In the endopiasmic reticuium of the pulmonary artery.A comparison of these fractions with the previously characterized fractions from porcine gastricsmooth muscle reveals important differences in the basal Mg -̂ATPase activity, in the ratio of the(Cai+,Mgi+)-ATPase of the plasmaJemma to that of the endopiasmic reticuium, and in the ratio ofthe (Na+,K+)-ATPase activity to the plasmalemmal (Ca2+,Mg2+)-ATPase activity. These differencescan be ascribed in part to the species and in part to the tissue. These data suggest that in the bovinepulmonary artery the Ca2+ extrusion via the ATP-dependent Ca2+ pump may have a less predominantrole, and that the Ca2+ uptake by the endopiasmic reticuium, and possibly also the Ca2+ extrusion viathe Na+-Ca2+ exchanger could be more important in this tissue than in the porcine stomach.(Circulation Research 1988;62:266-278)

C hanges in the intracellular Ca2+-concentration([Ca2+L,) regulate the contraction-relaxationcycle or smooth muscle. To counteract the

Ca2+ influx through the plasma membrane (PM) and theCa2+ release from the endopiasmic reticuium (ER), thesmooth muscle cell possesses two different types ofCa2+-transport ATPases, one located at the PM, theother at the ER.1 However, the relative contribution ofeach Ca2+-transport mechanism to the cellular Ca2+

homeostasis, their regulation by second messengers,and the role of other mechanisms such as the Na+-Ca2+

exchanger have not yet been unequivocally specified.A major obstacle in tackling these problems is thedifficulty in separating the membranes containing theseCa2+-transport systems. Although the isolation of PMfrom vascular smooth muscle has been reported,2"4 nopurified ER has been obtained. Calcium-oxalate load-ing of ER vesicles has been used to obtain a purifiedER fraction from stomach smooth muscle.3 A majordisadvantage of this procedure is the interference ofintravesicular calcium-oxalate crystals with assays for(Ca2+,Mg2+)-ATPase activity, Ca2+ uptake, and Ca2+

release.

We have recently developed a method to isolate apurified ER fraction from visceral smooth muscle

From the Laboratorium voor Fysiologie, Universiteit Leuven,Campus Gasthuisbcrg, Leuven, Belgium.

J.A.E. is a research assistant of the Nationaal Fonds voorWetenschappelijk Onderzoek (NFWO, Belgium).

Address for correspondence: L Raeymaekers, Laboratorium voorFysiologie, Campus Gasthuisberg, B 3000 Leuven, Belgium.

Received May 7, 1987; accepted August 24, 1987.

without calcium-oxalate loading.6 This procedure hasnow been applied to vascular smooth muscle, and wereport the isolation of a purified ER fraction frombovine main pulmonary artery. The separation of ERand PM vesicles has allowed us to characterize in thistissue the two types of Ca2+-transport ATPases bymeans of their (Ca2+,Mg2+)-ATPase activity, theirphosphointermediate, the calmodulin binding, andantigenic properties. A comparison of the phosphoin-termediates in different types of smooth muscle alsoreveals important differences of the relative amounts ofboth Ca2+-transport ATPases.

Materials and MethodsPreparation of Membrane Fractions

Membrane fractions were prepared as described byRaeymaekers et al.6 Briefly, bovine main pulmonaryarteries (the truncal part between the pulmonary valveand the bifurcation) were obtained from a localslaughterhouse about 15 minutes after killing of theanimal and immediately cooled in ice. After removingthe adventitial layer, the arteries were chopped andpassed through a press as described by Wuytack et al.7

The mince (300 g) was then homogenized with anUltra-turrax (Stanfen, FRG) ( 3 x 1 0 seconds) in 3volumes of ice-cold 0.25 M sucrose containing 1 mMdithiothreitol and 0.5 mM phenylmethylsulfonylfluo-ride and centrifuged in a Sorvall GSA (Wilmington,Delaware) motor at I3,000gnmx for 30 minutes. Thepellet and the floating layer were discarded and thesupernatant, designated as the postmitochondrial su-



Eggermont et al Ca2+ Transport In Vascular Smooth Muscle 267

pernatant, was pooled. One hundred fifty milligramsdigitonin, dissolved in 10 ml ethanol, was added to 800ml of the supernatant. Solid sucrose and potassiumchloride were added up to a final concentration of 50%(wt/wt) and 0.6 M, respectively. This material wasloaded in a Beckman Til5 (Palo Alto, California) ora Kontron TZT 32 (Zurich, Switzerland) zonal rotorbelow 250 ml of a 15-45% sucrose gradient that waslinear with respect to volume and contained 0.6 M KC1and 1 mM dithiothreitol. The central part of the rotorwas filled with 150 ml 0.6 M KC1. Centrifugation wasperformed at 105, OOO^ for 20 hours at 4° C. Fixedvolume fractions were collected and each fraction wasassigned a percent sucrose value as determined by aZeiss (Oberkochen, FRG) hand refractometer. Thefractions were diluted three times with 0.6 M KC1containing 1 mM dithiothreitol and pelleted in aKontron TFT 45.94 rotor at 140,000^^ for 1 hour at4° C. The pellets were resuspended in 0.25 M sucrosecontaining 1 mM dithiothreitol and stored at - 80° C.In some preparations, no digitonin was added to thepostmitochondrial supernatant. In other experiments,the potassium chloride in the postmitochondrial super-natant and in the sucrose gradient was omitted.

Membrane fractions from porcine gastric smoothmuscle (antral part), porcine main pulmonary artery, orbovine thoracic aorta were prepared by the sameprocedure. Porcine tissues were obtained about half anhour after the killing of the animals. Inside-outplasmalemmal vesicles from pig erythrocytes andsarcoplasmic reticulum (SR) vesicles from pig skeletalmuscle were prepared as described previously.8 Bovinecardiac sarcoplasmic reticulum vesicles were preparedaccording to Jones et al.9

Enzyme AssaysAll enzyme activities were assayed as described

previously* by continuously monitoring the reactionrate at 37° C in a Beckman DU7 spectrophotometer(Irvine, California) or in an Aminco DW2 spectro-photometer (Silver Spring, Maryland). 5'-Nucleo-tidase was assayed using a Sigma kit (Sigma Chem-ical, St. Louis, Missouri). Reduced nicotinamidedinucleotide (NADH) or reduced nicotinamide dinu-cleotide phosphate (NADPH) cytochrome c reductase(rotenone-insensitive) activities were measured byfollowing the reduction of cytochrome c at 550 nm asdescribed.10 Basal Mg2+-ATPase activity was mea-sured by following the oxidation of NADH at 340 nmin the presence of a NADH coupled enzyme system.3

Because of its nonlinear time course," the basalMg2*-ATPase activity was arbitrarily defined as theactivity corresponding to the decrease in absorbancebetween time zero and 2 minutes. The (Na+,K+)-ATPase activity and the (Ca2+,Mg2+)-ATPase activitywere measured in the presence of a NADH coupledenzyme system as described previously.6 The(Na+,K+)-ATPase activity is defined as the ATPaseactivity that was inhibited by 10/uMdigitoxigenin. The(Ca2+ ,Mg2+)-ATPase activity is defined as the ATPaseactivity that was stimulated by the addition of Ca2+ (up

to a final free concentration of 10"5 M) to a Ca2+-free,EGTA (0.5 mM) containing medium. Because of thenonlinear basal Mg2+-ATPase activity, the (Na+,K+)-ATPase and (Ca2+ ,Mg2+)-ATPase activities were mea-sured with a double-beam spectrophotometer asdescribed.6 In the first part of the experiment, thesample and the reference cell were identical in com-position, resulting in an absorbance-difference signalthat was perfectly constant with time. After recordingthis baseline, digitoxigenin [for measuring (Na+,K+)-ATPase] or Ca2+ [for recording (Ca'+ ,Mg2+)-ATPase]was added to one of the cells, and the enzyme activitywas calculated from the slope of the curve. To obtainthe maximal specific activity of the (Ca2+,Mg2+)-ATPase of the plasmalemma, 0.6 piM calmodulin wasadded. Alamethicin (0.5 /ng//xg protein) was includedto expose latent (Na+,K+)-ATPase and (Ca2+,Mg2+)-ATPase activities.12

The distribution of the membrane markers in thesucrose gradient is presented in normalized histogramsas described by Beaufay et al.'3 The histograms areplotted on a density scale, expressed in percent sucrose,which is divided into zones corresponding to theequilibrium density of the collected fractions. Theordinate gives the frequency within the correspondingzone of the sucrose gradient. The frequency equalsa/(A-D%), where "a" is the activity recovered withina zone of the gradient, "A" is the sum of the activitiesrecovered in the whole gradient, and "D%" is thepercent sucrose spanned by that zone. The surface areaof each section of the diagram (frequency X D%)equals a/A and corresponds to the fraction of the totalactivity recovered in that zone. The total surface areaequals 1.0. The results of 5 experiments were averagedby superimposing the histograms of the 5 experimentsand calculating the mean frequency for every unevenpercent sucrose between 17% and 39% sucrose. Withthese data a new histogram was plotted.

Measurement of the Phosphointermediate Formationof the Ca2* -Transport ATPases

Membrane vesicles were phosphorylated in a me-dium (50 fi\ total volume) containing 120 mM KC1, 30mM imidazole (pH 6.9), 50 /AM CaCl2 or 0.5 mMEGTA, alamethicin (0.5 /xg//xg protein), and 1 mg/mlof membrane proteins. In some experiments, 50 /AMLaCl3 was added. The reaction was started by adding6 /iM ATP containing ATP[y-32P] at 10 ̂ Ci/nmol andwas allowed to proceed for 20 seconds at 4° C. It wasstopped with 150 /xl stop solution containing 10%trichloric acid (TCA), 50 mM H3PO4, and 0.5 mMATP. The samples were kept on ice for 10 minutes andthen transferred to a tube containing 10 ml 0.2 Msodium acetate at pH 5.3 without or with 0.2 Mhydroxylamine. In some experiments, the sampleswere transferred to a tube containing 10 ml stopsolution instead of the sodium acetate solution. After20 minutes at room temperature, the samples werefiltered on a glass microfiber filter (Whatman GFA orGFB, Maidstone, England). The filters were rinsedwith 4x7.5 ml stop solution, dried, and counted to



268 Circulation Research Vol 62, No 2, February 1988

determine the amount of MP retained on the filter. Thedifference between the phosphate incorporation in thepresence of Ca2 + and the incorporation in the Ca2+-free,EGTA-containing medium was taken as the Ca2+-dependent phosphorylation.

Phosphorylation of the Ca2+-transport ATPasesand separation of the phosphoprotein intermediates inacid gels were carried out as described previously.8

Phosphorylation with the catalytic subunit of thecAMP-dependent protein kinase (10 /ig/ml) in thepresence of ATP[y-32P] and subsequent gel electro-phoresis and autoradiography were carried out asdescribed previously.14

Electrophoresis and Western BlottingLaemmli-type SDS-polyacrylamide gel electropho-

resis (SDS-PAGE) was done on 7.5% or 12% slab gelsof 0.75 mm thickness.15 The gels were either stainedwith Coomassie brilliant blue or used for the electro-phoretical transfer of the separated proteins on anitrocellulose sheet by a Western blotting procedure.16

Calmodulin[123I] binding to membrane fractionsseparated by SDS-PAGE and electrophoretically trans-ferred to a nitrocellulose sheet was done according toa previously described method."

The 32-kDa calelectrin of porcine gastric smoothmuscle was purified as described previously.18 Rabbitpolyclonal antibodies against this protein wereraised in rabbits. Antibody binding and detec-tion of the immune complexes were carried out onmembrane fractions separated by SDS-PAGE andelectroblotted onto a nitrocellulose sheet. Boundanti-ribophorin I or anti-calelectrin antibodies werevisualized using peroxidase-conjugated secondaryantibodies.17

Antibodies against the 100-kDa ATPase were visu-alized by Protein A[123I] labeling and autoradiography."

Other AssaysCa2+ uptake was measured at 37° C in a medium

containing (in mM) KC1 150, NaN3 5 (to blockmitochondrial Ca2+ uptake), imidazole-HCl (pH 6.9)30, Tris-ATP 5, MgCl2 6,43Ca EGTA 1, with or without5 mM potassium oxalate or 50 mM potassium phos-phate. The filters were rinsed and the 45Ca remainingon the filters was counted.

Protein was measured by the method of Lowry et al20

using bovine serum albumin as a standard.

MaterialsCalmodulin and calmodulin[mI] were prepared as

described.' Digitonin was obtained from Merck, Darm-stadt, FRG. Alamethicin was from Sigma. Rabbitskeletal muscle lactate dehydrogenase and pyruvatekinase were obtained from Boehringer, Mannheim,FRG. The catalytic subunit of the cAMP-dependentprotein kinase was obtained from Sigma. Perox-idase-conjugated swine anti-rabbit and peroxidase-conjugated rabbit anti-mouse immunoglobulins weresupplied by DAKO-Patts, Copenhagen, Denmark.ATPfr-^P] (3,000 Ci/mmol), Protein A[123I] (30

mCi/mg) and 45Ca were obtained from Amersham,Little Chalfont, England. Mouse monoclonal antibod-ies against ribophorin I from canine liver were kindlyprovided by Dr. M. Hortsch, EMB Laboratory, Hei-delberg. Rabbit polyclonal antibodies against thecardiac SR Ca2+ pump were a gift from Dr. K. Gietzen,University of Ulm, Ulm, FRG. Rabbit polyclonalantibodies against phospholamban from canine cardiacSR were kindly provided by Dr. L.R. Jones, IndianaUniversity, Indianapolis, Indiana.

ResultsSubcellular Fractionation of the Membrane Vesicles(Figure 1 and Table 1)

Because digitonin forms an equimolar complex withcholesterol, it selectively increases the density ofplasma-membrane (PM) vesicles and only slightlyaffects the density of other membranes. This selectivedigitonin effect results from the higher cholesterolcontent of PM vesicles as compared with internalmembranes.21

Density-gradient centrifugation of a postmitochon-drial supernatant of bovine pulmonary artery in thepresence of digitonin results in a shift of the major partof membrane proteins so that 86% of the protein is re-covered between 28 and 40% sucrose (Figure 1, Panels1A-C). Concomitantly, almost all the PM vesicles arerecovered in this zone: 97% of the (Na+,K+)-ATPaseactivity, and 94% of the 5'-nucleotidase activity (Table1 and Figure 1, Panels 3B and C). Because it presentsthe highest specific activity of PM markers, the 28-40% sucrose zone will be referred to as the PM fraction,without implying that it is a totally pure PM fraction(see below). The distribution of the basal Mg2+-ATPase activity correlates very well with that of the(Na+,K+)-ATPase (Figure 1, Panels 3B and C), con-firming the plasmalemmal localization of the Mg2+-ATPase activity.

The distribution of the putative ER markers NADHand NADPH cytochrome c reductase (rotenone-insen-sitive)22 is bimodal (Figure 1, Panels 2B and C). Thepredominant part of the total activity of these enzymesis contained within the PM zone (Table 1). This maybe due to contamination with ER vesicles or to anenzyme activity associated with PM vesicles or withouter mitochondrial membranes.23 However, approxi-mately one third of their total activity is situated at adensity lower than 28% sucrose, and the highestspecific enzyme activities of the ER markers NADHand NADPH cytochrome c reductase are observed inthe 16-24% sucrose zone. The material recovered inthis zone will be referred to as the ER fraction. Thisfraction is practically free of plasma membranesbecause the specific activity of the PM marker(Na+ ,K+)-ATPase is 17 times lower than that in the PMfraction (Table 1).

Ninety-four percent of the total enzyme activity of5'-nucleotidase is localized in the PM fraction (Table1). However, the specific activity in the PM fractionover that in the ER fraction (PM/ER ratio) amounts onlyto a factor 4, whereas a seventeenfold and an elevenfold



Eggermont et al CaJ+ Transport in Vascular Smooth Musde 269

CiMgATPm :

B

i 1

I i

j — i — ,

H

c

J \

Jr'

\

\

FIGURE 1. Histogram of the frequencydensity distribution of protein content(J), endoplasmic reticulum markers (2),plasma membrane markers (3),(Ca2* .Mg2*)-ATPase activity (4), andphosphointermediate of the (Ca1*,Mg2* )-ATPases (4). Column A, resultsof a typical experiment without digito-nin; column B, results of a typicalexperiment with digitonin; column C,average of 5 experiments with digitonin.Histograms are constructed as ex-plained in "Materials and Methods."The surface area of each section of thehistogram equals the fraction of totalactivity that is recovered in the corre-sponding zone of the sucrose gradient.

« 22 28 3i U> K 22 16 22 K 3i M

% Sucrose

PM/ER ratio is noted for (Na+,K+)-ATPase andMg2+-ATPase respectively (Table 1). This differencemay be explained by the presence of a small amountof 5'-nucleotidase in intracellular membrane vesiclesthat is not shifted by digitonin or by the adsorption ofa soluble form of the enzyme to the ER membranes.The presence of an enzyme associated with intracel-lular membranes24 and of the soluble enzyme23 has beendemonstrated in hepatocytes.

The total recovery in our fractions of the membranemarkers has been calculated using the postmitochon-drial supernatant as a reference material. Recoveries of5'-nucleotidase and NADH cytochrome c reductase(rotenone-insensitive) amount only to between 16 and28%, because a soluble form of these enzymes alsooccurs25 and because they might also be partiallyextracted by the treatment with high potassium chlorideand digitonin. In contrast, recoveries between 65 and100% are obtained if (Na+ ,K+)-ATPase activity is usedas a parameter, indicating that the majority of the PM

vesicles are recovered. Neither the recovery of the(Ca2+,Mg2+)-ATPase activity nor that of the Ca2+-dependent phosphointermediate could be determinedbecause in the postmitochondrial supernatant the basalMg2+-ATPase activity and the Ca2+-independent,hydroxylamine-insensitive phosphoprotein level weretoo high to allow an accurate measurement of the(Ca2+,Mg2+)-ATPase activity or phosphointermediatelevel, respectively. The recovery of the oxalate-stim-ulated calcium uptake is only 20%. However, we havepreviously shown that the oxalate-stimulated calciumuptake is very sensitive to centrifugation,26 and, there-fore, this parameter underestimates the recovery of thecorresponding ER-type ATPase.

Finally, it should be mentioned that the boundarybetween the ER and PM zones, deduced from thedistribution of the total enzyme activity of the PMmarkers, was always very sharp (see Figure 1, Panel3B). However, its position in the sucrose gradientvaried in different experiments from 29 to 31.5%




Table 1. Distribution of Protein Content and Total Activities of Membrane Markers, (Ca2+, Mg2+)-ATPaseand Phosphoin termed late in the Sucrose Gradient and Specific Activities of These Enzymes in the EndopiasmicReticulum and Plasma Membrane Fraction

Protein

NADPH cyt c reductase

NADH cyt c reductase

5'-Nucleotidase

Mg2+-ATPase

(Na+, K+)-ATPase

(Ca2 + , Mg2+)-ATPase

Phosphoprotein

% of total

16-28%Sucrose

14.1+5.2

30.9±10.2

37.6±11.6

5.9 + 1.9

3.2±0.8

2.8±1.0

14.3±3.1

29.3±8.1

activity*

28-40%Sucrose

85.9±5.2

69.1 + 10.2

62.4 ±11.6

94.1 ±1.9

%.8±0.8

97.2 ±1.0

85.7 + 3.1

70.7 ±8.1

ER

61.2± 15.4

9,502 ±2,914

235 ±79

359 ±134

26±18.6

358 ±87.0

63.5 ±2.5

Specific activitiestPM

14.2±4.0

1,379 ±169

932 + 399

4,218 ±1,457

445 ± 245

282.5±84.0

13.0±2.9

Ratio

4.3 (ER/PM)

6.9 (ER/PM)

4.0 (PM/ER)

11.7 (PM/ER)

17.1 (PM/ER)

1.3 (ER/PM)

4.9 (ER/PM)

•Given values represent percent of total activity recovered in endopiasmic reticulum (ER) fraction (16-28% sucrose)and in plasma membrane (PM) fraction (28-40% sucrose). Values are mean + SEM of five experiments.

tSpecific activities are expressed in nmol/min/mg protein except for phosphoprotein formation, which is expressed inpmol phosphate/mg protein. Values are mean±SEM of five experiments.

sucrose. This variability explains the gradual transitionof the ER and PM markers when the average of 5experiments is plotted (Figure 1, Panel 3C).

Characterization of Membrane-Associated ProteinsInclusion of 0.6 M KC1 in the postmitochondrial

supernatant and in the density gradient results in a moreefficient removal of extrinsic proteins.6 As demon-strated by SDS-PAGE (Figure 2), fractions treated with0.6 M KC1 contain smaller amounts of polypeptides ofM, 205, 68, 37, 35, and 32 kDa. The 205-kDapolypeptide is probably the heavy chain of myosin sinceit comigrates with this protein (data not shown). Theamount of actin (43 kDa) that is associated with themembranes is hardly influenced by the addition ofpotassium chloride. The other polypeptides belong toa group of proteins that are associated with membranesin a Ca2+-dependent way but of which the function isstill unknown. They have been named calelectrins orcalcimedins. (For review, see Geisow and Walker27 andMoore.28) Polypeptides of this family with a MI in SDSgels of 68, 37, 35, and 32 kDa have been demonstratedin isolated plasma membranes from pig stomachsmooth muscle.18 Polypeptides of bovine pulmonaryartery comigrate with the calelectrins previously de-scribed in stomach smooth muscle and can readily beidentified (Figure 2A). The presence of these proteinsis diminished by potassium chloride treatment of thePMS (Figure 2A), as previously observed in stomachsmooth muscle."

The potassium chloride-treated membranes frombovine pulmonary artery contain a larger amount ofcalelectrins than the potassium chloride-treated mem-branes of pig stomach. This conclusion is based on acomparison of the gels stained with Coomassie blue(Figure 2A) and of the immunoblottings with antibod-ies directed against the purified 32-kDa polypeptidefrom pig stomach smooth muscle (Figure 2B). Theseantibodies not only bind to the immunizing antigen of32 kDa but also react in bovine pulmonary artery withthe 37- and 35-kDa calelectrins. The crossreaction of

anti-calelectrin antibodies with other members of thisprotein family has also been observed in otherlaboratories.27 The 37-kDa calelectrin is preferentiallyassociated in the pulmonary artery with the PM fractionas observed for the calelectrin-like proteins in othercells.27 It is not clear at present whether the differencesin calelectrin content of membranes of pig stomach andbovine pulmonary artery reflect differences in theamount of calelectrin in vivo, or whether it is due to adifference in the extraction of these proteins by amedium of high ionic strength.

Ribophorin I is a membrane protein that binds theprotein synthesizing ribosomal complex to the roughER membrane and is exclusively localized in therough ER.29 A monoclonal antibody directed againstthis protein selectively recognizes one protein inmembranes from porcine gastric smooth muscle andbovine pulmonary artery. This protein is mainlypresent in the ER-enriched fraction. Omission ofpotassium chloride from the preparative solutionsresults in a more equal distribution over the wholesucrose gradient (Figure 2B).

Phospholamban, a 22-kDa protein that regulates theCa2+ -transport ATPase of cardiac SR,30 has recentlyalso been demonstrated in smooth muscle ER.14 This22-kDa protein can be phosphorylated by the catalyticsubunit of cAMP-dependent protein kinase and isdecomposed by boiling in SDS. Figure 3 shows sucha protein, localized predominantly in the ER-enrichedfractions, suggesting the presence of phospholambanin the ER of bovine pulmonary artery. Besides phos-pholamban, various other polypeptides of the ER ofvascular smooth muscle are phosphorylated by cAMP-dependent protein kinase, while under identical con-ditions very little phosphorylation of plasma-mem-brane proteins is observed (Figure 3).

Subcellular Localization of Ca2* -Transport ATPasesCa2+ uptake. Both ER and PM vesicles contain an

ATP-dependent, azide-insensitive Ca2+-uptake system(Figure 4). Ca2+-precipitating anions, such as oxalate



Eggermont et al Ca"+ Transport in Vascular Smooth Muscle 271

% sucrose

bovine pulm artery(*KCl)

ER PM

bovine pulm. arteryt - KCII

22 25 27 31 3i 37

pig stomachI-KCI I

ER PM

'19 21 24 '27 29 32 35

*- ~ vz n n

MrxlOOO

— 205

975

68

FIGURE 2. SDS-PAGE and immunoblotting of different membrane fractions from bovine main pulmonary artery and porcine stomach.All fractions were prepared with digitonin with or without 0.6 M KCI as described in "Materials and Methods." Endoplasmic reticulum(ER)- and plasma membrane (PM)- enriched fractions are indicated. A, Coomassie blue staining pattern of SDS-PAGE gel; B,immunoblot of a gel identical to that shown in A, but sequentially treated with monoclonal antibody to ribophorin I and with antiserumto the 32-kDa calelectrin. The antibody to ribophorin I stains at a 68-kDa band and the antiserum against calelectrin at 32, 35, and37 kDa.

at 5 mM and phosphate at 50 mM, markedly enhancethe Ca2+ uptake in the ER fraction, whereas theirstimulatory effect is less pronounced in the PMfraction, as previously observed in pig stomach smoothmuscle.36 Oxalate and phosphate are equally potentstimulators of the Ca2+ uptake in the PM fraction of thebovine pulmonary artery. This is in contrast to ourprevious findings on the PM fraction of porcine gastricsmooth muscle where oxalate was less effective thanphosphate.3 It is therefore possible that the bovinepulmonary artery PM fraction contains a relativelylarger amount of the oxalate-stimulated Ca2+-trans-porting system of the ER type than the pig stomach PMfraction. This hypothesis is supported by our findingson the relative amount of the phosphorylated interme-

diate of the 100-kDa Ca2+-transport ATPase in thesetwo types of smooth muscle (see below). However, itshould also be noted that the observed Ca2+ uptake inthe PM vesicles is only a small fraction of the Ca2+

pump activity because a large part of the vesicles maynot have been resealed in the inside-out orientation andalso because digitonin is known to increase thepermeability of the PM. For this reason the(Ca2+ ,Mg2+)-ATPase activity is a much better index ofCa2+ pump activity than the Ca2+ uptake.

(Ca2+,Mg2+)-ATPase activity. The active transport isenergized by the hydrolysis of ATP into ADP andinorganic phosphate (P,) and the Ca2+-stimulated,Mg2+-dependent ATPase activity of membrane frac-tions therefore reflects the turnover of the Ca2+-




A

Boil

6SR ER PM ER PM

FIGURE 3. A, Autoradiogram of membrane fractions phosphorylated in the presence of ATP[y-nP] and the catalytic subunit ofcAMP-dependent protein kinase. Endoplasmic reticulum (ER) and plasma membrane (PM) fractions from bovine pulmonary arterymembrane were collected from the same regions of the density gradient as those shown in Figure 2. Following solubilization in SDS,membrane fractions were ( + )or were not ( — ) boiled and separated by SDS-PAGE (12% slab gels). The ER fraction of smooth muscleas well as the cardiac SR contain a phosphoprotein ofMr 22 kDa (upper arrowhead) that is decomposed in 5-kDa polypeptides (lowerarrowhead) when the membrane-SDS mixture is boiled. This phosphoprotein is poorly represented in the PM fraction. B, lmmunoblotof a parallel gel of that shown in A. The electroblot was incubated with antibodies directed against purified phospholamban fromcanine cardiac muscle. Fixed antibodies were visualized using Protein A[I23I]. Partially decomposed phospholamban is also indicatedby arrowheads.

transport ATPase. To measure the maximal(Ca2+,Mg2+)-ATPase activity, we routinely added cal-modulin (10 fig/ml) because the (Ca2+,Mg2+)-ATPaseof the PM is a calmodulin-dependent enzyme. Tounmask latent enzyme activities, we also includedalamethicin, a channel-forming antibiotic. This sub-stance makes the membranes permeable for ATP,exposes the intravesicular membrane side of closedvesicles to the reaction medium, and prevents theaccumulation of Ca2+ inside the vesicles.12 Suchintravesicular Ca2+ concentration would inhibit the(Ca2+,Mg2+)-ATPase activity.

Fourteen percent of the total (Ca2+,Mg2+)-ATPaseactivity is found in the ER zone of the density gradient(Table 1). The distribution of the total (Ca2+,Mg2+)-ATPase activity (Figure 1, Panels 4B and C) isintermediary between that of the PM markers (Figure1, Panels 3B and Q and that of the ER markers (Figure1, Panels 2B and Q . The specific (Ca2+ ,Mg2+)-ATPaseactivity of the PM and ER fraction does not differappreciably (Table 1). These findings are compatiblewith the presence of two types of Ca2+-transportATPase that are differently distributed (see below).

Phosphorylation experiments. DISTRIBUTION OF THEPHOSPHORYLATED INTERMEDIATES IN THE SUCROSE GRADI-

ENT. The formation of an alkali-labile, hydroxylamine-sensitive acylphosphate bond is an essential step in thecatalytic cycle of both the ER and PM Ca2+-transportATPase. Phosphorylation of the membrane vesicles for20 seconds at 4° C in the presence of ATP[y-32P], 50IJM Ca2+, and alamethicin (0.5 /ig/^g protein) andsubsequent determination of the total incorporated 32Pallow us to quantify the Ca2+-dependent incorporationof phosphate in both ER and PM vesicles. More than90% of the incorporated phosphate is hydrolyzed bytreatment with hydroxylamine, indicating that anacylphosphate bond was formed (results not shown).

The distribution of the total amount of phosphopro-tein (Figure 1, Panels 4B and C) closely resembles thatof the putative ER markers (Figure 1, Panels 4B andC). The ER/PM ratio of the phosphointermediate levelis about 5 (Table 1). This observation suggests that theformed phosphoprotein corresponds to the phosphoin-termediate of the Ca2+-transport ATPase of the ER.This is congruent with the autoradiograms obtainedafter phosphorylation and gel electrophoresis that showonly one phosphointermediate of 100 kDa, which ispredominantly localized in the ER (see p. 273).

VISUALIZATION OF TWO DIFFERENT PHOSPHOPROTEIN IN-

TERMEDIATES BY GEL ELECTROPHORESIS. The phosphoin-



Eggermont et al Ca*+ Transport in Vascular Smooth Muscle 273

I

2000

1600

1200

« 800c 600

100

200

iOO

200

PM

10 20 30 min

termediates of the Ca2+-transport ATPases can bevisualized by phosphorylation of the membrane vesi-cles with ATPfy-32!*] and subsequent electrophoresis inacid medium.8 In the presence of micromolar [Ca2+],a phosphointermediate with a similar molecular mass( ± 100 kDa) as that of the SR of skeletal muscle andwith a predominant localization in the ER-enrichedfraction is revealed (Figure 5). La3+ (50 juM) reducesthe phosphorylation level of the 100-kDa ATPase, butenhances the formation of the phosphointermediate ofM, 130-140 kDa,1 which then becomes visible in thePM-enriched fraction (Figure 8).

Because ER fraction of bovine pulmonary artery ispractically devoid of any contaminating PM vesicles,all (Ca2+,Mg2+)-ATPase activity in this fraction mustbe catalyzed by the 100-kDa enzyme. Under the givenexperimental conditions, this ER fraction can thereforebe used to determine the ratio (R) of the (Ca2+ ,Mg2+)-ATPase activity (A) to the level of phosphoproteinintermediate (P) of the 100-kDa enzyme, (R = A/P)(see Figure 5). No 130-kDa intermediate can bedetected in the PM fraction if the phosphorylation iscarried out in the absence of La3+ (Figures 5 and 8).Therefore, to measure the amount of phosphointerme-

ER

FIGURE 4. The time course of the Ca2* uptake ina fraction enriched in endoplasmic reticulwn (ER)and in plasma membrane (PM) vesicles. Ca2*uptake was performed in the absence of ATP (o),in the presence of 5 mM ATP without precipitatingonions (o), in the presence of 5 mM ATP and 50mM phosphate (•) and in the presence of 5 mMATP and 5 mM oxalate (•).

10 20 30 min

diate corresponding to the 100-kDa Ca2+-ATPase, itsuffices to phosphorylate the membrane fractions in aLa3+-free medium and to calculate the 32P incorporatedin the membrane fraction (see "Materials and Meth-ods"). No separation between the 100- and 130-kDaphosphoprotein is required as the 130-kDa phosphoin-termediate cannot be observed under the given exper-imental conditions (La3+ free). By multiplying theamount of phosphointermediate in each fraction by theratio R, a value for the (Ca2+,Mg2+)-ATPase activitycatalyzed by the 100-kDa enzyme can be estimated foreach fraction. By subtracting this value from the actualmeasured (Ca2+ ,Mg2+)-ATPase activity, a value for the(Ca2+,Mg2+)-ATPase activity catalyzed by the 130-kDa enzyme for each fraction can be estimated. If weassume that both Ca2+-ATPases are recovered to asimilar extent, these estimates suggest that in themembranes prepared by our density gradient proce-dure, 43 ± 8% (n = 3) of the total (Ca2+ ,Mg2+)-ATPaseactivity, can be ascribed to the 100-kDa ATPase andthat the remaining 57% of the (Ca2+,Mg2+)-ATPaseactivity is catalyzed by the 130-kDa enzyme, althoughthe level of its phosphorylated intermediate is muchlower than that of the 100-kDa ATPase.

PM RBC SR

% Sucrose 18 22 25 28 31 3U 37 UO

pmd

(Ca2*

(nmol

Pmg-'

Mg2*)ATPase

mg~l mini)

52338

61381*

36305

20212

10 92tf 261

10170

10231

Ratio 65 63 8.5 10.6 2U 29 17 23.1

FIGURE 5. Autoradiogram of a dried pol-yacrylamide gradient gel (5-15%) showing thephosphointermediates of the Ca2* -transportATPase in different subcellular fractions ofbovine pulmonary artery. Phosphorylation wasperformed in the presence ofATP[y-nP] andSO(JLM Ca2* without La1*. The phosphointerme-diates ofSR of porcine skeletal muscle (SR) andof inside out vesicles of porcine red blood cells(RBC) are also shown. % sucrose refers to thezone in the sucrose gradients from which thesamples were taken. Under the autoradiogram,the phosphointermediate level (pmolplmg), the(Ca2*,Mg2*)-ATPase activity (nmollmglmin),and the ratio of(Ca2*,Mg2*)-ATPase to phos-phointermediate level (10'lmin) are shown foreach fraction. The underlined ratios were usedto calculate the (Ca2*,Mg2*)-ATPase activitycatalyzed by the 100-kDa enzyme in the otherfractions, as explained in the text.




pulmonary artery

ER PMcardiac

SR

TiV*

FIGURE 6. Autoradiogram of a nitrocellulose blot of theendoplasmic reticulum (ER) and plasma membrane (PM)fraction of bovine pulmonary artery and of bovine cardiacsarcoplasmic reticulum (SR). Blots were incubated with poly-clonal antibodies against the porcine cardiac SR Co**-transportATPase. Fixed antibodies were labeled with Protein A['al].

Further characterization of the Ca2*-transportATPases. Rabbit polyclonal antibodies elicited againstthe 100-kDa Ca2+-transport ATPase of porcine cardiacSR bind in a highly specific way to a polypeptide of thesame M, that is mainly associated with the ER-enrichedfractions (Figure 6).

Binding of calmodulin[123I] can be studied on nitro-cellulose blots of membrane fractions separated bySDS-PAGE.' Figure 7 shows the binding of calmodulin

[125I] to the plasmalemmal (Ca2+,Mg2+)-ATPase. Aspreviously observed on stomach smooth muscle, mostbinding occurs with 3 polypeptides presenting a M, of128-124 kDa. At least one of them corresponds to theCa2+-transporting enzyme.6 An 86-kDa calmodulin-binding polypeptide is also associated with the PMfraction, while a 62-kDa polypeptide can be detectedin the ER fraction. No binding occurs at the 100-kDa(Ca2+,Mg2+)-ATPase.

The visualization of the phosphorylated intermedi-ates by gel electrophoresis and autoradiography (Figure5) reveals only one phosphoprotein of 100 kDa in themembrane fractions of the bovine pulmonary artery.This result clearly differs from our previous observa-tions on membranes prepared from porcine stomachfollowing the same method, in which a 130-kDaphosphoprotein in addition to the one of 100 kDa couldbe easily visualized. The isolated membrane fractionsfrom both tissues are compared in parallel experimentsin Figure 8. To differentiate further the two ATPasesin addition to their molecular mass and to facilitate thedetection of the 130-kDa phosphointermediate, phos-phorylation was also carried out in the presence ofLa3+. La3+ suppresses the phosphoprotein formation ofthe 100-kDa ATPase but enhances that of the 130-kDaATPase.' Figure 8 clearly shows that in the ER and PMfractions of porcine stomach, the ratio of the 130-kDato the 100-kDa phosphointermediate is much higherthan in the corresponding fractions from bovine pul-monary artery. Consequently, these data suggest that inbovine pulmonary artery and aorta, the contribution ofthe ER-type Ca2+-transport ATPase to the total(Ca2+,Mg2+)-ATPase activity is larger than it is inporcine gastric smooth muscle. Even in the presence ofLa3+, the PM-enriched fraction from bovine pulmonaryartery presents more 100 kDa phosphorylation than 130kDa, whereas in porcine stomach, relatively muchmore phosphorylation is seen at 130 kDa. Figure 8 also

ER PM% sucrose 19 22 25 27 30 32 35 Mr

"1000*-200

•92.5

•69

-4(5

FIGURE 7. Autoradiogram showing calmodulin[mI]binding of bovine pulmonary artery membrane fractionsseparated by SDS-PAGE (10%) and electroblotted on anitrocellulose sheet. Most of the calmodulin['"l] is boundto a group of polypeptides (Mr: 128,000-122,000) thatis preferentially associated with the high-density frac-tions, which are most enriched in plasma-membranemarkers.



Eggermont et al Ca2+ Transport in Vascular Smooth Muscle 275

La,3*

BMPAER

B.MPAPM

— 4-

PSTER

— +

PSTPM

— +

PMPAPM_ +

B.AOPM_ +

MrxlOOO

# • • • • • • •130

FIGURE 8. Autoradiogram of dried polyacry-lamide gradient gels (5-15%) showing thephosphointermediates of the 100-kDa and 130-kDa Ca2*-transport ATPases in membranefractions of different types of smooth muscle:Endoplasmic reticulum (ER) vesicles andplasma membrane (PM) fractions of bovinemain pulmonary artery (B.MPA), porcinestomach smooth muscle (P.ST), porcine mainpulmonary artery (P.MPA), and bovine aorta(B.AO) were phosphorylated in the presence of6yMATPand5yMCa2*, withoutLa3* orwith50 yM La1* as indicated, and separated bySDS-PAGE in an acid medium. In porcinestomach, the 130-kDa phosphoprotein is visi-ble in the absence of La3 *. In bovine pulmonaryartery, a faint La1*-induced 130-kDa band canbe seen, whereas in bovine aorta no 130-kDaphosphoprotein could be visualized.

shows that the bovine aorta presents a pattern of theother extreme. Its PM fraction, although enriched to asimilar degree in (Na+,K+)-ATPase activity (data notshown), presents only the 100-kDa ATPase and the130-kDa ATPase cannot be detected, even if La3+ isincluded. To find out whether the difference observedbetween the bovine vascular tissues and the porcinestomach is due to species or tissue differences, mem-brane fractions isolated by the same procedure fromporcine pulmonary artery were included. They pre-sented an intermediate distribution pattern of 100- and130-kDa phosphoprotein intermediates (Figure 8),suggesting that tissue was well as species differencesare contributing. These different phosphorylation pat-terns cannot be explained by the presence of factorsinfluencing the phosphorylation such as a difference inthe basal Mg2+-ATPase activity between bovine andporcine PM fractions. This possibility could be ruledout because mixing of plasma membranes of porcinegastric smooth muscle with PM vesicles of bovinepulmonary artery did not affect the phosphointerme-diate formation (data not shown).

DiscussionWe have previously shown that a combination of

potassium chloride extraction, addition of digitonin,and application of the sample underneath a sucrosedensity gradient is an efficient procedure to obtain fromporcine gastric smooth muscle a membrane fractionenriched in ER and to eliminate contaminating cytos-keletal and contractile proteins.6 We have now appliedthis method to a vascular smooth muscle to characterizeits Ca2+

:transporting enzymes. This work confirms ourprevious results on gastric smooth muscle but alsoreveals important differences between various types ofsmooth muscles.

The material recovered between 16 and 24% sucroseis considered as a purified ER fraction. However,because of the diversity of the intracellular organellesthe term "ER fraction" may be a misnomer and therelative contribution of rough and smooth ER, Golgi

and coated vesicles or lysosomes to this fractionremains to be determined. This low density fractiondefinitely contains membranes derived from the roughER, as indicated by the presence of ribophorin I. Thisis not unexpected since treatment with high potassiumchloride is known to reduce the number of boundribosomes.31 The selective enrichment in the ER-type100-kDa Ca2+-transport ATPase and in phospholam-ban indicates that this fraction also contains mem-branes involved in the Ca2+ storage. The very lowequilibrium density of these membrane vesicles may bean intrinsic feature or it may be due to the loss of someassociated proteins by the treatment with high potas-sium chloride. However, because of its good separationfrom plasma membrane vesicles, this fraction is atpresent the most suitable for the study of the 100-kDatransport ATPase.

The PM-enriched fraction at 28-40% sucrose stillcontains an appreciable amount of putative ER markersand of the 100-kDa Ca2+-transport ATPase of the ERtype. This contamination may be explained by thepresence of cholesterol-containing intracellular mem-branes that are also shifted by digitonin or by thepresence of ER vesicles that are somehow connectedto PM vesicles. This association may be an artifactinduced by the homogenization or it may have amorphological correlate because junctional ER vesi-cles have been observed to be linked to the plasmamembrane by means of "bridging structures."32

An interesting difference between the PM vesiclesfrom bovine pulmonary artery and porcine gastricsmooth muscle is that the (Na+ ,K+)-ATPase activity ishigher and that the (Ca2+,Mg2+)-ATPase activity islower in the pulmonary artery (present results) than ingastric smooth muscle.6 Because the activity of the(Na+ ,K+)-ATPase sets an upper limit to the Ca2+ effluxthat can be mediated by a Na+-Ca2+ exchanger, thebovine pulmonary artery may, in contrast to the pigantrum, present the proper conditions for Na+-Ca2+

exchange playing an important role in Ca2+ extrusion.However, it remains to be determined whether the




Na+-Ca2+ exchanger plays a more important rolein the regulation of the intracellular Ca2+ concentrationin bovine pulmonary artery than it does in porcineantrum.

Our results demonstrate the presence of two differenttypes of Ca2+-transport ATPase in bovine main pul-monary artery, which is in agreement with our previousobservations in porcine gastric smooth muscle.1 A130-kDa, calmodulin[l23I]-binding protein that is as-sociated with the PM-enriched fraction representsthe plasmalemmal type of Ca2+-transport ATPase,whereas the 1 OO-kDa protein that reacts with polyclonalantibodies against the (Ca2+,Mg2+)-ATPase of porcinecardiac SR, that does not bind calmodulin['aI] and thatoccurs mainly in the ER fraction corresponds to the ERCa2+ pump. Phospholamban is also present in the ERfraction and may have a similar function as in cardiacSR30 being a cAMP-dependent and or a Ca2+ + cal-modulin-dependent regulator of the ER Ca2+ pump.

Whereas the 1 OO-kDa phosphointermediate is easilyvisualized in the bovine pulmonary artery and aorta,the 130-kDa phosphointermediate can only be ob-served in bovine pulmonary artery if La3+ is includedin the phosphorylation medium and it cannot bevisualized in the bovine aorta even if La3+ is included.The distribution and the relative enrichment of thephosphointermediate levels in the isolated fractions donot coincide with these of the (Ca2+,Mg2+)-ATPaseactivity. This inconsistency can be explained by as-suming that the phosphointermediate level of the130-kDa Ca2+-transport ATPase of the plasmalemmais, for the same (Ca2+,Mg2+)-ATPase activity, muchlower than that of the 1 OO-kDa ATPase. The obser-vation that the ratio of ATPase activity to phospho-protein level is much higher for the 130-kDa ATPasethan for the 1 OO-kDa ATPase is not surprising in viewof the known kinetic behavior of the 1 OO-kDa ATPaseof skeletal muscle SR33 and of the 130-kDa enzyme oferythrocytes.34 The phosphointermediate of the 100-kDa enzyme can reach a level close to the maximalnumber of catalytic sites33 whereas the phosphointer-mediate of the 130-kDa enzyme amounts at most to10% of the enzyme.34 For this reason, the 130-kDaphosphointermediate is not a suitable PM marker, e.g.,for assessing the contamination of ER vesicles by PMvesicles, although it has been used by several authorsfor that purpose. Because in bovine aortic smoothmuscle no 130-kDa phosphoprotein was observed,Sumida et al proposed that the mechanism of regulationof cytoplasmic Ca2+ in aortic smooth muscle might beanalogous to that in skeletal and cardiac muscle.35 Also,Chiesi et al36 did not observe a phosphoprotein of 130kDa in bovine aortic microsomes and therefore con-cluded that these microsomal preparations were en-riched in ER vesicles. However, in view of our findings,these results have to be reinterpreted.

We have made a semiquantitative measurement ofthe (Ca2+,Mg2+)-ATPase activity of the 130-kDa en-zyme by calculating the activity of the 100-kDaATPase that contaminates the PM fraction, and bysubtracting this activity from the actually measured

Ca2+-stimulated ATPase activity. According to thiscalculation the 100-kDa ER-type ATPase would rep-resent 43% of the total (Ca2+,Mg2+)-ATPase activity,and the remaining 57% would be catalyzed by the130-kDa PM-type (Ca2+,Mg2+)-ATPase.

It should be pointed out that the calculated ratio ofthe 100-kDa to the 130-kDa Ca2+-transport ATPasemay not represent the Ca2+-transporting capacity of theER and PM membranes in the living tissue. Forinstance, the 100-kDa Ca2+-transport ATPase of the SRof skeletal muscle has a stoichiometric ratio (Ca2+

transported/ATP hydrolyzed) of 2 ," whereas this ratioequals 1 for the 130-kDa enzyme of smooth muscle.37

If these values are applied to the smooth muscleenzymes, the contribution of the 100-kDa enzymewould increase from 43% to 57%. In addition, therelative importance of the 100-kDa to the 130-kDaenzyme will depend on many factors, such as theirstimulation or inhibition by second messengers and onthe kinetics of activation of the 130-kDa ATPase bycalmodulin.38 The Ca2+-transport capacity of the ERmay also be influenced by the presence of intraluminalCa2+-binding proteins, such as calsequestrin.39 Inaddition, definite conclusions about the relative im-portance of both Ca2+-transport enzymes cannot bedrawn without a precise knowledge of their recoveryin the isolated fractions.

Differences in the ratio of the ER-type ATPase to thePM-type ATPase between different smooth musclescan be demonstrated by comparing the relative amountof the two phosphorylated transport intermediates. Inboth ER and PM membrane fractions of bovinepulmonary artery, the ratio of the 100- to the 130-kDaphosphointermediate is much higher than in the cor-responding porcine gastric smooth muscle membranefractions. The relative contribution of the 100-kDaCa2+ transport ATPase to the cellular Ca2+-transportcapacity may, therefore, be appreciably larger in bovinepulmonary artery than in gastric smooth muscle. Thisdifference is partly species-dependent and partly tissue-dependent because porcine pulmonary artery presentsan intermediate pattern of phosphorylated ATPases. Inbovine aorta, no 130-kDa phosphoprotein intermediatecould be detected, although its presence has beendemonstrated previously by calmodulin affinitychromatography.40 In this tissue, calcium transport inthe ER may be even more important than in thepulmonary artery. The way in which the intracellularCa2+ homeostasis is realized may, therefore, differaccording to the type of smooth muscle, and thecontribution of the ER may be relatively more impor-tant in vascular smooth muscle than in visceral smoothmuscle. This hypothesis is in agreement with morpho-logical data showing a well-developed ER system (upto 7.5% of the nucleus- and mitochondria-free cellvolume) in large elastic arteries such as the rabbit mainpulmonary artery, whereas in visceral smooth muscle,only 2% of the cell volume is occupied by the ER.41

However, even between smooth muscle presenting asimilar ER content, such as the guinea pig portal vein(2% of cell volume42) and the guinea pig taenia coli



Eggermont et al Ca*+ Transport in Vascular Smooth Muscle 277

(1.5% of cell volume43), differences in Ca2+-handlingproperties may exist.42

AcknowledgmentsWe thank Dr. K. Gietzen (Department of Pharma-

cology and Toxicology, University of Ulm) for the giftof antiserum to the Ca2+ pump of cardiac SR and Dr.M. Hortsch (EMBL, Heidelberg) for the gift ofmonoclonal antibodies to ribophorin I.

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KEY WORDS • vascular smooth muscle • Ca!+-transportATPase • endoplasmic reticulum • plasma membrane



Ca2+-transport ATPases of vascular smooth muscle

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