Expression of Ca2+ binding proteins of the sarcoplasmic reticulum of striated muscle in the endoplasmic reticulum of pig smooth muscles

Cd caldmn (lcle3) 14, 681-689 0 LcngnmnGroupUK Lid 1883

Expression of Ca*+ binding proteins of the sarcoplasmic reticulum of striated muscle in the endoplasmic reticulum of pig smooth muscles L. RAEYMAEKERS, J. VERBIST, F. WUYTACK, L. PLESSERS and R. CASTEELS

Physiological Laboratory, KU Leuven, Leuven, Belgium Abstract - The Ca*’ binding proteins in the lumen of intracellular Ca*+ stores differ betwean muscle and non-muscle cells, indicating a specific role of these proteins in intracellular Ca*+ regulation. Since smooth muscle cells possess both muscle and non-muscle characteristics,we have studied the presence and the differantial expreaaion of the muscle-type Ca*+ binding proteins - calsequestrin,sarcalumenin,and the hiatldtnarich Ca*’ binding protein (HCP) - in several smooth muscle tissues from the pig. Western blot analysis showed that among the smooth muscles studied, the cardiac isoform of calsequestrin Is expressed at the highest levels in the stomach. Calsequestrinwas present at lower levels in ileum and trachea, whereas this protein was undatectable in aorta and main pulmonary artery. The total amount of calsequestrtnin the stomach was estimaM to be Zo-3g-times lower than in the pig heart. Whereas cakequesMn from pig presented the same apparent MI in sodium dodecyl sulphate polyauylamide gels as the well ckuactertred protein from rabbit, the apparent Mr of both sarcalumeninand HCP was lower in pig than in rabbit. The presence of HCP was demonstrated in pig stomach and ileum, while sarcalumenln was detacted only in the stomach. These results demonstrate further biochemical differences between smooth muscle cells of large blood vessals and those of the digestive tract. The present findings on the differentialdistributionof musdetypa Ca*+ binding proteins are discussed in relation to biochemical and functional differences between these smooth muscle cells.

freecytosolic Ca2+ concentrationis the primaq determinantfor contractileactivationin both striated and smoothmuscle and is also known to play a key role in the regulationof a varietyof cellularfunctions in non-muscle cells. Depending on the cell type, the kind of signal and the physiologicalactiv-

The

ity, the activatorCa2+enters from tbe extracellular spaceat&r is n4eased fromintracehlar orgauellar stoles. The seqw%ation of Ca2+ill the intracellular store is thought to be facilitatedby luminal Ca2+ bindingproteiusthat lower the concentrationof free Ca2’ in tie stores. Recentwork ou the intraluminal

cELLcALcIuM

932

Ca2+binding proteins of the endoplasmic reticulum (ER) of non-muscle cells has shown that these proteins differ from the corresponding proteins of the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle (see Fliegel et al. 111and Mihrer et al. [2] for reviews). The Ca2+ binding proteins identified in the SR of striated muscle fibres am calsequestrin, sarcalumenin, the histidine-rich Ca2’ binding protein (HCP) and the high affinity Ca2’ binding protein, mom recently named calreticulin (see [2] for review). Calsequestrin exists as cardiac or fast-twitch skeletal muscle isoforms, which are the products of two different genes [3]. Calreticulin is a minor component in striated muscle cells. It is, however, also present in non-muscle cells and in smooth muscle cells where it represents one of the major luminal Ca2’ binding proteins of the ER [2,4]. In mammalian tissues, the expression of calsequestrin, sarcalumenin and HCP has been detected almost exclusively in striated muscle. The only exception is the demonstration of the cardiac isofomr of calsequestrin in the ER of smooth muscle cells of the pig stomach [S]. The occurrence of particular Ca binding proteins in striated muscle suggests that they may play an important and specific role in the regulation of the function of these contractile cells. Therefore, we have investigated whether besides calsequestin, also sarcalumenin and HCP are expressed in smooth muscle. In addition, in view of the marked functional heterogeneity of different smooth muscle tissues, we have explored the heterogeneity in expression of striated muscle type Ca2+ binding proteins in several vascular and non-vascular smooth muscle tissues. We show that calsequestrin levels, as measured by means of Western blot analysis, differ markedly between various types of smooth muscle. The highest levels were found in the smooth muscle of the stomach and ileum, whereas calsequestrin could not be detect& in the vascular smooth muscles of pulmonary artery and aorta. We furthermore show that HCP and sarcalumenin am also expressed in smooth muscles of the digestive tract These results su est that the muscle-type proteins involved in Ca% uptake and Ca2+ storage am mainly expressed in smooth muscles which express desmin and not vimentin as the major type of intermediate filament protein.

Mater-la&and methods Preparationof membrane vesicles

Endoplasmic reticulum was prepared from the stomach (an&al part), longitudinal ileum, trachea, pulmonary artery and aorta, all from pig, as described by Raeymaekers et al. [a]. By this method a separation of ER from plasma membranes is obtained by using digitonin to increase the density of plasma membrane and by centrifugation in a gradient solution of high ionic strength. Vesicles of ER equilibrating between 18-2546 (w/w) sucrose were pooled. SR from pig cardiac ventricles, pig intercostal skeletal muscle, or from the white back muscles of the rabbit were isolated by differential centrifugation. The SR obtained between 10 000 g for 15 min, and 100 000 g for 60 min was washed once in 0.6 M KCl and resuspended in 0.25 M sucrose, 10 mM Tris pH 7.5. Protein concentrations were determined by a bicinchoninic acid (BCA) protein assay reagent kit (Pierce, Rockford, IL, USA). Proteinpurifscation

Pig skeletal muscle HCP was purified as described by Hofmann et al. 171. Rabbit skeletal muscle 160 kD glycoprotein (sarcalumenin) and the 53 kD glycoprotein were copurified on Concanavalin-A Sepharose as described by Campbell and MacLennan 181. Pig cardiac calsequestIin was purified according to the method of Slupsky et al. [9], including the optional step of chromatography of the ion exchange fractions on phenyl-Sepharose. Preparationand characterizationof antibodies

Polyclonsl antibodies against pig cardiac calsequestrin and pig skeletal HCP were prepared by immunizing rabbits with 4 injections of 100 pg of puritled protein at 4 week intervals. Animals were bled 7 days after the final booster. For the production of monoclonal antibodies against the 53 kD and 160 kD glyceproteins, female balb/c mice (10 weeks old) were immunized by intraperitoneal injection of f 100 M of the copurifed antigens. 2 booster injections of 50 pg were given 7 and 21 days later.

Ca’+-BINDING PROTEINS IN HR OF PIG SMOOTH MUSCLES

583

Spleen cells were fused with Sp 210 Ag 14 mouse myeloma cells by using PEG 1500 as described by Hybridoma colonies were Gm et al. [lo]. screened for antibody production using Western blots of the 53 kD and 160 kD glycoproteins. For detection of cross-reactive proteins, unfractionated culture supematant was used. Western blots wefe prepared from sodium dodecyl sulphate (SDS) polyacrylamide gels (7.5% (w/v) acrylamide) using ImmobilonP membranes (Millipore Corp., Bedford, MA, USA) and immune-stained using horseradish peroxidase coupled secondary antibodies as described previously [l 11. Monoclonal antibodies specific for desmin or vimentin were obtained from Sigma. Rabbit antibodies directed against rat caheticulin were kindly provided by Dr Soling (Gottingen, Gemrany).

lysed according to the Pica-tag method (Waters Chromatography Divisions, Milford, CT, USA).

Results

Expression of calsequestrinin smoothmuscle

Calsequestrin was purified from porcine cardiac muscle (Fig. 1) and used as immunogen in rabbits. Figure 2 shows that the antibodies recognized pig cardiac calsequestrin (Mr 55 000) but reacted only very weakly with calsequestrin in pig skeletal SR (Mr 63 000). Figure 2 also shows that in ER enriched fractions of smooth muscle the antibodies

cs

CompetitiveELISA

The wells of microtiter plates (Nunc, Denmark) were coated with 100 p.l purified cardiac calsequestrin dissolved in coating buffer (15 mM NaaCO3, 34.5 mM NaHC03, pH 9.8) at a concentration of 1 pg/ml by overnight incubation at 4°C. Non-specific protein binding sites were blocked by incubating with 3% milk powder in coating buffer. Washing buffer was PBS pH 7.4, containing 0.05% v/v Tween 20 throughout the assay. Rabbit anti-calsequestrin antibodies at 2000-fold dilution were preincubated with the extracts from heart or stomach or with purified calsequesnin for 2 h at 37°C. and incubated in the coated wells for 2 h at 37°C. Secondary antibodies were swine anti-rabbit conjugated with horseradish peroxidase (Dakopatts, Denmark). Incubation was carried out at 37°C for 1 h. Staining was performed for 30 min at room temperature in phosphate buffer, pH 6.0 containing 0.4 mg/ml ophenylenediamine and 0.03% H202. The reaction was stopped by 2.5 N HCl and the absorbance was read at 490 MI. (Soft Max mIcroplate reader, Molecular Devices Corporation, CA, USA.) Amino acid composition Purified HCP from rabbit or pig was hydrolsed in

HCl-phenol (gas phase) at 110°C for 24 h. Phenylisothiocyanate derivatized amino acids were ana-

HCP

Mr

-

Fig. 1 Polyacrylamide gel electrophoresis and protein s&i&gof pmilted antigena used for the production of mhsemm in mbbits. HCP: histidinc-rich Cal’ binding proteinpurifii fromporciue skeletal muscle. Cs: calsequatrin purifiutfmm porc& cardiac muscle.

584

CELL CALCIUM

SK

CA

ST

IL A0

PA

TR

M,x103

in ileum and in the trachea (lanes ST, IL and TR, respectively. Calsequestxinis not detectable in the purified ER of the pulmonary artery or aorta. In contrast, all the ER fractions examined showed a very similar content of calreticulin, which migrates with a higher apparent molecular weight than cardiac calsequestrin (Fig. 3). To exclude the possibility that the apparent absence of calsequestrin in the RR of some smooth muscles could be due to the loss of this protein from the vesicles during purification, we also tested for the presence of calsequestrin using a method for purification of calsequestrin from cardiac muscle described by Slupsky et al. [9]. This method does not SK

Fig. 2 Immunological identification of calsequestrin in ER-enriched membrane fractions from smooth muscle tissues. SR and ER membrane proteins were separated by SDS-PAGE on 7.5% polyacrylamide gels, transferred to Immobilon-P membranes, and incubated with aoti-calsequestrin antiserum at 1:lOO dilution. 40 p.g of protein was loaded ou each lane: skeletal muscle SR (lane SK), cardiac muscle SR (lane CA), smooth muscle EX from stomath (ST), ileum (IL), aorta (AO), pulmonary artery (PA), and trachea (TR). The anuw indicates the position of the cardiac form of calsequestrin (CS);

bind to a protein with the same apparent molecular weight as cardiac calsequestrin. It has been shown previously for the RR of pig stomach that a protein of the same mobility presented the biochemical characteristics of cardiac calsequestrin [5]. The present results show that the amount of calsequestrin in the purified tractions of FB varies between the different smooth muscle tissues. Among the smooth muscles stndied, calsequesti is most abundant in the ElRfrom stomach. Smaller amounts am present

CA ST

IL A0

PA

TR

CRT

Fig. 3 Immunological identification of calmticulin in ER enriched membmoe fractions of smooth muscle+ SR and ER membrane proteins were separated by SDS-PAGE on 7.5% polyaayhunide gels, transferred to Immobilon-P filtexs, and incubated with anticalmtictin antiserum at MOO dilution. 40 pg of protein was loaded on each lane: pig skeletal muscle SR (lam SK), cardiac muscle SR (lane CA), smooth muscle ER from stomach (ST), ileum (IL). putnonary srtay (PA), aorta (AO), and =hea WI). The armw indicates cah-eticulin &I, 63 kD).

Ca”-BINDING PROTEINS IN ER OF PIG SMOOTH MUSCLES

585

the isolation of the ER vesicles. The muscle is homogenized in ammonium sulfate (65% saturation) and the 65-8596 ammonium sulfate kaction is further fractionated on an ion exchange column. We also found that the fractions of the ion exchange column of a pulmonary artery extract were negative for calsequestrin by Western blotting (results not shown), whereas calsequestrin could be readily detected in a parallel extract of pig stomach smooth muscle (Fig. 2). We also investigated whether the expression of calsequestrin in large blood vessels is species dependent. Calsequestrin of cardiac samoplasmic reticulum of cow and dog cross-reacted with the antiserum directed against the porcine protein. The vascular tissues of both dog and cow were negative for calsequestrin when tested on isolated ER or on fractions of the ion exchange column described above (data not shown).

Comparison of the calsequestrin content of cardiac and smoothmuscle

require

Calsequestrin containing ammonium sulfate fractions were prepared from pig heart and stomach and fractionated by ion exchange chromatography as described by Slupsky et al. [9]. For both tissues the purification was started from the same weight of tissue. Calsequestrin containing fractions eluting between 0.5-0.75 M NaCl were pooled and compared for calsequestrin content by competitive ELISA. The antiserum was pm-incubated with these extracts and the concentration required for half-maximal inhibition of the binding to purilied calsequestrin was determined. For the stomach extract, a 20-30-times high= concentration of the extract was required than for the cardiac extract (Fig. 4A). The specificity of the antiserum in this test is shown on Western blots of the extracts (Fig. 4B).

protein (rglml) FYg. 4 Comparison of the calsequestrin content of cardiac muscle and the smooth muscle of the stomach by competitive ELBA. Fractions obtained from an ion exchange c&mm WQCobtained f?om pig cardiacmuscleor film pig stcmach smooth muscle as described in the Results section. (A) Difkent amountsof &se extmcts given as pg proteiplm in the abscissa were incubated with the sntkrum at 1:2000 dilution and subsequmtly applied to calsap~=trin coated miczotite~plates. (Filled cklcs) pure cardiac calaquestrin; (fikd triangles) extract from pigvmtricular mu8ck;(filledsqusrecl) extmctfnm pigstomach (anti) smooth muscle. Half-maximal inhibition of tbc reaction by the extmct from smooth muscle was observed at much higba protein caacentratiau than inhibitia~ by tbecardiecexhact. AAercomctionforthediffermttorslproteincontcntoftbcssmples,avrkreofabout~isobtainedfortheRtioof calsequestrincontent in cardiac mu& over that in stomach smooth muscle. (B) The sam extmcts as used in (A) wem applied to 7% SDS polyacrylamide gela and subsequa~tly analysed by Weatem bktiag using anti-calsequeskin antisenan. The extract fnrm stomach smooth muscle was used undiluted whexeas the cardiacextmct was diited 204mea

586

Expression of HCP in smooth muscle

CELL CALCIUM

porcine skeletal muscle to investigate the presence of this HCP-like protein in ER enriched fractions of smooth muscle. Western blot analysis (Fig. 4) shows that a cross-reacting protein of the same apparent Mr 115 000 was present in isolated ER from smooth muscle. Figure 5, furthermore, shows that this immunoreactive protein could only be detected in ER from antrum and longitudinal ileum and not in ER from tracheal or vascular smooth muscle. As observed for calsequestrin, the HCP content of smooth muscle ER was lower than that of SR of cardiac or skeletal muscle.

In order to detect a possible expression of HCP in pig smooth muscle tissues, we raised antibodies against HCP purified from the SR of pig skeletal muscle according to the purification method described for rabbit skeletal HCP by Hofinann et al. [7] (Fig. 1). The application of this purification protocol to porcine skeletal muscle yielded a protein of apparent Mr 115 Ooo in SDS polyacrylamide gels, instead of Mr 165 000 as seen for rabbit HCP (Fig. 5). The fact that both proteins were purified by an identical purification procedure is an important argument in itself for the homology of these pro- Expression of sarcalumenin and the 53 kD teins. This procedure includes extraction of the sar- glycoprotein in smooth muscle coplasmic reticulum in detergent at 100°C. The protein from pig was retained in such an extract and on Sarcalumenin of SR of rabbit skeletal muscle is a subsequent ion exchange chromatography it eluted Ca2’ binding glycoprotein of apparent Mr 160 000 at an ionic strength very similar to that for rabbit _ SK, SK CA ST IL A0 PA TR HCP (430 mM and 410 mM NaCl, respectively). The most unusual characteristic of rabbit HCP is its very high content of histidine (12.7%) as determined from cDNA sequencing 1121. Therefore, we also compared the amino acid composition of the purified porcine and rabbit protein. The experimentally determined percentage of histidine was 11.7% for the porcine protein and 15.2% for the rabbit protein. Since the histidine residues mainly occur in a repeated sequence [12], the slightly lower histidine content could be due to a smaller number of these repeats in the porcine protein. Like the rabbit pro45tein, HCP from pig also demonstrated a high content of aspartic acid and glutamic acid residues. In line with this observation is the finding that the protein from pig stains metachromatically blue with the cationic dye Stains-All (data not shown), as seen for the rabbit protein [7]. It should also be mentioned that the actual difference in molecular weight beCn2+ tween rabbit and pig HCP would probably be smal- Fig. 5 hnmuoologicd identificationof the histibridt binding protein in ERuuiched membnme fractions from smooth ler than that suggested by the difference in migration in SDS gels, because the predicted molecular muck tissuea. SR and ER mcmbmne pmteins wem wpacatedby weight of rabbit HCP Ovr,96 116) is much lower SDS-PAOEon 7.5% polyacrykmide gek, tmnsfared to ImmobiIon-P membmmx, and incubated with anti-HCP antisaum at than the value obtained from SDS gels 17, 121. The 1:2OOdihtion. 4Ojtgofpmtcinwaskadedoncachknc&ektal discrepancy between the predicted and the observed muscle SR (lane SK),cardiac muack SR (lane CA), omootb values may be caused by the protein conformation, muscle EjR from etom8d1 (ST), ileum (IL), pulmomuy atay post-translational modifiition and/or the presence (PA), aorta (AO), and tmchea tutmothmuscle (TR). The urows of acidic residues preventing the binding of SDS. indicate the histidine-rkl~Ca% binding proteia in mbbit (M, 165 We have used the antibodies against HCP from kD) and pig (& 130 kD).

Ca’+-BINDING PROTEINS IN ER OF PIG SMOOTH MUSCLES

SK,

SK

CA

ST

IL A0

PA

TR M,x103

q_ ---* SL -

- 116 - 96.4

- 66

c

- L5

Fig. 6 Immunological identification of sarcalumenin in ER-enriched membrane fractions from smooth mucle tissues. SR and ER membrane proteins were separated by SDS-PAGE on 7.5% polyacrylamide gels, transferred to Immobiion-P filters, and incubated with monoclonal anti-sarcalumenin culture supematant. 40 lg of protein was loaded on each lane: pig skeletal muscle SR (lane SK), cardiac muscle SR (lane CA), smooth muscle ER from stomach (ST), ileum (IL), pulmonary artery (PA), aorta (AO), and trachea (TR). The upper and lower armws indicate sarcahanenin in, respectively, rabbit (M, 160 kD) and pig (I& 130 kD) skeletal SR.

587

porcine skeletal and cardiac muscle. Porcine samalumenin presents, however, a Mr of 130 000, unlike the Mr 160 000 sarcalumenin from rabbit. The homology of the 130 kD protein from pig with sarcalumenin from rabbit was further demonstrated by Stains-All staining on SDS polyacrylamide gels. The 160 kD sarcalumenin of rabbit SR, which stained blue with Stains-All, was not present in porcine SR, and the latter preparation possessed a 130 kD blue-staining band which was absent in rabbit SR (data not shown). In addition, the 130 kD porcine protein copurified with the 53 kD glycoprotein on a Concanavalin-A agarose column (data not shown), as is the case for the 160 kD protein from the rabbit 181. In the pig stomach, antibody HlOD7 recognized a small amount of the 53 kD glycoprotein. On the original blots, a weak reaction at the 53 kD position was also seen in ileum, whereas the other tissues were negative (Fig. 6). Antibody GlOD7 mcognixed a protein of similar mobility as skeletal sarcalumenin in the ER fraction from stomach only (Fig. 6). The reaction was very weak, only a faint band being visible after photographic reproduction. No reaction was seen in the other smooth muscles. Sarcalumenin and the 53 kD protein are therefore other constituents of the ER of pig stomach smooth muscle. However, their relative amount as compared with cardiac muscle appears to be lower than that of calsequestrin and HCP.

Discussion

on SDS gels and of 102 000 kD as determined from amino acid and carbohydrate content [13]. It consists of two major domains. The -NH2 terminal domain is highly acidic and binds Ca2’. The C terminal domain is identical to the 53 kD glycoprotein, a quantitatively mote important component of the SR. Both glycoproteins copurify on a Concanavalin-A column [81. Our monoclonal antibody GlOD7 raised against the rabbit proteins recognized both the 53 kD and 160 kD glycoproteins of rabbit skeletal muscle, indicating that this antibody reacts with the C terminal domain (Fig. 6). The antibody also retognizes both proteins on Western blots of SR of

In this study we have demonstrated that the expmssion of the Ca2’ binding proteins HCP, samalumenin and cardiac calsequestrin is not contined to striated muscle but that these proteins are also present in the ER of several smooth muscles. The largest amount of HCP, samalumenin and cardiac calsequestrin was detected in the pig stomach. This tissue also expresses the 53 kD glycoprotein, which is in line with an earlier report [14] mentioning the presence of the 53 kD glycoprotein in smooth muscle cells from rabbit, In ER from pig ileum we could detect calsequesttin, HCP and the 53 kD glycoprotein, whereas sarcal~enin, if present, remained below the detection limit. In the trachea, there was a cross-matting

588

band with the anti-calsequestrin serum only. In the ER from pulmonary artery and aorta, there was no reaction with any of the antibodies used. Another negative result on calsequestrin in smooth muscle has been reported by Milner et al. [4]. These authors did not detect calsequestrin in isolated membranes from porcine uterus, and only trace amounts in ammonium sulfate extracts of the same tissue. Assuming an equally effective extraction of calsequestrin from stomach and from the heart, our results indicated that the amount of calsequestrin present in pig stomach smooth muscle was 2&30fold lower than in pig cardiac (ventricular) muscle. This relatively low amount is not surprising in view of the low content of ER Ca2+ pump in smooth muscle 1111. Furthermore, it should be taken into account that the volume occupied by muscle cells relative to the volume of tissue is smaller in the stomach than in the heart Also, the total volume of ER in gastrointestinal smooth muscle cells [15] is smaller than in the heart [16]. In addition, a considerable fraction of the ER of smooth muscle is made up of rough ER It can be expected that the lumen of rough RR contains non-muscle type Ca2’ binding proteins which may participate in the translocation of nascent proteins, protein folding and sorting of secretory proteins [17, 181. It has indeed been shown that a major Ca2’ binding protein of the ER of smooth muscle is caheticulin, a typical nonmuscle protein [4J, and this protein was present in about equal amounts in the ER preparations investigated in the present study. These ER proteins typically possess the C-terminal KDEL sequence for retention in the ER [l, 191. The fact that calsequestrin, samahunenin, the 53 kD glycoprotein and HCP do not possess this sequence [l, 21 suggests the existence of a separate non-KDEL compartment in smooth muscle, similar to the SR of skeletal muscle although less extensively developed. The Ca2’ stored in the ER of smooth muscle is known to play an important role in excitation-contraction coupling, especially durhi the initial phase of the contraction 1201. The Ca2g in the E(S)R is mainly bound to low-affinity Ca’+ binding proteins. The fact that the SR of striated muscles and the RR of non-muscle cells contain a different set of these proteins points to important functional differences which may be related to the different mechanisms of

CELLCALCIUM

initiation of Ca2+release operating in these tissues. In this respect it is interesting to compare the heterogeneous distribution of the SR-type Ca2+binding proteins in smooth muscle cells to their contractile behaviour. Our results suggest that in large blood vessels, SR-type Ca2’ binding proteins are either not expressed at all or are expressed at a level much lower than in stomach or intestinal smooth muscle. Smooth muscle cells of large blood vessels present slow tonic contractions, whereas smooth muscle cells of the digestive tract present relatively faster phasic contractions. In this context it is important to mention that smooth muscle cells which present phasic contractile activity mainly express desmin as the major intermediate filament, whereas tonic smooth muscles express vimentin [21, 221. Desmin is characteristically found in sarcomeric muscles whereas vimentin is expressed in non-muscle cells. The predominant expression of desmin in the gastrointestinal smooth muscles investigated in this study and of vimentin in the vascular muscles was ascertained by Western blotting (data not shown). During the preparation of this manu8cript,Pathak et al. [23] reported the presence of HCP in smooth muscle cells of small but not of large blood vessels. Since small blood vessels present relatively fast contractions as compared to conductance vessels and express desmin [21], the results of Pathak et al. are also in accordance with a correlation between the type of contractile activity and the expression of sarcomer&-type proteins in smooth muscle. The exact function, if any, of the SR-type Ca2’ binding proteins in smooth muscle cells remains to be established. One possibility is that the Ca2’ binding properties of these proteins (capacity, affinity, kinetics) are better suited to the functional requirements than those of non-muscle Ca2’ binding proteins. In addition, a specific function has been postulated for calsequestrin besides the binding of Ca2’. Ikemoto et al. I241 have proposed that the binding of Ca2’ to calsequestrin would modulate directly or indirectly the ryanodine sensitive Ca2+ channel and thereby also the Ca2’ release from the SR. In striated muscle, calsequestrin is indeed localized in the same subcompartment of the SR where the ryanodine sensitive Ca2’ release channel is located. It would, therefore, be of interest to determine whether there is also a correlation in ex-

Ca2+-BINDING PROTEINS IN ER OF PIG SMOOTH MUSCLES

589

pression and localization between SR-type Ca2’ release channels and Ca2’ binding proteins in smooth muscle.

Biochem. J., 271,649-653. 12. Hofmenn SL. Goldstein JL. Or& K. Ahmaw CR. Slaughter CA. Brown MS. (1989) Mokcular cloning of a histidine-rich &+-binding protein ofsarcoplasmic mticulum that contains highly conserved mpeated ekments. J. Biol. C-hem.,264,18083-18090. 13. LebemrE.ChamkJHM.GmenI’JM,Ma&umanDH. (1989) Mokcular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcopbkmic mticulum. Pmt. Natl, Acad. Sci. USA, 86.60476051. 14. Pepper DR Raab CR. Campbell KP. (1985) Ideatificntioar and chawmriaatiott of the 53,000 Da and 160,000 Da glycoproteins and the Ca-ATPase of samoplasmic reticulum in red and white.skeletal, smooth, and cardiac muscks. (Abstract). Biophys. J., 47,344a 15. Gabella 0. (1989) Handbook of Physiobgy, Section 6, The GastrointestinalSystem Vol I Part 1. &hulk Xl. Wood JD. Ratmer BB. (eds). American Physiological Society, pp. 103-139. 16. Page E. McCallister LP. (1973) Quantitativeekctnm microscopic &scription of heart muscle cells. Applkation to normal, hypertmphied and thymxin-stimulated heart Am. J. Cat&l., 31, 172-181. 17. Gething MJ. San&took J. (1992) Pmtein folding in the 1~11. Nature, 355,33-45. 18. Koch G. Smith M. Mater D. Webster P. Mortam R. (1986) Endoplasmic reticulum contains a common, abundant calcium-binding glycopmtein, endoplasmin. J. Cell Sci, 86, 217-232. 19. Munro S. Pelham RB. (1987) A C-termim11signal prevents secmtion of luminal ER proteins. Cell, 48,899-m. 20. Casteels R. Wuytack F. Himpens B. Raeymaekers L. (1986) Regulatory systems for the cytoplasmiccalcium concenttation in smooth muscle. Biomed. Biochim Acta, 45, Sl47S152. 21. Frank ED. Warren L. (1981) Aortic smooth musck cells contain vimentin instead of desmin. Pmt. Natl. Acad. Sci. USA, 78,3020-3024. 22. Gabbiani G. Schmid E. Winter S. et al. (1981) Vascular smooth muscle cells differ from other smooth muack cells: Predominance of vimentinfilammts anda speoifii &type actin. Pmt. Natl. Acad Sci. USA, 78,298-302. 23. PatbakRK. AndemonRCJW. HetinanuSL. (19%) Histidine-rich calcium binding protein, a samoplasmic mticulum protein of striated muscle, is also abundant in arteriolar smooth musck cells. J. Musck Res. Cell Motil., 13.366-376. 24. Ikemoto N. Roajat M. Meszams L. Koshita M. (1989) Postulated role ofcalaequestrin in the regulation of calcium release from samoplasmic mticulum. Biochemistty, 28. 67646771.

Acknowledgements We thank Mr Val& Feytons (Department of Biochemistry, Campus Gasthuisberg, KU Leuven, Belgium) for carrying out the amino acid analysis and Dr Ming (Gbttingen. Germany) for providing calreticulin specific antibodies.

References 1. Pliegel L. Burns K. Wlasiclmk K. Michalak M. (1989) Peripheral membrane proteins of samoplasmic and endoplasmic mticulum. Comparison of carboxyl-tetmkal amino acid sequences. Biocbam.Cell Biol., 67.696-702. 2. Milner RE. Famulski KA. Michalak M. (1992) Calcium binding pro&ins in the sarcoplasmicA?ndoplasmicreticulum of muscle and nonmuscle cells. Mol. Cell. Bicchem., 112, 1-13. 3. Scott BT. Simmerman HKB. Collins JH. Nadal-Ginard B. Jones LR. (1988) Complete amino acid sequence of canine cardiac cakequest& deduced by cDNA cloning. J. Biol. Chem., 263.8958-8964. 4. Milner RE. Baksh S. Shemanko C. et al. (1991) Cabeticnlin, and not calsequestrin, is the major calcium binding protein of smooth muscle samoplasmic reticulum and liver endoplasmic teticulum. J. Biol. Chem., 266,7155-7165. 5. Wuytack F. Raeymaekem L. Verb&J. Jones LR. Casteels R (1987) Smooth-muscle endcplasmic reticnlum contains a cardiac-like form of calsequestrin. B&him. Biophys. Acta. 899, 151-158. 6. Raeymaekers L. Wuytack F. Casteels R (1985) Subccllular fiwionatbn of pig stomach smooth muscle. A study of the distribution of the (Ca*+~~ATPase activity in pkamalemma and endcplasmic mticulum. B&him. Biophys. Acta, 815,441-458. 7. Hofmann SL. Brown MS. Lee E. Patlak RK. Anderson RGW. Goldstein JL. (1989) Purifkation of a sarcoplasmic mticulum protein that binds Ca*+ and plasma Iipopmteim. J. Biol. Chem., 264.8260-8270. 8. Campbell KP. Mw%cmmn DH. (1981) PuriEcation and chamcterization of the 53,OOOdaltonglyccprotein frcns the samoplaamic reticulum. J. Biol. Chem., 256,4626-4632. 9. Slupsky JR, Ohnishi M Carpenter MR. Reithmeier RAF. (1987) Characterization of cardiac calsequestrin. Biochemistry, 26.65396544. IO. Galfnl G. Howe SC. Milstein C. Butcher GW. Howard JC. (1977) Antibodisa to major histocompatibility antigens produced by hybrid cell lines. Nature, 266.550-552. 11. Eggermont JA. Wuytack F. Verbist J. Casteels R (1990) Express&t of endoplasmic reticulum Ca2+-pumpisoforms and of phospholamban in pig smooth-muscle tissues.

Please send reprint requests to : Dr L. w, Physio logical Laboratory, KU Leuven, Campus Gasthuiaberg, Hemstmat 49, B-3000 Leuven, Belgium Received : 24 November 1992 Revised : 4 February 1993 Accepted :8 February 1993