Ce//Cafcium(1990)11. 261-268 0
LOi?Q”W
GrcupUKUd1990
Effects of cyclic nucleotide dependent protein kinases on the encloplasmic reticulum Ca*+ pump of bovine pulmonary artery L. RAEYMAEKERS,
J.A. EGGERMONT,
Latwratorium voor Fysioogie, K. Ueuven,
F. WUYTACK
and Fi. CASTEELS
Leuven, Belgium
Abstract - This pa r describes the stimulation by cyclic nucleotide dependent protein JY uptake by isolated endoplasmic reticulum (ER) vesicles from the kinases on the Ca bovine main pulmonary artery. This ER fraction has previously been shown to be highly enriched in phospholamban, a protein kinase substrate that has been well characterized in cardiac sarcoplasmic reticulum (SR), where its phosphorylation is accompanied by an increased rate of Ca2+ uptake [for review see 351. As previously observed for the phosphorylation of phospholamban [IO], the stimulation of the rate of Ca uptake was as high with cGMP dependent protein kinase as with CAMP dependent protein kinase. The effect of phosphorylation of the ER membranes from smooth muscle on the Ca2+ uptake was smaller than that seen in cardiac SR, and it was only observed if albumin was included during the isolation of the membranes. This relatively small effect is probably not due to a lower ratio of phospholamban to Ca2+-transport enzyme in the ER membranes as compared to cardiac SR. Several alternative explanations are discussed. Considerable progress has been made recently in the characterization of the Ca2t-transport ATPases of smooth muscle (for review see [l, 23). The presence of two types of Ca2t-transport ATPases, one of 100 kDa in the endoplasmic reticulum and one of 130 kDa in the plasma membrane, has been well documented 13-61. The Ca” pump of the endoplasmic reticulum is functionally and structurally related to the Ca2+-pump proteins expressed in the sarcoplasmic reticulum of skeletal and cardiac muscle. It has been shown by biochemical, immunological 171 and cDNA-sequencing techniques [8, 91 that the Ca2+ pump of smooth muscle is more related to that of cardiac muscle (and to that of non-muscle cells, see Discussion) than to that of fast skeletal muscle.
Interestingly, there are two more parallels which discriminate the cardiac- and smooth-muscle Ca2+-transport and storage system from that of fast skeletal muscle. Phospholamban, a phosphorylatable protein that regulates the Ca2+ pump in the cardiac sarcoplasmic reticulum but not in fast skeletal muscle, was detected in smooth muscle [ 10, I I]. Calsequestrin, the main Ca2+-binding protein of the lumen of the sarcoplasmic reticulum, was also found in smooth muscle. The calsequestfin isoform expressed in smooth muscle is similar to that of cardiac muscle, but it differs from that of fast skeletal muscle [ 121. It has been proposed that the Ca2t-transport ATPases of smooth muscle are subject to regulation by protein kinases and that this mechanism may
261
262
contribute to a decrease of the Ca2’ concentration and to relaxation induced by substances that increase the level of cyclic nucleotides, especially cyclic GMP [13-161. In biochemical studies stimulation of the Ca2+ pump of the plasmalemma by cGMP-dependent protein kinase has been observed, as well on isolated plasmalemmal vesicles [17, 181 as on the purified Ca2’ pump [lS, 191. An une uivocal demonstration of a stimulation of the 29 pump of the endoplasmic reticulum has Ca hitherto not been possible, because of the difficulty of purifying this cell organelle from smooth muscle. However, experiments carried out on intact tissues [20, 211 and on skinned cells [15, 22, 231 have provided evidence for the existence of such a of mechanism. In addition, the 2presenee phospholamban suggests that the Ca pump of the endoplasmic reticulum of smooth muscle could be a target for regulation by second messengers [lo]. We have previously demonstrated the phosphorylation of phospholamban of isolated membranes by CAMP-dependent and exogenous endogenous protein kinase [lo] and by exogenous cGMPdependent protein kinase [24]. We have now studied the functional effect of phosphorylation on the Ca2’ uptake by an endoplasmic reticulum fraction purified from the bovine main pulmonary artery. This fraction has been characterized previously. It has been shown to contain little plasma-membrane marker enzymes and to be highly enriched in phospholamban [6]. In this paper we report that the Ca2’ uptake in these endoplasmic reticulum vesicles is stimulated by exogenous CAMP- and by cGMPdependent protein kinases. The possible role of phospholamban in this effect is discussed. Materials and Methods Preparation of membranes A membrane fraction enriched in endoplasmic reticulum was prepared from the bovine main pulmonary artery as described previously [6], except that albumin (1 mg/ml) was included in the sucrose density gradient. Briefly, about 150 g of muscle was homogenized in 6 volumes of 0.25 M sucrose containing 1 mM dithiothreitol and 0.5 mM PMSF and centrifuged at 20 000 x gma for 20 min in a
CELL CALCIUM
Sorvall GSA rotor. The supematant was supplemented with 0.6 M KCl (final concentration), 120 mg digitonin dissolved in 20 ml H20, and sucrose (50% final concentration). The supematant was then pumped into a Kontron TZT32 zonal rotor underneath a sucrose density gradient extending between 15 and 45% sucrose which also contained 0.6 M KC1 and 1 mg/ml albumin. Following overnight centrifugation at 26000 rpm the endoplasmic reticulum membranes were recovered between 18 and 25% sucrose. Tqtxin-digestion Phosphorylated (see below) or unphosphorylated endoplasmic reticulum membranes (0.6 m&nl) were treated with the indicated concentrations of trypsin at 30°C for 3 min. The medium contained 50 mM Na-HEPES (pH 6.9) and 0.5 mM EGTA. The digestion was stopped by adding an at least three fold excess (weight/weight) of soybean trypsin inhibitor, with incubation for 2 min before making any further additions. Phosphorylationand gel electrophoresis Membranes (0.6 mg/ml) were phosphorylated at 3O’C in a medium containing 50 mM Na-HEPES @H 6.9), 0.5 mM EGTA, 5 mM MgCl2, 5 mM NaN3, 100 pM [y-3%1 ATP, and the indicated concentrations of the catalytic subunit of CAMP-dependent protein kinase. After 2 min the membranes were either treated with trypsin, as described above, or the reaction was stopped by mixing with an equal volume of solubilization buffer for SDS polyacrylamide gel electrophoresis. Membranes solubilized in SDS were warmed at 100°C for 3 min or at 37°C for 10 ruin, as indicated. The Laemmli-type gels of 12% polyacrylamide were fixed, dried and autoradiographed, or blotted onto Immobilon paper (Millipore) as described in [25]. 4SCa uptake Untreated membranes or membranes treated with trypsin were preincubated for 3 min at 30°C in the same medium as used for phosphorylation (except for the presence of 1 mM cold ATP), with or
263
EFFECTS OF PROTEIN KINASFS ON ER Cd+ PUMP
B
A b: G-kinase O: A-kinase
12: a 0
/
/
‘i-j:p
lo-
0 0
time Fig.
I
I
2
4
I
I
0.080.11
%04
mm
I
I
I
I
0.15
024
038
1
1
27
ICaz>pfl
1 Tbe effect of A-kinase and G-kinase on tbe rate by Ca2’ uptake by isolated ER vesicles fkom bovine main pulmonary artcq.
[Ca2’] was buffered with EGTA at 0.4 pM. (A) Time came
of the “Ca2’
uptake in contml conditions (fiied circles) and in the
presence of 50 nM of the catalytic subunit of A-kinaae (open circles) or in the presence of 50 nh4 G-kinase and 2 ph4 B-bmmo cGh$P (open triangles). (B) The depemlence of the rate of 45Ca2+uptake+by ER vesicles on the Ca2’-concentration (ti* Control (filed circles).
buffemd by EGTA).
In the presence of 50 nM of the catalytic subunit of A-kiaase (open cimles). Values shown an the mean of 4
observations. SE. of the mean was smaller than the size of the symbols
without the indicated concentration of the cGMF-dependent protein kinase or the catalytic subunit of cAMPdependent protein kinase. The preincubated membranes were diluted 20 times in an uptake medium at 30°C containing 100 mM KCl, 5 mM NaN3, 30 mM imidazole-HCl @H 6.9), 6 mM MgCh, 5 mM Na-ATF, 5 mM K-oxalate and 45Ca2+ buffered at the desired free Ca2+ concentration with 1 mM total EGTA. Aliquots were removed at the indicated times, filtered on 0.45 pm nitrocellulose filters and rimed two times with 4 ml 0.25 M sucrose containing 2 mM EGTA. The radioactivity on the filters was counted.
Materials Trypsin and trypsin inhibitor were obtained from Boehringer (Maunheim, FRG). G-kinase was prepared from bovine lung as described in (261. The catalytic subunit of CAMP-dependent protein kinase and 8 bromo-cGMF were obtained from Sigma. Monoclonal antibody 2D9 to canine cardiac phospholamban was kindly provided by Dr Larry R. Jones, Indiana University, USA.
Results
We have previously described a membrane fraction from smooth muscle that is enriched in marker enzymes for the endoplasmic reticulum and in 100 kDa Ca2+-transport AT&se 15, 61. The resence of phospholamban suggests that the Ca 2P PumP of these vesicles, as the corresponding Ca2+ pump of cardiac SR, may be subject to regulation by c clic nucleotide-dependent protein kinases, Ca2Y + cahnodulin-dependent protein kinase and by protein kinase C. The presence of phospholamban was demonstrated by its phosphorylation and by its reaction with antibodies against pllritied phospholamban from cardiac muscle. This paper describes the functional modification of the Ca2’ pump activity in the endoplasmic reticulum vesicles concomitant with the phosphorylation of phospholamban. A stimulation of the Ca2’ uptake by cyclic nucleotide dependent protein kinase was seen in vesicles prepared by the modified (inclusion of albumin in the density gradient) procedure (Fig. l), but not by the original (no albumin added) procedure (data not shown). As shown in Figure lB, pretreatment of the vesicles with the catalytic subunit of CAMP-dependent protein kinase
264
CELL CALCIUM
Mr
403
Fig. 2
Automdiogram
of an SDS-PAGE gel of isolated Ek
membranes
phosphorylated
[r3%]-ATP.
Prior to electrophoresis, the SDS-solubilized sample
by A-k&se
in the presence
of
was warmed at a temperature of 37’C in order to preserve the 25 kDa pentameric form of phcmpholamban [34]. ER membranes of bovine pulmonary artery at 0.5 mg/ml were phosphorylated for 2 min at 3O’C in the presence of 50 oh4 of the catalytic subunit of A-k&se.
ER protein (10 pg) was applied per lane. Lane 1:
control. Lane 2: ER treated with 4 pg/ml trypsin for 3 min prior to phosphorylation. Lane 3: EX membranes phosphorylated prior to exposure to trypsin
(A-kinase) augmented the Vmax by 13% and decreased the IQ.5 for Ca2’ from 0.2 to 0.14 pM. Similar results were obtained with cGMP-dependent protein kinase (G-kinase), as shown in Figure 1A at a Ca2+-concentration of 0.4 pM. A similar efficiency of A- and G-kinase for phosphorylating phospholamban of smooth muscle ER and cardiac SR and for stimulating the Ca uptake of cardiac SR has previously been described [26]. The magnitude of stimulation by protein kinase of the 45Ca2+ uptake in the smooth muscle endoplasmic reticulum vesicles is relatively small
and appears to be smaller than that seen in cardiac SR (compare e.g. Fig. 1A with Fig. 1 in [26]). There are several possible explanations for this difference (see Discussion), e.g. it could be due to the presence of a higher phosphatase activity in the smooth-muscle membranes as compared to cardiac However, the SR. phosphorylation of phospholamban was not altered if the phosphatase inhibitor NaF (10 mM) was included in the assays. Another possibility is that phospholamban was already partially phosphorylated in vivo and would remain so during the isolation of the vesicles. This possibility is unlikely because of the absence of ATP during the purification and because of the long duration of the procedure. Another reason could be a partial proteolysis of phospholamban during the isolation procedure. It has indeed been shown that limited trypsinization of isolated cardiac SR results in a stimulation of the Ca uptake, which is similar to that induced by phosphorylation by A-kinase [27]. However, we could not find a larger effect on the Ca2’ uptake in ER preparations purified under conditions to minimize proteolytic breakdown (i.e. inclusion in the homogenization medium of 4 mM EGTA to inhibit calpain and addition of the additional proteolytic inhibitors aprotinin (5 pg/ml), benzamidine (0.5 mM), leupeptin (2.5 p.g/ml), pepstatin (2.5 pg/ml) and soybean lrypsin inhibitor (IO Wml). In our isolated ER preparation from smooth muscle, many polypeptides besides phospholamban are substrates for cyclic nucleotide-dependent protein kinases (Fig. 2, see also [lo, 261). Phosphorylated phospholamban of cardiac muscle is resistant to trypsinization than the more unphosphorylated protein. Figure 2 shows that phospholamban in smooth-muscle ER presents the same property and that it is the only phosphorylatable protein in the ER presenting this As in cardiac SR, limited characteristic. trypsinization was accompanied by a stimulation of the rate of Ca2+ uptake. The Ca2+ uptake was augmented with increasing trypsin concentrations up to 10 pg/ml (Fig. 3A). Higher concentrations were inhibitory (data not shown). The percentage of stimulation of the Ca2+ uptake by the optimal trypsin concentration of 10 pg/ml is shown in Figure 3B. The stimulation seen with trypsin
265
EFFECl-S OF PROTEIN KINASES ON ER Ca2+ PUMP
A
B
160 ‘;
P
140g
/
:
120 -
30 2
C .z
2
2
q ct;
20 ,O
,\” I 4
2 time Fig. 3 The effect of trypsinization
A
min
TR
t TR
on the tate of ca2” uptake by isolated ER vesicles from bovine main pulmonary artery. (A) Time
coume of the Ca*’ uptake by ER vesicles treated with different conceutrations of hypsiu for 3 min at 3o’C. ER vesicles at 600 @ml were incubated without try-p& (filled circles), or treated with 2 (filled triangles), 5 (filled squares) or 10 (open squares) uml
trypsin.
Proteolysis was stopped by the addition of excess trypsin inhibitor and 2 min later the suspension was diluted 20 fold in the ‘%a*‘uptake medium.
Data points are the mean of 4 determinations.
rate of Ca*+ uptake (A) by 40 nM A-k&se
(catalytic
The result shown is the mean of 4 determinations
S.E. were smaller than the size of the symbols.
subunit), (TR) by 10 @ml
on one preparation.
treatment is somewhat smaller than that seen with A-kinase. Trypsin treatment largely prevented further stimulation by phosphorylation via A-kinase, i.e. their effects were not additive (Fig. 3B). In order to understand the regulation of the Ca2’ transport in the intracellular membranes of smooth muscle, it is important to assess the stoichiomehy between phospholamban and the Ca2’-transport ATBase. This parameter is however difficult to even in SR where determine, cardiac phospholamban is the only phosphorylatable protein and the Ca2+-pump constitutes the largest fraction of the total membrane protein 1281. This task is even more complicated for the ER from smooth muscle because of its low content of Ca2+-pump and of phospholamban and because of its more heterogenous composition. Therefore, we have not attempted to determine this ratio but we have instead tried to get an idea of the ratio between the Ca2+-transport ATPase in smooth muscle ER and that in cardiac SR, and we have compared this ratio with the corresponding ratio determined for phospholamban. The amount of phospholamban in the internal membranes from cardiac and from smooth muscle was compared from the amount of 32P-incoipo ration into the monomeric 5 kDa band in SDS polyacrylarnide gels of solubilized samples treated at 100°C. By using different ratios of
(B) Stimulation
trypsin, or (A + TR) by trypsin followed
of the
by A-kinase.
Similar results were obtained on a second preparation
catalytic subunit of CAMP-dependent protein kinase to ER vesicles, it was ascertained that maximal levels of phosphorylation were obtained. In five separate experiments, each with a different ER- as well as SR-preparation, the ratio of phospholamban in cardiac SR to that in smooth-muscle ER was 7.1 +_0.75 (mean + S.E., n = 5). The validity of this approach was confirmed by comparing immunoblots of cardiac SR and smooth ER fractions. Serial dilutions of the membrane tractions were applied to SDS polyacrylamide gel, blotted to Immobilon (Millipore) paper and reacted with monoclonal antiphospholamban antibody. Fractions of SR which were diluted 4 to 6 times more than the smooth muscle ER yielded approximately equally intense staining as judged by eye (data not shown). The ratio of the amount of Ca2+-transport ATPase in cardiac and smooth muscle membranes was determined from the Ca2+-dependent ATPase activity. This ratio was 8.6 f 1.6 (mean f S.E., n = 4), which is therefore a value similar or only slightly higher than the ratio found for phospholamban. Discussion The ER fraction from bovine pulmonary artery used in this study has previously been characterized. It is enriched in the 100 kDa Ca2+-transport ATPase
266
which is immunologically similar to that of cardiac SR but not to that of fast skeletal muscle SR. It also contains phospholamban, as demonstrated by the phosphorylation by A-kinase of polypeptides of Mr 5 and 25 kDa in SDS gels [6]. Phospholamban has previously been shown to be present in the E!R of several but not all smooth muscles examined [lo]. It was detected in membranes from pig stomach smooth muscle and dog and rabbit aorta [lo], and in bovine main pulmonary artery 161. It was detected immunocytochemically in dog ileum and ileac artery and in pig coronary artery 1111. Phospholamban could not be detected in membranes from pig aorta 181 and membranes from pig coronary artery presented only a very weak signal (Vrolix M. & Raeymaekers L., unpublished observations). As judged from 3%-incorporation, the ER membranes from bovine pulmonary artery used in this work presented a higher content of phospholamban than the fractions which we have isolated so far from other smooth-muscle tissues. Because of the presence of phospholamban in the ER membranes from smooth muscle, it is expected that the Ca uptake in these membranes would be stimulated by A-kinase, in analogy to cardiac SR. Since we have shown that phospholamban is also a good substrate for G-kinase [24], this kinase can be expected to have a similar functional effect as A-kinase. The Ca2’ uptake by the ER fraction as originally isolated was unresponsive to protein kinases. However, the Ca2’ uptake by the ER vesicles prepared by the modified procedure (inclusion of albumin in the density gradient) was significantly stimulated by added A-kinase or G-kinase. The mechanism of the protective effect of albumin is not clear at present. One possibility is that free fatty acids, which are complexed by albumin, could be inhibitory factors causing damage to the regulatory system of the Ca2+ pump. However, it should also be mentioned in this respect that muscle albumin stimulates the Ca2’ uptake by isolated SR membranes from cardiac muscle. This effect is due is3interaction of muscle albumin with some types of actin, which inhibit the Ca uptake by activating a Ca2’ release pathway [29, 301. If, however, as is most likely, the effect of phosphorylation of SR or ER membranes is mediated by an enhanced activity of the Ca pump
CEZL CALCIUM
rather than by an inhibition of the Ca efflux, then this mode of action of albumin cannot explain our results. It is quite possible that the protective effect of albumin on the regulatory system of the Ca2+-transport ATPase of the isolated smooth-muscle ER vesicles is only partial. This would explain the apparent lower effect of phosphorylation of phospholamban in smooth muscle membranes. The degree of stimulation of the Ca uptake by the ER fraction indeed appears to be smaller than that seen in isolated SR preparations from cardiac muscle. However, in theory several other explanations are possible for this low magnitude of stimulation. The presence of a high endogenous kinase activity in the ER preparation, resulting in a partial phosphorylation in the control experiments, can be eliminated because the ER preparation from bovine pulmonary artery, similar to that of pig stomach [lo], contains very little endogenous kinase. Furthermore, this activity is only ap arent in the presence of CAMP (not cGMP) or R Ca + calmodulin, while the experiments were conducted without the addition of these agents. Alternatively, the FR membranes could contain a higher phosphatase activity than the cardiac SR. However, this explanation is unlikely, since NaF did not influence the degree of phosphorylation of phospholamban or other substrates in the ER or SR fractions. Also the possibility that phospholamban of smooth muscle ER would be partially phosphorylated and remain so during isolation of the membranes is very unlikely. Another possibility is partial proteolysis of phospholamban during the purification of the ER vesicles. As shown for cardiac phospholamban [27], and confirmed for smooth muscle in this paper, the effect of trypsinization of phospholamban is simiIar to the effect of phosphorylation. Therefore, proteolysis of phospholamban could result in Ca2’ uptake which can not be further stimulated by phosphorylation. However, we did not find any experimental evidence for partial proteolysis since the purification of the ER vesicles in the presence of a mixture of proteolytic inhibitors did not result in a larger stimulation of the Ca2’ uptake by A-kinase. Another argument against proteolytic damage is the ratio of phosphorylatable phospholamban to Ca2+-transport
EFFECTS
OF PROTEIN
KINASES
267
ON ER Ca2+ PUMP
ATPase (see below). Since the amino acid sequence of phospholamban of smooth muscle is identical to that of cardiac muscle [31], a difference in the sttucwe of phospholamban can also not explain the observed differences between Cardiac and smooth muscle. The expression in orcine smooth muscle of two forms of ER-type Ca 8 -transport ATPases has been detected from sequencing of mRNAderived cDNA clones [8]. There is also evidence for a third Ca2+ pump which may also be expressed in smooth muscle tissues [32]. These isoforms differ from the Ca2t-transport ATPase of SR of fast skeletal muscle. One isoform is very similar that the Ca2t-transport ATPase expressed in the SR of cardiac and slow skeletal muscle, another isoform is very similar to the Ca2+-transport ATPase found in non-muscle cells. It is possible that the small effect of phosphorylation on the ER Ca2+ pump is related to the simultaneous presence of these different since it is quite feasible that isoforms phospholamban would exert different effects on different isoforms, or that phospholamban would be associated with only a fraction of the total number of Ca2’ pump units. Although our experiments did not provide any evidence for a significantly lower ratio of phospholamban to Ca2’ transport ATPase in the ER fraction from smooth muscle as compared to cardiac SR, such a possibility cannot be excluded because the determination of this ratio may be sub’ect to artefacts. As an index of the amount of C$’ pump > we used the Ca2’ stimulated ATPase activity which is at present, to the best of our knowledge, the only feasible approach for the fractions concerned. However, a different degree of deterioration of the ATPase activity during the isolation procedure between smooth muscle and cardiac muscle would invalidate the use of ATPase activity ratios. It remains to be determined whether the phosphorylation of phospholamban of smooth muscle cells also occurs in vivo and which role this phenomenon would play in the regulation of cytoplasmic Ca2+. Recently, Huggins et al. studied the phosphorylation of phospholamban in isolated membrane vesicle and in intact cells [33]. They confirmed our observations [24] that phospholambau is a good substrate for G-kinase in vitro. In intact
3%loaded tissues they were unable to demonstrate the phosphorylation of phospholamban. It is quite possible, however, that in their experiments phospholamban remained below the detection limit, since control experiments showing phosphorylation of phospholamban by exogenous kinases subsequent to isolation from the 3%-leaded tissues had not been included. This problem therefore requires further study. In conclusion, we have shown that exogenous A-kinase and G-kinase stimulate the rate of Ca uptake in a smooth-muscle endoplasmic reticulum fraction highly enriched in phospholamban. The magnitude of stimulation depended on the conditions of isolation of the vesicles, but was always smaller than the stimulation seen in cardiac SR. Several possible explanations for this smaller effect have been discussed, but at present it cannot be decided which of these is correct, It remains to be investigated whether the small effect of protein kinases on the ER Ca2’ pump of smooth muscle is an artefact of the isolation procedure, or whether it is due to differences in the function of phospholamban or of the Ca2t-transport ATPases between smooth muscle and cardiac muscle. Acknowledgements We thank Dr F. Hofmann (Univereitit des Saarlandes, Homburg, FRG) for the gift of cGMP-dependent protein Kim and Dr Larry R. Jones (University of Indiana, Indiana, USA) for the gif? of monoclonal anti-phospholamban antibody. J.A.E. is a Research Assistent of the National Fund for Scientific Research (N.F.W.O.), Belgium.
References 1. Wpack F. Raeymaekers L. Casteels R. (1985) The Ca -tranqort AT&es in smooth muscle. Experientia, 41, 900-905. 2. Wgtack F. Racymaekers L. Verbist J. Caste& R. (1987) Ca -transport ATPases in tbe plasma membrane and endoplasmic reticulum. In: Aqhileri LJ. cd. The role of calcium in biological systems. Vol 4. Florida, CRC Ress. 3. Wuytack F. Raeymaelmrs L. De Schutter G. Caste& R. (1982) Demonstration of the phosphorylated intermediates of the Ca”-lnmsport ATPa& in a~m&osomal &action and in a (Ca2’-Mg2’)-ATPase wrified tirn smootb muscle bv mea& of calmodulin &i&y chromatogaphy. Biocbim.Biophys. Acta, 693.45-52. 4. WuNk F. Raeymaekers L. Verbist J. De Smedt H. Caste& R (1984) Evidence for the presence in smooth muscle of two typea of Ca”-transport ATPase. Biochem. J.,
268 224,445451. 5. Raeymaekers L. Wuytsck F. Casteels R. (1985) Subce.l~lar fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2++Mg2’)-ATPase activity in plasmalemma and endoplasmic reticulum. B&him. Biophys. Acta. 815,441-454. 6. Eggermont JA. Vmbx M. Raeymaekers L. Wuytack F. Caste& R. (1988) Ca2’-tranaport ATPases of vascular smooth muscle. Circ. Res.. 62,266278. 7. Wuytack F. Kanmum Y. Eggemxmt JA. et al. (1989) Smooth muscle exnresses a cardia&low muscle isoform of the Ca’+-tmnsport ’ATPase in its endoplasmic mtictdum. Biochem. J., 257,117-123. 8. Eggermont JA. Wuytack F. De Jaeger S. Nelles L. Caste& R. (1989) Evidence for two isofonns of the endoplasmic reticulum Ca’+-pump in porcine smooth muscle. B&hem. J., 260,757-761. 9. Lytton J. Zarain-Herzberg A. Periasamy M. MacLennan DH. (1989) Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. J. Biol. Chem., 264,7059-7065. 10. Raeymaekers L. Jones LR. (1986) Evidence for the presence of phospholamban in the endoplasmic reticulum of smooth muscle. B&him. Biophys. Acta, 882,258-265. 11. Ferguson DG. Young EF. Raeymaekem L. Kmnias EG. (1988) Localization of phospholamban in smooth muscle using immunogold electron microscopy. J. Cell Biol., 107, 555-562. 12. Wuytack F. Raeymaekers L. Verbist J. Jones LR. Casteels R. (1987) Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin. Bicchim. Biophys. Acta, 899, 151-158. 13. Kobayashi S. Kanaide H. Nakamura M. (1985) Cytosolic free calcium transients in cultured vascular smooth muscle cells: microfluorometric measurements. Science, 229, 553-556. 14. Rashatwar S. Comwell TL. Lincoln TM. (1987) Effects of 8-bromo-cGMP on Ca2’ levels in vascular smooth muscle cells: possible regulation of Ca2’-ATPaae by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 84,5685-5689. 15. Itoh T. Kanmura Y. Kuriyama H. Sasaguri T. (1985) Nitroglycerineand isoprenahne-induced vasodilatiom assessment from the actions of cyclic nucleotides. Br. J. Pharmacol., 84,393-w. 16. Furukawa KI. Tawada Y. Shigekawa M. (1988) Regulation of the plasma membrane caz”-pump by cyclic nucleotides in cultured vascular smooth muscle cells. J. Biol. Chem., 263, 8058-8065. 11. Suematsu E. Hiram M. Kuriyama H. (1984) Effects of CAMP- and cGMP-dependent protein kinase and cahnodulin on Ca2’ uptake by highly purified sarcolemmal vesicles of vascular smooth muscle. Biochim. Biophys. Acta, 773, 83-90. 18. Vrolix M. Raeymaekem L. Wuytack F. Hofmann F. Casteels R. (1988) Cyclic GMP-dependent protein k&se stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol. B&hem. J., 255, 855863. 19. Fumkawa K. Nakamura H. (1987) Qclic CMP regulation of the plasma membrane (Ca’++Mg”)-ATPase in vascular smooth muscle. J. Bicchem., 101,287-290. 20. Casteels R. Raeymaekem L. (1979) The action of
CELL CALCIUM
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
3 1.
32.
33.
34.
35.
acetylcholine and catecholaminea on an intracellular Ca store in the smooth muscle cells of the guinea-pig taenia coli. J. Physiol.. 294, 51-68. Itoh T. Ixumi H. Kuriyama H. (1982) Mechanisms of relaxation induced by activation of ~adrenoceptom in smooth muscle cells of the guinea pig mesentetic artery. J. Physiol., 326.475-493. Saida K. Van Bmemen C. (1984) Cyclic AMP modulation of adrenoreceptor-mediated arterial smooth muscle contractions. J. Gen. Physiol., 84, 307-318. Twort CHC. van Breemen C. (1988) cyclic yanosine monophosphate-enhanced sequestration of Ca + by sarcoplasmic mticuIum in vascular smooth muscle. Circ. Rea., 62,961-964. Raeymaekers L. Hofnuum F. Casteels R (1988) Cyclic GMP-dependent protein kinase phosphorylatea phospholamban in isolated sarcoplasmic reticuhun t%om cardiac and smooth muscle. B&hem. J., 252,269-273. Wuytack F. De Schutter G. Verbist J. CasteeIs R (1983) Antibodies to the calmodulin-binding Ca2’-transport A’IPase from smooth muscle. FEBS L&L. 154, 191-195. Hofmann F. Flcckemi V. (1983) Characterixation of phosphorylated and native cGMP-dependent protein kinase. Eur. J. B&hem., 130,599~603. Kirchberger MA. Bon&man P. Kasinathan C. (1986) Proteolytic activation of canine cardiac sarcoplasmic reticulum calcium pump. Biochemistry, 25,54845492. Louis CF. Turnquist J, Jarvis B. (1987) Phospholamban stoichiometry in canine cardiac muscle sarcoplasmic reticulum. Nemo&em. Res., 12,937-941. Chiesi M. Guerini D. (1987) Characterization of heart cytosolic proteins capable of modulating calcium uptake by the sarcoplasmic reticulum. I. Isolation of a protein with protective activity and its identification as muscle albumin. Eur. J. Biochem., 162.365-370. Chiesi M. Schwaller R. (1987) Chamcterixation of heart cytosolic proteins capable of modulating calcium uptake by the sarcoplasmic reticuhmt. 2. Identification of actin isoforms with inhibitory activity. Eur. J. B&hem., 162, 371-377. Verboomen H. Wuytack F. Eggermont JA. et al. (1989) cDNA cloning and sequencing of phospholamban from pig stomach smooth muscle. Biochem. I., In press. Burk SE. Lytton J. MacLe~an DH. ShuII GE (1989) cDNA cloning, functional expression, and mRNA tissue distribution of a third organelhu Ca2+ pump. J. BioI. Chem., In press. Huggins JP. Cook EA. Piggott JR. Mattinsley TJ. England PJ. (1989) Phosnholamban is a good substrate for cyclic GM&dependem protein kinase m vitro, but not in intact cardiac or smooth muscle. Biochem. J. 260, 829-835. Kimhberger MA. Antonetz T. (1982) Phospholambarr dissociation of the 22,000 molecular weight protein of cardiac sarcopIasmic reticulum into 11,000 and 5,500 molecular weight forms. B&hem. Biophys. Res. Commun., 105,152-156. Tada, M. bud, M. (1983) J. Mol. Cell. Cardiol. 15. 565
Please send reprint requests to Fysiologie. voor Laboratorium Gasthuisberg, B-3000 Leuven, Belgium. Received : 2 August 1989 Revised : 20 October 1989 Accepted : 25 Gctober 1989
: Dr L. K.U.Leuven,
Raeymaekers, Cempus
LOi?Q”W
GrcupUKUd1990
Effects of cyclic nucleotide dependent protein kinases on the encloplasmic reticulum Ca*+ pump of bovine pulmonary artery L. RAEYMAEKERS,
J.A. EGGERMONT,
Latwratorium voor Fysioogie, K. Ueuven,
F. WUYTACK
and Fi. CASTEELS
Leuven, Belgium
Abstract - This pa r describes the stimulation by cyclic nucleotide dependent protein JY uptake by isolated endoplasmic reticulum (ER) vesicles from the kinases on the Ca bovine main pulmonary artery. This ER fraction has previously been shown to be highly enriched in phospholamban, a protein kinase substrate that has been well characterized in cardiac sarcoplasmic reticulum (SR), where its phosphorylation is accompanied by an increased rate of Ca2+ uptake [for review see 351. As previously observed for the phosphorylation of phospholamban [IO], the stimulation of the rate of Ca uptake was as high with cGMP dependent protein kinase as with CAMP dependent protein kinase. The effect of phosphorylation of the ER membranes from smooth muscle on the Ca2+ uptake was smaller than that seen in cardiac SR, and it was only observed if albumin was included during the isolation of the membranes. This relatively small effect is probably not due to a lower ratio of phospholamban to Ca2+-transport enzyme in the ER membranes as compared to cardiac SR. Several alternative explanations are discussed. Considerable progress has been made recently in the characterization of the Ca2t-transport ATPases of smooth muscle (for review see [l, 23). The presence of two types of Ca2t-transport ATPases, one of 100 kDa in the endoplasmic reticulum and one of 130 kDa in the plasma membrane, has been well documented 13-61. The Ca” pump of the endoplasmic reticulum is functionally and structurally related to the Ca2+-pump proteins expressed in the sarcoplasmic reticulum of skeletal and cardiac muscle. It has been shown by biochemical, immunological 171 and cDNA-sequencing techniques [8, 91 that the Ca2+ pump of smooth muscle is more related to that of cardiac muscle (and to that of non-muscle cells, see Discussion) than to that of fast skeletal muscle.
Interestingly, there are two more parallels which discriminate the cardiac- and smooth-muscle Ca2+-transport and storage system from that of fast skeletal muscle. Phospholamban, a phosphorylatable protein that regulates the Ca2+ pump in the cardiac sarcoplasmic reticulum but not in fast skeletal muscle, was detected in smooth muscle [ 10, I I]. Calsequestrin, the main Ca2+-binding protein of the lumen of the sarcoplasmic reticulum, was also found in smooth muscle. The calsequestfin isoform expressed in smooth muscle is similar to that of cardiac muscle, but it differs from that of fast skeletal muscle [ 121. It has been proposed that the Ca2t-transport ATPases of smooth muscle are subject to regulation by protein kinases and that this mechanism may
261
262
contribute to a decrease of the Ca2’ concentration and to relaxation induced by substances that increase the level of cyclic nucleotides, especially cyclic GMP [13-161. In biochemical studies stimulation of the Ca2+ pump of the plasmalemma by cGMP-dependent protein kinase has been observed, as well on isolated plasmalemmal vesicles [17, 181 as on the purified Ca2’ pump [lS, 191. An une uivocal demonstration of a stimulation of the 29 pump of the endoplasmic reticulum has Ca hitherto not been possible, because of the difficulty of purifying this cell organelle from smooth muscle. However, experiments carried out on intact tissues [20, 211 and on skinned cells [15, 22, 231 have provided evidence for the existence of such a of mechanism. In addition, the 2presenee phospholamban suggests that the Ca pump of the endoplasmic reticulum of smooth muscle could be a target for regulation by second messengers [lo]. We have previously demonstrated the phosphorylation of phospholamban of isolated membranes by CAMP-dependent and exogenous endogenous protein kinase [lo] and by exogenous cGMPdependent protein kinase [24]. We have now studied the functional effect of phosphorylation on the Ca2’ uptake by an endoplasmic reticulum fraction purified from the bovine main pulmonary artery. This fraction has been characterized previously. It has been shown to contain little plasma-membrane marker enzymes and to be highly enriched in phospholamban [6]. In this paper we report that the Ca2’ uptake in these endoplasmic reticulum vesicles is stimulated by exogenous CAMP- and by cGMPdependent protein kinases. The possible role of phospholamban in this effect is discussed. Materials and Methods Preparation of membranes A membrane fraction enriched in endoplasmic reticulum was prepared from the bovine main pulmonary artery as described previously [6], except that albumin (1 mg/ml) was included in the sucrose density gradient. Briefly, about 150 g of muscle was homogenized in 6 volumes of 0.25 M sucrose containing 1 mM dithiothreitol and 0.5 mM PMSF and centrifuged at 20 000 x gma for 20 min in a
CELL CALCIUM
Sorvall GSA rotor. The supematant was supplemented with 0.6 M KCl (final concentration), 120 mg digitonin dissolved in 20 ml H20, and sucrose (50% final concentration). The supematant was then pumped into a Kontron TZT32 zonal rotor underneath a sucrose density gradient extending between 15 and 45% sucrose which also contained 0.6 M KC1 and 1 mg/ml albumin. Following overnight centrifugation at 26000 rpm the endoplasmic reticulum membranes were recovered between 18 and 25% sucrose. Tqtxin-digestion Phosphorylated (see below) or unphosphorylated endoplasmic reticulum membranes (0.6 m&nl) were treated with the indicated concentrations of trypsin at 30°C for 3 min. The medium contained 50 mM Na-HEPES (pH 6.9) and 0.5 mM EGTA. The digestion was stopped by adding an at least three fold excess (weight/weight) of soybean trypsin inhibitor, with incubation for 2 min before making any further additions. Phosphorylationand gel electrophoresis Membranes (0.6 mg/ml) were phosphorylated at 3O’C in a medium containing 50 mM Na-HEPES @H 6.9), 0.5 mM EGTA, 5 mM MgCl2, 5 mM NaN3, 100 pM [y-3%1 ATP, and the indicated concentrations of the catalytic subunit of CAMP-dependent protein kinase. After 2 min the membranes were either treated with trypsin, as described above, or the reaction was stopped by mixing with an equal volume of solubilization buffer for SDS polyacrylamide gel electrophoresis. Membranes solubilized in SDS were warmed at 100°C for 3 min or at 37°C for 10 ruin, as indicated. The Laemmli-type gels of 12% polyacrylamide were fixed, dried and autoradiographed, or blotted onto Immobilon paper (Millipore) as described in [25]. 4SCa uptake Untreated membranes or membranes treated with trypsin were preincubated for 3 min at 30°C in the same medium as used for phosphorylation (except for the presence of 1 mM cold ATP), with or
263
EFFECTS OF PROTEIN KINASFS ON ER Cd+ PUMP
B
A b: G-kinase O: A-kinase
12: a 0
/
/
‘i-j:p
lo-
0 0
time Fig.
I
I
2
4
I
I
0.080.11
%04
mm
I
I
I
I
0.15
024
038
1
1
27
ICaz>pfl
1 Tbe effect of A-kinase and G-kinase on tbe rate by Ca2’ uptake by isolated ER vesicles fkom bovine main pulmonary artcq.
[Ca2’] was buffered with EGTA at 0.4 pM. (A) Time came
of the “Ca2’
uptake in contml conditions (fiied circles) and in the
presence of 50 nM of the catalytic subunit of A-kinaae (open circles) or in the presence of 50 nh4 G-kinase and 2 ph4 B-bmmo cGh$P (open triangles). (B) The depemlence of the rate of 45Ca2+uptake+by ER vesicles on the Ca2’-concentration (ti* Control (filed circles).
buffemd by EGTA).
In the presence of 50 nM of the catalytic subunit of A-kiaase (open cimles). Values shown an the mean of 4
observations. SE. of the mean was smaller than the size of the symbols
without the indicated concentration of the cGMF-dependent protein kinase or the catalytic subunit of cAMPdependent protein kinase. The preincubated membranes were diluted 20 times in an uptake medium at 30°C containing 100 mM KCl, 5 mM NaN3, 30 mM imidazole-HCl @H 6.9), 6 mM MgCh, 5 mM Na-ATF, 5 mM K-oxalate and 45Ca2+ buffered at the desired free Ca2+ concentration with 1 mM total EGTA. Aliquots were removed at the indicated times, filtered on 0.45 pm nitrocellulose filters and rimed two times with 4 ml 0.25 M sucrose containing 2 mM EGTA. The radioactivity on the filters was counted.
Materials Trypsin and trypsin inhibitor were obtained from Boehringer (Maunheim, FRG). G-kinase was prepared from bovine lung as described in (261. The catalytic subunit of CAMP-dependent protein kinase and 8 bromo-cGMF were obtained from Sigma. Monoclonal antibody 2D9 to canine cardiac phospholamban was kindly provided by Dr Larry R. Jones, Indiana University, USA.
Results
We have previously described a membrane fraction from smooth muscle that is enriched in marker enzymes for the endoplasmic reticulum and in 100 kDa Ca2+-transport AT&se 15, 61. The resence of phospholamban suggests that the Ca 2P PumP of these vesicles, as the corresponding Ca2+ pump of cardiac SR, may be subject to regulation by c clic nucleotide-dependent protein kinases, Ca2Y + cahnodulin-dependent protein kinase and by protein kinase C. The presence of phospholamban was demonstrated by its phosphorylation and by its reaction with antibodies against pllritied phospholamban from cardiac muscle. This paper describes the functional modification of the Ca2’ pump activity in the endoplasmic reticulum vesicles concomitant with the phosphorylation of phospholamban. A stimulation of the Ca2’ uptake by cyclic nucleotide dependent protein kinase was seen in vesicles prepared by the modified (inclusion of albumin in the density gradient) procedure (Fig. l), but not by the original (no albumin added) procedure (data not shown). As shown in Figure lB, pretreatment of the vesicles with the catalytic subunit of CAMP-dependent protein kinase
264
CELL CALCIUM
Mr
403
Fig. 2
Automdiogram
of an SDS-PAGE gel of isolated Ek
membranes
phosphorylated
[r3%]-ATP.
Prior to electrophoresis, the SDS-solubilized sample
by A-k&se
in the presence
of
was warmed at a temperature of 37’C in order to preserve the 25 kDa pentameric form of phcmpholamban [34]. ER membranes of bovine pulmonary artery at 0.5 mg/ml were phosphorylated for 2 min at 3O’C in the presence of 50 oh4 of the catalytic subunit of A-k&se.
ER protein (10 pg) was applied per lane. Lane 1:
control. Lane 2: ER treated with 4 pg/ml trypsin for 3 min prior to phosphorylation. Lane 3: EX membranes phosphorylated prior to exposure to trypsin
(A-kinase) augmented the Vmax by 13% and decreased the IQ.5 for Ca2’ from 0.2 to 0.14 pM. Similar results were obtained with cGMP-dependent protein kinase (G-kinase), as shown in Figure 1A at a Ca2+-concentration of 0.4 pM. A similar efficiency of A- and G-kinase for phosphorylating phospholamban of smooth muscle ER and cardiac SR and for stimulating the Ca uptake of cardiac SR has previously been described [26]. The magnitude of stimulation by protein kinase of the 45Ca2+ uptake in the smooth muscle endoplasmic reticulum vesicles is relatively small
and appears to be smaller than that seen in cardiac SR (compare e.g. Fig. 1A with Fig. 1 in [26]). There are several possible explanations for this difference (see Discussion), e.g. it could be due to the presence of a higher phosphatase activity in the smooth-muscle membranes as compared to cardiac However, the SR. phosphorylation of phospholamban was not altered if the phosphatase inhibitor NaF (10 mM) was included in the assays. Another possibility is that phospholamban was already partially phosphorylated in vivo and would remain so during the isolation of the vesicles. This possibility is unlikely because of the absence of ATP during the purification and because of the long duration of the procedure. Another reason could be a partial proteolysis of phospholamban during the isolation procedure. It has indeed been shown that limited trypsinization of isolated cardiac SR results in a stimulation of the Ca uptake, which is similar to that induced by phosphorylation by A-kinase [27]. However, we could not find a larger effect on the Ca2’ uptake in ER preparations purified under conditions to minimize proteolytic breakdown (i.e. inclusion in the homogenization medium of 4 mM EGTA to inhibit calpain and addition of the additional proteolytic inhibitors aprotinin (5 pg/ml), benzamidine (0.5 mM), leupeptin (2.5 p.g/ml), pepstatin (2.5 pg/ml) and soybean lrypsin inhibitor (IO Wml). In our isolated ER preparation from smooth muscle, many polypeptides besides phospholamban are substrates for cyclic nucleotide-dependent protein kinases (Fig. 2, see also [lo, 261). Phosphorylated phospholamban of cardiac muscle is resistant to trypsinization than the more unphosphorylated protein. Figure 2 shows that phospholamban in smooth-muscle ER presents the same property and that it is the only phosphorylatable protein in the ER presenting this As in cardiac SR, limited characteristic. trypsinization was accompanied by a stimulation of the rate of Ca2+ uptake. The Ca2+ uptake was augmented with increasing trypsin concentrations up to 10 pg/ml (Fig. 3A). Higher concentrations were inhibitory (data not shown). The percentage of stimulation of the Ca2+ uptake by the optimal trypsin concentration of 10 pg/ml is shown in Figure 3B. The stimulation seen with trypsin
265
EFFECl-S OF PROTEIN KINASES ON ER Ca2+ PUMP
A
B
160 ‘;
P
140g
/
:
120 -
30 2
C .z
2
2
q ct;
20 ,O
,\” I 4
2 time Fig. 3 The effect of trypsinization
A
min
TR
t TR
on the tate of ca2” uptake by isolated ER vesicles from bovine main pulmonary artery. (A) Time
coume of the Ca*’ uptake by ER vesicles treated with different conceutrations of hypsiu for 3 min at 3o’C. ER vesicles at 600 @ml were incubated without try-p& (filled circles), or treated with 2 (filled triangles), 5 (filled squares) or 10 (open squares) uml
trypsin.
Proteolysis was stopped by the addition of excess trypsin inhibitor and 2 min later the suspension was diluted 20 fold in the ‘%a*‘uptake medium.
Data points are the mean of 4 determinations.
rate of Ca*+ uptake (A) by 40 nM A-k&se
(catalytic
The result shown is the mean of 4 determinations
S.E. were smaller than the size of the symbols.
subunit), (TR) by 10 @ml
on one preparation.
treatment is somewhat smaller than that seen with A-kinase. Trypsin treatment largely prevented further stimulation by phosphorylation via A-kinase, i.e. their effects were not additive (Fig. 3B). In order to understand the regulation of the Ca2’ transport in the intracellular membranes of smooth muscle, it is important to assess the stoichiomehy between phospholamban and the Ca2’-transport ATBase. This parameter is however difficult to even in SR where determine, cardiac phospholamban is the only phosphorylatable protein and the Ca2+-pump constitutes the largest fraction of the total membrane protein 1281. This task is even more complicated for the ER from smooth muscle because of its low content of Ca2+-pump and of phospholamban and because of its more heterogenous composition. Therefore, we have not attempted to determine this ratio but we have instead tried to get an idea of the ratio between the Ca2+-transport ATPase in smooth muscle ER and that in cardiac SR, and we have compared this ratio with the corresponding ratio determined for phospholamban. The amount of phospholamban in the internal membranes from cardiac and from smooth muscle was compared from the amount of 32P-incoipo ration into the monomeric 5 kDa band in SDS polyacrylarnide gels of solubilized samples treated at 100°C. By using different ratios of
(B) Stimulation
trypsin, or (A + TR) by trypsin followed
of the
by A-kinase.
Similar results were obtained on a second preparation
catalytic subunit of CAMP-dependent protein kinase to ER vesicles, it was ascertained that maximal levels of phosphorylation were obtained. In five separate experiments, each with a different ER- as well as SR-preparation, the ratio of phospholamban in cardiac SR to that in smooth-muscle ER was 7.1 +_0.75 (mean + S.E., n = 5). The validity of this approach was confirmed by comparing immunoblots of cardiac SR and smooth ER fractions. Serial dilutions of the membrane tractions were applied to SDS polyacrylamide gel, blotted to Immobilon (Millipore) paper and reacted with monoclonal antiphospholamban antibody. Fractions of SR which were diluted 4 to 6 times more than the smooth muscle ER yielded approximately equally intense staining as judged by eye (data not shown). The ratio of the amount of Ca2+-transport ATPase in cardiac and smooth muscle membranes was determined from the Ca2+-dependent ATPase activity. This ratio was 8.6 f 1.6 (mean f S.E., n = 4), which is therefore a value similar or only slightly higher than the ratio found for phospholamban. Discussion The ER fraction from bovine pulmonary artery used in this study has previously been characterized. It is enriched in the 100 kDa Ca2+-transport ATPase
266
which is immunologically similar to that of cardiac SR but not to that of fast skeletal muscle SR. It also contains phospholamban, as demonstrated by the phosphorylation by A-kinase of polypeptides of Mr 5 and 25 kDa in SDS gels [6]. Phospholamban has previously been shown to be present in the E!R of several but not all smooth muscles examined [lo]. It was detected in membranes from pig stomach smooth muscle and dog and rabbit aorta [lo], and in bovine main pulmonary artery 161. It was detected immunocytochemically in dog ileum and ileac artery and in pig coronary artery 1111. Phospholamban could not be detected in membranes from pig aorta 181 and membranes from pig coronary artery presented only a very weak signal (Vrolix M. & Raeymaekers L., unpublished observations). As judged from 3%-incorporation, the ER membranes from bovine pulmonary artery used in this work presented a higher content of phospholamban than the fractions which we have isolated so far from other smooth-muscle tissues. Because of the presence of phospholamban in the ER membranes from smooth muscle, it is expected that the Ca uptake in these membranes would be stimulated by A-kinase, in analogy to cardiac SR. Since we have shown that phospholamban is also a good substrate for G-kinase [24], this kinase can be expected to have a similar functional effect as A-kinase. The Ca2’ uptake by the ER fraction as originally isolated was unresponsive to protein kinases. However, the Ca2’ uptake by the ER vesicles prepared by the modified procedure (inclusion of albumin in the density gradient) was significantly stimulated by added A-kinase or G-kinase. The mechanism of the protective effect of albumin is not clear at present. One possibility is that free fatty acids, which are complexed by albumin, could be inhibitory factors causing damage to the regulatory system of the Ca2+ pump. However, it should also be mentioned in this respect that muscle albumin stimulates the Ca2’ uptake by isolated SR membranes from cardiac muscle. This effect is due is3interaction of muscle albumin with some types of actin, which inhibit the Ca uptake by activating a Ca2’ release pathway [29, 301. If, however, as is most likely, the effect of phosphorylation of SR or ER membranes is mediated by an enhanced activity of the Ca pump
CEZL CALCIUM
rather than by an inhibition of the Ca efflux, then this mode of action of albumin cannot explain our results. It is quite possible that the protective effect of albumin on the regulatory system of the Ca2+-transport ATPase of the isolated smooth-muscle ER vesicles is only partial. This would explain the apparent lower effect of phosphorylation of phospholamban in smooth muscle membranes. The degree of stimulation of the Ca uptake by the ER fraction indeed appears to be smaller than that seen in isolated SR preparations from cardiac muscle. However, in theory several other explanations are possible for this low magnitude of stimulation. The presence of a high endogenous kinase activity in the ER preparation, resulting in a partial phosphorylation in the control experiments, can be eliminated because the ER preparation from bovine pulmonary artery, similar to that of pig stomach [lo], contains very little endogenous kinase. Furthermore, this activity is only ap arent in the presence of CAMP (not cGMP) or R Ca + calmodulin, while the experiments were conducted without the addition of these agents. Alternatively, the FR membranes could contain a higher phosphatase activity than the cardiac SR. However, this explanation is unlikely, since NaF did not influence the degree of phosphorylation of phospholamban or other substrates in the ER or SR fractions. Also the possibility that phospholamban of smooth muscle ER would be partially phosphorylated and remain so during isolation of the membranes is very unlikely. Another possibility is partial proteolysis of phospholamban during the purification of the ER vesicles. As shown for cardiac phospholamban [27], and confirmed for smooth muscle in this paper, the effect of trypsinization of phospholamban is simiIar to the effect of phosphorylation. Therefore, proteolysis of phospholamban could result in Ca2’ uptake which can not be further stimulated by phosphorylation. However, we did not find any experimental evidence for partial proteolysis since the purification of the ER vesicles in the presence of a mixture of proteolytic inhibitors did not result in a larger stimulation of the Ca2’ uptake by A-kinase. Another argument against proteolytic damage is the ratio of phosphorylatable phospholamban to Ca2+-transport
EFFECTS
OF PROTEIN
KINASES
267
ON ER Ca2+ PUMP
ATPase (see below). Since the amino acid sequence of phospholamban of smooth muscle is identical to that of cardiac muscle [31], a difference in the sttucwe of phospholamban can also not explain the observed differences between Cardiac and smooth muscle. The expression in orcine smooth muscle of two forms of ER-type Ca 8 -transport ATPases has been detected from sequencing of mRNAderived cDNA clones [8]. There is also evidence for a third Ca2+ pump which may also be expressed in smooth muscle tissues [32]. These isoforms differ from the Ca2t-transport ATPase of SR of fast skeletal muscle. One isoform is very similar that the Ca2t-transport ATPase expressed in the SR of cardiac and slow skeletal muscle, another isoform is very similar to the Ca2+-transport ATPase found in non-muscle cells. It is possible that the small effect of phosphorylation on the ER Ca2+ pump is related to the simultaneous presence of these different since it is quite feasible that isoforms phospholamban would exert different effects on different isoforms, or that phospholamban would be associated with only a fraction of the total number of Ca2’ pump units. Although our experiments did not provide any evidence for a significantly lower ratio of phospholamban to Ca2’ transport ATPase in the ER fraction from smooth muscle as compared to cardiac SR, such a possibility cannot be excluded because the determination of this ratio may be sub’ect to artefacts. As an index of the amount of C$’ pump > we used the Ca2’ stimulated ATPase activity which is at present, to the best of our knowledge, the only feasible approach for the fractions concerned. However, a different degree of deterioration of the ATPase activity during the isolation procedure between smooth muscle and cardiac muscle would invalidate the use of ATPase activity ratios. It remains to be determined whether the phosphorylation of phospholamban of smooth muscle cells also occurs in vivo and which role this phenomenon would play in the regulation of cytoplasmic Ca2+. Recently, Huggins et al. studied the phosphorylation of phospholamban in isolated membrane vesicle and in intact cells [33]. They confirmed our observations [24] that phospholambau is a good substrate for G-kinase in vitro. In intact
3%loaded tissues they were unable to demonstrate the phosphorylation of phospholamban. It is quite possible, however, that in their experiments phospholamban remained below the detection limit, since control experiments showing phosphorylation of phospholamban by exogenous kinases subsequent to isolation from the 3%-leaded tissues had not been included. This problem therefore requires further study. In conclusion, we have shown that exogenous A-kinase and G-kinase stimulate the rate of Ca uptake in a smooth-muscle endoplasmic reticulum fraction highly enriched in phospholamban. The magnitude of stimulation depended on the conditions of isolation of the vesicles, but was always smaller than the stimulation seen in cardiac SR. Several possible explanations for this smaller effect have been discussed, but at present it cannot be decided which of these is correct, It remains to be investigated whether the small effect of protein kinases on the ER Ca2’ pump of smooth muscle is an artefact of the isolation procedure, or whether it is due to differences in the function of phospholamban or of the Ca2t-transport ATPases between smooth muscle and cardiac muscle. Acknowledgements We thank Dr F. Hofmann (Univereitit des Saarlandes, Homburg, FRG) for the gift of cGMP-dependent protein Kim and Dr Larry R. Jones (University of Indiana, Indiana, USA) for the gif? of monoclonal anti-phospholamban antibody. J.A.E. is a Research Assistent of the National Fund for Scientific Research (N.F.W.O.), Belgium.
References 1. Wpack F. Raeymaekers L. Casteels R. (1985) The Ca -tranqort AT&es in smooth muscle. Experientia, 41, 900-905. 2. Wgtack F. Racymaekers L. Verbist J. Caste& R. (1987) Ca -transport ATPases in tbe plasma membrane and endoplasmic reticulum. In: Aqhileri LJ. cd. The role of calcium in biological systems. Vol 4. Florida, CRC Ress. 3. Wuytack F. Raeymaelmrs L. De Schutter G. Caste& R. (1982) Demonstration of the phosphorylated intermediates of the Ca”-lnmsport ATPa& in a~m&osomal &action and in a (Ca2’-Mg2’)-ATPase wrified tirn smootb muscle bv mea& of calmodulin &i&y chromatogaphy. Biocbim.Biophys. Acta, 693.45-52. 4. WuNk F. Raeymaekers L. Verbist J. De Smedt H. Caste& R (1984) Evidence for the presence in smooth muscle of two typea of Ca”-transport ATPase. Biochem. J.,
268 224,445451. 5. Raeymaekers L. Wuytsck F. Casteels R. (1985) Subce.l~lar fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2++Mg2’)-ATPase activity in plasmalemma and endoplasmic reticulum. B&him. Biophys. Acta. 815,441-454. 6. Eggermont JA. Vmbx M. Raeymaekers L. Wuytack F. Caste& R. (1988) Ca2’-tranaport ATPases of vascular smooth muscle. Circ. Res.. 62,266278. 7. Wuytack F. Kanmum Y. Eggemxmt JA. et al. (1989) Smooth muscle exnresses a cardia&low muscle isoform of the Ca’+-tmnsport ’ATPase in its endoplasmic mtictdum. Biochem. J., 257,117-123. 8. Eggermont JA. Wuytack F. De Jaeger S. Nelles L. Caste& R. (1989) Evidence for two isofonns of the endoplasmic reticulum Ca’+-pump in porcine smooth muscle. B&hem. J., 260,757-761. 9. Lytton J. Zarain-Herzberg A. Periasamy M. MacLennan DH. (1989) Molecular cloning of the mammalian smooth muscle sarco(endo)plasmic reticulum Ca2+-ATPase. J. Biol. Chem., 264,7059-7065. 10. Raeymaekers L. Jones LR. (1986) Evidence for the presence of phospholamban in the endoplasmic reticulum of smooth muscle. B&him. Biophys. Acta, 882,258-265. 11. Ferguson DG. Young EF. Raeymaekem L. Kmnias EG. (1988) Localization of phospholamban in smooth muscle using immunogold electron microscopy. J. Cell Biol., 107, 555-562. 12. Wuytack F. Raeymaekers L. Verbist J. Jones LR. Casteels R. (1987) Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin. Bicchim. Biophys. Acta, 899, 151-158. 13. Kobayashi S. Kanaide H. Nakamura M. (1985) Cytosolic free calcium transients in cultured vascular smooth muscle cells: microfluorometric measurements. Science, 229, 553-556. 14. Rashatwar S. Comwell TL. Lincoln TM. (1987) Effects of 8-bromo-cGMP on Ca2’ levels in vascular smooth muscle cells: possible regulation of Ca2’-ATPaae by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA, 84,5685-5689. 15. Itoh T. Kanmura Y. Kuriyama H. Sasaguri T. (1985) Nitroglycerineand isoprenahne-induced vasodilatiom assessment from the actions of cyclic nucleotides. Br. J. Pharmacol., 84,393-w. 16. Furukawa KI. Tawada Y. Shigekawa M. (1988) Regulation of the plasma membrane caz”-pump by cyclic nucleotides in cultured vascular smooth muscle cells. J. Biol. Chem., 263, 8058-8065. 11. Suematsu E. Hiram M. Kuriyama H. (1984) Effects of CAMP- and cGMP-dependent protein kinase and cahnodulin on Ca2’ uptake by highly purified sarcolemmal vesicles of vascular smooth muscle. Biochim. Biophys. Acta, 773, 83-90. 18. Vrolix M. Raeymaekem L. Wuytack F. Hofmann F. Casteels R. (1988) Cyclic GMP-dependent protein k&se stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol. B&hem. J., 255, 855863. 19. Fumkawa K. Nakamura H. (1987) Qclic CMP regulation of the plasma membrane (Ca’++Mg”)-ATPase in vascular smooth muscle. J. Bicchem., 101,287-290. 20. Casteels R. Raeymaekem L. (1979) The action of
CELL CALCIUM
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
3 1.
32.
33.
34.
35.
acetylcholine and catecholaminea on an intracellular Ca store in the smooth muscle cells of the guinea-pig taenia coli. J. Physiol.. 294, 51-68. Itoh T. Ixumi H. Kuriyama H. (1982) Mechanisms of relaxation induced by activation of ~adrenoceptom in smooth muscle cells of the guinea pig mesentetic artery. J. Physiol., 326.475-493. Saida K. Van Bmemen C. (1984) Cyclic AMP modulation of adrenoreceptor-mediated arterial smooth muscle contractions. J. Gen. Physiol., 84, 307-318. Twort CHC. van Breemen C. (1988) cyclic yanosine monophosphate-enhanced sequestration of Ca + by sarcoplasmic mticuIum in vascular smooth muscle. Circ. Rea., 62,961-964. Raeymaekers L. Hofnuum F. Casteels R (1988) Cyclic GMP-dependent protein kinase phosphorylatea phospholamban in isolated sarcoplasmic reticuhun t%om cardiac and smooth muscle. B&hem. J., 252,269-273. Wuytack F. De Schutter G. Verbist J. CasteeIs R (1983) Antibodies to the calmodulin-binding Ca2’-transport A’IPase from smooth muscle. FEBS L&L. 154, 191-195. Hofmann F. Flcckemi V. (1983) Characterixation of phosphorylated and native cGMP-dependent protein kinase. Eur. J. B&hem., 130,599~603. Kirchberger MA. Bon&man P. Kasinathan C. (1986) Proteolytic activation of canine cardiac sarcoplasmic reticulum calcium pump. Biochemistry, 25,54845492. Louis CF. Turnquist J, Jarvis B. (1987) Phospholamban stoichiometry in canine cardiac muscle sarcoplasmic reticulum. Nemo&em. Res., 12,937-941. Chiesi M. Guerini D. (1987) Characterization of heart cytosolic proteins capable of modulating calcium uptake by the sarcoplasmic reticulum. I. Isolation of a protein with protective activity and its identification as muscle albumin. Eur. J. Biochem., 162.365-370. Chiesi M. Schwaller R. (1987) Chamcterixation of heart cytosolic proteins capable of modulating calcium uptake by the sarcoplasmic reticuhmt. 2. Identification of actin isoforms with inhibitory activity. Eur. J. B&hem., 162, 371-377. Verboomen H. Wuytack F. Eggermont JA. et al. (1989) cDNA cloning and sequencing of phospholamban from pig stomach smooth muscle. Biochem. I., In press. Burk SE. Lytton J. MacLe~an DH. ShuII GE (1989) cDNA cloning, functional expression, and mRNA tissue distribution of a third organelhu Ca2+ pump. J. BioI. Chem., In press. Huggins JP. Cook EA. Piggott JR. Mattinsley TJ. England PJ. (1989) Phosnholamban is a good substrate for cyclic GM&dependem protein kinase m vitro, but not in intact cardiac or smooth muscle. Biochem. J. 260, 829-835. Kimhberger MA. Antonetz T. (1982) Phospholambarr dissociation of the 22,000 molecular weight protein of cardiac sarcopIasmic reticulum into 11,000 and 5,500 molecular weight forms. B&hem. Biophys. Res. Commun., 105,152-156. Tada, M. bud, M. (1983) J. Mol. Cell. Cardiol. 15. 565
Please send reprint requests to Fysiologie. voor Laboratorium Gasthuisberg, B-3000 Leuven, Belgium. Received : 2 August 1989 Revised : 20 October 1989 Accepted : 25 Gctober 1989
: Dr L. K.U.Leuven,
Raeymaekers, Cempus
