Calmodulin-dependent Ca2+-pump ATPase of human smooth muscle sarcolemma

Cell

Calcium

4 : 219 - 235,

CALMODULIN-DEPENDENT SMOOTHMUSCLESARCOLEMMA

L .M. Popescu

and P.

1983

Ca

2+

-PUMP

ATPase

OF HUMAN

Ignat

Department of Cell Biology & Histology, Faculty of Medicine, P.O.Box 35-10, Romania (reprint requests to LMP)

Bucharest

35,

ABSTRACT 2-l-stimulated ATPase has been iso ted from the An enzymatically active Ca l& sarcolemmal sheets of human smooth muscle (myometrium). Ca -ATPase activity was quantitated in an assay medium which simulated the characteristic free ionic concentrations of the cytosol. New computer programs for calculating the composition of solutions containing metals (Ca, Mg, Na, K) and ligands (EGTA, ATP), based on the updated stability T+onstants, were -affinity exused. In detergent-soluble for-9 the enzyme has a high Ca pressed by an apparent K (Ca +) of 0.25 +_ 0.04 pM. The maximum specific activity (about 20 r~+molof?‘./mg protein/min) was found in the micromolar domain of free-Ca concenltrations, the same levels required fo?Tormal maximal contractions in smooth muscle. The variation of free-Ca concentration in the assay medium over 4 orders of magnitude (pCa 9 to pCa 5) resulted in a sigmoidal dependence of enzymatic ac tjyity , with a Hill coefficient of 1.4, which suggested the regulation of Ca -ATPase by allosteric effecters. The presence and the activator role of endogenous calmodulin in smooth muscle sarcolemma was proved by calmodulin-depletion experiments and by using suitable anticalmodulinic concentrations of trifluoperazine. The addition of exogenous calmodulin restored the enzyme activity. Apparently, the concentration of calmodulin in isolated smooth muscle sarcolemma is about 0.1% of sarcolemmal proteins , as deduced from the comparison of calmodulin-depletion and calmoduli qqreaddition experiments. Calmodulin -affinity and V by a factor of increased significantly the enzyme Ca 4 about 10). At vasiance with the sarcoplasmic the reticulum m?% +-ATPase, sarcolemmal Ca -ATPase is extremely sensitive to orthovanadate, halfma imal inhibition being observed at 0.8 PM vanadate. In conclusion, the I+ appears very similar Ca -ATPase isolate $t+from smooth muscle sarcolemma -pump ATPases of erythrocyte membray+, heart to the well-known Ca sarcolemma or axolemma. We suggest “94 this high-affinity Ca -ATPase represents the calmodulin-regulated Ca -extrusion pump of the smooth muscle sarcolemma. 219

INTRODUCTION Sarcolemma of syzoth muscle cells is a biological barrier between 9~ extracellular Ca concentration of 2-3 mM and an intracellu l,jjr Ca concentration of less than 1 PM. To maintain the intracellular Ca homeostasis ‘cromolar domain, all smooth muscle cells must extrude the inni;ss;;-% that has leaked into the cell or has2eftered the cell during the excitation-contraction coupling, because the Ca accumulation ability of the cellular organelles is inevitablg+limited. Electron probe X-ray microanalysis of smooth rny%cle after Ca -overloading showed that the maximum storage capacity (Ca accumulation f binding) of the sarcoplasmic reti sjylum , mitochondria and even the nucleus is in the range of 100-200 mmoles Ca /kg dry weight (l-5). 2+ Two possible mechanisms are currently considered for Ca extrusion through the smooth muscle sarcolemma %gaingt+ the tremendous concentration gradient: either an electrogenic Na /Ca exchanq+system (for a quite exhaustive review see ref. 6) or an ATP-dependent C.a ex@m,ion (2, 712). From the molecular point of view, if an ATP-driven Ca eJection exists in the surface membrane of smoy+th muscle cell, our present knowledge would re yjre the involvement of a Ca -transport ATPase like the well-pumping ATPase of the erythrocyte membrane, described by 2+ known Ca omogeneity by Niggli et al. (14). Such Ca Schatzmann (13) and purified to 4+ transport ATPases as active Ca pumps were found, during the last few years, in the plasma membrane of at least 20 different cell types ! (for the most recent and comprehensive review see ref. 15). We eport here the isolation and characterization of a sarcolemmal high ca2$ . . -affinity ATPase from human myometrial smooth muscle. It was our intention to study the properties of the detergent-solubilized enzyme in an assay medium which has an ionic composition that simulated the in situ microenvironment of the enzyme. To minimize the in situ/in vitro discrepancy we took advantage of the new computer programs developed by Fabiato and Fabiato (16) for calculating the composition of solutions containing metals (Ca, Mg, Na, K) and ligands (EGTA, ATP), based on the updated stability The results obtained in our experimental conditions show that the co y+tants. has kinetic characteristics -ATPase of smooth musclf+ sarcolemma Ca -ATPases similar to the high affinity Ca isolated from the erythrocyte membrane, (17, 18), the heart sarcolemma (19) or the plasma membrane of other excitable cells (15, 20, 21). To asses y+hether or not calmodulin -ATPase we considered the regulates the activity of the sarcolemmal Ca most recent formulation of Cheung’s criteria (22) for calmodulin-regulated the enzyme appeared calmodulin depenbiological reactions , and, indeed, dent. Some

of the results

presented

in this report

in a short preliminary form (23).

220

have recently

been communicated

MATERIALS

AND METHODS

Pieces of normal human myometrium were obtained from the Gynaecologic Surgery, during the operations for hysterectomy, and were brought to the laboratory in ice-cold Krebs solution. The composition of the Krebs solution was (mM): NaCl, 127.0; KCl, 4.7; CaCl 5.6; pH adjus?Jd2t06i .l$CJll&‘~~l~io~~~~? ’ 25.0; KH2P04, 1.3; glucose, Isolation

of smooth muscle

cell

sarcolemma:

Our method for isolating myometrial sarcolemmae (Fig. 1) is evolved from the method described by Oliveira and Holzhacker (24) for the isolation of ileum smooth muscle cell membrane. In essence the technique consists in the evacuation of the intracellular components from broken smooth muscle cells. Then, the sarcolemmae can be readily sediomented, washed and collected. All procedures were performed at 0.4 C, unless otherwise indicated. Light microscopy was regularly used to evaluate the separation as well as the removal of the intracellular content. and disruption of cells, An amount of about 2 g of myometrium is required to obtain the sufficient quantity of final product, Homogenization was done in an all-glass PotterElvehjem homogenizer at high speed, until a homogenous suspension appeared. The homogenization medium was: 20 mM Tris-maleate, 250 mM sucrose and 1 mM EDTA. The resultant suspension was filtered through a plastic cheesecloth to remove gross connective tissue, and the residue was discarded. This process was repeated three times until the effectivity of cell separation and disruption was found to be adequate. The filtrate was then centrifuged and the resultant pellet was again homogenized. The next centrifugation resulted in the sedimentation of “cell ghosts”: cell envelopes still containing some “islets” of intracellular components (Fig. 2A). After the extraction of the residual cellular content and repeated centrifugations the final sarcolemmal fraction was obtained. The final product ranges from typical long sarcolemmal tubules (Fig. 2B) to small sarcolemmal sheets (sarcolemmal fragments). Electron microscopy was used to verify the purity of sarcolemmal sheets (Fig. 3). Preparation

of calmodulin-depleted

isolated

sarcolemmae:

Carafoli et al. (25) originally reported a method for obtaining a calmodulinfree heart sarcolemmal preparation by using a hypotonic treatment, immediately followed by a hypertonic wash in the presence of EGTA and by several hypotonic washes in the absence of the chelator. To obtain the calmodulin-depleted sarcolemmae we used an adaptation of the procedure described by Caroni and Carafoli (19). Sarcolemmal pellet was diluted: 1:lOO in 20 mM Tris , pH 7.4, kept 30 min on ice, and made hypertonic by the addition of the same volume of 1.2 M KCl, 20 mM Tris and 4 mM EC lA, pH 7.4. The hypertonic suspension was centrifuged for 30 min at 4000 x g and the pellet was washed three times with 20 mM Iris. The final pellet was resuspended in 0.16 M KC1 and 20 mM Tris, pH 7.4, and immediately used.

221

MYQMETRIUU I i”

3x:

20 mM 250 mM 1 mM

HOMOGENIZATION

+

TRIS SUCROSE EDTA

FILTRATION -0.

I I

\

Residue

FlItrate

-

CENTRIFUGATION 65Oxg. lOmln

)-----7

:

Pellet 1 HOMOG;NIZATION

; . :.

CENTRIFUGATION 160xg , 5mln

.**

7x

-*.

CELI

-------

1

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S &“W

&iOSTS 1

REMOVAL Of RESIDUAL r(CI 0.6M

CELLU.CAR CONTENT 30mln ,37’C 1

CENTRlFUGArlON

-------1

5 mln

16Oxg.

6 x i” . -0.

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I Supernatant --

-----T

JOOOxg , Smin

I I

4 Pe l/et # CENTRIFUGATION 4OOOxg,5

Supernatant -------I

min

/ Supernatant I

SARCOLEMMAl

Fig.

1.

Procedure

for

SHEETS

isolating

222

myometrial

sarcolemma

Fig.

2.

Light

myometrium. ganelles. tubule

microscopy. The

B_, Isolated with

A_, Isolated

black

spots

are

sarcolemma,

membrane

smooth-muscle

clusters which

cell

ghost

from

humal

of residual intracellular or appears as an empty flattened

veils.

-2ooam Fig.

3.

Electron

colemmal

sheet.

muscle

sarcolemma

represent metry versus

microscope Note

with

plasma

the morphological

of sarcolemma the extracellular

image

the typical

membrane

marker

enables side

(cross

section)

ultrastructural

and external

of sarcolemma

the recognition (ES).

223

of an isolated

appearance

of the

lamina.

and the

sar-

of the

smooth

Caveolae

caveolar

intracellular

side

asym(IS)

Calmodulin, from bovine purchased from Sigma. Determination

heart

(4390 U/mg prot;

was

of protein:

Protein was determined by the method of Lowry serum albumin as a standard. Solubilization

lot 31F-9615))

of isolated

sarcolemmae

et al. (26)

using bovine

in SDS:

Klingenberg (27) estimated the solubilizing power of an extensive series of detergents (e.g. SDS, Triton X-100, deoxycholate, etc) as a function of the detergent-to-protein ratio and reported that SDS was the most efficient. We prefered to solubilize the sarcolemmae with a high concentration of SDS for a short time period. Sarcolemmal sheets were partially solubilized in 100 mM SDS, 20 mM Tris , pH 7.4, for 5 min at room temperature. Sarcolemmal residues were removed by centrifugation. The solubilisate contained about 0.1 mg of protein per ml. Determination

of Ca

2+

-ATPase

activity:

The Ca2+ -ATPase activity was measured spectrophotometrically. ATP hydrolysis was monitored by following the production of P. from ATP. All assays were conducted at a temperature of 37OC, with lo-15 ug of membrane protein present in 2 ml of reaction medium. The inorganic phosphate was measured according to Roos et al. (28). 2+ The medium used for Ca -ATPase assays was prepared in an attempt to simulate, as much as possible,the intracellular composition. For calculating the tot cone ntrat.ons nec$ssary to obtain specified free concentra-+!!+ Zl+ tions of Ca , Mg , K and Na in the presence of EGTAand ATP we used a Fortran computer program and the stability constants used were the same as those used by the Fabiatos (16). We used basically two types of assay c nditions: either a standard medium (pCa 6) or media with various !?+ free Ca concentrations, the other ionic species being kept constant.*+The , free ionic2$oncentra$ons in th+” standard assay medium were (mM): Ca -0.001; Mg 0.4; pH 7.4; ionic strength , 0.5; K , 74; Na , 7.6; ATP, 0.13 M. It should be noted that the commercially available CaC12 was never used because of the uncertainty of the hydratation degree of this salt. Instead of CaC12, CaC03 was used as suggested by Ashley and Moisescu (29).

RESULTS Ultrastructure

and purity

of isolated

sarcolemma:

The identity, purity and morphologic integrity of the isolated sarcolemmae were suggested by light microscopy (Fig. 2B) and proved by electron microscopy (Fig. 3). Sarcolemmal sheets isolated from human uterine smooth muscle consist, exactly like the in situ sarcolemma, of non-caveolar regions (portions of sarcolemma devoid of caveolae) alternating with

224

caveolar domains. The caveolae represent the only reliable morphological marker for sarcolemma because the intracellular membranes are completely devoid of caveolae. Our procedure for separating the sarcolemma resulted in the isolation of plasma membrane together with the external lamina (glycocalyx) as shown in Fig. 3. The average protein yield of the sarcolemma1 fraction was l-2$ of the initial homogenate. Since the ratio of cell surface area to cell volume is 5-10 times greater for uterine smooth muscle cells than for cardiac or skeletal muscle fibers, the contamination of sarcolemmal preparations with intracellular components is ultimately a minor problem under our working conditions. In any case, the electron microscopy or recognizible constituents showed the absence of nuclei, mitochondria, of the contractile apparatus (e. g. myofilaments , dense bodies) in the final sarcolemmal pellet. Contamination with sarcoplasmic reticulum elements unlikely because of the sedimentation (microsomal vesicles) seems, a priori, procedure (Fig. 1). To asses the pusification of isolated sarcolemmae the enzymatic activity of sarcolemmal Ca -ATPase was measured in cell homogenates, cell ghosts and final sarcolemmal fraction, at pCa 6, using the standard assay medium (see Materials and Methods). An increase of the enzyme specific activity paralleled the separation of sa.rco~+ mmae during the isolation procedure. If we consider the high-affinity Ca as a marker for sarcolemma -ATPase then the sarcolemmal fraction appeared purified 12-fold over the cell homogenate and 4-fold over the cell ghosts. Identification

of the high Ca

2+

-affinitg

sarcolemmal

ATPase:

Fig. 4A shows that the activity of detergent-solubilized ATPase, determined in an assay medium which mimics the in situ microenvironment of the enzyme, 2+ is strongly dependent on the free Ca The maximum specific concentration. activity of the enzyme, about $,Onmol of Pi/mg of protein/min, was found between 0.8 and 5 uM free Ca , which ‘s functionally relevant for the 2: ,p;;;,utT,t;v;;;eez;t; ;;3yrne as a Ca -extrusion pump. The high Ca2+F+ -ATPase is expressed by its apparefl+t K (Ca ) of 0.25 -+ 0.04 pM (Fig. 4B). The dependence of sarcolemmal Ca -AyPase activity on the ATP concentration was. determined at pCa 6. When the specific activity was plotted against the free ATP concentration, varied from 10 PM to 400 pM, a rectangular hyperbola was yielded. Hanes plots resulted in an apparent Km(ATP) of 20 PM. 2+ no specific inhibitor of plasma membrane Ca -ATPase is availait may be useful to mention here that the inhibition by low concenis now generally accepted as a functional marker for of plasma membranes (17). Particularly for my$le cells, orthovanadate is useful in distingu’ y+hing the sarcolemmal Ca -ATPase from the sarcoplasmic reticulyy Ca -ATPase because the orthovanadate sensitivity of sarcolemmal Ca -ATPas2+is much higher, with a K. of about 0.6 pM 09). Fig. 5 shows that Ca -ATPase of smooth muscle sarcolemma is very sensitive to vanadate, half maximal inhibition being observed at 0.8 phj+vanadate. The concentration of 2 pM vanadate inhibited about 70% of Ca -ATPase activity and 10 PM vanadate (not shown in Fig.5) Although ble (15),

225

0.2

.

. :i/

.

-9A et+ 1 Ill 0.25

u.5

c

2+ Fig. 4A. Dependence of sarcolemmal Ca -ATPase activity on pCa. samples were averaged to obtain each point. Pig. 4B. Linew5$ver-Burk plot corresponding to points in Fig. 4A. apparent Km(Ca ) of 0.25 + 0.04 uM can be calculated.

100

Five An

. \

,’ ..= =

1 50 ;-_ .z CL

l \ ; ‘\.

.

\ 1

‘L. I

I

1

2

3

G

I

I

2+ -ATPase. The enzyme Fig. 5. Vanadate inhibition of sarcolemmal Ca activity was estimated in the standard assay medium (pCa 6) supplemented the average of 3-5 samples. The 100% with Na VO . Points represent activity3cor?esponds to 1.4 umol Pi/mg protein/h.

226

2.4 -ATPase of skeletal muscle sarproduced the complete inhibition. Ca vana(vanadate) of 10 PM (30) and the K coplasmic ?e+ticulum has a K uptake by sarc 161 p asmic reticulum vesicles isolated Gr! date) of Ca smooth muscle is 12 pM (12). Sarcolemmal

high Ca

2+

-affinity

ATPase

versus

interfering

ATPases:

2+ 2+ To differentiate the sarcolemmal Ca -ATPase and the “basal” MJ_ ATPase the enzymatic activity was measured in the virtual absence of (pCa 9) but in the presence of a totp+Mg concentration of 3.95 mM ;;;zeC$;+ : 0.5 mM). The remaining basal Mg -dependent ATPase activity was lo-12 nmol of P. eleased/mg of protein/h, namely less than 1% of p+e da’+ -ATPase act9+ity at pCa 6. Apparently, the Mg fully stimulated dependent ATPase (responsive to Mg alone), if present in the smooth muscle sarcolemma as a separate enzyme (9, 24, 31), was not extracted the short SDS treatment that we used to solubilize :;;~“,th~o~~~nbr;J;a% -+,TPase. The possible existence of a nonspecific loqyaffinity Ca or Mg ATPase did not interfere with the assay of high ATPas 2+because we used (very) low concentrations of free Ca *+-affinity and/or free Mg to satisfy the high-affinity enzyme without activating Ca the low-affinity enzyme (s) (32). Th: ou+abain-sensitive component of the ATPase activity was defined as Na -K -ATPase. However, ouabain 0.1 mM did not produce a significant inhibition of the ATPase activity. In 4 experiments (n=l2) the inhibitp+n was 6 2 1.8%. This was routinely subtracted to obtain the “true” Ca ATPase activity. The ATPase activity was not affected by 5 mM NaN and, therefore, the mitochondrial ATPase contamination (if any) was negliiible. Also, mypsin ATPase contamination was unlikely because the sarcolemmal high CaLr -affin&y ATPase activity of smooth muscle has a pH optimum of 7.4 and the Mg -ATPase activity of smooth muscle myosin exhibits two pH optima at acidic (pH 5) and alkaline (pH 9.5) pH values (33). Presence

and effects

of calmodulin

in smooth muscle

sarcolemma:

Fig. 6 s.&ows the effect, over 4 orders of mag$tude, of increasing the concentration on the solubilized Ca free-Ca -ATPase. The sigmoidal resultant suggests the participation of (an) allosteric effector( Anyway, on the basis of current knowledge, it seems attractive to determine the effect of calmodulin on this ATPase. Table I provides evidence for the existence of calmodulin in smooth muscle sa colemma and for the activator role of calmodulin on the sarcolemmai r?+ Ca -ATPase. Although there is very recent evidence that trifluoperazin5+ at high concentrations (100 uM or more) can inhibit the calmodulin-free Ca ATPase (17, 34), the fact should be noted that we used low concentrations of trifluoperazine, which are anticalmodulinic and do not inhibit the enzyme itself (17, 34). Data in Table I suggests that the concentration of calmodulin in (isolated) smooth muscle sarcolemma would be about 0.1% of sarcolemmal proteins.

227

7i I

/*-

.

/

\

..

.

.

tl 0 -

0.5 -

I

/

,_-•‘.

-2

ILL 8

7

6

5 I

1

8

1

6

5

ICI

2+ Fig. 6. Ca -ATPase activity of sarcolemmal solubilisate expressed as a function of pCa: v, ATPase activity ft+given pCa; V, maximal ATPase activity obtained at saturating free-Ca concentration. The same type of experiment as shown in Fig. 4A, but extended. Three samples were averaged to obtain each point. A Hill plot of the data is inserted: ordinate, log v/V-v; abscissa, pCa. The slope resulted in a Hill coefficient of 1.4.

Table I. Effects of calmodulin (CaM) removal, CaM addition and of anti-C% concentrations of trifluoperazine (TFP) on the activity of sarcolemmal Ca ATPase Ca Conditions

2+

-ATPase activity nmol Pi/mg/h

S arcolemma Sarcolemma + 25 nM TFP Sarcolemma + 50 nM TFP CaM-depleted sarcolemma CaM-depleted sarcolemma + CaM 14 ng/lOO ng protein Cal&depleted sarcolemma + CaM 87 ng/lOO pg protein

172 74 21 26

57 88 85

45

74

156

9

2+ -ATPase activity was determined in the standard assay medium at pCa Ca 6. The specific activities of 3 sarcolemmal preparations were averaged.

228

-

Free

ca*+p

2+ -ATPase solubiliaed from Fig. 7. Calmodulin (CaM) activation of Ca smooth muscle sarcolemma. Enzyme activity was measured in the presence (0) and in the absence (A) of endogenous calmodulin. Standard errors of media from 8-10 determinations on 5 different samples are’indicated. When trifluoperazine (TFP) 25 uM (.) was used as a calmodulin antagonist, only 3 determinations were averaged. 2+ Calmodulin activates the Ca -ATPase also after solubilization from sarcolemma (Fig. 7), a finding that suggests a direct interactio q+between the -ATPase activator and the enzyme. The analogous behaviour of the Ca activity of the isolated sarcolemma (Table I) and of the detergent-solubilized enzyme (Fig. 7) shows that the two activities are expressions of the same enzyme located in the sarcolemma. Calmodulin had a marked effect on t&e kinetic parameter3+of Ca2+-ATPase by shifting the enzyme from low Ca -affinity to high Ca -affinity and increa@g the V In the absence of endogenous calmodulin, the apparent K (Ca ) of 0.2?&.04 uM was shifted to about 1 PM and the V was conce%?%tion d&-eased by about10 times. The use of anticalmodulinic (25 PM) of trifluoperazine confirme d2ihe specific involvement of calmodulin in the regulation of sarcolemmal Ca -ATPase. Last but not least, the activator role of calmodulin was clearly suppoted by control experiments with exogenously added calmodulin. The addition of exogenous calmodulin 150 &ml to c*odulin-depleted preparations of solubilized enzyme increased the Ca -ATPase activity about 6 times, at pCa 5. It is noteworthy that our findings meet clearly the set of criteria proposed by Cheung (22) . e whether calmodulin regulates a certain biological reaction. Thus, :;,“r,c,i;+ -ATPase of human smooth muscle sarcolemma ap ears as a member 9+ of the now well-established group of plasma membrane Ca -pump ATPases which interact directly with calmodulin.

229

DISCUSSION results reported here resolve the presumptive Ths+ -extrusion pump ATPase in the smooth muscle Ca by our first report (2). Comparison plasma

of smooth muscle

membrane

Ca

2+

sarcolemma

-ATPases

Ca

well-known

2+

logical existence of a sarcolemma, suggested

-ATPase

as Ca

2+

with other

-extrusion

pumps:

2+ Table II shortens the “Discussion ” because it shows clearly that Ca ATPase isolated from smooth y+uscle sarcolemma has the properties yay+cted for the. functional Ca -extrusion pump req ired by the intracellular homeostasis in smooth must lo?+.Similar high Ca ‘+-affinity sarcolemmal (Ca ) of 0.25 + 0.17 uM or 0.31 20.07 uM ATPases with an apparent were isolated from the muscle of nonpregnant monkey myometrium and from the smooth muscle of pregnant monkey myvetrium, respectively (36). Wuytack and Casteels (37) demonstrated a Ca -ATPase activity in the microsomal fraction of porcine coronary artery smooth muscle: the enzyme

Table II. Characteristics sarcolemma ’ compar’son % as active Ca -pumps +4

Human erythrocyte membrane

Par,j!+neters of -ATPase Ca Km(Ca2+)

uM

Substitutes

for

Hill

Ca

uM

coefficient

Direct interaction with calmodulin Kl/2

of vanadate

Dog heart sarcolemma

0.3 2+

Specific activity (nmol Pi/mg/min) Km (ATP)

2+ of Ca -ATPase from human smof+th muscle with other plasma membrane Ca -ATPases

uM

pH optimum +) The characteristics are tes - 13,15,17,18,20,25,35 *) Parameter not discussed

0.2

Human smooth muscle s arcolemma

- 0.4

0.25

No

No

No

22

142

20

*

30

20

1.3

*

1.4

Yes

Yes

Yes

0.6

0.7

0.8

7.4

7.3

7.4

taken from the following references: ; heart sarcolemma - 17,19,20. in the above-mentioned references.

230

known

erythrocy-

had a sp$cific activity of abot& 20 nmol ATP/mg protein/min, a relatively -affinity (K for Ca 1.17 + 0.15 pM) and a Hill coefficient of low Ca 1.23. However, thes? authors did not consider the possible effect of calmodulin. High-affinity

Ca

in smooth muscle

2-l-ATPase

and ATP-driven

Ca

2+

transport -

sarcolemma:

to define According to Pennisto@5) there are three fundamental criteria -pump: 1) that p+e pump be of plasma membrane a plasma membrane Ca origin; 2) that the pump have a 3) that the pump show a high Ca Penniston noted (15L+that , in practice, it is not alwfzs possible -affinity ATPase and high Ca s&ate both high Ca of the literature on plasma membrane Ca ~~~~~~~~~~~~y~~~~a~~~~sh~~~t~~~~~~f~~~~s~~+finding of transport -was reported. This is also our case, 2+ 2-t transport (Ca uptake) has been detected in various An ATP-driven Ca microsomal fractions isolated from smooth muscles (8-12, 37-39). A.l&hough there is a large variation of the kinetic parameters reported p+r Ca transport in smooth muscle microsomes (the initial rate of Ca uptake in in the range of 0.8-10 nmol,$ftmgprotein/min and the half maximal activation the domain of l-10 uM Ca ) a reasonable agreement with our findings is obvious. Moreover, reLc+ently , Morel et al. (39) suggested that the cal-pump associated with the microsomal fraction modulin-s timulated Ca isolated from rat aorta is derived from the plasma membrane. Calmodulin

regulation

of sarcolemmal

Ca

2+

-pump ATPase:

2+ involvement of calmodulin in the activation of sarcolemmal Ca ests the existence of a self-regulating mechanism of the homeos&sis in smooth muscle. Calmodulin is activated ~ZZ1,“,5~lZeCYaY re ches or exceeds micromolar;F+oncentration when the intracellular Ca % (22), thereby activating the Ca -extrusion ATPase. As Ca returns to the submicromolar steady-state level by pumping through the sarco&mma or by uptake into sarcoplasmic reticulum the active calmodulin-Ca thus decreasing the enzyme activity to its ATPase complex dissociates, basal level. The direct

REFERENCES I)

Popescu, L .M. & Diculescu, I. (1975). Calcium in smooth muscle sarcoplasmic reticulum in situ. Conventional and X-ray analytical electron microscopy. J. Cell Biol. 67, 911-918.

231

2)

Popescu, L.M. (1977). Cytochemical study of the intracellular calcium distribution in smooth muscle. In Excitation-Contraction Coupling in Smooth Muscle (ed. R. Casteels, T. Godfraind, J.C. Ruegg), pp. 1323, Elsevier/North-Holland, Amsterdam.

3)

Somlyo, A.P., Somlyo, A.V. & Shuman, H. (1979). Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm. J. Cell Biol. 81, 316-335.

4)

Popescu, L.M., de Bruijn, W.C., Zelck, U. & Ionescu, N. (1980). Intracellular distribution of calcium in smooth muscle: facts and artifacts. Correlation of cytochemical, biochemical and X-ray microanalytical findings. Morphology and Embryology (Bucharest) 26, 251258.

5)

Popescu, L .M. & de Bruijn, W.C. reticulum of smooth muscle. X-ray muscle fibres. In 10th International vol. 3, pp. 385-386, Hamburg.

6)

van Breemen, C . , Aaronson, P. & Loutzenhiser, calcium interactions in mammalian smooth muscle. 30, 167-208.

7)

Casteels, R. & van Breemen, C. (1975). Active and passive across cell membrane of the guinea-pig taenia coli. Pflugers 359, 197-207.

8)

M.G. & Worcel, M. (1976). Calcium Rangachari, P. K. , Pernollet, uptake by myometrial membranes: effect of A23187, a calcium ionophore. Eur. J. Pharmacol. 40, 291-294.

9)

Janis, R. A. , Cy+nkshaw , D.J. & Daniel, E.E. (1977). Control intracellular Ca activity in rat myometrium. Am. J. Physiol: Endocrinol. Metab. Gastrointest. Physiol. 1, C50-C58.

(1982). Calcium in the sarcoplasmic microanalysis of oxalate-treated Congress on Electron Microscopy,

R. (1979). Pharmacol.

SodiumRev.

Ca fluxes Arch.

of

10)

Ackerman, stimulated is blocked

K.E.O. & WikstrBm, M.K.F. (1979). (Ca*++ Mg2+)ATPase activity of rabbit myometrium plasma membrane by oxytocin. FEBS Lett. 97, 283-287.

11)

Wuytack, F. , De Schutter, G. & Casteels, R. (1980). F+he eff2$t of calmodulin on the active calcium-ion transport and (Ca + Mg )dependent ATPase in microsomal fractions of smooth muscle compared with that in erythrocytes and cardiac muscle. Biochem. J. 190, 827831.

12)

Wibo, M., Morel, N . & Godfraind, T . (1981). Differentiation pumps linked to plasma membrane and endoplasmic reticulum microsomal fraction from intestinal smooth muscle. Biochim. Acta 649, 651-660.

232

2+ of Ca in the Biophys.

13)

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