Determination of calcium transport and phosphoprotein phosphatase activity in microsomes from respiratory and vascular smooth muscle

223

Biochimica et Biophysica Acta, 500 (1977) 223--234 © Elsevier/North-Holland Biomedical Press

BBA 28352

D E T E R M I N A T I O N OF CALCIUM T R A N S P O R T AND P H O S P H O P R O T E I N PHOSPHATASE ACTIVITY IN MICROSOMES F R O M R E S P I R A T O R Y AND V A S C U L A R SMOOTH MUSCLE

HOWARD SANDS, JAMES MASCALI and ELISABETH PAIETTA

Division of Molecular and Cellular Biology, National Jewish Hospital and Research Center, 3800 E. Cotfax Avenue, Denver, Colo. 80206 (U.S.A.) (Received April 18th, 1977)

Summary 1. Calcium transport into microsomal vesicles of respiratory (tracheal) smooth muscle was characterized. This calcium transport was ATP dependent and stimulated b y the presence of the oxalate ion. The magnitude of transport was similar to that reported for microsomes from other types of smooth muscle. 2. Bovine and rabbit, heavy and light microsomes were isolated from respiratory (tracheal) and vascular (aortic) smooth muscle. Preincubation of these vesicles with cyclic AMP and protein kinase did not alter the transport of calcium into the vesicles. There was no evidence of phosphate incorporation into microsomal membrane proteins. Similar results were obtained if phosphorylase b kinase replaced the combination of cyclic AMP and protein kinase during the preincubation. 3. The phosphoprotein phosphatase activity of cardiac sarcoplasmic reticulum and smooth muscle microsomes was determined. The activity of this enzyme was found to be several-fold less in the cardiac sarcoplasmic reticulum than in various smooth muscle microsome preparations.

Introduction

The mechanisms b y which drugs with ~-adrenergic activity (such as epinephrine and isoproterenol) produce relaxation of smooth muscle and positive inotropic and chronotropic effects in cardiac muscle are unknown. The involvement of cyclic AMP in mediating the action of these drugs has been suggested [1]. The hypothesis that many, if not all, of the actions of cyclic AMP in mammalian systems are mediated via activation of cyclic AMP dependent-protein Abbreviation : EGTA, ethyleneglycol-bis(fl-aminoethylether)N,N'-tetracetic acid.

224 kinases [2] has stimulated a search for physiologically significant substrates for these enzymes. Recently it has been suggested by Kirchberger and co-workers that cyclic AMP stimulated phosphorylation of cardiac sarcoplasmic reticulum results in the enhanced transport of calcium into the vesicles [3--8]. This enhanced calcium transport into the sarcoplasmic reticulum causes an increase in the rate of relaxation of the myocardium which characterizes the cardiac response to epinephrine [9]. Kirchberger and co-workers also found similar effects of cyclic AMP and protein kinase in slow skeletal muscle (a tissue whose rate of relaxation is also increased by epinephrine) but not in fast skeletal muscle (a tissue whose rate of relaxation is decreased by epinephrine) [8]. However, Schwartz et al. [10] while confirming the observations on cardiac and slow skeletal muscle did find phosphorylation and enhanced calcium transport in fast skeletal muscle. These same workers found that the enzyme phosphorylase b kinase also phosphorylated and enhanced calcium transport into the sarcoplasmic reticulum of cardiac and slow as well as fast skeletal muscle. They suggest that a complex relationship exists between relaxation, calcium uptake, phosphorylation and the glycogenolytic enzyme system. The role cyclic AMP plays in calcium transport into smooth muscle is unclear. Cyclic AMP has been reported to: increase the binding of calcium in aortic microsomes [11,12]; have no effect [ 13] or stimulate the calcium uptake of myometrial microsomes [14]; and stimulate the uptake of calcium in intestinal smooth muscle microsomes [15]. Recently Clyman et al. [16] reported that cyclic AMP and protein kinase did not alter calcium uptake into, or catalyze the phosphorylation of microsomes prepared from human umbilical artery, and Allen has reported a similar lack of an effect of protein kinase and cyclic AMP on calcium binding of canine aortic microsomes [17]. This report describes studies on the effect of cyclic AMP and protein kinase on calcium transport into vascular and respiratory smooth muscle microsomes prepared from bovine and rabbit tissues. In no case did the combination of cyclic AMP and protein kinase enhance calcium transport. We also measured the phosphoprotein phosphatase activity of the smooth muscle microsome preparations and found it to be significantly higher than that measured in cardiac sarcoplasmic reticulum. Methods I. Sources and preparation o f tissue a. Bovine tracheal and aortic s m o o t h muscle. Bovine trachea and aorta were obtained within half an hour of slaughter of animals and transported to the laboratory in ice-cold isotonic sucrose. The smooth muscle layers of the trachea were stripped off, minced and washed in isotonic sucrose. All preparative steps were carried out at 0--4 ° C. The tracheal smooth muscle was disrupted in isotonic sucrose with a Potter-Elvehjem homogenizer using a motor-driven loosefitting Teflon pestle. Bovine aorta was trimmed free of loose connective tissue and adherent fat, minced and washed in isotonic sucrose. The tissue was homogenized for 15 s three times in a Polytron at a rheostat setting of 5 with a rest interval of 15 s. The resulting homogenates from the trachea and aorta were each filtered through two layers of cheesecloth. Microsomal vesicles were prepared from these suspensions.

225

b. Rabbit tracheal and aortic smooth muscle. Large male New Zealand white rabbits were killed by anoxia, achieved by exposing the rabbit to 100% CO2 in an enclosed chamber. The trachea and aorta were removed and immediately placed in isotonic sucrose. The tissues were stripped free of any adherent fat and loose connective tissue and washed in isotonic solution. Aorta and tracheal tissues were minced and placed in isotonic sucrose and homogenized for 15 s three times in a Polytron with a rheostat setting of 5 with a rest interval of 15 s. The homogenates were filtered through two layers of cheesecloth and microsomal vesicles prepared from the resulting suspensions. c. Rabbit cardiac muscle. Rabbit heart tissue was prepared in the same manner as rabbit trachea and aorta, except that 10 mM sodium bicarbonate with 5 mM sodium azide, pH 7.0, was used as the washing and homogenizing solution. H. Preparation o f microsomes a. Light microsomes from smooth muscle. Microsomal vesicles were prepared by a modification of the m e t h o d of Fitzpatrick and co-workers [ 18,19]. Aortic and tracheal suspensions were centrifuged at 1500 X g for 20 min and the supernatant fraction then centrifuged at 27 000 X g for 10 min yielding a second supernatant fraction which was centrifuged at 27 000 rev./min for 70 min in a type 30 Spinco rotor. The resulting pelleted microsomal vesicles are subsequently referred to as the "light microsomal" fraction. Microsomes were suspended in 3--6 ml of isotonic sucrose (Dounce tissue grinder, with a tight fitting pestle). b. Heavy microsomes from s m o o t h muscle. The procedure used is a modification of that described by Harigaya and Schwartz [20]. Aortic and tracheal suspensions were centrifuged at 8700 X g for 20 min, yielding a supernatant fraction which was centrifuged at 37 000 X g for 30 min. The resulting pelleted microsomal fraction was termed "heavy microsomes". The pellet was suspended in the same manner as the light microsomal pellet and was ready for immediate use. c. Sarcoplasmic reticulum from cardiac muscle. The procedures of Harigaya and Schwartz [20] were used for the preparation of heart sarcoplasmic reticulum. III. Assay o f calcium transoort Calcium transport was measured at 37°C in an incubation mixture containing 20 mM histidine (pH 7.2), 5 mM MgC12, 110 mM KC1, 10 mM NaC1 and 5 mM ATP {sodium salt). When present in the incubation the sodium oxalate concentration was 3.75 mM and that of sodium azide was 5 mM. Present in the incubation were either 20 pM CaC12 (referred to as "high calcium") or a C a . EGTA buffer that gave a final concentration of ionized Ca 2÷ of 0.75 /aM [4], {referred to as "low calcium"). Approximately 2 pCi of 4SCa were added to the incubation mixture. The calcium transport reaction was started by the addition of microsomal protein to a final concentration of 160--1400 pg per 10 ml. Calcium transport activity was measured by passing aliquots of the reaction mixture through millipore filters (type HA) at appropriate time intervals. Filters were washed im-

226 mediately with 2 ml of 40 mM histidine (pH 7.2), 100 mM KC1 and 10 mM NaCl. Filters were dissolved in ethylene glycol m o n o m e t h y l ether before the addition of Aquasol (NEN). Radioactivity was measured in a liquid scintillation counter. Non-specific 4SCa2+ accumulation was calculated by filtering samples immediately after addition of protein to the reaction mixture, and these counts were subtracted from each sample.

IV. Preincubation conditions Microsomal protein (160--1400 pg protein) was incubated for 10 min at 30°C in a 0.75 ml final volume of an incubation mixture containing 0.026 mM sucrose, 20 mM histidine (pH 7.2), 5 mM MgC12,55 mM KC1, 10 mM NaC1 and 5 mM Na • ATP, in either the presence or absence of 10 -3 mM cyclic AMP and either 50 pg of cyclic AMP-dependent protein kinase (isolated from either bovine tracheal smooth muscle or bovine cardiac muscle); or 108 pg of phosphorylase b kinase, according to the method of Schwartz et al. [10]. Upon termination of the 10 min preincubation period the calcium transport reaction was started by the addition of the pre-incubated microsomes to the reaction buffer as previously described. V. Detection of 32p incorporation into microsomes Microsomes were incubated with protein kinase according to the method of Sands et al. [21]. The ATP concentration of the incubation mixture was 5 mM and included 2 pCi of [~/-32P]ATP. Similar experiments were performed using phosphorylase b kinase [10] instead of protein kinase. The incorporation of 32p into the trichloroacetic acid precipitable proteins of the microsomes was determined by the method of Sands et al. [21]. Polyacrylamide gel electrophoresis according to Weber and Osborn [22] was utilized for the detection of the incorporation of 32p into the sodium dodecyl sulfate solubilized proteins. VI. Phosphoprotein phosphatase assay Protein phosphatase activity was measured on the basis of the release of radioactive orthophosphate from 32P-labelled histone as substrate. For the preparation of phosphatase substrate, calf t h y m u s mixed histone at a concentration of 5 mg/ml was phosphorylated with [~,-32P]ATP (4000 cpm/ pmol) by partially purified cyclic AMP-dependent protein kinase. 1.5 ml of the incubation mixture contained 4 mg of histone, 135 pg of protein kinase, 50 mM sodium glycerol/phosphate (pH 6.5), 10 mM sodium fluoride, 2 mM theophylline, 3.3 mM EGTA, 10 mM magnesium chloride and 10 -3 mM cyclic AMP. After a 30 rain incubation at 30°C, the reaction mixture was dialysed overnight against a total volume of 12 1 deionized water at 4°C, with three changes of dialyzing buffer. The protein phosphatase assay was performed according to the method of Maeno and Greengard [22], in a total volume of 0.3 ml, containing the preincubation buffer used in the calcium transport assay (0.708 mM sucrose, and 15--180 pg of microsomal protein). The reaction was started by addition of 133 #g phosphorylated histone (containing 2--2.5 • l 0 s cpm) and was terminated after 0, 2, 5 and 10 min incubation at 37°C by addition of 0.2 ml 30% trichloroacetic acid. After the addition of 0.3 ml 0.625% bovine serum albu-

227 min as a carrier for the precipitation, the protein was removed by a 2 min centrifugation at 12 000 X g. The molybdate extraction procedure was used to separate the orthophosphate from the deproteinized supernatant fraction [24]. The radioactivity in 1 ml of the organic solvent phase was determined in a liquid scintillation spectrophotometer. Blank values, i.e., the counts obtained in zero-time samples, were subtracted. The specific activity of the radioactive ATP used as a precursor in the protein phosphorylation reaction was used for the calculation of the a m o u n t of inorganic phosphate released from the substrate protein. The specific activity of the phosphatase was defined as pmol of 32p released per mg of protein per minute.

VII. Other m e t h o d s Protein was determined by standard biochemical procedures [25,26]. Experimental observations were compared by the Student's t test or, when appropriate, the paired Student's t test. The results are reported as the means and the standard errors of the means. P values of 0.05 and less were regarded as significant. VIII. General c o m m e n t s All experiments were repeated at least three times; there was some quantitative variability but very little qualitative variation. All assays of calcium transport were done at eight time points; however, in order to simplify presentation only the data from a single time point are presented in the tables. Because there are uncertainties as to the nature of the nonmitochondrial subcellular preparations derived from smooth muscles [27] the term microsome is employed rather than sarcoplasmic reticulum. IX. Sources o f chemicals and e n z y m e s Tracheal smooth muscle protein kinase was prepared according to the procedure of Sands et al. [21]. Phosphorylase b kinase and bovine cardiac protein kinase were obtained from Sigma, as was Type II histone. The [~t-32P]ATP was purchased from ICN Chemical and Radioisotope Division and the 4SCa from New England Nuclear Corp. Results Characterization o f calcium transport in bovine respiratory and vascular s m o o t h muscle microsomes The rate of calcium transport into the light microsomes obtained from bovine tracheal smooth muscle is illustrated in Fig. l a . Calcium transport was ATP dependent and markedly enhanced by the addition of oxalate. The addition of sodium azide, an inhibitor of calcium transport in mitochondria, had little effect. Essentially similar results were obtained using heavy microsomes obtained from bovine tracheal smooth muscle. While the qualitative pattern was the same, (Fig. l b ) the specific activity was considerably higher than that found for the light microsomes. Experiments designed to characterize the calcium transport in light and heavy microsomes obtained from bovine aortic smooth muscle yielded qualitatively similar results (data n o t shown).

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TABLE

I

EFFECTS OF CYCLIC AMP AND PROTEIN KINASE TRACHEAL SMOOTH MUSCLE MICROSOMES

ON CALCIUM

TRANSPORT

INTO

BOVINE

Heavy and light microsomes were prepared from bovine tracheal smooth muscle and preincubated with and without cyclic AMP and smooth muscle protein kinase as described under Methods. Calcium transp o r t i n t o t h e m i e r o s o m e s w a s m e a s u r e d i n t h e p r e s e n c e o r a b s e n c e o f 3 . 7 5 m M s o d i u m o x a l a t e . T h e calcium concentration d u r i n g t h e t r a n s p o r t a s s a y w a s e i t h e r 2 0 p M o r 0 . 7 5 t i M . T h e d a t a a r e r e p o r t e d as t h e mean ± S.E.; values are nmol Ca2+/mg protein per 15 rain, and n = the number of observations. Light microsomes 2 0 o M C a 2+

0 . 7 5 p M C a 2+

2 0 ]JM C a 2+

0 . 7 5 p M C a 2+

29.9 (± 5 . 1 )

38.5 (± 1 0 . 6 )

63.8 (-+ 1 3 . 6 )

101.5 (+_ 2 4 . 3 )

Preincubation

13.0 (+- 3 . 5 )

26.8 (+- 8 . 6 )

51.1 (-+ 7 . 5 )

78.2 (_+ 2 2 . 7 )

Preincubation + cyclic AMP + protein kinase

13.7 (-+ 2 . 0 )

25.2 (± 9 . 5 )

53.4 (± 8 . 2 )

80.1 (_+ 2 4 . 8 )

--

17.0 (± 4 . 0 )

30.8 (± 5 . 5 )

34.8 (± 9 . 0 )

Preincubation

8.0 (± 2 . 7 )

14.3 (+- 3 . 5 )

25.5 (± 3 . 6 )

25.9 (_+ 6 . 9 )

Preincubation + cyclic AMP + protein kinase

9.0 (± 2 . 4 )

14.1 (+_ 4 . 7 )

22.7 (± 5 . 6 )

31.8 (± 1 3 . 1 )

3

3

4

4

Plus oxalate

Minus oxalate

n

Heavy microsomes

229 T A B L E 11 EFFECTS OF CYCLIC AMP AND PROTEIN AORTIC SMOOTH MUSCLE MICROSOMES

KINASE ON CALCIUM TRANSPORT

INTO BOVINE

Heavy and light microsomes were prepared from bovine aortic smooth muscle and preineubated with and without cyclic AMP and smooth m u s c l e p r o t e i n k i n a s c as d e s c r i b e d u n d e r M e t h o d s . C a l c i u m t r a n s p o r t i n t o t h e m i c r o s o m c s w a s m e a s u r e d in t h e p r e s e n c e o r a b s e n c e o f 3 . 7 5 m M s o d i u m o x a l a t e . T h e c a l c i u m c o n c e n t r a t i o n d u r i n g t h e t r a n s p o r t a s s a y w a s 0 . 7 5 DM. T h e d a t a axe r e p o r t e d as t h e m e a n ± S.E. o f t h r e e o b s e r v a t i o n s . V a l u e s are n m o l C a 2 + / m g p r o t e i n p e r 1 5 r a i n .

Plus o x a l a t e Preincubation

Preincubation + cyclic AMP + protein kinase Minus oxalate Preincubation Freincubation

+ cyclic AMP + protein kinase

Light microsomes

Heavy microsomes

3 6 . 4 (± 1 5 . 7 ) 2 6 . 3 (-+ 1 0 . 2 ) 2 7 . 1 (+ 2 . 2 5 )

7.9 (± 0 . 5 ) 7.1 (± 0 . 3 ) 7.3 (+ 1 . 2 )

4 . 0 (-+ 1 . 8 ) 1 1 . 1 (-+ 5 . 8 ) 1 0 . 4 (± 5 . 7 )

2 . 4 (± 0 . 1 ) 3.8 (± 0 . 8 ) 3.9 (± 1 . 0 )

Effect o f cyclic A M P and protein kinase on clacium uptake in bovine smooth muscle microsomes The effects of preincubating tracheal smooth muscle microsomes with cyclic AMP and protein kinase are shown in Table I. The results were qualitatively sin~ ilar regardless of whether light or heavy microsomes were used, and were independent of the calcium concentdation used in the transport assay. Preincubation alone resulted in a loss of calcium transport when compared with the nonpreincubated control. The presence of cyclic AMP and protein kinase during the preincubation did not alter the transport of calcium into the vesicles either in the presence or absence of oxalate. Calcium transport in bovine aortic smooth muscle light and heavy microsomes was also not altered by preincubation with cyclic AMP and protein kinase (Table II). In the vascular smooth muscle microsomes the decrease in calcium transport after preincubation in the absence of cyclic AMP and protein kinase was not as evident and in the absence of oxalate it appears that preincubation slightly increased the calcium transport. Another difference between the calcium transport of bovine tracheal and aortic smooth muscle was that the heavy aortic microsomes transported less calcium than did the light aortic microsomes. The opposite was seen in the tracheal preparations. Effect of cyclic A M P and protein kinase on calcium transport into rabbit cardiac and smooth muscle microsomes The ability of cyclic AMP and protein kinase to stimulate the transport of calcium into rabbit cardiac microsomes is shown in Table III. An increase of 15--300% was seen in every experiment. As a l r e a d y d e m o n s t r a t e d for the bovine tissues, calcium transport in microsomes isolated from rabbit vascular and respiratory smooth muscle was n o t affected by preincubation with cyclic AMP and protein kinase. Similar results were obtained using heavy and light microsome preparations. Effect of phosphorylase b kinase and bovine cardiac protein kinase on calcium transport into bovine aortic and tracheal smooth muscle microsomes The effects of phosphorylase b kinase and bovine cardiac protein kinase on

230

TABLE

III

EFFECT OF CYCLIC AMP AND PROTEIN KINASE ON CALCIUM TRANSPORT INTO CARDIAC SARCOPLASMIC RETICULUM AND TRACHEAL AND AORTIC SMOOTH MUSCLE MICROSOMES IN THE RABBIT Rabbit cardiac sarcoplasmie reticulum and heavy and light microsomes from aortic and tracheal smooth muscle were prepared and preincubated with and without cyclic AMP and smooth muscle protein kinase as d e s c r i b e d u n d e r M e t h o d s . C a l c i u m t r a n s p o r t i n t o t h e m i c r o s o m e s w a s m e a s u r e d i n t h e p r e s e n c e o f 3 . 7 5 mM sodium oxalate. The calcium concentration d u r i n g t h e t r a n s p o r t a s s a y w a s 0 . 7 5 laM. T h e d a t a a r e r e p o r t e d as t h e m e a n +_ S . E . ; n = n u m b e r o f o b s e r v a t i o n s , n.s. = n o t s i g n i f i c a n t . C a 2+ (nmol/mg protein per 5 min) Cardiac sarcoplasmic reticulum

Ca + (n mol/mg

protein per 15 rain)

Heavy microsomes

Light microsomes

Aorta

Trachea

Aorta

Trachea

l>reincubation

553 (+ 1 6 5 )

56 (± 2 4 )

35 (± 9 )

33 (± 1 0 )

45 (± 1 0 )

Preincubation + cyclic AMP + protein kinase

852 (+_ 2 2 4 )

51 (-+ l S )

32 (+ 1 4 )

41 (± 4 )

45 (+- 7)

n

7

3

3

3

3

Pvia paired l test

0.02

n.s.

n.s.

n.s.

n.s.

calcium transport into light microsomes isolated from bovine tracheal smooth muscle are shown in Table IV. Preincubation with these enzymes, like smooth muscle protein kinase, did not alter the calcium transport into smooth muscle mierosomes.

Phosphory lation experiments Experiments designed to detect the incorporation of 32p from [7-32p]ATP into microsomal proteins were performed. Smooth muscle microsomes were incubated with either protein kinase and cyclic AMP or with phosphorylase b kinase. After 10 min the reaction was terminated by the addition of trichloro-

TABLE

IV

EFFECTS OF PHOSPHORYLASE b KINASE AND CARDIAC PROTEIN KINASE TRANSPORT INTO BOVINE TRACHEAL SMOOTH MUSCLE LIGHT MICROSOMES

ON

CALCIUM

Light microsomes were prepared from bovine tracheal smooth muscle and preincubated with either cyclic A M P a n d c a r d i a c p r o t e i n k i n a s e o r p h o s p h o r y l a s e b k i n a s e as d e s c r i b e d u n d e r M e t h o d s . C a l c i u m t r a n s p o r t into the microsomes was measured in the presence or absence of 3.75 mM sodium oxalate. The calcium concentration d u r i n g t h e t r a n s p o r t a s s a y w a s 2 0 t i M . T h e d a t a a r e r e p o r t e d as t h e m e a n ± S . E . o f t h r e e o f four observations. Values are nmol Ca2+]mg protein per 15 rain. Cyclic AMP and cardiac protein kinase

Phosphorylase b kinase

Plus oxalate Preincubation + enzyme + preincubation

2 9 . 6 -+ 2 . 2 2 0 . 2 _+ 1 . 3 2 1 . 2 _+ 2 . 0

51.0 + 12.0 1 8 . 9 +_ 4 . 6 1 8 . 5 +_ 3 . 9

Minus oxalate Preincubation + enzyme + preincubation

17.1 + 0.0 10.4 ± 2.3 13.9 ± 1.0

1 2 . 2 _+ 1 . 7 14.1 ± 3.8

231

A. Light Microsomes

B. Heavy Microsomes

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Fig. 2. Phosphoprotein phosphatase activities f o u n d in r a b b i t s m o o t h m u s c l e m i c r o s o m e s a n d c a r d i a c s a r c o p l a s m i c r e t i c u l u m . E n z y m e a c t i v i t y w a s m e a s u r e d w i t h p h o s p h o r y l a t e d h i s t o n e as the substrate using the p r o c e d u r e s d e s c r i b e d u n d e r M e t h o d s . T h e d a t a are r e p o r t e d as t h e m e a n of t h r e e e x p e r i m e n t s -+ S.E. o, A o r t a ; ~, t r a c h e a ; e , H e a r t ; * p ~ 0 . 0 5 w h e n c o m p a r e d w i t h h e a r t .

acetic acid or sodium dodecyl sulfate. The trichloroacetic acid precipitate was collected and washed on a millipore filter. No increase in the incorporation of 32p into the precipitate after incubation with either enzyme could be detected (data n o t shown). The protein in the sodium dodecyl sulfate terminated reaction mix was resolved on sodium dodecyl sulfate polyacrylamide gels, the gels sliced into one mm sections and the 32p incorporated measured b y liquid scintillation counting. Again no increase in the incorporation of 32p into microsomal proteins by cyclic AMP and protein kinase could be detected (data not shown).

Phosphoprotein phosphatase activity The phosphoprotein phosphatase activity of the various microsomal preparations was measured under the same conditions as those of the phosphorylation preincubations. The results are illustrated in Figs. 2 and 3. As shown in Fig. 2, microsomes from cardiac muscle, as well as respiratory and vascular smooth muscle of the rabbit all contain phosphoprotein phosphatase activity. However, the phosphoprotein phosphatase activity was several fold higher in the smooth muscle microsomes than in cardiac microsomes. Similar or even higher levels of phosphoprotein phosphatase were found in bovine respiratory and vascular smooth muscle microsomes (Figs. 3a and b).

Inhibitors of the phosphoprotein phosphatase activity The phosphoprotein phosphatase activity of the smooth muscle microsomes could be inhibited b y 50 mM m o l y b d a t e (100%) and 20 mM phosphate (63%). The protein phosphatase inhibitor found in skeletal muscle [ 28] did not inhibit the smooth muscle microsomal phosphoprotein phosphatase when 10/~g were added to 8 pg of microsomal protein. Unfortunately, these agents could not be used to further the study of the effects of cyclic AMP and protein kinase on the

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F i g . 3. P h o s p h o p r o t e i n phosphatase a c t i v i t i e s f o u n d in b o v i n e s m o o t h m u s c l e m i c r o s o m e s . E n z y m e activity was measured with phosphorylated h i s t o n e as s u b s t r a t e u s i n g t h e p r o c e d u r e d e s c r i b e d u n d e r M e t h o d s . T h e d a t a are r e p o r t e d as t h e m e a n o f t h r e e e x p e r i m e n t s + S . E . o , A o r t a ; ;\ t r a c h e a .

calcium transport of smooth muscle microsomes. The divalent cationic inhibitors markedly interfered with the calcium transport process. Phosphate (20 mM), while inhibiting the phosphatase activity, could not substitute for oxalate in the calcium transport assay, and the protein inhibitor, besides being ineffective on the microsomal enzyme, also drastically decreased the activity of the exogenous smooth muscle protein kinase (data not shown). Discussion The data reported here represent an extension to smooth muscle of the work reported by a number of other investigators on the rble of cyclic AMP and protein kinase on calcium transport into both skeletal and cardiac sarcoplasmic reticulum [3--8,10]. It has been suggested that phosphorylation of sarcoplasmic reticulum membranes of cardiac and slow skeletal muscle is a mechanism to regulate calcium transport [3--8]. The major goal of this study was to extend the observation made on cardiac and skeletal muscle sarcoplasmic reticulum to smooth muscle systems. We present here a survey of the effects of the cyclic AMP dependent protein kinase in smooth muscle microsomes (heavy and light) from two types of smooth muscle (respiratory and vascular) of two species (bovine and rabbit). Since the characteristics of calcium transport of a respiratory smooth muscle have not previously been reported, it was first necessary to study these parameters. Our data indicate that the calcium transport into both heavy and light microsomes of bovine tracheal smooth muscle is essentially similar to that reported for the light microsomes of vas-

233

cular [18] and intestinal [19] smooth muscle. Once the characteristics of the calcium transport had been established for the various microsomal preparations, the effects of cyclic AMP and protein kinase could be explored. Preincubation with cyclic AMP and protein kinase did not affect the rate of calcium transport in any of the smooth muscle preparations studied: i.e., bovine and rabbit; aortic and tracheal; heavy and light microsomes. This observation was independent of the calcium concentration during the calcium transport assay and the tissue source of the protein kinase. The stimulating effect of cyclic AMP and protein kinase on calcium transport of rabbit cardiac sarcoplasmic reticulum served as a "positive c o n t r o l " in that we confirmed the results reported by others [3--8,10]. Calcium transport into smooth muscle microsomes was also n o t affected by phosphorylase b kinase, in contrast to the effect of this enzyme on cardiac and skeletal muscle sarcoplasmic reticulum [ 10]. In other experiments no evidence for the transfer of 32p from [7-32p]ATP to the microsoma! proteins by either protein kinase or phosphorylase b kinase could be demonstrated. These data from the several preparations reported here agree with those of Clyman et al. from the human umbilical artery [ 16]. The presence of phosphoprotein phosphatases in cardiac sarcoplasmic reticulum has been reported by Kirchberger and coworkers [29,30]. The data presented here indicate that smooth muscle microsomes contain more phosphoprotein phosphatase activity than cardiac sarcoplasmic reticulum. This phosphatase activity was inhibited by m o l y b d a t e and phosphate and not by the protein phosphatase inhibitor of skeletal muscle. Unfortunately we were not able to use these agents to selectively block phosphatase activity whilst maintaining calcium transport and protein kinase activity. To date no substance has been found which will affect only the phosphatase. At this time the physiological significance of the apparent lack of an effect of cyclic AMP and protein kinase, as well as the high phosphatase activity is not known.

Acknowledgement This work was supported b y research grant HL-14964 from the National Institutes of Health, United States Public Health Service.

References 1 2 3 4 5 6 7 8 9 10

R o b i n s o n , G . A . , B u t c h e r , R . W . a n d S u t h e r l a n d , E.W. ( 1 9 7 1 ) Cyclic AMP, A c a d e m i c Press, New Y o r k K u o , J . F . a n d G r e e n g a r d , P. ( 1 9 6 9 ) Proc. Natl. A c a d . Sei. U.S. 6 4 , 1 3 4 9 - - 1 3 5 5 K i r c h b e r g e r , M.A., T a d a , M., R e p k e , D.I. a n d K a t z , A.M. ( 1 9 7 2 ) J. Mol. Cell. Cardiol. 4 , 6 7 3 - - 6 3 0 K i r c h b e r g e r , M.A., T a d a , M. a n d K a t z , A.M. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 6 1 6 6 - - 6 1 7 3 T a d a , M., K i r c h b e r g e r , M.A., R e p k e , D.I. a n d K a t z , A.M. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 6 1 7 4 - - 6 1 8 0 T a d a , M., K i r c h b e r g e r , M.A. a n d K a t z , A.M. ( 1 9 7 5 ) J. Biol. Chem. 2 5 0 , 2 6 4 0 - - 2 6 4 7 K i r c h b e r g e r , M.A. a n d C h u , G. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 1 9 , 5 5 9 - - 5 6 2 Kixchberger, M.A. and Tada, M. ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 7 2 5 - - 7 2 9 Wiggers, C.J. a n d K a t z , L.N. ( 1 9 2 2 ) A m . J. Physiol. 5 8 , 4 3 9 - - - 4 7 5 S c h w a r t z , A., E n t m a n , M.L., Kaniike, K., Lane, L.K., van Winkle, W.B. a n d Bornet, E.P. ( 1 9 7 6 ) Biochim. Biophys. Acta 426, 57--72 11 Webb, R.C. a n d Bhalla, R.C. ( 1 9 7 6 ) J. Mol. Cell. Cardiol. 8 , 1 4 5 - - 1 5 7 12 B a u d o u i n - L e g r o s , M. a n d Meyer, P. ( 1 9 7 3 ) Br. J. P h a r m a c o l . 4 7 , 3 7 7 - - 3 8 5 13 Batra, S.C. a n d Daniel, E.E. ( 1 9 7 1 ) C o m p . B i o c h e m . Physiol. 3 8 A , 2 8 5 - - 3 0 0

234

1 4 Krall, J . F . , S w e n s e n , J . L . a n d K o r e n m a n , S.G. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 4 8 , 5 7 8 - - 5 8 8 15 A n d e r s s o n , R . a n d N i l s s o n , K. ( 1 9 7 2 ) N a t u r e 2 3 8 , 1 1 9 - 1 2 0 16 C l y m a n , R . I . , M a n g a n i e l l o , V . C . , L o v e l l - S m i t h , C.J. a n d V a u g h a n , M. ( 1 9 7 6 ) A m . J. P h y s i o l . 2 3 1 1074--1081 17 Allen, J . C . ( 1 9 7 7 ) B l o o d Vessels 1 4 , 9 1 - - 1 0 4 18 F i t z p a t r i c k , D . F . , L a n d o n , E . J . , D e b b a s , G. a n d H u r w i t z , L. ( 1 9 7 2 ) S c i e n c e 1 7 6 , 3 0 5 - - 3 0 6 19 H u r w i t z , L . , F i t z p a t r i c k , D . F . , D e b b a s , G. a n d L a n d o n , E . J . ( 1 9 7 3 ) S c i e n c e 1 7 9 , 3 8 4 - - 3 8 6 2 0 H a r i g a y a , S. a n d S c h w a r t z , A. ( 1 9 6 9 ) C i r c u l a t i o n R e s . 2 5 , 7 8 1 - - 7 9 4 21 S a n d s , H., M e y e r , T . A . a n d R i c k e n b e r g , H . V . ( 1 9 7 3 ) B i o e h i m . B i o p h y s . A c t a 3 0 2 , 2 6 7 - - 2 8 1 2 2 W e b e r , K. a n d O s b o r n , M. ( 1 9 6 7 ) J. Biol. C h e m . 2 4 4 , 4 4 0 6 - - 4 4 1 2 23 M a e n o , H. a n d G r e e n g a r d , P. ( 1 9 7 2 ) J. Biol. C h e m . 2 4 7 , 3 2 6 7 - - 3 2 7 7 24 P l a u t , G . W . E . ( 1 9 6 3 ) in M e t h o d s in E n z y m o l o g y ( C o l o w i e k , S.P. a n d K a p l a n , N . O . , eds.), V o l . 6, p 3 1 9 , A c a d e m i c Press, N e w Y o r k 2 5 L a y n e , E. ( 1 9 5 7 ) in M e t h o d s in E n z y m o l o g y ( C o l o w i c k , S.P. a n d K a p l a n , N . O . , eds.), Vol. 3, p. 4 4 7 A c a d e m i c Press, N e w Y o r k 2 6 I t z h a k i , R . F . a n d Gill, D.M. ( 1 9 6 4 ) A n a l . B i o c h e m . 9 , 4 0 1 - - 4 1 0 27 F o r d , G . D . ( 1 9 7 6 ) F e d . P r o c . 3 6 , 1 2 9 8 - - 1 3 0 1 2 6 C o h e n , P., N i m m o , G . A . a n d A n t o n i n , J . F . ( 1 9 7 7 ) B i o c h e m . J. 1 6 7 , 4 3 5 - - 4 4 4 29 T a d a , M., K i r e h b e r g e r , M . A . a n d Li, H.-C. ( 1 9 7 5 ) J. C y c l i c N u c l e o t i d e R e s . 1 , 3 2 9 - - 3 3 8 3 0 K i r c h b e r g e r , M . A . a n d R a f f o , A . ( 1 9 7 7 ) J. C y c l i c N u c l e o t i d e R e s . 3, 4 5 - - 5 3