Journal

of Molecular

and Cellular

Cardiac Adenylate of a Subcellular

Cardiology

7, 685695

Cyclase. I. Preparation and Characterisation Fraction Containing Catecholamine-Sensitive Adenylate Cyclase

B. JARROTT* Department

(1975)

of Physiology, (Received

AND GWYNNETH

Monash 28 August

University,

Clayton,

1974, accepted

M. PICKEN 3 168, Victoria,

4 November

Australia

1974)

B. JAXLROTT AND G. M. PICKFJN. Cardiac Adenylate Cyclase. I. Preparation and Characterisation of a Subcellular Fraction Containing Catecholamine-Sensitive Adenylate Cyclase. Journal of Molecular and Cellular Cardiology (1975) 7, 685-695. Differential centrifugation of homogenates of rat ventricles yielded subcellular fractions which were assayed for adenylate cyclase activity, as well as enzymes associated predominantly with certain intracellular structures. The distribution of adenylate cyclase activity paralleled only that of the marker enzyme for sarcolemma, 5’-AMPase. However, the heterogeneity of the particulate fractions made it impossible to ascertain the exact intracellular localization of adenylate cyclase. Histological examination of the fraction (PI) containing the bulk of the adenylate cyclase activity revealed large numbers of isolated myofibres. Purification of this fraction was effected by homogenizing it in a large volume of isotonic sucrose to disperse and dissolve the myofibrillar matrix. The resultant homogenate was then subjected to differential centrifugation. A pellet (P4) was obtained containing only slight activity of marker enzymes for mitochondria and sarcoplasmic reticulum, but which was enriched in 5’-AMPase activity, plasma membranes (as revealed by its cholesterol/phospholipid ratio) and possessed a reduced muscle protein content. Histological examination of this fraction revealed an absence of myofibres. The specific activity of the basal, isoprenaline-and fluoridestimulated adeynlate cyclase in P4 was doubled relative to Pi and this fraction contained approximately half of the total adeynlate cyclase activity of PI. It was concluded that the bulk of the catecholamine-sensitive adenylate cyclase activity of the cardiac muscle homogenate was localized in the sarcolemma. The described procedure for the isolation of a purified sarcolemma fraction is rapid and does not require the use of an ultracentrifuge or density gradients. KEY WORDS: Cardiac cholamine-stimulated

muscle; Subcellular adenylate cyclase.

fractionation;

Sarcolemma

membranes;

Cate-

1. Introduction

It has been hypothesized that the P-adrenoceptor of cardiac muscle is a regulatory component of adenylate cyclase [ZO, 231 and that adenosine 3’5’-monophosphate (cyclic AMP), the enzyme product, initiates the intracellular events leading up to the characteristic inotropic and metabolic responsesof cardiac muscle to @-adrenoagonists. If this hypothesis is correct, then it opens the way for the isolation and characterization of the P-adreno-receptor as a biochemical entity. * Present address: Clinical Austin Hospital, Heidelberg,

Pharmacology 3084, Victoria,

and Therapeutics Australia.

Unit,

University

of Melbourne,

686

B.

JARROTT

AND

G.

M.

PICKEN

In all mammalian tissues, adenylate cyclase is a particulate enzyme and is generally considered to be localized in the plasma membrane [5, ,?I]. In some tissuessuch asheart [8], skeletal muscle [22], adrenal cortex [3Z] and thyroid gland [34], the enzyme has been found in mitochondrial and microsomal fractions, but it was not ruled out unequivocally that this was due to the contamination of these fractions by plasma membranes. In the present study we have sought a method for the preparation of cardiac catecholamine-sensitive adenylate cyclase that is rapid, high yielding and relatively free of myosin ATP-ase. An analytical approach has been adopted in that the enzyme activity was not assumedto be present in any particular membrane such as sarcolemma, sarcoplasmic reticulum, etc., but that a subcellular fraction containing the bulk of the catecholamine-stimulated adenylate cyclase would be isolated and characterized. A preliminary account of this work has been published

1141.

2. Materials

and Methods

Subcellular fractionation

of homogenate

A male Wistar rat (200 to 300 g) was injected i.p. with pentobarbitone (100 mg/kg) and heparin (1000 i.u.). When anaesthetized, the heart was excised and perfused retrogradely through the aorta with 20 ml of ice-cold sucrose (0.25 M) containing dithiothreitol (2 mM) and Tris-HCI (10 mM, pH 7.2) to wash out blood. The atria and large vesselswere excised and discarded. The ventricles were chopped finely and transferred to a smooth glasshomogenizer tube (i.d. 12.70 mm) containing 5 ml of the sucrosemedium. Homogenization was effected by 10 upand-down strokes with a Dounce-type smooth glasspestle (diameter 12.36 mm), followed by 5 strokes with another pestle (diameter 12.64 mm) and then 3 strokes with a motor-driven perspex Potter-Elvehjem-type pestle (diameter 12.64 mm). The resultant homogenate was filtered under suction through a stainlesssteel gauze (mesh 40) into a glass test tube (100 x 12.7 mm). This suspension (H) was centrifuged at 8000 g min in an International refrigerated centrifuge (model PR-2). The supernatant was kept and the pellet resuspendedin 5 ml of homogenizing medium and re-centrifuged at 8000 g min. This procedure was repeated once more, and the final pellet (Pi) was resuspended in 2 ml of medium. The resulting supernatants were combined and centrifuged at 300000 g min in a MSE superspeed 65 ultracentrifuge to yield a pellet (Pz). The supernatant from this spin was recentrifuged at 1.26 x 107g min to yield a pellet (Ps) and final supernatant (S). All of the above procedures were carried out at 4°C and the subcellular fractions kept on ice until analysed.

ADENYLATE

CYCLASE

ACTIVITY

Purijkation

IN

SARCOLEMMA

FRACTION

687

of PI fraction

Aliquots (2 ml) of combined, resuspended pellets (PI) from several animals were added to 200 ml of ice-cold homogenizing medium and re-homogenized for various times (10 to 30 s) in a stainless steel Sorvall Omnimixer. Each 202 ml homogenate was recentrifuged in a glass tube at 60000 g min in the International refrigerated centrifuge, and the resultant pellets (P 4) were resuspended in 4 ml of homogenizing medium. These suspensions, and fraction Pr were examined by phase contrast microscopy to determine the degree of dispersion and dissolution of myofibrils. In some experiments, Pr and Pd fractions were stirred with an equal volume of sodium iodide (4 M), cysteine (40 nm), ATP (3 mM), EDTA (5 mM) and MgCIz (5 mM) (final pH adjusted to 7.4 with Tris base) for 30 min at 0°C and membranes isolated as described by Tashima et al. [30].

Enzyme assays Adenylate cyclase activity was assayed using the standard incubation mixture of Drummond and Duncan [7] except that, (a) twice the amount of pyruvate kinase (1.54 i.u.) was used, (b) tissue added contained 40 to 60 pg protein, and (c) the final incubation volume was 50 ~1. When adenylate cyclase of fractions Pr and P4 was determined, phosphatidylinositol (4 kg) was included in the incubation mixture [18] and (-) isoprenaline bitartrate (final concentration lo-4w), or sodium fluoride (final concentration 8 mM) were added in a volume of 5 ~1. Hs-cyclic AMP was isolated as described by Krishna and Birnbaumer [17]. All adenylate cyclase assays were performed within 20 min of preparation of the subcellular fractions. Subcellular fractions were also assayed for 5’-AMPase [1.5], Mg2+-dependent, (Na+ + K+) stimulated ATPase [%I, esterase [ 131, succinate dehydrogenase [33], lactate dehydrogenase [16] and total @-glucuronidase [6]. Preliminary experiments were performed to determine optimal amounts of subcellular fractions and incubation conditions.

Chemical determinations Aliquots (generally 1 ml) of particulate fractions were centrifuged at 120000 g min and the pellet extracted with 2 ml of chloroform/methanol (2: 1) as described by Folch et al. [IO]. The chloroform/methanol extract was evaporated to dryness under Nz and the cholesterol [2.5] and phospholipid [1] content of the residue determined. Phospholipid was measured as inorganic phosphorus after perchloric acid digestion of the residue and a factor of 25 used to convert pg phosphorus to pg phospholipid.

688

B. JARROTT

AND

G. M.

PICKEN

Total protein content of subcellular fractions was determined by the method of Campbell and Sargent [3]. The muscle and stroma protein content of pellets from various subcellular fractions was determined by the method of Helander [II].

Materials

Phosphalidylinositol, ex yeast, was obtained as a 25 mg/ml solution in chloroform/ methanol from Koch-Light (U.K.). A n aliquot (0.32 ml), was evaporated to drynessunder Ns and then dispersed in 10 ml of Tris-HCI (25 mM), EDTA (1 mM), pH 7.6 by sonication. This solution was kept for up to 2 weeks at 4°C. ATP, AMP, 2-phosphoenol pyruvate (tri-sodium salt), cyclic AMP (sodium salt), bovine serum albumin (fatty acid-free), P-naphthyl acetate and ouabain were purchased from Sigma. Pyruvate kinase was obtained from Calbiochem. Adenosine-2-Hs-5’triphosphate was obtained from The Radiochemical Centre (U.K.) and diluted with unlabelled ATP in Tris-HCI (10 mM, pH 7.6) to a specific activity of 50 mCi/ mmol. All other reagents were of the highest purity available.

3. Results

Differential centrifugation of homogenates of rat ventricles revealed that the bulk of the adenylate cyclase activity (83%) sedimented in the PI fraction and only small amounts (9% and 8%) were found in Pz and Ps respectively. However, the relative specific activity ofthe adenylate cyclase activity in Pswas much greater than the relative specific activity of this enzyme in PI (Figure 1) and indicates that Ps was a purer preparation of adenylate cyclase. This distribution of adenylate cyclase was remarkably similar to that of 5’-AMPase, a marker for sarcolemma [24], (Figure 1). Although PI contained the bulk of the adenylate cyclase and 5’-AMPase activity, it also contained a high proportion of succinate dehydrogenase (45%) (a marker for mitochondria [32]), and esterase(7 1%) (a marker for sarcoplasmicreticulum [13]). In fact, the only enzyme absent was lactate dehydrogenase, a marker for soluble cytoplasm [IS]. Examination of P1 by phase contrast microscopy [Plate 1(a)] revealed that the fraction consisted mainly of isolated myofibrils and the biochemical study of PI suggested that mitochondria and sarcoplasmic reticulum were still enmeshed amongst the myofibres within the myofibrils. Thus it was not possible to draw any firm conclusions about the intracellular localization of adenylate cyclase except to note that its distribution closely paralleled that of the marker enzyme for sarcolemma, 5’-AMPase. A number of procedures were then attempted in an effort to dissolve the contractile proteins in PI, and then re-fractionate this pellet. Stirring with 2~ sodium

ADENYLATE

CYCLASE

ACTMTY

IN SARCOLEMMA

689

FRACTION

8-1 765>r .Z .? 4t 0 3.5! ; 22 IO) .z GO m a 5432IO-

AIL

n

SDH

0-o

0-510

Percent

of total

cr2i-zmoo

proteins

FIGURE 1. Distribution patterns of adenylate cyclase activity and marker subcellular fractions of rat ventricle homogenates. Ordinate: relative specific (percentage of total activity recovered&ercentage of total proteins recovered). protein content of fractions PI, Ps, Ps and S (cumulatively from left to right).

enzyme activity

activities in of fractions

Abscissa: relative

iodide according to the procedure of Tashima et al. [30] led to the disappearance of the myofibres and the appearance of (Naf AZ K+)-stimulated ATPase activity this procedure resulted in a of 2.3 pmol Pi released/h/mg protein. However, marked loss of AMPase activity and basal, isoprenaline (lo-4 M)-and fluoride (8 mM)-stimulated adenylate cyclase activity. An attempt was then made to dissolve the contractile protein in a large volume of ion-free medium. Homogenisation of PI with a Sorvall Omnimixer in 200 ml of isotonic sucrose for increasing times, produced a greater degree of dissolution of the myofibrils as seen under the phase contrast microscope. By 30 s at full speed [Plate l(b)] the myofibrils were totally dispersed and only a very few small membrane elements were visible. Concurrently, basal and isoprenaline-stimulated adenylate cyclase activities of Pr were depressed and the ability of (-) isoprenaline (10-4~) to activate the enzyme was completely destroyed by 20 s homogenization in the Omnimixer. Addition of 4 p.g phosphatidylinositol to the incubation mixture restored the ability of isoprenaline to activate adenylate cyclase in fractions

690

B. JARROTT

AND

G. M. PICKEN

prepared by up to 20 s homogenization. designated as that pellet obtained following in 200 ml of isotonic sucrose. Analysis of enzyme markers in P4 revealed of the 5’-AMPase activity of the homogenate dehydrogenase, esterase and /3-glucuronidase TABLE

1. Enzyme

markers

during

5’-AMPase

Homogenate

171 & 10.6* (100%) 108 f 2.3 (63%) 84 j= 8.8

P4

that it contained approximately 5Oq:, but only small amounts of succinate (Table 1). The specific activity of of sarcolemmal

Succinate dehydrogenase

Fraction

Pl

preparation

In subsequent experiments Pa was 15 s full speed homogenization of PI

Esterase

& 18.7 (100%) 4.7 136 f (39%) 20 & 3.2

* Mean f S.E. of 3 to 4 preparations. h/g wet wt of ventricular muscle.

All enzyme

(P?)

P-Glucuronidase

163 & 6.2

351

0.88

(lw%) 0.12 f

(54%) 13 f

(13%) 0.001 * 0.0002

1.0

(8%) activities

& 0.03

(100%) 88 3 11.8

(6%)

(49%)

fraction

are expressed

0.03

(1%) in pmol products

formed/

5’-AMPase activity increased approximately three-fold and, on the assumption that this enzyme was a valid marker for the sarcolemma, this indicated that the procedure led to a purification of sarcolemma membranes. When examined for adenylate cyclase activity, this fraction contained about half the basal activity of Pr but twice its specific activity (Table 2). TABLE

2. Adenylate

cyclase

activity

Basal Total*

Pl P4

428 212 * Pmol cyclic AMP 7 sp. act. = specific

sp. act.? 11.5 22.8

during

preparation

of sarcolemmal

Isoprenaline-stimulated Total* sp. act.? 1538 490

formed/mm/g wet weight. activity = pmol cyclic AMP

41.2 52.7

formed/min/mg

fraction

(P4)

Fluoride-stimulated Total* sp. act.? 8237 2346

220.8 252.3

protein.

An analysis of the muscle protein content of H, Pi, and PJ, revealed that there was a marked but not complete reduction of muscle protein content in P4 (Table 3). The molar ratio of cholesterol to phospholipid was then determined and found to be higher in Pa than in PI and H (Table 4).

ADENYLATE CYCLME ACTIVITY IN !MRCOLeMMA FRACTION

691

TABLE 3. Stroma and muscleprotein contentof subcellularfractions Fraction H Pl P4

Stroma protein

Muscle protein

Total protein

54.4 f 3.0* 36.5 & 2.5 7.5 f 0.9

75.6 f 4.3* 47.1 f 3.2 12.5 & 1.8

130.0 f 7.2* 83.6 f 4.5 20.0 f 2.6

* mg/g wet weight, Resultsare the mean& S.E.(n = 3).

TABLE 4. Lipid compition of subcellularfractious Fraction H Pl

PC

cholesterol (nmol/mg protein) 8.4 f 0.79 17.1 f 2.32 29.7 f 1.66

Phospholipid (nmol/mg protein) 107.5 f 8.11 161.4 f 6.37 148.8 f 28.7

Molar Ratio

0.078 f 0.008 0.1p5 f 0.010 0.214 f 0.038

Results are the mean f S.E.for 3 determinations.The molar quantity of phospholipid was calculated by assuming a molecular weight of 700.

4. Discussion Thezresults of these studies lend support to the hypothesis that a major proportion of adenylate cyclase activity of rat heart homogenatesis closely associatedwith the outer cell membrane (sarcolemma). We have demonstrated (a) that the distribution of adenylate cyclase in various subcellular fractions paralleled only that of the marker enzyme for sarcolemma (5’- AMPase), and (b) that further puri6cation of the fraction containing the bulk of the plasma membranes resulted in concentrating its basal, isoprenaline- and fluoride-stimulated adenylate cyclase activities. On the basis of the norepinephrine-sensitive adenylate cyclase content of a microsomal fraction prepared from dog heart, Entman et al. [8, 91, postulated an intracellular localization of this enzyme in sarcoplasmic reticulum. However, cardiac ventricular muscle has a high connective tissue content, a rigid intracellular organization, and extensive invagination of the sarcolemma (T-tubules) into the cell interior. For these reasons, preparation of homogeneoussubcell&r fractions from cardiac muscle by differential centrifugation is lesssuccessfulthan from other tissues such as liver [I,?]. Furthermore, it has been shown that conventional preparations of cardiac microsomal fractions contain vesicles from both the invaginated sarcolemma (T-system) and the sarcoplasmicreticulum [Z], and Entman et al. [S, 91 did not rule out unequivocally that the adenylate cyclase activity in their microsomal fraction was due to such sarcolemrnal contamination. A knowledge of the exact intracellular localization of adenylate cyclase is essential if one is to propose a role for the enzyme product, cyclic AMP as intracellular mediator

692

B. JARROTT

AND

G. M.

PICKEN

between catecholamine interaction and enhanced contractility of cardiac muscle [9, 27, 281. The present studies have not unequivocally placed adenylate cyclase in the sarcolemma, but have greatly strengthened the case supporting this view. Earlier studies in our laboratory f14] had established that gentle homogenization of cardiac tissue was essential for the preservation of both basal and isoprenalinestimulated adenylate cyclase activity. Thus, gentle homogenization techniques were employed with a view to minimizing the shearing forces between pestle and homogenizer tube, which could lead to vesicularization of the sarcolemmal invaginations. Such vesicles formed from fragments of these T-tubules could sediment with both mitochondria and sarcoplasmic reticulum. In order to assess the accuracy of the observed intracellular localization of adenylate cyclase, it was essential to examine the degree of heterogeneity of the fractions “Pi,” “Ps,” “P3,” and “S”. This was established by examining the distribution within these fractions of enzymes of known intracellular localization. S-AMPase was used as a marker for sarcolemma [24]; succinate dehydrogenase for mitochondria [32] ; esterase for sarcoplasmic reticulum [Z3] ; total g-glucuronidase for intact lysosomes [6] and lactate dehydrogenase for soluble cytoplasm [IS]. There was a striking similarity between the distributions of adenylate cyclase and the sarcolemmal marker, 5’-AMPase. The distribution of lactate dehydrogenase indicates that virtually all cells were broken during the preparation of “PI,” “Ps,” “Ps,” and “S” fractions. However, no firm conclusions regarding the localization of adenylate cyclase could be drawn, because of the biochemical heterogeneity of fraction PI, which contained not only the bulk of protein, 5’-AMPase and adenylate cyclase, but also significant amounts of succinate dehydrogenase and esterase. It became clear after microscopic examination of “PI” that its observed biochemical heterogeneity was very likely due to the enmeshing of intracellular structures such as mitochondria, sarcoplasmic reticulum, and even elements of T-tubules in the large, intact clumps of myofibrillar protein. A method was therefore sought for dissolution or dispersion of these myofibrils and isolation of sarcolemmal membranes. Conventional techniques for isolating cardiac sarcolemmal membranes containing (Naf + K+)-stimulated ATPase activity involve extracting a suspension of myofibres (obtained similarly to fraction Pi) with strong salt solutions for periods of 1 to 20 h to dissolve the myonbrillar proteins [29, 301. However, in the present study, we found that such sarcolemmal membranes, while enriched in (Na+ f K+)-ATPase activity, were totally devoid of adenylate cyclase activity even after the addition of phospholipid. This suggests that salt solutions of high ionic strength are either inactivating adenylate cyclase or solubilizing the enzyme from the membranes. McCollester [Z9] has developed a mild procedure for preparing sarcolemmal membranes from skeletal muscle homogenates which essentially involves extracting myofibres with large volumes of ion-free water. Whilst this procedure for preparing sarcolemmal membranes from skeletal muscle did not prove entirely successful in our hands when using cardiac

ADENYLATE

CYCLASEACTIVITY

IN SARCOLEMMAFRACTION

693

muscle, we found that the myofibrillar proteins could be dissolved by homogenizing the myofibres at high speed in a large volume of isotonic sucrose. After centrifugation of this solution, the particulate fraction (Pd) contained approximately half of the AMPase activity, approximately one fourth of the protein content and only a small fraction of the succinate dehydrogenase, esterase and /3-glucuronidase activity of fraction Pi. This suggests that this fraction is enriched in sarcolemmal membranes. Chemical analysis of Pd showed that it contained a higher cholesterol/ phospholipid ratio than Pi which is indicative of a purification of plasma membranes [4]. Fraction Pa also contained approximately 50% of the basal adenylate cyclase activity of Pi but no isoprenaline-stimulated enzyme activity. However, the addition of phosphatidylinositol but not phosphotidyl serine to the incubation mixture resulted in restoration of the catecholamine-stimulated activity. Interestingly, phosphatidylinositol is one of the more water soluble phospholipids and it is possible that the extraction procedure for the myofibrillar proteins may also have leached phosphatidylinositol from the sarcolemmal membrane. In conclusion, we report a rapid and simple procedure for the isolation of the bulk of the catecholamine-sensitive adenylate cyclase activity of rat cardiac muscle homogenates.

Acknowledgements This work was supported by a grant from the National Heart Foundation of Australia. The excellent technical assistance of Mr Gary Nolan is gratefully acknowledged.

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New York: Academic Press(1967). COLEMAN, R. & FINEAN, J. B. Preparationsand propertiesof isolatedplasmamembranesfrom guinea-pigtissues.Biochimica et biojdysica acta 125, 197-206(1966). 5. DAVOREN, P. R. & SUTHERLAND, E. W. The cellular location of adenyl cyclasein the pigeonerythrocyte. 3ournulof Biological Chemistry 238, 3016-3023(1963). 6. DE Duvr, C., PRESSMAN, B. C., GIANE?TO,R., WA~UX, R. & A~PELMANS, F. Tissue fractionation studies 6. Intracellular distribution patterns of enzymes in ratliver tissue.Biochemical 30urna160, 604-617 (1955). 4.

7.

DRUMMOND, G. I. & DUNCAN, L. Adenyl cyclase in cardiac tissue. journal of Biological Chemistry 245,976-983 (1970).

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21. 22. 23. 24. 25. 26. 27. 28.

B. JARROTT AND G. M. PICKEN ENTMAN, M. L., LEVEY, G. S. & EPSTEIN, S. E. Demonstration of adenyl cyclase in canine cardiac sarcoplasmic reticulum. Biochemical and Biophysical Research Communications 35, 728-733 (1969). ENTMAN, M. L., LEVEY, G. S. & EPSTEIN, S. E. Mechanism of action of epinephrine and glucagon on the canine heart. Circulation Research 25, 429-438 (1969). FOLCH, J., LEES, M. & SLOANE-STANLEY, G. H. S. A simple method for the isolation and purification of total lipides from animal tissues. 3ournd of Biological Chemistry 226, 497-509 (1957). HELANDER, E. On quantitative muscle protein determinations. Acta physiologica scandinavica 41, Supplement 141, l-99 (1957). HULSMANS, H. A. M. Microsomes in heart muscle homogenates. Biochimica et biophysics acta 54, 1-14 (1961). IMAI, K., OYUFU, T. & SATO, R. Biochemical characterisation of microsomes isolated from heart and skeletal muscles. Journal of Biochrmist7y 60, 274-285 (1966). JARROTT, B. & PICREN, G. M. Regulatory role of adenyl cyclase and cyclic AMP. Advances in Cardiology 12, 149-161 (1974). KAULEN, H. D., HENNING, R. & STOFPEL, W. Comparison of some enzymes of the lysosomal and the plasma membrane of the rat liver cell. Ho#e-Sgler’s