Journal

of Molecular

and Cellular Cardiology

(1975)

7, 27-37

Catecholamine Stimulated Myocardial Adenylate Cyclase : Effects of Phospholipase Digestion and the Role of Membrane Lipids ROBERT Division

of Cardiology,

J. LEFKOWITZ

Department of Medicine and Department of Biochemistry, Medical Center, Durham, North Carolina 27710, U.S.A. (Received

31 December

1973,

and accepted

1 February

Duke

University

1974)

R. J. LEFKOWITZ. Catecholamine Stimulated Myocardial Adenylate Cyclase: Effects of Phospholipase Digestion and the Role of Membrane Lipids. Journal of Molecular and Cellular Cardiology (1975) 7, 27-37. Phospholipase digestion of myocardial membranes causes a reduction in basal and fluoride stimulated adenylate cyclase and an abolition of catecholamine stimulated activity. The effects were seen with phospholipases A, C and D, although A was most potent. The effects of phospholipase A could be prevented by the synthetic phospholipase A inhibitor 2,3 distearoyloxpropyl (dimethyl) P-hydroxyethylammonium acetate or by the calcium chelator Ethyleneglycolbis-(e-aminoethylether) N, N’tetra-acetic acid (EGTA). Several lipids were tested for their abihty to restore catecholamine responsiveness to the phospholipase treated membranes. Only the total lipids extracted from the membranes with chloroform methanol were effective. Reconstitution was not achieved with pure phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, lecithin or lysolecithin. Optimal restoration of hormone response occurred at a lipid concentration of 20 ug/ml. WORDS: Adenylate cholamines; Membranes;

KEY

cyclase; CAMP;

Beta adrenergic Phospholipase;

receptor; Myocardial; Isoproterenol.

Lipids;

Cate-

1. Introduction Catecholamines have pronounced effects on cardiac contractile force and rate. Many, if not all of these effects appear to be mediated by stimulation of the membrane bound enzyme adenylate cyclase, and the production of cyclic adenosine 3’, 5’ monophosphate (CAMP) [Z]. Binding of catecholamines to beta adrenergic receptors is followed by activation of adenylate cyclase and biological processes. The mechanisms involved in coupling receptor binding to enzyme are unknown. Recent observations from several laboratories have indicated that membrane lipids play a crucial role in the process by which hormones stimulate adenylate cyclase [7-9, 15, 16J. These studies have shown that when membranes containing adenylate cyclase are dispersed with either detergents, such as Lubrol-PX or digitonin [7-9, 1.51, or treated with phospholipases [15, 161, there is a disproportionate loss of hormone sensitive as opposed to fluoride sensitive enzyme activity. Addition of exogenous lipids to these systems has led to partial “reconstitution” of

28

R. J.

LEFKOWITZ

hormone response. As has been pointed out [15], lipids may be acting at several points, e.g., binding of hormone to membrane receptors, transmission of receptor binding to adenylate cyclase activation, or at multiple sites in this sequence. Another as yet unresolved point, is the specificity of the lipid requirement for hormonal response. In order to further explore the role of membrane lipids in mediating catecholamine stimulation of myocardial cyclase, the effects of phospholipase digestion of myocardial membranes on isoproterenol-stimulated adenylate cyclase have been studied. The results indicate that: (1) catecholamine activation of the enzyme is much more sensitive than fluoride activation to the effects of phospholipase digestion, and (2) catecholamine sensitivity can be restored to the lipase treated membranes by addition of total lipids previously extracted from myocardial membranes but not by any of a number of purified lipids.

2. Materials

and

Methods

Phospholipase A (Vipera Russelli) 5 units/mg, in 50% glycerol, phospholipase A (bee venom), Phospholipase C and D, lecithin, lysolecithin, phosphatidylethanolamine, EGTA, Folch fraction I, myokinase, phosphoenolpyruvate kinase, phosphoenopyruvate and isoproterenol bitartrate were purchased from Sigma; phospholipase A (Crotahs terrzjkus terrzjicus) and d, 1-2, 3-distearoyloxpropyl(dimethyl) P-hyd roxethylammonium acetate from Calbiochem and phosphatidylL-Serine from Mann. Phosphatidylinositol (plant) was obtained from Analabs; phosphatidylinositol (Bovine brain) from General Biochemicals. Chloroform and methanol were reagent grade, J. T. Baker Co. [c92P] ATP (l-10 Ci/mmol) was obtained from New England Nuclear Co.

Myocardial

membranes

These were prepared by homogenization of canine ventricular myocardium in 0.25 M-sucrose as previously described [5, 61. Membranes sedimenting at 10 000 x g were washed twice with sucrose, then used for adenylate cyclase assays.

Adenylate cyclase assay This was performed using a modification of the method of Krishna et al. [4, 61 which follows the conversion of [z- saPI ATP to [sap] CAMP in the presence of an ATP regenerating system. Incubation mixtures contained: ATP 1.5 mM, Tris-HCl buffer, 30 mM (pH 7.5) ; MgC12, 5 mM, cyclic AMP, 0.1 InM, phospho-

MYOCARDlAL

ADENYLATE

CYCLASE

: LIPIDS

29

enolpyruvate 5 mM; phosphoenolpyruvate kinase, 40 pggiml; and myokinase, 20 pg/ml, in a volume of50 ~1. Incubations were performed for IO min at 37°C. As reported previously [S], under these conditions CAMP formation is linear for at least 20 min and over a wide range of protein concentrations. In most assays approximately 50 pg of membrane protein were present. [ssP] CAMP was isolated by chromatography on Dowex AG-50W-X2 as described by Krishna et al. [4] and quantitated by liquid scintillation spectrometry.

Phospholipase

digestion

This was performed by incubating myocardial membranes (2 to 3 mg/ml) with the indicated concentration of enzyme for 5 min at 37°C. Since Gas+ markedly inhibits myocardial adenylate cyclase [13], it was not added to these incubations. Although phospholipase A has a Gas+ requirement, sufficient Ca2+ was present in our reagents to permit lipase effects as demonstrated by complete inhibition of phospholipase effects by prior addition of the Ca s+ chelator EGTA (see below). Wells has shown that very low concentrations of Gas+ are needed to activate phospholipase A (10-5~) [19] and Mayer has also found significant Ca2+ in reagents commonly used for adenylate cyclase assays [14]. After completion of the initial phospholipase digestion, enzyme assays were performed without washing the membranes. It was demonstrated in con&o1 experiments that glycerol in the concentrations added with phospholipase A (0.1 to 0.5%) did not affect adenylate cyclase activity. Unless otherwise indicated, phospholipase A refers to the enzyme from Vipera russelli.

Preparation

of” Total Lipid”

fraction

Five to ten grams of ventricular myocardium were homogenized in 0.05 M-T&HCI-1 mM EDTA pH 7.5 in an Omnimixer for 10 s followed by homogenization in a Potter homogenizer. After centrifugation at 30 000 x g for 10 min, followed by a buffer wash the pellet was suspended in 10 mls of 0.05 Tris-HCl pH 7.5 and 60 mls of chilled chloroform-methanol (2 :l). The mixture was homogenized with 10 strokes of a Potter homogenizer and placed in a separatory funnel. The lower layer was removed and centrifuged 15 min at 3000 x g in a glass tube. Aliquots of this solution were evaporated to dryness under a stream of Ns in tared tubes at 4%.

Preparation All lipids were suspended in 0.01 M-Tris-Hcl, Model LS75 Sonicator (usually for 2 min).

of lipids pH 7.5 and sonicated

with a Branson

30

R. J. LEFKOWITZ

Protein determinations These were

by the method

of Lowry

et al. [lo].

Expression of results stimulated adenylate cyclase” refers to the In figures and tables, “ Isoproterenol increment of activity above basal levels caused by isoproterenol at 5 x 10-5~. Enzyme activity is expressed as pmol of CAMP generated/mg/lO min.

3. Results Phospholipase digestion of myocardial membranes caused a decrease in basal, fluoride and catecholamine stimulated adenylate cyclase activity. Effects on hormone stimulated activity were much greater than those on basal or fluoride activity (Figure 1). All three phospholipases A, C and D had similar effects. At enzyme

;;;‘- ;;;q lIrK 0

50

100

0 50 Phospholipose

100 (pg/ml)

0

50

100

FIGURE 1. Effects of phospholipase digestion of myocardial membranes on adenylate cyclase activity. Values are mean of duplicate determinations. In this and subsequent figures, “isoproterenol stimulated adenylate cyclase” refers to the increment in enzyme activity above basal levels caused by isoproterenol 5 x lCF5~ur; (a) Basal, Phospholipase A (m----m), Phospholipase C (@---a), Phospholipase D (A----A), (b) Fluoride stimulated, (c) Isoproterenol stimulated.

concentrations of 100 pg/ml, hormone response was virtually abolished. Because it was most potent, phospholipase A was selected for further study. Figure 2 shows results obtained with phospholipase A from three different sources. Although differences in potency were apparent, results were qualitatively similar with each. As noted under “Methods”, exogenous Gas+ was not added to the phospholipase A incubations. Not surprisingly, sufficient Ca2+ was present in reagents used for reasonable enzyme activity [14, 191. Inasmuch as phospholipase A has an absolute Ca2+ requirement, it seemed of interest to test the effect of the Ca2f chelator, EGTA, on the enzyme effect (Table 1). As has been noted by others, Ca2f present in reagents inhibits myocardial adenylate cyclase and activity is increased by EGTA

MYOCARDIAL

ADENYLATE

CYCLASE

L---Phospholipase

31

: LIPIDS

A-

FIGURE 2. Effects of several phospholipase A preparations on isoproterenol stimulated myocardial adenylate cyclase. Values are means j= S.E.M. 3 determinations from 3 different membrane preparations. ‘Control” refers to adenylate cyclase activity in membranes which were incubated without phospholipase A. The source of each phospholipase A preparation is Vipera russelli (@), Crotolus temifcus tertijcw (Hi), Bee venom ( n ) .

[14]. When EGTA was added to the membranes prior to phospholipase A, the effect of the enzyme was entirely blocked. However, if enzyme digestion was allowed to proceed before addition of EGTA, full enzyme effects were seen. 2,3 distearoyloxpropyl (dimethyl) P-hydroxyethylammonium acetate is a synthetic lipid quarternary ammonium salt which is known to be a potent inhibitor of phospholipase A [17]. As noted in Figure 3, it was capable of partially inhibiting the effect of phospholipase A on isoproterenol stimulated cyclase in the myocardial membranes. An even more striking inhibition of the phospholipase was noted with the total lipid fraction. TABLE

1. Effect of EGTA

on phospholipase

digestion of myocardial

Isoproterenol stimulated adenylate cyclase*

Addition

(P mol CAMP/IO None

1050

Phospholipase A-100 pg/ml EGTA-1 mM EGTA, then Phospholipase A Phospholipase A, then EGTA

2010 2050

* Isoproterenol digestions

were

was present as described

under

membranes

at 5

X

methods.

1k5M

in all incubations. Values are means -&

min/mg protein) f 50 0 * 80 -& 60 0

Conditions of phospholipase four determinations.

S.E.M.

A

32

R. J. LEFKOWITZ

Tot01 lipid

FIGURE 3. Inhibition ofphospholipase A effects on isoproterenol stimulated myocardial adenylate cyclase by synthetic phospholipase A inhibitor and total lipid fraction. The inhibitor or total lipid was added to tb.e membranes at the indicated concentration prior to addition of phospholipase A. Concentrations are those present during the phospholipase A digestion. The concentration of lipid or inhibitor present during the subsequent adenylate cyclase assay was 2.5 times lower. Phospholipase A inhibitor alone was without effect on the adenylate cyclase. Values are means + S.E.M. of 3 determinations from 3 different membrane preparations.

The effects of lipids on adenylate cyclase activity in phospholipase treated membranes were tested in two ways. In one set of experiments lipids were added to the membranes prior to the phospholipase to determine if they could block the effects of phospholipase digestion on catecholamine sensitive adenylate cyclase, (top panel, Figure 4). In other experiments, lipids were added after completion of phospholipasedigestion to evaluate whether catecholamine sensitivity of the adenylate cyclase could be restored, (lower panel, Figure 4). Of the materials tested, only the total lipids extracted from myocardial membraneswere effective in either blocking the phospholipaseeffect or in restoring catecholamine responseafter incubation with phospholipaseA. In addition to the lipids indicated in the figure, negative results have also been obtained with phosphatidylethanolamine, phosphatidylinositol of plant origin, lecithin and lysolecithin. Figure 5 indicates that optimal restoration of hormone responseoccurred at a total lipid concentration of 20 pg lipid/ml incubation, (1 pg lipid per incubation tube).

4. Discussion

A crucial role for membrane phospholipids has previously been established for (Na+ and KS-)-dependent ATPase and certain microsomal enzymes [11]. Sutherland et al. [18] were the first to observe that “solubilized” adenylate cyclase

MYOCARDIAL

Lipids

Lipids

01

ADENYLATE

added

added

CYCLASE

33

: LIPIDS

before

after

:$$j;~ _ i:::+:.:.:,> Control Phosphoiipase

Phosphotic . . . A

Tdtal Lipid

Phosbhatidyl serine

FIGURE 4. Inhibition and reversal of phospholipase A effect on isoproterenol stimulated myocardial adenylate cyclase by lipids. In the experiments represented in the top panel the lipids were added to the membranes prior to addition of phospholipase A. In the second panel the lipids were added after completion of the 5 min phospholipase incubation. “Control” refers to isoproterenol stimulated adenylate cyclase activity in membranes incubated without phospholipase A. When lipids were added first their concentration was 200 pg/ml during the lipase incubations (80 pg/ml during the subsequent cyclase incubation). When lipids were added after the lipase digestion, their final concentration was 40 pg/ml in the cyclase incubation. Control experiments indicated that total lipids alone did not significantly affect isoproterenol stimulated adenylate cyclase. Values are mean & S.E.M. of 4 determinations from 4 membrane preparations. In both panels, the upper dashed line refers to isoproterenol stimulated cyclase in the absence of phospholipase A, and the lower dashed line the activity in the presence of phospholipase A.

preparations lost their hormone responsiveness. The first systematic study of a lipid requirement for a hormone sensitive adenylate cyclase was reported by Pohl et al. [1.5]. These authors noted that treatment of liver plasma membranes with either digitonin or phospholipase A caused a marked reduction in glucagon sensitive activity. Addition of the total lipids extracted from the membranes partially restored the hormone response. Further studies revealed that several pure lipids as well as cruder lipid fractions had similar effects, although phosphatidylserine was clearly most potent. Interestingly, 1251-glucagon binding to the membranes was also decreased by digitonin or lipase treatment. Lipids partially restored the binding. There was a correlation between the ability of lipids to restore hormone sensitive adenylate cyclase activity and to restore glucagon binding. It was concluded that the lipids were involved at multiple sites in the

34

R. J. LEFKOWITZ

500

--------

-----_

2 $0 ?!,il:

Control ______

----------

0

+z!~ zoo& 2 0 0 a3 “2 E --------------------------------2 IOO-

Ol

I IO Totol

Phospholipase

I 100 lipid odded (ag/ml)

A

I 1000

FIGURE 5. Effects of varying lipid concentration on isoproterenol stimulated adenylate cyclase of phospholipase A treated myocardial membranes. The two dashed lines represent the activity in control membranes or membranes to which phospholipase A, 100 pg/ml had been added. Lipid was added after the phospholipase A digestion. Concentrations refer to those actually present in the cyclase assay. Values are means of determinations from 2 separate experiments.

system, e.g., the hormone binding site, sites involved in transmission of the hormonal signal, etc. Using liver membranes prepared in a somewhat different fashion, Rethy et al. [16] noted profound decreases in basal, fluoride, glucagon, and epinephrine stimulated cyclase after lipid extraction with organic solvents or phospholipase treatment. In agreement with Pohl et al. [ 151 phosphatidylserine was found to partially restore the glucagon and fluoride responses. Phosphatidykerine was even more potent in restoring the epinephrine effect. Phosphatidylinositol restored basal activity but not hormonal or fluoride stimulated activity. Phosphatidylethanolamine and phosphatidylcholine were inert. Pohl et al. [15] had previously reported that phosphatidylethanolamine and phosphatidylcholine, although not as potent as phosphatidylserine partially restored the glucagon response [15]. Using solubilized preparations derived from cat ventricular myocardium, Levey has reported an apparently more specific lipid requirement. Thus, addition of phosphatidylserine restored glucagon [7] and histamine [9] responsiveness of the enzyme but not the epinephrine response. Pure monophosphatidylinositol restored the catecholamine response but not that to histamine or glucagon [8]. A very recent report suggests that phospholipase treatment of thyroid membranes selectively eliminates TSH responsiveness without altering basal or fluoride stimulated activity [12]. Phosphatidylcholine was said to restore the hormone response. The results reported in this communication confirm the crucial role of membrane

MYOCARDIAL

ADENYLATE

CYCLASE

: LIPIDS

35

phospholipids in hormone sensitive adenylate cyclase. However, the specific individual lipids necessary for restoration of catecholamine responsiveness could not be identified. Only the total membrane lipids were successful. In this regard, several apparent discrepancies in the literature should be noted. Thus, Levey’s data suggested that specific lipids may mediate the effects of specific hormones, e.g., phosphatidylinositol for catecholamines, phosphatidylserine for glucagon, etc., [7-91. The findings of Rethy et al. [16] h owever, suggested less specificity in that phosphatidylserine restored both glucagon and epinephrine responses. The observations of Pohl et al. [15j also suggested less specificity in that a number of different lipids enhanced glucagon responsiveness. As noted, we were unable to restore hormone response with any single lipid. There are several possible explanations for these apparent discrepancies. The first is that markedly different experimental techniques were used in the various studies. Alteration of membrane lipids achieved by solubilization, lipase treatment, or solvent extraction are likely to be rather different, More specific experimental differences are also present. Thus, whereas phospholipase digestion was carried out for 45 min by Rethy et al. [16] only a 5 min incubation was performed by Pohl et al. [1.5] and in the current studies. Secondly, purified lipid preparations are highly sensitive to oxidation and the purity of commercially available “pure” lipid preparations is often questionable. Only one highly purified preparation of monophosphatidylinositol was capable of restoring the catecholamine response in Levey’s experiments, whereas several other commercial preparations were inert [S]. Finally, species and organ differences may explain some of the apparent discrepancies in lipid requirements. An as yet unresolved question is the site or sites at which the lipids are acting. Current concepts of hormone responsive adenylate cyclase systems include a number of interacting units (1) : 1) so-called receptors or hormone binding sites; 2) the catalytic unit; 3) “modulator” units which somehow translate hormone binding into changes in the activity of the catalytic unit. Studies reported to date for glucagon sensitive adenylate cyclase suggest multiple sites of action for the lipids. The catalytic unit appears to be involved in that fluoride sensitive activity decreases [1.5, 16j. The hormone binding sites are also involved inasmuch as 12sI.-glucagon binding was decreased by digitonin or phospholipase treatment [15]. Recent findings by Levey have shown high affinity glucagon binding sites in solubilized preparations of cat myocardium [3]. These preparations also contain adenylate cyclase which is unresponsive to glucagon until lipids are added. These findings suggest that lipids are involved in “coupling” hormone-receptor binding to adenyIate cyclase activation. It seems amply documented that membrane lipids are crucially involved in hormonally responsive adenylate cyclase systems. In particular, lipids clearly are involved in the coupling of beta adrenergic receptors to adenylate cyclase in the heart. Further studies will be necessary to document the sites and mechanisms by which these lipids are functioning.

36

R.J. LEFKOWITZ Acknowledgements

The author wishes to thank Dr Edgar Haber in whose laboratory (Massachusetts General Hospital) much of this work was performed, and Dr Donald O’Hara for many helpful suggestions. This work was supported by United States Public Health Service Grant HE-5196, SCOR HE-14150, H. E. W. grant #l ROl HL16037-01 and by a grant-in-aid from the American Heart Association with funds contributed in part by the North Carolina Heart Association. Dr Lefkowitz is an Established Investigator of the American Heart Association.

REFERENCES 1.

2.

BIRNBAUMER, L., POHL, S. L. & RODBELL, M. The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. II. Comparison between glucagon and fluoride-stimulated activities. Journal of Biological Chemistry 246, 1857-1860 (1971). EPSTEIN, S. E., LEVEY, G. S. & SKELTON, C. L. Adenyl cyclase and cyclic AMP, Biochemical links in the regulation of myocardial contractility. Circulation 43, 437-450 (1971).

3.

KLEIN, I., FLETCHER, M. A. & LEVEY, G. S. Evidence for a dissociable glucagon binding site in a solubilized preparation of myocardial adenylate cyclase. Journal of Biological

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(1973).

(1971). G. W. G. & HABER, E. Specific binding of beta-adrenergic catecholamines to a subcellular fraction from cardiac muscle. Journal of Biological Chemistry 248, 342-349 (1973). LEVEY, G. S. Restoration of glucagon responsiveness of solubilized myocardial adenyl cyclase by phosphatidylserine. Biochemical and Biophysical Research Communication 43, 108-l 13 (1971).

68, 1773-1777

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responsiveness of solubilized myocardial of Biological Chemistry 246, 7405-7410

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myocardial adenylate cyclase-restoration of responsiveness by phosphatidylserine. Journal of Clinical Investigation 51, (1972). LOWRY, 0. H., ROSEBROUGH, N.J., FARR, A. L. & RANDALL, R.J. Protein measurement with the folin-phenol reagent. Journal of Biological Chemistry 193, 265-271 (1951). IGARTONOSI, A., DONLEY, J. & HALPIN, R. A. Sarcoplasmic reticulum. III. The role of phospholipids in the adenosine triphosphatase activity and Casf transport. Journal of Biological Chemistry 243, 6 l-70 ( 1968). MASHITER, K., YAMASHITA, K. & FIELD, J. B. The role of phospholipids in thyroidstimulating hormone (TSH) stimulation of adenylate cyclase in thyroid plasma membranes. Journal of Clinical Investigation 52, 54a (1973). MAYER, S. E. Adrenergic receptors for metabolic responses in the heart. Federation Proceedings 29, 1367-1372 (1970).

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KRISHNA, G., WEISS, B. & BRODIE, B. B. Simple, sensitive method for the assay of adenyl cyclase. Journal of Pharmacology and Experimental Therapeutics 163,379-388 ( 1968). LEFKOWITZ, R. J. & HABER, E. A fraction of the ventricular myocardium that has the specificity of the cardiac beta-adrenergic receptor. Proceedings of the National Academy of

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S. E. Effects of adrenergic agonists and antagonists on adenylate cyclase of dog heart and liver. Journal of Pharmacology and Experimental l’7wafieutic.s 181, (1972). POHL, S. L., KRANS, H. M.J., KOZYREFF, V., BIRNBAUMER, L. & RODBELL, M. The glucagon-sensitive adenyl cyclase system in plasma membranes of rat liver. III. Evidence for a role of membrane lipids. Journal of Biological Chemistry 246, 4447-4454 (1971). RETHY, A., TOMASI, V., TREVISANI, A. & BARNABEI, 0. The role ofphosphatidylserine in the hormonal control of adenylate cyclase of rat liver plasma membranes. Biochimica et bio@hysica acta 200, 58-59 (1972). ROSENTHAL, A. F. & GEYER, R. P. A synthetic inhibitor of venom lecithinase A. Journal of Biological Chemistry 235, 2202-2206 (1960). SUTHERLAND, E. W., RALL, T. W. & MENON, T. Adenyl cyclase. I. Distribution, preparation and properties. Journal of Biological Chemistry, 237, 1220-1227 (1962). WELLS, M. A. A kinetic study of the phospholipase As (Crotalus adamanteus) catalyzed hydrolysis of 1,2-dibutyryl-sn-glycero-3-phosphorycholine. Biochemistry 11, 1030-1041 (1972).

Catecholamine stimulated myocardial adenylate cyclase: effects of phospholipase digestion and the role of membrane lipids.

Journal of Molecular and Cellular Cardiology (1975) 7, 27-37 Catecholamine Stimulated Myocardial Adenylate Cyclase : Effects of Phospholipase Dig...
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