Adenylate cyclase in cardiac microsomal fractions

j%urnal

of Molecular

and Cellular

Adenylate

Cardiology

Cyclase

GEORGE

(1978)

10, 317-331

in Cardiac

I. DRUMMOND

Microsomal AND JEAN

Fractions

DUNHAM

Biochemistry Group, Defiartment of Chemistry, University of Calgary, Calgary, l2.N 1Jv4, Canada (Received

10 May

1977,

accepted 5

July

1977)

G. I. DRUMMOND

AND JEAN DUNHAM. Adenylate Cyclase in Cardiac Microsomal Fractions. and Cellular Cardiofogp (1978) 10, 317-331. Adenylate cyclase was examined in microsomal fractions of rabbit heart and compared with activity sedimenting at lower gravitational forces (washed particle fractions). Enzyme activity in the two fractions differed markedly in several respects. The microsomal enzyme (which comprised 5% of the total activity) was dramatically stimulated by low concentrations of the nonionic detergents Lubrol PX and Triton X-100, while basal activity in the washed particle fraction was only modestly stimulated and the fluoride-activated enzyme was inhibited by detergent. Both basal- and fluoride-stimulated activity in the microsomal fraction was enhanced by incubation of the membranes with phospholipase A, whereas basal activity in the washed particle fraction was decreased by this treatment and fluoride activation was unaffected. The rate of activation of adenylate cyclase by the GTP analog, guanylylimidodiphosphate, was much slower at 20°C in the washed particle fraction than in the microsomal preparation. Epinephrine dramatically increased the rate of activation by guanine nucleotide in washed particle membranes, but had a much more modest effect on the microsomal enzyme. It is concluded that adenylate cyclase is present in microsomal fractions of the ventricular myocardium and can be distinguished from the plasma membrane enzyme by virtue of these differences. Since microsomal fractions of the heart constitute membranes of the sarcoplasmic reticulum, it is suggested that a fraction of the heart’s adenylate cyclase resides within these intracellular membranes.

Journal of Molecular

KEY WORDS: Adenylate cyclase; lemma; Plasma membranes.

Heart;

Microsomes;

Sarcoplasmic

reticulum;

Sarco-

1. Introduction It is widely accepted that adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1. l] resides within the plasma membrane of eukaryotic cells. In the heart, evidence exists that this enzyme is also present in the sarcoplasmic reticulum. Some years ago Entman et al. [5l reported that a microsomal fraction from canine cardiac muscle that actively accumulated Gas+ contained adenylate cyclase. Similar results were reported by Katz et al. [9]. Sulakhe and Dhalla [2.5] have reported that 2 to 4% of the total adenylate cyclase in dog and rabbit hearts could be recovered in the microsomal fraction. In all these instances the enzyme was stimulated by epinephrine and F-. Microsomal preparations from heart muscle are considered to

318

G. I. DRUMMOND

AND

J. DUNHAM

represent predominantly membranes of the sarcoplasmic reticulum. The possibility, however, exists that such microsomal fractions are contaminated with fragments of plasma membrane, which could account for small amounts of adenylate cyclase present. Such contamination could arise from excessive disruption of the sarcolemma during homogenization. Thus, definitive evidence that the enzyme is present in the sarcoplasmic reticulum has not been available. During work in our laboratory on solubilization and purification of adenylate cyclase from rabbit ventricle, it became necessary to examine microsomal preparations. We found that adenylate cyclase was indeed present in these fractions; moreover it differed noticeably in several respects from that in the easily sedimentable (plasma membrane) fraction. These findings, which point to a discrete adenylate cyclase in microsomal membranes of the heart, are described herein.

2. Materials

and

Methods

White female rabbits (2.5 to 3 kg) were anesthetized with sodium pentobarbital, and their hearts were removed and perfused with warm aerated Krebs-Ringer bicarbonate solution to thoroughly remove blood. Atria and large vessels were removed, the ventricles were cleared of fat and connective tissue and placed in ice. Ventricular muscle (usually 4 g from one heart) was finely minced and homogenized at 4°C in 5 vol. of 0.25 M sucrose, 20 mM Tris-HCI, 1 mM dithiothreitol, 5 mM MgCla, 1 mM EDTA, pH 7.5 in a Polytron PT 10 homogenizer for 30 s at a rheostat setting of 4 (to thoroughly disperse the tissue) and then for 2 s at maximal velocity. The suspension was filtered through a 250 pm nylon mesh under light suction, diluted with an equal volume of medium and centrifuged at 25 000 g for 10 min in a Sorvall RC-2 centrifuge. The supernatant fluid was removed and kept on ice. The pellet was suspended in 10 vol. of medium (based on initial tissue wt) and homogenized for two 15 s intervals at maximum velocity in a Polytron homogenizer. The suspension was centrifuged as before. The pellet was suspended in 10 vol. of medium (based on initial tissue wt) and constituted the washed particle fraction. The resulting supernatant fluid was combined with that from the first extraction and centrifugation was carried out at 100 000 g for 1 h in a Beckman Model L ultracentrifuge. The resulting pellet was suspended in a volume of medium equivalent to l/25 that used for the initial homogenization; this constituted the 100 000 g particle or microsomal fraction. Fresh preparations were used for each experiment and were assayed immediately.

Adenylate cyclase assay Adenylate cyclase was determined modifications. The assay medium

by the method of Salomon et al. [20] with minor contained in a final vol. of 150 ~1, 40 mM Tris-

CARDIAC

MICROSOMAL

ADENYLATE

CYCLASE

319

HCl, pH 7.5, 8 mM theophylline, 8 mM MgS04, 5.5 mM KCl, 20 mM phosphoenol pyruvate, 1 mM cyclic AMP, 170 pg/ml pyruvate kinase, 1 rnM [@P]ATP (40 d/min/pmol) and membranes (5 to 300 pg protein). ATP was added to start the reaction and incubations (unless otherwise indicated) were conducted at 20°C for 10 min. To measure F--stimulated activity, membranes were incubated with 8 mM NaF in the complete assay mix (ATP absent) for 20 min at 20°C prior to assay. The reaction was then started by addition of ATP as usual. Reactions were terminated by addition of 100 ~1 of “stop solution”, and [saP]cyclic AMP was isolated and quantitated as described by the above authors [ZU]. Specific activity was defined as pmol cyclic AMP formed per mg protein per min. Enzyme activity was proportional to protein concentration and time under all conditions used. Protein was determined by the method of Lowry et al. [17].

Materials [aazP]ATP, triethylammonium salt (20 Ci/mmol), and [aH(G)] adenosine 3’, 5’cyclic phosphate, ammonium salt (35 C-11 mmol) were purchased from New England Nuclear Corp. ; 5’-guanylylimidodiphosphate, sodium, was obtained from PL Biochemicals Inc. ; Lubrol PX from ICI Products Group, Montreal, and Triton X-100 from BDH Chemicals, Montreal, Canada. Pyruvate kinase (Type II from rabbit muscle), phosphoenol pyruvate, trisodium salt, and phospholipase A (from Vipera russelli) were purchased from Sigma Chemical Co., St Louis, Missouri, U.S.A.

3. Results Specific activity of adenylate cyclase in the washed particle fraction was 25 and 155 pmol per min per mg for basal and F--stimulated activity respectively (mean of five preparations, assay temperature 20°C). The corresponding values fort he microsomal fraction were 40 and 200 pmol per min per mg. Based on the original homogenate, the recovery of activity in the washed particle and microsomal fraction was 70% and 5 y0 respectively.

E$ect

of non-ionic

detergents on adenylate cyclase

in washed particle and microsomal fractions Adenylate cyclase in washed particle and 100 000 g particle fractions responded differently to low concentrations of Lubrol PX in the assay. Figure 1 shows the results of several preparations assayed in the presence of varying concentrations of

320

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(mM)

FIGURE 1. Effect of Lubrol PX on adenylate cyclase activity. Lubrol was present in the assay at the concentrations indicated. Top panels, 100 000 g particle; bottom panels, washed particle. (a) and (c), Basal activity; (b) and (d), F--stimulated activity. Each symbol represents a different membrane preparation; protein present in the assay (pg) was (0) 25; (V) 28; (A) 46; (0) 64; (0) 293; (A) 247; 0) 89.

detergent up to 2 mM [an average molecular weight of 600 for Lubrol PX was used; a concentration of 0.33 nm is equivalent to 0.02% (w/v)]. In each experiment both the basal [Figure 1 (a)] and F--activated enzyme [Figure 1 (b)] in the 100 000 g particle fraction was strongly stimulated (4- to 6-fold) by low concentrations of the detergent, whereas basal activity in the washed particle preparation [Figure 1 (c)] was only modestly increased and the F--activated enzyme in this fraction was inhibited [Figure 1 (d)]. In the studies depicted in Figure 1, a rather wide range of protein was present in the assay in the several experiments (see legend to Figure 1). We considered it possible that the difference in response of the two preparations to detergent might simply reflect, or at least be affected by, the ratio of detergent to protein in the assay. Figure 2 shows data from several experiments in which enzyme activity was examined with regard to this ratio (expressed as pmol detergentjmg protein in the assay). The same pattern of response to detergent in the two preparations emerged. The microsomal enzyme was stimulated maximally by a deter,gent/protein ratio of about 2; basal activity declined at ratios higher than this, Modest stimulation of activity in the washed particle fraction occurred at a

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FIGURE 2. Adenylate cyclase as a function of detergent-protein ratio. Four separate preparations of the 100 000 g particle (top panels) and of the washed particle (bottom panels) were assayed for was assayed at constant basal (a) and (c), and F--stimulated activity (b) and (d) . Each preparation protein concentration, but the amount varied among the different preparations from 25 to 300 pg. Detergent concentration was varied as in Figure 1. Detergent-protein ratio was calculated as detergent (pmol)/protein (mg) in the assay.

detergent/protein ratio of 0.5, while the F--stimulated enzyme in this fraction was inhibited at ratios above this. These data show a remarkable difference in the response of adenylate cyclase in the two preparations to Lubrol PX. The effect of another non-ionic detergent, Triton X-100, was examined. This detergent, at low concentrations, also produced a striking increase in both basal and F--stimulated activity in the microsomal fraction [Figure 3(a)]. In contrast, basal activity in the washed particle preparation was only modestly increased while the F--stimulated activity was depressed by concentrations as low as 0.02% w/v (0.31 mM) [Figure 3(b)]. The pattern of behaviour to detergent was not confined to rabbit heart particles. When guinea pig ventricle was fractionated in an analagous manner, the response of the enzyme in the microsomal and washed particle fractions to Lubrol PX (as shown in Figure 4) was virtually identical to that of rabbit heart. It was considered that the differing effect of detergent might simply reflect a

322

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AND

J.

DUNHAM

(0)

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3. Effect of Triton X-100 on adenylate cyclase. Conditions are as in Figure 1, except X-100 was present at the concentrations indicated. (a) 100 000 g pellet, 70 p,g protein; particle, 69 pg protein. (0) Basal; (0) F--stimulated activity.

difference in particle size in the two fractions. To test this, washed particle preparations were exhaustively homogenized in medium containing 1.25 M KC1 which effectively disrupted all membrane structures and dissolved much of the contractile protein. Following sedimentation and resuspension, such preparations responded to detergent identically to the particles from which they were derived. In the studies described thus far, all adenylate cyclase assays were conducted at 20°C. This was done because earlier experiments (data not shown) revealed that the cardiac enzyme was highly labile in the presence of detergents at physiological temperatures. In Figure 5, both preparations were assayed in the absence and presence of Lubrol PX (0.05% w/v, 0.825 mu) at 20, 28, 32 and 37°C. It is seen that stimulation of basal activity in the 100 000 g pellet fraction was still in evidence at 28”C, but at temperatures above this activity fell drastically [Figure 5(a)]. Basal activity in the washed particle fraction [Figure 5(c)] was even more labile to detergent, slight stimulation occurred only at 20°C and above this temperature the detergent greatly reduced activity. In both preparations, F--stimulated activity was much more stable in the presence of detergent; activity increased with tempera-

CARDIAC

o-

MICROSOMAL

0

0.4

ADENYLATE

0.8

Lumbrol

FIGURE 4. Assay of guinea pig heart adenylate from four female guinea pigs were fractionated Methods). (a) 100 000 g particle, 68 yg protein; (0) F--stimulated activity.

1.2

CYCLASE

323

1.6

PX (ITIM)

cyclase in the presence of Lubrol PX. Ventricles as described for rabbit hearts (Materials and 8Opg protein. (0) Basal; (b) washed particle,

ture up to 37°C. Thus the stimulatory effect of the detergent on the F--activated enzyme in the microsomal fraction was seenat all temperatures examined [Figure 5(b)] and the inhibitory effect of the detergent on this activity in the washed particles [Figure 5(d)] was also seenthroughout the temperature range examined.

Effect of ~ho.spholi@se A treatment on adenylate cyclase

In search for other differences between enzyme in the microsomal and washed particle fractions, the effect of phospholipasetreatment was examined. Membranes were incubated with varying amounts of phospholipaseA for 30 min at 30°C prior to assay, and after sedimentation and washing, were assayedfor adenylate cyclase. The data in Figure 6 reveal a striking difference between the two preparations with respect to this membrane perturbing agent. Both basal and F--stimulated adenylate cyclase in the 100 000 g particle fraction were increased following treat-

324

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I. DRUMMOND

AND

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DUNHAM

I

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(b) g particle

.

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particle

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20 temperature

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FIGURE 5. Effect of Lubrol PX on adenylate cyclase at various temperatures. Standard assay conditions were used except that the temperature was varied as indicated and Lubrol PX concentration, where present, was 0.825 mu (0.05%). Top panels, 100 000 g particle, 65 pg protein; bottom panels, washed particle, 72 pg protein. Basal activity, (a) and (c) ; F--stimulated activity, (b) and (d). Open symbols, no detergent; closed symbols, + detergent.

ment of membranes with small amounts of phospholipaseA; this declined when larger amounts were present. In contrast, basal activity in the washed particle fraction was unaffected by small amounts of phospholipase, and was reduced by larger amounts. F--stimulated activity in the washed particle fraction was unaltered by phospholipaseA treatment.

Activation

of adenylate Gyclase by guanyl nucleotide and efiinephrine

Guanine nucleotide, specifically GTP, is required for hormonal stimulation of adenylate cyclase in eukaryotic cells. Rodbell and his associates[16] introduced and extensively studied the role of a metabolically stable analog of GTP, guanylylimidodiphosphate, (Gpp(NH)p) [18, 211 in glucagon activation of the liver enzyme. This analog also activates the cardiac enzyme [13, 1.51.We examined the

CARDIAC

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ADENYLATE

100

000

Washed

0

2

5

15 2550120 Phospholipase

(b)

g port1cle

(dl

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CYCLASE

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FIGURE 6. Effect of incubating membranes with phospholipase A on adenylate cyclase. Phospholipase A was added to 0.5 ml aliquots of membrane suspensions in homogenizing medium (prepared without EDTA and containing 100 mM KC1 and 0.2 mM CaQs) to produce the final concentrations indicated. The tubes were incubated in a shaking bath for 30 min at 30°C. EGTA was then added to a final concentration of 0.2 mn and the suspensions were centrifuged at 100 000 g for 1 h. The pellets were suspended in 0.5 ml of homogenizing medium and assayed for basal and F--stimulated adenyiate cyclase. Values are the means from three different membrane preparations, except (d) in which values are the means of duplicate preparations. Vertical bars represent the average deviation from the mean. Top panels, 100 000 g particle using 45 to 73 pg protein; bottom panels, washed particle fraction using 48 to 55 pg protein in the assay. (a) and (c), Basal; (b) and (d), F--stimulated activity.

effect of Gpp(NH)p in the absence and presence of epinephrine on adenylate cyclase in both membrane fractions. In both preparations, epinephrine alone produced barely detectable stimulation of the enzyme (Figure 7). When added directly to the assay, Gpp(NH)p (0.1 mM) produced greater stimulation of the microsomal enzyme than of the washed particIe enzyme (Figure 7). Gpp(NH)p and epinephrine together caused dramatic stimulation of the washed particle enzyme, while the stimulatory effect of the two agonists together on the microsomal enzyme was not markedly greater than that caused by guanyl nucleotide alone. These data indicated that activation of the washed particle enzyme by Gpp(NH)p at 20°C

326

G.

I.

DRUMMOND

AND

J.

DUNHAM

C

GEG

(b)

t

z 2 0.8 FG a 0.6 .o 0 u” 0.4 t

0

CGE

G G

GG + E

+ E

FIGURE 7. Activation of adenylate cyclase by Gpp(NH)p and epinephrine. In one set of experiments (open bars), Gpp(NH)p, 0.1 nm (G), epinephrine, 50 PM (E), or Gpp(NH)p plus epinephrine (G+E) were added directly to the assay (C, control). In another set (shaded bars) membranes (70 to 165 pg protein) were incubated with 0.1 mu Gpp(NH)p in the complete assay system without ATP for 20 min at 20°C. ATP was then added to start the assay, the time of incubation in all cases was 5 min at 20°C. In the first set (open bars) values are means from three separate membrane preparations (70 to 165 pg protein per assay) ; vertical bars represent the average deviation from the mean. In the second set (shaded bars) values are means of two determinations. (a) washed particle; (b) 100 000 g particle.

might be significantly slower than that in the microsomal fraction. To test this, membranes were incubated with 0.1 mM nucleotide for 20 min prior to assay. Indeed, Figure 7 (shaded bars) reveals that after this time interval only slight activation of the washed particle enzyme had occurred, and a dramatic synergism was seen with epinephrine and the GTP analog. In contrast, the microsomal enzyme was almost fully activated by 20 min incubation with the analog prior to assay and the addition of epinephrine caused only a modest further increase. More detailed studies on the rate of activation of the enzyme in both preparations by guanine nucleotide in the presence and absence of epinephrine is shown in Figure 8. Under the conditions used, the washed particle enzyme was slowly activated by Gpp(NH)p and the catecholamine produced a dramatic increase in the rate of activation. The microsomal enzyme was more rapidly activated by Gpp(NH)p alone and epinephrine produced only a modest acceleration of rate.

CARDIAC

MICROSOMAL

ADENYLATE

(al

I IO

I 20

I 30 Prior

J 0 incubation

327

1

(b)

l -.-

I 0

CYCLASE

I-I

I IO

I 20

I 3(

(min)

FIGURE 8. Rate of activation of adenylate cyclase by Gpp(NH)p and Gpp(NH)p plus epinephrine. Membranes (70 to 165 pg protein) were incubated with 0.1 mn Gpp(NH)p (A, A), 0.1 rnM Gpp(NH)p plus 50 FM epinephrine (0, w), or without additions (controls) (0, @) at 20°C for the times indicated. ATP was then added to begin the assay which was conducted for 5 min at 20°C. Values are the means of three separate experiments; the vertical bars are the average deviation from the mean. (a) Washed particle; (b) 100 000 g particle. 4. Discussion These studies confirm that adenylate cyclase is present in microsomal fractions prepared from heart. They provide evidence that the activity residing there is distinctly different from that in the more easily sedimentable (plasma membrane) fraction. Thus, the microsomal enzyme in both rabbit and guinea pig ventricle was greatly stimulated by low concentrations of the non-ionic detergents Lubrol PX and Triton X-100, whereas the washed particle enzyme was only slightly enhanced and the F--activated enzyme in this fraction was inhibited by both detergents. In addition, another membrane-perturbing agent, phospholipase A, increased both basal and F--stimulated activity in the 100 000 g particle fraction while causing a decrease in basal activity and having no effect on F--stimulation of the enzyme in the washed particle preparation. The enzyme in the two preparations also differed significantly with respect to Gpp(NH)p and epinephrine activation. Lefkowitz [14] has reported that incubation of canine heart membranes

328

G. I. DRUMMOND

AND

J. DUNHAM

sedimenting at 10 000 g with phospholipase A for 5 min at 37°C caused a diminution in basal and F--stimulated adenylate cyclase. Sulakhe and Dhalla [25] found that incubation of their microsomal preparation with phospholipase A at 23°C for 10 min caused a small increase in basal and a marked increase in F--stimulated activity. Our results are generally in accord with both of the above studies and point to a distinct difference in adenylate cyclase in the two particulate fractions. Evidence has been provided from a variety of studies that stimulation of adenylate cyclase by Gpp(NH)p is a time- and temperature-dependent process [18, 191 and that the primary effect of hormone is to increase the rate of guanine nucleotide activation [I, 8, 13, 18, 19, 21, 221. In our study, activation of the enzyme by Gpp(NH)p at 20°C occurred more slowly in the washed particle fraction than in the microsomal preparation. In both preparations, epinephrine increased the rate of guanine nucleotide activation, but the effect of the catecholamine was much more dramatic in the washed particle preparation. We do not have an explanation for these observations but they point, at least, to quantitative differences in catecholamine activation of the enzyme in the two fractions. Our method of preparing the microsomal fraction differs from the widely used method of Harigaya and Schwartz [a. We used an isotonic medium (0.25 mM sucrose) because cardiac adenylate cyclase is more stable under these conditions than in hypotonic medium. The procedure we used closely resembles that of Stam et al. [23] which was considered to yield membranes with improved Gas+ accumulating activity. These fractions possess active Gas+ binding and accumulating systems and are considered to contain primarily fragmented membranes of the sarcoplasmic reticulum. The problem of resolving the precise cellular locale of adenylate cyclase in the heart has been difficult. Numerous studies involving fractionation of homogenates by differential centrifugation and preparation of at least partially purified plasma membranes [3, 7, 24, 26, 291 provide strong evidence that the enzyme is predominantly in the surface membrane. These approaches are always complicated by poor resolution of any one membrane type from other membrane fragments and it is virtually impossible to ascertain the absolute purity and yield especially of plasma membrane and sarcoplasmic reticulum. The possibility has thus been real that adenylate cyclase present in microsomal fractions does not arise from the sarcoplasmic reticulum but rather represents contamination with plasma membrane. Indeed the kinetic properties of the enzyme in the microsomal fraction described by Sulakhe and Dhalla [25] (X, for substrate, & for divalent cation, pH profile, and activation by F- and catecholamine), were remarkably similar to those described earlier [Z] for the enzyme in particles sedimented at low gravitational forces. In the studies of Entman et al. [5j and Katz et al. [9] the fraction of total adenylate cyclase in the microsomal fraction was not reported. Sulakhe and Dhalla [25] found 2 to 4% of the activity in rabbit and dog heart homogenates in this fraction. When a correction was applied to account for the yield of Gas+-accumulating

329

CARDIAC MICROSOMAL ADENYLATE CYCLASE

vesicles this value became 9 to 15% [.2.5]. The fraction of total adenylate cyclase in our microsomal preparations was also small, 5%. The marked differences in this activity from that in the more easily sedimented fraction, provide strong evidence that it does not originate as a contamination by plasma membrane, but that it represents a discrete membrane-bound form of the enzyme. Our data should not be interpreted to indicate two kinetically distinct enzyme species. The differences noted, particularly the effect of membrane-perturbing agents, may more properly reflect differences in structure and composition of the membranes in which the catalytic unit residues. Our data allow the suggestion that a small fraction of the adenylate cyclase in the heart is located in the sarcoplasmic reticulum. Recently much interest has centered on a role of cyclic AMP in Caa+ transport within the sarcoplasmic reticulum of the heart. Kirchberger et al. [IO, 14 and LaRaia and Morkin [12] have found that cardiac microsomes were phosphorylated by cyclic AMP dependent protein kinase and the phosphorylated membranes accumulated larger amounts of Ca sf than control preparations. A specific 22 000 dalton protein appeared to be the site phosphorylated [28]. Membrane phosphorylation and increased Ca2+ accumulation also occurred when the membranes were treated with epinephrine [I4 implying the presence of catecholamine sensitive adenylate cyclase in the microsomal membranes. These investigators [II, 271 have interpreted their findings that catecholamine interaction with adenylate cyclase in the sarcoplasmic reticulum results in cyclic AMP stimulated phosphorylation of membrane sites (via cyclic AMP dependent protein kinase) leading to enhanced Caa+ accumulation. This could account for the well known physiological effects of beta adrenergic amines on the heart, namely abbreviation of systole and increased contractile force. These and other recent studies [4j point to the presence of an internal beta receptor-adenylate cyclase complex in the sarcoplasmic reticulum. Although the fraction of the total adenylate cyclase we have found in this site is small, it could perform a critical physiological function, that of forming cyclic AMP to regulate Ca2+ transport in these intracellular membranes.

Acknozerledgements This work was supported by grants from the Medical and the Alberta Heart Foundation.

Research

Council

of Canada

REFERENCES 1.

2.

CUATRECASM, P., JACOBS, S. & BENNETT, V. Activation of adenylate cyclase by phosphoramidate and phosponate analogs of GTP. Possible role of covalent enzymesubstrate intermediates in the mechanism of hormonal activation. Proceedings JVational AcademyofSciences (U.S.A.) 72, 1739-1743 (1975). DRUMMOND, G. I., SEVERSON, D. L. & DUNCAN, L. Adenyl cyclase. Kinetic properties

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G. I. DRUMMOND AND J. DUNHAM and nature of fluoride and hormone stimulation. Journal of Biological Chemistry 246, 41664173 (1971). ENGELHARD, V. H., PLUT, D. A. & STORM, D. R. Subcellular location of adenylate cyclase in rat cardiac muscle. Biochemica et biophysics acta 451,4861 (1976). ENTMAN, M. L., GOLDSTEIN, M. A. & SCHWARTZ, A. The sarcoplasmic reticulumglycogenolytic complex, an internal beta adrenergic receptor. Life Sciences 19, 16231630 (1976). ENTMAN, M. L., LEVEY, G. S. & EPSTEIN, S. E. Demonstration of adenyl cyclase activity in canine cardiac sarcoplasmic reticulum. Biochemical and Biophysical Research Communications 35,728-733 (1969). HARIGAYA, S. & SCHWARTZ, A. Rate of calcium binding and uptake in normal animal and failing human cardiac muscle. Circulation Research 25,781-794 (1969). HUI, E. C., DRUMMOND, M. & DRUMMOND, G. I. Calcium accumulation and cyclic AMP-stimulated phosphorylation in plasma membrane enriched preparations of myocardium. Archives Biochemistry and Biophysics 173,415427 (1976). JACOBS, S., BENNETT, V. & CUATRECASAS, P. Kinetics of irreversible activation of adenylate cyclase of fat cell membranes by phosphonium and phosphoramidate analogs of GTP. Journal of Cyclic Nucleotide Research 2,205-223 (1976). KATZ, A. M., TADA, M., REPKE, D. I., IORIO, J. & KIRCHBERGER, M. A. Adenylate cyclase: its probable location in sarcoplasmic reticulum as well as sarcolemma of canine heart. Journal of Molecular and Cellular Cardiology 6,73-78 ( 1974). KIRCHEIERGER, M. A., TADA, M., REPKE, D. I. & KATZ, A. M. Cyclic adenosine 3’,5’monophosphate-dependent protein kinase stimulation of calcium uptake by canine cardiac microsomes. Journal of Mole&w and Cellular Cardiolopv 4,673-680 ( 1972). KIRCHBERGER, M. A., TADA, M., REPKE, D. I. & KATZ, A. M. Adenosine 3’,5’monophosphate-dependent protein kinase catalyzed phosphorylation reaction and its relationship to calcium transport in cardiac sarcoplasmic reticulum. Journal of Biological Chemistry 249,6 166-6 173 ( 1974). LARAIA, P. J. & MORKIN, E. Adenosine 3’,5’-monophosphate-dependent membrane phosphorylation. Circulation Research 35,298306 (1974). LEFKO~ITZ, R. J. Stimulation of catecholamine-sensitive adenylate cyclase by 5’guanylyl-imidodiphosphate. 3ournal of Biological Chemistry 249,6119-6124 (1974). LEFKO~ITZ, R. J. Catecholamine-stimulated myocardial adenylate cyclase. Effects of phospholipase digestion and the role of membrane lipids. Journal of Molecular and Cellular

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7,27-37

(1975).

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CARDIACMICROSOMALADENYLATE 21. 22. 23.

24. 25. 26. 27. 28.

29.

CYCLASE

331

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