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Phospholipids, adenylate cyclase, and the heart
Journal
of Molecular
and Cellular
Phospholipids,
Cardidogy
(1972) 4, 283-285
Adenylate GERALD
Diticna
of Endocrinology
Department (Received
S. LEVEY*
and Metubolimn, of Medicine,
16 January
Cyclaee, and the Heart
University of Miami School of Medicine, Miami, Florida, U.S.A.
1972, and accepted
18 January
1972)
G. S. LEVEY. Phoepholipids, Adenylate Cyolaze, and the Heart. Journd of Molecdar and Celluk Cardidogy (1972) 4, 283-285. Phospholipida are neoezaary components of all hormone-sensitive adenylata oyclaee systems. We have previouzly described the preparation of a solubiIized myooardial adenylate oyolaae whioh is unresponsive to hormonal stimulation. Phosphatidylzerine restored histamine and gluoagon aotivation and phoephetidylinositol restored norepinephrine aotivation of the solubilized enzyme freed of detergent by DEAE-aelluloze chromatography. The possible relationship of phoapholipids to oaloium tramport and hormone-induced adenylate cyoleze a&iv&ion in aarooplasmio reticulum is discussed.
Since Sutherland and coworkers first postulated that adenylate cyclase was a lipoprotein [la], a great deal of evidence has accumulated demonstrating that lipids, particularly phospholipids, are critical for all hormone-sensitive adenylate cyclase systems. Detergents, including Triton [14], digitonin [I], and Lubrol-PX [6], abolish hormone-mediated activation of adenylate cyclase. Phospholipases have been shown to abolish the glucagon activation of liver adenylate cyclase [I] and the binding of glucagon to isolated liver membranes [13]. Pohl et al. found that the addition of pure phosphatidylserine partially restored glucagon-binding and activation of adenylate cyclase in phospholipase A-treated liver membranes [12]. We have reported the preparation of a solubilized myocardial adenylate cyelase which is unresponsive to hormonal-stimulation [6]. The addition of pure phosphatidylserine restores responsiveness of the enzyme, freed of detergent by DEAE-cellulose chromatography, to glucagon and histamine [7’, 91 but not norepinephrine [Table 1(a)]. Chromatographioally-pure monophosphatidylinositol restores responsiveness to norepinephrine [fl, but not to glucagon or histamine [Table l(b)]. It is of considerable interest that the concentration of norepinephrine producing half-maximal activation of the enzyme was 8 x lo-8 M which is less than 1% of that required in the particulate (membrane-bound) preparations of the adenylate cyclase and approximating that found in physiologic preparations. Furthermore, the addition of monophosphatidylinositol to the partioulate preparation itself lowers the concentration of norepinephrine producing half-maximal activation toward that found in physiologic preparations, suggesting that the * Investigator,
Howard
Hughes Medical
Institute. 283
284
0. 9. LEVEY
TABLE
1. Effect of phospholipida on hormone-responsiveness of solubilized myocardial adenylate cyclase freed of detergent by DEAE-cellulose chromatography* Cyclic 3’,5’-AMP accumulated -per 6 min per mg protein Phosphatidylserinet Absent Present
(4
Control
469&27
Glucagon (1 x 10-5 M) Histamine (8 x IO-5 M) Norepinephrine (6 x 10-J M)
441f50 540f45 442536
(b)
Phosphatidylinositol$ Absent Present
Control Glucagon (1 X 10-s rd) Histamine (8 x 10-S M) Norepinephrine (2 x 10-s M)
250*20 238&40 286+ 10 240f40
726&28 1150f35 14ao+45 765&-56
300f50 300*40 150&86 850f 75
* Experimental details are cited in referenoes 7 and 8. t Phosphatidylserine present at 8 pg/incubation. $ Phosphatidylinoaitol present at 6 &inaubation.
process of homogenization alone has the capacity to alter the lipid+mzyme relationship resulting iu a decreased sensitivity of the enzyme to hormonal stimulation. These data may be pertinent to other processes such as hormone-induced calcium accumulation and release in sarcoplasmic reticulum since this process may require a hormone-sensitive adenylate cyclase system [3]. Hawthorne and Kemp suggested that the accumulation of divalent cations by mitochondria is dependent on the presence of membrane phosphoiuositides [5]. A number of studies have been reported demonstratiug the presence of phosphatidylinositol and other phospholipids in cardiac microsomes, the most recent being in this Journal [15, 41. Furthermore a series of investigations by Martonosi et al. have served to emphasize the critical role of phospholipids on ATPase activity and calcium transport in sarcoplasmic reticulum [lo, 111.Specific studies of the effects of phospholipids on the cardiac microsomal adenylate cyclase system have not been reported. However, apparent discrepancies concerning the effects of hormones on calcium accumulation in isolated sarcoplasmic reticulum [3, 21 may be related to alterations in specific phospholipids in these membranes produced by the individual techniques of preparation. It seems likely, on the basis of the recent data outlined above that studies of the role of phospholipids on adenylate cyclase activity and calcium accumulation in cardiac microsomes in the presence and absence of hormones such as the catecholamines, are of major theoretical and practical importance in both normal and disease states.
PHOSPHOLIPIDS,
ADENYLATE
CYCLASE,
AND
THE
285
HEART
These studies supported in part by the National Institutes of Health, Grant No. l-R01 HE 13715-01, and Florida Heart Association, Grant No. 71 AG 20. REFERENCES 1. BIRNBAUMER, L., POHL,
2.
3.
4.
6. 6. 7.
S. L. & RODBELL, M. The gluoagon-sensitive adenyl cyclaae system in plasma membranes of rat liver. II. Comparison between glucagonand fluoride-stimulated activities. Journal of Biological Chemi.&y 246, 1857-1860 (1971). DHALLA, N. S., S ULAKHE, P.V., KHANDELWAL, R.L. &HAMILTON, I.R.Excitation contraction in heart. II. Studies on the role of adenyl cyclase in the calcium transport, by dog sarcoplasmic reticulum. Li$e Sciences 9, 625-632 (1970). ENTIUN, M. L., LEVEY, G. S. & EPSTEIN, S. E. Mechanism of action of epinephrine and glucagon on the heart: evidence of a cyclic 3’,5’-AMP mediated increase in sarcotubular calcium stores. CircuZattin Rerrearch 25, 429438 (1969). GLOWER, J. & HARRIS, P. The lipid composition of subcellular fractions from different chambers of the dog’s heart. Journal of Molecular and Cellular Card
Communication.3 43, 108-113 (1971). 8. LEVEY, G. S. Restoration of norepinephrine-responsiveness of solubilized myocardial adenylate cyclase by phosphatidylinositol. Journal of Biological Chemistry 246, 7405-7407 (1971). 9< . LEVEY, G. S. & KLEIN, I. Solubilized myocardial sdenylate cyclase: Restoration of histamine-responsiveness by phosphatidylserine. Journal of Clinical Investigation (In press). 10. MARTONOSI, A. The activating effect of phospholipids on the ATPase activity and Ca2+ transport of fragmented sarcoplasmic reticulum. Biochemical and Biophysical 13, 273-278 (1963). Research Communicationa 11. MARTONOSI, A., DONLEY, J. & HALPIN, R. A. The role of phospholipids in the adenosine triphosphataae activity and Ca2+ transport. Journal of Biological Chemistry 243, 61-70 (1968). 12. POHL, S. L., KRANS, H. M. J., KOZYREFF, V., BIRNBAUMER, L. & RODBELL, M. The glucagon-sensitive adenyl cyclaae system in plasma membranes of rat liver. VI. Evidence for a role of membrane lipids. Journal of Biologic& Chemietry 246, 4447-4454 (1971). 13. RODBELL, M., KRANS, H. M. J., POHL, S. L. t BIRNBAUMER, L. The glucctgonsensitive adenyl cyclase system in plasma membranes of rat liver. III. Binding of glucegon: method of assay and specificity. Journal of Biological Chemistry 246,
1861-1871 (1971). SUTHERLAND, E. W., RALL, T. W. & MENON, T. Adenyl Cycke I. Distribution, preparation and properties. Journal of Biological Chemistry 237, 1220-1227 (1962). lipid 15. WEOLICKI, W. B., Jr., STAM, A. C., JR. & SONNENBLICK, E. H. Structural 14.
content
Molecular
of
organelles and Cellular
of
the
well-oxygenated
Cardiology 1, 131-142
canine
(1970).
myocardium.
Journal
qf
of Molecular
and Cellular
Phospholipids,
Cardidogy
(1972) 4, 283-285
Adenylate GERALD
Diticna
of Endocrinology
Department (Received
S. LEVEY*
and Metubolimn, of Medicine,
16 January
Cyclaee, and the Heart
University of Miami School of Medicine, Miami, Florida, U.S.A.
1972, and accepted
18 January
1972)
G. S. LEVEY. Phoepholipids, Adenylate Cyolaze, and the Heart. Journd of Molecdar and Celluk Cardidogy (1972) 4, 283-285. Phospholipida are neoezaary components of all hormone-sensitive adenylata oyclaee systems. We have previouzly described the preparation of a solubiIized myooardial adenylate oyolaae whioh is unresponsive to hormonal stimulation. Phosphatidylzerine restored histamine and gluoagon aotivation and phoephetidylinositol restored norepinephrine aotivation of the solubilized enzyme freed of detergent by DEAE-aelluloze chromatography. The possible relationship of phoapholipids to oaloium tramport and hormone-induced adenylate cyoleze a&iv&ion in aarooplasmio reticulum is discussed.
Since Sutherland and coworkers first postulated that adenylate cyclase was a lipoprotein [la], a great deal of evidence has accumulated demonstrating that lipids, particularly phospholipids, are critical for all hormone-sensitive adenylate cyclase systems. Detergents, including Triton [14], digitonin [I], and Lubrol-PX [6], abolish hormone-mediated activation of adenylate cyclase. Phospholipases have been shown to abolish the glucagon activation of liver adenylate cyclase [I] and the binding of glucagon to isolated liver membranes [13]. Pohl et al. found that the addition of pure phosphatidylserine partially restored glucagon-binding and activation of adenylate cyclase in phospholipase A-treated liver membranes [12]. We have reported the preparation of a solubilized myocardial adenylate cyelase which is unresponsive to hormonal-stimulation [6]. The addition of pure phosphatidylserine restores responsiveness of the enzyme, freed of detergent by DEAE-cellulose chromatography, to glucagon and histamine [7’, 91 but not norepinephrine [Table 1(a)]. Chromatographioally-pure monophosphatidylinositol restores responsiveness to norepinephrine [fl, but not to glucagon or histamine [Table l(b)]. It is of considerable interest that the concentration of norepinephrine producing half-maximal activation of the enzyme was 8 x lo-8 M which is less than 1% of that required in the particulate (membrane-bound) preparations of the adenylate cyclase and approximating that found in physiologic preparations. Furthermore, the addition of monophosphatidylinositol to the partioulate preparation itself lowers the concentration of norepinephrine producing half-maximal activation toward that found in physiologic preparations, suggesting that the * Investigator,
Howard
Hughes Medical
Institute. 283
284
0. 9. LEVEY
TABLE
1. Effect of phospholipida on hormone-responsiveness of solubilized myocardial adenylate cyclase freed of detergent by DEAE-cellulose chromatography* Cyclic 3’,5’-AMP accumulated -per 6 min per mg protein Phosphatidylserinet Absent Present
(4
Control
469&27
Glucagon (1 x 10-5 M) Histamine (8 x IO-5 M) Norepinephrine (6 x 10-J M)
441f50 540f45 442536
(b)
Phosphatidylinositol$ Absent Present
Control Glucagon (1 X 10-s rd) Histamine (8 x 10-S M) Norepinephrine (2 x 10-s M)
250*20 238&40 286+ 10 240f40
726&28 1150f35 14ao+45 765&-56
300f50 300*40 150&86 850f 75
* Experimental details are cited in referenoes 7 and 8. t Phosphatidylserine present at 8 pg/incubation. $ Phosphatidylinoaitol present at 6 &inaubation.
process of homogenization alone has the capacity to alter the lipid+mzyme relationship resulting iu a decreased sensitivity of the enzyme to hormonal stimulation. These data may be pertinent to other processes such as hormone-induced calcium accumulation and release in sarcoplasmic reticulum since this process may require a hormone-sensitive adenylate cyclase system [3]. Hawthorne and Kemp suggested that the accumulation of divalent cations by mitochondria is dependent on the presence of membrane phosphoiuositides [5]. A number of studies have been reported demonstratiug the presence of phosphatidylinositol and other phospholipids in cardiac microsomes, the most recent being in this Journal [15, 41. Furthermore a series of investigations by Martonosi et al. have served to emphasize the critical role of phospholipids on ATPase activity and calcium transport in sarcoplasmic reticulum [lo, 111.Specific studies of the effects of phospholipids on the cardiac microsomal adenylate cyclase system have not been reported. However, apparent discrepancies concerning the effects of hormones on calcium accumulation in isolated sarcoplasmic reticulum [3, 21 may be related to alterations in specific phospholipids in these membranes produced by the individual techniques of preparation. It seems likely, on the basis of the recent data outlined above that studies of the role of phospholipids on adenylate cyclase activity and calcium accumulation in cardiac microsomes in the presence and absence of hormones such as the catecholamines, are of major theoretical and practical importance in both normal and disease states.
PHOSPHOLIPIDS,
ADENYLATE
CYCLASE,
AND
THE
285
HEART
These studies supported in part by the National Institutes of Health, Grant No. l-R01 HE 13715-01, and Florida Heart Association, Grant No. 71 AG 20. REFERENCES 1. BIRNBAUMER, L., POHL,
2.
3.
4.
6. 6. 7.
S. L. & RODBELL, M. The gluoagon-sensitive adenyl cyclaae system in plasma membranes of rat liver. II. Comparison between glucagonand fluoride-stimulated activities. Journal of Biological Chemi.&y 246, 1857-1860 (1971). DHALLA, N. S., S ULAKHE, P.V., KHANDELWAL, R.L. &HAMILTON, I.R.Excitation contraction in heart. II. Studies on the role of adenyl cyclase in the calcium transport, by dog sarcoplasmic reticulum. Li$e Sciences 9, 625-632 (1970). ENTIUN, M. L., LEVEY, G. S. & EPSTEIN, S. E. Mechanism of action of epinephrine and glucagon on the heart: evidence of a cyclic 3’,5’-AMP mediated increase in sarcotubular calcium stores. CircuZattin Rerrearch 25, 429438 (1969). GLOWER, J. & HARRIS, P. The lipid composition of subcellular fractions from different chambers of the dog’s heart. Journal of Molecular and Cellular Card
Communication.3 43, 108-113 (1971). 8. LEVEY, G. S. Restoration of norepinephrine-responsiveness of solubilized myocardial adenylate cyclase by phosphatidylinositol. Journal of Biological Chemistry 246, 7405-7407 (1971). 9< . LEVEY, G. S. & KLEIN, I. Solubilized myocardial sdenylate cyclase: Restoration of histamine-responsiveness by phosphatidylserine. Journal of Clinical Investigation (In press). 10. MARTONOSI, A. The activating effect of phospholipids on the ATPase activity and Ca2+ transport of fragmented sarcoplasmic reticulum. Biochemical and Biophysical 13, 273-278 (1963). Research Communicationa 11. MARTONOSI, A., DONLEY, J. & HALPIN, R. A. The role of phospholipids in the adenosine triphosphataae activity and Ca2+ transport. Journal of Biological Chemistry 243, 61-70 (1968). 12. POHL, S. L., KRANS, H. M. J., KOZYREFF, V., BIRNBAUMER, L. & RODBELL, M. The glucagon-sensitive adenyl cyclaae system in plasma membranes of rat liver. VI. Evidence for a role of membrane lipids. Journal of Biologic& Chemietry 246, 4447-4454 (1971). 13. RODBELL, M., KRANS, H. M. J., POHL, S. L. t BIRNBAUMER, L. The glucctgonsensitive adenyl cyclase system in plasma membranes of rat liver. III. Binding of glucegon: method of assay and specificity. Journal of Biological Chemistry 246,
1861-1871 (1971). SUTHERLAND, E. W., RALL, T. W. & MENON, T. Adenyl Cycke I. Distribution, preparation and properties. Journal of Biological Chemistry 237, 1220-1227 (1962). lipid 15. WEOLICKI, W. B., Jr., STAM, A. C., JR. & SONNENBLICK, E. H. Structural 14.
content
Molecular
of
organelles and Cellular
of
the
well-oxygenated
Cardiology 1, 131-142
canine
(1970).
myocardium.
Journal
qf