protein and polypeptide hormones · protein and polypeptide hormones proceedings of the...
TRANSCRIPT
Λ
P R O T E I N AND P O L Y P E P T I D E HORMONES
P R O C E E D I N G S O F T H E I N T E R N A T I O N A L S Y M P O S I U M L I E G E , M A Y 19-25, 1968
Honorary President
M . D U B U I S S O N
Rector o f the University o f Liege
President
H . V A N C A U W E N B E R G E
Professor o f Internal Medicine, University o f Liege
Editor
M . M A R G O U L I E S
Liege
1969
EXCERPTA MEDICA FOUNDATION Amsterdam
C O N T E N T S
HENRY V A N CAUWENBERGE: General Introduction xvn
P A R T 1
M A I N R E P O R T S A N D I N V I T E D P A P E R S
I . Radioimmunoassay of Protein and Polypeptide Hormones
S. A. BERSON: Introduction to symposium on radioimmunoassay 3
Session 1: Specificity of Radioimmunoassay
W . M . H U N T E R : Control of specificity in the radio-immunoassay 5 R. FRÄSER, C. L O W Y and A . H . RUBENSTEIN: Radioimmunoassay on urine samples . . 1 4 H.-J . QUABBE: Sources of error in the immunoprecipitation system of radioimmunoassays 21 G . D . B R Y A N T , M . HUXSTER and F.C. GREENWOOD: Immuno-reactive fragments of endo
genous protein hormone in plasma . . 2 6 F . A . STEPHENSON and F . C . GREENWOOD: A new hormone of lactation or an artefact of
radioimmunoassay 28 J . L A N D O N , J . G I R A R D and F . C . GREENWOOD: The specificity of a radioimmunoassay for
human plasma A C T H 29 B . R. WEBSTER and F . C . GREENWOOD: Relative measurements of H-TSH obtained by
radioimmunoassay 3 2 J.P. MONJARDINO, G . D . B R Y A N T , F . L . STEPHENSON and F . C . GREENWOOD: Density
gradient analysis of labelled and unlabelled peptide hormones and of exogenous plasma hormones 34
Session 2: Labelling—Separation of Free Labelled Hormone from Antibody-bound Hormone
R.S. Y A L O W and S.A. BERSON: Topics on radioimmunoassay of peptide hormones 36 K . J . C A T T and G . W . TREGEAR: Solid-phase radioimmunoassay 45 C. R. M O R G A N : A two antibody system for radioimmunoassay of protein hormones . 4 9 V. HERBERT: Coated charcoal separation of free labelled hormone from hormone bound
to antibody 55 L . E . M . M I L E S and C . N . HALES: A n immunoradiometric assay of insulin 61
Session 3: Special Problems in the Radioimmunoassay of Small Polypeptides
R.S. Y A L O W and S.A. BERSON: Special problems in the radioimmunoassay of small polypeptides 71
ν
CONTENTS
K . J . C A T T , M . D . C A I N and J . P. C O G H L A N : Radioimmunoassay of angiotensin I I in blood 77 S.M. G L I C K , P. KUMARESAN, A. K A G A N and M . WHEELER: Radioimmunoassay of oxy
tocin 81 R . H . UNGER and A . EISENTRAUT: Special problems of the radioimmunoassay for glu
cagon 8 4 R. ASSAN, G. ROSSELIN and G. TCHOBROUTSKY: Glucagon in blood and immunological
specificity of glucagon molecule 8 7 T H . L . GOODFRIEND: Radioimmunoassay of bradykinin and angiotensin 9 1 R.C. T A L A M O , K . F . AUSTEN and E. HABER: Effect of carrier and method of coupling on
the immunogenicity of bradykinin 9 3
Session 4: Assays of Gonadotropins
P. FRANCHIMONT: Radio-immunoassay of gonadotropic hormones 9 9 A . R . M I D G L E Y , JR. and L . E . REICHERT, JR.: Specificity studies on a radioimmunoassay
for human follicle stimulating hormone 1 1 7 W . D . O D E L L , L . E . REICHERT and R . W . BATES: Pitfalls in the radioimmunoassay of car
bohydrate containing polypeptide hormones 124 R.J . R Y A N and C H . F A I M A N : Radioimmunoassay of human follicle stimulating hormone:
A comparison of several FSH antisera 129 W . R . B U T T and S.S. L Y N C H : Some observations on the radioimmunoassay of follicle
stimulating hormone 134
Π. Lipolysis and Lipogenesis. Mode of Action of A C T H
E . W . SUTHERLAND and R . W . BUTCHER: Introduction to chapter on protein and polypeptide hormone and metabolism 141
Session I and 2 Lipolysis — Lipogenesis
L . A . CARLSON: Lipolysis of adipose tissue triglycerides. Regulation, physiological and clinical significance 143
C . H . HOLLENBERG and A . VOST: Regulation of adipose tissue mass. Origin of adipose lipid and control of fat cell formation (See Part 2 )
R . W . BUTCHER and E . W . SUTHERLAND: The role of cyclic A M P in the lipolytic and anti-lipolytic actions of hormones on adipose tissue 150
P.R. B A L L Y and K . L . T I L B U R Y : Hormone-induced fatty acid (FFA)-release from isolated fat cells: Role of calcium ion concentration in the incubating medium 154
K . STOCK and E. WESTERMANN: Interactions between A C T H , adrenolytic drugs and prostaglandin Ej in the lipolytic system 159
J. ÖSTMAN: Hormones and l ipid metabolism of human adipose tissue 162 E. W A L A A S , O. W A L A A S and A . W I C K : Effect of ouabain on insulin stimulation of 3 2 P
labelling of adenosine triphosphate and guanosine triphosphate in the isolated rat diaphragm 164
J.L . SKOSEY: Influence of adrenocorticotropin upon lipogenesis by isolated adipose tissue 166
VI
CONTENTS
Session 3: Mode of Action of ACTH
S . B . K O R I T Z : On the mechanism of action of adrenocorticotropin 171 R . W . BUTCHER and E . W . SUTHERLAND: The role of cyclic A M P in the steroidogenic
actions of A C T H and L H 176 R . V . FARESE: ACTH-induced changes in the steroidogenic activity of cell-free prepara
tions of the rat adrenal 181 J . J . FERGUSON, JR.: Studies on the steroidogenic effect of adrenocorticotropin . . . 185 L . D . G A R R E N : Studies on the possible role of protein synthesis in the regulation of steroi
dogenesis by A C T H 189 M . STAEHELIN and R . M A I E R : The effect of inhibitors of protein synthesis on various para
meters of A C T H action in the adrenal 193
I I I . Relationship of Structure to Activity of Protein and Polypeptide Hormones
C . H . L i : Remarks on the studies of structure-function relationships of protein and polypeptide hormones 199
R . SCHWYZER: Relationship of structure to activity of polypeptide hormones . . . . 2 0 1 J. LEONIS: Protein conformation and biological activity 208 B . T . P ICKERING: A comparative endocrinologist's approach to structure-action relation
ships 2 1 4 J. RUDINGER, I . KREJCI , I . POLÄCEK and B . K U P K O V A : Neurohypophysial hormone anal
ogues wi th inhibitor properties: Structure-activity relationships 2 1 7 F . M O R E L , S. JARD, J . BOURGUET and F . BASTIDE: Neurohypophysial peptides. Relation
of molecular structure to activity 2 1 9 B . BERDE: Relationship of structure to activity of neurohypophysial hormones . . . 2 2 2 E. SCHNABEL and A. OBERDORF: Analogues of physalaemin derivatives and their activities 2 2 4 P. DE LA LLOSA and M . JUTISZ: Quaternary structure and biological activity of ovine
pituitary interstitial-cell stimulating hormone (ICSH or L H ) 229 R . GRATTAROLA: Effects of human gonadotropins on ovulation in women 2 3 4 E. A. K O L L I , A . L . SINITSYNA and Υ υ . M . K F D A : The effect of enzymatic hydrolysis and
aggregation on the biological activity of the bovine growth hormone 239
IV . Mode of Action of Protein and Polypeptide Hormones on the Mechanisms of Transport
T. R . RIGGS: Introduction to the chapter on transports 2 4 5
Session 1: Transport of Ions
H . RASMUSSEN: Influence of polypeptide hormones upon ion transport and ion binding 2 4 7 F . BASTIDE, J . BOURGUET, S. JARD and F . M O R E L : Mode of action of neurohypophysial
hormones on active sodium transport by isolated frog skin 257 J. CRABBE: Stimulation of active sodium transport by insulin 2 6 0 I . L . SCHWARTZ and R. W A L T E R : Neurohypophyseal hormone-calcium interrelationships
in the toad bladder 2 6 4
V I I
CONTENTS
Session 2: Transport of Glucids
R . K . CRANE: Comments on insulin and the membrane transport of sugars 270 M . RODBELL: A possible mechanism for the primary action of insulin 277 W . D . STEIN: A possible cytoplasmic form of the permease concerned in galactoside trans
port in Escherichia coli 280 Τ. CLAUSEN, J . LETARTE and M . RODBELL: Cations, glucose transport, and insulin action 282
Session 3: Transport of Amino Acids
I . G . W O O L : Insulin and amino acid transport in muscle 285 K . L . MANCHESTER: Contribution to the discussion on hormones and amino acid trans
port 296 E. SCHOFFENIELS: The fluxes of amino acids across the intestinal epithelium of the tortoise 300 B . JEANRENAUD, R . J . Ho and M . T O U A B I : Aspects of intermediary metabolism in the
isolated fat cells. I . Lipolysis and antilipolysis. I I . Amino acid metabolism . . . 303
Session 4: Transport of Lipids
A . A N G E L : The composition and transport of newly synthesized lipid in isolated white adipose cells 307
T . J . F IDDLER and I . R . FALCONER: Effect of prolactin on mammary gland lipoprotein lipase activity 320
J . W I N A N D , J . FURNELLE and J . CHRISTOPHE: The heterogeneity of the N E F A pools in
epididymal and liver tissue of normal mice 324
P A R T 2
F R E E C O M M U N I C A T I O N S
i. Radioimmunoassay of Protein and Polypeptide Hormones
Session 1: Specificity of Radioimmunoassay
R.P. E K I N S and G . B . N E W M A N : The optimisation of precision and sensitivity in the radioimmunoassay method 329
J. G I R A R D and F . C . GREENWOOD: Radioimmunoassay for human growth hormone in urine. Aspecific factors imitating the presence of growth hormone 332
J . l . T H O R E L L : Improved accuracy of standard curves in radioimmunoassay 335 P.H. W R I G H T and W . J . MALAISSE: A routine method for the assay of insulin antibodies
and some applications 337 T H . LEMARCHAND-BERAUD and A. V A N N O T T I : Variations of human plasma TSH in
thyroid diseases by a radioimmunoassay and study of its specificity 340 J. M . LOWENSTEIN, A.S. B L U M , F .S . GREENSPAN and J. R. H A R G A D I N E : Immunofluores-
cent assay of TSH and LATS 343 N . F . C U N N I N G H A M : The radioimmunoassay of bovine and ovine growth hormones . 345
VIII
CONTENTS
P. FREYCHET, G. ROSSELIN and J. D O L A I S : Radioimmunological study of cross-reactions
presented by the human thyrotropin 348
Session 2: Labe Hing—Separation of Free Labe lied Hormone from Antibody-bound Hormone
A . E . FREEDLENDER: Practical and theoretical advantages for the use of I 1 2 5 in radioimmunoassay 351
M . L . M I T C H E L L , S. COLLINS, J. B Y R O N and M . ERNESTI: Immunoassay of human growth
hormone or insulin by enzyme proteolysis 354 H . B R A U M A N : Technical observation on radioimmunoassay of H . G . H 356
Session 3: Special Problems in the Radioimmunoassay of Small Polypeptides
E. HABER, Μ. B. V A L L O T T O N , A . K I M U R A and L . B. PAGE: Immunoassay for Angiotensin
I I 359 G. W. B O Y D , J. L A N D O N and W.S. PEART: Some problems encountered in the radio
immunoassay of circulating angiotensin I I 365 H . J.G. HOLLEMANS, J. V A N DER M E E R and J. L. TOUBER: A radioimmunoassay of
angiotensin, its use in the measurement of renin activity in plasma 370 J.-P. FELBER and M . L . A U B E R T : Study on the specificity of antisera used for the radio
immunological determination of A C T H . Measurement of the circadian rhythm of plasma A C T H 373
A. G A L S K O V : Problems in the determination of corticotropin by the double antibody radioimmunochemical method 377
V . K . V A N C E , J.J. SCHNURE and M . R E I C H L I N : Induction of antibodies to porcine A C T H in rabbits with nonsteroidogenic polymers of BSA and A C T H 380
J.E. S O K A L : Bioassay of glucagon 385 R . M . B U C K L E , G . D . A U R B A C H and J.T. POTTS: The use of radioimmunoassay of
parathyroid hormone in the assessment of adaptation of parathyroid glandular activity 389
Session 4: Assays of Gonactofrophins
S.W. ROSEN, S. SCHLAFF and J. R O T H : Anti-human follicle stimulating hormone: Complete cross reactivity with three other human glycoprotein trophic hormones, luteinizing hormone, human chorionic gonadotropin and thyrotropin 396
R.B. SNOOK: Immunological and biological properties of antiserum to bovine L H . . 398 P . M . STEVENSON and A . C . S P A L D I N G : Experiences with a radio-immunoassay for
luteinising hormone 401 R.E. D O L K A R T , B. H A L P E R N , E. TOROK, J . I . BREWER, J.H. SKOM and A . B . GERBIE:
Parallel immuno-assay and bio-assays on 76 patients with choriocarcinoma . . . 404 P. G. CROSIGNANI, F . P O L V A N I and R. SARACCI: Characteristics of a radioimmunoassay
for H C G - L H 409 H . G . BURGER, J.B. B R O W N , K . J . C A T T , B. H U D S O N and J.R. STOCKIGT: Physiolo
gical studies on the secretion of human pituitary luteinising hormone and gonadal steroids 412
H . K A R G and D . SCHAMS: Attempts to determine L H in bovine blood 415
IX
CONTENTS
I I . Lipolysis and Lipogenesis. Mode of Action of A C T H
Sessions 1 and 2: Lipolysis—Lipogenesis
C.H. HOLLENBERG and A. VOST: Regulation of adipose tissue mass. Origin of adipose lipid and control of fat cell formation 421
C. N . H A L E S , Τ . M . CHALMERS, M . C . PERRY and D . R . W A D E : Investigations of the
hormonal control of lipolysis 432 M . B E N U Z Z I - B A D O N I and J.-P. FELBER: Purification of a factor from guinea-pig serum
which potentiates the lipolytic activity of A C T H in isolated fat cells 444 E. G O T H , G . CSEH, J. F Ö V E N Y I and L . G R A F : The effect of epinephrine and human
pituitary polypeptides on adipose tissue of obese subjects 448 G . POZZA, A . G H I D O N I and G . SORGATO: Insulin effect on lipogenesis in human adipose
tissue in vitro 451 A. E L E W A U T , J. PARIJS, A . DE WEERDT and F . BARBIER: Influence of insulin on lipid
metabolism 455
Session 3: Mode of Action of ACTH
M . K . B I R M I N G H A M , S. HUBERMAN and L . R I V E N : Stimulation of aerobic glycolysis in mouse adrenal glands by A C T H and by adenosine-3',5'-monophosphate 458
R.A. L E V I N E : Studies on the mechanism of the steroidogenic response to exogenous cyclic 3 ',5 ' -A MP in man 461
E . K . M A T T H E W S and M . SAFFRAN: Effect of A C T H and of ions on the membrane potential of adrenocortical cells 465
M . MARGOULIES, P. C O N I N X and G . PLOMTEUX: Some observations on the effects of calcium, potassium and chloramphenicol on the adrenal response to A C T H in vitro 467
P.C. SCRIBA and O.A. M Ü L L E R : On the adrenal subcellular distribution of 3 H - A C T H . 472
I I I . Relationship of Structure to Activity of Protein and Polypeptide Hormones
T.C. W u u and M . SAFFRAN: A neurohypophysial polypeptide that binds lysine vasopressin and oxytocin 477
D . BRANDENBURG and H . A . OOMS: Des-glycineA l-des-phenylalanineB 1-insulin and
related insulin derivatives 482 H . A. OOMS, Y . A R N O U L D and J . R . M . FRANCKSON: Comparison between the immuno
logical and metabolic behaviours of crystalline insulin and labelled iodo-insulins . 485 A. WALSER and T H . M Ü L L E R : The adrenocorticotropic effect in humans of several
synthetic peptides related to ß 1 - 2 4 corticotrophin 487 P.A. DESAULLES, B . R I N I K E R and W. R I T T E L : High corticotrophic activity of a short-
chain synthetic corticotrophin analogue 489 L . SZPORNY, G Y . T. HAJOS, SZ . SZEBERENYI and G Y . FEKETE: Comparative pharma
cology of some synthetic human A C T H sequences 492 G . LUGARO, M . M . CASELLATO, M . M O T T A , F . PIVA and L . M A R T I N I : Cerebral cortex
principles and the control of A C T H secretion 495 G . CSEH and L . G R A F : Species characteristics in lipid mobilizing peptides of pig
pituitary 498
χ
CONTENTS
A. PEKKARINEN and U . K . R I N N E : Bioassays of prolonged corticotropin preparations on living guinea pig and their clinical comparison in the two-day corticotropin test in man 500
A . SAKAMOTO and K . N . PRASAD: The newly-observed catecholamine-like actions of ß-melanocyte-stimulating hormone 503
L . O L I V E R and A . STOCKELL HARTREE: Amino acid sequences in horse growth hormone 505 F. J .A . PROP: Action of prolactin and human placental lactogen ( H P L ) on human
mammary gland in vitro 508 G . H E N N E N and J . G . PIERCE: Further characterization of the human chorionic thyroid-
stimulating factor (HCTSF) 511
I V . Mode of Action of Protein and Polypeptide Hormones on the Mechanisms of Transport
Session I : Transport of Ions
R.S. SNART, N . N . S A N Y A L and T. D A L T O N : Adsorption of octapeptide hormones onto artificial membranes in relation to their effect on ion transport 519
J . H . CORT, B . L I C H A R D U S , V . PLISKA, V . U H R I N , T. B A R T H and J . R U D I N G E R : The
origin, nature and mechanism of action of natriuretic hormone 523
Session 2: Transport of Glucids
J .R. CARTER, JR. and D . B . M A R T I N : The effect of N-ethylmaleimide on insulin-mediated glucose transport in isolated fat cells 526
L . L . M A D I S O N , R. L U F T and W . A . SEYFFERT, JR. : Acute effect of physiologic and
pharmacologic doses of growth hormone on hepatic glucose output and peripheral glucose utilization 529
J. M . SOWERBY: Effect of luteinising hormone on the in vitro penetration of D-xylose into luteinised rat ovary 539
Session 3: Transport of Amino Acids
W . J . POZNANSKI: Hormonal influences on plasma amino acid(s) levels in man 542 E. BERNARD-WEIL, C. DA L A G E , C. PIETTE and L . O L I V I E R : Action of lysine vasopressin
on the protein content of HeLa cell culture and on the R N A and D N A concentration of tissue incubations 547
P A R T 3
D I S C U S S I O N S
Session 1: Specificity of Radioimmunoassay
A. Non-hormonal non-specificity Interference with primary hormone-antibody reaction by salts and other solutes 554 Damage to labelled hormone, preparation damage, incubation damage . . . . 562
XI
CONTENTS
Damage to endogenous hormones 571 B. Hormonal non-specificity
Cross reacting biologically distinguished hormones 573 Isohormones 582 Hormonal fragments, derivatives, metabolic products, etc 585
C. Species specificity 589
Session 2: Labelling and Separation
A. Preparation and stability of labelled preparations Methods 608 Limitations of specific activity 612 Purification 621 Incubation damage 624
B. Separation methods
Double antibody 627 Charcoal 633 Solid-phase 639 Labelled antibody 646 Comparative study of methods 646
Session 3: Special Problems in the Radioimmunoassay of Small Polypeptides
A. Discussion of guest speakers' contributions 653 B. Immunization procedures 665 C. Problems of sensitivity with special reference to optimal conditions
Concentrations of tracer and antiserum, time and temperature of incubation, volume of incubation, etc 672 Extraction of hormones from plasma 682
D . Specificity. Cross-reactivity of isohormones 684 E. Generation of hormones in plasma 685 F. Correlation of neutralizing and binding activity of antisera 689
Session 4: Assays of Gonadotropins
A. State of purification of gonadotropins: evidence for contamination (biochemical and biological) 693
B. Immunochemical cross-reactivities of FSH, L H , H C G , TSH Reaction with antigenic determinants common to all hormones or contamination in the system ? 697 Antigenic determinants, carbohydrate or peptide? 701
C. Suppression of cross-reactions by addition of loading quantities of other hormones. Selection of antisera for enhanced specificity 705
D . Immunochemical identity or lack of identity of glandular, plasma and urinary gonadotropins 714
X I I
CONTENTS
Ε. Correlation of bioassay and immunoassay results. Dissociation of biological and immunochemical activities 718
F. Separation techniques for bound and free hormones 719 G. Physiological evidence for specificity of radioimmunoassay. Appropriate responses
of plasma hormone to suppression by target hormones, e.g., thyroid, estrogens, etc. . 723
Session 5: Lipolysis
A. Why do so many hormones stimulate lipolysis? 735 B. D o all lipolytic agents work through cyclic A M P ? 736 C. D o various hormones act on the same or on different sites? 737 D . Prostaglandins. A role in physiologic lipolysis? 739 E. Role of the ions, chiefly Κ and Ca 740 F. Species differences: human versus experimental animals? 741
Session 6: Lipogenesis
A . What is the importance of in situ fatty acid synthesis versus deposition from plasma ? 747 B. The cell duplication question 749 C. By what mechanism is insulin a 'lipogenetic' factor? 750 D . Other lipogenetic factors or antilipolytic factors, including I L A 752 Ε. Questions of compartmentalization 754
Session 7: Mechanism of Action of ACTH
A. Outer membrane - A C T H interaction Formation of 3 ' ,5 ' -AMP 759 A C T H and electrolytes 760 A C T H penetration in the adrenal cell 764
B. Action on steroidogenesis Increased protein synthesis 766 Enzyme activation, steroid enzyme and N A D P H formation 768 Cholesterol ester and fatty acids 773 Increased transformation of cholesterol into pregnenolone and mitochondrial membrane phenomena 774
Session 8: Structure—Activity
A . General topics 777
B. Neurohypophyseal hormones 780
C. Insulin-proinsulin 782
D . Physealamin 784
E. ACTH-MSH 785
XIII
CONTENTS
F. G H , lipotropin, HCS, HCT 792 G. Gonadotropins 798
Session 9: Ion Transport
A. Properties of membranes 807 Β. Comments on properties of membranes and ion transport 808
Session 10: Glucid Transport
A. Discussion of Dr . R .K . Crane's report 818 B . On the suggested insulin receptors
Hexokinase? 822 Adenyl-cyclase ? The effects on other nucleotides and sugar phosphates 826 SH-groups involved? 832 Interaction of insulin with membrane lipids 832
C. Insulin-like effects of ions 834 D . Effect of insulin on the activity of glucose-6-phosphate dehydrogenase, guanosine-
triphosphatase and ribonuclease in the liver mitochondria of alloxan-diabetic animals 837
E. Action of other polypeptide hormones on sugar metabolism Growth hormone 840 Luteinizing hormone 840
Session 11: Amino Acid Transport
A. Relation to protein synthesis 844 B . Interrelations of amino acid transport with other transports
Relation of amino acid transport to ions 856 Relation to other transports 859
C. Problems of specificity Tissue specificity 861 Specificity of transport systems 862
Session 12: Lipid Transport
A. Discussion of Dr . A. Angel's report 867 B . The role of lipoprotein lipase in transport 869 C. The fatty acid acceptor in the interstitial space 871 D. The rate of triglyceride disappearance from the blood 872
Stop Press Session 1
I . F Ö L L I N G and N . N O R M A N : Circulating insulin antibody in a case without previous insulin treatment. Plasma protein studies 877
M . RODBELL and L . BIRNBAUMER: Adenyl-cyclase: a single enzyme activated via separate hormone-sensitive receptor molecules 878
XIV
CONTENTS
R.A. LEVINE: Stimulation of plasma insulin and growth hormone in man by cyclic
3 ' ,5 '-AMP 879
K . L . MANCHESTER: Action of insulin and intracellular pH 882
A. S. L U Y C K X and P.J. LEFEBVRE: Release of glucagon or a glucagon-like immuno-reactive material by rat jejunum incubated in vitro 884
L . A. FROHMAN, J.E. SOKAL and M . R E I C H L I N : Inhibition of biologic activity of glucagon by anti-glucagon serum 887
J.S. INCZE and H . ANTONIADES: Relation between morphologic changes in pancreatic islets and insulin levels in blood of alloxan-diabetic normal and hypophysectomized rats 888
W . D . O D E L L , W . D . D A V I D S O N , C. CHARTERS and J.C. THOMPSON: Radioimmunoassay
for human gastrin using unconjugated gastrin as an antigen 894
G.W. B O Y D , A.E. F I T Z and W.S. PEART: The measurement of plasma renin by radioimmunoassay of generated angiotensin I 896
Stop Press Session 2
J.-G. RAUSCH-STROOMANN, G. TRENKNER and R.PETRY: The influence of human chori
onic gonadotropic hormone (HCG) on metabolism of obese persons on restricted diet 903
B . L . PIMSTONE, S.J. SAUNDERS, J . D . L . HANSEN and B . BUCHANAN-LEE: Observations on
the serum albumin and plasma amino acids in association with the elevated growth hormone levels found in protein deficiency states 906
G. NISWENDER, L . REICHERT Jr. and A.R. M I D G L E Y Jr.: Radioimmunoassay for lutein
izing hormone in cattle and sheep 908
N . M U N T E A N U IVÄNUS, C. LEPÄDATU and B . SARA: Interrelation between insulin and
lipolysis 909
G. HENNEN: Thyroid-stimulating activity in serum of pregnant women 911
Addendum
L . K . STAROSELTSEVA: A study of insulin activity in healthy persons, patients with diabetes and patients with insulinoma 917
M . I . BALABOLKIN: Acromegaly in relation to protein and lipid metabolism 918
E.A. VASIUKOVA, G.S. ZEFIROVA, M . L B A L A B O L K I N and A.S. VDOVICHENKO: Changes in
growth hormone secretion under the influence of anabolic steroids in certain endocrine diseases 920
Subject Index 922
Authors Index 937
xv
ON T H E A D R E N A L S U B C E L L U L A R D I S T R I B U T I O N O F 3 H - A C T H *
P.C. SCR1BA and O .A. M Ü L L E R
I I . Medizinische Kl in ik der Universität, Munich, German Federal Republic
Introduction
Studies performed on the biochemical mechanism of stimulation of adrenal protein synthesis (Scriba and Reddy, 1965) revealed that after A C T H treatment of rats a protein factor of the soluble cell fraction from the adrenals (105,000 χ g supernatant) was doubled in activity. This factor was shown to be non-dialysable, heat labile (55 °C), inactivated by trypsin and rate limiting for 1 4 C-glycine incorporation by the 15,000 χ g soluble cell fraction of adrenal homogenates (Farese and Reddy, 1963; Scriba and Reddy, 1965) and by adrenal polysomes (Scriba and Fries, 1967). This protein factor is probably aminoacyltransferase (Scriba and Reddy, 1965). The stimulation by A C T H in vivo of the adrenal protein factor, rate limiting adrenal protein synthesis in vitro and the stimulation of corticosterone synthesis in vivo appeared to be dissociable 30 hours and 8-14 days after hypophysectomy (Scriba, Fries and Kluge, 1967). Such dissociation after hypophysectomy has been reported for other adrenal effects of A C T H (Harding and Nelson, 1964; Staehelin, Barthe and Desaulles, 1965). With regard to mechanisms of actions of A C T H , it therefore appeared attractive to leave the investigations of events in the adrenal following A C T H and to turn to the problem of the subcellular distribution of 3 H - A C T H in the adrenal. Information about adrenal receptor(s) for A C T H might possibly be obtained from such studies.
Methods
Synthetic ß{ ~ 2 3corticotropin-23-amide-acetate catalytically labelled with tr i t ium was generously supplied by Farbwerke Hoechst A G . The material was purified using dextran gel filtration (v. Werder, Schwarz and Scriba, 1968) resulting in parallel elution of radioactivity and biological activity upon rechromatography. Purified 3 H - A C T H , 300-450 m U =3-4.5 μg Ξ Ξ 1.1-1.65 μ Ο , in 1.0 ml saline (0.025 Ν HCl) was infused for 95 sec. into the thoracic aorta of rats 90 min. after hypophysectomy. Corticosterone increment in adrenal vein plasma was assayed fluorimetrically (Scriba et al., 1966), and found to be maximal from 4 to 11 min. after the start of the 3 H - A C T H infusion. At times indicated (Table I ) both adrenals were removed, carefully cleaned and homogenized for 3 x 5 sec. (Potter-Elvehjem) at 26 mg/ml 0.25 Μ sucrose-electrolyte solution (Scriba and Reddy, 1965) within 5 min. Preparation of subcellular fractions was achieved by differential centrifugation (Schneider and Hogeboom, 1951) of homogenates: supernatants: nuclear fraction (15 min. χ 1,000 χ g sed.), mitochondrial fraction (10 min. χ 15,000 χ g sed.), microsomal fraction (90 min. χ 105,000 χ g sed.) and soluble cell fraction (SN). Nuclear fractions, averaging 54% of the total adrenal weight, contained 76% 'intact' nuclei, 7% ruptured nuclei, 14% nuclei with adherent cellular material, 3% erythrocytes and practically no unbroken cells.
* Supported by Deutsche Forschungsgemeinschaft and Studienstiftung des Deutschen Volkes.
472
ON THE ADRENAL SUBCELLULAR DISTRIBUTION OF 3 H - A C T H
T A B L E I
Adrenal subcellular distribution of ^H-ACTH a. Activity (CPM) of subcellular fractions given as
b. % of total adrenal activity. Results given as mean ±S.E.
Minutes after start o f 3 H - A C T H infusion 3 12 40 120
Number o f experiments (N) 19 17 17 17
T o t a l activity ( C P M ) per pair of adrenals 407.8 ± 5 4 . 0 337.6 + 78.0 215.7 + 31.8 220.0+18.4
Nuclear fraction a. C P M 105.3 + 13.6 84.1 + 12.9 85.4+16.8 78 .5+ 5.5 b. % 27.37 + 2.38 30.86 + 2.42 37.95 + 2.05 37.41+2.41
Mitochondr ia l fraction a. C P M 48 .2+ 9.3 52 1 + 10.8 35 .0+ 5.6 17.7+ 2.5 b. % 13.69 + 3.04 17.01+1.89 16.54+1.63 8.49+1.16
Microsomal fraction a. C P M 77.2+18.5 38 .7+ 7.1 16.9+ 2.1 6 . 9 + 1.1 b. % 17.93 + 3.13 12.97 + 0.84 8.85 + 1.14 3.39+0.54
Soluble cell fraction a. C P M 177.2 + 39.5 162.7 + 58.2 78.4+14.2 116.8 + 16.3 b. % 41.02+4.21 39.15 + 3.32 36.67 + 2.68 50.71+3.03
Colorimetric determination (Scriba and Reddy, 1965) showed an R N A / D N A ratio (Sie-bert, 1966) of 0.52, and no detectable D N A in the 1,000 χ g SN. Subcellular fractions were solubilized with Ν CS and analyzed by liquid scintillation counting at 24% efficiency using the LS. method for quench correction.
Results
Distribution of adrenal radioactivity was first studied 3 min. after the beginning of 3 H -A C T H infusion, corticosterone release into adrenal vein being already stimulated at this time. Subsequently, 12,40 and 120 min. intervals were chosen, corticosterone release being maximally stimulated at 12 and still elevated at 120 min. The yield of radioactivity in both adrenals, calculated as % of infused radioactive A C T H , was remarkably low and showed a continuous decline (0.134-0.034%) with time. Surprisingly, the actual activities (CPM) per pair of adrenals remained constant from 40-120 min., indicating prolonged retention of the label from 3 H - A C T H .
In nuclear fractions a remarkably large amount of radioactivity was found. The possibili ty that the radioactivity in the nuclear fraction (54% of adrenal weight) was due to contamination wi th soluble cell fraction can be excluded, since the latter was diluted approx. 1:50 during the homogenization-centrifugation procedure. Init ial (3 min.) radioactivity in the nuclear fraction may be falsely high due to microsomal contaminations, the radioactivity of the latter however decreased significantly wi th time. Nuclear radioactivity (CPM) was constant from 12-120 min., but showed a relative (%) increase wi th time. The % values at 40 and 120 min. were significantly higher than at 3 min. (p<0.0025 and p<0.05, respectively).
Mitochondrial radioactivity decreased continuously from 12-120 min. (ρ < 0.0025) both absolutely (CPM) and relatively {%) . The same decrease was found in the microsomal fraction (3 vs. 12 min.: p<0.05; 12 vs. 40 min.: p<0.005; 40 vs. 120 min.: p<0.0005). Microsomal radioactivity was surprisingly high at 3 min. Since microsomal material may well be lost to nuclear and mitochondrial fractions, microsomal radioactivity appears to be at this time in excess of microsomal contribution to cell volume. The decline of radioactivity (CPM) in the soluble cell fraction parallels the decrease of total adrenal radioactivity and probably represents removal of label from the adrenals. The relative radioactivity (%) remained there-
4 7 3
P.C. SCRIBA A N D O.A. MÜLLER
fore approximately constant from 3-40 min. Slight increases ( ρ < 0.05) of radioactivity (CPM) in soluble cell fractions at 120 min. may be due to decreases in microsomal and mitochondrial counts.
Discussion
The results present, among others, the following unanswered questions:
1. Is the radioactivity found in adrenal cell fractions (e.g. nuclei) still part of 3 H - A C T H or of fragment(s) of A C T H ?
2. What is the zonal distribution of 3 H - A C T H throughout the adrenal cortex? 3. What is the relation of binding sites and of site(s) of action of 3 H - A C T H ? 4. Are there more than one adrenal cellular receptor for A C T H and what is their nature?
Summary
Subcellular distribution of 3 H - A C T H in the adrenals after infusion for 95 sec. in hypo-physectomized rats, was studied 3, 12, 40 and 120 min. after the start of the infusion, using homogenization and differential centrifugation. Considerable amounts of radioactivity (CPM) were still found 40 and 120 min. after 3 H - A C T H infusion in nuclear fractions. Nuclear radioactivity (%) increased relatively from 3-40 min. A comparably large amount of radioactivity was detected 3 min. after the start of the 3 H - A C T H infusion in the microsomal fraction. The results are to be discussed in relation to mechanisms and sites of actions of A C T H .
REFERENCES
FARESE, R . V . and R E D D Y , W.J . (1963): Effect of adrenocorticotrophin on adrenal protein synthesis. Endocrinology, 73, 294.
H A R D I N G , W . B . and NELSON, D . H . (1964): Effect o f hypophysectomy on N A D P and N A D P H concentration and corticosteroid secretion in rat adrenal. Endocrinology, 75, 501.
SCHNEIDER, W . C . and HOGEBOOM, G . H . (1951): Cytochemical studies o f mammalian tissues: The isolation of cell components by differential centrifugation: A review. Cancer Res., II, 1.
SCRIBA, P.C. and FRIES, M . (1967): Aminoacyltransferase stimulation o f protein synthesis by pig adrenal polysomes. Nature {LoncL), 214, 91 .
SCRIBA, P.C., FRIES, M . and K L U G E , F. (1967): Dissociated stimulation by A C T H of adrenal cort icosterone and protein synthesis. Acta endocr. (Kbh.), Suppl. 119, 178.
SCRIBA, P.C., H A C K E R , R . , DJETERLE, P., K L U G E , F., HOCHHEUSER, W. and S C H W A R Z , Κ. (1966): ACTH-Bestimmungen im Plasma aus dem Bulbus cranialis venae jugularis. Klin. Wschr., 44, 1393.
SCRIBA, P.C. and R E D D Y , W.J . (1965): Adrenocorticotrophin and adrenal protein synthesis. Endocrinology, 76, 745.
SIEBERT, G. (1966): Gewinnung und Funktion isolierter Zellkerne. Z . klin. Chem., 4, 93. S T A E H E L I N , M . , BARTHE, P. and DESAULLES, P .A. (1965): On the mechanism o f the adrenal gland
response to adrenocorticotrophic hormone in hypophysectomized rats. Acta endocr. (Kbh.), 50, 55. v. W E R D E R , Κ. , S C H W A R Z , Κ. and SCRIBA, P.C. (1968): Übe r Serumproteinbindung von A C T H . Klin.
Wschr., in preparation.
474