Neurobiology of Vasopressin

209 Pages·1985·5.897 MB·English

by  D. Richter (auth.), Dr. Detlev Ganten M.D., Ph.D., Dr. Donald Pfaff Ph.D. (eds.)

Current Topics
in Neuroendocrinology
Volume 4
This collection of studies was conceived as part of a two volume review
of the subject. The contents of Volume 6 are listed below.
Neurobiology of Oxytocin
Biosynthesis of Oxytocin
By D. Richter
Regulation of Oxytocin Release
By M. L. Forsling
Proteolytic Conversion of Oxytocin, Vasopressin and Related
Peptides in the Brain
By J. P. H. Burbach
Electrophysiology of Oxytocin
By J. Dreifuss
Oxytocin and Behavior
By G. L. Kovacs
Oxytocin Effects on the Cardiovascular System
By M. Petty
Oxytocin in Labour and Lactation
By A. Robinson
Oxytocin as an Ovarian Hormone
By D. C. Wathes
Subject Index
Neurobiology
o f Vasopressin
Editors
D. Ganten and D. Pfaff
Contributors
G. Clarke R.E. Lang M.l McKinley L.P. Merrick
W Rascher D. Richter M. Sofroniew Th. Unger
A.Weindl
With 53 Figures
Springer-Verlag
Berlin Heidelberg New York Tokyo
Editors
Dr. DETLEv GANTEN, M.D., Ph.D.
Pharmakologisches Institut
UniversiHit Heidelberg
1m Neuenheimer Feld 366
6900 HeidelbergjFRG
Dr. DONALD PFAFF, Ph.D.
Rockefeller University
York Avenue, and 66th Street
New York, NY 10021jUSA
The picture on the cover has been taken from Nieuwenhuys R., Voogd J., van Huijzen Chr.:
The Human Central Nervous System. 2nd Edition. Springer· Verlag Berlin Heidelberg
New York 1981
ISBN-13:978-3-642-68495-1 e-ISBN-13:978-3-642-68493-7
DOl: 10.1007/978-3-642-68493-7
Library of Congress Cataloging in Publication Data. Main entry under title: Neurobiology of
vasopressin. (Current topics in neuroendocrinology; v. 4) Includes bibliographies and index.
I. Vasopressin. 2. Neurobiology. I. Ganten, D. (Detlev), 1941-. II. Pfaff, Donald W., 1939-.
III. Clarke, G. IV. Series. [DNLM: I. Vasopressins-physiology. WI CU82Q v. 4/WK 520 N4936]
QP572.V3N48 1985 599'.0188 85-4797
ISBN-13:978-3-642-68495-1 (U.S.)
This work is subject to copyright. All rights are reserved, whether the whole or part of the material is
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© by Springer-Verlag Berlin Heidelberg 1985
Softcover reprint of the hardcover I st edition 1985
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2121/3130-543210
Contents
Biosynthesis of Vasopressin
By Do Richter
With 8 Figures
0 0 0 0 0 0
Electrophysiological Studies of the Magnocellular Neurons
By Go Clarke and L. Po Merrick
With II Figures 17
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Volume Regulation of Antidiuretic Hormone Secretion
By Mo Jo McKinley
With 5 Figures 61
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Vasopressin, Cardiovascular Regulation and Hypertension
By Wo Rascher, Ro Eo Lang, Tho Unger
With 13 Figures 101
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Neuroanatomical Pathways Related to Vasopressin
By A. Weindl and Mo Sofroniew
With 16 Figures 137
Subject Index 197
0
Biosynthesis of Vasopressin
D. RICHTER!
Contents
1 Historical Account. . . . . . . . . . . . . . . . . . 1
1.1 Analysis by Pulse-Chase Experiments. . . . . . . . 2
l.2 The Neurophysin Precursors. . . . . . . . . . . . 3
1.3 Biosynthesis and Structure of the Hormone Precursor. 3
1.3.1 Translation of Hypothalamic mRNA 3
1.3.2 Tryptic Peptide Mapping . . . 6
1.3.3 Cloning and cDNA Sequencing. 6
1.4 Relevance of the Precursor Structure . 6
2 Structural Organization of the Gene . . . 8
2.1 Structure of the Rat Vasopressin Gene . 8
2.2 Vasopressin Deficiency in Brattleboro Rats 10
3 Expression in Heterologous Systems . . . . . 10
3.1 In Vitro Conversion and Glycosylation . . 10
3.2 Synthesis and Conversion in an Oocyte System 11
3.3 Expression in Bacteria . . . . . . . . . . . 12
4 Comparison of Vasopressin and Oxytocin Precursor Structures 13
5 Concluding Remarks . 14
References. . . . . . . . . . . . . . . . . . . . . . . . . 15
1 Historical Account
Since the first reports by van Dyke and co-workers (reviewed in Acher 1979) on
the isolation of a protein complex then called the "van Dyke protein" and later
identified as being composed of the hormone vasopressin and its corresponding
carrier protein, known as neurophysin numerous publications have appeared
dealing with the synthesis of vasopressin in the hypothalamus and its axonal
transport to the neurohypophysis (reviewed in Sachs 1969; Valtin et al. 1974;
Breslow 1979; Acher 1979; Brownstein et al. 1980; Pickering 1983).
In 1964 Sachs and Takabatake presented evidence that the nonapeptide vaso
pressin might be synthesized on ribosomes via a biologically inactive precursor.
A few years later, Sachs proposed a remarkable hypothesis for the biosynthetic
pathway of this hormone (Sachs et al. 1969):
According to this model (precursor model) the biosynthesis of the peptide bonds in
vasopressin would occur solely in the perikaryon on ribosomes, via pathways common to
Institut fUr Physiologische Chemie. Abteilung Zellbiochemie, UniversiHit Hamburg,
Martinistr. 52, D-2000 Hamburg 20
Current Topics in Neuroendocrinology, Vol. 4
© Springer-Verlag Berlin Heidelberg 1985
2 D. Richter
the biosynthesis of other peptide chains; initially however, the hormones would be con
structed as part of a macromolecule (probably a protein). The release of the octapeptide
from the precursor molecule presumably takes place during the formation and maturation
of the NSG (neurosecretory granules). It is unknown whether or not neurophysin biosyn
thesis occurs via a similar mechanism or whether the hormone and neurophysin share a
common precursor. The biosynthesis of vasopressin and neurophysin nevertheless appear
closely related.
It was this pioneering concept of Sachs which finally led to the structure of
the vasopressin and oxytocin precursors (Land et al. 1982, 1983). The early at
tempts to identify the proposed vasopressin precursor were principally based on
pulse-chase experiments carried out in vivo in normal and mutant rat strains
(Brattleboro rats); the latter lack biologically active vasopressin and hence pro
vide a model for studying the coordinate synthesis of vasopressin and its respec
tive carrier protein, neurophysin (reviewed in Valtin et al. 1974).
With refined techniques Brownstein, Gainer, and associates (Brownstein et al.
1980) succeeded in the isolation of high-molecular-weight proteins from rat hy
pothalami which were composed of vasopressin- or oxytocin-like peptides and
their corresponding neurophysins.
The final resolution of the structure of the vasopressin precursor was achieved
by a combination of cell-free translation studies and recombinant DNA tech
niques from which the amino acid sequence could be deduced (reviewed in
Richter 1983 a). These data proved unambiguously that the vasopressin precursor
is composed of several distinct peptides, the hormone, its carrier protein neuro
physin, and a glycoprotein.
This article concentrates on the attempts leading to the elucidation of the
biosynthetic pathway and the structure of the vasopressin precursor. Since oxy
tocin is structurally similar to vasopressin, overlapping data are included where
relevant; in a separate section the structures of the two hormone precursors are
compared.
1.1 Analysis by Pulse-Chase Experiments
The early work by Sachs and co-workers showed that 35S-labeled vasopressin was
synthesized in the hypothalamus after injection of 35S-cysteine. Subsequent pulse
chase experiments indicated that the hormone synthesis took place on ribosomes,
since it could be inhibited by the antibiotic puromycin (Sachs and Takabatake
1964). On the other hand, biologically active vasopressin was not associated with
the ribosomal fraction; this led to the suggestion that vasopressin was initially
synthesized as an inactive precursor on ribosomes, and later converted into the
mature hormone at a site different from the ribosomes.
Extending his studies on the biosynthesis of the neurophysins, Sachs then pre
sented evidence that synthesis of vasopressin and its respective carrier protein,
neurophysin, may be correlated (Sachs 1969). This assumption was based on the
observations that vasopressin and its neurophysin were found in the same subcel
lular fractions, and that neurosecretory granules contained both peptides, which
are released simultaneously from organ cultures of the neurohypophysis.
Biosynthesis of Vasopressin 3
1.2 The Neurophysin Precursors
Direct application of 35S-cysteine to the supraoptic nucleus ofthe brain, sampling
of the tissue by the micropunch technique, and subsequent analysis by electrofo
cusing led to the identification of precursors to the neurophysins (Brownstein et
al. 1980). One precursor could be converted by limited trypsinization into neuro
physin and an oligopeptide which, although not identical with vasopressin,
showed features similar to vasopressin as "indicated by immunoreactivity and af
finity chromatography; the other precursor gave rise to oxytocin-associated
neurophysin and an oxytocin-like peptide.
Although these data suggested the existence of a composite precursor, as pro
posed by Sachs, this model was still debated. It was not clear why the potential
precursors failed to cross-react with antibodies against vasopressin or oxytocin.
Possibly, the failure of the immunological identification was due to the vasopres
sin or oxytocin antibodies used, which discriminated between acidic and ami
dated forms ofthe hormones, a specificity ofthe antibodies unsuitable for detect
ing potential hormone precursors.
Although partial trypsinization of the precursor gave rise to neurophysin it
was not possible to identify authentic vasopressin1_ 8, which ought to have been
released if the hormone was present. This discrepancy was probably due to the
limited trypsinization conditions applied in these experiments.
Extraction of hypothalamic tissue and identification by specific antibodies re
vealed a glycosylated precursor to neurophysin and vasopressin, suggesting that
this proh ormone is a glycoprotein (Lauber et al. 1979). Further studies by Cohen
and associates (Beguin et al. 1981; Lauber et al. 1981) indicated the existence of
a protein even larger than the vasopressin prohormone, with immunologic deter
minants for vasopressin, neurophysin, ACTH, and p-endorphin. This group pro
posed a model whereby the larger composite precursor serves as "mother ship",
for all the other hormones referred to. Direct sequence analysis is needed to
understand the nature of this precursor.
1.3 Biosynthesis and Structure of the Hormone Precursor
The complete structural organization of the postulated vasopressin precursor be
came evident by a combination of cell-free translation studies and recombinant
DNA technology. The experimental concept adopted included (a) translation of
hypothalamic mRNA and immunological identification of the products; (b)
tryptic peptide mapping; and (c) cloning and sequencing of the cDNA encoding
the hormone.
1.3.1 Translation of Hypothalamic mRNA
Cell-free translation systems in general offer a simple way to study the immediate
translation product of an exogenous mRNA. As with other hormones, vasopres
sin is synthesized as a primary translate, the preprohormone which contains a so
called signal peptide at its NH2 terminus, an essential marker for transportation
4 D. Richter
into the lumen of the endoplasmic reticulum (Kreil 1981). The signal peptide is
cleaved off during synthesis, a process which can be simulated in a cell-free system
by complementation with membranes prepared from the endoplasmic reticu
lum.
The in vitro biosynthesis of vasopressin or oxytocin was studied in cell-free
systems programmed with hypothalamic mRNA from mouse, rat, or calf (re
viewed in Chaiken 1983; Gainer 1983; Richter 1983 a). The cell-free systems used
were wheat-germ extracts, rabbit reticulocyte lysates, or Xenopus laevis oocytes,
with 35S-cysteine or tritiated amino acids as radioactive markers. The translation
products were identified by immunoprecipitation followed by electrophoresis in
dodecyl sulfate on polyacrylamide gels, and visualized by autoradiography.
The cell-free synthesized products were initially identified by immunological
means. By this method precursors with apparent molecular weights of 21 000-
25000 and 16000-19000 were obtained with antisera raised against the respective
neurophysins.
In order to identify antigenic determinants for vasopressin or oxytocin within
these precursors, specific antisera were applied which did not discriminate be
tween the acid or deamidated form of the hormones; an essential prerequisite in
the search for potential vasopressin or oxytocin precursors. By means of sequen
tial immunopecipitation it became evident that the initially identified precursors
also contained antigenic determinants for vasopressin and oxytocin: the bovine
~ ~
I I
II
I
-19 1 913 1071)9 147
I I I I II I
NHz ~"'\~_ I:::::::::::::::::::::::::::::::::::::::::::::::::':':':':::::::::::::::::::i~-(OOH
Signal 13 107
Peptide t.::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::1
Neurophysin II 1Il'wI .=,n47
1 10
I I
.-Gly
r
1• 9 109 127
I I I I
l3l 147
Arginine ( Im iI
Vasopessin 109 118 134 147
~ ~
Fig. I. Scheme of the calf vasopressin precursor. Hatched bar represents signal peptide; the
solid bars, vasopressin; Stippled bars, neurophysin; cross-hatched bars, glycoprotein and its
fragments; open bars, amino acids involved in processing. The arrangement of cysteine res
idues, glycine residue for amidation, the basic amino acids lysine and arginine, and leucine
residues as signals for proteolysis is shown at the top of the figure; C, carbohydrate chain
Biosynthesis of Vasopressin 5
-23
MET LEU ALA MET MET LEU ASN THR THR LEU SER ALA CYS
RAT AGCAGAGCAGAGCTGCACGCA-GTGCCCACCT ATG CTC GCC ATG ATG CTC AAC ACT ACG CTC TCT GCT TGC
GCACAGTA********CA****G*A*GT******---- *C* *GT *** *G* *** *C* G** G*C **A **G C*C **C ***
CALF - PRO Asp ALA -- PRO --
-1 +1 VASOPRESSIN
PHE LEU SER LEU LEU ALA LEU THR SER ALA CYS TYR PHE GLN ASN CyS PRO ARG GLY
TTC CTG AGC CTG CTG GCC CTC ACC TCT GCC TGC TAC TTC CAG AAC TGC CCA AGA GGA
*** **C *** *** *** *** T** *** *** **T *** *** *** *** *** *** *** **G **C
---------- PHE-------------------
NEUROPHYS I N 20
ALA THR SER Asp MET GLU LEU ARG GLN CYS LEU PRO CYS GLY PRO GLY GLY Lys GLY ARG
GCC ACA TCC GAC ATG GAG CTG AGA CAG TGT CTC CCC TGC GGC CCT GGC GGC AAA GGG CGC
*** *TG *** *** C** *** *** *** *** *** *** *** *** *** **C **G *** *** **C ***
- MET -- LEU ----------------------
40
CYS PHE GLY PRO SER I LE CYS CYS ALA Asp GLU LEU GL Y CYS PHE LEU GLY THR ALA GLU ALA LEU
TGC TTC GGG CCG AGC ATC TGC TGC GCG GAC GAG CTG GGC TGC TTC CTG GGC ACC GCC GAG GCG CTG
*** *** *** **C *** *** *** *** *G* *** *** *** *** *** *** G** *** **G *** *** *** ***
----------- GLY VAL --------
60
ARG CYS GLN GLU GLU ASN TYR LEU PRO SER PRO CYS GLN SER GL Y GLN Lys PRO CYS GLY SER GL Y
CGC TGC CAG GAG GAG AAC TAC CTG CCC TCG CCC TGC CAG TCT GGC CAG AAG CCT TGC GGA AGC GGA
*** *** **A *** *** *** *** *** **G *** *** *** *** **C *** *** *** **C *** **G *** **G
80
GLY ARG CYS ALA ALA ALA GLY I LE CYS CYS SER Asp GLU SER CYS VAL ALA GLU PRO GLU CYS ARG
GGC CGC TGC GCT GCC GCG GGC ATC TGC TGC AGC GAT GAG AGC TGC GTG GCC GAG CCC GAG TGT CGA
*** *** *** **C *** **C *** *** *** *** *A* *** *** *** *** *** A** *** *** *** **C **G
---------------- ASN THR ---------
GLYCOPROTE I N
GLU GL Y PHE PHE ARG LEU THR ALA ARG GLU GLN SER ASN ALA THR GLN LEU
GAG GGT TTT TTC CGC CTC ACC --- -- GCT CGG GAG CAG AGC AAC GCC ACG CAG CTG
"A •• , G'C GG' TT' 'C' CG' CGC GTT **C AAC **C *G* *** *** **G **C *T* ***
-- VAL GLY PHE PRO ARG ARG VAL --ASN Asp ARG LEU -
120
Asp GLY PRO ALA ARG GLU LEU LEU LEU ARG LEU VAL GLN LEU ALA GLY THR GLN GLU SER VAL Asp
GAC GGG CCA GCC CGG GAG CTG CTG CTT AGG CTG GTA CAG CTG GCT GGG ACA CAA GAG TCC GTG GAT
*** *** **G AG* G** *CC T** T** **G C** *** **G *** *** **G *** G*G *CG *** C** *C* **G
------ SER GLY ALA ALA PRO - PRO ALA GLU
140
SER ALA Lys PRO ARG VAL TYR STOP
TCT GCC AAG CCC CGG GTC TAC TGA GCCATGC-------CCCCCCACGCCTCCCCCCTACAGCATGGAAAATAAAC-TT
C*C *** C** *** G*C *** *** *** *G*GCGCCCCCCCC*T******C***G-***TGG*****C*A*********G**
PRO __ GLN _ GL Y ______
TT AAAAAA (POL Y A)
••••• GGC (POL Y A)
3'
Fig. 2. Comparison of the nucleotide and amino acid sequences of calf and rat vasopressin
precursors. The positive numbers indicate the positions of the amino acids; the negative
numbers, amino acids of the signal sequence. Identical nucleotides in both sequences are
shown by asterisks, identical amino acids by solid lines; broken lines indicate a lack of cor
responding sequences of either precursor; absence of an amino acid is shown by a gap
21 OOO-dalton preprohormone was composed of vasopressin and neurophysin II,
the other, with a molecular weight of 16500, of oxytocin and neurophysin I
(Schmale and Richter 1981 a).
Cell-free translation of mRNA extracted from either supraoptic or paraven
tricular hypothalamic nuclei of salt-treated rats indicated that vasopressin precur
sor synthesis was considerably increased with mRNA derived from the su-