Proceedings of the 1984 Laurentian Hormone Conference

659 Pages·1985·12.426 MB·English

by  Roy O. Greep (Eds.)

RECENT PROGRESS IN
HORMONE RESEARCH
Proceedings of the
1984 Laurentian Hormone Conference
Edited by
ROY O. GREEP
VOLUME 41
PROGRAM COMMITTEE
G. D. Aurbach I. A. Kourides
J. D. Baxter A. R. Means
J. C. Beck J. E. Rail
H. Friesen N. E. Schwartz
R. O. Greep J. L Vaitukaitis
P. A. Kelly W. Vale
1985
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LIST OF CONTRIBUTORS AND DISSCUSSANTS
J. Abramowitz P. F. Hall
R. N. Anderson W. Hansel
D. R. Aquilano J. D. Hildebrandt
G. D. Aurbach J. A. Holt
C. W. Bardin K. B. Horwitz
K. L. Barker R. Iyengar
E. Battenberg L. Joshi
J. D. Baxter D. Keefe
N. Ben-Jonathan P. Kelly
H. A. Bern K. Kim
L. Birnbaumer M. A. Kirschner
F. E. Bloom E. Knobil
A. Borofsky Y. Kobayashi
D. D. Brandon D. T. Krieger
G. Callard B. A. Larson
I. Callard W. Leavitt
G. T. Campbell K. Lederis
M. G. Caron R. J. Lefkowitz
R. A. Cerione D. A. Leong
M. C. Chang H. A. Levey
T. Chen R. Levine
G. P. Chrousos D. W. Lincoln
J. Cidlowski G. A. Lincoln
J. H. Clark M. B. Lipsett
J. Codina B. A. Littlefield
S. Cohen D. L. Loriaux
R. B. Constant K. L. MacCannell
W. F. Crowley J. A. Magner
C. N. d'Arville J. R. Mancillas
D. Dluzen G. B. Martin
O. V. Dominguez R. Mattera
P. Donahoe S. M. McCann
R. Edgren A. S. McNeilly
R. M. Evans J. Meites
C. E. Farin M. M. Menezes-Ferreira
A. Ferron R. J. Milner
M. Filicori C. Monder
T. A. Fitz R. Moudgal
H. M. Fraser T. G. Muldoon
J. Fryer C. S. Nicoll
M. C. Gershengom R. S. Nishioka
N. Gesundheit G. D. Niswender
G. Gibori R. Osathanondh
K. A. Gregerson A. H. Payne
H. Grotjan D. Pearson
ix
X CONTRIBUTORS
P. Petrick J. Stalvey
P. G. Quinn B. S. Stannard
M. H. J. Raj K. Sterling
V. D. Ramirez R. L. Stouffer
C. S. S. Rani T. Sunyer
J. Rivier J. G. Sutcliffe
J. Robbins L. Swanson
F. J. Rojas T. Taylor
C. Ronin E. Terasawa
M. C. Rosenfeld M. O. Thorner
K. Ryan M. Tomita
N. A. Samaan J. L. Vaitukaitis
N. F. Santoro J. D. Veldhuis
H. R. Sawyer D. L. Vogel
R. H. Schwall M. R. Walters
N. Schwartz L. L. Wei
S. M. Sedlacek B. D. Weintraub
A. Segaloff J. Weisz
M. Seimanoff P. Wise
G. Siggins K. L. Wong
M. S. Smith N. Woo
D. I. Spratt
PREFACE
This volume, as are all the others of Recent Progress in Hormone
Research, is based on the proceedings of a Laurentian Hormone Confer-
ence. This one was held in late August 1984 at the Homestead in Hot
Springs, Virginia.
Comprehensive coverage of progress in this very broad area of biomé-
dical science was not intended, instead the aim was to select areas cur-
rently under intensive investigation. They are explored in great depth in
papers by leading authorities, and are followed by penetrating and critical
discussions.
The present volume reexamines at the molecular level the hormone-
receptor interactions and the subsequent series of intracellular events that
have provided many important new insights made possible by recent
methodological advances and improved instrumentation. First, the bio-
synthesis, secretion, metabolism, and mechanism of action of the steroid
hormones are examined anew with strikingly rewarding results. In like
manner the mechanisms of action of TSH and TRH are revealed at a level
of sophistication hitherto undreamed of. On another front, the molecular
characterization of a brain specific mRNA represents a great step forward
in understanding the interaction of hormones and cortical neurons. Addi-
tionally, important clarification of the factors affecting changes in fre-
quency and amplitude of GnRH pulses and the resulting functional conse-
quences in various mammals including humans with disorders of fertility
are reported in great detail. Topping off these fundamental advances are
two reports on the biological heritage of mammalian endocrinology.
These detail the structure and activities of urotensin I and urotensin II as
derived from the urophysis of a variety of fishes and tested in both the
lower vertebrates and mammals.
Headlong advances on all these fronts, including perhaps foremost of
all the interspecies transfer of genes that control the secretion of specific
hormones, open fantastic vistas of effective therapeutic achievements
that surely lie ahead. The reading of these triumphs in our understanding
of the role of hormones as chemical communicators in the total neuro-
physiology of the living organism will attest that the future of hormone
research has never before loomed so bright.
I take this opportunity to thank Drs. Geula Gibori, Christopher
Longcope, Wendell W. Leavitt, Paul A. Kelly, Dorothy T. Krieger,
Samuel M. McCann, Ian P. Callard, and Jacob Robbins for their skillful
XI
Xll PREFACE
service in chairing the several sessions. I also wish to express to Lucy
Felicissimo and Linda Carsagnini our appreciation for their speedy tran-
scribing of the taped discussions. To our Executive Secretary, Martha
Devin I am deeply grateful for her effective handling of the multitudinous
affairs that help to make this annual Conference and the published pro-
ceedings a successful enterprise. To the staff of Academic Press goes my
praise and gratitude for their devoted attention to detail that yields such a
satisfying publication.
Roy O. Greep
RECENT PROGRESS IN HORMONE RESEARCH, VOL. 41
Trophic Stimulation of Steroidogenesis: In Search of the
Elusive Trigger1
PETER F. HALL
Worcester Foundation for Experimental Biology,
Shrewsbury, Massachusetts
It is a great honor to be asked to deliver the Gregory Pincus Memorial
Lecture. My contact with Gregory Pincus was entirely indirect, but for
me it was certainly significant. I heard him deliver a characteristically
flamboyant lecture on adrenal steroidogenesis at the Royal Society of
Medicine, London, in 1954. In the course of this presentation, he sug-
gested that ACTH stimulates steroid synthesis at some early point in the
pathway—probably before pregnenolone. It was the first time I had cause
to consider the idea that a hormone could stimulate specific steps in a
biosynthetic pathway. As things turned out, I came to work at the Foun-
dation which Pincus helped to create but by that time he was no longer
alive, so that I was destined never to meet him.
When my time came to start a laboratory, I considered the problem of
the mechanism by which LH stimulates steroidogenesis in rabbit testis. It
seemed to me useful to break this question into two experimentally more
manageable questions: namely, which step(s) in the pathway is(are) stim-
ulated by the hormone and how are these steps stimulated, i.e., what is
the molecular basis of stimulation? At Pittsburgh, I met Seymour Koritz
who was working with ACTH and we decided to join forces. We sus-
pected that LH and ACTH probably act by the same, or similar mecha-
nisms, and that we could use adrenal, testis, and corpus luteum as the
needs of our experiments dictated, because each tissue offers certain
advantages. The question of where stimulation is exerted was approached
by incubating tissue with radioactively labeled substrates with and with-
out the trophic hormone added in vitro, to measure the production of
radioactive steroid hormone (Fig. 1). With [7a-3H] cholesterol as sub-
strate, the trophic hormones stimulated incorporation of 3H into the end-
products of the steroidogenic pathway. When we used [3H]pregnenolone
as substrate, we noticed two differences: conversion was an order of
1 The Gregory Pincus Memorial Lecture.
1
Copyright © 1985 by Academic Press, Inc.
All rights of reproduction in any form reserved.
ISBN 0-12-571141-7
2 PETER F. HALL
Acetate *- Melavonate ·» Squalene
Corticosteroids
Androgens ^.
^_______ Pregnenolone ^Cholesterol
Estrogens **~^^
Gestagens
FIG. 1. The steroidogenic pathway.
magnitude greater and the trophic hormone was without effect on this
conversion (1-4). The conclusion seemed inescapable that both trophic
hormones in all three tissues stimulate steroid synthesis beyond choles-
terol and before pregnenolone; in short, it is the side-chain cleavage of
cholesterol that is stimulated. We found no evidence of stimulation before
cholesterol although we could not exclude the possibility that under some
conditions the synthesis of cholesterol from acetate could be accelerated
by the trophic hormone. These studies confirmed the earlier work of
Stone and Rechter with the perfused adrenal (5).
I. Within the Mitochondrion
The fact that the side-chain cleavage system is confined to mitochon-
dria made the source of energy for side-chain cleavage a subject of consid-
erable interest. It was found that in beef adrenal the side-chain cleavage
reaction can use reversed electron transport and transhydrogenation as a
source of reducing equivalents in the form of NADPH for the cleavage
reaction (6, 7). Moreover mitochondrial malic enzyme, which was sug-
gested as a source of NADPH for llß-hydroxylation (8), could not ac-
count for the greater consumption of NADPH required for side-chain
cleavage. As it turned out this information did not reveal how ACTH
regulated the rate of production of pregnenolone; no evidence was found
to suggest that ACTH or cyclic AMP increased the production of NADPH
by the adrenal. Purification of the side-chain cleavage enzyme from beef
adrenal revealed important properties of the enzyme (9): the stoichiome-
try of the reaction (10), the involvement of heme in the cleavage of the
20,22 bond (11) and aggregation of the enzyme to an active form com-
posed of 16 subunits (9). However these findings did not clarify the mech-
anism of action of ACTH.
At this point it was decided to make use of an adrenal cell tumor line
developed by Sato and co-workers (12) and studied in detail by Kowal
(13)—the so-called Y-l cell. This cell line has been shown to respond to
ACTH and cyclic AMP even better than normal adrenal (13) and mono-
layer cultures offer advantages for some of the experimental approaches
to be described. The tumor cell line does not synthesize the usual cortico-
TROPHIC STIMULATION OF STEROIDOGENESIS 3
TABLE I
Production of Pregnenolone by Isolated
Mitochondria from Y-l Cells"
Pregnenolone
Additions to cells (nmol/min/mg protein)
— 32.1 ± 2.9
ACTH 29.9 ± 3.3
db cyclic AMP 30.6 ± 2.4
a Y-l cells were incubated for 30 minutes with
the additions shown. Mitochondria were pre-
pared and incubated for 20 minutes at 30°C.
Pregnenolone was extracted from mitochondria
plus incubation medium and measured by radio-
immunoassay (14).
steroid hormones but steroidogenic activity can be measured by determin-
ing the rate of production of 20a-dihydroprogesterone (14). The use of Y-l
cells soon brought two important findings: (1) It was decided to use this
system to test directly the hypothesis that ACTH stimulates side-chain
cleavage of cholesterol by isolating mitochondria from cells incubated
with and without ACTH to measure the rate of production of pregneno-
lone by the isolated mitochondria. To our disappointment, no difference
could be detected between the production of pregnenolone by the two
groups of mitochondria (Table I) (14). This experiment has been repeated
by more than 8 workers in my laboratory with the same result. Moreover
addition of cholesterol to the isolated mitochondria did not influence the
outcome of such experiments. These negative observations cast doubt on
the cherished hypothesis that ACTH stimulates side-chain cleavage of
cholesterol.
(2) When Y-l cells were incubated with radioactive amino acids with
and without ACTH, we observed, by gel electrophoresis of mitochondrial
extracts, two labeled proteins of molecular weights of 27,000 and 13,000
(15) (Fig. 2). With the aid of inhibitors of the synthesis of protein and
RNA (including chloramphenicol), we concluded that these proteins are
made on stable mRNA (Fig. 2) by cytoplasmic ribosomes and transported
to mitochondria (15).
II. Source of Mitochondrial Cholesterol
Although the first of these two observations (failure to stimulate mito-
chondrial production of pregnenolone) was disturbing, it was consistent
with the findings of Garren and co-workers who found that cycloheximide
inhibits the response to ACTH at some step before side-chain cleavage
(16). When cholesterol (as opposed to acetate) is the source of the steroids
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FIG. 2. Y-l cells were incubated with [3H]leucine without ACTH or with [14C]leucine
with ACTH for 1 hour. Mitochondrial protein from the cells incubated with the two isotopes
was analyzed by disc gel electrophoresis with sodium dodecyl sulfate. Gels were examined
by densitometry and then sliced for double-label counting by liquid scintillation spectrome-
try. The tracing of A was from control cells; that from treated cells was superimposable.
550
produced by the adrenal, the only steps before side-chain cleavage are
those associated with the movement of cholesterol to and within mito-
chondria. Garren et ai, on the basis of studies in vivo, made the prescient
suggestion that ACTH stimulates transfer of cholesterol to mitochondria
(16). This idea could explain our failure to find increase in the production
of pregnenolone by isolated mitochondria from cells stimulated by
ACTH—by removing the mitochondria from a continuing supply of cho-
lesterol in the cytoplasm, we prevented the mitochondrial enzyme system
from expressing the stimulating influence of ACTH (14). To test this
hypothesis we inhibited side-chain cleavage by incubating the cells with
aminoglutethimide, and incubated the cells with and without ACTH. Un-
der these conditions, steroidogenic cholesterol should accumulate in mi-
tochondria and would be available for side-chain cleavage when the inhib-
itor was removed. After various periods of time mitochondria were
prepared in the presence of the inhibitor, were washed to remove the
inhibitor, and then incubated in buffered medium for measurement of the
production of pregnenolone. The result was clear: production of preneno-