National Institute of Environmental Health Science.

Annual report : National Institute of Environmental Health Sciences (Volume 1985) online

. (page 57 of 114)
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to mimic in vitr o the conditions observed in vivo . If this approach proves
to be successful, then it could provide the means to evaluate the role of
steroids, peptides, and/or amines in modifying LHRH/LH release.


Johnston, C.A., Tesone, M., and Negro-Vilar, A.: Cellular mechanisms of
acute estrogen negative feedback on LH secretion: Norepinephrine, dopamine
and 5-hydroxytryptamine metabolism in discrete regions of the rat brain.
Brain Res. Bull. 13: 363-369, 1984.

Johnston, C.A., Tesone, M., and Negro-Vilar, A.: Steroid-monoamine feed-
back interactions in discrete brain regions using as a model the monosodium
glutamate (MSG)-lesioned rat. Life Sci . 34: 1287-1297, 1984.

Spinedi, E., Johnston, C.A., and Negro-Vilar, A.: Increased respon-
siveness of the hypothalamic-pitui tary axis after neurotoxi n-induced
hypothalamic denervation. Endocrinology 115: 267-272, 1984.

O'jeda, S.R. and Negro-Vilar, A.: Release of prostaglandin E 2 from the
hypothalamus depends on extracellular Ca + availability: relation to LHRH
release. Neuroendocrinology 39: 442-447, 1984.

Negro-Vilar, A., Tesone, M., Johnston, C.A., DePaolo, L., and Justo, S.N.:
Sex Differences in Regulation of Gonadotropin Secretion: Involvement of
Central Monoaminergic and Peptidergic Systems and Brain Steroid Receptors.
In Serio, M., Motta, M., Zanisi, M., and Martini, L. (Eds.): Sexual
Differentiation: Basic and Clinical Aspects . New York, Raven Press, 1984,
pp. 107-118.

Ojeda, S.R. and Negro-Vilar, A.: Prostaglandin E 2 induced luteinizing
hormone-releasing hormone release involves mobilization of intracellular
Ca + . Endocrinology 116: 1763-1770, 1985.

Negro-Vilar, A.: LHRH: Physiology, Pharmacology, and Role in Fertility
Regulation. In Paulson, J., Negro-Vilar, A., Lucena, E., and Martini, L.
(Eds.): Male Fertility and Sterility . New York, Academic Press (In
Press ).

Negro-Vilar, A., Spinedi, E., and Valenca, M.M.: Pulsatile Secretion of
ACTH: Characterization and Effect of a Glucocorticoid. Proceedings from
Pulsatility Conference. New York, Academic Press (In Press).


Z01 ES 70090-02 LRDT

Johnston, C.A., Tesone, M., and Negro-Vilar, A.: Cellular mechanisms of
acute estrogen negative feedback on LH secretion: Pituitary responsiveness
to LHRH and estradiol receptor kinetics in the pituitary, preoptic hypotha-
lamic area, and the caudal hypothalamic area of the rat brain. Brazi 1 i dn
J_. Med. Biol . Research (In Press).

Tesone, M. and Negro-Vi lar, A.: Estrogenic feminization of the LH response
to orchidectomy is associated with prolonged nuclear estradiol receptor
retention and induction of cytoplasmic progestin receptors in brain and
pituitary. J . Steroid Biochem . (In Press).

Ojeda, S.R., Capdevila, J., Snyder, G., Negro-Vilar, A., and Falck, J.R.:
Involvement of Arachidonic Acid Metabolites in the Control of Hypothalami c-
Pituitary Function. In Advances in Prostagla n din, T h romboxane, and
Leukotriene Research . New York, Raven Press, Vol. IT (In Press).




Z01 ES 70092-02 LRDT


October 1, 1984 to September 30, 1985

TITLE OF PROJECT (SO characters or less Title must fit on one line between the borders )

Cellular and Molecular Mechanisms Mediating Peptide Hormone Action

PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)

Pi : a. Negro-Vilar Head, Reprod. Neuroendocrinology Section LRDT NIEHS

Others: M. D. Culler

C. A. Johnston
M. M. Valenca

Staff Fellow
Staff Fellow
Visiting Fellow



University of North Carolina, Department of Anatomy

Yale University Medical School, Department of Obstetrics and Gynecology

The Wellcome Research Laboratories, Department of Molecular Biology


Laboratory of Reproductive and Developmental Toxicology


Reproductive Neuroendocrinology Section


NIEHS, NIH, Research Triangle Park, North Carolina 27709








□ (a) Human subjects

□ (a1) Minors

□ (a2) Interviews

□ (b) Human tissues E (c) Neither

SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided )

Analysis of the cellular and molecular mechanisms mediating peptide hormone
action constitute an important component of the research efforts of the
Reproductive Neuroendocrinology Section. The close interrelationships
mediating neuroendocrine responses within the hypothalamic-pi tuitary-gonadal
systems offer an excellent opportunity to analyze some unique characteristics
of peptide-peptide, peptide-amine, and peptide-amine-steroid interactions.
Studies using pituitary cell cultures are directed to evaluate the precise
mechanisms through which peptidergic or aminergic secretagogues enhance or
suppress peptide hormone release. Protocols are designed to evaluate charac-
teristics of hormone-receptor interactions, post-receptor as well as
transmembrane events involved in the hormone-release process, and definition of
the specific intracellular messengers transducing the action of key hypothala-
mic peptides involved in pituitary hormone release. Other studies are designed
to explore the intriguing peptide-amine interactions (such as serotonin-AVP)
previously reported by our group, to better understand the nature and physio-
logical significance of those interactions.

At the testicular level, studies are designed to determine the intratesticul ar
effects of LHRH-analogs, known to adversely affect both the endocrine and the
gametogenic functions of the testis. Since some of these analogs are presently
being tested for use in human contraception, an understanding of their site(s)
and mechanisms of action is of obvious significance. The interaction of these
LHRH-A with intrinsic peptidergic systems within the testis, such as the
proop.iomelanocortin-derived peptides, is also being explored. The results may
provide very significant advances to our knowledge of paracrine and/or
autocrine effects of gonadal peptides.

PHS 6040 (Rev, 1/84)

GPO 91 4-8 1 S

Z01 ES 70092-02 LRDT



Nature of Problem

Great advances have been made in recent years to further our understanding
of some of the intrinsic mechanisms of hormone action. Several basic
systems of intracellular messengers coupled with membrane receptors or of
cytosolic receptors, which upon nuclear translocation affect the genome,
have been defined in a variety of tissues. Within the hypothalamic-
pituitary-gonadal system, a diversity of aminergic, peptidergic, and
steroidal hormones exert profound hormonal effects by evoking a multitude
of cellular responses. In each individual case, the hormone-cell interac-
tion acquires special characteristics according to the chemical nature of
the hormone involved, and to the type and location of the target cell.
Most hormones exert specific effects at selected sites, and this specifi-
city usually involves stimulation (or sometimes inhibition) of key intra-
cellular messengers after interaction with a specific cellular receptor.
Definition of the sequence and type of events mediating the effects of dif-
ferent peptides or amines within the hypothalamic-pi tuitary-gonadal axis
is, therefore, of paramount importance toward our understanding the inti-
mate cellular and subcellular mechanisms mediating major reproductive

Object! ves

The primary objectives of this research project are the following:

(1) To evaluate and characterize the mechanism(s) and specific cellular and
subcellular site(s) of action of certain key peptidergic and aminergic
neurohormones known to be secreted from the hypothalamus into the portal
circulation, which modulate the release of different pituitary hormones
clearly involved in reproductive functions. In addition, special attention
will be given to peptide-amine interactions that may well represent an
important mode of combined hormonal regulation. Finally, possible
paracrine and autocrine actions of peptide hormones will also be evaluated.

(2) Much evidence indicates that most hormones are secreted in a pulsatile
pattern and that this episodic mode of secretion plays an important role

in receptor activation and hormone response. We plan to analyze the effect
that mode of stimulation (i.e., amplitude, interpulse interval, pulse
height, etc.) or input deprivation (i.e., absence of pulsatile stimulation)
have on peptide-induced hormone release from cultured dispersed cell
systems. Attempts will be made to induce up or down regulation of peptide
receptors and to evaluate the changes in intracellular messengers that may
mediate those modifications in cell response.

(3) In support of the previous studies, we intend to evaluate the mecha-
nisms underlying the enhanced cellular responsiveness at the hypothalamic


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and pituitary level that is observed in adult animals after neonatal neuro-
toxin treatment. Elucidation of these mechanisms may provide some fun-
damental answers toward our understanding of the factors that regulate eel 1
responsiveness under physiological or pathophysiological conditions.

role of peptides in
is as a model to
id location, within
several peptidergic
snt in the testis of
several species. These studies will, in addition, attempt to define the
physiological role of these peptides, as well as the particular circumstan-
ces in which their action is clearly expressed.

Ex perimental Approach and Scientific Justification; Recent Accomplishments
and Si gni f icance

(1) Work carried out by scientists presently at the Reproductive Neuroendo-
crinology Section has established and characterized specific pituitary
membrane receptors for hypothalamic peptides such as angiotensin II (All)
and arginine vasopressin (AVP). In both cases, high affinity, low capacity
membrane receptors were identified, with K^'s in the low nanomolar range,
values found compatible with the doses of peptide required to elicit a nor-
mal physiological response. In the case of AVP, the use of 3 H-1 i gand
enabled us to evaluate both the binding characteristics as well as the
biological activity (enhanced ACTH and B-endorphin release) using intact,
dispersed anterior pituitary cells. Specific receptor blockers, like sara-
lasin, blocked the action of All, and other agonists of either All or AVP
showed binding affinities comparable with their respective biological
activities. Since other groups have shown that corticotropin releasing
factor (CRF) acts also on specific membrane receptors, it is clear that
three different peptides that stimulate ACTH/g-endorphin secretion do so by
acting on specific and independent receptors. Moreover, while
CRF-stimulated ACTH release involves enhanced cAMP formation the effect of
AVP is not mediated by activation of that nucleotide, strongly suggesting
that other intracellular messengers might be involved in the action of AVP
and, perhaps, of All.

Additional studies from our group have clearly established an intriguing
peptide-amine interaction between AVP and serotonin to enhance ACTH release
from the pituitary. Several studies involving the use of specific seroto-
nin receptor blockers, serotonin synthesis inhibitors, or tissues obtained
from rats (Brattleboro DI ) with a genetic deficiency in AVP synthesis,
indicate that the AVP-serotoni n interaction can be observed in vivo and
might, therefore, constitute an important physiological mechanism in the
regulation of ACTH release.

(2) We have recently explored the role of two specific intracellular
activators of protein kinase C, di acyl glycerol and a phorbol ester (phorbol
12, 13 dibutyrate; PDBu) on anterior pituitary hormone release in vitro.


Z01 ES 70092-02 LRDT

Using a dispersed cell system, we established that both compounds enhance
the release of all anterior pituitary hormones (ACTH, e-End, GH, TSH, PRL,
LH and FSH) in a concentration-and time-dependent manner. Certain hor-
mones, like GH, ACTH and 3-End, were particularly sensitive to the actions
of these secretagogues. The results suggest that stimulus-mediated activa-
tion of PI turnover may result in the intracellular formation of diacylgly-
cerol and the subsequent activation of the Ca activated, phospholipid
dependent protein kinase C. The ensuing protein phosphorylation may be an
important component of the processes leading to peptide hormone release.

(3) Recent studies in our Section have established the presence of
proopiomelanocortin (POHC)- derived peptides in testicular interstitial
fluid (TIF). Using highly sensitive and specific radioimmunoassays, we
established the presence of f5-endorphin and ACTH in TIF from both intact
and hypophysectomized animals. The latter observation clearly indicates
that the peptides found in TIF are not derived from the peripheral plasma,
since after removal of the pituitary gland plasma levels of both
POMC-peptides are undetectable. Authenticity of the peptides measured was
further established by chromatography and parallelism of serial dilutions
with the standard curve in the radioimmunoassays. Recent experiments indi-
cate that stimulation with either LH or HCG both in intact or hypophysec-
tomized animals can profoundly affect levels of these peptides in TIF
suggesting a relationship between gonadotropin stimulation and the
POMC-derived peptides. Quite recently, we have obtained evidence that
another neural peptide, angiotensin II, is present in large concentrations
in human follicular fluid. These observations, coupled with recent reports
describing POMC-like peptides in gonads and reproductive tract of both
sexes, as well as the demonstration of the expression of a POMC-like gene
in testis and epididymis indicate local synthesis and regulation of these
peptides and suggest a paracrine or autocrine role for these factors in
endocrine and/or gametogenic functions.

(4) Another series of studies have been conducted to elucidate the effects
of superactive luteinizing hormone-releasing hormone (LHRH) analogs on
testicular function. Chronic and continuous administration of different
LHRH-A has been shown to suppress both the endocrine and gametogenic func-
tions of the testis, representing the basis for the potential use of these
compounds as contraceptive agents. Studies in the Reproductive
Neuroendocrinology Section have been directed at analyzing the direct
intratesticular effects of these analogs of LHRH on testosterone secretion
and also their possible interactions with other peptidergic systems present
in the testis. Preliminary results indicate that an interaction between
LHRH and POMC-derived peptides in the testis may be involved in the regula-
tion of testosterone secretion. The results also suggest that LHRH analogs
have biphasic effects upon Leydig cell function, with short-term stimu-
latory and long-term inhibitory effects being observed.


Z01 ES 70092-02 LRDT


Several experimental approaches will be employed to evaluate peptide/amine
effects at the cellular/subcel lular level in the anterior pituitary. Most
of the studies will continue to require the use of in vitro systems and a
combination of either short-term or long-term tissue or cell cultures. In
the case of the pituitary gland and due to the heterogeneity of the cell
types, attempts will be made to process enzymati cal ly dispersed cells
through an elutriation procedure which may provide useful enrichment of
certain cell types. In addition, cell cultures will be submitted to speci-
fic stimulation by hypothalamic peptide hormones, and attempts will be made
to mimic the pulsatile rate of delivery of these neural hormones with the
use of recently developed computerized cellular engineering perifusion
systems .

These approaches will enable us to search for answers to the following

(1) Role of certain intracellular messengers on the peptide-i nduced
anterior pituitary hormone release. Using specific and selected pep-
tidergic secretagogues, such as AVP, All, CRF, LHRH, or TRH , attempts will
be made to characterize the cellular events leading to the exocytotic
secretion of hormones. Particular attention will be given to the possible
effects of peptides on membrane phospholipids. This will be accomplished
by using P-32 labeled cells to study the breakdown and resynthesis of
phosphatidyl i nositol , by measuring activation of the phosphol ipi d-dependent
protein kinase C, and by employing certain specific probes such as

diacyl glycerol , phorbol esters, and ionophores. Since either inositol
biphosphate (IP 2 ) or inositol triphosphate (IP 3 ) may function as
messenger(s) to transmit the signal expressed by surface receptors to the
interior of the cell, we will explore the possibility that one of these
factors may be involved in the action of some of the above peptides. In
addition and in light of recent evidence that we and others have provided
indicating that arachidonic acid and/or arachidonate metabolites can
enhance pituitary hormone release, experiments will be conducted to evaluate
the linkage between the receptors regulating arachidonic acid turnover and
the PI cycle. The role of Ca 2 on the above events as well as the possible
direct role of arachidonic acid as a second messenger will similarly be

(2) Peptide-amine and peptide-peptide interactions at the anterior
pituitary level. Combined stimulation with peptides using selected pulsa-
tile delivery patterns in a continuous perifusion system will be employed
to evaluate peptide-peptide interactions. Similar approaches will be used
to analyze peptide-amine interactions. Cells will be obtained from animal
models in which the secretion of the appropriate pituitary hormone is
selectively enhanced or suppressed or after experimental manipulations

Z01 ES 70092-02 LRDT

designed to alter the in vivo input of a given peptide or amine. When
appropriate, autoradiographic studies will be performed to analyze the
uptake and intracellular handling of peptidergic or aminergic ligands.

The specific objectives of these experiments are to determine (a) at
the physiological level, how important are these peptide-peptide and
peptide-amine interactions in a given integrated biological response
and (b) at the cellular and subcellular level, what intracellular
systems participate in these interactions. As an example, we would
ask the question: is the AVP-serotonin interaction that results in an
enhanced secretion of ACTH/B-Endorphin a physiologically relevant phe-
nomenon? Further, is that interaction mediated by a common intracellu-
lar messenger resulting in a potentiation of the effects or is it
expressed by the independent stimulation of two different messenger
systems, perhaps converging later in a final common pathway?

(3) Role of peptides in testicular function. Recently developed
superagonists of LHRH, currently being evaluated for clinical use in
contraception, will be employed to determine the direct effects at the
testicular level as well as possible indirect effects at central
(hypothalamic) and pituitary levels. Specific parameters to be eval-
uated at the testicular level will be testosterone secretion into
testicular interstitial fluid and peripheral plasma, as well as
testosterone secretion from whole testis in short-term in vitro
cultures. The possible interaction between LHRH -A and other testicu-
lar peptides, particularly 6-endorphin and ACTH, will also be eva-
luated. In selected experiments, blockade of opioid receptors will be
achieved by naloxone or naltrexone pretreatment to determine the role
of endogenous testicular opioids on the endocrine function of the


Spinedi, E. and Negro-Vi lar, A.: Angiotensin II increases ACTH release in
the absence of endogenous arginine-vasopressin (AVP). Life Sci . 34:
721-729, 1984.

Spinedi, E. and Negro-Vilar, A.: Evaluation of the corti cotropic activity
in vitro . Acta Bid. Clin. Latinamer . 18: 399-409, 1984.

Negro-Vilar, A., Snyder, G., Falck, J.R., Manna, S., Chacos, N., and
Capdevila, J.: Involvement of eicosanoids in release of oxytocin and
vasopressin from the neural lobe of the rat pituitary. Endocri nology 116:
2663-2668, 1985.

Negro-Vilar, A., Spinedi, E., and Johnston, C.A.: Monoamine-Peptide
Interactions in the Regulation of Pituitary Cell Function. In Labrie, F.
and Proux, L. (Eds.): Endocrinology . Amsterdam, Elsevier, 1985, pp.


Z01 ES 70092-02 LRDT

Negro-Vilar, A. and Valenca, M.: Testicular Peptides Regulating Gonadal
Function. In Sairam, M. (Ed.): Gonadal Proteins and Peptides and Their
Biological Significance . Singapore, World Science Publishing Company,
1985, pp. 352-366.

Fernandez, L., Tarlatzis, B.C., Rzasa, P.J., Caride, V.J., Negro-Vilar,
A., De Cherney, A.H., and Naftolin, F.: Renin-like activity in ovarian
follicle fluid. Fert. Steril . (In Press).

Valenca, M.M. and Negro-Vilar, A.: Pro-opiomelanocortin-deri ved peptides
in testicular interstitial fluid: Characterization and changes in secre-
tion after HCG or luteinizing hormone-releasing hormone analog treatment.
Endocri nolog y (In Press).

Negro-Vilar, A. and Lapetina, E.: 1,2-Didecanoyl glycerol and phorbol
12, 13-dibutyrate enhance anterior pituitary hormone secretion in vitro .
Endocrinology (In Press).

Negro-Vilar, A. and Culler, M.D.: Computer-Controlled Perifusion System for
Neuroendocrine Tissues: Development and Applications. In Conn, P.M.
(Ed.): Methods in Emzymology; Hormone Action: Neuroendocrine Peptides,
Part B. New York, Academic Press (In Press).





£01 ES 70094-01 LRDT


October 1, 1984 to September 30, 1985

TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders-)

Neuroendocrine Regulation of Prolactin and Pro-Opiomelanocortin-Deri ved Peptides

PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator ) (Name, title, laboratory, and institute affiliation)

PI: C. A. Johnston Senior Staff Fellow LRDT NIEHS

Others: A. Negro-Vilar

Head, Reprod. Neuroendocrinology Section LRDT NIEHS


University of California-San Francisco, School of Medicine


Laboratory of Reproductive and Developmental Toxicology


Reproductive Neuroendocrinology Section


NIEHS, NIH, Research Triangle Park, North Carolina 27709







□ (a) Human subjects

□ (a1) Minors

□ (a2) Interviews

□ (b) Human tissues xH (c) Neither

SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.)

Experiments by the Reproductive Neuroendocrinology Section focus on the
identification and development of cellular and subcellular mechanisms involved
in the regulation of pituitary hormone secretion, in particular, prolactin
(PRL), adrenocorticotropic n (ACTH), and 3-endorphin (3-end). Specific studies
are designed to elucidate the role and interactions of monoaminergic
neurotransmitters and peptide FeTeasi ng~and release-i nhTpi ting hormones in the
secretory control of these hormones Trom the anterior (AP) and neurointer-
mediate (NIL) lobes of the pituitary, the sequential arrangement and possible
connectivity o f that neuronal circuitry , and the developmental maturation of"
that circuitry^ Furthermore, various stressful stimuli not orPly dramatically
affect reproductive function as well as the secretion of PRL, ACTH, and 3-end,
but the stress-induced increase in plasma PRL has also been shown to require a
developmental maturation postnatally. Therefore, this paradigm has been uti-
lized to selectively examine the neuronal mechanisms involved in the stress-
induced increase in PRL secretion. In addition, drugs which interact
selectively with specific neurotransmitter or neuropeptide neuronal systems are
utilized to determine the individual contributions of specific neuronal systems
to the regulatory secretion of PRL, ACTH, and 3-end, and to aid in elucidating
the neuronal connectivity involved in that regulation. Lastly, specific
lesions and surgical procedures are empl oyed _t£ determi ne the source of various

Online LibraryNational Institute of Environmental Health ScienceAnnual report : National Institute of Environmental Health Sciences (Volume 1985) → online text (page 57 of 114)