Hypothalamic hormones aka hypothalamic releasing factors

18 Pages·2005·0.49 MB·English

11
STARLING REVIEW
. .
Hypothalamic hormones a.k.a. hypothalamic releasing
factors
Roger Guillemin
SalkInstituteforBiologicalStudies,10010N.TorreyPinesRoad,LaJolla,California92037,USA
(RequestsforoffprintsshouldbeaddressedtoRGuillemin;Email:[email protected])
(cid:1)(cid:2)˜(cid:1) (cid:3)(cid:4)´ µ(cid:4) (cid:5)(cid:6)(cid:7)(cid:4)(cid:8)(cid:5)(cid:9)(cid:2)˜(cid:10)’ ’(cid:6)´(cid:11)(cid:9)(cid:12)(cid:9)(cid:13) µ(cid:6)(cid:11)(cid:6)(cid:14)(cid:9)˜(cid:8)(cid:13) (cid:4)’(cid:5)(cid:4)´(cid:4)(cid:10)(cid:10)(cid:8)(cid:1) ‘(cid:1)´(cid:2)µ(cid:3)(cid:4)’ (cid:4)’(cid:13) (cid:5)(cid:9)´(cid:11)(cid:4)µ(cid:9)(cid:1).*
Homer, Epic, Iliad, Book 6 line 337.
Abstract
Originallysearchedforandeventuallyisolatedasfactorsof functions of both soma and psyche. This review will be
hypothalamic origin controlling anterior pituitary secre- composedofbriefessaysoncurrentknowledgeofeachof
tions, these hypophysiotropic peptides are now a chapter the original ‘hypothalamic hormones’, TRH, GnRH,
of physiology and medical endocrinology of their own. somatostatin,GHRHandcorticotropinreleasinghormone
Defying the concept of ‘neuropeptides’ they and their and will close on possible and probable futures.
receptors are now known to be ubiquitous and to have JournalofEndocrinology(2005)184,11–28
subtle as well as profound effects on a large number of
Introduction go to Geoffrey W Harris working at the Maudsley
Hospital in London in the 1950s, and later as head of the
A short history and the tribulations of nomenclature Department of Physiology in Cambridge.
ThecentralproposalofHarris’neurohumoralcontrolof
It was with pleasure that I read and answered Stephen G
pituitary function was that the unusual capillary system
Hillier’s invitation to contribute a review-style article on
existing between the ventral hypothalamus and the
‘HypothalamicReleasingFactors’fortheJournal’scelebration
anterior lobe of the pituitary would be the conduit
of the introduction of the word ‘hormone’ by Ernest H
for substances of hypothalamic origin that would act as
Starling in his 1905 Croonian Lectures (Starling 1905a).
releasersofeachandallpituitaryhormonesuponreaching
ThencameanolessgraciousletterfromJulianDaviswith
the parenchyma of the gland. There were powerful
allsortsofpracticaleditingconsiderationsforthewritingof
doubters, such as Solly Zuckerman. I will only refer here
a review-style article on ‘Releasing Hormones’. I will have
tothevolumewrittenbyGWHarrisin1955forasurvey
more to say on nomenclature below.
of the literature to that date (Harris 1955).
The proposal that the brain is somehow involved in
It was not until 1969 that the concept was finally
controllingpituitaryfunctionsgoesbacktothe1930s–see
validated by the isolation and structural characterization
the 1936 Croonian lecture by FHA Marshall (1936) and
of one of these postulated messengers, a totally novel
the imposing volume ‘The Hypothalamus and Control
molecule,thyrotropinreleasingfactor(TRF)inextractsof
Levels of Autonomic Function’ as the proceedings of a
ventralhypothalamictissues(Burgusetal.1969,Nairetal.
symposium organized in New York in 1939 by the
1970) and ultimately (ten years later) the actual demon-
Association for Research in Nervous and Mental Diseases
stration and quantitative measurements of several of that
(Fulton et al. 1940).
(andother)hypothalamicmessengersinbloodobtainedby
While others were also involved, the major credit for
catheterization of the hypothalamo–pituitary vessels
establishing the concept of a hypothalamic neurohumoral
(Wilber & Porter 1970).
control of anterior pituitary secretions must undoubtedly
Immunohistochemistry showed the anatomical distri-
bution of the hypothalamic neurones involved in the
*‘‘Now, however, my wife, persuading me with the right words, synthesis and neuro-secretion of these newly discovered
urgesmetobattle.’’ substances, a releasing factor for the secretion of
JournalofEndocrinology(2005)184,11–28 DOI:10.1677/joe.1.05883
0022–0795/05/0184–011 (cid:1)2005SocietyforEndocrinology PrintedinGreatBritain Onlineversionviahttp://www.endocrinology-journals.org
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12 RGUILLEMIN · Hypothalamicreleasingfactors
thyrotropin (TSH) and simultaneously of prolactin (in the early 1950s to 1969, manipulating literally several
most species), another for the secretion of both gonado- millionsoffragmentsofsheep(inmylaboratory)orpig(in
tropins - luteinizing hormone (LH) and follicle stimulat- Schally’s laboratory) hypothalami, from tons of sheep or
ing hormone (FSH), another for the secretion of pig brains (Fig. 1), eventually to recognize, isolate and
growth hormone (GH), and another for the secretion of characterize the first of these hypothalamic hormones,
adrenocorticotropin (ACTH). TRF, the thyrotropin releasing factor, isolating less than
Thesenovelsubstancesofhypothalamicorigincameto 1mg of the substance, and eventually characterizing its
bereferredtoas‘releasingfactors’(RF)(Saffran&Schally molecular structure (in 1969) by early mass spectrometry
1956);hencethyrotropinreleasingfactor(TRF),luteiniz- asthatofa(simple!)tripeptide(Fig.2)(Burgusetal.1969,
ing hormone (gonadotropin) releasing factor (LRF/ Nair et al. 1970). The growth hormone releasing factor
GnRF),growthhormonereleasingfactor(GRF),cortico- (GRF, GHRH) was eventually, 20 years after the dem-
tropinreleasingfactor(CRF),anomenclature(‘factors’)in onstration of its existence (Reichlin 1960), isolated and
keeping with the fact that their molecular structure had characterized as a 44 residue peptide (Guillemin et al.
notbeenestablished.Whenthatwasachieved,theywere 1982), not from the hypothalamus, but from a rare tumor
then referred to as ‘releasing hormones’ (RH) or hypo- of the pancreas which had induced acromegaly in the
thalamic hormones. They certainly fulfilled the definition absence of a pituitary tumor. The substance of (human)
and use of the word as proposed by EH Starling. hypothalamic origin was shown later to be identical
Personally, I am still somewhat partial to the nomen- (Bohlen et al. 1983). The corticotropin releasing factor,
clature‘releasingfactors’.Referringtothem,asagroupor the existence of which was shown in the early 1950s
individually, one immediately thinks ‘hypothalamo– (Guillemin & Rosenberg 1955, Saffran & Schally 1955),
pituitary’.Indeed,inthecaseof‘growthfactors’(fibroblast was not fully characterized until 1981 (Vale et al. 1981).
growth factor (FGF), epidermal growth factor (EGF), For today’s practicing biologist this seems hard to
nervegrowthfactor(NGF),platelet-derivedgrowthfactor believe.Noneoftheearlyseparationmethodswouldwork
(PDGF)....), we think immediately of a series of sub- onmicrogramquantitiesofextracts,thesubstancessought
stanceswhichnooneproposedtocall‘growthhormones’ couldonlyberecognizedintheeffluentoftheseseparation
when their molecular structures were established. How- methods (ion exchange or paper-chromatography, the
ever, with the unexpected observation (Brazeau et al. variousphasesofcounter-currentdistribution)bycomplex
1973) and characterization in hypothalamic extracts of an and time consuming bioassays, HPLC of (underivatized)
inhibitory factor of the secretion of growth hormone native peptides was not available until 1975 with the
whichInamedsomatostatinweshouldfeelmorecomfort- isolation of (cid:6)-endorphin, and sequencing of somatostatin
ablewiththereferencetothese‘hypothalamichormones’, in 1973 by manual Edman degradation was at the rate of
wellinkeepingwiththeuseofthewordsbyStarlingwho one residue/day. An automated peptide sequencer was
described, indeed, activating and inhibiting effects of available in 1982 for GRF and mass spectrometry was
his tissue extracts in his 1905 Croonian lecture series. At becoming far more available. All of the above is like the
the end of this review, after the many new facts it will archeology of what will follow in this short review. For
encompass, I will come back to that terminology. those of us who were the early plodders of that era, the
Anotherexampleofproblemswithnomenclatureisthe current state and course of things are as humbling as they
now totally accepted use of the word ‘agonist’ when are rewarding.
referringtoanalogsofGnRH(seebelow)whicharemany But far more than revolutionary developments in
times more potent than the native molecule, but are used methodologyhashappenedinthe50yearsorsosincethe
as inhibitors of the secretion of gonadotropins through original isolation and characterization of these hypo-
mechanismsnowwellunderstoodandthatwewilldiscuss thalamic molecules. The original hypophysiotropic
below. But a molecule which is 1/100th as active as the hypothalamic peptides with which we were dealing have
nativesequenceisalsoanagonistofGnRH.Referringto been found to be widely distributed throughout the
the analogs which are so much more powerful than the central nervous system in well-defined locations. More-
nativesequence,theeffectsobservedanddesiredareactu- over, what we originally recognized as neuropeptides are
allyofreceptordown-regulationandinhibition(seebelow nowknowntobeexpressedandfunctionalintissuesofthe
inthesectiononGnRH)leadingtodecreasedfunction.It gastrointestinal tract, the lungs, the heart, the hemato-
wouldbemuchmoresatisfactorytorefertothesemolecules poietic system, and the immune system. And, more
as‘superagonists’,awordIhadsuggestedandusedasearly importantly, the same is true of the multiple receptors for
as1978(withobviouslynosuccess)(Rivieretal.1978),the these same peptides.
wordtobeassociatedwiththeultimateinhibitoryactivity; As we will see in the next sections, molecular biology
just one more example of these vagaries of nomenclature with its powerful methodology has expanded exponen-
where bad habits are sure to be winners. tially the original knowledge we referred to above, both
Letmeclosethisintroductionbothtothisreviewandto for basic research and clinical medicine. A search on the
this field with a short historical recounting. It took from websiteoftheLibraryofMedicine,Washington,DC,USA
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Hypothalamicreleasingfactors · RGUILLEMIN 13
Figure1 In1965atBaylorCollegeofMedicine,Houston,Texas.Ourfirststepof
purificationonSephadexG25.Thecolumnwas2mtall,0·20mdiameterandloadedwith
3·5kgSephadexG-25indiluteaceticacid(RaymondGivens,laboratoryassistantnextto
‘his’column).Inatypicalrun,whichwouldlast24–36h,wewouldapplythecrude2M
aceticacidextractof200–250000fragmentsofsheephypothalamus.Anactivefraction
(asperbioassays...)wasthenprocessedonseveralsuccessivestepsofcounter-current
distribution,ionexchangechromatographyetc...,allfollowedbybioassays.Comparethis
withtoday’smethodologyusingPCRortherecentlyproposed‘peptidomics’,isolatingand
sequencinginasinglepassover100peptidesfromonehalfofonemousepituitaryora
fewmgsfromanytissue,bycombinednanoscaleliquidchromatography,electrospray
ionizationmassspectrometryandfittingwithGenBank’sESTdatabase.
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14 RGUILLEMIN · Hypothalamicreleasingfactors
Figure2 FirstinscriptionofthestructureofTRFbyRogerBurguswithlocationsoffragmentationionsfromtheearlyresultsofmass
spectrometryincollaborationwithDominicMDesideriofromtheLipidResearchCenter,BaylorCollegeofMedicine,Houston,
Texas.RogerBurgusdiedin1998afteralongillness.
for publications dealing with the original molecules Thesequenceoftheseessayswillfollowthatofthedates
we isolated has yielded well over 100000 references. It is (year) on which each of these hypothalamic peptides was
thus quite impossible to produce a complete critical originally isolated, characterized, and reported.
review of that now immense literature within the con-
straints of this Starling Review, and more importantly
because of the very limitations of this reviewer. I will TRF, TRH
thus limit myself to selected topics/issues which, in my
opinion, represent some of the most intriguing observa- A very comprehensive review on TRH, its overall biol-
tions made over the years in as much as they departed ogy,includingitsnon-hypophysiotropicactivities,biosyn-
from or expanded the original way of thinking about thesis, and biology of non-TRH fragments from the
one or other of these hypothalamic peptides. When a precursor, was published in 1999 (Nillni & Sevarino
comprehensive review on a particular subject has been 1999).
written, I will make reference to it and will discuss only The small tripeptide, pGlu-His-Pro-NH , is indeed
2
recent reports which would have appeared after that processed from a larger precursor (29kDa, 255 residues),
review. I am well aware that I will not touch here on all through now classic cleavage mechanisms, C-terminal
the expanding observations which have been reported amidation,andcyclizationoftheN-terminalGlntopGlu.
in the field, and I do not intend to discuss or describe in Pro-TRH mRNA has been located in several brain areas
detailthemethodologiesleadingtotheseobservationsand other than the paraventricular nucleus (olfactory bulb,
conclusions. lateralhypothalamicarea,medianeminence,preopticarea,
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Hypothalamicreleasingfactors · RGUILLEMIN 15
periaqueductalgray,brainstemanddorsalvagalcomplex) signaling pathways through STAT-3 and CREB binding
and in non CNS tissues (pancreatic (cid:15)-cells where TRF sites that mediate leptin and melanocortin responses
and somatostatins show opposing paracrine effects on respectively (Kim et al. 2002, Guo et al. 2004).
glucagon secretion, the whole gastrointestinal tract where Otherrecentpublicationsareextendingthecomplexity
TRF may modulate contractions, the genitourinary sys- ofwhatappearstobeanintegratedmechanismcontrolling/
tem, ventral prostate, Leydig cells, epididymis, seminal regulating the overall homeostasis (or should we say allo-
vesicles). Multiple physiological activities of TRH on the stasis) of energy production and expenditure of the whole
functions of these various organs have been reported. Of organism. All peptides known to be involved in appetite
earlyclinicalinterestwasthereportedpresenceofTRHin controlorresponsetofeedingorfastingarenowknownto
several locations of the ventral horn of the spinal cord, bepartofthemechanismscontrollingTSHrelease(galanin
either in motor neurons or in extensive projections from (Wittmannetal.2004b),neuropeptideY(NPY)(Broberger
bulbospinal neurons to motor neurons. TRH enhances etal.1999),(cid:6)-MSH(Feketeetal.2000,Kimetal.2002),
motor neuron firing leading to increased muscle tone, leptin,asalreadyquotedabove).Thereisalsorecentevidence
contractibility and spinal reflexivity. Clinical studies have that catecholamines originating from medullary (C1–3)
repeatedlyreportedbeneficialeffectsofi.v.administration adrenergic neurons and shown to be co-localized with
ofTRHinseveralmotorneurondiseases.Theseimprove- CART (cocaine-and-amphetamine-regulated-transcript)-
ments, for some unexplained reason, are of short duration proteinsmakemultipleconnectionswiththehypophysio-
(days to weeks) (Brooke 1989). tropicTRHneuronsintheparaventricularnucleusandare
Some remarkable recent observations have been made involved in the rapid release of TRH in response to cold
that lead one to consider that the hypothalamic TRH- exposure (Wittmann et al. 2004a).
secreting neurons are under multiple controlling mecha- Alltheseremarkableobservationsoffunctionalrelation-
nisms which all are otherwise involved in the control of ships, combined with morphological demonstrations of
appetite regulation, food intake, along with TSH-release co-locationsofthesepeptidesandtheirreceptorsinspecific
andultimatelythyroidfunction,insomesortofwhatcould neurons, are the vindication of the early proposal of the
be considered as a general regulatory-system determining existenceofpeptidergicneuronsfirstininsects,andlaterin
energy expenditure and adaptive thermogenesis. It has birds and mammalians by the remarkable morphologist,
long been known that during starvation in rodents, the BertaScharrer(Fultonetal.1940,Scharrer1978).
pituitary–thyroid axis is down-regulated leading to low With the recent demonstration that leptin can directly
levels of circulating TSH and thyroid hormones. Several target the TRH promoter in vivo in neurons of the
reports have shown that leptin is involved in regulating paraventricular nucleus (Guo et al. 2004) and that it can
release of TRH from a defined population of TRH invitropromoteneuriteoutgrowthfromarcuateneuronsin
neurons in the paraventricular nucleus. Low levels of well controlled situations (Bouret et al. 2004, Pinto et al.
circulatingleptinareaccompaniedbyadecreaseinTRH- 2004)withoppositeeffectsofghrelin(seebelow),wemay
mRNA in TRH-secreting neurons; transfection data and begin to understand the role(s) of these peptides in the
in vivo studies show that leptin can regulate TRH gene functioning and the structural evolution and/or mainten-
expressionviaactivationofintracellularsignal-transducer- ance of the neurons in which they co-locate with one of
and-activator-of transcription 3 (STAT-3) proteins in the classic neuromediators.
TRH neurons, with evidence that the STAT-3 binding The rapid inactivation of TRH by plasma and tissue
siteontheleptinreceptorisalsorequiredfortheeffecton incubates has been extensively studied by several groups
TRH release (Guo et al. 2004, Huo et al. 2004). More- (see Bauer (2004) for review). I will only mention the
over, it appears that the leptin-responsive TRH neurons remarkable substrate specificity of an enzyme designated
are concentrated in the caudal region of the medial and TRH-degrading ectoenzyme (TRH-DE) which selec-
periventricular parvocellular subnucleus of the para- tively inactivates TRH, a unique property among
ventricular nucleus (Huo et al. 2004), i.e. only in a neuropeptide-inactivatingenzymes.Atthepituitarylevel,
subpopulationoftheentirenucleusthathasbeenshownto the activities and the mRNA levels of TRH-DE are
bespecificallyinvolvedinthehypophysiotropicactivityof stringently regulated by thyroid hormones as well as by
TSH release leading to thyroid activation. Indeed, leptin estradiol, strongly indicating that on adenohypophyseal
does not activate STAT-3 in TRH neurons located in targetsitesthisenzymeservesregulatoryfunctionsforthe
otherregionsofthehypothalamus,suchastheperifornical release of pituitary hormones. In the brain, the activity of
and lateral hypothalamus (Huo et al. 2004). TRH-DE is not influenced by peripheral hormones but
TheTRHneuronsintheparaventricularnucleushave the high activity of this enzyme and the distinct locali-
also been shown to express receptors for (cid:6)-MSH zationofTRH-DEinrelationtotheotherelementsofthe
(melanocortin-4R), a peptide involved in appetite and TRH-signalingsystem(Heueretal.2000)stronglysuggest
satietycontrol,suggestingthatbothsignalingsystemsmay thatthispeptidasemayactasaterminatorofTRHacting
regulate TRH gene expression. In cell cultures the TRH in extrahypothalamic brain areas as a neuromodulator
promoter has been shown to respond to both these and/or neurotransmitter.
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16 RGUILLEMIN · Hypothalamicreleasingfactors
LRF, LHRH, GnRH express a GnRH-receptor the activation of which is
associated with rapid and dose-dependent increase in
Early isolation and sequencing showed that porcine and cytoplasmic Ca2+ concentration and GnRH release, indi-
ovineLRF(GnRH)havethesamedecapeptidesequence, cating an autocrine regulation of GnRH (Cesnjaj et al.
including pyro-Glu N-terminal and amidated Gly as 1993). The firing and release of GnRH by GT-I cells is
C-terminal. Very rapidly it was shown that the molecule estrogen sensitive, in keeping with the well-known
was active in all species studied, from mammalians to proestrous surge of gonadotropins.
birds,fish,andamphibianstostimulatereleaseofgonado- There is good evidence that GnRH controls activation
tropinsusuallyleadingtoovulation,egglaying,spawning. ofthegeneexpressionforthe(cid:6)-and(cid:15)-subunitsofLHand
Inmammalians,includinghumans,abolusi.v.injectionof FSH, dimerization of the (cid:6)-(cid:15)-submits, and glycosylation,
the decapeptide released simultaneously LH and FSH. inaccordwiththefrequencyofthepulseavailabilityofthe
PerfusionofGnRH,however,afteranimmediateincrease peptide (GnRH).
in LH and FSH plasma concentration, showed a gradual Inallthesestudies,FSH,asmeasuredbyhighlyspecific
and persistent fall in LH and FSH secretion that was not immunoassayforthe(cid:15)-chain(soastoavoidfalsepositives
explainedbydepletionofpituitarystores.Inaremarkable due to the fact that LH and FSH share an identical
series of elegant physiological studies in the monkey, (cid:6)-chain) would usually accompany LH in its secretory
Knobil and his collaborators demonstrated that the endo- peaks but not always or consistently. Pulse administration
genous hypothalamc release of GnRH was pulsatile, with of GnRH with pretreating or acute combined admini-
a frequency of 60–90min depending on the animal, each stration of activin, follistatin, with or without (cid:6)-estradiol
GnRH pulse being correlated with electrical activity of never consistently produced a pattern of specific, singular
similar frequency in the arcuate nucleus of the hypo- secretion of FSH. Electrical stimulation, localized stereo-
thalamus, and each GnRH pulse being followed in tacticlesionsoftheparaventricularnucleus-dorsalanterior
secondsbysecretionofLHmeasuredinthejugularveinor hypothalamus i.e. a region different from the region
better still in hypophysial superficial veins. In pituitary involvedinGnRHsecretion(inrodents,asomewhatloose
stalk sectioned animals peripheral intravenous injection of network referred to as the septo-preoptic-infundibular
GnRH in a pulsatile pattern would regularly stimulate pathway) (Silverman et al. 1987) have been claimed to
secretion of LH for any length of time so long as the modify FSH release without altering LH pulsatility. A
pulsatile mode was maintained (Knobil & Hotchkiss report from the laboratory of McCann, who was the first,
1988). in 1964, to propose the existence of an FSH-controlling
The question remained as to the controlling origin of center in the hypothalamus, of a partial purification of an
the periodic firing of the medial basal hypothalamus FSH-releasing factor from sheep hypothalamus extracts
neurons releasing GnRH in the hypothalamo–pituitary stoppedshortofreportingisolationandcharacterizationof
vessels. An in vitro perifusion system of human fetal suchasubstance.Thatwasthestatusuntilacoupleofyears
mediobasal hypothalamus (20–23 weeks of gestation) and ago (Padmanabhan & McNeilly 2001).
fromadultbrainsshowedthatthefragmentswouldrelease Relativelyearlyaftertheisolationandsequencingofthe
GnRH in discrete pulses with a periodicity of about porcineandovineGnRHs,itwasrealizedthattheGnRHs
60–100min. The secretion was Ca2+-dependent and isolatedfromseveralclassesofvertebratesshowedmultiple
could be suppressed by morphine, an effect that was substitutions in their sequence when compared with the
reversedbynaloxone(Rasmussenetal.1989).Butwasthe pig or sheep GnRH. It was also realized that these struc-
GnRHpulsegeneratedanintrinsicpropertyoftheGnRH turalvariantsofGnRHweredistributedinawiderangeof
neurons or was it influenced by other neurons in the tissues (midbrain, sympathetic ganglia, immune T and B
surroundingtissueofthemedialbasalhypothalamus?That lymphocytes, breast and prostate cancers), where they
question was answered by the development of immortal- wereshowntohavespecificbiologicalactivitiesinrelation
ized GnRH secreting neurons (GT-I neurons) by geneti- to the particular tissues and later were correlated with
cally targeted tumorigenesis (Mellon et al. 1990). These the presence of GnRH receptors (see below). In most
cellsexpressneuronalbutnotglialcellmarkers.TheGT-I vertebrate species two or three forms of GnRH could be
neurons express GnRH mRNA, release GnRH in re- demonstrated,suggestingearlygeneduplications.
sponse to depolarization, or spontaneously in a pulsatile One of these variants, originally isolated in chicken
manner.Theyalsorespondtoaseriesofagentsforwhich brain(nowreferredtoaschickenGnRHIIorGnRHII)
they have receptors - estrogens, opiates, (cid:15) -adrenergic- was found to be universally present and totally conserved
1
and D -dopaminergic-receptors, tyrosine kinase- in all vertebrates from fish to humans (Millar 2003, Neill
1
receptors, activators including insulin-like growth factor et al. 2004). GnRH II is widely distributed in tissues
(IGF)-I and -II, FGF2, and insulin. Thus, the GnRH suggesting that it has acquired significant functions
neurons per se appear to constitute the GnRH pulse through the phylogeny. While the genomic and mRNA
generator. Probably the most remarkable observation structuresofGnRHIandIIarecloselyrelated,GnRHII
made with the GT-I cells was that they were shown to is expressed at much greater levels outside its brain locus,
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Hypothalamicreleasingfactors · RGUILLEMIN 17
particularly in the kidney, bone marrow and prostate, ChangesinGnRHcontentofbrainareas,cellnumberand
leading one to suggest that the peptide has specific cell size in response to visual, olfactory and other stimu-
functions in these tissues. Molecular phylogenetic analysis lants of sexual behavior have been observed in many
showsthatthegenecodingforGnRHIIislikelytheresult species from fish to mammals (Rissman et al. 1997).
of a duplication before the appearance of vertebrates and Neill and collaborators (2004) have recently reported
predicts the existence of a third GnRH form in humans thesuccessfulidentificationofageneencodingtheGnRH
and other vertebrates (White et al. 1998). Indeed, a third II receptor in humans, and its cloning and sequencing
form,GnRHIIIwasrecentlycharacterizedinthelamprey from monkey and pig tissues. They also reported that
andithasbeenreportedbyimmunofluorescencealsotobe GnRH II mRNA’s expression, as measured using an
located in the GnRH neurons of rat and human hypo- antisense riboprobe, is totally ubiquitous (21 different
thalamus more particularly involved in the secretion of tissues of human origin with highly variable degrees of
FSH (Lumpkin et al. 1989, Dees et al. 1999). GnRH III expression)(Neilletal.2001).Thislargedistributionofthe
has been proposed (Yu et al. 2002) as the long sought GnRHIIreceptorinhumantissuesparallelsthatobserved
FSHRF, but there is no consensus on that proposal earlier for GnRH I receptor. Such findings suggest the
(Kovacsetal.2002).ThedemonstrationofaspecificFSH existenceinhumansofawidespreadparacrineorautocrine
releasing factor is still open. regulatory system (Neill et al. 2004).
AsIsaidabovethecDNAforthereceptorofGnRHII Expression of the GnRH II receptor has been demon-
has been cloned in amphibians, its homologs in primates strated in cell lines derived from human endometrial
have been sequenced (Millar et al. 2001, Neill et al. and ovarian cancers. Proliferation of these cell lines was
2001), and their locations ascertained, thus leading to reduced by GnRH II in a time- and dose-dependent
an investigation of functions of GnRH II other than manner, at equimolar doses much lower than a GnRH I
hypophysiotropic ones. superagonist.Sinceallendometrialandovariancancercell
The type II receptors have partial identity with the lines express GnRH II receptors and GnRH II has more
human type I receptor, both having a characteristic potent antiproliferative properties on tumor cell growth
G-protein coupled receptor (GPLC) structure though thanGnRHI,evenmorepowerfulanalogsofGnRHIIor
withsomedifferencesintheproteinC-terminalsequence, non-peptidicmimicsmaybeworthexploringasantitumor
suggesting an early evolutionary gene duplication (Millar agents (Grundker et al. 2002, Tang et al. 2002).
2003). GnRH II was found to be about 400 times more GnRH receptors have been reported in biopsies of
potent than GnRH I in binding that receptor. GnRH II glioblastoma, melanomas (van Groeninghen et al. 1998),
has been reported to be present in extracts of amphibian andintwomelanomacelllineswithevidencethatGnRH
sympatheticgangliawhere,likeGnRHI,itappearstobe superagonists inhibited proliferation and invasiveness
involved in synaptic transmission in the sympathetic (metastatic potential) in an in vitro model (Moretti et al.
ganglia via a receptor highly specific for GnRH II. 2002). Labeled analogs of GnRH have also been used to
Because the peptide facilitates fast excitatory transmission scan possible dissemination (metastases) of various tumors
by neurotransmitters (Bosma et al. 1990), this could when a biopsy showed binding of GnRH I or II.
representageneralmechanismforGnRHIItofunctionas
a neuromodulator in its multiple locations in the nervous
system. In many of the CNS areas (midbrain, supraoptic GnRH analogs as superagonists or antagonists
nucleus)boththetypeIIreceptorandthepeptideGnRH
II have been demonstrated in the same neurons, often As is the case for all these native hypothalamic peptides,
overlapping with early developing mammalian GnRH I half-livesareveryshort(min)astheyarerapidlydegraded
cells, at least in early embryo development. Neurons that by various enzymes in blood or tissue fluids. Very early
express the mammalian gene for GnRH I in the preoptic after the sequencing of porcine and ovine GnRH a series
area and periventricular region of the hypothalamus of agonist analogs were reported with increased potency
stained with type II GnRH receptor antiserum (Rhesus either due to metabolic stability or greater receptor
monkey), suggesting that GnRH II might regulate affinity.Todate,averylargenumberoftheseanalogshave
mammalian GnRH I neurons (Millar 2003). These been produced in academia and in industry. It was also
multiple examples of the peptide GnRH I and/or II and early recognized that these agonist analogs with greater
the GnRH I receptor being present in neurons alongside specific activity than the native decapeptide, the super-
classic neurotransmitters and being able to facilitate agonists,producedanimmediateincreaseinLHandFSH
(modulate)synaptictransmissionmaybeanotherexample secretion, which was consistently followed by a state of
of what we mentioned earlier regarding leptin and, as we inhibition of secretion of the gonadotropins and gonadal
will see below, probably also that regarding CRF and steroids, a typical example of the ligand-induced down-
urocortin and glutamatergic transmission. regulation of the receptors. Such GnRH superagonists
ItwouldbeinterestingtoseewhetherGnRHIIcould have been successfully used in the treatment of endo-
stimulate and maintain dendrite growth and structures. metriosis,primaryprecociouspuberty,prostatecancerand
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18 RGUILLEMIN · Hypothalamicreleasingfactors
as a pre-operative treatment of large leiomyomas. When remain in the nasal cavity, including the olfactory and
used in long-term therapies it has been customary to vomeronasal mucosa, in the adult including humans
include so-called ‘add back’ regimens during which syn- (Wirsig-Wiechmann 2001). These peripheral GnRH
theticsexsteroidsareadministeredinwellcontrolleddoses processes terminate within the lamina propria of the
along with the superagonist to overcome the side effects chemosensory mucosa. GnRH receptors are also ex-
such as hot flashes, bone resorption and osteopenia. pressedbychemosensoryneurons.Thus,paracrinerelease
A series of true GnRH antagonists are also available ofGnRHmaywellinfluencethefunctionofthechemo-
which bind to the receptor without activating it and sensoryneuronsaspatchclamprecordingtechniqueshave
preventbindingofthenormalligandGnRH.Theobvious shown,inrodents,thatGnRHincreasessodiumconduct-
advantage of these analog antagonists is that they produce ance in olfactory neurons (Wirsig-Wiechmann 2001).
an immediate decrease in circulating gonadotropins and GnRH may thus be involved or responsible for the
gonadalsteroidlevels,easilyreversibleandneverpreceded traditional reports of increased sexual activity (arousal)
by the surge seen with the superagonists. Unexpectedly, induced by body odors at some times of the menstrual
some of the early GnRH antagonists were shown to cycle in women, and in the overall functions of phero-
stimulatereleaseofhistaminebymastcells,anundesirable monesingeneral.Theelucidationofthisuniqueoriginof
side effect. Newer compounds have practically overcome the GnRH neurons before they reach their ultimate
theproblem.TheseGnRHantagonistsarecurrentlyused hypothalamic location and function explains both the
inmedicineforthesameindicationsmentionedabovefor anosmia either complete or selective and the isolated
the superagonists, with the additional advantage that the gonadotropin deficiency in patients with one form or
initial flare-up in gonadotropin secretion is avoided. One another of Kallmann’s syndrome and related develop-
of these antagonists is currently approved for use in mental abnormalities (Lieblich et al. 1982). See also a
preventing premature LH surge in ovulation induction in recent report (Rugarli et al. 2002) implicating a unique
in-vitro fertilization programs. In patients with cancer of protein(KAL),thegeneproductofKAL-I,intheterminal
the prostate long-term use of the antagonists leads to well steps of olfactory axon guidance, connections with the
controlledloweringofplasmaandrogenslevels(Yen2004, earlyGnRHneuronsandpossiblyotherperipheralaxonal
Yen & Jaffe 2004). More recently, some nonpeptidic mappings.
antagonists have been synthesized with the idea that they
could be active orally. Two such compounds are in early
clinicaltrialswithsatisfactoryresults.Inlong-termtherapy Somatostatin
judicious dosing of the antagonist would eliminate ‘add
back’ regimens. When we first isolated, sequenced and synthesized
In closing this section on GnRH, I will review briefly somatostatin from hypothalamic tissues in 1972 (Brazeau
thecurrentknowledgeabouttheontogenyoftheneurons etal.1973)itwasaphysiologicallyunexpectedinhibitorof
which eventually control the sex life of all vertebrates. the pituitary secretion of growth hormone. Indeed, we
During early development, at about the same time as were searching for a growth hormone releasing factor.
gonadotropins appear in the progenitor cells of the Rapidly, it was shown by Charlie Gale in Seattle to
pituitary, neurons expressing GnRH are found in the producehypoglycemiaintheirbaboons,relatedtoinhibi-
epitheliumoftheolfactoryplacode(Wrayetal.1989)and tion of the secretion of glucagon and insulin, a fact
inthenervusterminalis(alsoreferredtoascranialnervezero confirmedbySamYeninacromegalicpatients;Duboisin
or terminal nerve), a differentially organized set of nerve France and Rolf Luft and Thomas Høkfelt in Stockholm
fibers comingled with chains of GnRH-containing showedthatsomatostatinwassynthesizedinthedeltacells
neuronal cell bodies extending peripherally to the lamina of the endocrine pancreas. Rapidly again, the group
propriaofthenasalchemosensorymucosaandcentrallyto around Michael Besser in London was to show effects of
the ventral forebrain where they eventually establish an somatostatinonthesecretionofgastrointestinalpeptidesas
adult-like distribution. Progenitors of neurons expressing wellasimmunolocalizationofsomatostatininthepertinent
GnRH–mRNA and peptide product (only GnRH1) and secretory elements (Albinus et al. 1975).
GnRH receptors, and destined for the preoptic area, And in a Starling Review, it is only fitting to mention
migrate from the olfactory placode, crossing into the thatsecretinwasshowntobeapowerfulstimulatorofthe
septal-preoptic area apparently following gradients of secretion of pancreatic somatostatin in a perfused dog
the adhesion molecule neural cell adhesion molecule pancreas preparation (Ipp et al. 1977).
(NCAM) and the axonal growth promoting protein, The early observations on multiple activities of the
netrin (Schwarting et al. 2004). Gamma-aminobutyric peptide, unrelated to growth hormone secretion, were
acid (GABA), which is also found in a population of followed by the synthesis both in academia and the
these GnRH neurons, has also been implicated in this pharmaceuticalindustryofaverylargenumberofanalogs
migratorydevelopment(Tobetetal.1996).Recentstudies of the native structure of somatostatin with the aim of
have shown that some of the early GnRH neurons dissociating the multiple activities of the peptide and
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Hypothalamicreleasingfactors · RGUILLEMIN 19
improving on the short half-life of the native molecule. excellent reviews have recently appeared (Reubi 2003,
Availability of these analogs led to the characterization Weckbecker et al. 2003) describing the native sequences
of somatostatin receptors in multiple tissues. We know of somatostatin in various species, somatostatin analogs,
now that there exist 5 different receptors, with isoforms, mechanisms of action, the receptors (Weckbecker et al.
and their tissue distribution is now well known (Raulf 2003),theuseoflabeledanalogsandtheiruseindiagnostic
et al. 1994). They have been cloned and expressed and and therapeutic opportunities (Reubi 2003).
knockout animals have been prepared. Scintigraphy with labeled analogs of somatostatin has
Thus,thereisnowextensiveevidencethatsomatostatin shown the presence of several types of receptors in
alsooriginatesfromtheimmunesystemandhassignificant pathological peripheral tissues, including the retro-orbital
immunomodulatory activity. The thymic epithelial and tissueinGrave’sdisease.Accumulationoftheradionuclide
dendritic cells synthesize somatostatin and express type 2 is correlated with the presence in the orbital tissue of
somatostatin receptors. Somatostatin is now recognized activated lymphocytes, and treatment with octreotide has
to be part of an immunoregulatory circuit that inhibits been reported to improve the ophthalmopathy (Krassas &
production of interferon-gamma, tumor necrosis factor (cid:6) Kahaly1999).Thesametechniqueofscintigraphywitha
(TNF(cid:6)), CRH and Substance P at sites of (chronic) labeledanalogsuchasoctreoscanhasledtotherecognition
inflammation (Karalis et al. 1994, Weinstock & Elliott that many cancers express specific somatostatin receptors.
2000). Locally released somatostatin has been reported Somatostatin receptors scintigraphy in primary breast
to be involved in the classic anti-inflammatory action cancer has been reported to correlate with expression of
attributed to glucocorticoids (Karalis et al. 1995). A the subtype 2A and type 5 receptors, including in
recently discovered variant of somatostatin, named metastases (Schulz et al. 2002). Malignant melanomas
cortistatinbecauseofitsprevalenceinthebraincortex,has including uveal melanomas have been shown to express
also been shown to be expressed in human monocytes, somatostatin receptors (SST-R), particularly SST-R2 and
monocyte-derivedmacrophagesanddendriticcellsbothin -R5. Somatostatin analogs have been proposed in the
the normal state and when activated by various lipopoly- treatment of these tumors, either alone particularly as
saccharides.Bothcortistatinandsomatostatinbindtotype high-energyradioisotope-labeledpeptide(octreotide)oras
2 somatostatin receptors (Elliott et al. 1999, Weinstock & adjuvant therapy. Similar observations and conclusions
Elliott2000),morespecificallytheisoform2A.Tcellsand have been made and proposed in cases of small cell lung
Bcells,likemacrophages,expressmRNAforthereceptor cancer and bronchial carcinoid disease (O’Byrne et al.
type 2. Thus, somatostatin can be considered as an 2001).Infact,allso-calledneuralcrestorneuroendocrine
immunoregulatory cytokine that regulates inflammation. tumors (insulinomas, glucagonomas, gastrinomas) have
Intra-articular injection of somatostatin or one of the been shown to express somatostatin receptors and to
analogs has been reported to reduce synovitis and inflam- respond,sometimesdramatically,tolong-actinganalogsof
mation in patients with rheumatoid arthritis (Coari et al. somatostatin. A large number of reports deal with adeno-
1995). It also reduces the concomitant pain possibly carcinomaoftheprostate.Ithasbeenknownforyearsthat
because, in a new twist and as recently demonstrated, all prostatic tumors contain cellular elements known as
somatostatin also binds to the mu-opioid receptor. Mu- neuroendocrinecellsastheyarepositiveinimmunocyto-
opioid receptors in the skin, particularly in the keratino- chemistry for a variety of peptides such as bombesin,
cytes, are involved in the perception of peripheral pain. TRH, somatostatin, chromogranin A and also serotonin
Analogs of somatostatin such as octreotide and lanreotide (Hansson & Abrahamsson 2001). The degree of differen-
havemuchgreateraffinitytothemu-opioidreceptorthan tiation and proliferation of these cells appears to correlate
the native peptide. inversely with tumor progression and the appearance of
A few words about cortistatin, the neuropeptide with the androgen independent state. It has been proposed
highhomologywithsomatostatinexpressedinmosttissues (Zapata et al. 2002) that cancerous epithelial cells become
along with somatostatin, but primarily in brain cortex, responsive to paracrine and autocrine factors from the
hence its name. Cortistatin-14 inhibits GH release in neuroendocrine cells by up-regulation of receptors for
response to GHRH at the same doses as somatostatin-14. these neuropeptides. In cell lines such as PC3, analogs of
Cortistatin binds to all somatostatin receptors, but unlike somatostatinbindingtotype2andtype5receptorsinhibit
somatostatin, also binds to the ghrelin receptor (see the proliferation. Thus the correct analog of somatostatin in
section on GHRH below). Both peptides at equivalent relation to the particular receptor expressed may become
doses similarly inhibit GH release induced by ghrelin partofthetherapyofsomeofthesetumors(Weinstock&
(Broglio et al. 2002). Elliott 2000, Mosca et al. 2004).
Somatostatin analogs have been reported to inhibit
angiogenesis in vitro and in vivo through receptors present CRF, CRH
on endothelial cells and also through inhibition of para-
crine growth factor secretion such as IGF-I, vascular In keeping with the historical nomenclature vagaries I
endothelial growth factor (VEGF), and FGF-2. Two mentioned above, the originally named CRF is still CRF
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20 RGUILLEMIN · Hypothalamicreleasingfactors
inmuchoftheliteratureratherthanCRH,butbothterms affinityforCRF-R1thanforCRF-R2,whileurocortinI
are used. When in the 1950s Hans Selye was calling binds with equal high affinity to both receptors, in all
attention to the importance of ‘the first mediator’ of the locations; in contrast urocortin II and III are selective for
ACTH response upon exposure to stress, which was CRF-R2.BothreceptorsarefoundthroughouttheCNS,
finally characterized as a 41 residue peptide 30 years later CRF-R1 being prevalent; it is found throughout the
bythegroupledbyWVale(Valeetal.1981),hehadno cerebral cortex, cerebellum, olfactory bulb, medial sep-
suspicionoftheextraordinarymultiplicityofactionsofthat tum, hippocampus, amygdala and the pituitary. CRF-R2
molecule which is involved in the normal response(s) to is found primarily in hypothalamus, lateral septum, and
stress besides the activation of the secretion of ACTH. also in the choroid plexus. CRF and CRF-mRNA are
Two major reviews have been published on CRF, its found in the cerebellum of several species (Cummings
variants,multiplereceptors,mechanismofaction,multiple et al. 1994), particularly in the two primary afferent
physiologicalfunctionsandclinicalimplications(DeSouza systems, i.e. the climbing fibers and the mossy fibers
& Nemeroff 1990, Bale & Vale 2004). (Palkovits et al. 1987) and the two CRF receptors
Ascouldbeexpectedforamoleculeofthatphysiologi- (CRF-R1 and CRF-R2) have been demonstrated in
calimportance,theprimarysequencesoftheseveralCRF similarlocations(Bishopetal.2000).CRFpotentiatesboth
variants and related molecules (urocortins I, II, III) share spontaneous and glutamate-induced activity of Purkinje
major identities from amphibians to several mammalians cells (Bishop & King 1992). CRF has been found to be
including humans. essential for induction of long-term depression in the
The action of CRF and CRF-related molecules are cerebellum, a mechanism which has been proposed for
mediated through G-protein coupled receptors derived consolidating motor learning (Ito 2001). There is now
fromtwodistinctgenes,CRF-type1(CRF-R1)andCRF- evidence that the amounts of CRF that can be measured
type 2 (CRF-R2) receptors. Both receptors have different invariousregionsofthecerebellumvaryindirectrelation
anatomicaldistributionsandphysio-pharmacologicalprop- to the degree of electrical stimulation that can be applied
ertiesandexistinseveralisoforms(Ehrhart-Bornsteinetal. to those regions of the cerebellum, thus implying
1998). that CRF is a necessary neuromodulator component of
In situ hybridization for CRF-mRNA has shown both cerebellar function. CRF and its mRNA have been
message and peptide (CRF-41) in a subpopulation (the localizednotonlyinvaricositiesoftheclimbingfibersbut
parvocellular neurons) of the paraventricular nucleus, also in primary axons (Tian & Bishop 2003).
surrounding the vasopressin synthesizing neurons (Grino That report also showed the presence of CRF in radial
et al. 1989, De Souza & Nemeroff 1990), the central glial cells and astrocytes both in the granule cell layer and
nucleus of the amygdala, the midbrain (substantia nigra within white matter and stated that the amount of the
and periaqueductal gray) and the dorsal vagal complex of peptidewassignificantlychangedfollowingstimulationsof
themedulla.Intheselocations,CRFisprimarilyinvolved two afferent regions of the cerebellum. Increased c-fos
in ACTH-stress response and, as could be expected, its expression was also observed in the same loci following
levels are modified by adrenalectomy and glucocorticoid stimulation. While the authors were particularly cautious
administration as well as by acute or chronic stress in constantly referring to CRF-like immunoreactivity
exposure. CRF-41 has been found in hypophysial portal (CRF-LIR) in all these studies in case other peptides
vessels together with vasopressin but not urocortin. Simi- knownorunknownmightberecognizedbytheantibody,
larly, mice deficient in urocortin I (knock-out) have these results are remarkable as they show CRF to be
normal levels of plasma ACTH and show a normal directly involved in the actual functioning of cerebellar
responsetostressentirelymediatedbyCRF(Bale&Vale neuronal systems. Parallel results have been reported for
2004). CRF neurons in hippocampal and cortical regions of the
ACRF-bindingprotein(37kilodaltons)(CRF-BP)has immature rat brain (Brunson et al. 2002) following stress
been characterized and is expressed in the brain, the exposure.
pituitaryandperipheraltissues(liver,kidney,spleen).The In an extensive series of experiments with brain
ligandsiteforthebindingproteinisdifferentfromthatof slices maintained in vitro, Liu et al. (2004) have recently
themainreceptor,CRF-R1forCRF.CRF-BPmayplay demonstrated an even more refined implication of CRF
a role in limiting the distribution or duration of action of and urocortin I in modulating excitatory glutamatergic
CRF. transmission in two limbic nuclei (the central nucleus of
Urocortin I is expressed in neurons of the Edinger- theamygdalaandthelateralseptummediolateralnucleus),
Westphal nucleus of the midbrain, the gastrointestinal with evidence of the functional demonstration of the two
tract, testes, cardiac myocytes, thymus, skin, and receptorsCRF-R1andCRF-R2onneuronsofthesetwo
spleen, while urocortin II and III are also found in the nucleiwhichareknownalsotoexpressthepeptidesorto
hypothalamus. beinnervatedbyfiberscontainingthepeptides.Activation
Recent and still ongoing studies on the CRF receptors ofoneortheotherreceptorbythepeptideligandleadsto
and ligand have shown that CRF has a tenfold higher opposite effects on overall glutamatergic transmission in
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