.Brain Research 841 1999 114122www.elsevier.comrlocaterbres
Effects of D9-THC on VIP-induced prolactin secretion in anterior pituitarycultures: evidence for the presence of functional cannabinoid CB receptors1
in pituitary cellsFernando Rodrguez de Fonseca a,), Tibor Wenger b, Miguel Navarro a, Laura L. Murphy c
a Instituto Complutense de Drogodependencias, Departamento de Psicobiologa, Facultad de Psicologa, Uniersidad Complutense, 28223 Madrid, Spain b Second department of Anatomy, Semmelweis Uniersity School of Medicine, Budapest, Hungary
c Department of Physiology, School of Medicine, Southern Illinois Uniersity, Carbondale, IL, USAAccepted 29 June 1999
.Peripheral administration of cannabinoid CB receptor agonists to laboratory rats induce a brief rise in plasma prolactin PRL levels1followed by a prolonged decrease in PRL secretion from the pituitary. While the inhibitory component of this biphasic response dependson the cannabinoid-induced activation of dopamine release from hypothalamic terminals located in the median eminence, theneurobiological mechanisms underlying the activation phase of PRL release remains to be explained. In the present study the possible
9 .direct effect of the cannabinoid receptor agonist D -Tetrahydrocannabinol THC on prolactin secretion and cAMP accumulation was . .examined in anterior pituitary cultures. THC 0.1 and 1 mM increased cAMP levels, and induced PRL release 1 and 10 m . THC did not
.affect vasoactive intestinal peptide VIP, 0.5 mM induced cAMP accumulation in pituitary cultures, showing additive effects at THC 1mM concentration. However, THC did prevent VIP-dependent increases in prolactin secretion. These results indicate that THC, through adirect pituitary action, activates both the synthesis of cAMP and PRL release and interferes with intracellular mechanisms involved inPRL secretion by VIP. These actions could be mediated through cannabinoid CB receptors which were found to be present in anterior1pituitary cells, including lactotrophs, as revealed by immunocytochemistry with a specific polyclonal antibody raised against the CB1receptor protein. q 1999 Elsevier Science B.V. All rights reserved.
.Keywords: Rat; Pituitary; Cannabinoid; Vasoactive intestinal peptide VIP ; Prolactin; cAMP; Lactotroph; CB receptor1
9The acute administration of natural D -tetrahydro-. .cannabinol, THC or synthetic HU-210, CP 55,940
w xcannabinoid CB receptor 7 agonists to laboratory ro-1dents produce a well characterized set of effects in anterior
w xpituitary hormone secretion 4,11,23,21 . These actionsinclude decreases in the release of gonadotropins, thyroid-stimulating hormone and growth hormone and a sharp rise
w xin adrenocorticotropic hormone 21,24,27 . The administra-tion of anandamide, one of the endogenous ligands for
w x w xCB receptors 8 results in similar effects 11,41,42 . Most1of these anterior pituitary effects of cannabinoids are pro-
) Corresponding author. Fax: q 34-91-3943189; E-mail:firstname.lastname@example.org
duced by cannabinoid-induced changes in the activity ofhypothalamic neurotransmitters controlling pituitary hor-
w xmone synthesis and release 17,18,21,28,35,40 . Moreover,CB receptors were reported to be present in several1hypothalamic nuclei implicated in anterior pituitary regula-tion such as the paraventricular and arcuate nucleiw x 14,23,37 . These findings indicate a central hypo-
.thalamic site of action for cannabinoids to elicit its neu-roendocrine effects, although a direct action on pituitary
w xcells cannot be excluded 25 .Prolactin secretion has been proposed as a model for
studying the direct effects of cannabimimetics on thew xpituitary 25 . The stimulation of cannabinoid CB recep-1
tors induces a characteristic biphasic response on plasma .prolactin PRL secretion in laboratory rodents: a brief
w xincrease in plasma PRL levels 11,21 followed by aw xprolonged inhibition of its release 28,38 . While the in-
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. .PII: S0006-8993 99 01809-0
( )F. Rodrguez de Fonseca et al.rBrain Research 841 1999 114122 115
hibitory actions of cannabimimetics on PRL secretion hasbeen explained on the basis of the activation of the release
of dopamine the main inhibitory modulator of PRL re-w x. w xlease 2 into the portal vasculature 4,12,17,38 , the acti-
vation component of this response lacks of a consistentexplanation. Hypothetically, PRL activation response tocannabimimetics could be mediated by a direct pituitaryaction. Although it has been proposed that cannabinoidsmay interfere with the steroid hormone signaling mecha-
w xnisms within the pituitary 25,26 , as it does in the hip-w xpocampus 9 , a more plausible hypothesis is a potential
cannabinoid CB receptor-mediated effect. However, the1presence of CB receptors in anterior pituitary cells re-1mains to be conclusively determined, although sparse CB1receptors have been described throughout the anterior pitu-
w xitary using low resolution autoradiography techniques 14 .In addition, preliminary findings indicate that anandamidecan increase PRL secretion in anterior pituitary cell cul-
w xtures 41 . This effect can be blocked by the CB receptor1antagonist SR 141716A. Taking in account these prelimi-
.nary reports, the aims of the present study were: 1 toinvestigate the effects of the cannabinoid receptor agonist,THC, on PRL release from anterior pituitary cells in
.culture; 2 to evaluate the effects of THC on the actions .of vasoactive intestinal peptide VIP , a neuromodulator
that acts in the lactotroph cell to stimulate PRL release,through receptors coupled to signal transduction mecha-nisms similar to that described for the CB receptor; and1
.lastly, 3 to identify the presence of CB receptors in1pituitary cells by immunocytochemistry using specific anti-bodies raised against the CB receptor.1
2. Materials and methods
In vivo experiments were carried out with adult male .SpragueDawley rats 300350 g . In vitro studies were
carried out with pituitary cells obtained from randomlycycling retired breeder female SpragueDawley rats. All
animals were housed under controlled photoperiod lights. .on: 08002000 h and temperature 23"18C . They had
.free access to standard food Panlab, Barcelona and water.
THC was obtained through the National Institute on .Drug Abuse NIDA, Bethesda, MD . It was dissolved in
sesame oil as vehicle, and made up daily to the appropriateconcentration to be administrated orally in 0.1 ml. For invitro studies, THC was dissolved in culture medium con-taining 0.001% ethanol. VIP was obtained through Penin-sula Labs. and dissolved to the appropriate concentrationin culture medium.
2.3. Anterior pituitary cell culture
Pituitary glands from retired breeder female rats weredissected free of the posterior lobe, and the anterior portionof the gland was minced under sterile conditions and
placed in DMEM-F12 media containing antibiotics 10,000Urml penicillin, 10 mgrml streptomycin and 25 mgrml
.amphotericin maintained at 48C. Cell dispersion wasachieved by incubation of tissue with media containingantibiotics, 0.1% of bovine serum albumin and trypsin .1:250 for 120 min in a Dubnoff shaking water bath at378C under a steady stream of 95% oxygen, 5% carbondioxide. Pituitary fragments were gently agitated by re-peated aspiration with a siliconized Pasteur pipet every 45min during dispersion. Dispersed cells were then cen-trifuged at 200=g for 15 min and the pellet was resus-pended at a concentration of approximately 2=105cellsrml in DMEM-F12 containing antibiotics and 5%fetal calf serum and 10% horse serum prior to plating. The
.cells were plated in 35-mm culture dishes Corning previ-ously coated with poly-L-lysine and allowed to incubatefor 72 h in a water-jacketed tissue culture incubator main-tained at 378C under 95% room air and 5% carbon dioxideat saturated humidity. Culture media was replaced every36 h.
2.4. Prolactin, THC and cAMP determinations
PRL levels in plasma and culture media were deter-mined using a double antibody RIA kit supplied by the
.NIADDK Bethesda, MD, USA . Data were expressed asngrml of reference preparation rPRL-RP3. Plasma levelsof THC were measured using a radioimmunoanalysis kitsupplied by NIDA. Data were expressed as ngrml andconverted to mM concentrations. Details on the methods
w xhave been published elsewhere 12,38 . Individual ethanolextracted cAMP levels were measured using a commer-cially available specific double-antibody RIA prepared by
.Advanced Magnetics Cambridge, MA .
.Male SpragueDawley rats Charles River, Hungaryweighing 240260 g were used. Animals were perfused
with fixative 4% paraformaldehyde and 0.04% picric acidin 0.1 M, pH 7.4, phosphate buffer following deep anes-
.thesia sodium pentobarbiturate, 60 mgrkg , using previ-w xously described methods 42 . After perfusion, the pitu-
itaries were quickly removed and immersed for postfixa-tion in the same solution for 1 h. The pituitaries were cuton a cryostat or embedded in paraplast and subsequentlysectioned. In both cases, adjacent fine sections of pituitarygland were collected to facilitate the identification of thelocalisation of cannabinoid receptors and PRL immuno-
( )F. Rodrguez de Fonseca et al.rBrain Research 841 1999 114122116
reactivity in the same zone or cells within the pituitary, tobe able to compare the distribution of the immunopositivematerial. No differences were registered between paraplastand cryostat sections. The sections were cut 56 mm andindividually mounted on slides. After deparaffination theadjacent sections were washed in buffer and incubated
with anti-rat PRL antibodies Dilution 1:2000, kindly pre-.pared by Dr. G.M. Nagy , or anti-CB or anti-CB poly-1 2
.clonal antibodies Dilution 1:5000 kindly provided byprofessor Allyn Howlett Department of Pharmacology, St.
.Louis University, St. Louis, MO, USA . CB antibody was1raised against the CB amino terminus 114, modified by1a cysteine at the carboxyl terminus to facilitate coupling toprotein carrier. CB antibody was raised against the 1162
w xterminus 16 . The specificity of the CB and CB antibod-1 2w xies was determined as described 16 . All primary antibody
dilutions contained 0.2% Triton X-100. The incubationwas done at room temperature for 24 h, followed by anadditional 24-h incubation at 48C. Biotin-conjugated goatanti rabbit IgG was used as second antibody in 1:2500dilution for 2 h, followed by peroxidase-conjugated
streptavidin Both purchased from Jackson Immunore-.search, USA . The second antibody and the streptavidin
conjugate were diluted 1:2000 for at room temperature for1 h. The reaction was developed with DAB-nickel-glu-cose-oxidase method.
The observations and photography of the slides wereperformed using a Zeiss Axiophot microscope.
. . .Fig. 1. Effect of THC 5 mgrkg, in sesame oil, p.o. on plasma prolactin levels upper panel and THC concentrations lower panel 30, 60 and 180 minafter its administration. Values are means"S.E.M. of at least seven independent measurements. U : P-0.05 vs. control value.
( )F. Rodrguez de Fonseca et al.rBrain Research 841 1999 114122 117
2.6. Experimental designs
In a first experiment, in order to establish the temporaleffects of THC on PRL secretion and the working concen-tration for the subsequent in vitro studies, male animals
9 .were treated orally with D -THC 5 mgrkg or vehicle.Animals were sacrificed for collecting trunk blood andplasma samples obtained at 30, 60 and 180 min aftertreatment, for measuring plasma levels of PRL and THC.In a second experiment, a time-course of VIP effects onPRL secretion and cAMP accumulation was performed inorder to optimize the conditions for the VIP-THC interac-tion study. To this end, after the initial 72 h of incubation,
the plating medium was aspirated and each dish washedtwice with serum-free DMEM-F12 before the addition of
.medium containing VIP 500 nM . Control dishes weretreated with media only. The cells were incubated for 5, 20or 60 min. Medium was harvested for PRL determinationand cells were extracted with ice-cold absolute ethanol andstored at y208C for cAMP measurements. Based on thisexperiment, an incubation time of 15 min was selected forfurther experiments. In a third experiment, the interactionof THC and VIP on PRL secretion and cAMP accumula-tion was analyzed in pituitary cultures. After the initial 72h of incubation, the plating medium was aspirated andeach dish washed twice with serum-free DMEM-F12 be-
. . .Fig. 2. Time-course of the effects of VIP 500 nM on cAMP accumulation upper panel and PRL secretion lower panel at 5, 20 and 60 min afterexposure to the neuropeptide. Data are means"S.E.M. of at least four independent measurements. U : P-0.05 vs. control group.
( )F. Rodrguez de Fonseca et al.rBrain Research 841 1999 114122118
fore the addition of medium containing THC 0.1, 1 or 10. .mM or vehicle medium containing 0.001% ethanol .
After 15 min incubation medium was aspirated and savedfor PRL assay. Fresh media containing vehicle, THC or
.THCqVIP 500 nM was added and incubated for an-other 15 min. Medium was harvested for PRL measure-ments and cells were extracted with ethanol for cAMPdeterminations.
.Data were assessed by analysis of variance ANOVA .Following a significant F value, post hoc analysis New-
.manKeuls test was performed for assessing specificgroup comparisons. Calculations were performed using thestatistical package BMDP.
The treatment of the rats with an acute dose of THCproduced time-dependent biphasic effects on PRL secre-
tion; a short stimulation of PRL release at 30 min post-treatment and a significant decrease at 60 min P-0.05,
.NewmanKeuls test . Peak levels of THC were detectedin plasma samples from the 30 min treatment group rang-
.ing from 0.23 to 0.54 mM , which were decreased at 60 .and 180 min Fig. 1 . In vitro experiments with pituitary . .cell cultures Fig. 2 revealed that VIP 500 nM increased
cell contents of cAMP and PRL secretion into the media at .5 and 20 min P-0.01, NewmanKeuls test . When
pituitary cells were incubated in the presence of THC 0.1.and 1 mM , cAMP levels increased significantly over basal
conditions, whereas PRL secretion was enhanced at 1 and10 mM THC concentrations. VIP significantly stimulatedcAMP production when administered alone or together
.with THC, showing additive effects with THC Fig. 3 .Thus, the percentage of increase in cAMP by VIP overbasal levels was not different in control and THC-treatedcells, despite the higher levels of cAMP induced by thecannabinoid. However, when administered concomitantlywith THC, VIP did not stimulate PRL release. The per-centage increase in PRL release was significantly greaterin controls than in THC-treated cells. The higher the THC
.Fig. 3. Upper panels: effect of THC treatment on the cAMP response to basal conditions and VIP 5...