Protein kinase C activators stimulate betaendorphin secretion from hypothalamic cells

Brain Research Bulletin,Vol. 29, pp. 553-557, 1992 Printed in the USA. All rights reserved.

0361-9230/92 $5.00 + .oo Copyright 0 I992 Pergamon Press Ltd.

Protein Kinase C Activators Stimulate BetaEndorphin Secretion From Hypothalamic Cells LEONARD

P. KAPCALA,’ CHING-FENG

WENG

AND HORNG-HENG

JUANG2

department of Medicine, Division O~Endocr~no~o~y, Universityof Maryland, School o~~edici~e and hospital, ~a~t~~ore,MB ZI20f Received 29 January 1992; Accepted 26 April 1992 KAPCALA, L. P., C.-F. WENG AND H.-H. JUANG. Protein kinase C activators stir&ate hypofhala~~c ceiis.BRAIN RES BULL 29f5) 553-557,

ber~-endor~h~~ secrelion from

I992.-Relatively little is known about the regulation of secretion of

h~othalamic ~~-endo~hin, the potent opioid that is believed to play a variety of physiological roles in brain, Previous work has shown that arginine vasopressin (AVP), which acts in brain primarily via activation of the phosphoinositol (PI) second messenger system, stimulates secretion of hypothalamic beta-endorphin. To test the hypothesis that activators of protein kinase C (PKC), which is activated following PI hydrolysis, stimulates secretion of beta-endorphins from hypothalamus, we studied the separate effects of stimulators of PKC including phorbol ester 12-myristate- 13-acetate (PMA) and I -oleolyl-2-acetyl glycerol (OAG - a diacyl glycerol analogue) on secretion of immunoreactive (IR-) beta-endorphin (measured by RIA) from dissociated fetal rat hy~thalamic cell cultures. We also studied AVP and angiotensin II (Ang II). hypothalamic peptides which activate the PI second messenger pathway, and intemctions of PMA and forskolin (FSK), an activator of the cyclic AMP/protein kinase A (PKA) pathway. PMA, OAG, AVP, and Ang Ii stimulated IR-beta-endorphin secretion. The stimulatory effect of both PMA and FSK on IR-beta-endorphin secretion was greater than that of PMA or FSK alone and was essentially additive. From these results we conclude that (a) hypothalamic beta-endorphin secretion can be stimulated by activating PKC: (b) AVP and Ang II are potential physiological regulators of hypothalamic beta-endorphin secretion; (c) coupling of PKC activation and stimulation of hypothalamic IR-beta-endorphin secretion which is presumed at maturity (adulthood) originates during early development (fetal life); (d) stimulating hypothalamic beta-endorphin secretion via PKC activation utilizes a distinct, different mechanism than stimulation of hypothalamic eta-endo~hin secretion via PKA activation. Beta-endorphin secretion Regulation Angiotensin II Arginine vasopressin

Hypothalamus

Protein kinase C

BETA-ENDORPHIN (&END) is an important opioid produced in pituitary, brain and several peripheral sites following processing of pro-opiomelanocortin (POMC)(25). Interest in P-END as an important neuroregulator in brain is supported by many studies suggesting that it is involved in regulating a variety of physiological events including central reproductive neuroendocrine function (via effects on gonadotropin-releasing hormone) (I l), homeostatic processes such as control of ventilation and blood pressure (21), and specific behaviors and stress responses(8,9). The major site of brain synthesis of POMC is ventral-lateral hypothalamus which contains highest concentrations of POMC peptides( 18). POMC is subsequently processed to @END and related peptides and other derivatives including adrenocorticotropin and cu-melanocyte stimulating hormone. These derivatives may undergo post-tmnslational modifications to enhance or diminish biological potency (25) and are dist~buted to specific brain regions via axoplasmic transport (6). Modulation of hypothalamic P-END secretion can increase or decrease its biological effects. Although there is limited in-

Second messenger

formation, the physiological reguIation of hypothalamic &END secretion is not well understood. A major stimulatory candidate for hypothalamic @-END secretion is corticotropin-releasing hormone (CRH), which is the major stimulator of synthesis and secretion of P-END and other POMC-related peptides in anterior pituitary and which is also a regulator of these peptides in intermediate lobe (20). In anterior pituitary, CRH and arginine vasopressin (AVP) stimulate POMC-related peptide secretion by activating the cyclic AMP (CAMP) and phosphatidyl inositol (PI) pathways, respectively (1). In stimulating the latter pathway, two second messengers, diacyl glycerol (DAG) and inositol triphosphate, facilitate signal transduction by stimulating protein kinase C (PKC) and mobilizing intracellular calcium, respectively. Several groups (5,6,15) using different in vitro models have demonstrated that CRH stimulates hypothalamic B-END secretion and we have recently demonstrated that activation of the CAMP/protein kinase A (PKA) pathway stimulates P-END secretion from fetal hypothalamic cells in culture ( 16). In addition,

’ Requests for reprints should be addressed to Leonard P. Kapcala, M.D., University of Maryland School of Medicine, Bressler Research Building, Room 8-O17,655 West Baltimore Street, Baltimore, MD 2 1201. * Current Address: Physiology Department, University of Maryland School of Dentistry, Baltimore, MD 2 1201.

553

KAPCALA,

554

AVP has been shown to stimulate hypothalamic P-END secretion (6.26). It has been suspected that activating the PI pathway in hypothalamus may be involved in stimulating hypothalamic pEND secretion (6) considering that stimulation of hypothalamic P-END by AVP (6) is believed to interact with VI type brain receptors (23) coupled to the PI pathway and that AVP stimulates pituitary via this pathway (1). However, there are no studies showing that activating steps in the PI pathway directly stimulates hypothalamic P-END secretion. Consequently, we tested the hypothesis that PKC activators stimulate hypothalamic P-END secretion and measured P-END responses to recognized stimulators of PKC and PI hydrolysis in dissociated fetal hypothalamic cells. Furthermore, previous work has shown that POMC-related peptide secretion can be stimulated from anterior pituitary by activating the PKA or PKC pathways via different mechanisms and that simultaneous stimulation of both pathways results in additive effects (1). Therefore, we also sought to determine whether a similar phenomenon occurred in hypothalamic cells with respect to immunoreactive (IR)-P-END secretion. METHOD

Muterids

Timed-pregnant Sprague-Dawley rats were housed under a 12L: 12D h cycle and fed ad libitum laboratory chow and water. Pregnant rats were anesthetized with pentobarbital and fetuses (day 17- 18 gestation) were removed by laparotomy. Brains were dissected under a stereo-microscope and diencephalon was isolated. Because hypothalamus is a major part of the diencephalon, cells dissociated from this region were termed hypothalamic cells. Briefly, tissues were dissociated using mechanical-enzymatic techniques previously described (14). Dissociated cells (0.250.5 X lo6 cells/cm2) were plated on 25 cm2 Corning flasks which had been coated with poly-D-lysine (10 F/ml). Medium consisting of a 1:1 mixture of Dulbecco’s Modified Eagles Medium and Ham’s Fi2 (GIBCO) containing 15 mM Hepes, 33 mM NaHC03, 10% heat inactivated fetal calf serum (GIBCO), penicillin (5 h/ml), streptomycin (5 h/ml), and Fungizone (0.5 F/ ml) was used initially for 1 day. Subsequently, medium was changed twice weekly and contained the same components except that chemically-defined supplements (4) consisting of 3 X lO-‘M selenium, 10v6M transfenin, 2 X lo-‘M progesterone, 104M putrescine, and 5 p/ml insulin were substituted for serum. A chemically-defined serum free medium was used to avoid the potentially complicating nature of serum whereby uncharacterized substances and/or concentrations of substances which can vary in different batches of sera can unknowingly influence cellular responses. Cultures, consisting of neurons and glia (3) were maintained in a humidified air/CO2 (95%:5%) incubator at +37”c. Experiments were conducted between 9- I3 days in culture because previous studies had shown cells contained significant quantities ( 12) of IR-P-END which can be secreted at this time. Medium was changed prior to each experiment and cells were incubated in Krebs-Ringer’s buffered solutions (KRB-G 118.5 mM NaCI, 5 mM KCl, 1.18 mM MgS04, 1.18 mM KH2POI, 2.5 mM CaCl,, 25 mM NaHC03, 5 mM dextrose, pH 7.35) containing 0.1% bovine serum albumin, lOaM ascorbic acid, and bacitracin (300 r/ml) and experimental substances. After an initial 20-min equilibrating incubation, cells underwent serial 20-min incubations utilizing 2 ml of control KRB-G solution or KRB-G containing 60 mM KCl, AVP (Peninsula), angiotensin II (Ang II) (Peninsula), phorbol ester 12-myristate- 13-acetate (PMA) (Sigma). I-oleolyl-2-acetyl glycerol (OAG) (Sigma), for-

WENG

AND

JUANG

skolin (FSK) (Sigma) or FSK plus PMA. Dimethyl sulfoxide was the diluent for PMA, OAG, and FSK and was also contained in respective controls. Cells were washed with control KRB-G solution after each experimental treatment. Radioimmunoassay (RIA) was performed using the H-7 flEND antisera (generously provided by Dr. George ChrousosNICHD/NIH, Bethesda, MD) which has previously been described (14). Sample volume was 500 p. The standard curve of the RIA also contained a similar volume of control KRB-G to minimize the possibility of nonspecific interference from media components. Samples below the limit of assay sensitivity (typically 2-4 fmol/tube) were assigned the limit of assay sensitivity to prevent potential overestimation of stimulated secretion. Intraand inter-assay variation were 8% and 2 l%, respectively. Statistical differences were determined by comparing the immediately preceding control incubation with the experimental incubation and also in some instances the subsequent epoch using the paired t test. Multiple comparisons were analyzed by analysis of variance (ANOVA) followed by Duncan’s multiple range test using p < 0.05 as the criterion for statistical significance. RESULTS

We employed a screening protocol to study whether PMAstimulated hypothalamic P-END secretion. Serial exposure of cells to increasing doses of PMA showed a statistically significant stimulation of P-END release with 10e6M PMA (Fig. l), a concentration shown to stimulate secretion of other neuropeptides (2,22) maximally. Increased IR-P-END release persisted through the next epoch but release returned to baseline in the following epoch. A depolarizing concentration (60 mM) of KC1 also stimulated P-END secretion. OAG ( 10m4M), an analogue of diacyl glycerol, which stimulates PKC. and AVP and Ang II ( 10e6M), hypothalamic peptides recognized to activate PI hydrolysis, were also studied. All of these substances stimulated IR-P-END release (Fig. 2). Although significant stimulation of IR-P-END secretion occurred during incubation with AVP and Ang II, the pattern of stimulation produced by OAG was different. IR-P-END secretion increased immediately during incubation with OAG but this increment was not statistically significant. In contrast, a statistically significant @ < 0.05) and greater increment occurred in the epoch immediately following removal of OAG. In the subsequent epoch (not shown), IR-@-END secretion was decreasing toward baseline. When cells were simultaneously exposed to both forskolin (FSK-stimulator of adenylate cyclase/PKA pathway) and PMA. each ofwhich individually stimulated IR-P-END secretion from hypothalamic cells, stimulation of IR-P-END secretion was greater (Fig. 3) than that produced by either substance alone. Considering the responses of each individual flask with regard to its respective pretreatment control, ratios of treatment and posttreatment epochs relative to control showed that responses of cells exposed to both PMA and FSK were greater than those of cells exposed to PMA or FSK alone (Table 1). Recognizing the variability of response of individual flasks, these responses essentially suggested an additive stimulation. We have observed a similar additive pattern of responses in other experiments. DISCUSSION

Our studies indicate that exposure of hypothalamic cells to PMA and OAG, agents recognized to stimulate PKC (I), results in stimulation of IR-P-END secretion and that the stimulatory effect of these activators can be sustained beyond the time of exposure to cells. AVP and Ang II also stimulated IR-P-END secretion. AVP has been shown to stimulate IR-P-END secretion from adult hy-

BETA-ENDORPHIN

SECRETION

555

10%

FW

6or+l KG

1

180

2OO(min)

FIG. 1. Effect of serial PMA stimulation on IR-P-END secretion from fetal hypothalamic cells on culture day 13. Following a 20-min equilibration, cells were serially incubated (20 mitt/epoch) in 2 ml control KRB-G solution (open bars) or KRB-G solution containing 1W8, 10dM PMA or 60 mM KC1 (shaded bars). Each bar represents mean f SEM for 6 replicate flasks. Statistical difference by the paired t test is represented by ‘p < 0.05 using the immediately preceding epoch as the control.

pothalamic explants (5,26) and is believed to be an endogenous stimulator of hypothalamic B-END secretion. However, to our knowledge, Ang II stimulation of hypothalamic &END secretion has not previously been reported. These results further support the view of qualitatively similar regulation of POMC-related peptide secretion from anterior pituitary (1) and hypothalamus by endog-

100

]

1

n

lo4

enous peptides including CRH, AVP, and Ang II. Although we have not necessarily demonstrated that ANG II or AVP stimulates POMC neurons directly, immunocytochemical identification of neuronal fibers containing Ang II (17) and AVP (7) throughout hypothalamus particularly near regions containing POMC penkarya support their potential role as physiological regulators of POMC-

Control

M cru;

1Od II AVP

10-%ANG

II

FIG. 2. Effect of OAG, AVP, and Ang II on IR-B-END secretion from fetal hypothalamic cells on culture day 13. Following a 20-min equilibration, cells were serially incubated (20 min/epoch) for two more successive control epochs in 2 mls KRB-G solution. Results (mean f SEM for 5 replicate flasks) are presented for epochs immediately preceding, during, and immediately following incubation with experimental substance. Difference by the paired t test is represented by ‘p < 0.05 or “*p < 0.01 using the control epoch immediately preceding the experimental substance as the specific control (shown above).

556

KAPCALA,

related peptide secretion. Activation of the PI pathway and specifically stimulation of PKC has been shown to participate in the stimulation of POMC-related secretion from anterior pituitary and is presumably at least one of the mechanisms by which AVP and Ang II stimulate POMC peptide secretion from anterior pituitary (1). In contrast, CRH activates the cAMP/PKA pathway in pituitary and brain ( I .6). Direct studies showing that activation of the PI pathway is involved in stimulating POMC-related peptide secretion from hypothalamus are not available. Our work is the first to show that acute activation of PKC stimulates IR-P-END secretion from hypothalamus. Sarkar et. al (24) reported that incubation of hypothalamic neurons with DAG for 4 h resulted in increased IR-&END secretion. However. the design of their experiment using a long incubation (4 h) did not allow one to distinguish whether secretion was directly stimulated by activating the PKC pathway or perhaps indirectly stimulated by activating the PKC pathway which could have increased POMC synthesis, P-END cellular content and ultimately the releasable pool of P-END that could be secreted. In contrast, our stimulation, using relatively short incubation periods (20 min) during which it was unlikely that significant alterations in POMC synthesis occurred, indicates that brief activation of the PKC pathway stimulates IR-/%END secretion from hypothalamus as it does from anterior pituitary (27). Furthermore, simultaneous activation of the PKA and PKC pathways by FSK plus PMA produced greater stimulator-y effects (than PMA or FSK alone) on IR-P-END from hypothalamic cells as has previously been described in anterior pituitary (I ). These responses were essentially additive and suggest that different mechanisms are involved in the stimulation produced by simultaneously activating PKA and PKC pathways. Considering difficulties in studying hypothalamic secretion of &END in vivo, various in vitro hypothalamic models consisting

m m

TABLE

WENG

I

EFFECT OF PMA, FSK, AND PMA PLUS FSK ON IR+END FROM FETAL HYPOTHALAMIC CELLS

Post-z;e;ent

(~)

RELEASE

PMA

FSK

199 & 26

185 z? 16

304 + 44+

I51 +- 54

119&42

442 +- 52**

PMA + FSK

Results depict ratios (mean + SEM) of IR-/3-END release during experiment depicted in Fig. 3. * = PMA + FSK > PMA or FSK, (p c 0.05). ** = PMA + FSK > PMA or FSK, (p < 0.01). Statistical analysis by ANOVA and Duncan’s multiple range test. of slices (6). pieces (5). or whole blocks (15)of hypothalami and dissociated neonatal ( 19) and fetal (I 3.14) hypothalamic cells have been used. Each model exhibits distinct advantages and disadvantages. Furthermore, other laboratories have used dissociated hypothalamic or cortical cells to stimulate secretion of several neuropeptides, CRH (IO). somatostatin ( 10,22), and growth-hormone releasing hormone (2) by a PKC stimulator (phorbol ester). However. we ( 14) have recently pointed out a limitation of studying IRP-END secretion from dissociated hypothalamic cell cultures by showing that CRH which stimulates P-END secretion from adult ( 15) and fetal hypothalamic explants (14) was unable to stimulate secretion in cultured cells (despite our demonstration of CRH receptors and CRH-induced stimulation of CAMP accumulation in fetal hypothalamic cell cultures). We concluded that this inability to respond was related to culture conditions and/or the absence of specific cytoarchitectural relationships which occur with intact hypothalamic tissues (I 4). In view of this, we emphasize a caveat

pre-treatment treatment post-treatment

TT

lOaM FSK

AND JUANG

lO-bMA

10lbM FSK +10 Y PYA

FIG. 3. Effect of stimulating concentration of PMA, FSK. and PMA plus FSK on IR+END secretion from fetal hypothalamic cells on culture day 9. Experimental design is similar to that previously described in Fig. 2. Results depict mean t SEM IR-P-END secretion of 4 replicate flasks in epoch (20 min) preceding, during, and after treatment with experimental substance. Treatment in all cases produced a statistically significant @ < 0.05) increment (treatment vs. pretreatment by paired I test) in IR-P-END secretion.

BETA-~N~RPHIN

SECRETION

557

which should be kept in mind with this model, demonstrating alterations in secretion may suggest a substance as a potential physiological regulator, but an absent response to a specific substance does not necessarily exclude that substance as a potential physiological regulator. In contrast, our results stimulating IR-B-END secretion from hypothalamic cells in culture by activating signal transduction at a receptor level (AVP, Ang II) and at a p&receptor level via stimulation of PKA and PKC pathways clearly demonstrates that this model can he useful for learning about specific mechanisms regulating hypothalamic @-END secretion. These resuits demons~ting stimulation of h~~~~~ic &END secretion from fatally derived cells by AVP and postreceptor activation of PKA and PKC pathways also indicate that signal transduction

pathways suspected of participating in the regulation of hypothalamic @-END secretion in adulthood (6) function in a similar way in fetal life.

In summary, we have shown that direct stimulators of PKC as well as AVP and Ang II, putative activators of the PI pathway

in brain, stimulate IR-@END secretion from dissociated fetal hypothalamic ceils. These results also show that activation of this pathway, which is presumably coupled to stimulation of hypothalamic P-END secretion at mature stages (adulthood) begins at early stages of development (fetal life). Furthermore, these observations demonstrate that studying dissociated fetal hypothalamic cell cultures can provide insight into regulatory relationships involved in hypothalamic P-END secretion and suggest that activation of the PKC pathway stimulates hypothalamic fiEND secretion by a different mechanism than that of stimulating @-END secretion by activating the PKA pathway.

We thank Connie Ball for her secretarial assistance. This work was supported by NIH grant NS-233 17 from NINDS. Part of this work was presented at the 20th Annual Neuroscience Meeting in November 1990 in St. Louis, MO.

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