Stimulated secretion of pro-opiomelanocortin-related peptides in hypothalamic cells

Peptides, Voi. 6, Suppl. 1, pp. 1--4,1985.©AnkhoInternational Inc. Printed in the U.S.A.

0196-9781/85$3.00 + .00

Stimulated Secretion of Pro-Opiomelanocortin-Related Peptides in Hypothalamic Cells 1 L E O N A R D P. K A P C A L A , 2 T H O M A S A. G R I F F I N A N D T A M Y R A M. B I R K

D e p a r t m e n t o f Medicine, Case Western R e s e r v e University, School o f Medicine, Cleveland, O H 44106

KAPCALA, L. P., T. A. GRIFFIN AND T. M. BIRK. Stimulated secretion ofpro-opiomelanocortin-related peptides in hypothalamic cells. PEPTIDES 6: Suppl. 1, 1--4, 1985.--Although it has been suggested pro-opiomelanocortin (POMC) related peptides in brain may be neurotransmitters or neuromodulators, little is known about their secretion from neurons because it is difficult to study neurosecretion with an in vivo model. To demonstrate the possibility that POMC peptides may be neuroregulators which can be secreted in response to specific stimuli, we studied the secretion of immunoreactive (IR-) adrenocorticotropin (ACTH) and IR-/3-endorphin from dissociated hypothalamic cells during potassium-induced depolarization. Significant increments (p <0.025) in secretion of IR-ACTH (267%) and IR-fl-endorphin (88--172%) over basal secretion were stimulated by 60 mM KCI in the presence of calcium. Conclusion: Stimulated secretion of POMC peptides from hypothalamic cells by potassium and calcium follows classical neurosecretory mechanisms and suggests these neuropeptides could be neuroregulators in brain. Pro-opiomelanocortin

Adrenocorticotropin

/3-Endorphin

T H E widespread brain distribution of immunoreactive (IR-) adrenocorticotropin (ACTH) [7] and IR-/3-endorphin [15], peptides derived from processing of pro-opiomelanocortin (POMC), and their multiple behavioral, biochemical and homeostatic effects produced in the CNS [3] suggest potential roles as neuromodulators or neurotransmitters. Nevertheless, relatively little is known about the regulation of secretion of POMC peptides from neurons because it is difficult to study secretion of brain peptides with an in vivo model. Isolated reports using in vitro models have demonstrated that release of IR-/3-endorphin [4, 5, 10, 17] and IRa-melanotropin (MSH) [18] can be stimulated from hypothalamic fragments or synaptosomes by potassium-induced depolarization. However, the stimulated release of IR-ACTH has not been reported. Increasing numbers of reports have demonstrated the utility of studying secretion of a neuropeptide, somatostatin, with dissociated monolayer brain cell cultures [11-14]. This relatively simple model suggests several advantages. These include tissue homogeneity, direct secretion into medium, immediate contact between cells and nutrients, gases, and regulatory substances, and removal of the regulatory influences one brain region might have on another region. Nevertheless, monolayer brain cell cultures have only rarely been employed for studying secretion of POMC peptides in brain [2,8]. To demonstrate the possibility the POMC peptides

Hypothalamus

Stimulated secretion

may be neuroregulators capable of being secreted from neurons in response to specific stimuli, and to demonstrate the utility of studying their regulation of secretion in dissociated brain cell cultures, we studied the secretion of POMC peptides from dissociated hypothalamic ceils during potassium-induced depolarization. METHOD

Cell Cultures Timed pregnant Sprague-Dawley rats (Harlan SpragueDawley Inc., Madison, WI) were housed in our laboratory using a 12-hr light, 12-hr dark lighting schedule and were fed ad lib with water and rat lab chow. Fetuses were removed on gestational day 17 from mothers which had been deeply anesthesized with pentobarbital (36 mg). Fetal heads were transsected at the supranrbital margin, yielding the brain, which was dissected under sterile conditions using a stereomicroscope. The diencephalon which is primarily composed of the hypothalamus was isolated and placed in iced Hank's Balance Salt Solution. Cells were dissociated using mechanical-enzymatic techniques as described by Vaccaro and Messer [ 16] and modified by Robbins et al. [ 14]. Cell suspensions were counted with a Coulter counter and plated at 10 and 20x 10~ cells/25 cm ¢ tissue culture flask which had been previously coated with poly-L-lysine (10

'Presented at the Fifth Annual Winter Neuropeptide Conference, January, 1984. 2Requests for reprints should be addressed to Leonard P. Kapcala, M. D., Endocrinology Division, Cleveland Metropolitan General Hospital, 3395 Scranton Road, Cleveland, OH 44109.

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FIG. 1. Effects of potassium, calcium and EGTA on IR-ACTH (femtomoles/dish) released from hypothalamic cells (2{)x 106 cells/dish). Studies were performed on day 4. Bars represent the mean (-SEM) of IR-ACTH released from 3 dishes. Bars without SEM depict replicates that were at or below the limit of RIA sensitivity, which is illustrated by an arrow. KRB-G containing the specified concentrations of K ÷, Ca ÷+ and EGTA was the incubation medium, p values for the differences between the means of each control and experimental epoch were calculated using the paired Student's t-test and are illustrated for each pair when the difference was statistically significant (p<0.05).

/zg/ml). Minimal Essential Medium (MEM) (GIBCO) containing penicillin (10,000 units/ml), streptomycin (10,000 /zg/ml) and 10% synthetic " s e r u m , " based on the serum free medium developed by Bottenstein et al. [1] and modified by R. Robbins (personal communication), was added to cells. A chemically defined supplement was used because horse and fetal calf serum (GIBCO) were found to contain approximately 3000 and 250 pg/ml of IR-ACTH respectively and also significant quantities of IR-/3-endorphin. The synthetic " s e r u m " consisted of selenium (3 x 10-6 M), corticosterone (3x10 -8 M), 17-fl-estradiol (10 -6 M), transferrin (10 -9 M), L-tri-iodothyronine (3x 10-1° M), insulin (10 -6 M), and putrescine (10 -4 M). This chemical supplement has been shown to facilitate the development of morphological differentiation (cellular attachment to the dish, aggregation of cells, development of various cell types, and neurite formation) as in cultures supplemented with serum and also production of somatostatin by dissociated brain cells (personal communication, R. Robbins). Similar morphological differentiation has also been observed in our cultures. Media were changed at 1-3 day intervals and cultures were maintained in a waterjacketed, air-CO2 (95%:5%) incubator at 100% humidity and 37°C. Radioimmunoassays Radioimmunoassays (RIAs) were performed essentially as previously described [6]. The ACTH antisera [6], raised against synthetic ACTHl_24 which had been coupled to bovine serum albumin is directed toward the mid-portion of ACTH1-39 and does not show significant molar immunoreactivity (<0.01%) with /3-hendorphin, /3-hLPH, oCRF, or c~-MSH. The/3-endorphin antisera (generously provided by Dr. G. Mueller) was raised against synthetic/3-hendorphin

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FIG. 2. Effects of potassium, calcium and EGTA on IR-/3-endorphin (femtomoles/dish) released from hypothalamic cells plated at 10x 106 cells/dish (n=5) and 20x106 cells/dish (n=3). Studies were performed on day 4. Bars represent the mean (-SEM) of IR-/3endorphin released. Bars without SEM depict replicates that were at or below the limit of RIA sensitivity, which is illustrated by arrows. KRB-G containing the specificied concentration of K +, Ca ++ and EGTA was the incubation medium. Statistical analysis was performed as specified in Fig. 1. Significant differences are illustrated.

coupled to thyroglobulin [9] and shows equimolar reactivity with B-hendorphin, purified /3-hLPH, /3-LPH61-a7 (8-endorphin), N-acetyl-/3-endorphin, N-acetyl ~-endorphin, and no significant reactivity with ACTHl_39, a - M S H , or oCRF. Intra/inter-assay variations in the A C T H and /3-endorphin RIAs were approximately 5%/20% and 7%/18% respectively. Typical sensitivity in the ACTH and /3-endorphin assays were approximately 1.5 and 5 fm per tube respectively. A volume of 500 /zl was used for samples and standards to maximize RIA sensitivity per dish. To mediate against the possibility of interference of salts in the RIAs, the standards for each RIA were diluted with a control solution of Krebs-Ringer buffer which had not been exposed to cells. The various concentrations of ions and E G T A in control KRB-G solutions did not alter specific RIA binding. Secretion Experiments Potassium stimulated depolarization studies were performed on day 4 in the following manner. After decanting the original media, 4 ml of Krebs-Ringer buffered solutions (KRB-G: 118.5 mM NaC1, 1.18mM MgSO4, 1.18 mM KH2PO4, 25 mM NaHCO3, 5 mM dextrose---pH 7.35), containing various concentrations of KC1, MnCI2, CaCI2, and EGTA, were serially added to flasks. The first equilibrating ePoch was 30 minutes and thereafter solutions were changed at 15 minute intervals with 2 control epochs preceding each experimental epoch. Epochs without CaCI2 (2.5 mM) contained MnC12 (2.5 mM) as a substitute divalent cation. When depolarization was induced with 60 mM KC1, NaC1 was lowered to 63 mM to maintain isomolarity. Initially, 1 mM E G T A was used to chelate calcium, but subsequent studies used 2.5 mM E G T A which was equimolar to calcium. KRB-G solutions were decanted from each flask at the end

H Y P O T H A L A M I C S E C R E T I O N O F POMC PEPTIDES of each epoch, placed in a boiling water bath for 10 minutes and stored frozen (-20°C) until assay. Replicates from all epochs were assayed directly in either the same ACTH or /3-endorphin RIA. Immunoreactive quantities were converted to molar equivalents (molecular weight ACTH1_3a---4500 daltons; /3-endorphin--3500 daltons), which were then expressed in terms of the number of cells plated. Statistical Analysis

Experimental replicates in the release experiments were compared to the appropriate control, the immediately preceding control epoch in the potassium depolarization studies. Data were analyzed using the paired Student's t-test. To prevent overestimating a stimulated increase in peptide release, replicates from release experiments showing values below the limit of sensitivity in the RIAs were arbitrarily assigned the value at this limit. RESULTS Potassium stimulated depolarization without calcium did not induce release o f either IR-ACTH or IR-/3-endorphin (Figs. 1,2). In contrast, potassium stimulated depolarization in the presence of calcium did produce significant increments in secretion of POMC peptides (Figs. 1, 2). When twice as many cells were plated and subsequently stimulated with 60 mM postassium in the presence of calcium, basal release was similar but stimulated release increased proportionately to the increased number of cells plated (Fig. 2). A lesser but significant increment in secretion of IR-ACTH was also produced by 60 mM KCI, and 2.5 mM calcium in the presence of 1 mM E G T A (Fig. 1). However, when the concentration of E G T A was made equimolar (2.5 mM) to calcium, secretion of IR-/3-endorphin was not stimulated (Fig. 2). Thus IR-ACTH and IR-fl-endorphin could be secreted from hypothalamic cells by the classical stimulus, potassiuminduced depolarization. Furthermore, the calcium dependent nature of this phenomenon was illustrated by the absence of increased secretion in the absence of calcium, and the attenuated secretion or absence of increased secretion in the

3 presence of calcium, 60 mM potassium and EGTA, a chelator which binds clacium and prevents the influx of calcium. DISCUSSION Our fmdings using hypothalamic cells in the depolarization studies are in accord with the findings observed by others studying IR-fl-endorphin [4, 5, 10, 17] or IR-a-melanotropin [18] secretion from hypothalamic slices or synaptosomes. Our results also indicate that secretion of IR-ACTH can similarly be stimulated. Induction of release of POMC peptides by depolarizing concentrations of potassium requires the presence of free calcium and follows classical neurosecretory mechanisms. Thus, the demonstration that POMC peptides can be released by chemically induced membrane depolarization provides evidence that they could indeed be neuroregulators promoting communication within the brain. Furthermore, the similarity of results between dissociated cells and other in vitro models (slices, synaptosomes) which have disadvantages such as heterogeneous cellular exposure to medium and partially intact interconnecting neural pathways, or absence of intact cells, emphasizes the potential utility of studying the regulation of POMC peptide secretion in a dissociated cell culture system. Further secretory studies of POMC peptides in hypothalamic cells ought to indicate whether or not the theoretical advantages suggested by dissociated cell cultures are in fact real.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. G. Mueller for the fl-endorphin antisera, to Dr. Richard Robbins for assistance in developing the cell culture system, to Dr. Henry Strade of Organon Pharmaceuticals (West Orange, New Jersey) and Ciba-Geigy (Basel, Switzerland) for ACTH fragments used to develop the RIA, to the National Pituitary Agency for ACTH1-a9, to Dr. Randall Travis for suggestions in the review of this manuscript, to Miss Fran Apltauer for excellent secretarial assistance, and to the Department of Medicine (Cleveland Metropolitan General Hospital) for support in this study.

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KAPCALA, GRIFFIN AND BIRK 16. Vaccaro, D. and A. Messer. Preparation of fetal rat hypothalamus cells in primary monolayer culture. Tissue Cult Assoc Manual 3: 561-563, 1977. 17. Vermes, I., G. H. Mulder, F. Berkenbosch and F. J. H. Tilders. Release of 13-1ipotropin and 13-endorphin from rat hypothalami in vitro. Brain Res 211: 248-254, 1981. 18. Warberg, J., C. Oliver, A. Barnea, C. R. Parker and J. C. Porter. Release of immunoreactive c~-MSH by synaptosomeenriched fractions of homogenates of hypothalami. Brain Res 175: 247-257, 1979.