Release of NPY-like immunoreactive material from primary cultures of chromaffin cells prepared from bovine adrenal medulla

Neuropharmacology Vol. 24, No. 7, pp. 693-695, Printed in Great Britain. All rights reserved

RELEASE OF NPY-LIKE

Laboratory

1985 Copyright

0

002X-3908/85 $3.00 + 0.00 1985 Pergamon Press Ltd

IMMUNOREACTIVE MATERIAL FROM PRIMARY CULTURES OF CHROMAFFIN CELLS PREPARED FROM BOVINE ADRENAL MEDULLA

of

Y. Kataoka, E. A. Majane and H.-Y.T. Yang Preclinical Pharmacology, National Institute of Mental Health Saint Elizabeths Hospital, Washington, D.C. 20032, USA

Sumnary In primary cultures of bovine chromaffin cells, the cell of NPY imnunoreactive material was found to be stable for When these cells were transferred to the Locke’s days. from the culture medium, NPY immunoreactive material was Addition of potassium (45 mM) resulted in spontaneously. increase in the rate of NPY release in a calcium dependent NPY imnunoreactivity in chromaffin cells is composed of lesser imnunoreactive pept ide and two very itnnunoreactivities. The same imnunoreactive peptides were by 45 mM potassium.

contents about 4 solution released a 3-fold fashion. NPY, a minor released

INTRODUCTION Neuropeptide Y (NPY) was isolated from porcine brain and characterized biochemically by This peptide is now known to be located in several neuronal Tatemoto et al. (1982). and peripheral nervous system. It is also present in populations of the central norepinephrine containing chromaffin cells and nerve fibers in the adrenal glands of many In the bovine adrenal gland, NPY content is higher in the species (Allen et al., 1983). NPY is stored in chromaffin medulla than in the cortex. Furthermore, in the medulla, HPLC granules indicating the possibility that NPY is released from adrenal glands. analysis of NPY immunoreactivity in bovine adrenal chromaffin granules has detected imnunoreactivity multiple molecular forms of NPY-like peptides including NPY, one lesser We report here the release of these NPY-like and two other very minor peaks. itnsunoreactive peptides from isolated bovine chromaffin cells kept in culture. MATERIALS AND METHODS Bovine adrenal glands were purchased from a local slaughter house and transported to the Chromaffin cells were isolated from these glands according to the laboratory on ice. method of Kilpatrick et al. (1980) with minor modifications. The modifications made are: by Greenberg and Zinder I) perfusion of adrenal glands was carried out as described The isolated and 2) the Percoll gradient centrifugation step was eliminated. (1982), chromaffin cells were plated onto culture dishes (Falcon 3003), I07 cells per 100 ~HII dish containing ID ml of Dulbecco modified Eagle medium (GIBCO), and cultured under the The content of NPY in chromaffin cells conditions as reported by Kataoka et al. (1984). To study the release of NPY, chromaffin during the culture was determined over 6 days. cells attached to the culture dishes were washed with phosphate buffered saline and After 30 min incubated in 5 ml Locke’s solution at 37O C in a 5% CO2-95% air atmosphere. medium was separated from the chromaffin cells by gentle aspiration incubation, the in The clear incubation medium was placed followed imnediately by centrifugation. centrifuged and passed through octadecylsilyl polypropylene tubes, boiled for IO min, The c-18 cartridges were washed with 10 ml H2D and then silica cartridges (Sep-Pak-C-18). The samples were eluted with 4 ml 60% acetonitrile containing 0.1% trifluoroacetic acid. For the dried in a vacuum centrifuge (Savant Instruments) and radioimnunoassayed for NPY. the chromaffin cells were scraped from the culture dishes measurement of NPY ccl 1 content, washed once with 5 ml phosphate buffered and transferred into 15 ml polypropylene tubes, To the cells, 1 ml IN CHqCOOH was added, and saline by gentle mixino and centrifuoation. The clear sup&nates were passed the cell; -were sonicated, heated and centrifuged. eluted as described for the through the c-18 cartridges and NPY imnunoreactivity releasate. The NPY was radioimnunoassayed as previously described (Majane et al., 1985).

693

Preliminary

694

Notes

The immunoreactive materials in the chromaffin cells as well as in the releasate were The releasate and the extract from the chromaffin cells were treated as analyzed by HPLC. described above and then chromatographed on reverse-phase HPLC columns, BioSil ODS 5 (250 The reverse-phase column was eluted with a linear gradient of CH3CN, 20-60% in x 4 mm). 0.1% trifluoroacetic acid over a period of 60 min. The flow rate was I ml/min. One ml fractions ware collected, lyophilized and analyzed for NPY imnunoreactivity as previously described. The cell content of NPY immunoreactive material was found to be constant for four days (Table I). After 4 days, a sharp decrease in NPY content was observed (Table I). Thus, the chromaffin cells were used for the release study two days after isolation and culture. The results of the release study are shown in Fig. I. When the chranaffin cells were incubated in Locke’s solution for 30 min, a spontaneous release of 0.78 f 0.1 pmol NPY per I07 cells was observed; this is about 5% of the cell’s NPY content. The addition of potassium (45 mM) to the incubation medium was found to increase the release of NPY to 2.320.8 pmoi/lO7 cells; this is equal to about 13% of the NPY content in the chromaffin cells. NPY release induced by 45 mM KCI was reduced to 1.220.6 pmol/l07 cells by the omission of Ca2+ from the incubation medium (Fig. I). RESULTS AND DISCUSSION Table

I

NPY IMMUNOREACTIVITY IN CULTURED BOVINE ADRENAL CHROMAFFIN CELLS AS A FUNCTION OF DAYS IN CULTURE DAYS IN CULTURE

TOTAL NPY-IR pmol/l07 cells

NPY- IR % of Day 0

7.00

0 I

100

7.92 7.61 5.28 4.97 4.36 2.97

: 4 2

113 109 75.4 71 .o 62.2 42.4

Chromaffin cells were cultured, extracted and assayed for NPY-IR as described in the methods section. The percoll gradient step was included. Each time point is the average of duplicates from two different experiments. Figure

I

Release of endogenous NPY imnunoreactivity from two day old cultured chromaffin cells: effect of the high (49 mM) K+ and Ca2+ ions. Dishes containing I07 ccl Is each were washed free of media with phosphate buffered saline and exposed for 30 min to Locke’s solution containing the appropr i ate ionic composition. NPY imnunoreactive material from both releasate and cells was extracted and analyzed by radioimnunoassay described in the methods section. Eazt value corresponds to the mean+S.E.M. of 6 dishes. Statistics were done by the paired student’s t test.

,” 6 “0 2 ‘; t 4 ‘h

SmMK+ 45mMK+45mNK+ control +Ca++ -Ca++

++

p <.OOl

. . . . control

p K.002

. . . . (45mMK++Ca++) vs (45mMK+-Ca++)

YS

45mMK+ Ca

Enkephalins in adrenal gland are known to be stored in chromaffin granules (Stine et al., and can be released from isolated bovine chromaffin cells by stimulation of 1981) nicotinic receptors. Although enkephalins coexist with NPY in the adrenal chromaffin ceils of many species, whether nicotinic receptor stimulation can result in NPY secretion still remains to be determined. This study seems to suggest that primary cultures of bovine chromaffin cells may be a convenient model for exploring the regulation of NPY secretion and also the possible interaction between NPY, enkephalins and catecholamines. However, it-should be noted that in the bovine adrenal glands NPY and enkephalins are detected in separate chromaffin cells (Majane et al., 1985).

Preliminary

Notes

695

Figure

Fraction

2

High pressure I iquid chromatography of releasate from normal (A) and high (B) K+ treated chrcmaff in ccl Is. NPY immunoreact i ve material from two-day old cultures as shown in Figure I was combined and dried by vacuum centrifuge (Savant). Each sample containing material released from 3.5.107 cells, was analyzed by HPLC using a Biosil ODS 5 column (4x250 mm). The reverse phase column was eluted at a flow rate of I ml/min with a linear gradient of 60% CH3 CN containing 0.1% 20 to trifluoroacetic acid over a period of 60 min. One ml fractions were collected, lyophilized and radioimnunoassayed for NPY immunoreactivity.

(Ml )

By HPLC (Fig. 2), NPY imnunoreactivity in chromaffin cells was shown to consist of NPY, a I esser immunoreactive peptide and two very minor imnunoreactivities. The same imnunoreactive peaks were also detected when the NPY imnunoreactivity released by 45 mM K+ was analyzed by HPLC (Fig. 2). The nature of the NPY like immunoreactive peaks which are distinct from NPY still remains to be studied. The functional role of NPY in the adrenal that NPY may have a modulatory effect on catecholamines. Using the primary cultures can now be explored.

gland is not yet known, it the release or on the effects of bovine chromaffin cells,

has been proposed of the released this possibility

REFERENCES Neuropeptide Y (NPY) in the Allen, J.M., Adrian, T.E., Polak, J.M., Bloom, S.R. (1983). adrenal gland. J. Autonomic Nerv. System 9: 559 Greenberg, A. and Zinder, 0. (1982). Alphaand beta-receptor control of catecholamine secretion from isolated adrenal medulla cells. Cell Tissue Res. 226: 655-665 Kataoka, Y., Gutman, Y., Guidotti, A., Panula, P., Wroblewski, J., Cosenza-Murphy, D., Wu, J.Y. and Costa, E. (1984). Intrinsic GABAergic system of adrenal chromaffin cells. Proc. Natl. Acad. Sci. USA 81: 3218-3222 Kilpatrick, D.L., Ledbetter, F.H., Carson, K.A., Krishner, A.G., Slepetis, R. and Kirshner, N. (1980). Stability of bovine adrenal medulla cells in culture. J. Neurochem. 35: 679692 H.-Y.T. (1985) Neuropeptide Y in Majane, E.A., Al ho, H., Kataoka, Y., Lee, C.H. and Yang, bovine adrenal glands: distribution and characterization. Endocrinologgy, in press Stine, S.M., Yang, H.-Y.T. and Costa, E. (1980). Release of enkephalin-like immunoreactive material from isolated bovine chromaffin cells. Neuropharmacology 19: 683-685 Tatemoto, K., Carlquist, M., Mutt, V. (1982). amino acid sequence Neuropeptide Y: complete of the brain peptide. Proc. Natl. Acad. Sci. USA 79: 2513