High molecular weight somatostatin secretion by cultured rat brain cells

Brain Research, 239 (1982) 463-477 Elsevier Biomedical Press

463

H I G H M O L E C U L A R W E I G H T S O M A T O S T A T I N SECRETION BY C U L T U R E D RAT B R A I N CELLS

ROBERT A. P E T E R F R E U N D and WYLIE VALE*

Peptide Biology Laboratory, The Salk lnstHute, P.O. Box 85800, San Diego, CA 92138 and ( R.A.P.) Department of Neurosciences, University of California, San Diego La Jolla, CA 92093 (U.S.A.) (Accepted October 15th, 1981)

Key words: somatostatin secretion - - cell culture - - somatostatin-28 - - somatostatin-14 - - hypothalamus - - cerebral cortex - - phorbol esters - - 8-Br-cAMP

INTRODUCTION

The tetradecapeptide somatostatin (SS-14) was originally isolated from extracts of sheep hypothalamus based on its ability to inhibit growth hormone secretion by cultured cells of the anterior pituitary 1. SS-14 has a wide species distribution 2s, and is found in cerebral cortex, amygdala, spinal cord, pancreatic islet D cells, stomach and small intestine 29. In addition to regulating growth hormone secretion, the peptide inhibits the secretion of a variety of other hormones including insulin, glucagon and TSH. Investigations into the biology of somatostatin have employed in vitro model systems. Robbins et al. 20 and Ensinck et al. 6 reported biosynthesis of SS-14 in dispersed rat cell cortex cultures and rat hypothalamic slices respectively. Biosynthesis from high molecular weight precursors in pancreas has also been described 16. Several groups have established in vitro systems to study somatostatin secretion using organ cultures a3,19,23 or dispersed cell tissue culturell, 13. These workers characterized somatostatin-like immunoreactivity (SSLI) as identical to SS-14 and implicated growth hormone z2, GABA H, substance P and neurotensin 23, and acetylcholine 19 as regulatory agents. Recently, a 28 amino acid, N-terminally extended form of SS-14 has been isolated and characterized from extracts of ovine hypothalamus7,26,27 and porcine gut 7. A 25 amino acid peptide has also been isolated from ovine hypothalamus v and a somatostatin-like peptide of apparent molecular weight 4000 has been described in extracts of rat hypothalamus 25. The high molecular weight, N-terminally extended somatostatins have biological activity similar to SS-14. Synthetic analogues of SS-28 bind with high affinity to brain tissue and also have specific biological activities",14,1s, 31

*To whom all correspondence should be sent at San Diego. O006-8993/82/(RRD-O000/$02.75 © Elsevier Biomedical Press

464 We now report secretion of an amino terminally extended peptide with SSLI from fetal rat brain tissue in long term dispersed cell culture.

MATERIALSAND METHODS Dissection: Pregnant Sprague-Dawley rats were purchased from Zivic Miller (Alison Park, PA), received on the 16th day of gestation and housed with free access to food and water on a 12 h light, 12 h dark schedule. On the morning of the 18th day of gestation, mothers were etherized and the fetuses removed individually or in pairs through an abdominal incision. The fetal brain was exposed by peeling away the membranous skull after severing the cervical spinal cord. A superficial slice of rostral lateral cerebral cortex was removed with iridectomy scissors or with a pair of fine forceps. Shallow cuts were made bilaterally in the hypothalamic sulci, rostral to the optic chiasm and rostral to the mammillary bodies for removing hypothalamic tissue. The hypothatamic and cortical tissues were separately placed into room temperature Hepes Dissociation Buffer (HDB) containing 0.1 ~ bovine serum albumin (BSA)(Fraction V, Calbiochem). Eleven to twelve fetuses from each pregnant mother was a typical yield. Dissection of fetuses from 8 mothers generally required less than 60 min. o/ BSA and Tissue culture: Tissue was washed once with 37 °C HDB-0.1/,, transferred to spinner flasks containing 0.4~ collagenase II (Worthington Diagnostics, Freehold, N J) and 10/zg/mi DNAase II (Sigma, St. Louis, MO) dissolved in HDB. Approximately 1 ml of enzyme solution was used per hypothalamus and 45 ml for the pooled cortex tissue. Digestion proceeded for 2.5-3 h at 37 ~'C in a water bath with constant gentle stirring. Tissue chunks were sheared by gentle passage through a silanized Pasteur pipette at 45 min intervals. At the end of the digestion period, cells were pelletted, resuspended in a small volume of 0.1 ~ BSA-HDB and layered over 20 ml of HDB containing 4 ~ BSA, and centrifuged. The supernatant was discarded and the pellet washed 3 times with culture medium. Cell yield was determined with a Coulter counter. Cells were plated at a density of 107 cells per 60 mm tissue culture dish (Falcon 3002) or 5 × 106 cells per 35 mm dish (Falcon 3001). All dishes were coated with sterile poty-o-lysine (Sigma) at 20 #g/ml in water for 4-6 h a t room temperature and washed once with culture medium immediately prior to plating. Cells were maintained in 15 ~o CO2, 85 ~o air, 100 ~o humidity at 37 °C. Medium was changed on the fifth day in culture to HMEM + 5 ~ Horse serum (HS) + 5 ~o fetal calf serum (FCS) + mycostatin. Medium was changed every fourth day thereafter. Buffers and media: Hepes Dissociation Buffer (HDB) was composed of 137 mM NaCI, 5 mM KC1 and 0.7 mM Na~ HPO4, buffered with 25 mM HEPES, and brought to pH 7.35. Gentamycin sulfate (Sigma) was added to a final concentration of 100 /zg/ml. The solution was autoclaved. Collagenase solution was made from HDB to which was added 0.4~o (w/v) final concentration collagenase II (Worthington Diagnostics), DNAase II (t0 /tg/ml; Sigma), 0.4% BSA and 0.2~ D-glucose. The solution was filtered and stored frozen.

465 Hepes-Krebs-Ringer-bicarbonate-glucose solution (HKRBG) consisted of 11 l mM NaCl, 4.7 mM KCI, 2.5 mM CaCI2, 1.2 mM MgSO4.7 H20, 1.2 mM KH~PO4, 24.8 mM Na2HCO3 and 11.1 mM D-glucose buffered with 15 mM HEPES and brought to pH 7.35. Prior to an experiment Bacitracin (Calbiochem, La Jolla, CA) was added to a final concentration of 30 #g/ml along with 0.1 ~ w/v BSA (Crystalline, Pentex, Miles Labs). The mixture was gassed and filtered. H K R B G , with 10 times the regular potassium concentration (59 mM), was prepared by substituting KCI for NaC1. Culture Medium. Dulbecco's Modified Eagle's medium was buffered with HEPES and supplemented with 50 units/ml of penicillin and 0.0001 ~ streptomycin (H M E M). Horse serum (10 ~ v/v) (HS, Gibco, Long Island, N Y) and fetal calf serum (10','~o v/v) (FCS, Gibco) were added along with mycostatin (Calbiochem) to a final concentration of 20 #g/ml. Photography. Phase contrast photography was performed with an Olympus IMT microscope and an Olympus C35A camera using Kodak Panatomic X film. Release experiments. Cells were washed 3 times for 5 rain at 37 °C in 10~/o CO2-90~'.~I air atmosphere in H K R B G . Volumes were 0.75 ml for 35 mm dishes and 1.0 ml for 60 mm dishes. Cells were then equilibrated in H K R B G for 1 h then incubated in H K R B G for 1 h with or without drugs, peptides or 59 mM potassium and finally in high potassium H K R B G for 1 h. Cells were washed with H K R B G once for 5 rain between the second and third hour of incubation. At the end of each incubation, the media was collected into an equal volume of ice-cold 4 or 2 N acetic acid, heated for 5-7 rain at greater than 90 °C in a water bath, cooled and dried in a Savant Speed Vac. Samples were frozen until assayed. For chromatography samples, the media was collected into an equal volume of ice-cold 4 N HOAc containing 200 units/ml Trasylol and 20 mM EDTA. The mixture was then heated to 90-95 °C in a water bath for 5 rain, frozen in liquid nitrogen, and lyophilized. An aliquot was removed prior to freezing for determination of total SSLI. Radioimmunoassay. (RIA) was performed by the method of Vale et al. 3o using the centrally directed antiserum $201 and [1251]Tyr-AIa-SS or with the amino terminally directed antiserum $39 using [125I]Tyrll-SS (ref. 28). Lyophilized samples were redissolved in cold assay buffer containing phenol red and neutralized with NaOH.

Gel fihration chromatography Procedure one: samples for chromatography were resuspended in 0.75 ml 6 M guanidine-HCl in 3 N HOAc, diluted to 3 M guanidine-HCl in 3 N HOAc and applied to a BioRad 1.75 c m x 100 cm column packed with 90 cm of Sephadex G-50 (fine) capped with an 8.5 cm plug of Sephadex G-10 (Pharmacia). The column was eluted with 3 N HOAc at room temperature. Samples of 2.0 ml were collected at a flow rate of 6.6 ml h, dried in a Savant Speed Vac and assayed. Procedure two : samples were resuspended in 1.0 ml 6 M guanidine-HCl in 3 N HOAc, heated for 2 min in a boiling water bath, diluted to4 M guanidine-HCl in 3 N HOAc, heated again for 2 min and applied to a column eluted with 3 N HOAc containing 0.01 ~ BSA at room temperature.

466

plating. b:-20 h after plating, c: 4 days after plating, d: 7 days after plating.

High performance liquid chromatography (HPLC) : solvents for HPLC were composed of 1.15'~/o formic acid and 0.70'~/o triethylamine, pH 3.0 (solution A) and 60% acetonitrile --40~o solution A (solution B). Samples were redissolved in 1.5 ml solution A and applied to a Waters C-18 column. The column was eluted using an Altex gradient liquid chromatograph Model 332 with 3 5 ~ B, 6 5 ~ A for 2 min

467 followed by a gradient to 5 0 ~ B over 15 rain. Flow rate was 1.5 ml/min at 1300 psi. Fractions were collected every 0.5 rain in glass tubes and lyophilized prior to assay with antiserum $201. Culture medium extractions. Culture medium was removed from the culture dishes and placed immediately into glass tubes on ice. Samples were stored frozen until extracted. Three milliliters of extraction mixture consisting of a 9:1 mixture of methanol and 1 N acetic acid/0.1 N HCI was added to 1 ml of culture medium and samples were vortexed. Following centrifugation, the pellet was discarded and the supernatant partially dried under nitrogen gas in a 37 °C water bath. Final drying occurred in a Savant Speed Vac. Samples were solubilized for RIA as described above. Statistical analysis. Three programs in the Salk Institute VAX computer system provided statistical analyses. The program RIA 8 calculated and statistically evaluated radioimmunoassay data. The program Bioprog 9 performed parallel-line analyses. EXBIOL, a statistical package prepared by Dr. E. Sakiz, carried out logarithmic transformations and statistical tests for evaluating release experiments. RESULTS

(1) Cell morphology Digestion of tissue with the collagenase/DNAase solution for 2.5 h yielded a few undissociated fragments and an opaque supernatant. After washing, 1.4-2.0 × 106 cells per hypothalamus were recovered. Immediately after plating, the cells typically appeared round or ovoid and floated individually or in pairs in the culture medium. Significant sticking of cells to substrate occurred within 2-3 h after plating. Aggregates or chains of cells were frequently observed with the appearance of processes extending from some of the cells (Fig. l a). By 18-24 h after plating, attachment to substrate was essentially complete with extensive process formation. Many of these processes branched or arched and appeared to make contact with other cells (Fig. lb). Neural appearing cells had a variety of shapes and branching patterns. The bipolar, spindleshaped cell and a triangular cell with two or three processes were most commonly observed. Within 5 days of plating, a growth of flat, lightly refractile material began to dominate the culture and obscure the morphology of the more highly refractile neuralappearing elements (Fig. lc). This lightly retractile cell growth became confluent within 7-10 days and appeared to surround or underlie the neural-appearing elements (Fig. ld). Cell adherence to the substrate and subsequent morphologic differentiation appeared to depend on the presence of serum at the time of plating. Addition of cytosine arabinoside (Ara c; 10-,~ M) for days 1-5 of plating diminished the appearance of flat cells but Ara c-treated neural elements sloughed from the substrate faster than non-treated cells in the same preparation. There were no obvious morphologic differences between hypothalamus and cortex cells (Fig. 2a, b). However, proliferation of lightly refractile cells was often less rapid in cerebral cortex cultures than in hypothalamus cultures.

468

]

(II) Culture medium extractions On the fifth day in culture, medium was removed from cells plated at 1.3 x 107 cells per 60 mm dish (hypothalamus) or 1.0 × 107 cells per 60 mm dish (cortex) and extracted as described in Methods. Hypothalamus culture medium extracts contained greater than 2700 fmol of SSLI per dish. Extracts of culture medium from cortex culture dishes contained 2000 fmol per dish. Control extractions of an equal volume media never exposed to cells possessed less than 130 fmol SSLI. Recovery of synthetic SS-14 added to culture medium was greater than 90~. The samples extracted from cortex culture medium and hypothalamus culture medium were separately pooled and assayed in several dilutions for SS immunoreactivity. Displacement curves paralleled dilutions of synthetic SS within 95 % confidence limits (Fig. 3).

(III) Secretion experiments On the 10th or 1lth day in culture, cells were treated with drugs, hormones or high potassium buffer in secretion experiments. Samples were concontrated 1.5-2.0 times for radioimmunoassay. Buffer only samples and buffer plus test peptide samples were always included in radioimmunoassays to control for non-specific interference of tracer binding due to salt concentration or peptide cross-reaction. Hypothalamus and cortex cells incubated in high potassium HKRBG for 1 h released up to 16 and 12 times the amount of SSLI secreted during 1 h Gontrol

469

SYNTHETIC

5000

SSI4

HYPOTHALAMUS CORTEX

4000 z D 0

m 3000 z

2000

I000

I [0

I I N~N~ " [ 20 50 I00 200 DILUTION FACTOR

Fig. 3. lmmunoactivity of dilutions of extracted culture medium. Culture medium was removed from cells plated at 1 × 107 cells per 60 mm dish and extracted as described in Methods. Dilutions of the extracts were assayed with antiserum $201 and displacement of trace was compared to displacement by synthetic SS-14. An unweighted linear regression analysis and the Student's t-test evaluated displacement data in the computer program BIOPROG. Displacement lines were parallel at the 95 % confidence level.

incubations respectively (Table I). Typical maximum secretion was 3-10 times the control rate. Secretion in response to 59 mM K + was prevented when calcium was removed from the medium or when cobalt was administered in a calcium free medium (Fig. 5). Following washing, cells responded normally to 59 mM K ÷. The depolarizing agent, veratridine, at levels greater than 10-s M also induced release of SSLI into the incubation medium. Material released from cells was assayed in serial dilutions using antiserum $201 and immunoreactivity was compared to dilutions of synthetic SS-14. Displacement curves for synthetic SS-14, cortex secretion samples and hypothalamus secretion

TABLE I

S S L I secretion by cell cultures Basal secretion (fmol) 35 mm dish Hypothalamus Cortex

< 50 < 50

60 mm dish Hypothalamus Cortex

100-200 100-200

Maximal secretion (fmol)

700 400 1600 1200

470 6000 SYNTHETIC S S I 4 HYPOTHALAMUS

5000

CORTEX 4000

3000

2000

I000

I

I0

20 DILUTION

I

I

50 FACTOR

I00

Fig. 4. Immunoreactivity of high potassium medium after a 1 h incubation of cultures, Brain cells at 1 )< 107 cells per 60 mm dish were treated for 1 h with high potassium H K R B G on day I1 in culture. Medium was collected as described for ehromatrography samples (see Methods). Dilutions of the lyophilized medium were assayed with antiserum $201 and evaluated as described in Fig. 3. Displacement lines were parallel at the 95 ~ confidence level.

samples were parallel, with greater than 95 ~ confidence as determined by the t-test (Fig. 4). Cells from both cortex and hypothalamus responded to administration of 5 mM 8-Br-cAMP. Lesser response was seen at 0.1 mM. IBMX at levels greater than 0,1 mM also produced SSLI release (Fig. 5). We were unable to demonstrate increased release of SSLI with isoproterenol (100 #M), phenylephrine (100 /zM), sodium fluoride (1 raM), thyrotropin releasing hormone (1 #M), bradykinin (10 #M), bombesin (10 #M) or rat growth hormone (100 ng/ml). Phorbol 12-myristate 13-acetate (PMA, Sigma) was found to release SSLI from brain cell cultures in a dose-dependent manner (Fig. 6). Control experiments demonstrated that appropriately diluted ethanol, the solvent for PMA, slightly depressed release of SSLI. Viability of cells following depolarizing stimulus was demonstrated by returning cells to serum supplemented culture medium for 2 days and then repeating treatment with high potassium HKRBG. Cells were able to release significant quantities of SSLI in the second experiment. Cells exhibited minimal trypan blue uptake (0.05 ~o trypan blue in HDB) during a 10 min incubation at 37 °C immediately following treatment with high potassium HKRBG. Morphologic appearance of the cells after a release experiment was unremarkable.

(I. V Gel filtration chromatography) On day 11 in culture, high potassium HKRBG incubation medium from cells

471

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59 m'idK"

Fig. 5. Release of SSLI by brain cell cultures. Results o f several experiments expressed as a fraction of maximum secretion elicited by 59 m M potassium in H K R B G using dishes not exposed to drugs. Maximum secretion in 59 m M K + corresponds to approximately 400 fmol/dish. A : response of hypothalamus cells to administration of 59 m M K ÷ in medium lacking added calcium. After equilibration, cells were washed once with H K R B G or calcium-free H K R B G . Cells were then treated for I h with 59 m M K ~ in the presence or absence of calcium or in calcium-free media containing cobalt. B : response of cortex cells to 59 m M K +. Procedure is the same as in A. C: response of hypothalamus cells to I B M X and 8Br-cAMP. **, different from control P < 0.01 ; *, different from control P ~ 0.05. n.s., not significantly different from control. N u m b e r in parentheses number of treated dishes.

~Wr

==

N=8 IOO '~

80

60 ~ F-

~

4o 2o

N=4

N=3

N=3

CONTROL I0 nM I00 nM PMA

PMA

I.M

5 9 rnM

PMA

K ¢"

Fig. 6. Response to PMA. Cells were incubated first in H K R B G for 1 h (control) and then in experimental medium for 1 h. A stock solution of 200 # M P M A in ethanol was diluted for treatment. Results are expressed as a fraction of m a x i m u m release elicited by high potassium (59 raM) H K R B G . In control experiments, equivalent ethanol concentrations slightly depressed SSLI secretion. **different from control (P < 0.01) by Multiple Range Test of Duncan. N indicates the number of treated dishes; bars represent standard error.

59mid K"

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SS 28

800 o

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700

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400

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=

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300

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30

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40

45

50

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55

60

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FRACTION NUMBER

Fig. 7. Gel filtration chromatography of secreted SSLI, Incubation media from cells treated with high potassium H K R B G on the 1 l t h day in culture was collected as described in Methods andapplied to a freshly poured Sephadex G-50 (fine) column caplx-~l with a plug o f Sephadex G-10 (see Methods). Samples from hypothalamus cells, cerebral cortex cells and a mixture of synthetic S.'S~28 and SS-14 were run in succession on the same column. Top: elution profile as determined by the centrally directed antiserum $201. Bottom: elution profile as determined by the amino terminal directed antiserum $39; arrows indicate migration position of synthetic SS-28 a n d SS-14 o n the ~ column. It is not clear why $39 and $20t recorded different quantitities of immunoreactive SS-14 for the hypothalamus sample.

473 treated for 1 h was collected as described in Methods. Pooled samples from 13 dishes of hypothalamus cells and pooled samples from 17 dishes of cerebral cortex cells were divided into two portions. One half of each pool was chromatographed using Procedure One and the remainder with Procedure Two. For Procedure One, samples from hypothalamus, cerebral cortex and a mixture of synthetic SS-28 and SS- 14, prepared in the same fashion as the cell samples, were run in succession on the same Sephadex G-50 column. The elution profile determined with the centrally directed antiserum $201 revealed 3 peaks ofimmunoreactivityfor both hypothalamus and cerebral cortex samples (Fig. 7, top). A minor peak elutedjust after the dextran marker for void volume (V0). This peak was not seen in the profile of synthetic somatostatins. A major peakeluted at Ka 0.380.50 in both cell sample profiles. This peak corresponded exactly to the peak identified as SS-28 in the profile of synthetic peptide. A third peak comigrated with synthetic SS-14 at a Ka of 0.71-0.79. The amino terminally directed antiserum $39, which does not detect amino terminally modified somatostatins including SS-28, failed to detect the peak at the void volume and failed to detect the peak at Ka 0.38-0.50. $39 detected the immunoreactivity at Ka 0.71-0.79 (Fig. 7, bottom). Recovery of synthetic SSLI migrating as SS-28 was 39~ o11 a molar basis; 38 °Jo of synthetic SS-14 was recovered. For media from hypothalamus cultures, approximately 28~',~ of total immunoreactivity applied to the column was recovered, as computed from aliquots made prior to lyophilization. Twenty-two percent of this immunoreactivity migrated as SS-28. Recovery of cerebral cortex immunoreactivity / was 3 0...,, of applied material. Twenty-one percent migrated as SS-28 (Table II). A similar gel filtration profile was obtained for sample prepared using Procedure Two. Recovery was unchanged for the fraction migrating as SS-14 and slightly increased for the fraction migrating as SS-28. Two days after the initial secretion experiment, the cells which supplied the chromatographic samples were treated again with high potassium HKRBG. Average yield of SSLI was 1247 fmol per dish for hypothalamus samples and 450 fmol per dish for cerebral cortex cultures. ( V) HPLC.

Samples from the peaks corresponding to SS-28 from the gel filtration profile TABLE 11 Reco very of SSLI ariel"gel filtration Sample

Applied (Jmol)

Recovered ~fmol)

% SS-14

°o SS-28

°o AT Vo

Hypothalamus Cortex Synthetic SS-28 SyntheticSS-14

8.000 I0,500 110,000 250,000

2,300 3,300 44,000 95,000

72 70

22 21

6 9

474 500 e = HYPOTHALAMUS = = CEREBRAL CORTEX SYNTHETIC SS 28

400

300 .-"

200 100 2---

---------

0

-

5

lO

15

20

25

ELUTION TIME, MINUTES Fig. 8. B P L C profile o f gel filtration peak. Pooled fractions f r o m the peaks corresponding to SS-28

from gel filtration by Procedure Two were applied to a Waters C-18 column and eluted as described in Methods. Antiserum $201 was used for assay. 0 - - 0 = hypothalamus samples; H = cerebral cortex samples; A - - A "= synthetic SS-28 peak from Sephadex G-50 chromatograph.

using Procedure Two were further characterized with HPLC. A Waters C-18 column was precalibrated with synthetic SS-28 and carefully washed prior to application of samples from hypothalamus, cerebral cortex and SS-28 from gel filtration. Retention times for the samples were identical (Fig. 8). Recovery was greater than 90~o for hypothalamus samples and 60-70 oj~ for cortex samples. DISCUSSION

Production and secretion of CNS peptides may be advantageously studied in dispersed cell culture because intact cells survive for long periods; test substances such as transmitters and hormones have direct access to tissue, and direct measurement of functional parameters is simplified. Collagenase dispersal of fetal rat brain tissue yields a variety of celt types, many of which have the appearance of neural elements. These cells undergo rapid morphologic differentiation and appear to contact one another. The proliferation of nonneural-appearing elements is a significant problem for long-term brain cell culture. The lightly refractile growth appears to obscure the fine branching patterns of cell process. It is possible that the potentiated differentiation associated with Ara c treatment reported by Gamse et al. 11 is a function of increased visibility of fine processes in the absence of an obscuring background rather than any direct alteration of the development of neural-appearing cells. Serial dilution of depolarizing incubation buffer applied to cultures confirms that hypothalamus cells in dispersed culture secrete high levels of a substance immunologically similar to SS-14. As has been previously described, this secretion appears to be mediated by cell membrane depolarization and is calcium dependent. The present findings indicate that cerebral cortex cells in dispersed culture also exhibit a calciumdependent release of SSLI into depolarizing incubation media. It is interesting that cells exhibit a detectable secretion rate of SSLI into cell

475 culture medium in the absence of any administered secretagogues; this confirms the observation of Dells et al. 3. It is possible that there is inhibitory and stimulatory physiologic regulation of somatostatin secretion. Results from repetition of release experiments separated by 48 h indicates continued viability of the cells. Release of SSLI is therefore not attributable to generalized cell destruction. The secretory response to the phosphodiesterase inhibitor IBMX and the analogue 8-Br-cAMP indicate that the cyclic nucleotide system may have a physiologic role in regulation of somatostatin secretion in the intact animal. However, a physiologic substance whose action could be mediated by a cyclic nucleotide second messenger has not yet been identified. It is possible that fetal brain tissue is insufficiently functionally differentiated to respond to regulatory substances implicated in adult studies. Alternatively, loss of response to drugs and transmitters may be an artifact of cell culture. A mechanism of action for PMA-induced secretion has not been defined. Specific receptor binding has been reportedS, 12 and changes in cell membrane have also been described10,1~. The effects seen with cultured brain cells broaden the activity spectrum of the phorbol ester family of compounds which are also potent secretagogues for anterior pituitary hormones. At least two forms of SSLI are recoverable from depolarizing incubation media. One form comigrates with SS-14 and is the major recovered form on a molar basis. A second form of SSLI comigrates with synthetic SS-28 in gel filtration and HPLC and appears to be an amino terminally extended form of SS-14. These data provide evidence for physiologic secretion of high molecular weight somatostatin by the central nervous system and are consistent with a previous finding using an acute preparation of tissue fragments az. Other groups analyzing SSLI secretion by in vitro systems may have lost SS-28-1ike immunoactivity in fractions at or near the void volumes of Sephadex G-25 or Bio gel P4 columns in chromatographic procedures. Since synthetic SS-28 possesses biologic activity qualitatively similar to SS-14, and in view of the high potency of SS-28 in biological assays and in binding studies, it is possible that SS-28 or a related peptide has a physiological role and therefore that SS-14 and SS-28 could be separate regulatory entities. ACKNOWLEDGEMENTS This work was supported by NIAMDD Grants AM-20917 and AM-26741, NIAAA Grant AA-03504 and a grant from the March of Dimes Birth Defect Foundation. Research conducted in part by The Clayton Foundation for Research, California Division. Dr. Vale is a Clayton Foundation Investigator. R. Peterfreund is the recipient of a Medical Scientist Training Program Award PHS GM 07198. Carolyn Douglas, Karen von Dessonneck, Joan Vaughan, Mildred Newberry and Gayle Yamamoto are thanked for expert technical assistance; Susan McCall is thanked for preparation of the manuscript.

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Note added in proof Recently a report has appeared describing calcium dependent release of SSLI from cultured cerebral cortex cells: Robbins, R. J., Sutton, R. E. and Reichlin, S., Endocrinology 1l0 (1982) 496-500. The same group has also developed a perfusion system for dispersed brain cells: Scanlon, M. F., Robbins, R., Bo[affi, J. L. and Reichlin, S., Endocr. Soc. ,4bstr., 199 (1981).