[30] Somatostatin release from dissociated cerebral cortical cell cultures

412

Q U A N T I T A T I O N O F N E U R O E N D O C R I N E SUBSTANCES

[30]

homogenates gives one a factor of 4- or 5-fold lower detection limits compared to conventional LCEC analysis of these homogenates. Even in cases where the lower limit of detection is not necessary microbore LCEC offers the advantages of decreased cost of columns and decreased solvent consumption. The injection of a small volume leaves sufficient sample for several replicate injections in most cases. Acknowledgment This work was supported by a grant from the Scottish Rite Schizophrenia Research Foundation.

[30] S o m a t o s t a t i n R e l e a s e f r o m D i s s o c i a t e d C e r e b r a l C o r t i c a l Cell C u l t u r e s

By ROXANNE M. LANDON and RICHARD J. ROBmNS Somatostatin (SS) was first reported to be present in the cerebral cortex by Brownstein and co-workers, j Immunohistochemical studies demonstrated that the majority of the immunoreactive somatostatin (IRS) was contained in a unique subset of cortical interneurons. 2,3 A putative role for SS in the cortex was further strengthened by the demonstration of specific SS receptors on cortical membranes, 4 by definition of electrophysiologic effects on cortical neurons] and by reports of the effects of SS on cortical neurotransmitters. 6-s To date, however, a physiologic role for SS in the cortex remains undefined. The biology of the cortical somatostatinergic neuron has been examined from several aspects using primary cell culture techniques. 5 DissociM. Brownstein, A. Arimura, H. Sato, A. V. Schally, and J. S. Kizer, Endocrinology 96, 1456 (1975).

2 j. McDonald, J. Parnevelas, A. Karamanlidis, N. Brecha, and J. Koenig, J. Neurocytol. 11, 825 (1982). S. Shiosaka, K. Takatsuki, M. Sakanaka, S. lnagaki, H. Takagi, E. Senba, Y. Kawai, H. lida, H. Minagawa, Y. Hara, M. Takashi, and M. Tohyama. J. Comp. Neurol. 204, 21 I (1982).

4 C. B. Srikant and Y. C. Patel, Proc. Natl. Acad. Sci. U.S.A. 78, 3930 (1981). 5 j. Delfs and M. Dichter, J. Neurosci. 3, 1176 (1983). 6 S. Tanaka and A. Tsujimoto, Brain Res. 208, 219 (1981). 7 A. Tsujimoto and T. Shokichi, Life Sci. 28, 903 (1981). 8 j . G a r c i a - S e v i l l a , T. M a g n u s s o n , a n d A . C a r l s s o n , Brain Res. 155, 159 (1978).

METHODS IN ENZYMOLOGY, VOL. 124

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

[30]

SOMATOSTATIN RELEASE FROM CORTICAL CULTURES

413

ated monolayer cultures of rat cerebral cortical cells have been used to define posttranslational events in the biosynthesis of SS, 9 to study receptor populations on cortical cells, to examine neurotransmitter turnover, and to quantitate peptide release under basal and stimulated conditions.10.~l This chapter will focus on the methods which we have used to characterize the release of IRS from cultured rat cerebral cortical cells. We will discuss our methods for (1) dispersal of cells from fetal brain tissue, (2) plating and maintenance of the cultured cells, (3) performing acute release experiments, and (4) measuring peptide content by radioimmunoassay. Cell Culture Methods Establishment of primary cerebral cortical cell cultures by enzymatic or mechanical dispersal of adult mammalian brain tissue has been unsuccessful, in general, due to irreversible membrane damage to neurons during preparation, and to the rapid overgrowth of nonneuronal cells in these cultures within 3 to 4 days. We have chosen to employ 16-day-okt fetuses as our source of brain tissue because many of the neurons have just completed their final division ("last birthday") and because there are relatively few glial precursors present at this age. The disadvantages of these cultures include a lack of adult differentiation of the cells, absence of normal intercellular synaptic and nonsynaptic connections, and necessity for an artificial hormonal and growth factor environment. These deficiencies are balanced by the ability to plate large numbers of identical cultures, rapid exchange of medium nutrients, gases, secretogogues, and secreted products, and the power to completely control the constituents of the medium or of release buffers. The major caveat in interpreting the results lies in the fact that one is defining potential capacities of the cells. No attempt to extrapolate results to the in vivo state should be undertaken, nor is it necessary. The method for dispersing neuronal tissues by a combination of enzymatic and mechanical techniques was first described by Moscona. ~2 A number of successful dissociation methods for fetal brain tissue have been developed.13-17 The present technique is a modification of the method of Vaccaro and Messer.13 9 R. t0 R. ~1 R. 12 A. ~3 D. ~4 M.

J. Robbins and S. Reichlin, Endocrinology 113, 574 ([983). J. Robbins, R. E. Sutton, and S. Reichlin, Brain Res. 234, 377 (1982). J. Robbins, R. E. Sutton, and S. Reichlin, Endocrinology 110, 496 (1982). A. M o s c o n a , Exp. Cell Res. 3, 535 (1952). Vaccaro and A. Messer, Tissue Cult. Assoc. Manual 3, 561 (1977). A. Dichter, Brain Res. 149, 279 (1978).

414

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[30]

Solutions and Media Hanks' balanced salt solution (HBSS; GIBCO, Grand Island, NY) without calcium or magnesium is purchased sterile and stored in glass bottles at 25 °. Phosphate-buffered saline (PBS, 0.01 M, pH 7.2-7.4) consists of 145.5 mM NaCI, 2.7 mM NaEHPO4, and 7.4 mM NaH2POa'H20. This solution is autoclaved and stored at 4 ° in glass bottles. Papain-protease-DNase solution (PPD) consists of 0.01% (w/v) papain (Worthington, Freehold, NJ), 0.1% neutral protease (Dispase II; Boehringer Mannheim, Indianapolis, IN), and 0.01% deoxyribonuclease I (Worthington, LSOO 02139, from bovine pancreas) in HBSS supplemented to 12.4 mM MgSO4. MEM-deoxyribonuclease (MEM-DNase) consists of 0.01% deoxyribonuclease I in base medium (see below). PPD and MEM-DNase are prepared at 4 ° and are stored at - 2 0 ° in 10-ml aliquots. The aliquots are thawed and sterile-filtered through a 0.20-txm filter just prior to use. Culture medium consists of a standard medium with 10% serum (v/v). The base medium is HEPES buffered (10 mM, Caibiochem-Behring, La Jolla, CA) minimum essential medium (Eagle) with Earle's salts and glutamine (GIBCO). This solution is prepared from powdered stock. It is then supplemented to 33 mM glucose, and penicillin-streptomycin (GIBCO, 2000 U-2000/xg/liter) is added. The medium is stored at - 2 0 ° in glass bottles and is sterile-filtered through 0.20- or 0.45-1xm filters, respectively, after adding 10% fetal calf serum or 10% heat-inactivated horse serum (MEM-FCS, MEM-HIHS; GIBCO). Trypan blue vital stain consists of 0.01 M PBS with 0.4% (w/v) trypan blue. The solution is boiled to dissolve the trypan blue, cooled, and the pH is adjusted to 7.25.

Preparation of Dispersed Cortical Cells Although strict sterile surgical procedure is not necessary, the work area must be very clean and the instruments should be sterile. A timed pregnant female Sprague-Dawley rat (Charles River Co., Wilmington, MA) is anesthetized with pentobarbital sodium (0.5 ml of a 65 mg/ml 15 A. Faivre-Bauman, E. Rosenbaum, J. Puymirat, D. Greuselle, and A. Tixier-Vidal, Dev. Neurosci. 4, 118 (1981). ~6 W. J. Shoemaker, R. A. Peterfreund, and W. Vale, this series, Vol. 103, p. 347. 17 S. Reichlin, in "Brain Peptides" (D. T. Krieger, M. J. Brownstein, and J. B. Martin, eds.), p. 711. Wiley, New York, 1983.

[30]

SOMATOSTATIN RELEASE FROM CORTICAL CULTURES

415

solution) on the sixteenth day of gestation. After cleansing the abdomen with 70% ethanol, a longitudinal incision (3-4 cm) is made and the uterus with the fetuses intact is removed and placed in a sterile tissue culture dish (Corning 25020, 100 mm) containing 5 ml cold PBS. The placental membranes are dissected away and the embryos are transferred to a second tissue culture dish of PBS on ice. Each fetus is placed on its left side in PBS under a dissecting microscope. The fetal brain is isolated by firmly grasping the fetus by the neck with a curved forceps and making a horizontal incision just above the eye and ear vesicles with an iridectomy scissors, cutting just to the position of the ear vesicle. By reopening the scissors slightly and pulling gently with the forceps, the brain can be separated from the spinal cord and the skull. The brain is then placed on its dorsal surface and bilateral cuts are made along the lateral diencephalic sulci with a scalpel. The lateral telencephali are placed in a sterile 15 ml conical centrifuge tube (Falcon 2099) containing 5 ml of iced HBSS. Two or three females are used at one time and the total number of fetuses can vary from 15 to 40. The total dissection time should not exceed 1 hr. The tissue from up to 20 fetuses can be collected into 5 ml HBSS. The procedure continues under a laminar flow hood. The tissue is washed by gently tapping the tube to suspend the brain pieces. The HBSS is aspirated with a sterile 23-cm Pasteur pipet once the tissue has been allowed to resettle to the bottom of the tube. After removing the initial collection HBSS, the tissue is washed three times with 5 ml HBSS. The final wash is aspirated and 5 ml of PPD is added to the tube. The tissue is triturated with a sterile 23-cm cotton plugged Pasteur pipet that has been fire polished to remove the sharp, cut-glass edges of the tip and to reduce the size of the opening from 2 to l mm. For each trituration, the tissue is gently drawn into the pipet and expulsed slowly to avoid cell shearing and bubble formation. The tissue is triturated five times, and then the tube is capped tightly and incubated at 37° for 30 min. The tissue is again triturated 10 times and incubated an additional 30 min. To then change enzyme solutions, the suspension is triturated five times, capped tightly, and centrifuged at 1500 g for 5 min. The PPD solution is removed from the cell pellet, 5 ml of MEM-DNase is added, and the tissue is triturated 10 times. After a 15 min incubation and another l0 triturations, the cell suspension is centrifuged at 1500 g for 5 min, and the MEM-DNase is aspirated. The cells are resuspended in 10 to 15 ml MEM-FCS by gentle trituration, and 100/.d of the well-mixed suspension is removed for counting. The cell sample is prepared for counting by mixing 80/~1 of HBSS, 10 ~1 trypan blue vital stain, and 10/~1 of cells. A 10-/~l aliquot of this mixture

416

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[30]

is counted on a Bright-Line hemacytometer (American Optical, Buffalo, NY). The cell yield typically ranges from 10 to 12 million cells per fetus, and cell viability as determined by trypan blue exclusion is greater than 95%. The cells are plated into 25 cm 2 disposable plastic tissue culture flasks (Corning 25100) at a density of 107 cells and the final volume is adjusted to 5 ml with additional MEM-FCS. Cultures are maintained at 37° and 100% humidity in a 5% CO2 water-jacketed incubator, and are fed with 5 ml MEM-HIHS on the fourth, seventh, and eleventh days in vitro. Immediately after plating, the suspension of neurons and nonneuronal elements (glial, ependymal, vascular, and meningeal cells) float as indistinguishable, spherical cells. Within hours, some neurons begin to reaggregate and adhere to the flask along with the nonneuronal cells (see Fig. 1A). As the cultures mature, the nonneuronal cells proliferate to confluence and the neuronal complexes tend to flatten. The supportive cells form a fiat, phase dark background for the phase bright complex of neuronal cells and interconnecting processes (see Fig. IB). After 14 days in vitro, the neurons begin to disperse and lose the characteristic aggregate

FIG. 1. Phase contrast photomicrographs of dispersed cerebral cortical cells. (A) Two to 6 hr after plating m a n y cells still appear rounded, s o m e reaggregation has occurred. (B) Typical appearance of cells from 7 to 12 days.

[30]

S O M A T O S T A T I RELEASE N FROM CORTICAL CULTURES

417

morphology, possibly due to the continued proliferation of the support cells. The release experiments are performed on the twelfth or thirteenth day of culture, at which point cellular and media IRS content is stable. 9 Somatostatin Release Experiments

Somatostatin is found within secretory granules of neurons and is released from nerve endings in response to depolarization.~7 The secretion of SS following chemical depolarization by high extracellular potassium depends upon normal sodium and calcium flux through the cell membrane. ~ In vitro preparations that have been successfully employed to study the regulation of cortical SS secretion include cerebral cortical slices, lsA9 monolayer cultures, 2°,21 and capillary membrane perfusion of cultured cells. 22 Measuring the release of this peptide from the intact neurons of monolayer cortical cultures has provided insights into the potential role of SS in the central nervous system. 1°,11,2°,23-25 Reagents Kreb's Ringer bicarbonate solution26 (KRB): 118.6 mM NaCl, 4.8 mM KC1, 2.5 mM CaC12, 1.2 mM KHzPO4, 1.3 mM MgSO4, 24.6 mM NaHCO3, 33 mM D-glucose. KRB is prepared from stock solutions and allowed to equilibrate overnight in a 5% CO2 incubator. The pH at equilibration should be 7.4; this may be expedited by directly bubbling the solution with 5% COJ95% air. All chemicals to be tested are solubilized in KRB for use. High potassium Kreb's Ringer bicarbonate solution (KRB-K+): same formulation as above except the KCI is increased to 60 mM and the NaC1 is reduced to 63.4 mM to maintain isoosmolarity. ~8 L. L. Iversen, S. D. Iversen, F. Bloom, C. Douglas. M. Brown, and W. Vale, Nature (London) 273, 161 (1978). ~9 M. Berelowitz, D. Dudlak, and L. Frohman, J. Clin. Invest. 69, 1293 (1982). 2o j. Delfs, R. Robbins, J. L. Connolly, M. Dichter, and S. Reichlin. Nature (London) 283, 676 (1980). 21 R. A. Peterfreund and W. W. Vale, Brain Res. 239, 463 (1982). 22 M. F. Scanlon, R. J. Robbins, J. L. Bolaffi, 1. M. D. Jackson, and S. Reichlin, Neuroendocrinology 37, 269 (1983). 23 R. J. Robbins, J. Neurochem. 40, 1430 (1983). 24 R. J. Robbins and R. M. Landon, Brain Res. 273, 374 (1983). 2~ R. J. Robbins and R. M. Landon, Brain Res. 332, 161 (1985). 26 H. F. DeLuca, in "Manometric and Biochemical Techniques" (W. W. Umbreit, R. H. Burris, and J. F. Stauffer, eds.), p. 146. Burgess, Minneapolis, Minnesota, 1972.

418

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[30]

Acetic acid, 1 M (1 M ACOH): to extract somatostatin from harvested cells. Acetic acid, 10 M (10 M ACOH): to acidify medium and KRB, mixed at a 1 : 10 ratio (10 N ACOH : sample).

Experimental Protocol 1. On the twelfth or thirteenth day in vitro, remove culture flasks from the incubator and pour off the medium. 2. Wash each culture three times with 2 to 5 ml equilibrated KRB. To avoid washing the cells off the culture surface, the flask should be turned over so that the injected KRB hits the opposite side of the vessel. A repeating pipettor that can deliver large volumes (Eppendorf Repeater 4780, Brinkman Instruments, Westbury, NY) facilitates rapid processing throughout the experiment. 3. Add 4 ml KRB to each flask and incubate cultures for 30 rain at 37°, 5% CO2 and 100% humidity. This preincubation is an optional step to provide for a more stable baseline. 4. Remove cultures from incubator and wash three times as in step 2. 5. Add 4 ml KRB and incubate 10 min. (This is the first epoch of the experiment.) 6. Pour incubate from each flask into a 13 x 100 mm disposable glass culture tube containing 0.4 ml 10 M ACOH, heat the samples at 90° for 10 rain to inactivate peptidases, and freeze the samples at -20 °. 7. Repeat steps 5 and 6 three times. 8. Add 4 ml of experimental KRB (KRB containing the substance to be tested) to each flask, incubate l0 min, and repeat step 6. 9. The cells may be harvested by adding 2 ml 1 M ACOH to the flask and scraping the cells off the culture surface with a rubber policeman. The samples are centrifuged in 13 x 100 mm tubes at 2000 g for 10 min and the supernatents are transferred to clean tubes and are heat-inactivated and frozen along with the incubates. This protocol has been used successfully to test the effect of a single test substance on the release of SS (Fig. 2). For each release experiment, one set of cultures serves as a parallel control group and does not receive the test substance, each I0 min epoch acts as a consecutive control for the following incubation, and each culture can function as its own control if the SS release is expressed as a percentage of epoch 4 (baseline) release. This specific paradigm may be changed in an infinite number of ways to suit the experimenter. Ultimately, under any paradigm, it is important to achieve a stable rate of baseline release, allow enough time for each epoch

[30]

SOMATOSTATIN RELEASE FROM CORTICAL CULTURES 250

419

.T_

2OO

.~

iiiiii

E 50

1

2

3

4

5

I

2

3

4

5

Epoch

FI6. 2. Characteristic IRS release from cortical cultures (10v cells per flask, day 13 in vitro). Bars represent the mean (-+SEM) of IRS released during each epoch of 10 rain. (A) The cultures were bathed in normal KRB for the first four epochs, and then the cells were exposed to KRB supplemented with 0.1 mM glutamic acid for the fifth epoch. (B) The cultures were bathed in KRB without calcium for four epochs and then the cells were exposed to KRB minus calcium plus 0.1 mM glutamic acid during the fifth epoch.

to yield measurable levels of SS, and to be able to demonstrate that the culture is viable posttreatment (e.g., high potassium will stimulate SS release above baseline, cells appear viable by trypan blue exclusion, or cultures will release basal levels of SS hours after the experiment). Measuring Somatostatin Content A r a d i o i m m u n o a s s a y (RIA) for m e a s u r i n g i m m u n o r e a c t i v e s o m a t o s t a t i n (IRS) w a s d e v e l o p e d initially b y A r i m u r a a n d c o - w o r k e r s 1.27a n d t h e n b y o t h e r s z8,29 (also see r e v i e w b y Patel a n d Reichlin3°). T h e s e n s i t i v e a n d specific R I A u s e d in this l a b o r a t o r y 29 r e c o g n i z e s m o r e t h a n o n e f o r m of IRS: the s m a l l e s t t e t r a d e c a p e p t i d e , a 2 8 - a m i n o acid p e p t i d e , a n d larger p r o h o r m o n e f o r m s . 9,2° A n y r e f e r e n c e to IRS thus implies m e a s u r e m e n t of 27A. Arimura, H. Sato, D. H. Coy, and A. V. Schally, Proc. Soc. Exp. Biol. Med. 148, 784 (1975). 28 S. Kronheim, M. Berelowitz, and B. L. Pimstone, Clin. Endocrinol. 5, 619 (1976). 29y. Patel and S. Reichlin, Endocrinology 102, 523 (1978). 30 y. C. Patel and S. Reichlin, in "Methods of Hormone Radioimmunoassay" (B. M. Jaffe and H. R. Behrman, eds.), p. 77. Academic Press, New York, 1979.

420

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[30]

all of the above forms. The principal requirements for the assay are radiolabeled trace and suitable antiserum.

Preparation of SS Trace lodination Reagents Column: Sephadex G-25 (bead size 20-80/zm) in a 2.5 x 20 cm glass column, maintained at 4 ° . Column buffer: 0.1 M acetic acid, 0.1% bovine serum albumin (BSA), degassed, and stored at 4 °. Peptide: 2.5 p,g (Tyrq-somatostatin 14 (Peninsula, San Carlos, CA) in 10/zl column buffer. The peptide can be solubilized, aliquoted and stored at - 2 0 ° for 6 months. Phosphate buffer: 0.5 M PO4 buffer (0.5 M Na2HPO4, pH adjusted to 7,5 with 0.5 M NaH2PO4). 0.1 M acetic acid, 5% BSA. ~2~I: 1 mCi sodium iodide-125, carrier-free, 350-600 mCi/ml (Amersham, Arlington Heights, IL). Chloramine T: 10/zg of 10/zg/10 ml dH20 solution. Iodination Procedure. Under an approved fume hood, (TyrJ)-SS is iodinated by a modified Chloramine T method. 3j The peptide is mixed with 10/,d 0.5 M PO4 buffer and 1 mCi NaJ25I in a 0.5 ml microcentrifuge tube, and the tube is covered with Parafilm. The Chloramine T is added, the Parafilm is replaced, and the reaction vial is tapped gently for 20 to 30 sec. The reaction is stopped by adding 100/.d 0.1 M acetic acid with 5% BSA. The mixture is immediately applied to the G-25 column and eluted with column buffer. Iodinated albumin and aggregate labeled peptide combine to elute as a void volume peak (Vo) and unreacted iodide follows as a salt volume peak (Vi), Labeled somatostatin (J25I-labeled SS) interacts with the Sephadex beads and elutes beyond the Vi as a broad peak (see Fig. 1, Patel and Reichlin29). Suitable trace for RIA is taken from the descending shoulder of this peak. Several fractions are pooled and 100-p,1 aliquots are stored at - 2 0 ° for 6 to 8 weeks for routine use. The quality of the radioligand may be assessed by the t a l c - r e s i n - T C A test. 32 Usable SS trace should meet the test criteria of <25% resin binding, >90% talc adsorption, and >90% TCA precipitation. 3~G. C. Greenwood, W. M. Hunter, and J. S. Glover, Biochem. J. 89, 114 (1963). 32B. B. Tower, M. B. Sigel, R. E. Poland, W. P. VanderLaan, and R. T. Rubin, this series, Vol. 70, p. 322.

[30]

SOMATOSTATIN RELEASE FROM CORTICAL CULTURES

421

Radioimmunoassay of SS RIA Reagents RIA buffer: 0.05 M PO4 buffer, 0.5 M PO4 buffer (see lodination Reagents) diluted 1:10 with dH20, 0.1% sodium azide, 0.1% RIA grade BSA, 0.25 M EDTA (disodium form). Adjust pH to 7.5, store at 4°. Charcoal slurry: 0.07 M barbital buffer (0.012 M barbital plus 0.058 M sodium barbital, pH 8.6), 0.025% RIA grade BSA, 0.05% Norit " A " charcoal. Store at 4° . SS trace: 125I-labeled SS is prepared as described above. It is diluted in cold RIA buffer and free ~25I is removed by mixing 300 mg Dowex-1, chloride form (1X8-400, Sigma, St. Louis, MO) with every l0 ml diluted trace. The trace-Dowex is mixed by vortexing and is centrifuged at 2000 g for 7 min. The supernatent should yield 4000-5000 cpm per 50-pA aliquot. SS antibody: Rabbit antiserum #693 is provided in lyophilized form courtesy of Dr. Seymour Reichlin, Tufts-New England Medical Center, Boston, MA (see Patel and Reichlin 29 for details of development of antisera). One capsule of 100 pA lyophilized #693 is diluted to 10.0 ml (1 : 100) 0.05 M PO4, 0.1% BSA, 0.1% sodium azide. It is stored at -20 ° in 100-tA aliquots. At time of assay, each aliquot is diluted in 10 ml (1 : 10,000 final dilution) of cold RIA buffer. The final titer in the assay tube is ! :55,000. SS standards: Synthetic cyclic somatostatin 14 (Peninsula) is dissolved in 0.01 M acetic acid, 0.1% BSA to 200 ~g/ml. Standards are diluted in 0.05 M PO4 buffer, 0.1% BSA, 0.!% sodium azide, pH 7.5, to concentrations ranging from 40 to 2560 pg/ml. The standards are f~rozen in 500- to 600-/A aliquots for one-time use. RIA Procedure. The samples are prepared in duplicate for RIA by thawing and aliquoting the optimal volume into 12 × 75 mm glass tubes. The samples, along with tubes for the standards (containing the corresponding volume of acidified KRB) are lyophilized in a Speed Vac concentrator (Savant Instruments, Farmingdale, NY). On the first day of the assay, the RIA buffer, standards, antibody, and trace are added to the assay tubes according to Table i. All tubes are vortexed and refrigerated for 60-72 hr. On the fourth day, 1 ml of charcoal slurry (the slurry should be mixed continuously) is added to all samples except total counts. The tubes are incubated 20-45 min and centrifuged at 2000 g, 4°, for 15 min. The supernatent is aspirated from each tube and the charcoal pellets are counted in a gamma counter. Results are calculated using a log/logit RIA program available for a Hewlett-Packard HP-41C calculator (Fig. 3). The intraassay coefficient of variation is determined using replicate measurements

422

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[30]

TABLE |

SOMATOSTATIN RADIO1MMUNOASSAY C o n t e n t s of i n c u b a t i o n t u b e s (/~l) Sample

R I A buffer

Anti-SS

Trace

Standard

Total counts Nonspecific binding Standards Unknowns

-400 350 400

--100 100

50 50 50 50

--50 --

(n= 10) over the range of standards normally used in the assay and is <2% over the range of 2-128 pg/tube. The interassay coefficient of variation, as calculated for three standards is 3% for 4 pg/tube (B/Bo of 0.775), 5% for 16 pg/tube (B/Bo of 0.488), and 13% for 64 pg/tube (B/Bo of 0.207) (n=8 for each calculation). The effects of all solutions and experimental substances on the RIA must be tested. Measuring the effect on nonspecific binding and total binding may be considered sufficient, but determining the effect over the entire range of standards proves to be more informative. When assaying release incubates, tubes for the standard curve are prepared by drying +3.0

,2.0

+1.0 o O.0

1.O

-2.0

-3.0 4

8

1

32

128

Log ConcentrationIN/tubal

FIG. 3. L o g / l o g i t plot of a t y p i c a l s o m a t o s t a t i n s t a n d a r d c u r v e u s i n g s y n t h e t i c c y c l i c s o m a t o s t a t i n for s t a n d a r d s .

[30]

SOMATOSTATIN RELEASE FROM CORTICAL CULTURES

423

down the volume of acidified KRB corresponding to that of the unknowns (see RIA procedure). Thus, any interference with binding due to the KRB salts or the tested substance is accounted for in every tube. An experimental substance may interfere severely enough to increase the nonspecific binding to a higher than acceptable value or it may shift the standard curve in a nonparallel manner. The volume of sample necessary to obtain measurable IRS levels may be restrictive because of the increased amount of salts remaining in the tube after lyophilization. Serum-containing medium also causes interference when volumes exceeding 100/~1 are to be assayed. In all the aforementioned situations, a preassay sample cleanup is warranted. Reverse-phase liquid chromatography using octadecasilyl silica (Sep-Pak Cjs, Waters Associates, Milford, MA; TABLE II DEVELOPING A PRIMARY CULTURE SYSTEM FOR MEASURING THE RELEASE OF A PEPTIDE

Phase Primary culture

Release experiment

Radioimmunoassay ,,.b

Factors of concern Age of embryo Brain region to isolate for culture Dispersal technique Growth conditions Choice of medium, serum, culture surface Age of culture Experimental paradigm Preincubation Length of incubation epochs Controls: parallel and consecutive Choice of incubation medium Sample treatment Acid extraction Heat inactivation Producing suitable antibody lodination of peptide Choice of iodination method Choice of gel for separation Evaluation of trace Titer of antibody to use in assay Incubation of assay tubes: time and temperature Time of addition of trace Separation method

" J. I. Thorell and S. M. Larson, "Radioimmunoassay and Related Techniques." Mosby, St. Louis, Missouri, 1978. b W. D. Odell and P. Franchimont, "Principles of Competitive Proteinbinding Assays." Wiley, New York, 1983.

424

QUANTITATION OF NEUROENDOCRINE SUBSTANCES

[31]

Bond-Elut Cl8, Analytichem International, Harbor City, CA) selectively adsorbs peptides. 33 This procedure can be employed to remove interfering substances and/or concentrate large volume samples. Summary Primary monolayer cultures of dispersed fetal cerebral cortical cells can be used to measure the release of the neuropeptide, somatostatin. Three to five percent of cellular IRS is released basally into KRB in 10 rain. Basal release is stable for at least 60 rain and stimulated levels of release can be induced by introducing ionophores, lj neurotransmitters, 1°,24 or peptides, z5 The peptide content of the incubation samples is readily measured by a well-characterized, sensitive RIA. Table II summarizes the major factors that must be taken into consideration when developing this system for measuring peptide release. Acknowledgments This research was supported by N1H Grant AM 20451. The authors wish to thank Dr. Seymour Reichlin for the gift of antiserum and Dr. John W. Leidy, Jr. for his advice regarding radioimmunoassay. We further recognize the secretarial assistance of Ms. Wendy Hall. 33 H. P. J. Bennett, A. M. Hudson, L. Kelly, C. McMartin, and G. E. Purdon, Biochem. J. 175, 1139 (1978).

[31] M e a s u r e m e n t o f P h o s p h o l i p i d T u r n o v e r in C u l t u r e d H o r m o n e R e s p o n s i v e P i t u i t a r y Cells

By THOMAS F. J. MARTIN Increased turnover of inositol phospholipids results from occupancy of receptors for numerous hormones and neurotransmitters. The proximal event induced by hormone receptor binding appears to involve activation of polyphosphoinositide hydrolysis by a phospholipase C-type reaction. The products [(diacylglycerol (DG) and inositol trisphosphate (Ins P3)] of phospholipid degradation have been implicated in second messenger functions. The polyphosphoinositides (PtdIns-4-P and PtdIns-4,5-P2) are minor constituents ( - ! % of phospholipids) and studies of hormone-regulated turnover frequently necessitate the use of radioisotopic methods. As METHODS IN ENZYMOI.OGY. VOL. 124

Copyright ct) 1986by Academic Press, lnc. All rightsof reproduction in any form reserved.