0013-7227/79/1056-1382$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society

Vol. 105, No. 6 Printed in U.S.A.

Concentrations of Epidermal Growth Factor, Nerve Growth Factor, and Submandibular Gland Renin in Male and Female Mouse Tissue and Fluids* YUKIO HIRATA AND DAVID N. ORTH Departments of Medicine and Physiology and the Cancer Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

ABSTRACT. The submandibular gland of the mouse contains

difference, and neither radioimmunoassayable NGF nor SGR

a variety of hormone-like substances and enzymes, such as epidermal growth factor (EGF), nerve growth factor (NGF), and submandibular gland renin (SGR). Using specific RIAs for each of these proteins and a radioreceptor assay (RRA) for EGF, the present study was performed to examine the concentrations of these three proteins and the molecular size of EGF in the submandibular glands and biological fluids of normal male and female mice. Submandibular glands of male mice contained far more radioimmunoassayable EGF, NGF, and SGR than those of female mice (66-, 92-, and 30-fold, respectively). Male saliva also contained far higher concentrations of radioimmunoassayable EGF, NGF, and SGR than that of the female (38-, 56-, and 49fold, respectively). Furthermore, the molar concentrations of these proteins appeared to be similar to one another in submandibular gland tissue and in saliva. In contrast, although high concentrations of radioimmunoassayable and radioreceptor assayable EGF were found in urine, there was no significant sex

were detectable in the urine of either sex. Milk contained smaller

T

HE SUBMANDIBULAR glands of mice contain a variety of biologically active peptides and peptidases: 1) epidermal growth factor (EGF; mol wt, 6,045), a potent stimulator of growth in a variety of tissues (13); 2) /?-nerve growth factor (NGF; a dimer with a mol wt of 26,000), which stimulates growth and maintenance of sympathetic neurons and development of sensory neurons (4-6); 3) submandibular renin-like enzymes (SGR; mol wt, 37,000), which can elevate blood pressure by specifically cleaving renin substrate to yield angiotensin I (7); and 4) specific arginine esteropeptidases, two different examples of which (mol wt, 26,000 and 29,000, respectively) are found associated with EGF and NGF in high molecular weight (HMW) complexes (8-10). Submandibular gland production of all of these substances appears to be under androgenic control, since levels in Received January 16, 1979. Address requests for reprints to: Dr. David N. Orth, Room A4215 Medical Center, Vanderbilt University, Nashville, Tennessee 37232. * This work was supported in part by NIH Research Grants 1-R01AM-19739 and 5-P17-HL-14214 and National Cancer Institute Research Grant 5-R25-CA-19429.

amounts of these three proteins than did saliva. Gel exclusion chromatography revealed that the major component of EGF in the glands and fluids had equal RIA and RRA activities and was similar in molecular size to mouse EGF standard (mol wt, 6045). A high molecular weight EGF was observed in the tissue extracts and urine and appeared, at least in tissue, to be EGF noncovalently bound to other proteins. These data confirm the sexual dimorphism of the submandibular gland of the normal mouse with respect to its content of EGF, NGF, and SGR and indicate that all three proteins are secreted into saliva in concentrations reflecting their concentrations in the submandibular gland. The roughly equimolar concentrations of these proteins in the gland and saliva suggest that they may be derived from a common precursor whose synthesis is regulated by a single gene. (Endocrinology 105: 1382, 1979)

normal males greatly exceed those in normal females, levels in castrated males fall to those of normal females, and levels in androgen-treated females quickly reach those of normal males (11-15). However, the exact interrelationships between synthesis and secretion of EGF, NGF, and SGR remain unclear. The present study was performed to confirm the sex dependency of production of these three submandibular gland proteins, to examine the concentrations of NGF and SGR relative to those of EGF in the submandibular glands and biologicalfluidsof normal male and female mice, and to determine the molecular size of EGF in mouse tissues and fluids. Materials and Methods Animals Adult male, female, and lactating 10-day postpartum Swiss albino mice, weighing 35-40 g, were studied. Collection of samples Submandibular glands were excised from five adult male and female mice after decapitation. The combined tissues were

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EGF, NGF, AND SGR IN MOUSE TISSUE AND FLUIDS homogenized in saline (9 ml/g wet tissue) in a Waring blender (Waring Products Division, Dynamics Corp. of America, New Hartford, CT) and centrifuged at 16,000 X g for 20 min at 4 C. The supernates were stored at —70 C until assayed. Urine was collected via spontaneous voiding which occurred during routine handling of the animals. Saliva was collected after hypersalivation was induced by ip injection of 5 /xg carbachol in animals that had been lightly anesthesized with sodium pentobarbital. Saliva was collected in a microcapillary tube placed between the tongue and the floor of the mouth. Milk was collected from lactating mice after they had been removed from their litters for 12 h and injected sc with 0.1 U Pituitrin (Parke-Davis, Detroit, MI). Ten minutes later, their teats were cleaned with alcohol and sucked with a vacuum milking device. All specimens were diluted with RIA standard diluent [50 mM Na2HPO4/KH2PO4 and 77 mM NaCl buffer, pH 7.4 (PBS), containing 0.1 mg/ml merthiolate and 5 mg/ml bovine serum albumin (BSA)] and stored at —70 C until assayed. RIA and radioreceptor assay (RRA) of EGF RIA was performed as previously described (11), except that separation of antibody-bound from free 125I-labeled mouse EGF (mEGF) was achieved by the dextran-coated charcoal method (16) rather than by paper electrophoresis or the solid phase system. Highly purified mEGF (kindly supplied by Dr. S. Cohen) (2) was used both as standard and as labeled tracer (SA, ~200 juCi/ju,g). The rabbit anti-mEGF serum was the same as that previously described (11). It was used at a final dilution of 1:800,000. Total volume of the incubation mixtures was 0.4 ml, which contained 0.1 ml standard or sample, 0.2 ml antiserum and ~8,000 cpm labeled mEGF, all in RIA standard diluent. Incubation was carried out at 4 C for 3 days, after which 1 ml of ice-cold standard diluent was added to each tube, followed immediately by another 1 ml ice-cold standard diluent containing 20 mg charcoal (Norit A, Fisher Scientific Co., Waltham, MA) and 5 mg dextran (Dextran T-70, Pharmacia Fine Chemicals, Piscataway, NJ). The tube was agitated vigorously on a vortex mixer and centrifuged at 6,000 X g at 4 C for 20 min. The supernate was decanted, and the precipitate, which contained the free hormone, was counted in a y-scintillation spectrometer (MS-588, Micromedic Systems, Inc., Horsham, PA). Results were calculated using a standard RIA program on a Wang 2200 programmable calculator (Wang Laboratories, Inc., Tewksbury, MA) equipped with a tape reader. The interassay and intraassay coefficients of variation were 13.7% and 12.9%, respectively. RRA of EGF was performed using a human placental membrane fraction, as previously described (17). The same mEGF preparation employed in RIA was used as both standard and 125 I-labeled tracer. The RRA standard diluent was 50 mM TrisHC1 buffer, pH 7.4, containing 2.5 mg/ml BSA. Total volume of the incubation mixtures was 0.3 ml, which contained 0.1 ml standard or sample, 0.1 ml suspended membrane fraction (25 ju.g protein), and ~ 100,000 cpm 125I-labeled mEGF, all in standard diluent. Incubation was carried out at 24 C. After a 10-min preincubation, labeled tracer was added to each tube and the mixture was further incubated for 40 min. After incubation, the

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tube was centrifuged, and the pellet containing the bound [125I]mEGF was washed once in standard diluent and counted in a y-scintillation spectrometer. Specific binding (~10% of total radioactivity) was defined as the difference between the radioactivity in the assay tube and that in a control tube containing excess unlabeled mEGF (0.1 — 1 jug); nonspecific binding was always less than 1% of the total binding. Interassay and intraassay coefficients of variation were 14.4% and 9.7%, respectively. RIA of NGF Highly purified mouse /?NGF (mNGF), 12f)I-labeled mNGF (SA, 60 ju.Ci/jug), and rabbit anti-mNGF serum were obtained from Collaborative Research, Inc. (Waltham, MA). The procedure for NGF RIA was similar to that of EGF, except that the double antibody separation method was used. Total volume of the incubation mixtures was 0.4 ml, which contained 0.1 ml standard or sample, 0.2 ml antiserum (final dilution, 1:4,000), and -10,000 cpm 125I-labeled mNGF, all in RIA standard diluent containing 1% normal rabbit serum. Incubation was carried out at 4 C for 3 days, after which 0.1 ml goat antirabbit yglobulin serum was added to each tube, and the mixture was further incubated overnight. After completion of incubation, 1.5 ml ice-cold standard diluent were added, and the tube was centrifuged at 6,000 x g at 4 C for 20 min. The supernate was carefully decanted, and the precipitate, which contained the bound [125I]mNGF, was counted in a y-scintillation spectrometer. Interassay and intraassay coefficients of variation were 17.5% and 10.6%, respectively.

RIA of submandibular gland renin The RIA procedure for mouse submandibular gland renin (mSGR) was similar to that reported by Michelakis et al. (13). Highly purified mSGR (kindly supplied by Dr. T. Inagami) (7) was used both as standard and labeled tracer. The partially purified y-globulin fraction of sheep anti-mSGR serum, obtained by DEAE-cellulose chromatography (Dr. T. Inagami), was dissolved in distilled water (1 mg protein/ml). Total volume of the incubation mixtures was 0.4 ml, which contained 0.1 ml standard or sample, 0.2 ml antiserum (final dilution, 1:80,000), and -10,000 cpm 125I-labeled mSGR (SA, 140 jLiCi/jttg), all in RIA standard diluent containing 0.02% normal sheep plasma. Incubation was carried out at 4 C for 3 days, after which 0.1 ml rabbit antisheep y-globulin serum was added to each tube, and the mixture was further incubated overnight. The remaining steps were identical to those for NGF RIA. Interassay and intraassay coefficients of variation were 10.7% and 10.4%, respectively. Sephadex G-50 gel exclusion chromatography To evaluate the molecular size of mEGF in submandibular gland extracts and biological fluids, a 0.9 X 58-cm column was packed with Sephadex G-50 fine resin (Pharmacia) equilibrated and developed at 4 C with PBS containing 0.1 mg/ml merthiolate. Samples were applied to the column and eluted by descending flow (15 ml/h; 40 cm hydrostatic pressure); 1-ml fractions were collected. Concentrations of mEGF in the eluates

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Endo • 1979 Vol 105 • No 6

HIRATA AND ORTH

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were determined both by RIA and RRA. Recovery of labeled mEGF added to the column was 80-90%.

125

I-

Results

EGF in submandibular gland extracts and biological fluids The RIA competitive binding curves generated by extracts of the submandibular glands of male and female mice and by mouse saliva, urine, and milk were all parallel to that of standard mEGF (Fig. 1A). Mouse NGF, SGR, or other known hormones did not react in the EGF RIA (data not shown). As shown in Fig. IB, the competitive binding curves of the submandibular gland extracts and biological fluids were also parallel to that of standard mEGF in the RRA. There existed significant correlation between the values obtained by RIA and RRA (Table 1) of male urine (r = 0.916; P < 0.01; n = 9), female urine (r = 0.933; P < 0.01; n = 9), male saliva (r = 0.886; P < 0.01; n = 6), and milk (r = 0.568; P < 0.05; n = 10).

However, the HMW mEGF in the submandibular gland extract was not observed when the column was developed with 0.05 M ammonium bicarbonate, pH 8.0 (data not shown), suggesting that the apparent HMW form of i.o SUBMANDIBULAR GLAND ( ? )

0.8

0.6 STANDARD mEGF

0.4

O.2

0

•r i.o 0.8

0.6

Molecular size of EGF in submandibular gland extracts and biological fluids The elution profiles of an extract of male submandibular gland and of male urine, male saliva, and milk from the Sephadex G-50 fine gel column are shown in Fig. 2. The major component in both tissue and fluids, measured both by RIA and RRA, coeluted with mEGF standard (mol wt, 6,045). In submandibular gland and urine, a minor mEGF component eluted in the void volume of the column (mol wt, >30,000). This HMW mEGF appeared to have equal activity in both RIA and RRA.

0.4

0.2 •

0.02

0.05

0.1

0.2

0.5

I

2

5

10

20

50

ng mEGF, p\ FLUID, OR /jg WET TISSUE ADDED PER TUBE

FIG. 1. Competitive binding curves generated by mouse submandibular gland extracts and biological fluids in mEGF RIA (A) and EGF RRA (B). The same tissue extracts and fluid specimens (O), standard mEGF (•), and l25 I-labeled mEGF tracer were used in both assays; the amounts added per assay tube are plotted on a logarithmic scale.

TABLE 1. Concentrations of EGF, NGF, and SGR in mouse tissue extracts and biological fluids

EGF

Submandibular gland" Male (5) Female (5) Urine Male (9)" Female (9) Saliva Male (6) Female (6) Milk (10)

NGF

SGR

RIA

RRA

RIA

RIA

0.331 0.005

0.529 0.005

0.461 0.005

0.451 0.015

0.235 ± 0.033'' 0.304 ± 0.069

0.356 ± 0.066 0.374 ± 0.084