Biosynthesis of /3 Nerve Growth Factor in Mouse Submaxillary Glands*

The biosynthesis of /3 nerve growth factor (PNGF) was studied in isolated mouse submaxillary glands incubated with ~+Wcystine. of anti-/3NGF immunoprecipitates labeled homogenates a single major peak of radioactivity, which purified was nearly completely precipitated the of equivalent amounts of anti+NGF, was absent from immunoprecipitates obtained by the addition of ferritin plus anti-ferritin. The cystine-containing tryptic peptides of the labeled species appeared identical with those of purified PNGF. to

The biosynthesis of /3 nerve growth factor (PNGF) was studied in isolated mouse submaxillary glands incubated with ~+Wcystine.
Sodium dodecyl sulfate gels of anti-/3NGF immunoprecipitates from labeled gland homogenates showed a single major peak of radioactivity, which comigrated with purified BNGF. This species was nearly completely precipitated by the addition of equivalent amounts of anti+NGF, but was absent from immunoprecipitates obtained by the addition of ferritin plus anti-ferritin. The cystine-containing tryptic peptides of the labeled species appeared identical with those of purified PNGF.
In submaxillary glands from adult male mice, labeling of /3NGF represented approximately 0.2% of the trichloroacetic acid-precipitable radioactivity. Castration reduced this value to one-third, while testosterone treatment of castrated animals restored the relative /3NGF synthesis to normal or more. No pNGF synthesis could be detected in glands from female animals.
Several tissues were examined for their ability to synthesize /3NGF in culture. Only the submaxillary gland incorporated detectable amounts of radioactivity into PNGF. Labeling of /3NGF could also be obtained by direct injection of isotope into the submaxillary gland in uiuo. The results are discussed in terms of the integration of /3NGF synthesis into neuronal development and maintenance. now been detected in a variety of vertebrates (1,2) and is generally recognized to play an important role in the development and maintenance of the sympathetic nervous system, and in the development of the sensory ganglia as well (1).
In the male mouse, the bulk of the NGF is found in the submaxillary gland (3) in the form of specific high molecular weight complex called 7 S NGF (4,5). The complex can be dissociated into three classes of subunits (a, p, and y) which have been purified (6). All the nerve growth-stimulating activity resides in the /3 subunit (PNGF), which is a dimer of two identical noncovalently associated chains (M, 13,259) (7-9) whose amino acid sequence has been determined (9). The y subunit is a potent arginyl esteropeptidase (10) and it has been proposed that it may function as a cleaving enzyme in the processing of a pNGF precursor (9,11). Support for this hypothesis has recently been obtained (12). No enzymatic activity has yet been found for the (Y subunit. Purified pNGF will elicit profound biochemical and morphological effects from responsive neurons. The well known stimulation of axonal outgrowth from cultured ganglia has proven useful as a bioassay for the factor (13), and pNGF has also been reported to influence the directionality of fiber outgrowth (14)(15)(16)(17). A variety of metabolic processes may be enhanced in the presence of PNGF, including cell enlargement and proliferation, glucose metabolism, RNA and protein synthesis, lipid synthesis Cl), adenosine 3':5'-monophosphate production (Ref. 18, but also see Refs. 19 and 201, uptake of certain small molecules (21), and assembly of neurotubules and neurofilaments (22). Despite this plurality of effects, it remains unclear how these diverse functions are integrated into the overall pattern of neuronal development and maintenance. Our knowledge of pNGF synthesis is particularly deficient, and the role of this factor cannot be fully ascertained without a precise description of where and when it is produced and how its synthesis and processing are regulated.
In this report, we show that isolated submaxillary glands synthesize pNGF de novo, and that the synthesis is regulated by steroid to minimize adsorption of pNGF to the glass and to the particulate fraction. The homogenates were centrifuged at 40,000 rpm in an A321 International rotor for 1 h at 4", and the resulting 105,000 x g supematants were analyzed by immunoprecipitation and trichloroacetic acid precipitation. For in viva labeling studies, the submaxillary gland was exposed and 1 mCi of L-[""Slcystine was injected directly into one lobe; 5 h later, the gland was excised and the injected lobe was homogenized and processed as described above. Isolation of pNGF by Immunoprecipitation-To determine the quantity of antiserum required to quantitatively precipitate 6NGF from tissue extracts, tracer quantities (2 to 3 ng) of l"SI-labeled pNGF were added to aliquots (20 ~1) of the 105,000 x g tissue supematants which were then titrated with increasing volumes of antiserum.
Nearly complete (90%) precipitation of '2sI-labeled pNGF was achieved at the optimal points, compared to precipitation of tracer 1~51-labeled pNGF added to solutions of purified pNGF in Buffer A.
For analysis of [Y?l]cystine incorporated into PNGF, aliquots (0.1 to 0.6 ml) of 105,000 x g supernatanta were diluted with 1 ml of Buffer A in siliconized conical glass centrifuge tubes. In cases where immunotitration indicated that the total quantity of 6NGF was less than 1 pg, purified pNGF (1 to 2 ng) was added as carrier.
The appropriate amount of antiserum was added and precipitation was allowed to proceed for at least 4 h at 4". The precipitates were collect,ed by centrifugation and washed once by resuspension and centrifugation in 1 ml of Buffer C (Buffer A supplemented with 10 pg/ml of L-cystine).
The precipitates were dissolved in 0.5 ml of 0.1 N NaOH and then neutralized with 0.5 ml of 0.1 N HCl. Then 0.11 ml of Buffer D (lo-fold concentrated suspension of Buffer A) was added, followed by a volume of antiserum identical with that used in the initial precipitation, plus an equivalent amount of unlabeled PNGF.
Following incubation for at least 4 h at 4", the immunoprecipitates were isolated by centrifugation and suspended in 1 ml of Buffer C. The suspensions were then quantitatively transferred to fresh siliconized tubes and centrifuged to obtain the washed immunoprecipitates.
This method resulted in a good recovery of /3NGF with a minimum of nonspecific precipitation and trapping (see "Results," Figs. 2 and 4, and Table I of the radioactivity could be immunoprecipitated at equivalence. (The residual 10% remained soluble even after the addition of more pNGF plus an equivalent volume of antiserum and thus probably represents pNGF molecules whose antigenicity was altered by the iodination procedure.) The extent of precipitation of tracer amounts of 12"I-labeled pNGF is therefore a valid measure of the precipitation of total /3NGF and can be used as a probe to determine the volume of antiserum required to quantitatively precipitate pNGF from tissue homogenates, and to calculate the recovery of pNGF on immunoprecipitation and washing. Comparison of Washing Procedures for Zmmunoprecipitates Obtained from Labeled Tissue Extracts- Fig.  2A indicates that anti+NGF antiserum added to a 35S-labeled submaxillary gland 105,000 x g supernatant precipitates a major peak of radioactivity which co-migrates on SDS gels with purified PNGF. There is, however, considerable radioactivity in other gel fractions, particularly in regions corresponding to the major labeled peaks in a trichloroacetic acid precipitate (Fig.  3). This radioactivity most likely represents nonspecific contamination by labeled submaxillary gland proteins, and its proximity to the ,f3NGF position prevents accurate determination of the radioactivity in BNGF. As shown in Fig. 2 and Table I, the background can be markedly reduced by (a) dissolving the immunoprecipitate in NaOH and then reprecipitating by neutralization followed by the addition of buffer and additional carrier pNGF plus antiserum, and (b) the use of siliconized tubes and the transfer of the immtmoprecipitate to a fresh tube prior to centrifugation during the final wash. The reduction in background contamination is accomplished with only a minor sacrifice of /3NGF recovery (Table I), and this washing method was therefore employed in the experiments described below. It should be noted that even with this procedure, a considerable portion (30%) of the total radioactivity in the immunoprecipitate is nonspecific, and therefore accurate determination of /3NGF synthesis requires analysis of the precipitated label by gel electrophoresis.
Specificity of Zmmunoprecipitation -The results shown in Fig. 4A provide further evidence that the labeled peak at the pNGF position is in fact PNGF. When an immunoprecipitate was obtained from the supernatant of the first precipitation by the addition of carrier pNGF plus equivalent antiserum, little radioactivity was observed at this position. The peak in the initial immunoprecipitate, therefore, represents a molecule which was nearly completely removed by the first addition of antiserum. This agrees with the calculated completeness of pNGF immunoprecipitation (90%) determined by the precipitation of tracer lz51-labeled /3NGF immunoprecipitated from a separate aliquot of homogenate, and provides further confirmation of the accuracy of the equivalence point determined by immunotitration of tracer 1251~labeled pNGF added to gland homogenates. In a second control, addition of ferritin plus anti-ferritin in amounts sufficient to give a larger immunoprecipitate than that obtained with anti-pNGF did not precipitate the labeled peak at the pNGF position (Fig. 4A).
When tracer '251-labeled pNGF was added to a separate aliquot of gland supernatant and immunoprecipitated in ex- actly the same way, 90% of the radioactivity appeared in four peak gel slices at the /3NGF position (Fig. 4B) As expected from the primary structure of pNGF (9), there were five major half-cystine-containing peptides.
We conclude that isolated submaxillary glands are capable of de nouo synthesis of PNGF. In a series of five separate experiments with submaxillary glands from castrated mice treated with testosterone (see "Methods"), the relative incorporation of label into pNGF was 0.25% f 0.03% (SE.) of the label incorporated into trichloroacetic acid-precipitable material. It should be noted that this may by an overestimate of the true relative rate of synthesis since pNGF is relatively rich in Gcystine (9), the amino acid used in these labeling studies.
pNGF in Culture Medium after Tissue Incubation -Following the incubation described in Fig. 4, bovine serum albumin was added to the culture medium (final concentration 1 mgl ml) which was then exhaustively dialyzed against Buffer A to has not yet been demonstrated.  pNGF Synthesis in Vivo -When L-[35S]cystine was injected directly into the submaxillary gland of a living mouse and the immunoprecipitate analyzed, radioactive pNGF was observed (data not shown). The label represented 0.45% of the trichloroacetic acid-precipitable radioactivity in the gland supernatant.

DISCUSSION
The submaxillary gland of the adult male mouse has long been recognized for its high NGF content (3), yet the source of this NGF has not been directly determined. Several lines of evidence argue against NGF uptake by the gland from the serum. (a) NGF injected directly into the circulation does not accumulate in the submaxillary gland (29, 34), (b) radioactive amino acids injected unilaterally into one lobe of the submaxillary gland preferentially label the NGF from the injected lobe (35), (c) the NGF activity in venous blood emuent from the gland exceeds that in arterial blood aflluent to the gland (33), and (d) daily injection of anti-NGF does not prevent the testosterone-induced rise in submaxillary gland NGF content in female or young male mice (34).
It is often assumed on the basis of these criteria that submaxillary gland NGF is produced in situ, but more direct evidence has thus far been lacking and the question is still controversial (36,37). Levi-Montalcini and Angeletti (35) have reported that addition of anti-NGF antiserum to homogenates of submaxillary glands labeled in culture precipitates radioactivity, but neither analytical characterization of the labeled material nor controls for nonspecific precipitation were presented. In view of the relatively large amount of label precip-itated (4 to 6% of the trichloroacetic acid-precipitable counts compared to our observations of approximately 0.2% for specific incorporation into PNGF), one is not convinced that the label in their immunoprecipitates represents NGF synthesized in vitro.
The findings presented herein provide the most conclusive evidence for de nova production of pNGF by mouse submaxillary glands, and the negligible levels of /?NGF synthesis in other tissues examined (Table III) suggest that most if not all of the submaxillary gland @NGF is produced in situ. As anticipated from previous kinetic studies (29), the hormonal modulation of gland BNGF levels (3,29,32,33) correlates with direct measurements of /?NGF synthesis (Table II).
Still, the physiological function of the submaxillary gland /3NGF remains unclear. The report of Hendry and Iversen (38) that sialectomy induces a transient drop in serum NGF levels suggested that the gland is the source of NGF for the circulation. However, Murphy et al. (39) were unable to detect this decline and proposed instead "that the serum factor arises from multifocal cellular secretion." In favor of this view is the observation that circulating NGF levels are eventually restored after sialectomy without concomitant regeneration of the gland (38), as well as the apparent ability of many primary (40-43) and transformed (43-47) cell types to produce NGF in culture. Clearly, the question of an endocrine role for the submaxillary gland in NGF production can be ascertained only when the effects of sialectomy on serum NGF levels are resolved. Alternatively, the possibility of an exocrine role for NGF secretion into the digestive tract has been considered (35) and given recent support by the finding of high NGF concentrations in mouse saliva (39,48). It should be recalled that snake venom is another rich source of NGF (49), and that the venom gland is the phylogenetic homologue of the mammalian salivary gland.
Perhaps the most significant question concerning NGF synthesis is the mechanism by which the factor is delivered to those neurons which depend upon it for growth and survival. The problem is complicated not only by the multiplicity of potential synthesis sites, but also by the possibility of changing physiological sources at different stages of development. The suggestion of a subordinate role for circulating NGF has been put forth (21,50), and alternate supply routes have been postulated. Mouse (46) and human (51) neuroblastoma can apparently produce the factor, and several workers have emphasized the potential importance of glial cells in NGF production (21,44,47,52), although this has been vigorously challenged (53). We were unable to detect pNGF synthesis in mouse brain or spinal cord (Table III) but have not yet examined synthesis by sensory or sympathetic ganglia. A provocative alternative is the potential involvement of target organs in NGF production. It is clear that NGF can be specifically taken up by both sympathetic (54, 55) and sensory (55) nerve terminals and transported retrogradely back to the cell body, suggesting the possibility of a direct NGF supply between effector organ and innervating neurons. Consistent with this, NGF production has been reported in mouse adrenals (42) and rat irides (40) in vitro, although our methods failed to detect synthesis in cultured mouse adrenals or vas deferens (organs which receive sympathetic innervation).
The question becomes particularly intriguing in view of the apparent ability of NGF to attract growing sympathetic and sensory fibers both in vivo (14,16) and in vitro (15,17), and it has been proposed that NGF synthesis and release by an effector organ might ultimately determine that organ's density and of p Nerve Growth Factor pattern of innervation (38,55). It therefore becomes imperative to test directly whether tissues which ultimately become innervated by sympathetic or sensory fibers synthesize and release NGF during the period of innervation.
A positive correlation would lend strong support to the concept of NGF as not only an essential growth and maintenance factor for responsive neurons, but also as a critical trophic messenger between target organ and growing axon.