Journal of Neuroscience Research 33:282-288 (1992)

Developmental Changes in Nerve Growth Factor Level in Rat Serum K. Murase, R. Takeuchi, E. Iwata, Y. Furukawa, S. Furukawa, and K. Hayashi Department of Molecular Biology, Gifu Pharmaceutical University, Gifu, Japan

In serum, nerve growth factor (NGF) forms a complex with a,-macroglobulin (a2M), which formation inhibits the immunoreactivity between NGF and its antibodies. For measuring the serum level of NGF, it is thus necessary to liberate NGF from the NGF-a2M complex and prevent reformation of such complex. The pretreatment of rat serum with 1 M guanidine hydrochloride for a few hours and operation of the enzyme immunoassay (EIA) in the presence of guanidine hydrochloride provided a reliable means for determination of the NGF level in serum. By this procedure we followed the serum NGF level in rats developmentally. It increased from prenatal day 2 to postnatal day 5 and decreased slightly at postnatal week 3, thereafter remaining constant throughout adulthood. In pregnant rats, the NGF level in serum increased threefold to fivefold before birth and then decreased rapidly. These data suggest that serum NGF level may reflect the demand for this molecule during establishment of the peripheral nervous system. 0 1992 Wiley-Liss, Inc. Key words: nerve growth factor, a2-macroglobulin, serum protein, guanidine hydrochloride, enzyme immunoassay

INTRODUCTION Nerve growth factor (NGF) is known to be an essential neurotrophic protein (Mr = 26,000) for the development and maintenance of peripheral sympathetic and sensory neurons (Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980) and magnocellular cholinergic neurons in basal forebrain nuclei in the central nervous system (Hefti, 1986; Kromer, 1986; Williams et al., 1986). NGF is synthesized in the areas to which these neurons project their axons, and is transported to the neuronal cell bodies by retrograde axonal transport. The amounts of NGF in the tissues are extremely low, and the two-site enzyme immunoassay (EIA) is the only method capable of detecting NGF quantitatively (Murase et al., 1990; Korshing and Thoenen, 1983). Besides its effect on the nervous system, NGF 0 1992 Wiley-Liss, Inc.

might also exhibit biological effects on nonneuronal cells. For example, NGF causes a significant stimulation of growth and differentiation of eosinophils and basophildmast cells (Matsuda et al., 1988), causes degranulation of rat peritoneal mast cells (Pearce and Thompson, 1986), induces shape changes in platelets (Gudat et al., 198l), activates the classical complement pathway by specific substitution for C1 (Boyle and Young, 1982), and stimulates chemotactic migration of polymorphonuclear leukocytes (Boyle et al., 1985). These observations indicate that NGF may play an accessory role in wound healing (Matsuda et al., 1988). The presence of NGF in the bloodstream, however, has been controversial, as the previous assay methods failed to detect circulating NGF. NGF in serum interacts with high molecular serum proteins that interfere with the detection of NGF by EIA (Hogue-Angeletti, 1969; Suda et al., 1978). One protein that binds NGF in serum has been identified as a,-macrogloblin or a,M (Ronne et al., 1979; Koo and Stach, 1989). Scatchard analysis indicates that NGF binds with mouse a2M at two different types of binding sites (Kd value = 2.9 5 0.8 nM and 1.2 2 0.1 nM) (Koo and Stach, 1989). As a2M is abundant in serum (1.5-4.2 mg in human sera) and NGF is in low concentration in the body, it is likely that NGF released into the blood stream is completely absorbed by a,M. Only in mice NGF is detected in serum or plasma by radioimmunoassay (Aloe et al., 1986; Lakshmanan, 1986) or EIA (Shinoda et al., 1988). In this case, the circulating factor is considered to be derived from the submaxillary gland, which gland in mice contains an extremely large amount of NGF. Recently, we developed a more sensitive EIA using the high affinity between biotinylated anti-NGF antibody and streptoavidin-P-D-galactosidase (Murase et al., 1990). This EIA system permitted the determination of 0.02 pg NGF/assay well. We therefore succeeded in detecting NGF in rat serum, although the recovery of NGF Received February 25, 1992; revised April 22, 1992; accepted May 7, 1992. Address reprint requests to Kyozo Hayashi, Department of Pharmaceutics, Gifu Pharmaceutical University, 5-6-1 Mitahora-Higashi, Gifu 502, Japan.

Developmental Changes in Nerve Growth Factor

was estimated to be only 15.6% (Murase et al., 1990). In the present study, we examined methods to liberate NGF from the NGF-a,M complex in order to obtain a reliable value for the level of NGF in rat serum. With our method we succeeded in measuring the developmental changes in the serum level of this growth factor.

283

t

it

h

al

. n

2 1000

m P

MATERIALS AND METHODS Materials Mouse submaxillary gland NGF and anti-NGF antiserum were prepared as previously reported (Murase et al., 1990). Human a,M was purchased from Sigma, and marker proteins for column calibration came from Pharmacia. Bovine serum albumin (BSA) was a product of Armour; and Bio-Gel P-100, of Bio-Rad. All other chemicals were reagent grade. Two-Site EIA The EIA was based on the sandwiching of antigen between anti-NGF antibody IgG coated on polystyrene plates and biotinylated anti-NGF antibody IgG. The bound antibody complex was quantified with streptavidin linked-P-D-galactosidase (Murase et al., 1990). Serum Samples Wistar rats (both sexes) were used. In the case of embryos and newborn rats, blood was collected by decapitation; and in older animals, from the tail. The blood was allowed to clot by standing for 2 hr at room temperature and was then kept overnight at 4°C. Serum samples were obtained after centrifugation of the coagulated blood.

500

0

t

1,Tube number

Fig. 1. NGF level of each fraction obtained by gel filtration of a mixture of human a,M and mouse NGF on a Bio-Gel P-100 column. Sixteen nanograms of mouse NGF and 0.5 mg of human a,M were mixed, incubated at 37°C for 24 hr, then applied to a Bio-Gel P-100 column (1 .O X 56.0 cm) equilibrated beforehand with Buffer A, and eluted with the same buffer. Fractions of 0.6 ml each were collected at a flow rate of 3. I ml/hr. After gel filtration, each fraction was treated with 10 mM 2-mercaptoethanol at room temperature for 5 hr (o),or with 1 M guanidine hydrochloride at 37°C for 5 hr (A),or left untreated ( 0 ) and then subjected to EIA. Column calibration was performed with blue dextran (Vo), ovalbumin (45,000), chymotrypsinogen (25,000), cytochrome C ( 1 2,500), and glucose (Vt) as molecular weight markers.

(106:1), the recovery of NGF dropped to 23.8 ? 3.44%. The recovery of NGF in the presence of a,M in a lo7fold excess (10 mg a,M/ng NGF) was 15.7 2 3.86%. Preparation of Homogenate From Placenta These data suggest that NGF in serum binds with a2M The placenta was sonicated in cold 0.1 M Tris-HC1 and that the immunoreactivity of NGF is thus decreased buffer pH 7.6 containing 1 M NaCl, 2% BSA, 2 mM to such an extent that the detection of NGF becomes very ethylenediamine tetraacetic acid (disodium salt), 80 difficult. It is known that reductants and denaturants are eftrypsin inhibitory units of aprotinidliter, and 0.02% NaN, at 5% wet tissue weight per volume. The solution fective reagents for disaggregation of protein complexes. was centrifuged at 10,OOOg for 30 min, and the super- For example, platelet-derived growth factor (PDGF) obtained from human platelets was reported to easily form natant obtained was assessed for NGF content. a complex with a,M through a disulfide exchange reaction (Huang et al., 1984). Thus, we examined the effect RESULTS of a reductant, 2-mercaptoethanol, and that of a denaturMethod for Liberation of NGF From ant, guanidine hydrochloride. We applied a mixture of NGF-aU,MComplex NGF and a,M (1 :lo5) to a Bio-Gel P- 100 column, subAs human a2M is a homologue of murine macro- sequently treated each fraction with 10 mM 2-mercapglobulin (Hudson et al., 1987), we used human a,M in toethanol or 1 M guanidine hydrochloride, and then asthe present study to search for a method to liberate NGF sessed each fraction with our EIA system (Fig. 1). from the NGF-a,M complex in rat serum. When a mix- Standard mouse NGF was also treated with 2-mercapture of 100 p,g a,M/ng NGF ( lo5:1) was applied to the toethanol or 1 M guanidine hydrochloride for preparation EIA, the recovery of NGF was 58.8 9.66%; and when of the calibration curve (Fig. 2). If mouse NGF was the ratio was increased further to 1 mg a,Mlng NGF treated with 1 M guanidine hydrochloride, the detection

*

'i,

Murase et al.

284

-

1000

0

94 c

i-

. . . ... I

I

I

1

. . . .... I

I

10

. . . .... I

I

100

...

*....I

1

Wml Concentration of PN G F

* . .

>....I

9

10

ng/ml

Fig. 2 . Effects of 2-mercaptoethanolor guanidine hydrochloTube number ride on EIA system for mouse NGF. Standard mouse PNGF was treated with 10 mM 2-mercaptoethanol (a),or with 1 M Fig. 3. Elution profile of a mixture of human a,M and mouse guanidine hydrochloride (A),or left untreated (0)and then NGF after treatment with guanidine hydrochloride. A mixture subjected to EIA. Each point indicates the mean 2 SE of four of 5 ng of mouse NGF and 0.15 mg of human a,M was determinations. incubated at 37°C for 24 hr, treated with 1 M guanidine hydrochloride at 37°C for 5 hr, then applied to a Bio-Gel P-100 column equilibrated with Buffer A . Conditions and calibration limit of EIA was slightly lower than that in the non- markers were the same as those given in the legend of Figure 1, except the flow rate was 4.0 ml/hr. treated case. But we could detect it as low as 1 pg/ml. When a mixture of NGF and human a,M was applied to the gel filtration column (Bio-Gel P-loo), the excess NGF, which was not complexed with a,M, gave shown in Figure 3 , the elution profile of the mixture a similar elution profile as standard NGF alone, although pretreated with the denaturant was identical to that of some NGF-like immunoreactivity (NGF-a,M complex) NGF in the absence of a,M, indicating that NGF trapped was observed at the void volume as a small shoulder by a2M was efficiently disaggregated by the treatment (Fig. 1, closed circles). When each fraction was treated with guanidine hydrochloride. The recovery of NGF with 10 mM 2-mercaptoethanol, the amount of NGF at from the column was almost 100%. In order to determine whether the treatment with its intrinsic position remained unchanged, but NGF in guanidine hydrochloride was efficient to recover NGF fractions in the void volume was detected in an amount from rat serum, we incubated 10% rat serum in 1 M about three times greater than that found for the untreated guanidine hydrochloride for 5 hr at 37"C, and then subfractions (Fig. 1, open circles). And when each fraction jected it to our EIA system. The recovery of a constant was assayed in the presence of 1 M guanidine hydro(10 pg/ml) of NGF exogeneously added to the amount chloride, the recovery of NGF in the void volume fracserum was 70-80%. Figure 4 shows the elution profile tions was tenfold higher than that of the untreated fracof rat serum pretreated with 1 M guanidine hydrochlotions (Fig. 1, closed triangles). When treated with both reductant and denaturant, the amount of NGF in the void ride. NGF was seen only at the expected position (Figure volume fractions was the same as that in the presence of 4, closed circles), suggesting that NGF in serum was guanidine hydrochloride only (data not shown). These liberated from endogenous NGF-a,M complexes by the results suggest that NGF bound a,M noncovalently to treatment with the denaturant. form an NGF-a,M complex from which each component dissociates in the presence of denaturant. Further, Figure Measurement of NGF Levels in Sera From I demonstrates that the immunoreactivity of NGF to anti- Developing Rats NGF antibody in the EIA system decreased remarkably Once we had found a good method to liberate immunoreactive NGF from NGF-a,M complexes, we then by formation of the complex with a,M. To ascertain that the increase in NGF content in the investigated the developmental changes in NGF level in void volume is due to liberation of NGF from NGF-a,M sera of male and female rats. Table I shows the NGF complexes, we treated the mixture of NGF and a,M with level for male and female rats from 2 days before birth 1 M guanidine hydrochloride before gel filtration. As (-2d) to postnatal week 15 (P15w). At the earlier de-

Developmental Changes in Nerve Growth Factor

+* L L

@%!

P 7 15 1

-

2.0

Tube number

Fig. 4. Elution profile of rat serum treated with guanidineHCl. One milliliter of rat serum was lyophylized and diluted with 0.5 ml of Tris-HC1 buffer, pH 8.3, containing 1 M NaCl, 2% BSA, 2 mM EDTA, 80 trypsin inhibitory units of aprotinin/liter, and 0.02% NaN,. After treatment with 1 M guanidine hydrochloride at 37°C for 5 hr, the mixture was applied to a Bio-Gel P-100 column (0.7 x 42.0 cm) equilibrated with the above buffer, and eluted with the same buffer. Fractions of 0.6 ml were collected at a flow rate of 3.1 ml/hr. Calibration markers were the same as those given in legend of Figure 1 . TABLE I. NGF Levels and Their Male/Female Ratio in Rat Serum Age - 2d

-Id

Pod P5d Plw P2w P3w P4w P5w P6w P7 w P8 w P9w Plow Pllw P12w P13w P14w P15w

Male (pg/ml)

Female (pg/ml)

Ratio of malelfemale

436.00 f 137.00 385.60 C 52.00 417.61 t 180.70 505.55 134.40 785.44 t 118.90 618.70 -t 172.40 499.95 2 248.20 390.57 136.23 411.60 133.60 456.05 5 419.10 311.50 2 161.80 260.00 -t 8.28 155.17 t 133.70 62.93 t 22.81 107.43 C 44.85 117.68 2 52.21 143.48 -+ 63.03 138.13 2 39.05 140.18 t 20.73 148.70 48.76 148.56 C 45.19 149.59 36.60 210.30 78.56 232.77 -+ 55.38 205.31 85.59 179.10 i 70.41 151.67 t 34.32 147.60 t 55.24 114.18 t 28.96 115.76 t 17.00 129.37 t 9.25 118.60 22.45 173.80 2 80.42 163.33 -+ 53.45 126.70 t 11.17 75.90 f 0.93

*

* *

* *

* *

*

0.83 1.27 1.28 0.90 1.20 2.46 0.91 1.03 0.94 0.99 0.90 1.15 1.03 0.99 1.09 1.06 1.64

Values represent the means t SEM of four determinations.

velopmental stages, it was impossible to obtain sufficient quantities of serum. At -2d and - Id we did not distinguish between male and female among the animals from which we obtained the serum. Prenatally (-2d and - Id), the average NGF level in serum was approximately 400 pg/ml. The NGF level in the serum increased

285

markedly from approximately 400 pg/ml at - Id to about 650 pg/ml at postnatal day 5 (P5d) and decreased steeply between P1w and P4w. The NGF level in serum of either sex remained virtually unchanged between P4w and P15w (male: 103.4 f 34.4 pg/ml; female: 128.0 ? 26.3 pg/ml), although a slight gradual increase in the level occurred at P9w and Plow. The male/female ratio of NGF content in serum was almost 1.0, indicating no significant sex difference. So the data were pooled, and are shown in Figure 5. Since the NGF level in serum of the embryo was relatively high, we also measured the NGF level in the serum and placenta of maternal rats. Figure 6A shows that for 4 days before birth the NGF level in serum from pregnant rats increased to approximately 400 pg/ml, a value 3-5 times higher than that for normal adult rats. After the delivery, the NGF level in serum decreased rapidly, reaching the level found in adult serum, within I week. In this prenatal period, the placental content of NGF was approximately 3 ng/g wet tissue, and this high NGF level in the placenta coincided in time with that in serum (Fig. 6B). Considering that the NGF level of lung, kidney, or liver in mice is approximately 200-500 pg/g wet tissue, the placenta contains a much larger amount of NGF. The drop in placental NGF just before birth closely paralleled that in serum (Fig. 6B).

DISCUSSION It has been questionable as to whether or not NGF may play a role as a humoral factor, since no one could measure it by radioimmunoassay , enzyme immunoassay , or bioassay except in the case of the mouse. Our present study has provided the first successful measurement of the physiological level of NGF in rat serum by EIA. We achieved this success by liberating NGF from the NGFa,M complexes. It appears that the EIA after the treatment of samples with 1 M guanidine hydrochloride provides a reliable means for determination of the NGF level in serum. Using this method, we measured the developmental change of NGF levels in rat serum. We showed that serum contains a substantial level of NGF from 2 days before birth to 2 weeks after birth, which gradually declines to a constant level (about 120 pg/ml) by adulthood (Fig. 5 ) . The male/female ratio of NGF contained in serum remained virtually unchanged throughout life (Table I). The time course of the developmental changes in the NGF level in serum agrees well with that of the development of peripheral tissues innervated by sympathetic neurons (Rubin, 1985; Cochard et al., 1979; Teitelman et al., 1979; Iverson et al., 1976). Although the NGF level was high before birth, along with synapse formation it decreased after birth. NGF is synthesized in

286

Murase et al. 1000

I

900

800

700

z . h

600

500

Y

B

400

300 200 100

0

El PO

P5

P10

P15

age (week) Fig. 5. Developmental time course of NGF levels in sera of male and female rats. Each point indicates the mean of triplicate assays. Each horizontal line in the figure indicates the mean value. Each horizontal dotted line indicates the standard deviation.

Fig. 6. Developmental time course of NGF level in serum (A) and placenta (B) of pregnant rats. Each point indicates the mean of triplicate assays. The horizontal lines in B indicate the mean values.

the target tissues of sympathetic neurons, released into the extracellular space, binds to its specific receptor on the formed neurons, and is then retrogradely transported to the neuronal cell body. A part of the NGF may avoid being taken by axons and enter into the blood. An alternative explanation is that NGF is synthesized specifically at the blood vessels de novo. Blood vessels are a tissue densely innervated by sympathetic neurons, and are known to be one of the dominant sites of NGF synthesis (Shelton and Reichardt, 1984). Recent observations in vitro showed that NGF exhibits various biological effects on mast cells or lymphocytes (Matsuda et al., 1988; Pearce and Thompson,

1986; Boyle et al., 1985) and that low-affinity NGF receptors are expressed on rat spleen mononuclear cells (Thorpe et al., 1987). Thus, one biological effect of NGF in serum might be on the immune system. Another role of NGF in serum may be regulation of the amounts of NGF available for neurotrophic effects. A part of the NGF may be secreted into the blood in order to lower the amount reaching the neuronal cell body. NGF in serum forms a complex with a 2 M as do some growth factors such as fibroblast growth factor (FGF) (Phillip et al., 1989) or platelet-derived growth factor (PDGF) (Raines et al., 1984; Huang et al., 1984). a 2 M , which is widely distributed in tissues and body

Developmental Changes in Nerve Growth Factor

287

linergic neurons after fimbrial transections. J Neurosci 6:2 155fluid, is likely to have different binding sites for pro2612. teases and bFGF (Phillip et d., 1989). It could serve as R (1969): Nerve growth factor (NGF) from snake a carrier protein to protect the NGF molecule from pro- Hogue-Angeletti venom and mouse submaxillary gland: interaction with serum teolysis (Starkey and Barret, 1977; Koo and Stach, 1989) proteins. Brain Res 12:234-237. and to deliver NGF in an active form to neurons or lym- Huang JS, Huang SS, Duel TF (1984): Specific covalent binding of platelet-derived growth factor to human plasma a,-macroglophocytes. The ganglion bioassay revealed that NGF bulin. Proc Natl Acad Sci USA 81:342-346. complexes with a2M has biological activity even though Iverson L, DeChamplain, Glowinsky J, Axelrod J (1976): Uptake, the activity is decreased a little (data not shown), and our storage and metabolism of norepinephrine in tissues of the degel filtration study revealed that the molecular weight of veloping rat. J Pharmacol Exp Ther 157509-516. NGF released from a 2 M is indistinguishable from that of Koo PH, Stach PW (1989): Interaction of nerve growth factor with a-macroglobulin. J Neurosci Res 22:247-26 1. native NGF (Fig. 4).As NGF has a higher affinity for its receptor than for a,M (Sutter et al., 1979; Landreth and Korshing S, Thoenen H (1983): Nerve growth factor in sympathetic ganglia and corresponding target organs of the rat: Correlation Shooter, 1980; Koo and Stach, 1989), NGF bound to with density of sympathetic innervation. Proc Natl Acad Sci a2M might be transferred from a,M and rebind to the USA 80135 13-3516. NGF receptor. Kromer LF (1986): Nerve growth factor treatment after brain injury neuronal death. Science 235:2 14-216. We also found that the serum level of NGF in pregnant rats is higher than that in normal adult female rats Lakshmanan J (1986): p-nerve growth factor measurement in mouse serum. J Neurochem 46:882-891. and suddenly decreases after delivery (Fig. 6 ) . Because Landreth GE, Shooter EM (1980): Nerve growth factor receptors on the NGF content in placenta is rather higher than that in PC 12 cells; Ligand-induced conversion from low-to-high affinother peripheral tissues, serum NGF in pregnant rats is ity states. Proc Natl Acad Sci USA 77:4751-4755. likely to be derived from the placenta. It is at present Levi-Montalcini R, Angeletti PU (1968): Nerve growth factor. Physiol Rev 48534-569. unclear whether the serum NGF in pregnant rats is corMatsuda H, Coughlin MD, Bienenstock J, Denburg JA (1988): Nerve related with that in their fetus or not. growth factor promotes human hemopoietic colony growth and In order to identify the cell(s) responsible for serum differentiation. Proc Natl Acad Sci USA 85:6508-6512. NGF and in order to clarify the physiological signifi- Murase K, Takeuchi R, Furukawa S , Furukawa Y, Hayashi K (1990): cance of serum NGF, we are now examining whether the Highly sensitive enzyme immunoassay for p-nerve growth factor (NGF): A tool for measurement of NGF level in rat serum. serum levels of NGF are correlated with certain neuroBiochem Int 22:807-813. logical disorders or nerve lesions.

ACKNOWLEDGMENTS This work was supported in part by Grant-in-Aids for Developmental Scientific Research and for Scientific Research on Priority Areas (molecular basis of neural connection) from the Ministry of Education, Science and Culture, Japan. REFERENCES Aloe L, Alleva E, Bohm A, Levi-Montalcini R (1986): Aggressive behavior induces release of nerve growth factor mouse salivary gland into the bloodstream. Proc Natl Acad Sci USA 83:6184618. Boyle MDP, Young M (1982): Nerve growth factor: Activation of classical complement pathway by specific substitution for component C1. Proc Natl Acad Sci USA 79:2159-2522. Boyle MDP, Lawman JP, Gee AP, Young M (1985): Nerve growth factor; A chemotactic factor for polymorphonuclear leukocytes in vivo. J Immunol 134:564-568. Cochard P, Goldstein M, Black IB (1979): Initial development of the noradrenergic phenotype in autonomic neuroblasts of the rat embryo in vivo. Dev Biol 71:lOO-114. Gudat F, Laubscher A, Otten U, Pletscher A (1981): Shape changes induced by biologically active peptides and nerve growth factor in blood platelets of rabbits. Br J Pharmacol 74533-538. Hefti F (1986): Nerve growth factor promotes survival of septa1 cho-

Pearce FL, Thompson HL (1986): Some characteristics of histamine secretion from rat peritoneal mast cells stimulated with nerve growth factor. J Physiol 372:379-393. Phillip AD, Olli S, Peter H, Daniel BR (1989): a,-Macroglobulin is a binding protein for basic fibroblast growth factor. J Biol Chem 264:7210-7216. Raines E, Bowen-Pope D, Ross R (1984): Plasma binding proteins for platelet-derived growth factor that inhibit its binding to cellsurface receptors. Proc Natl Acad Sci USA 81:3424-3428. Ronne H, Anundi H, Rask L, Peterson PA (1979): Nerve growth factor binds to serum alpha-2-macroglobulin. Biochem Biophys Res Commun 87:330-336. Rubin E (1985): Development of the rat superior cervical ganglion; Ganglion cell maturation. J Neurosci 5:673-684. Shelton DL, Reichardt LF (1984): Expression of the b-nerve growth factor gene correlates with the density of sympathetic innervation in effector organs. Proc Natl Acad Sci USA 81:79517955. Shinoda I, Takeuchi R, Kurobe M, Furukawa S , Hayashi K (1988): Molecular nature of p-nerve growth factor (NGF)-like immunoreactive substance(s) in mouse serum. J Clin Biochem Nutr 5: 135-144. Starkey PM, Barret AJ (1977): a,-Macroglobulin, a physiological regulator of proteinase activity. In Barrett AJ (ed): “Research Monographs in Cell and Tissue Physiology-Proteinases in Mammalian Cells and Tissues.” New York: North-Holland Publishing Co., pp 663-696. Suda K, Barde YA, Thoenen H (1978): Nerve growth factor in mouse and rat serum: Correlation between bioassay and radioimmunoassay determinations. Proc Natl Acad Sci USA 75:40424046.

288

Murase et al.

Sutter A, Riopelle RJ, Hams-WamckRM, Shooter EM (1979): Nerve growth factor receptors. J Biol Chem 254:5972-5982. Teitelman G, Baker H, Joh TH, Reis DJ (1979): Appearance of catecholamine-synthesizing enzymes during development of rat synthetic nervous system; Possible role of tissue environment. Proc Natl Acad Sci USA 76509-513. Thoenen H, Barde YA (1980): Physiology of nerve growth factor. Physiol Rev 60:1284-1335.

Thorpe LW, Stach RW, Hashim GA, Marchetti D, Perez-Polo JR (1987): Receptors for nerve growth factor on rat spleen mononuclear cells. J Neurosci Res 17:128-134. Williams LR, Varon S , Peterson GM, Wictorin K, Fischer W, Bjorklund A, Gage FH (1986): Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection. Proc Natl Acad Sci USA 83:9231-9235.

Developmental changes in nerve growth factor level in rat serum.

In serum, nerve growth factor (NGF) forms a complex with alpha 2-macroglobulin (alpha 2M), which formation inhibits the immunoreactivity between NGF a...
588KB Sizes 0 Downloads 0 Views