Submaxillary Gland Nerve Growth Factor Research Articles

Nerve growth factor synthesis by mouse submandibular gland cells in culture

Mouse submandibular gland (SMG) cells in culture rarely retian functional properties of SMG cells in vivo. We demonstrate that both primary SMG cells and the mouse SMG cell line SCA-9 secrete biologically active nerve growth factor (NGF). However, primary cells secrete 40-fold more NGF than SCA-9 cells, demonstrating that SCA-9 cells cannot substitute for primary SMG cells for the study of SMG NGF in vitro.

Thyroid hormones precociously increase nerve growth factor gene expression in the submandibular gland of neonatal mice.

The developmental regulation of the expression of nerve growth factor (NGF) was studied in the mouse submandibular gland (SMG). Having demonstrated that, in the neonatal mouse, maturation of the SMG can be accelerated by treatment with thyroid hormones, with the resulting induction in SMG content of NGF, studies were undertaken to further examine the locus of thyroid hormone action. Because of the sexual dimorphism of the SMG, both male and female neonatal mice were used. NGF messenger RNA levels were undetectable in SMGs from untreated immature mice, while hybridization to total RNA from T4-treated mice was easily observable for NGF complementary DNA. Treatment for 14 days compared to 7 days resulted in a 7-fold increase in SMG NGF mRNA levels. A signal was obtained in 21-day-old control mice using S1 nuclease protection analysis; T4 increased NGF mRNA levels by 100-fold in both male and female immature mice. Heteronuclear RNA levels were induced 20-fold by T4. No sex differences were readily observable. Determination of the effect of thyroid hormone treatment on SMG NGF gene expression by nuclear run-on assay demonstrated a significant transcriptional effect of T4. Initial experiments using the pmngf6 vector, which is a pBR322-derived probe containing the full length NGF cDNA, showed a 2.5-fold induction of gene transcription. When an internal fragment of pmngf6 was subcloned into pTZ18R, thus removing the dC/dG tails contained in pmngf6, the background hybridization was considerably reduced and a 12.5-fold induction in NGF gene transcription was obtained after T4 treatment of neonatal mice. The results show that thyroid hormones increase NGF gene expression in the SMG of the immature male and female mouse. This effect is due in part to a significantly enhanced rate of gene transcription.

Thyroid hormone and androgen regulation of nerve growth factor gene expression in the mouse submandibular gland

The nerve growth factor (NGF) content of the mouse submandibular gland (SMG) is under hormonal control and is modulated by both thyroid hormones (TH) and androgens. The sexual dimorphism of the gland is well documented. In the adult male mouse, the SMG contains 10 times more NGF compared to the female. Conversely, castration of male mice reduces the SMG NGF levels to those found in control females. In order to determine the locus at which androgens and TH exert their effect on NGF gene expression in the SMG, steady-state NGF mRNA levels were determined. Daily treatment of adult female mice with TH for 1 week increased NGF mRNA levels 6-fold. Androgen treatment produced a 20-fold increase in SMG NGF mRNA, which was comparable to levels detected in the control adult male SMG. The effect of TH on NGF mRNA levels was time-dependent and coincided with the increase in NGF protein concentrations. At 48 h after a single TH injection, NGF mRNA levels (measured in SMG total RNA) increased 2–4-fold, while heteronuclear (hn) RNA levels were increased 1.5–2-fold. The NGF gene transcription rate was determined by run-on assays following TH treatment. A small but significant 2-fold induction by TH of NGF gene transcription was found at 24–48 h. Cytoplasmic RNA prepared from the same SMGs used in the run-on experiments was tested by S1 nuclease protection; NGF cytoplasmic RNA was increased 7-fold in the SMGs of females treated with TH 48 h previously. These results demonstrate that the effect of TH on NGF gene expression is due in part to an induction of NGF gene transcription. The discrepancies observed between transcription rate and mRNA levels suggest that the major effect of TH is at the post-transcriptional level, possibly mRNA stabilization. The time required to observe an induction of TH on NGF gene transcription is suggestive of an indirect effect, possibly through the induction by TH of another protein which in turn activates the NGF gene.

Serine proteases promote human CFU-GM in methylcellulose culture systems.

Amino acid sequences and three dimensional structural analysis of serine proteases show sequence homology in a group of compounds, which include thrombin, Cl’ esterase, urokinase, trypsin, plasminogen, snake venom, kallikrein, and the γ subunit of mouse submandibular gland nerve growth factor. Common to the structure of these enzymes are active sites consisting of aspartic acid, histidine and serine. The similarities between the various serine proteases are striking in a number of details, including cellular location, chemical structure, molecular weight and function.

Regulation of nerve growth factor gene expression in primary brain monolayer cultures: effects of dibutyryl cyclic AMP and sodium butyrate

Primary brain cultures composed of a heterogeneous population of cells, most of which reacted with anti-fibronectin and only few with anti-GFAP antiserum, were found to synthesize a mRNA indistinguishable from mouse submandibular gland nerve growth factor (NGF) mRNA in hybridization characteristics and electrophoretic behavior in agarose gels. The levels of this mRNA were increased 2.5–10 fold by 1 mM dibutyryl cyclic AMP (db-cAMP) and sodium butyrate within 24 h, but not by 10 μM forskolin, and were unaffected by serum in the culture medium. These observations demonstrate that brain cells synthesize a NGF mRNA in primary culture and that the butyrate moiety of db-cAMP enhances NGF gene expression in these cells, probably by a modification of chromatin structure in and around the NGF gene.

Nerve growth factor-like immunoreactivities in rodent salivary glands and testis.

A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.

Neonatal hyperthyroidism causes impairment in submandibular gland-nerve growth factor (SMG-NGF) ontogeny in mice.

The effect of neonatal hyperthyroidism on submandibular gland (SMG) nerve growth factor (NGF) concentrations was studied in female Swiss-Webster mice. For this purpose, newborn female pups were treated with thyroxine (T4, 0.4 microgram/g body weight/day) from birth through day 6, while their littermate pups were treated with similar volumes of vehicle. Animals were sacrificed on days 7, 15, 21, 31 and 71. Somatic growth (body weight and nose to rump length), serum T4 and T3 concentrations, and SMG and serum-NGF concentrations were measured. The data indicate that neonatal hyperthyroidism a) causes inhibition of somatic growth; b) induces alterations in normal ontogenic patterns of serum T4 and T3; c) impairs the developmental rise in SMG-NGF concentration and d) transiently increases serum-NGF levels.

Thyroxine increases submandibular gland nerve growth factor and epidermal growth factor concentrations precociously in neonatal mice: evidence for thyroid hormone-mediated growth factor synthesis.

Thyroxine (T4) administration to adult female mice significantly increases submandibular gland (SMG) nerve growth factor (NGF) and epidermal growth factor (EGF) concentrations and does so in a time- and dose-dependent manner. Postnatal maturation of the SMG can be markedly accelerated by T4 treatment. We, therefore, performed a series of experiments to examine the effect of T4 on SMG NGF and EGF content and concentration as a function of postnatal age in neonatal mice. In experiment 1, male and female neonatal Swiss-Webster mice received daily subcutaneous injections of T4 (0.4 micrograms/g body weight) for 6, 13, or 20 days and were sacrificed 24 h after the last injection. Vehicle injected mice served as controls. SMG NGF and EGF content and concentration were measured by specific double antibody radioimmunoassay systems. Pools were made using either female or male SMGs. Since no significant differences were noted for NGF or EGF content using sex of the animal as the determining variable, the values were combined. At 7 days of age, mean SMG NGF and EGF content and concentration of control mice significantly exceeded those of T4-treated animals (p less than 0.05). At 14 days of age, mean SMG NGF and EGF content in T4-treated mice significantly exceeded those in control mice by 39- and 22-fold, respectively (p less than 0.001). At 21 days of age, these increases were 4100- and 2400-fold, respectively. In order to determine more precisely the time of onset of responsivity of the SMG to thyroid hormones, a second series of experiments was performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of developing sympathetic nervous system in thyroxine-mediated submandibular gland nerve growth factor and epidermal growth factor responses.

Thyroxine (T4) administration in mice during the 2nd wk of postnatal life elicits a precocious increase in submandibular gland-nerve growth factor (SMG-NGF) and epidermal growth factor (SMG-EGF) levels, but the mechanism(s) of T4 action has not been studied. The present report examines the role of the developing sympathetic nervous system (SNS) in the SMG-NGF and EGF responses to T4. For this purpose newborn mice were injected with T4 and/or 6-hydroxydopamine, a toxic congener of norephinephrine which causes selective destruction of sympathetic nerve terminals. The effectiveness of chemical sympathectomy was assessed by SMG-norepinephrine measurements using a sensitive radioenzymatic assay. The glandular norepinephrine contents were greatly reduced indicating that the dose and duration of 6-OHDA treatment were sufficient to cause a total sympathectomy in SMG tissue. In addition, the 6-OHDA treatment greatly reduced the wet weight and total protein content of the sympathetic superior cervical ganglia which innervate SMG tissue. SMG-NGF and EGF concentrations were measured by specific radioimmunoassays. 6-OHDA treatment alone did not affect the basal SMG-NGF and EGF concentrations. However, the maximal responses of SMG-NGF and EGF to T4 administration were greatly reduced by concurrent treatment with 6-OHDA. In summary, the data demonstrate a critical role for developing sympathetic nervous system in the T4-stimulated increase in SMG-NGF and EGF concentrations.

Serum and thyroid hormones T3 and T4 regulate nerve growth factor mRNA levels in mouse L cells

Mouse L cells synthesize and secrete a neurotrophic factor related to the β subunit of the submaxillary gland nerve growth factor (NGF) of male mice. Use of a cDNA probe which encodes the β-NGF mRNA demonstrated that L cells produce a transcript identical in size to that of the submaxillary gland. Moreover, target sites of restriction enzymes EcoRI, PstI and BamHI were not significantly rearranged in the β-NGF gene locus of these cells. The abundance of the β-NGF transcript was found to depend on culture conditions. Removal of serum depressed the cellular content of polyadenylated RNA by a factor of 1.7, and decreased specifically the pool of β-NGF transcript by an additional factor of 4. The presence of 10 −7 M testosterone in the serum-free medium did not modify the level of β-NGF mRNA, while addition of 10 −7 M T3 (or T4) increased this level by a factor of 1.5. These data provide the first evidence that the β-NGF mRNA of L cells is subjected to regulation, but in a way apparently different from that described for the submaxillary gland.

Differences and similarities in the neurotrophic growth factor requirements of sensory neurons derived from neural crest and neural placode.

This article reviews recent studies that have examined differences and similarities in the neurotrophic growth factor requirements of neural crest- and neural placode-derived sensory neurons of the developing chick embryo. From in vitro experiments using both explant and dissociated, neuron-enriched cultures of spinal and cranial nerve sensory neurons, it has been established that only sensory neurons of neural crest origin are responsive, at least in terms of survival and neurite outgrowth, to mouse submandibular gland nerve growth factor (NGF). Sensory neurons derived from neural placodes (neurons of the ventrolateral portion of the trigeminal ganglion and the entire neuronal population of the vestibular, geniculate, petrosal and nodose ganglia) are largely unresponsive to NGF throughout embryonic development, but do respond to neurotrophic activity present in extracts of brain and various peripheral 'end-organs', such as heart or liver. By incubation of neuron-enriched cultures with radiolabelled [125I]NGF, followed by autoradiographic exposure, it has been demonstrated that placode-derived neurons, in marked contrast to those of neural crest origin, are completely devoid of specific cell surface receptors for NGF. In contrast to differences in their requirement and responsiveness to NGF, both placode- and crest-derived sensory neurons are responsive to the survival and neurite-promoting activity of a recently purified brain-derived neurotrophic factor (BDNF). It is postulated that all primary sensory neurons have a dual growth factor requirement during development; their survival being dependent on a supply of both a peripheral and a central 'target'-derived neurotrophic factor. It appears that BDNF may act as common 'central target-derived' neurotrophic factor for both placode- and crest-derived sensory neurons, but that within peripheral tissues there are specific neurotrophic factors for each of these two classes of primary sensory neurons.

Nerve growth factor: a chemotactic factor for polymorphonuclear leukocytes in vivo.

Previous studies have shown that mouse submandibular gland nerve growth factor (NGF) stimulates chemotactic migration of human polymorphonuclear leukocytes in vitro. The results of the present study demonstrate that subdermal injection of NGF in mice also stimulates rapid and marked chemotactic recruitment of leukocytes. This property of NGF is manifest in the nanomolar range of concentrations, it requires the known serine class protease activity of the growth factor, and it does not require participation of the fifth component of complement. Another, as yet unrecognized, C5-independent pathway must be involved. Chemotactic stimulation of cells involved in the early inflammatory response to injury may help to explain earlier observations that NGF can accelerate the rate of contraction of experimentally induced wounds in mice.

Spinal cord contains neurotrophic activity for spinal nerve sensory neurons. Late developmental appearance of a survival factor distinct from nerve growth factor

Several tissues of the developing chick embryo have been reported to contain neurotrophic activity which can sustain the survival of sensory neurons maintained in culture. In a previous study, however, we noted that such nerve growth promoting activity was exceptionally low, if not absent, from extracts of spinal cord from chick embryos of up to 16 days incubation. Since then the combined results from a number of tissue culture studies have suggested that the central nervous system may be the source of a neurotrophic growth factor essential during the late development of sensory neurons. We have therefore carried out an extended range study of the neurotrophic properties of avian spinal cord. Extracts of spinal cord tissue prepared from chicks at stages between the last wk of embryogenesis and 12 wks after hatching were tested for their ability to promote survival and neurite outgrowth from both explant and dissociated neuron-enriched cultures of dorsal root, trigeminal, nodose and paravertebral chain sympathetic ganglia from chick embryos between 8 and 16 days old. We conclude from our results that spinal cord is a potent source of neurotrophic activity for sensory neurons, although this activity appears relatively late in development of the spinal cord. The predominant ontogenic increase in spinal cord neurotrophic activity was seen to occur during the first week after hatching. Sensory neurons from both spinal and cranial nerve ganglia were sustained in culture by spinal cord extracts, whereas sympathetic neurons did not respond. Neurons from older sensory ganglia (12–16 day old embryos) were much more responsive than similar neurons from young embryos (8 day). We have detected similar neurotrophic activity for sensory neurons in extracts of adult rat and adult human spinal cord. None of these activities are blocked by antiserum to mouse submandibular gland nerve growth factor.

Synthesis and partial maturation of the α- and γ-subunits of the mouse submaxillary gland nerve growth factor in Xenopus laevis oocytes

Sera raised against the α-, β- and γ-subunits of the mouse 7 S NGF were used to characterize translation products coded by submaxillary gland mRNAs microinjected into Xenopus oocytes. Anti-βNGF sera did not cross-react with any material. In contrast, the precursors of the α- and γ-subunits, as well as that of renin were identified. Use of tunicamycin, and a comparison of the translation products obtained in oocytes or in the reticulocyte lysate indicated that oocytes achieved the cleavage of signal sequences, the glycosylation of the α- and γ-precursors, and the subsequent secretion of the 3 proteins. In the submaxillary gland, however, the mature forms of αNGF, γNGF and renin are composed of peptides of smaller size than those produced by the oocytes. These latter appear to lack specific proteases involved in the terminal processing of the submaxillary gland proteins.

Acquisition of submandibular gland nerve growth factor (SMG-NGF) responsiveness to thyroxine administration in neonatal mice.

The growth of the submandibular gland (SMG) was studied in newborn mice from birth to 15 days of age. Progressive changes in wet weight were observed to accompany changes in biochemical constituents such as RNA, protein, and lipid. Thyroxine (T4) administration from days 0-6 produced changes in SMG growth and SMG accumulation of RNA, protein, and lipid components relative to control pups treated with a similar volume of vehicle. This hormone regimen produced no measurable changes in SMG nerve growth factor (SMG-NGF) concentration. T4 responsiveness also was studied from days 0-15. Three patterns of T4 injection (from days 0-6, 7-14, and 0-14) were found to elicit a differential response in the three biochemical constituents measured, but treatment from days 7-14 and 0-14 elicited precocious increments in SMG-NGF concentrations on day 15. The effect of T4 injection from birth was more effective in augmenting SMG-NGF concentration than hormone treatment initiated from days 7-14. A persistent T4 effect on SMG-NGF also was observed on day 21 following hormone treatment from days 7-14 or 0-14. In summary, the acquisition of SMG-NGF responsiveness to T4 appears to develop during the neonatal period. The administration of T4 during this period also precociously stimulates the mechanisms that govern the normal ontogeny of SMG-NGF at the time of weaning.

Ontogenesis of nerve growth factor and epidermal growth factor in submaxillary glands and nerve growth factor in brains of immature male mice: correlation with ontogenesis of serum levels of thyroid hormones.

Summary: Using specific and sensitive radioimmunoassays for nerve growth factor (NGF) and epidermal growth factor (EGF), we studied the developmental profile of NGF and EGF concentrations in male mouse submaxillary gland (SMG) from 2–60 days of age. We measured NGF concentrations in mouse cerebellum, cerebral cortex, and brain stem from ages 2–32 days. In addition, we assessed mouse serum thyroxine (T4) and triiodothyronine (T3) levels in seperate groups of animals ranging from 5–50 days of age. Mean SMG EGF content and concentration exceeded that of NGF between 2 and 18 days of age. At all subsequent ages, however, mean NGF and EGF content and concentration were similar. SMG NGF and EGF content were low at 2 days of age, decreased slightly through 8 days of age, and increased exponentially after 11 days of age with three phases of increase being apparent for both proteins. In the first phase, mean SMG NGF and EGF contents increased 2.4− and 2.3-fold respectively between 11 and 18 days of age. A second phase of increase was noted between 18 and 32 days when NGF and EGF increased 9845− and 7805-fold respectively. In phase three (32–60 days of age) more modest increases of 6.5-fold in mean NGF and EGF content were noted.Cerebellar NGF concentrations increased from 681 to 2241 pg/mg protein between ages 2 and 15 days and subsequently fell to a plateau level of 1304 pg/mg protein by age 18 days. Cerebral cortical NGF concentrations increased steadily from 251 to 1383 pg/mg protein between ages 2 and 32 days, whereas brain stem NGF concentrations maintained a plateau averaging 1100 pg/mg protein.Serum T4 concentrations in the mouse increased dramatically from 2.5 μg/dl at 5 days to a maximum of 8.1 μ/dl at 15 days, thereafter decreasing to a nadir 4.4 μ/dl by 40 days. Serum T3 concentrations followed a similar pattern, rising from 25 ng/dl on postnatal day 5 to 93 ng/dl on day 15, with a subsequent decline to 63 ng/dl by day 30. These results indicate a correlation between the increase in serum thyroid hormone levels in the mouse and the increases in SMG NGF and EGF levels and brain NGF levels between 5 and 32 days.Speculation: We and others have demonstrated that thyroid hormones increase brain NGF concentrations and stimulate submaxillary gland NGF and EGF synthesis. The present results are compatible with the hypothesis that the early increase in circulating thyroid hormone concentrations in the mouse play an important role in the increase in cerebral and cerebellar NGF levels during the first 3 wk and the increase in SMG NGF and EGF concentrations between 11 and 32 days of age. The known increase in mouse serum testosterone to adult male levels between 4–12 wk of age may be a factor in the late increase in NGF and EGF elevations after 32 days of age.

Nerve growth factor: activation of the classical complement pathway by specific substitution for component C1-.

The interaction of homogeneous preparations of mouse submandibular gland nerve growth factor (NGF) with the classical complement pathway was studied. NGF was found to be capable of carrying out the enzyme activities of the first component (C1-) of the classical complement pathway (i.e., the cleavage of zymogen C4 and C2). NGF would not substitute for any other classical pathway component, C2-C9. The C1(-)-like activity of NGF was inhibited by human C1- inactivator. This interaction of NGF with the complement system may account for the previously described ability of NGF to accelerate the rate of contraction of experimentally induced wounds.

Electrophoretic heterogeneity and polypeptide chain structure of the gamma-subunit of mouse submaxillary 7 S nerve growth factor.

The gamma-subunit of the high molecular weight form of mouse submaxillary gland nerve growth factor is known to be electrophoretically heterogeneous. The protein is a mixture of molecules containing either two or three chains, attached covalently by disulfide bonds, that arise from one or two endoproteolytic cleavages. Analysis of the amino acid composition and partial sequence of the resultant fragments has defined the location of the two cleavage sites. Further heterogeneity is generated by partial removal of the carboxyl-terminal basic residues located at the internal cleavage sites, presumably by an endogenous exopeptidase(s) with carboxypeptidase B specificity. Permutations of these endo- and exoproteolytic cleavages generate six possible microheterogeneous forms, which can account for the five major bands observed on electrophoresis at pH 7.05 and the three principal forms seen on isoelectric focusing.

Nerve growth factor zymogen. Stoichiometry of the active-site serine and role of zinc(II) in controlling autocatalytic self-activation.

Mouse submandibular gland nerve growth factor (NGF) is a 116 000 molecular weight protease with a high degree of specificity for certain lysyl and arginyl bonds. Thie protein can activate plasminogen and it is also a member of the general class of serine proteases [Orenstein, N. S., Dvorak, H. A., Blanchard, M. H., & Young, M. (1978) Proc. Natl. Acad. Sci, U.S.A. 75, 5497]. As isolated, NGF is an enzymically inactive zymogen. Upon dilution from high to very low protein concentrations or upon treatment with EDTA, the zymogen undergoes autocatalytic activation. Atomic absorption spectroscopy measurements reveal that NGF contains 1 g-atom of tightly bound Zn(II) per mol. Reaction of the fully autoactivated protease with [3H]DFP yields 1 mol of labeled serine per mol of enzyme. All results indicate that as long as Zn(II) remains bound to the zymogen, autocatalytic activation is inhibited. Removal of this ion, by dilution of the protein or by chelation, initiates autoactivation. The physiologic purpose of this unusual reaction is not known but it may be that Zn(II) serves to act as a control ion which keeps the protein in an inactive form (the zymogen) until it recognizes its naturally occurring substrate.

Epidermal Growth Factor in the Submandibular Gland and Serum of Mice with Muscular Dystrophy: Chemical Properties in Dilute Gland Extracts*

Epidermal growth factor (EGF) has been measured in extracts of submandibular glands from mice with hereditary muscular dystrophy. RIA results show that adult male and female dystrophic mice have significantly less submandibular gland EGF than do unafflicted controls. Despite the differences in gland content of the protein, serum levels of EGF are similar in both dystrophic and control animals. Furthermore, submandibular gland concentrations of amylase are normal in the dystrophic mice, indicating that not all proteins synthesized by the glands are affected. Gel filtration studied reveal that the elution properties of EGF in extracts of glands from dystrophic and control animals are indistinguishable. Unexpectedly, the chromatographic profiles indicate that most of the EGF in gland extracts elutes as a low molecular weight protein when the molecule is studied at low, biologically active concentrations; only a small portion of the protein is associated with a high molecular weight complex. Under the same experimental conditions, submandibular gland nerve growth factor maintains its association with other components in a high molecular weight form.