Regulation of sympathetic trophic factors in smooth muscle

The level and nature of trophic activity present in the chicken expansor secundariorum muscle has been shown to be altered by denervation. This muscle receives a dense, sympathetic innervation and contains high concentrations of trophic factors, which were found to be immunologically and functionally distinct from mouse Nerve Growth Factor. In young birds, denervation increased the number of neurons which could be supported by muscle extract. This difference was apparent with regard to E8 to E16 sympathetic neurons. Innervated but not denervated extract was additive with NGF in promoting neurite outgrowth. In contrast, when extracts of denervated and innervated muscle from mature birds were examined, no difference was seen in the number of neurons supported by each extract. However when the denervated and innervated extracts from mature birds were combined more neurons were supported than by a saturating dose of either extract alone. Furthermore, muscle from mature birds responded to denervation only between 2 and 9 days, whereas in young birds the effect was apparent for at least 3 weeks. Analysis of intact, control muscles during the first 8 weeks posthatch demonstrated that the number of neurons that could be supported by the individual extracts varied with the age of the bird. It is concluded that denervation does not in all instances lead to an increase in trophic activity, but does produce a change in the nature of the activity present, such that a different neuronal subpopulation may be supported.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of putative muscle-derived neurotrophic factors by muscle activity and innervation: in vivo and in vitro studies

The normal embryonic development of spinal cord motoneurons (MNs) involves the proliferation of precursor cells followed by the degeneration of approximately 50% of postmitotic MNs during the period when nerve-muscle connections are being established. The death of MNs in vivo can be ameliorated by activity blockade and by treatment with muscle extracts. Muscle activity and innervation have been suggested to regulate the availability of putative muscle-derived neurotrophic agent(s), and MNs are thought to compete for limited amounts of these trophic agents during normal development. Thus, activity and innervation are thought to regulate MN survival by modulating trophic factor availability. We have tested this notion by examining MN survival in vivo and ChAT development in spinal cord neurons in vitro following treatments with partially purified muscle extracts from normally active, paralyzed (genetically or pharmacologically), aneural, denervated, slow tonic, and fast-twitch muscles from embryonic and postnatal animals. Extracts from active and chronically inactive embryonic avian and mouse muscles were found to be equally effective in promoting the in vivo survival of MNs in the chick embryo. Similarly, extracts from fast-twitch and slow tonic postnatal avian muscles did not differ in their ability to promote both MN survival in vivo and ChAT activity in vitro. Although aneural and control embryonic muscle extract had similar effects on ChAT development in vitro, aneural muscle extract contained somewhat less MN survival-promoting activity when tested in vivo. By contrast, denervated postnatal muscle extract was more effective in promoting both MN survival in vivo and ChAT activity in vitro than age-matched control muscle extract.(ABSTRACT TRUNCATED AT 250 WORDS)     

Repression of the embryonic myosin heavy chain phenotype in regenerating chicken slow muscle is dependent on innervation.

Ventricular-like and fast myosin heavy chains (VL-MHC and FMHC) are transiently expressed during slow skeletal muscle development. The influence of innervation on repression of these MHC isoforms is investigated over an 84-day time course in: (1) normal anterior latissimus dorsi (N-ALD) muscles, (2) regenerating ALD (R-ALD) muscles, (3) denervated ALD (D-ALD) muscles, and (4) regenerating and denervated ALD (RD-ALD) muscles. Western blotting demonstrates that the VL-MHC is expressed in R-, D-, and RD-ALD muscles, but not in N-ALD muscles. Expression of the VL-MHC is transient in R-ALD muscles. In contrast, VL-MHC expression persists in RD-ALD muscles, and appears with time in D-ALD muscles. FMHC was not detected in N-ALD muscles by Western blotting. Two FMHCs are seen in R-ALD and RD-ALD muscles, and in 13-day embryonic ALD muscles. The slower migrating FMHC (FMHCA) comigrates with developmentally regulated FMHCs in fast pectoralis muscle, while the faster migrating FMHC (FMHCB) comigrates with the faster migrating FMHC in embryonic ALD muscle (13 days in ovo). FMHCB decreases in amount over the time course in R-ALD muscles, while FMHCA persists. In contrast, substantial levels of both FMHCs persist in RD-ALD muscles, and appear with time in D-ALD muscles. The cellular distribution of MHCs is followed by immunocytochemistry. Regenerating cells expressing VL-MHC and FMHC are replaced by a mature population in R-ALD muscles. Some of the mature myofibers in R-ALD muscles express FMHC, but not VL-MHC. In RD-ALD and D-ALD muscles, both regenerating and mature muscle cells are seen which express VL-MHC and FMHC. Our results indicate that innervation is required for the repression of VL-MHC and FMHCB during regeneration of slow muscle.     

Extension of sympathetic neurites in vitro towards explants of embryonic and neonatal mouse heart and stomach: ontogeny of neuronotrophic factors

In order to establish when target organs first produce neuronotrophic factors, extension of neurites in vitro from sympathetic ganglia (superior cervical and coeliac) of 1-day neonatal mice towards explants of 10-, 11-, 14- and 17-day embryonic and 1-day neonatal atrium and stomach was examined in co-cultures. Longer neurites extended from ganglia towards, than away from, atrial targets at all stages examined, and was most marked towards 17-day embryonic and neonatal explants. Treatment of atrial co-cultures with antiserum to nerve growth factor (NGF) almost totally blocked preferential neurite outgrowth. Directional growth of neurites towards stomach explants in co-cultures was not as pronounced as that towards atrium; extension of neurites was most marked when stomach was provided by 11-, 14- and 17-day embryos. Such outgrowth was only partially blocked by antiserum to NGF, significant preferential extension of neurites towards stomach persisting in the presence of the antiserum. These results indicate that atrium and stomach produce neuronotrophic factors from the earliest ages studied; the evidence indicates that in the case of atrium, NGF predominates but that stomach produces NGF as well as another factor immunologically distinct from NGF. It is of interest that both types of target explanted before they receive sympathetic innervation show evidence of producing NGF in culture.     

Motoneurone survival activity in extracts of denervated muscle reduced by prior stimulation of the muscle

Inactivation of skeletal muscle by denervation increases motoneurone survival activity in extracts of skeletal muscle. The present investigation shows that electrical stimulation of denervated muscle decreases motoneurone survival activity in extracts of these muscles. The result suggests that motoneurone survival is dependent on a factor(s) in muscle whose synthesis and/or release is regulated by muscle contraction.     

Myotrophic effects of muscle extracts obtained at different intervals after denervation

A study was made of the myotrophic effects of denervated muscle extracts on normal Wistar rat soleus muscle. Extracts obtained 1  h, 2, 4 and 7 days after sectioning of the sciatic nerve were administered intraperitoneally over five consecutive days. Soleus muscles were routinely processed for morphological and morphometrical analysis using light microscopic techniques. Quantitative differences were observed in the effects of different extracts on total muscle area, fibre cross-sectional area, mean minimum diameter and number of fibres/area. The greatest myotrophic response was elicited by extracts obtained at 2 and 4 days; differences with respect to controls and extracts obtained at 1 day were significant ( P <0.05) for all parameters studied. Statistically significant differences ( P <0.05) were also recorded for fibre cross-sectional area and mean minimum diameter between the 2- and 4-day groups and the 7-day group. It may thus be concluded that the time elapsing between denervation and extract obtention influences the effect of the extract on normal rat muscle.     

Nerve growth factor in medium conditioned by embryonic chicken heart cells

The present report demonstrates that embryonic chicken heart cells in culture release different nerve growth promoting factors to their culture medium, one which is biologically and immunologically similar to mouse gland beta NGF. Serum-free heart cell conditioned medium thus promoted neurite outgrowth from sympathetic and ciliary ganglia and supported survival of dissociated ciliary neurons. The addition of affinity purified antibodies against mouse beta NGF does substantially but not completely inhibit the fibre outgrowth from sympathetic ganglia, but does not to any extent diminish the effects on the parasympathetic neurons. The chicken NGF recovered from polyacrylamide gels after electrophoresis greatly enhanced sympathetic fibre outgrowth, an activity completely suppressive by anti-beta NGF antibodies. We conclude that a chicken NGF is being produced by the embryonic heart cells in culture, and that this factor may be produced also in the embryo to fulfill a role in heart innervation.     

Human beta nerve growth factor obtained from a baculovirus expression system has potent in vitro and in vivo neurotrophic activity

A baculovirus expression vector, which contains the coding sequences for human prepro (beta) nerve growth factor under control of the viral polyhedrin promoter, was constructed. Upon infection of insect cells with the recombinant virus, mature human beta nerve growth factor (rhNGF) was released into the culture fluid. The mature rhNGF was biologically active since rat pheochromocytoma (PC12) and human neuroblastoma (SH-SY5Y) cells were induced to extend neurites upon treatment with this material. This activity was abolished by treating with antiserum prepared against mature mouse beta NGF (mNGF). When compared with mNGF, rhNGF more rapidly elicited the differentiation response in both PC12 and SH-SY5Y cells. In an in vivo assay of cholinergic cell survival, rhNGF was nearly as potent as mNGF in protecting cholinergic neurons from degeneration following a fimbria-fornix lesion. These results show that the baculovirus expression system provides quantities of biologically potent human beta NGF suitable for a comprehensive program of research to ascertain beta NGF's potential as a therapeutic agent for the treatment of Alzheimer's disease.     

Sympathetic neuronal survival factors change after denervation

The increase in sympathetic neuronal survival factors in the hearts of rats chemically denervated with 6-hydroxydopamine has been examined after biochemical fractionation techniques. Affinity chromatography and gel filtration of extracts of control hearts have demonstrated the existence of two major components, neither of which is nerve growth factor (NGF). The concentrations of both of these components and of NGF are increased significantly after denervation.     

Purification of adult rat sciatic nerve ciliary neuronotrophic factor

The ciliary neuronotrophic factor (CNTF), a protein required for the survival of cultured avian embryonic parasympathetic ciliary ganglionic neurons, was recently purified from extracts of selected chick intraocular tissues. Here we report the purification of a mammalian CNTF activity from extracts of adult rat sciatic nerve using a fractionation procedure similar to that employed for isolating chick eye CNTF. About 2 micrograms of CNTF protein can be obtained from each 1.5 g batch of nerve tissue. Like the chick CNTF, the mammalian factor displays trophic activity for dorsal root and sympathetic as well as ciliary ganglionic neurons. The nerve CNTF activity differs from its chick counterpart in molecular weight and chromatographic behavior on ion-exchange columns. Unlike purified nerve growth factor (NGF), nerve CNTF activity is insensitive to anti-NGF antibodies and is unable to support the survival of 8-day chick embryo dorsal root ganglion neurons.     

Effect of clenbuterol on normal and denervated muscle growth and contractility

The reported anabolic action of some beta 2 agonists may have clinical applications in certain muscle wasting states. Administration of clenbuterol (2 mg/kg diet for 14 days) to rats resulted in a limited degree of hypertrophy of normal muscles; the effect was more pronounced on fast-twitch muscles than on slow-twitch muscles. The anabolic effect was greatest in denervated muscles, where it was significantly more effective on the slow-twitch type. Clenbuterol significantly improved the contractile properties of denervated slow-twitch muscle, reverting them toward normal, but had little effect on contractile properties of denervated fast-twitch muscle. Such differential effects of clenbuterol must be taken into consideration in the evaluation of any future human intervention study.     

Embryonic neurons transplanted into the tibial nerve reinnervate muscle and reduce atrophy but NCAM expression persists

OBJECTIVE: The aim of this study was to use the glycogen depletion technique to determine whether reinnervated muscle fibers could be distinguished from denervated muscle fibers by their size or by neural cell adhesion molecule (NCAM) expression. METHODS: Medial gastrocnemius muscles of five adult Fischer rats were reinnervated from embryonic neurons transplanted into the distal stump of the tibial nerve. Ten weeks later, the transplants were stimulated repeatedly to deplete reinnervated muscle fibers of glycogen. Areas of reinnervated (glycogen-depleted) muscle fibers were measured and assessed for NCAM expression. The areas of muscle fibers from reinnervated, denervated (n=5) and unoperated control muscles (n=5) were compared. RESULTS: Mean reinnervated muscle fiber area was significantly larger than the mean for denervated fibers (mean +/- SE: 40 +/- 6 and 10 +/- 1% of unoperated control fibers, respectively). NCAM was expressed in 55 +/- 7% of reinnervated fibers (mean +/- SE; range: 42-77%). The mean areas of reinnervated fibers that did or did not express NCAM were similar. NCAM was only expressed in some fibers in completely denervated muscles. DISCUSSION: Our data show that NCAM expression does not differentiate muscle denervation or reinnervation. Quantifying the area of large fibers did distinguish reinnervated muscle fibers from denervated fibers and showed that reinnervation of muscle from neurons placed in peripheral nerve is a strategy to rescue muscle from atrophy.     

NEUROGENIC CONTROL OF MUSCLE RIBOSOMAL PROTEIN SYNTHESIS

In vitro protein synthesis of ribosomes extracted from leg muscles of 20 Sprague-Dawley rats denervated 3 and 14 days (high sciatic transections) and 10 control rats was studied. The concentration/mg protein of total ribosomes extracted from the 14-day denervated muscle showed a significant increase. Distribution of muscle polyribosomes on sucrose density gradients was normal in all cases. Noncollagen protein synthesis of the heavy polyribosomes from both early and late denervated muscles showed a significant decrease. SDS polyacrylamide gels suggested that this decrease affected the synthesis of heavy chains of myosin, while the light chains of myosin, actin and tropomyosin had normal structure and amounts. Collagen synthesis of heavy polyribosomes showed slight to moderate increase in only 50 per cent of the cases. Supplementation of ribosomes extracted from denervated muscles with normal muscle soluble enzymes increased their noncollagen synthesis by 66 per cent. This suggests that the neurogenic control of ribosomal protein synthesis is accomplished by hormonal trophic substances contained in the muscle soluble enzymes.     

Regulation of beta-nerve growth factor expression by inflammatory mediators in hippocampal cultures

Substances which regulate expression of nerve growth factor (NGF) were examined in embryonic rat hippocampal cultures containing both neurons and glial cells. Both cell types expressed NGF mRNA when cultivated in vitro. Lipopolysaccharide, an activator of macrophages, elicited a significant increase in NGF mRNA. Interleukin-1 beta evoked a similar increase in NGF mRNA which was accompanied by a rise in NGF protein. The Il-1-induced increase was partially blocked by indomethacin, suggesting that prostaglandins might mediate this effect. Treatment of the cultures directly with prostaglandin E2 resulted in elevated levels of both NGF mRNA and protein. Thus, agents which promote inflammatory activity appear to increase NGF expression. Moreover, a suppressor of inflammation, dexamethasone, decreased NGF expression. Our observations indicate that a variety of immunomodulators regulate NGF expression in the hippocampus.     

Age-dependent requirements of sympathetic neurons in serum-free culture

Neurons dissociated from chick sympathetic ganglia of different embryonic ages can be cultured equally well in serum containing or serum-free (N1) medium on collagen polyornithine, and polyornithine pretreated with neurite-promoting factor (PNPF), in the presence of nerve growth factor (NGF). Quantitative analysis of nerve cell survival is best achieved in serum-free conditions and on PNPF-pretreated polyornithine. Under such conditions, 11-day embryonic (E11) sympathetic neurons showed a specific requirement for insulin, selenium and transferrin. Similar analyses at E8 and E16 revealed that the requirement for transferrin is developmentally regulated since it is not expressed by E8 nerve cells until after 3 days in vitro. These data strongly suggest that, besides NGF, other specific molecules are involved in the control of neuronal survival and that specific requirements may be displayed at different embryonic stages.     

In vivo survival requirement of a subset of nodose ganglion neurons for nerve growth factor

The sensory neurons of the nodose ganglion are the classic example of a population of peripheral nervous system neurons that do not require nerve growth factor (NGF) for survival during development but are dependent on other neurotrophins. We have re-examined this assertion by studying the development of the nodose ganglion of mice that have a null mutation in the NGF gene. Compared with wild-type embryos, the number of neurons undergoing apoptosis was elevated in NGF -/- mice, resulting in a significant reduction in the total number of neurons in the ganglion by the end of embryonic development. TrkA, the NGF receptor tyrosine kinase, was expressed in the nodose ganglion throughout development and there was a marked decrease in TrkA mRNA expression in the nodose ganglion of NGF -/- embryos. Although the in vitro survival of the majority of nodose neurons was promoted by brain-derived neurotrophic factor (BDNF), a minor proportion was supported by NGF in cultures established over a range of embryonic stages. These results clearly demonstrate that a subset of nodose ganglion neurons depends on NGF for survival during development. The finding that the expression of tyrosine hydroxylase (TH) mRNA was unaffected in the nodose ganglia of NGF-deficient embryos indicates that this NGF-dependent subset is distinct from the subset of catacholaminergic neurons in the nodose ganglion.     

Growth and denervation response of skeletal muscle fibers of newborn rats

The cross-sectional area of the fibers of hindlimb muscles of rats increased 10-40 times during the first 6 weeks after birth. Denervation at birth stopped the growth of the muscle fibers. The number of satellite cells decreased, and eventually all fibers vanished. Reinnervation, if any, was poor. Partial denervation did not induce collateral reinnervation. Some denervated gastrocnemius muscles were reinnervated and after 8-12 months contained hypertrophic fibers and signs of necrosis and regeneration. When soleus muscles were completely denervated and cross-reinnervated after 4 weeks by the peroneal nerve, only half as many fibers became reinnervated after neonatal denervation as compared to muscles denervated at the age of 4 weeks. The experiments suggest that immature muscle fibers are less apt to become reinnervated than mature fibers. The few reinnervated fibers may be overloaded and therefore hypertrophy and eventually necrotize. Regeneration is abortive because satellite cells are scarce. These results may be relevant for the understanding of neuromuscular disorders with early (fetal) onset.