Changes of nerve growth factor synthesis in nonneuronal cells in response to sciatic nerve transection

The intact sciatic nerve contains levels of nerve growth factor (NGF) that are comparable to those of densely innervated peripheral target tissues of NGF-responsive (sympathetic and sensory) neurons. There, the high NGF levels are reflected by correspondingly high mRNANGF levels. In the intact sciatic nerve, mRNANGF levels were very low, thus indicating that the contribution of locally synthesized NGF by nonneuronal cells is small. However, after transection an increase of up to 15-fold in mRNANGF was measured in 4-mm segments collected both proximally and distally to the transection site. Distally to the transection site, augmented mRNANGF levels occurred in all three 4-mm segments from 6 h to 2 wk after transection, the longest time period investigated. The augmented local NGF synthesis after transection was accompanied by a reexpression of NGF receptors by Schwann cells (NGF receptors normally disappear shortly after birth). Proximal to the transection site, the augmented NGF synthesis was restricted to the very end of the nerve stump that acts as a "substitute target organ" for the regenerating NGF-responsive nerve fibers. While the mRNANGF levels in the nerve stump correspond to those of a densely innervated peripheral organ, the volume is too small to fully replace the lacking supply from the periphery. This is reflected by the fact that in the more proximal part of the transected sciatic nerve, where mRNANGF remained unchanged, the NGF levels reached only 40% of control values. In situ hybridization experiments demonstrated that after transection all nonneuronal cells express mRNANGF and not only those ensheathing the nerve fibers of NGF-responsive neurons.     

Developmental changes of nerve growth factor and its mRNA in the rat hippocampus: Comparison with choline acetyltransferase

Previous experiments have demonstrated that in the septo-hippocampal system choline acetyltransferase (ChAT) is induced by nerve growth factor (NGF) (Gnahn et al. (1983) Dev. Brain Res. 9, 45-52) and that hippocampal NGF and mRNANGF levels are correlated with the density of cholinergic innervation (Korsching et al. (1985) EMBO J. 4, 1389-1393). In the present investigation we have compared the developmental changes of ChAT, NGF, and mRNANGF levels in this system. During the postnatal development of the hippocampus the time courses of NGF and ChAT were well correlated including the most rapid increase between P12 and P14. This increase in hippocampal NGF was preceded by a corresponding increase in mRNANGF. The developmental changes in hippocampal NGF levels were also closely reflected by corresponding changes in the septum. This, together with previous observations (Korsching et al., 1985) that the adult septum, in spite of relatively high NGF levels, does not contain measurable quantities of mRNANGF, suggests that the NGF levels in the septum are determined by the quantity of NGF transported retrogradely from the field of innervation rather than by local synthesis. During the prenatal period hippocampal NGF levels were relatively high, whereas the mRNANGF was below the level of detection. Since the ingrowth of septal fibers, and with that also the removal of NGF by retrograde transport, begins around birth, the relatively high prenatal NGF levels probably result from an accumulation produced by a small copy number of mRNANGF prior to the removal of NGF by retrograde axonal transport. It is concluded that the correlation of the developmental changes in NGF and mRNANGF with the ChAT activity in the hippocampus further supports the concept of a physiological role of NGF in the central nervous system.     

Regulation of nerve growth factor synthesis and release in organ cultures of rat iris

We studied the synthesis and release of nerve growth factor (NGF) in cultured rat iris with a two-site enzyme immunoassay by measuring the time course of NGF levels remaining in the iris and relased into the medium up to 72 h. For up to 3 h, the NGF levels in the iris did not change significantly. After that, they increased to a maximal level of 350 +/- 30 pg NGF/iris at 19 h, which is 200 times higher than the in vivo content. Between 20 and 72 h in culture, the NGF level decreased to 130 +/- 10 pg NGF/iris, whereas general protein synthesis did not change during that time period. Maximal rate of NGF production (203 pg NGF/h/iris) was seen between 9 and 12 h in culture. In the medium, NGF levels were first detectable after 6 h. Levels then increased with a time course similar to that seen within the iris, reaching a maximal level of 1,180 +/- 180 pg after 19 h in vitro, and then did not significantly change for up to 48 h. The NGF production of the densely sympathetically innervated dilator was three times higher than that of the predominantly cholinergically innervated sphincter. The NGF production was blocked by inhibitors of messenger RNA synthesis (actinomycin D) and of polyadenylation (9-beta-D-arabinofuranosyladenine) as well as by inhibitors of translation (cycloheximide). Monensin, which interferes with the transport of proteins through the Golgi apparatus, decreased NGF levels to 8-12% of controls in the medium, suggesting that the Golgi apparatus is involved in the intracellular processing of NGF.     

Relationship between levels of nerve growth factor (NGF) and its messenger RNA in sympathetic ganglia and peripheral target tissues.

We have developed a sensitive assay for the quantification of nerve growth factor mRNA (mRNANGF) in various tissues of the mouse using in vitro transcribed RNANGF. Probes of both polarities were used to determine the specificity of the hybridization signals obtained. Comparison of NGF levels with its mRNA revealed that both were correlated with the density of sympathetic innervation. Thus, vas deferens contained high levels of both NGF and mRNANGF, whereas skeletal muscle levels were barely detectable, indicating that in peripheral tissues NGF levels are primarily regulated by the quantity of mRNANGF and not by the rate of processing of NGF precursor to NGF. However, although superior cervical ganglia contained the highest levels of NGF, its mRNA was barely detectable. Thus, the high levels of NGF in sympathetic ganglia result from retrograde axonal transport rather than local synthesis. The quantity of NGF found in the submandibular glands of female animals was three orders of magnitude higher than expected from their mRNA levels. This observation is discussed in the context of the difference between the mechanism of storage and exocytosis of exocrine glands versus the constitutive release from other tissues.     

Nerve growth factor synthesis in cultured rat iris: modulation by endogenous transmitter substances

Organ cultures of rat iris show a characteristic change in the levels of both nerve growth factor (NGF) and its mRNA: a rapid but transient initial increase is followed by a smaller but persistently elevated NGF synthesis. This time course may be influenced by release of a factor(s) from degenerating nerve terminals and/or by the lack of some factor(s) repressing NGF synthesis in vivo. We therefore analyzed the influence of biogenic amine transmitter substances and putative neuropeptides on this elevation of NGF synthesis in cultured iris. The marked increase of NGF synthesis seen initially in culture was not completely mimicked by any of the substances tested. A specific increase in NGF production up to 150% of control was observed only with cGMP. We also obtained some evidence that reaction to trauma following the culture procedure could enhance NGF production: cutting of irides into small pieces increased NGF production in culture up to 250% of control and, vice versa, treatment with 1 microM dexamethasone decreased NGF production to about 60% of control. However, the sympathetic neurotransmitter norepinephrine (NE) decreased both NGF and its mRNA levels specifically in a dose-dependent manner (0.01-1 mM) to a minimum of about 25% of control. In situ hybridization with mRNA(NGF)-specific probes showed that in cultures of dissociated iris cells all cells were capable of expressing mRNA(NGF), but that 0.1 mM NE preferentially decreased expression of mRNA(NGF) in smooth muscle cells. Thus, our results indicate that the sympathetic transmitter NE is capable of downregulating NGF synthesis in the target cells of sympathetic neurons.     

THE ROLE OF NERVE GROWTH FACTOR IN THE RAMIFICATION OF SYMPATHETIC NERVE FIBRES INTO THE RAT IRIS IN ORGAN CULTURE

Small amounts of nerve growth factor (NGF) were present in the superior cervical ganglion and the iris of the rat. The observations that NGF content in each of the tissues was depleted during organ culture and that more NGF appeared in the media than was originally present in the tissues indicated that synthesis or activation of NGF had occurred in organ culture. Antibody to NGF or the depletion of endogenous NGF retarded growth of new sympathetic axons into irides in organ culture. Exogenously added NGF appeared to enhance the initiation of axonal sprouting and the rate of the ramification of nerve fibres.     

Levels of nerve growth factor and its mRNA in the central nervous system of the rat correlate with cholinergic innervation.

The levels of nerve growth factor (NGF) and its mRNA in the rat central nervous system were determined by two-site enzyme immunoassay and quantitative Northern blots, respectively. Relatively high NGF levels (0.4-1.4 ng NGF/g wet weight) were found both in the regions innervated by the magnocellular cholinergic neurons of the basal forebrain (hippocampus, olfactory bulb, neocortex) and in the regions containing the cell bodies of these neurons (septum, nucleus of the diagonal band of Broca, nucleus basalis of Meynert). Comparatively low, but significant NGF levels (0.07-0.21 ng NGF/g wet weight) were found in various other brain regions. mRNANGF was found in the hippocampus and cortex but not in the septum. This suggests that magnocellular cholinergic neurons of the basal forebrain are supplied with NGF via retrograde axonal transport from their fields of innervation. These results, taken together with those of previous studies showing that these neurons are responsive to NGF, support the concept that NGF acts as trophic factor for magnocellular cholinergic neurons.     

Developmental changes in the responses of rat chromaffin cells to neuronotrophic and neurite-promoting factors

This study describes the survival and neurite outgrowth behaviors of cultured adrenal medullary (chromaffin) cells obtained from postnatal rats 1 day (D1) to 100 days (D100) old in response to nerve growth factor (NGF), chick eye ciliary neuronotrophic factor (CNTF), and laminin. In the absence of trophic factors the 4-day survival of cultured chromaffin cells (relative to the number of cells attached at 2 hr) increased from one-third of the cells at D1 to 40% at D8 and 90-100% at D16 and older stages. At saturating concentrations NGF increased cell survival at D8 by 90%, but failed to support all chromaffin cells present at 2 hr. In contrast, CNTF supported the survival of all cells at D8. At D1 NGF and CNTF had only a very small effect on survival during the 4-day culture period, although both factors clearly enhanced the numbers of surviving cells after 8 days. Either NGF or CNTF also elicited neurite outgrowth from rat chromaffin cells, which amounted to approximately 15-20% at D1 and D8 and subsequently decreased to about 5-8% at D30 and virtually zero at D100. At this last age both factors applied together clearly elicited neurites. Such a potentiating effect of NGF and CNTF was also seen at earlier postnatal ages. Laminin did not affect neurite growth at D30 in the absence of trophic factors, as already described for D8 rat chromaffin cells. In the presence of NGF, however, it increased neurite length and branching during a 4-day culture period and even enhanced neurite recruitment at later culture times. These data suggest that rat chromaffin cells undergo age-related changes in their responses to NGF and CNTF and that laminin modulates their neurite outgrowth behaviors in the presence of trophic factors.     

Studies on the regulation of beta-nerve growth factor gene expression in the rat iris: the level of mRNA-encoding nerve growth factor is increased in irises placed in explant cultures in vitro, but not in irises deprived of sensory or sympathetic innervation in vivo

Beta-nerve growth factor (NGF) is a protein necessary for the survival and maintenance of sympathetic and sensory neurons that appears to be produced by the target tissues of these neurons in vivo. Both denervation and the culture of explants of one model target, the rat iris, leads to an increase in the NGF content, suggesting that innervating neurons may regulate a step in synthesis or turnover of NGF. To determine whether there is a change in synthesis controlled at the mRNA level, the rat iris has been assayed for its content of NGF mRNA after surgical and chemical denervation and after explant into culture. Using a sensitive blot hybridization assay, a large, rapid increase in the content of NGF mRNA was observed upon explant of the rat iris. The increase was readily detectable within 1 h, reached a maximum increase of 10- to 20-fold by 6 to 12 h, and was still evident after 3 d in culture. The distribution of NGF mRNA in different areas of the iris does not change during this time. This rapid increase in NGF mRNA is also seen in the fully innervated iris in vivo after trauma to the anterior chamber. In contrast, denervation to varying degrees in situ had no effect on NGF mRNA levels. Neither removal of sympathetic innervation by surgical or chemical methods nor combined surgical removal of sympathetic and sensory innervation detectably altered NGF mRNA content. Thus, denervation of the rat iris in situ does not cause the observed accumulation of NGF by increasing the level of NGF mRNA, and the increase in NGF content must be due to other factors.     

K-252a Promotes Survival and Choline Acetyltransferase Activity in Striatal and Basal Forebrain Neuronal Cultures

Abstract: The organic molecule K-252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration-dependent manner. A two- to threefold increase in survival was observed at 75 n M K-252a in both systems. A single application of K-252a at culture initiation prevented substantial (>60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5-h exposure of striatal or basal forebrain cells to K-252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5-h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K-252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K-252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin-3, NGF, brain-derived neurotrophic factor, interleukin-2, basic fibroblast growth factor), only brain-derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K-252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K-252a-responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neurodegenerative diseases.     

The Death Programme in Cultured Sympathetic Neurones Can Be Suppressed at the Posttranslational Level by Nerve Growth Factor, Cyclic AMP, and Depolarization

We have examined whether sympathetic neurones that have lost the potential to be rescued by protein and RNA synthesis inhibitors after a period of nerve growth factor (NGF) deprivation are irreversibly committed to die. We found that 15 h after withdrawal of NGF from 7-day cultures of neonatal rat superior cervical ganglion neurones, 50% of the neurones lost the potential to be rescued by cycloheximide but that NGF rescued most of the neurones. By 22 h after NGF withdrawal, only 10% of the neurones were rescued by inhibition of macromolecular synthesis with cycloheximide, puromycin, or actinomycin D, but as many as 60-80% of the neurones were rescued by NGF. This is after the time at which a DNA "ladder," consistent with cell death by apoptosis, was first detected (18 h). As long as 27 h of NGF withdrawal was required before 50% of the neurones lost the potential to be rescued by NGF. The survival-promoting agent 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP) or depolarization with 50 mM KCl (HK) rescued neurones with kinetics similar to those of NGF, and rescue by all three agents did not require protein synthesis. Thus, NGF, CPTcAMP, and HK can rescue neurones deprived of NGF at much later times than either protein or RNA synthesis inhibitors by acting at the posttranslational level, a finding suggesting that initiation of the cell death programme in sympathetic neurones is not an irreversible step.     

Comparison Between the Timing of JNK activation, c-Jun Phosphorylation, and Onset of Death Commitment in Sympathetic Neurones

Abstract: We have investigated the relationship between c-Jun N-terminal kinase (JNK) activity, apoptosis, and the potential of survival factors to rescue primary rat sympathetic neurones deprived of trophic support. Incubation of sympathetic neurones in the absence of nerve growth factor (NGF) caused a time-dependent increase in JNK activity, which became apparent by 3 h and attained maximal levels that were three- to fourfold higher than activity measured in neurones maintained for the same periods with NGF. Continuous culture in the presence of either NGF or the cyclic AMP analogue 4-(8-chlorophenylthio) cyclic AMP (CPTcAMP) not only prevented JNK activation from occurring, but also suppressed JNK activity that had been elevated by prior culture of the neurones in the absence of trophic support. When either NGF or CPTcAMP was added to cultures that had been initially deprived of neurotrophic support for up to 10 h, this resulted in complete suppression of total JNK activity, arrest of apoptosis, and rescue of >90% of the neurones that did not display apoptotic morphology by this time. However, when either agent was added after more protracted periods of initial neurotrophin deprivation (≥ 14 h), although this also resulted in near-complete suppression of total JNK activity and short-term arrest of apoptosis, not all of the neurones that appeared to be nonapoptotic at the time of agent addition were rescued. The lack of death commitment after 10 h of maintained JNK activity was not due to a late induction of c-Jun expression, because the majority of newly isolated sympathetic neurones had already been expressing high levels of c-Jun in their nuclei for several hours, yet were capable of being rescued by NGF. Elevation of JNK activity as a result of neurotrophic-factor deprivation was also associated with enhanced phosphorylation of c-Jun, assessed by immunoblot analysis and immunocytochemistry, and addition of NGF to cultures previously deprived of neurotrophic support resulted in a reversion of the state of phospho-c-Jun to that observed in cultures that had been maintained in the continuous presence of trophic support. We conclude that activation of JNK and c-Jun phosphorylation are not necessarily rate-limiting for apoptosis induction. In some neurones undergoing prolonged NGF deprivation, suppression of JNK activity and c-Jun dephosphorylation by NGF may be insufficient to effect their rescue. Thus, if c-Jun mediates death by increasing the expression of “death” genes, these must become effective very close to the death commitment point.     

Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation

We have developed an experimental paradigm to study the mechanism by which nerve growth factor (NGF) allows the survival of sympathetic neurons. Dissociated sympathetic neurons from embryonic day-21 rats were grown in vitro for 7 d in the presence of NGF. Neurons were then deprived of trophic support by adding anti-NGF antiserum, causing them to die between 24 and 48 h later. Ultrastructural changes included disruption of neurites, followed by cell body changes characterized by an accumulation of lipid droplets, changes in the nuclear membrane, and dilation of the rough endoplasmic reticulum. No primary alterations of mitochondria or lysosomes were observed. The death of NGF-deprived neurons was characterized biochemically by assessing [35S]methionine incorporation into TCA precipitable protein and by measuring the release of the cytosolic enzyme adenylate kinase into the culture medium. Methionine incorporation began to decrease approximately 18 h post-deprivation and was maximally depressed by 36 h. Adenylate kinase began to appear in the culture medium approximately 30 h after deprivation, reaching a maximum by 54 h. The death of NGF-deprived neurons was entirely prevented by inhibiting protein or RNA synthesis. Cycloheximide, puromycin, anisomycin, actinomycin-D, and dichlorobenzimidazole riboside all prevented neuronal death subsequent to NGF deprivation as assessed by the above morphologic and biochemical criteria. The fact that sympathetic neurons must synthesize protein and RNA to die when deprived of NGF indicates that NGF, and presumably other neurotrophic factors, maintains neuronal survival by suppressing an endogenous, active death program.     

Concentration-dependent regulation of neuronal gene expression by nerve growth factor

NGF is a neurotrophic protein that promotes the survival, growth, and differentiation of developing sympathetic neurons. To directly determine the effects of different concentrations of NGF on neuronal gene expression, we examined mRNAs encoding the p75 low-affinity NGF (LNGF) receptor, T alpha 1 alpha-tubulin (T alpha 1), and tyrosine hydroxylase (TH) in pure cultures of rat sympathetic neurons from postnatal day 1 superior cervical ganglia. Studies of the timecourse of gene expression during 2 wk in culture indicated that a 5-d incubation period would be optimal for the concentration-effect studies. Analysis of RNA isolated from neurons cultured in 2-200 ng/ml 2.5S NGF for 5 d revealed that, as the NGF concentration increased, neurons expressed correspondingly increased levels of all three mRNAs. Both LNGF receptor and TH mRNAs increased seven-fold, and T alpha 1 mRNA increased four-fold in neurons cultured in 200 versus 10 ng/ml NGF. In contrast, T26 alpha-tubulin mRNA, which is constitutively expressed, did not alter as a function of NGF concentration. When neurons were initially cultured in 10 ng/ml NGF for 5 d, and then 200 ng/ml NGF was added, LNGF receptor, T alpha 1, and TH mRNAs all increased within 48 h. The timecourse of induction differed: T alpha 1 mRNA was maximal by 5 h, whereas LNGF receptor and TH mRNAs first began to increase at 12 h after the NGF increase. These experiments show that NGF regulates expression of a subset of mRNAs important to neuronal growth and differentiation over a broad concentration range, suggesting that the effects of NGF may be mediated by more than just a single receptor operating at one fixed affinity. These results also suggest a mechanism for coupling neuronal synthesis of axonal proteins to increases in size of the innervated target territory during growth of the organism.     

Role of Nerve Growth Factor in Oxidanl Homeostasis: Glutathione Metabolism

Abstract— Free radicals are generated in the CNS by ongoing oxygen metabolism and biological events associated with injury and inflammation. Increased free radical levels may also persist in some chronic neurological diseases and in the aged. Nerve growth factor (NGF) is a member of the neurotrophin family of proteins that can regulate neuronal development, maintenance, and recovery from injury. NGF protected rat pheochromocytoma PC12 cells, an adrenal chromaffin-like NGF-responsive cell line, from the oxidant stress accompanying hydrogen peroxide treatment by stimulating GSH levels and enzymes in the GSH metabolism cycle and in the GSH/GSH peroxidase antioxidant redox system, a ubiquitous cellular antioxidant system. Specifically, NGF increased γ-glutamylcysteine synthetase (GCS) activity, the rate-limiting enzyme for GSH synthesis, by 50% after 9h and GSH levels by 100% after 24 h of treatment. NGF stimulated GSH peroxidase by 30% after 3 days and glucose 6-phosphate dehydroge-nase by 50% after 2 days. Treatment with NGF and cyclo-heximide, or actinomycin D, which inhibit protein and RNA synthesis, respectively, blocked the NGF stimulation of GCS and glucose 6-phosphate dehydrogenase. Increased GSH levels due to NGF treatment were responsible for the significant protection of PC12 cells from hydrogen peroxide-induced stress. Pretreatment of PC12 cells with NGF for 24 h rescued cells from the toxic effects of the extracellular hydrogen peroxide generated by the glucose/glucose oxidase system but did not rescue cells that were subjected to GSH deprivation due to treatment with 10 μMl -buthionine-(S,R)-sulfoximine, an inhibitor of GCS. However, treatment with 10 μMl -buthionine-(S,R)-sulfoximine alone did not affect PC12 cell viability, NGF stimulation of neurite extension, and NGF induction of GCS, GSH peroxidase, and glucose 6-phosphate dehydrogenase activity. When GSH levels were measured in PC12 cells that were treated for 24 h with other neurotrophins and growth factors, such as brain-derived neurotrophic factor, neurotro-phin-3, epidermal growth factor, insulin-like growth factor-I, and basic fibroblast growth factor, only epidermal growth factor was found to increase GSH levels by 30%. Whereas NGF increased GSH levels in the human neuro-blastoma SK-N-SH-SY5Y and the human melanoma A-875 in serum-free medium, addition of fetal calf serum to the medium abolished the NGF effects on GSH levels in the NGF-responsive cell lines, SK-N-SH-SY5Y, A-875, and the CNS C6 rat glioma subclone 2BD.     

Absence of the alpha and gamma subunits of 7S nerve growth factor in denervated rodent iris: immunocytochemical studies

Immunocytochemical studies were performed to determine if denervated rodent iris produces nerve growth factor (NGF) in a form chemically similar to that of the 7S NGF complex in mouse submandibular glands. Antisera to the alpha, beta, and gamma subunits of 7S NGF were raised in rabbits and characterized on immunoblots of SDS-containing polyacrylamide gels. Antisera were applied to stretch preparations of rat and mouse irides that were cultured for periods of 2 to 6 days or sympathetically denervated by superior cervical ganglionectomy and left in situ 4 days. Antibody binding was visualized by indirect immunofluorescence. In control studies done on plastic sections of mouse submandibular glands, antisera co-localized the three subunits of 7S NGF within secretory granules of granular tubule cells. In denervated rat iris, beta NGF immunoreactivity was evident in a cellular plexus that resembled in distribution and morphology nerve fibers in the normal iris, in agreement with a previous study (R.A. Rush (1984). Nature (London) 312, 364-367). Identical staining patterns were observed in mouse iris. In neither rat or mouse, however, did the nerve-like processes stain with antibodies suggests that the NGF-like protein in denervated rodent iris is not synthesized as part of the 7S NGF complex. Iris also did not react with antibodies to epidermal growth factor, a protein co-localized with NGF in mouse submandibular glands and in guinea pig prostate.     

Modulation of alkaline phosphodiesterase I in cultured rat hepatocytes

Alkaline phosphodiesterase I activity was measured in adult and foetal rat hepatocytes maintained in primary culture under various conditions. This enzyme was found to be expressed in both cell populations and could be resolved into two bands having apparent molecular weights of 130,000 and 250,000, respectively. Alkaline phosphodiesterase I activity was already at high levels in 15 day foetal liver and, as early as the 19th day of gestation, it reached adult levels. Alkaline phosphodiesterase I levels were well maintained during culture. In the absence of serum, its level continued to increase with time in foetal cells. It dramatically increased by days 4 and 5, in adult cells maintained on fibronectin and plastic, respectively. Dexamethasone stimulated alkaline phosphodiesterase I activity after a lag phase of 8 h, with a maximum reached after 40 h. As this induction was prevented by addition of actinomycin D or cycloheximide, it could be concluded that it required RNA and protein synthesis. Only the major Mr 250,000 form responded to dexamethasone and was sensitive to serum.     

Fate of intraocular chromaffin cell suspensions: role of initial nerve growth factor support

Summary Adrenal medullary tissue from adult rats was dissociated into cell suspensions and injected into the anterior chamber of the eye, where the cells were made to attach to the previously sympathectomized irides with the use of fibronectin. Short- and long-term survival of the chromaffin cells was examined in whole mounts of irides using Falck-Hillarp fluorescence histochemistry or indirect immunohistochemistry with antibodies against adrenaline and dopamine--hydroxylase (DBH). After 6 days in oculo all cells were immunoreactive for adrenaline; almost none displayed processes even if -nerve growth factor (NGF) was given at grafting. One month after weekly intraocular injections of NGF, many cells were surrounded by nerve fiber net-works and all cells were DBH-immunoreactive. Eight months postgrafting and 7 months after the last injection of NGF almost the entire iris was reinnervated and resembled a normal, sympathetically innervated iris. Both at 1 and 8 months, chromaffin cells, ganglion cells and transitional cell forms (chromaffin cells transforming towards ganglion-like cells) were found in irides from the NGF-treated eyes. The number of ganglion cells was remarkably increased with time by NGF, while the number of chromaffin cells decreased compared to controls. A single treatment with NGF at grafting had no marked effects as examined up to 3 months; at this time there was a certain outgrowth of nerve terminals, which, however, was not as pronounced as 1 month after repeated NGF injections. In conclusion, it is shown that some cells in a chromaffin cell suspension attach to the iris, transform to ganglion cells after an induction with exogenous NGF, and reinnervate the sympathically denervated iris. Such cells remain ganglion-like in character and continue to form processes even after cessation of exogenous NGF treatment.