Ontogeny of the nerve growth factor receptor in primary cultures of neural crest cells

Neural crest cells maintained as primary cultures for 4-7 days demonstrated specific binding of radioiodinated nerve growth factor ([125I]NGF); occasionally, significant binding of ([125I]NGF could be detected in 3-day cultures. Parallel cultures processed for indirect immunofluorescence revealed that the NGF receptor-positive neural crest cells represented a subpopulation of the total cells in culture. Cultures aged 4-7 days demonstrated fluorescent cells which were often grouped as aggregates. Some 3-day cultures contained faintly fluorescent cells. One- and two-day cultures were non-fluorescent. Melanocytes did not appear to bind NGF. Preliminary flow cytofluorometric analysis indicated that ca. 28% of neural crest cells in 5-day cultures possessed specific receptors for NGF.     

Catecholaminergic cells and support cell precursors in neural crest cultures differentially express nerve growth factor receptors

Long-term neural crest cultures grown in the continuous absence of exogenous nerve growth factor (NGF) contain a subpopulation of cells with NGF receptors exclusively of the low affinity subtype (Kd of approximately 3.2 nM). The current studies combined immunocytochemistry, using GIN1 (a support cell marker) or tyrosine hydroxylase antibodies, with radioautography following exposure to iodinated nerve growth factor (125I-NGF). The majority of cells specifically binding 125I-NGF were found to be immunoreactive for GIN1, indicating that the primary cell phenotype expressing receptors for NGF appear to be support cell precursors, at least under these conditions. These cells are likely to be responsive to and/or dependent upon NGF; the nature of this response or dependency remains to be determined. Some cells exhibiting silver grains were not immunoreactive for GIN1, suggesting that other cell phenotypes in neural crest cultures also have NGF receptors. In addition, some neural crest cells were found that stained with GIN1 and lacked 125I-NGF binding. Tyrosine hydroxylase-like immunoreactive cells apparently did not bind 125I-NGF under these culture conditions. Catecholaminergic sympathetic and sensory neurons from embryonic ganglia, derived from the neural crest, express both the high and low affinity forms of the NGF receptor. In order to determine whether the microenvironment played a role in the type of catecholaminergic cells appearing in culture, neural crest cells were grown in the continuous presence of exogenous NGF. Under these conditions, many tyrosine hydroxylase-like immunoreactive cells were found that specifically bound 125I-NGF. In addition, silver grains were still detected on these cells following a chase with nonradioactive NGF, designed to eliminate 125I-NGF bound to low affinity sites. Therefore, the catecholaminergic cells possess both the low and high affinity forms of the receptor. NGF's ability to modulate tyrosine hydroxylase activity, as it does in mature catecholaminergic neurons, was tested in this system. Surprisingly, there was no statistically significant difference in tyrosine hydroxylase activity in cultures grown in the absence or presence of exogenous NGF. This raises the possibility that embryonic catecholaminergic cells are unable to respond to NGF in this specific way, even though the receptors for the factor are present.     

Neuron-like cells in long-term neural crest cultures are not targets of nerve growth factor

It has been shown previously that a subpopulation of long-term (7-14 days) cultured neural crest cells undergoing differentiation possesses receptors for nerve growth factor (NGF). These cells are likely to be targets of NGF during the early stages of embryonic development. This study was conducted to determine whether cells exhibiting neuron-like characteristics (i.e. process formation, presence of putative neurotransmitters) in neural crest cultures have NGF receptors. This was accomplished by combining 125I-NGF radioautography and immunocytochemistry using antibodies against tyrosine hydroxylase, serotonin, and vasoactive intestinal polypeptide. Examination of light microscopic radioautographs revealed that none of the neuron-like cells with tyrosine hydroxylase-like, serotonin-like, or vasoactive intestinal polypeptide-like immunoreactivity bound 125I-NGF, and, therefore, do not possess NGF receptors. It is not known whether the lack of NGF receptors on neuron-like cells is due to the early developmental stage of these cells, or is caused by a difference in the microenvironment in vitro as compared to in vivo. The identity of the cultured neural crest cells which do possess NGF receptors remains to be determined.     

Selective expression of high-affinity nerve growth factor receptors on tyrosine hydroxylase-containing neuron-like cells in neural crest cultures

A subpopulation of cultured neural crest cells undergoing differentiation have receptors for nerve growth factor (NGF) that exhibit a binding constant similar to that of the low-affinity NGF binding site (3.2 nM). Recent studies have shown that NGF receptors are not present on neuron-like cells immunoreactive for tyrosine hydroxylase (TH), serotonin, or vasoactive intestinal polypeptide. Since tissues innervated by sympathetic neurons in vivo produce NGF, we sought to determine whether NGF deficits in the tissue culture microenvironment may be one parameter preventing the expression of NGF receptors on TH-containing neuron-like cells. Neural crest cultures were therefore grown in complete tissue culture medium (15% fetal bovine serum and 5% chicken embryo extract), with or without exogenous NGF (50 ng/ml). Examination of light-microscopic radioautographs following incubation with 125I-NGF revealed that, if the cultures were supplemented with NGF for 7 d, approximately 33% of neuron-like cells exhibiting TH-like immunoreactivity possessed NGF receptors. There were no obvious morphological differences between TH-containing cells that did or did not have NGF receptors. Scatchard analysis of cultures grown under these conditions again demonstrated the sole presence of the low-affinity form of the NGF receptor (Kd, 3.4 nM). Embryonic catecholaminergic sympathetic neurons exhibit both high- and low-affinity forms of the NGF receptor, raising the possibility that the Scatchard analysis may not have been sensitive enough to detect the high-affinity form of the receptor on a relatively small population of cells. Therefore we used a morphological approach that took advantage of the different dissociation rates of the 2 receptor types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve growth factor (NGF) receptor expression in chicken cranial development

In order to map the expression of receptors for nerve growth factor (NGF) during brain and cranial ganglia development, iodinated NGF (125I beta NGF) was used as a probe in an autoradiographical analysis performed between embryonic day 3 (E3) and posthatching day 3 (P3) of chicken development. Heavy autoradiographic labelling was observed at the classical NGF target sites, the proximal cranial sensory ganglia and the sympathetic superior cervical ganglion, throughout development and after hatching. In contrast, only weak labelling could be detected during a restricted time span in the vestibulocochlear (E4-E8) and the distal cranial sensory ganglia (E4-E10), the neurons of which originate from the otic and epibranchial placodes. Specific 125I beta NGF binding was also observed in various brain regions during early brain development. NGF receptor expression there followed a characteristic pattern. The neuroepithelial layer displayed very low levels of specific 125I beta NGF binding, while strong 125I beta NGF labelling was found in the mantle layer. Brainstem somatomotor nuclei, visceromotor columns, brainstem alar plate, cerebellar anlage, tectum, and basal forebrain (epithalamus, striatum) were found to be transiently labelled by 125I beta NGF in early development (E4-E12). Non-nervous tissues such as parts of the otic vesicle epithelium and skeletal muscle anlagen of the head were also labelled. These results, showing specific binding of 125I beta NGF to cranial cells of different origin (neural tube, neural crest, placode, and possibly mesoderm) strengthen the concept that NGF may have diverse functions in growth and differentiation of various tissues and cell types.     

Cultured rat Schwann cells express low affinity receptors for nerve growth factor

Schwann cell cultures prepared from postnatal Sprague-Dawley rat sciatic nerves were used to demonstrate the presence of specific receptors for the beta-subunit of nerve growth factor (NGF) on rat Schwann cells. Indirect immunofluorescence microscopy with a monoclonal antineuronal NGF receptor (NGFR) antibody indicated that NGFR antigen was expressed on the surface of Schwann cells but not of endoneurial fibroblasts. Studies with 125I-NGF confirmed this distribution of NGFR in the cultures and showed that the Schwann cell NGFR had a single NGF binding affinity (Kd of 1.8 x 10(-9) M). 125I-NGF binding by the cultured Schwann cells increased with time in vitro, reaching a plateau level on the 4th day, but decreased with increasing age, reaching 40% of the neonatal value in Schwann cells isolated from 12-day-old rats. Treatment of the cultures with NGF did not alter Schwann cell phenotype, survival or proliferation.     

Characterization of nerve growth factor binding to embryonic rat spinal cord neurons

The binding of iodinated beta-nerve growth factor, [125I]-NGF, to embryonic (E16) rat spinal cord cells, was investigated to characterize the binding properties and cellular distribution of nerve growth factor receptors. Spinal cord cells prepared without trypsin yielded two classes of NGF binding sites with Kd's of 3 x 10(-11) M and 4 x 10(-9) M. Fractionation of the cells by discontinuous gradients composed of 8%, 12%, and 17% metrizamide was used to separate motoneurons from other cell types. The motoneuron enriched fraction (8% metrizamide) contained approximately 10% of the cells and 64% of the choline acetyltransferase (ChAT) activity. In contrast, the 12% metrizamide fraction contained most (51%) of the cells and 36% of the ChAT activity, while the 17% metrizamide fraction contained the remainder of the cells and negligible amounts of ChAT activity. Characterization of [125I]-NGF binding to each metrizamide fraction showed that the motoneuron-enriched fraction exhibited both high and low affinity binding sites, while the other metrizamide fractions exhibited only the low affinity binding sites. These findings indicate that although low affinity NGF receptors appear to be relatively evenly distributed amongst embryonic rat spinal cord cells, high affinity NGF receptors are found primarily on motoneurons.     

Nerve growth factor receptors on normal and injured sensory neurons

The density and binding properties of receptors for nerve growth factor (NGF) in normal and injured sensory neurons have been analyzed by quantitative radioautography following incubation of tissue sections with radioiodinated NGF. The technique is designed to study binding sites that are half-maximally saturated by picomolar concentrations of NGF: Additional sites of lower affinity have not been emphasized. In normal adult rats, approximately half of lumbar sensory neurons have high-affinity receptors for NGF. One month after the sciatic nerve is cut, the mean number of high-affinity sites on heavily labeled neurons in the fifth lumbar dorsal root ganglion falls to less than 20% of normal values because of reduced receptor density and cell volume. Neurons with high-affinity receptors are more liable to atrophy after injury than those lacking such receptors. Receptors are lost not only in the cell bodies of sensory neurons but also on their peripheral and central processes. Delayed administration of NGF to the sciatic nerve 3 weeks after it is cut restores the receptor density to normal values and partially restores the neuronal cell volume. As part of the response to axonal injury and possibly because the cell body is deprived of NGF, fewer high-affinity receptors are displayed by sensory neurons. For at least 3 weeks after nerve transection, neurons that are atrophic and depleted of NGF receptors can be resuscitated by exogenous NGF.     

Uptake of nerve growth factor along peripheral and spinal axons of primary sensory neurons

To investigate the distribution of nerve growth factor (NGF) receptors on peripheral and central axons, [125I]NGF was injected into the sciatic nerve or spinal cord of adult rats. Accumulation of [125I]NGF in lumbar dorsal root ganglia was monitored by gamma emission counting and radioautography. [125I]NGF, injected endoneurially in small quantities, was taken into sensory axons by a saturable process and was transported retrogradely to their cell bodies at a maximal rate of 2.5 to 7.5 mm/hr. Because very little [125I]NGF reached peripheral terminals, the results were interpreted to indicate that receptors for NGF are present on nonterminal segments of sensory axons. The specificity and high affinity of NGF uptake were illustrated by observations that negligible amounts of gamma activity accumulated in lumbar dorsal root ganglia after comparable intraneural injection of [125I] cytochrome C or [125I]oxidized NGF. Similar techniques were used to demonstrate avid internalization and retrograde transport of [125I]NGF by intraspinal axons arising from dorsal root ganglia. Following injection of [125I]NGF into lumbar or cervical regions of the spinal cord, neuronal perikarya were clearly labeled in radioautographs of lumbar dorsal root ganglia. Sites for NGF uptake on primary sensory neurons in the adult rat are not restricted to peripheral axon terminals but are extensively distributed along both peripheral and central axons. Receptors on axons provide a mechanism whereby NGF supplied by glia could influence neuronal maintenance or axonal regeneration.     

Distribution of Radioiodinated Recombinant Human Nerve Growth Factor in Primate Brain Following Intracerebroventricular Infusion

The distribution of radioiodinated recombinant human nerve growth factor ([¹²⁵I]rhNGF) was evaluated in adult cynomolgus monkeys following unilateral intracerebroventricular (icv) administration. Animals were cannulated into the right ventricle and recovered for 7 days. Monkeys were infused with 1.2 μg of [¹²⁵I]rhNGF or [¹²⁵I]rhNGF with a 140-fold excess of rhNGF. Twenty-four hours after infusion, animals were anesthetized and transcardially perfused with an aldehyde fixative. Coronal brain sections were processed for quantitative film autoradiography or for cholineacetyltransferase immunohistochemistry and then emulsion dipped. Specific radiolabel was distributed bilaterally and, with equal density, throughout the basal forebrain and was colocalized with choline acetyltransferase-positive neurons. Specific labeling was also present in the superficial ventral cortex. Nonspecific binding was observed surrounding the ventricles and lining blood vessels. These results demonstrate that unilateral icv infusion is an effective approach for delivering NGF to basal forebrain cholinergic neurons in primates and represents a viable drug delivery strategy for the therapeutic use of NGF in Alzheimer's Disease.     

Retrograde transport of nerve growth factor in lesioned goldfish retinal ganglion cells

Previous experiments have shown that nerve growth factor (NGF) enhances regeneration of goldfish optic nerve after local application of NGF at the site of the lesion. However, the site and mechanism of action of NGF are not yet known. One possibility is that NGF is taken up at the site of the lesion and retrogradely transported to the cell bodies of the retinal ganglion cells and thereby exerts its trophic effects. The present work was carried out to assess the role of retrograde transport of NGF in this enhanced regeneration of goldfish retinal ganglion cells. In intact retinal ganglion cells of the goldfish, 125I-labeled NGF was found not to be retrogradely transported from the optic tectum to the retina, suggesting that retinal ganglion cells do not possess specific NGF receptors. However, if [125I]NGF was injected at the site of an optic nerve lesion at the time of lesion, [125I]NGF was retrogradely transported from the site of a lesion of the optic nerve to the cell body of retinal ganglion cells. The accumulated radioactivity was shown to be intact NGF by SDS-PAGE. The ability of NGF to decrease the time required for recovery of visual function was observed only when NGF was administered at the time of the injury. Likewise, no transport of [125I]NGF was observed when it was injected at the crush site 16 hr or longer after crush. Thus, there is a temporal correlation between the ability of intact [125I]NGF to be retrogradely transported from a lesion site to the retina and the regenerative effect of NGF. Autoradiography showed that the [125I]NGF accumulated only in retinal ganglion cells. The transport of NGF in the lesioned goldfish visual system was not specific for NGF in that other proteins (cytochrome c, bovine serum albumin) were transported equally well. Likewise, transport of [125I]NGF was not prevented by concomitant administration of excess unlabeled NGF. The retrograde transport of [125I]NGF therefore was not selective and did not appear to be mediated by specific NGF receptors in this system. This nonspecific transport of [125I]NGF did not occur in the axotomized spinal motor neurons in the neonatal or adult rat or in the newt. However, receptor-mediated transport is seen in lesioned sensory neurons in both species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Binding and retrograde transport of leukemia inhibitory factor by the sensory nervous system.

Leukemia inhibitory factor (LIF), a peptide growth factor with multiple activities, has recently been shown to support the generation and survival of sensory neurons in cultures of mouse neural crest and dorsal root ganglia (DRG). We have conducted binding experiments with 125I-LIF on cultures of DRG to determine the receptor distribution for LIF on these cells and found that at least 60% of the sensory neurons in the cultures bound 125I-LIF, all of which could be eliminated by the addition of unlabeled LIF. The other cells in the culture, which morphologically appeared to be Schwann cells, did not bind appreciable quantities of 125I-LIF. In order to investigate whether LIF is retrogradely transported to sensory neurons in vivo, 125I-LIF was injected into the footpads and gastrocnemius muscles of newborn and adult mice, following sciatic nerve ligation. Radioactivity accumulated in the distal portion of the sciatic nerve, indicating retrograde transport of LIF. Subsequent experiments on mice with unligated sciatic nerves showed that 125I-LIF is specifically transported into the sensory neurons of the DRG. There was no apparent transport of 125I-LIF into motor neurons in the spinal cord. These experiments demonstrate that LIF can specifically bind to and be transported by sensory neurons and further support the idea that LIF acts as a target-derived neurotrophic factor, analogous to NGF.     

Alpha 1-adrenergic receptors in the brain: characterization in astrocytic glial cultures and comparison with neuronal cultures

Binding of [125I]HEAT to membranes prepared from primary cultures of astrocytic glial cells was time-dependent and 70-85% specific. Various adrenergic agonists and antagonists competed for [125I]HEAT binding according to the potencies of prazosin greater than, yohimbine greater than or equal to, clonidine, norepinephrine (NE), and propranolol. Scatchard analysis showed the Bmax of 209 fmol/mg protein and a Kd of 184 pM for [125I]HEAT binding by astrocytic glial membranes. Pretreatment of astrocytes with NE resulted in a dose-dependent downregulation of [125I]HEAT binding sites with a maximal response observed after 8 h at 100 microM NE. Removal of NE from cultures after pretreatment resulted in a time- and protein synthesis-dependent recovery of binding sites to control levels within 120 h. Incubation of astrocytic glial cultures with NE stimulated phosphoinositide (PI) hydrolysis in a time- and dose-dependent manner with a maximal stimulation of 2-fold observed in 60 min by 100 microM NE. Clonidine expressed differential effects on alpha 1-adrenergic receptors of the neuronal and astrocytic glial cultures. Pretreatment with 10 microM clonidine caused a 40% decrease in the Bmax of [125I]HEAT binding without influencing the Kd value in neuronal cultures. This downregulatory effect of clonidine was associated with a reduction in the ability of NE to stimulate PI hydrolysis in clonidine pretreated cells. In contrast to neuronal cultures, clonidine neither downregulated [125I]HEAT binding sites nor stimulated PI hydrolysis in glial cultures.     

Localization of nerve growth factor bound to neurons growing nerve fibers in culture

The peptide hormone, nerve growth factor, is necessary for the normal growth and development of sensory and sympathetic neurons. In culture, this hormone is also need for the regeneration of fibers from sensory and sympathetic neurons. We have needed for the regeneration of fibers from sensory and sympathetic neurons. We have determine the distribution of the nerve growth factor receptors on cells cultured from embryonic dorsal root ganglia. The binding of the radiolabeled nerve growth factor is highly selective for neurons, though some saturable binding is present on non-neuronal cells, the density of binding is less than 10% of that on neurons. By incubating cultures with different concentrations of radiolabeled nerve growth factor, we were able to quantify the type I (high affinity) and type II (low affinity) sites on neuronal somata and along growing nerve fibers. We estimate that there are 5 times more type I sites on the nerve fibers than on the cell bodies. On the other hand, type II sites appear to be uniformly distributed on nerve fibers and cell bodies. The amount of radiolabeled nerve growth factor binding was directly correlated with the extent of nerve fiber growth. However, binding to growth cones was variable and was not obviously different from the main axis cylinder of the growing nerve fiber. Finally, the saturable binding found on non-neuronal cells appears to be the same as the type II sites on neuronal cells. Bases upon our calculations, the apparent equilibrium dissociation constants for the two types of cells are the same (approximately 2 X 10(-9) M).     

Retrograde axonal transport of [125I]nerve growth factor in ileal mesenteric nerves in vitro: effect of streptozotocin diabetes

The retrograde axonal transport of intravenously administered [125I]nerve growth factor ([125I]NGF) was examined in ileal mesenteric nerves maintained for short periods in vitro. [125I]NGF was injected systemically, and at various times thereafter mesenteric pedicles were ligated and incubated in vitro in Krebs-Henseleit medium under a number of different conditions. Retrogradely transported [125I]NGF began to accumulate distal to the ligature after an initial lag period and increased in a linear fashion for 3-4 h. The amount of retrogradely transported [125I]NGF was proportional to the length of the ileum innervated by each pedicle, which allowed for comparison of ileal segments of different lengths. Retrograde axonal transport of [125I]NGF was inhibited by vinblastine, colchicine and incubation in the cold, and was decreased by agents that interfere with oxidative or glycolytic metabolism. The accumulation of retrogradely transported [125I]NGF in ileal mesenteric nerves of 1-9 day streptozotocin diabetic animals placed in an in vitro bath containing normal (5.5 mM) glucose was decreased 40% compared to control animals. The induction of diabetes in vivo resulted in a greater decrease in the early phases of [125I]NGF export from ileal mesenteric nerve terminals compared to later phases. Ileal mesenteric nerve segments derived from untreated controls were incubated in vitro in media containing increased concentrations of glucose (27.5 and 50 mM) without reproducing the NGF transport defect found in diabetic animals.     

Regulation of Schwann cell nerve growth factor receptor by cyclic adenosine 3',5'-monophosphate

Previous studies indicated that Schwann cells in immature nerves express nerve growth factor (NGF) receptors, and that this expression is down regulated during development but re-induced by Wallerian degeneration. It was also shown that immature Schwann cells are induced to express galactocerebroside and other molecules characteristic of mature Schwann cells by either contact with an axon or treatment with the cyclic adenosine 3',5'-monophosphate (cAMP) analogues dibutyryl cAMP (dbcAMP) and 8-bromo cAMP or the adenylate cyclase activator forskolin. In the present study, NGF receptors on the surface of cultured Schwann cells were demonstrated by binding of an anti-rat NGF receptor monoclonal antibody or of radioiodinated NGF. Treatment of cultured Schwann cells with cAMP analogues or forskolin resulted in a progressive decrease in both immunoreactive NGF receptors and radioiodinated NGF binding. The cultured Schwann cells contained a polyadenylated RNA species homologous with human melanoma NGF receptor mRNA in sequence and size. The amount of this NGF mRNA was lower in cAMP analogue-treated than in untreated Schwann cells.     

Effect of neonatal treatment with capsaicin on the numbers and properties of cutaneous afferent units from the hairy skin of the rat

Carbamylcholine (CCh)-induced 22Na+ influx into clonal rat pheochromocytoma PC12h cells increased remarkably by culturing the cells in the presence of nerve growth factor (NGF) at a concentration of 50 ng/ml. After 2-4 days in culture with NGF, the CCh-induced 22Na+ influx into cells was enhanced 3- to 5-fold compared to the influx into NGF-untreated cells. No increase of CCh-induced 22Na+ influx was seen prior to 15 h. The ED50 value for NGF to increase the responsiveness was 4.6 ng/ml. This effect could not be mimicked by the addition of 1 mM dibutyryl cyclic AMP. Besides NGF, epidermal growth factor increased the CCh-induced 22Na+ influx into cells to a lower extent that NGF did. Insulin and dexamethasone had no effect. By contrast, the amount of [125I] alpha-bungarotoxin binding to PC12h cells was not changed when cultured in the presence of NGF. It is concluded that the functional nAChR and alpha-bungarotoxin binding sites of PC12h cells are controlled by different mechanisms.     

GABAergic and cholinergic neurons exhibit high-affinity nerve growth factor binding in rat basal forebrain

We have used dissociated, rat basal forebrain cultures to identify specific cell types that are potentially responsive to nerve growth factor (NGF). Expression of high-affinity NGF binding sites was examined. A subpopulation of cells containing choline acetyltransferase (CAT), the acetylcholine-synthesizing enzyme, exhibited high-affinity binding, employing combined immunocytochemistry and 125I-NGF radioautography. Unexpectedly, a gamma-aminobutyric acid (GABA)-containing cell group also expressed high-affinity binding. These cells that exhibit high-affinity binding appear to be neurons since they stain positively with the neuron marker, neuron-specific enolase, and negatively with the nonneuron marker, glial fibrillary acidic protein. Our observations suggest that NGF may regulate multiple brain systems and functions that have yet to be explored. Conversely, only subsets of cholinergic or GABA neurons expressed high-affinity binding, suggesting that these transmitter populations are composed of differentially responsive subpopulations.     

Interleukin-1 beta, but not IL-1 alpha, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor.

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1 beta (IL-1 beta). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1 alpha and IL-1 beta) and the antagonist (IL-1ra)-members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [(125)I] IL-1 beta to cultured astrocytes was saturable and of high affinity. Mean values for the K(D) and B(max) were calculated to be 60.7+/-7.4 pM and 2.5+/-0.1 fmol mg(-1) protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1 alpha (K(i) of 341.1 pM) and IL-1 beta (K(i) 59.9 pM), as well as the antagonist IL-1ra (K(i) 257.6 pM), displaced specific [(125)I] IL-1 beta binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [(125)I] IL-1 beta binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1 alpha and IL-1 beta revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1 beta (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1 alpha by itself had no stimulatory action on NGF release. The simultaneous application of IL-1 alpha and IL-1 beta elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1 beta. We concluded that IL-1 beta-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1 alpha and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.     

Differential regulation of SHC proteins by nerve growth factor in sensory neurons and PC12 cells

We have characterized some of the nerve growth factor (NGF) stimulated receptor tyrosine kinase (TrkA) signalling cascades in adult rat primary dorsal root ganglia (DRG) neuronal cultures and compared the pathways with those found in PC12 cells. TrkA receptors were phosphorylated on tyrosine residues in response to NGF in DRG neuronal cultures. We also saw phosphorylation of phospholipase Cγ1 (PLCγ1). We used recombinant glutathione-S-transferase (GST)–PLCγ1 SH2 domain fusion proteins to study the site of interaction of TrkA receptors with PLCγ1. TrkA receptors derived from DRG neuronal cultures bound preferentially to the amino terminal Src homology-2 (SH2) domain of PLCγ1, but there was enhanced binding with tandemly expressed amino- and carboxy-terminal SH2 domains. The most significant difference in NGF signalling between PC12 cells and DRG was with the Shc family of adapter proteins. Both ShcA and ShcC were expressed in DRG neurons but only ShcA was detected in PC12 cells. Different isoforms of ShcA were phosphorylated in response to NGF in DRG and PC12 cells. NGF phosphorylated only one whereas epidermal growth factor phosphorylated both isoforms of ShcC in DRG cultures. Activation of the downstream mitogen-activated protein (MAP) kinase, p42Erk2 was significantly greater than p44Erk1 in DRG whereas both isoforms were activated in PC12 cells. Blocking the MAP kinase cascade using a MEK1/2 inhibitor, PD98059, abrogated NGF dependent capsaicin sensitivity, a nociceptive property specific to sensory neurons.