Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

IgG response to intracerebral xenotransplantation: specificity and role in the rejection of porcine neurons.

Xenogenic fetal neuroblasts are considered as a potential source of transplantable cells for the treatment of neurodegenerative diseases, but immunological barriers limit their use in the clinic. While considerable work has been performed to decipher the role of the cellular immune response in the rejection of intracerebral xenotransplants, there is much still to learn about the humoral reaction. To this end, the IgG response to the transplantation of fetal porcine neural cells (PNC) into the rat brain was analyzed. Rat sera did not contain preformed antibodies against PNC, but elicited anti-porcine IgG was clearly detected in the host blood once the graft was rejected. Only the IgG1 and IgG2a subclasses were up-regulated, suggesting a T-helper 2 immune response. The main target of these elicited IgG antibodies was porcine neurons, as determined by double labeling in vitro and in vivo. Complement and anti-porcine IgG were present in the rejecting grafts, suggesting an active role of the host humoral response in graft rejection. This hypothesis was confirmed by the prolonged survival of fetal porcine neurons in the striatum of immunoglobulin-deficient rats. These data suggest that the prolonged survival of intracerebral xenotransplants relies on the control of both cell-mediated and humoral immune responses.

Normal feeding behavior, body weight and leptin response require the neuropeptide Y Y2 receptor.

Neuropeptide Y (NPY), a 36-amino-acid peptide widely expressed in the brain is involved in many physiological responses, including hypothalamic control of food intake and cardiovascular homeostasis. NPY mediates its effects through binding to the Y1, Y2 and Y5 G-protein-coupled receptors. Little is known of the role of the Y2 receptor in mediating the different NPY effects. We inactivated the Y2 receptor subtype in mice and found that these mice developed increased body weight, food intake and fat deposition. The null mutant mice showed an attenuated response to leptin administration but a normal response to NPY-induced food intake and intact regulation of re-feeding and body weight after starvation. An absence of the Y2 receptor subtype also affected the basal control of heart rate, but did not influence blood pressure. These findings indicate an inhibitory role for the Y2 receptor subtype in the central regulation of body weight and control of food intake.

New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy.

Adoptive cell transfer studies in regenerative research and identification of genetically modified cells after gene therapy in vivo require unequivocally identifying and tracking the donor cells in the host tissues, ideally over several days or for up to several months. The use of reporter genes allows identifying the transferred cells but unfortunately most are immunogenic to wild-type hosts and thus trigger rejection in few days. The availability of transgenic animals from the same strain that would express either high levels of the transgene to identify the cells or low levels but that would be tolerant to the transgene would allow performing long-term analysis of labelled cells. Herein, using lentiviral vectors we develop two new lines of GFP-expressing transgenic rats displaying different levels and patterns of GFP-expression. The "high-expresser" line (GFP(high)) displayed high expression in most tissues, including adult neurons and neural precursors, mesenchymal stem cells and in all leukocytes subtypes analysed, including myeloid and plasmacytoid dendritic cells, cells that have not or only poorly characterized in previous GFP-transgenic rats. These GFP(high)-transgenic rats could be useful for transplantation and immunological studies using GFP-positive cells/tissue. The "low-expresser" line expressed very low levels of GFP only in the liver and in less than 5% of lymphoid cells. We demonstrate these animals did not develop detectable humoral and cellular immune responses against both transferred GFP-positive splenocytes and lentivirus-mediated GFP gene transfer. Thus, these GFP-transgenic rats represent useful tools for regenerative medicine and gene therapy.

Activity-dependent regulation of tyrosine hydroxylase expression in the enteric nervous system.

The regulation of neuromediator expression by neuronal activity in the enteric nervous system (ENS) is currently unknown. Using primary cultures of ENS derived from rat embryonic intestine, we have characterized the regulation of tyrosine hydroxylase (TH), a key enzyme involved in the synthesis of dopamine. Depolarization induced either by 40 mm KCl, veratridine or by electrical field stimulation produced a robust and significant increase in the proportion of TH immunoreactive (TH-IR) neurons (total neuronal population was identified with PGP9.5 or Hu) compared to control. This increase in the proportion of TH-IR neurons was significantly reduced by the sodium channel blocker tetrodotoxin (0.5 microm), demonstrating that neuronal activity was critically involved in the effects of these depolarizing stimuli. KCl also increased the proportion of VIP-IR but not nNOS-IR enteric neurons. The KCl-induced increase in TH expression was partly reduced in the presence of the nicotinic receptor antagonist hexamethonium (100 microm), of noradrenaline (1 microm) and of the alpha(2)-adrenoreceptor agonist clonidine (1 microm). Combining pharmacological and calcium imaging studies, we have further shown that L-type calcium channels were involved in the increase of TH expression induced by KCl. Finally, using specific inhibitors, we have shown that both protein kinases A and C as well as the extracellular signal-regulated kinases were required for the increase in the proportion of TH-IR neurons induced by KCl. These results are the first demonstration that TH phenotype of enteric neurons can be regulated by neuronal activity. They could also set the basis for the study of the pathways and mechanisms involved in the neurochemical plasticity observed both during ENS development and in inflammatory enteric neuropathies.

Glioplasticity in irritable bowel syndrome.

BACKGROUND: Growing evidence indicates a wide array of cellular remodeling in the mucosal microenvironment during irritable bowel syndrome (IBS), which possibly contributes to pathophysiology and symptom generation. Here, we investigated whether enteric glial cells (EGC) may be altered, and which factors/mechanisms lead to these changes. METHODS: Colonic mucosal biopsies of IBS patients (13 IBS-Constipation [IBS-C]; 10 IBS-Diarrhea [IBS-D]; 11 IBS-Mixed [IBS-M]) and 24 healthy controls (HC) were analyzed. Expression of S100ß and GFAP was measured. Cultured rat EGC were incubated with supernatants from mucosal biopsies, then proliferation and Ca2+ response to ATP were analyzed using flow cytometry and Ca2+ imaging. Histamine and histamine 1-receptor (H1R) involvement in the effects of supernatant upon EGC was analyzed. KEY RESULTS: Compared to HC, the mucosal area immunoreactive for S100ß was significantly reduced in biopsies of IBS patients, independently of the IBS subtype. IBS-C supernatants reduced EGC proliferation and IBS-D and IBS-M supernatants reduced Ca2+ response to ATP in EGC. EGC expressed H1R and the effects of supernatant upon Ca2+ response to ATP in EGC were blocked by pyrilamine and reproduced by histamine via H1R. IBS supernatants reduced mRNA expression of connexin-43. The S100ß-stained area was negatively correlated with the frequency and intensity of pain and bloating. CONCLUSION AND INFERENCES: Changes in EGC occur in IBS, involving mucosal soluble factors. Histamine, via activation of H1R-dependent pathways, partly mediates altered Ca2+ response to ATP in EGC. These changes may contribute to the pathophysiology and the perception of pain and bloating in patients with IBS.

L. fermentum CECT 5716 prevents stress-induced intestinal barrier dysfunction in newborn rats.

BACKGROUND: Intestinal epithelial barrier (IEB) dysfunction plays a critical role in various intestinal disorders affecting infants and children, including the development of food allergies and colitis. Recent studies highlighted the role of probiotics in regulating IEB functions and behavior in adults, but their effects in the newborn remain largely unknown. We therefore characterized in rat pups, the impact of Lactobacillus fermentum CECT 5716 (L. fermentum) on stress-induced IEB dysfunction, systemic immune response and exploratory behavior. METHODS: Newborn rats received daily by gavage either L. fermentum or water. Intestinal permeability to fluorescein sulfonic acid (FSA) and horseradish peroxidase (HRP) was measured following maternal separation (MS) and water avoidance stress (WAS). Immunohistochemical, transcriptomic, and Western blot analysis of zonula occludens-1 (ZO-1) distribution and expression were performed. Anxiety-like and exploratory behavior was assessed using the elevated plus maze test. Cytokine secretion of activated splenocytes was also evaluated. KEY RESULTS: L. fermentum prevented MS and WAS-induced IEB dysfunction in vivo. L. fermentum reduced permeability to both FSA and HRP in the small intestine but not in the colon. L. fermentum increased expression of ZO-1 and prevented WAS-induced ZO-1 disorganization in ileal epithelial cells. L. fermentum also significantly reduced stress-induced increase in plasma corticosteronemia. In activated splenocytes, L. fermentum enhanced IFNγ secretion while it prevented IL-4 secretion. Finally, L. fermentum increased exploratory behavior. CONCLUSIONS & INFERENCES: These results suggest that L. fermentum could provide a novel tool for the prevention and/or treatment of gastrointestinal disorders associated with altered IEB functions in the newborn.

Phosphatidylcholine-phospholipase C mediates the induction of nerve growth factor in cultured glial cells.

Addition of phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) to cultured glial cells increased the levels of nerve growth factor (NGF) mRNA and the amount of cell-secreted NGF. The effect of PC-PLC was 2.5 times higher than that elicited by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate. In cells in which protein kinase C (PKC) was fully inhibited or downregulated, the effect of PC-PLC was reduced-though still evident-and similar to that exerted by sphingosine. Results thus indicate that PC-PLC induces the synthesis of NGF by glial cells by a PKC-dependent and PKC-independent mechanisms.

A theory that may explain the Hayflick limit--a means to delete one copy of a repeating sequence during each cell cycle in certain human cells such as fibroblasts.

A model that may explain the limited division potential of certain cells such as human fibroblasts in culture is presented. The central postulate of this theory is that there exists, prior to certain key exons that code for materials needed for cell division, a unique sequence of specific repeating segments of DNA. One copy of such repeating segments is deleted during each cell cycle in cells that are not protected from such deletion through methylation of their cytosine residues. According to this theory, the means through which such repeated sequences are removed, one per cycle, is through the sequential action of enzymes that act much as bacterial restriction enzymes do--namely to produce scissions in both strands of DNA in areas that correspond to the DNA base sequence recognition specificities of such enzymes. After the first scission early in a replicative cycle, that enzyme becomes inhibited, but the cleavage of the first site exposes the closest site in the repetitive element to the action of a second restriction enzyme after which that enzyme also becomes inhibited. Then repair occurs, regenerating the original first site. Through this sequential activation and inhibition of two different restriction enzymes, only one copy of the repeating sequence is deleted during each cell cycle. In effect, the repeating sequence operates as a precise counter of the numbers of cell doubling that have occurred since the cells involved differentiated during development.

The neuropeptide Y receptors, Y1 and Y2, are transiently and differentially expressed in the developing cerebellum.

Neuropeptide Y (NPY), a peptide widely expressed in the brain, acts through the protein G-coupled receptors Y1, Y2 and Y5. In the adult rat, this peptide modulates many important functions such as the control of energy balance and anxiety. Its involvement in brain development has been less investigated. In the present study, we have analysed the expression of Y1 and Y2 in the developing rat cerebellum using RNase protection assay. Both receptors were detected in the embryo but at very low levels. Their expression then increased, reaching a peak at postnatal day 10. At later stages, we observed a down-regulation of both Y1 and Y2 mRNA levels. This pattern of expression was delayed in hypothyroid rats, suggesting that the regulation of NPY receptors was strictly related to cerebellar development stages. In situ hybridisation and immunohistochemistry analyses revealed specific localisations of the receptors. Y1 was exclusively expressed by Purkinje cells while Y2 was found mostly in granule cells of the internal granule cell layer. These observations argue in favour of specific roles for Y1 and Y2 in the developing cerebellum. In an initial attempt to characterise these roles, we have determined the number of apoptotic cells in the developing cerebellum of Y2(-/-) mice and analysed the effects of NPY on primary cultures of cerebellar granule neurones. Our data showed that the absence of Y2 did not increase cell death in the internal granule cell layer of the developing cerebellum, and that NPY by itself did not prevent the death of differentiated granule cells cultured in serum-free medium. However, we found that co-treatment of the cells by NPY and neuromediators such as NMDA or GABA strongly promoted the survival of granule neurones. Taken together, these observations suggest an involvement of the NPY receptors in cerebellar ontogenesis that remains to be demonstrated in vivo.

Complementary and overlapping expression of Y1, Y2 and Y5 receptors in the developing and adult mouse nervous system.

Neuropeptide Y, a 36 amino acid peptide, mediates its biological effects by activating the Y1, Y2, Y5 and Y6 receptors, which are also receptors for the structurally related peptide YY. Different classes of receptors have been suggested to be involved in different neuropeptide Y functions. In this report, we have characterized the developmental regulation and compared the cellular localization of these receptors in the developing and in the adult central and peripheral nervous systems of the mouse. RNase protection assays revealed that Y1, Y2 and Y5 messenger RNAs were expressed very early in spinal cord, brain, cerebellum and dorsal root ganglion development and were often down-regulated at times corresponding to their acquirement of the adult function in neurotransmission. In situ hybridization of the adult brain showed that Y1 was widely expressed, Y2 displayed a more restricted pattern, Y5 was expressed at very low levels and only in a few brain nuclei and Y6 was not expressed. Virtually all areas containing neurons positive for Y5 also expressed Y1, whereas many Y1-positive cells clearly did not express Y5. In contrast, Y2 was not expressed by the neurons expressing Y1 or Y5. These findings suggest that neuropeptide Y signaling in the brain could be mediated by simultaneous Y1 and Y5 activation. Similar results were also obtained in peripheral sensory neurons. Furthermore, our results suggest that neuropeptide Y/peptide YY receptors play an important role in nervous system development and that selective receptor combinations are responsible for signaling the different effects of neuropeptide Y in the peripheral and central nervous systems.

Decreased choline acetyltransferase activity in nerve growth factor-transgenic mice during brain development.

Activity of the synthetic enzyme for acetylcholine, choline acetyltransferase was investigated during development and in adult nerve growth factor-transgenic mice. A conspicuous reduction of choline acetyltransferase activity was observed in the anterior brain of nerve growth factor-transgenic embryos from embryonic days 13 to 16 (E13 to E16). Choline acetyltransferase activity levels subsequently resumed to normal levels, with the exception of a 15% increase in the adult hippocampus. Nerve growth factor contents followed a similar time-course and regional distribution in normal and nerve growth factor-transgenic animals and displayed significantly higher values from E14 to the early postnatal period. Nerve growth factor contents were normal in the adult brain. In vitro experiments confirmed the involvement of nerve growth factor in the decrease of choline acetyltransferase activity levels observed in transgenic neurons during development. These results suggest a role for nerve growth factor in the initial phase of the phenotypic differentiation of cholinergic neurons. They show that nerve growth factor may, under specific development conditions, lead to a paradoxical down-regulation of choline acetyltransferase activity.

Cytotoxic effects of 1 alpha,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues on a glioma cell line.

1 alpha,25-Dihydroxyvitamin D3 (1 alpha,25(OH)2D3) has recently been reported to exert a toxic effect on both rat and human glioma cell lines. However the potential clinical use of 1 alpha,25(OH)2D3 in the treatment of glioma is impaired by its potent hypercalcemic effects. We have therefore investigated the effects on glioma cell growth of several vitamin D3 analogues which have previously been shown to be less calcemic in vivo than 1 alpha,25(OH)2D3. The present study shows that several analogues are able to induce, in vitro, the death of rat glioma cells (C6.9). The compound KH 1060 appears to be the most effective in the induction of cell death, while MC 1288 and CB 1093 are as potent as 1 alpha,25(OH)2D3. EB 1089 was somewhat less effective than 1 alpha,25(OH)2D3 and MC 903, which is currently used in the treatment of psoriasis, has only a weak activity on C6.9 cells. The effective doses used are around 10(-9) M for 1 alpha,25(OH)2D3 and 10(-10) M for KH 1060. Interestingly, the toxic effect exerted by 1 alpha,25(OH)2D3 and its analogues is accompanied by several of the biochemical features of apoptosis, such as DNA fragmentation and induction of the c-myc protooncogene. These findings, together with the fact that the therapies currently available for glioma are only palliative, suggest that 1 alpha,25(OH)2D3 analogues such as KH 1060, EB 1089 or CB 1093, alone or in combination with other therapeutic approaches, could be of potential interest in the treatment of brain glial tumors.

Regulation of NGF, BDNF and LNGFR gene expression in ROS 17/2.8 cells.

The secosteroid hormone 1.25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently shown to enhance the synthesis of NGF to mouse L929 fibroblasts. In view of the critical role of 1,25(OH)2D3 on bone metabolism, it has been investigated if ROS 17/2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH)2D3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH)2D3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH)2D3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant Kd approximately 10(-9) M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17/2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.

1,25-dihydroxyvitamin D3 regulates the synthesis of nerve growth factor in primary cultures of glial cells.

The effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2 D3) on nerve growth factor (NGF) synthesis was investigated in primary cultures of astrocytes prepared from brain of neonatal rats. 1,25-(OH)2 D3 elicited a dose-dependent increase of NGF mRNA with a maximal effect at 10(-7) M, which persisted for at least 48 h. Northern blot analysis revealed an expression of the vitamin D3 receptor (VDR) gene in primary glial cells. Treatment of cells with 1,25-(OH)2 D3 led to an increase in the VDR mRNA levels. Similar results were obtained in C6 glioma cells. Exposure of primary glial cells to 10(-8) M 1,25-(OH)2 D3 caused only a 2-fold increase of the levels of cell-secreted NGF after 3 days of treatment. However, a 5-fold increase was observed three days after a second addition of vitamin D3. Likewise, a pretreatment with lower doses of hormone such as 10(-10) M or 10(-9) M enhanced the responsiveness of the cells to a 24 h treatment with 10(-8) M hormone. It appears, therefore, that the duration of the treatment influences the level of synthesis of NGF, possibly as a consequence of the increase of the VDR gene expression. The specificity of 1,25-(OH)2 D3 is supported by the fact that a concentration of 10(-7) M of an another vitamin D3 metabolite, 24,25-(OH)2 D3, had no effect on NGF synthesis. Several lines of evidence indicate that astrocytes constitute the major cell type responsive to 1,25-(OH)2 D3 in primary cultures of glial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25-Dihydroxyvitamin D3 regulates the expression of the low-affinity neurotrophin receptor.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) is known to regulate the expression of neurotrophins [45,46]. Here, we report that 1,25-(OH)2D3 does not influence the expression of truncated or full-length forms of trkB and trkC receptors mRNAs in primary cultures of astrocytes and in C6 glioma cells. In contrast, low concentrations of 1,25-(OH)2D3 increased low-affinity neurotrophin receptor (P75NTR) mRNA and protein levels in C6 glioma cells. Putative vitamin D responsive elements (VDRE) in the P75NTR promoter have been investigated by transfecting plasmids containing sequences from P75NTR promoter fused to a cat reporter gene. A region between -610 and -860 bp upstream from the translation start codon was found to respond to 1,25-(OH)2D3. Interestingly, 1,25-(OH)2D3 does not regulate P75NTR in primary cultures of astrocytes even at concentration as high as 10(-7) M. Since long-term treatment of 1,25-(OH)2D3 induces cell death in C6 glioma cells but not in primary astrocytes [41], the possible involvement of P75NTR in 1,25-(OH)2D3-induced cell death is discussed. Finally, in-vivo studies show that treatment of 15-day-old and adult rats with 1,25-(OH)2D3 leads to a decrease in the level of P75NTR mRNA in the spinal cord but does not influence its expression in dorsal root ganglion or sciatic nerve. These results suggest that 1,25-(OH)2D3 may have a role in the specific regulation of P75NTR in vivo.

1,25-Dihydroxyvitamin D3, an inducer of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) has significant therapeutic potentials, in particular for neurodegenerative disorders. To determine factors that would enhance GDNF expression, we analysed the effect of 1,25-(OH)2 D3 in C6 glioma cells. Treatment of C6 cells with 10(-7) M, 1,25-(OH)2 D3 for 48 h elicited an 18.5-fold increase in the level of GDNF mRNA. In addition, our results indicate that 1,25-(OH)2 D3 is effective at concentrations as low as 10(-10) M and that retinoic acid has additive effects. These data indicate that 1,25-(OH)2 D3 is a potent inducer of GDNF expression and suggest that 1,25-(OH)2 D3 may contribute to the regulation of GDNF in vivo.

1,25-dihydroxyvitamin D3 regulates NT-3, NT-4 but not BDNF mRNA in astrocytes.

The effect of 1,25-dihydroxyvitamin D3 on neurotrophin mRNA expression was studied in primary cultures of astrocytes. In addition to its known effects on NGF expression, 1,25-dihydroxyvitamin D3 was shown to upregulate NT-3 mRNA levels, while NT-4 expression was slightly but significantly downregulated. No effect was observed on BDNF mRNA expression. These data clearly show a differential regulation of the four neurotrophins by 1,25-dihydroxyvitamin D3 in primary cultures of astrocytes and suggest that 1,25-dihydroxyvitamin D3 may participate in the expression of NGF, NT-3 and NT-4 in the central nervous system.

Expression of 25(OH) vitamin D3 24-hydroxylase gene in glial cells.

The expression of the 25(OH) vitamin D3 24-hydroxylase gene was studied in C6 glioma and rat primary glial cell culture. The expression of the 25(OH)D3 24-hydroxylase gene was not detected in C6 glioma or glial cells cultured in a serum-free medium. However, the 25(OH)D3 24-hydroxylase mRNA was induced in a dose-dependent manner in cells treated with 1,25(OH)2D3. These findings provide further evidence for an involvement of vitamin D3 metabolites in brain function.

Interactions between second messenger pathways influence NGF synthesis in mouse primary astrocytes.

Primary mouse brain astrocytes were stimulated with phorbol 12-myristate 13-acetate (PMA), serum, forskolin and ionophore A23187, in order to investigate the effect of distinct signalling pathways on the expression of the nerve growth factor (NGF) gene and of proto-oncogenes encoding transcription factors of the Fos and Jun families. PMA, and to a lesser extent serum, induced a marked accumulation of NGF transcripts, in agreement with published observations [Brain Res., 570 (1992) 316-322]. The effect of A23187 was less pronounced and that of forskolin barely detectable. No relationship was observed between the expression of NGF gene and that of c-fos, fos-B, fra-1, jun-B proto-oncogenes. In contrast, changes in the levels of NGF transcripts were associated with corresponding modifications of the levels of c-jun transcripts, a fact which suggests that the c-Jun protein exerts a regulatory role on the expression of the NGF gene. In these cells, however, the regulation of NGF synthesis appears complex, since a pretreatment with forskolin or ionophore A23187 interfered with the promoting effect elicited by PMA or serum in inducing an early decline of the levels of NGF transcripts. This phenomenon was accompanied by a corresponding decrease in the amounts of cell-secreted NGF in cells treated with forskolin and PMA. A23187 had a much more striking effect on the production of mature NGF since this compound maintained the level of cell-secreted NGF to basal values, irrespective of the presence of PMA. A similar inhibitory effect was observed with thapsigargin, another compound able to increase the cytosolic concentration of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)