Discovery of a Benzamide Derivative That Protects Pancreatic ß-Cells against Endoplasmic Reticulum Stress.

Endoplasmic reticulum (ER) stress-mediated pancreatic insulin-producing ß-cell dysfunction and death are critical elements in the onset and progression of both type 1 and type 2 diabetes. Here, through cell-based high throughput screening we identified benzamide derivatives as a novel class of ß-cell protective agents against ER stress-induced dysfunction and death. Through structure-activity relationship optimization, a 3-(N-piperidinyl)methylbenzamide derivative 13d markedly protects ß-cells against ER stress-induced dysfunction and death with near 100% maximum rescue activity and an EC50 of 0.032 µM. Compound 13d alleviates ER stress in ß-cells by suppressing ER stress-mediated activation of all three branches of unfolded protein response (UPR) and apoptotic genes. Finally, we show that 13d significantly lowers blood glucose levels and increases concomitant ß-cell survival and number in a streptozotocin-induced diabetic mouse model. Identification of ß-cell-protective small molecules against ER stress provides a new promising modality for the treatment of diabetes.

Metabolic Stress Induces Caspase-3 Mediated Degradation and Inactivation of Farnesyl and Geranylgeranyl Transferase Activities in Pancreatic ß-Cells.

BACKGROUND/AIMS: At least 300 prenylated proteins are identified in the human genome; the majority of which partake in a variety of cellular processes including growth, differentiation, cytoskeletal organization/dynamics and vesicle trafficking. Aberrant prenylation of proteins is implicated in human pathologies including cancer; neurodegenerative diseases, retinitis pigmentosa, and premature ageing syndromes. Original observations from our laboratory have demonstrated that prenylation of proteins [small G-proteins and γ-subunits of trimeric G-proteins] is requisite for physiological insulin secretion. Herein, we assessed the impact of metabolic stress [gluco-, lipotoxicity and ER-stress] on the functional status of protein prenylation pathway in pancreatic ß-cells. METHODS: Farnesyltransferase [FTase] and geranylgeranyltransferase [GGTase] activities were quantified by radioisotopic methods. Caspase-3 activation and FTase/GGTase-α subunit degradation were determined by Western blotting. RESULTS: We observed that metabolic stress activates caspase-3 and induces degradation of the common α-subunit of FTase and GGTase-I in INS-1 832/13 cells, normal rodent islets and human islets leading to functional defects [inactivation] in FTase and GGTase activities. Caspase-3 activation and FTase/GGTase-α degradation were also seen in islets from the Zucker diabetic fatty [ZDF] rat, a model for Type 2 diabetes. Consequential to defects in FTase/GGTase-α signaling, we observed significant accumulation of unprenylated proteins [Rap1] in ß-cells exposed to glucotoxic conditions. These findings were replicated in ß-cells following pharmacological inhibition of generation of prenylpyrophosphate substrates [Simvastatin] or catalytic activity of prenylating enzymes [GGTI-2147]. CONCLUSIONS: Our findings provide the first evidence to suggest that metabolic stress induced dysfunction of the islet ß-cell may, in part, be due to defective protein prenylation signaling pathway.

Discovery, Synthesis, and Evaluation of 2,4-Diaminoquinazolines as a Novel Class of Pancreatic ß-Cell-Protective Agents against Endoplasmic Reticulum (ER) Stress.

Pancreatic insulin-producing ß-cell dysfunction and death plays central roles in the onset and progression of both type 1 and type 2 diabetes. Current antidiabetic drugs cannot halt the ongoing progression of ß-cell dysfunction and death. In diabetes, a major cause for the decline in ß-cell function and survival is endoplasmic reticulum (ER) stress. Here, we identified quinazoline derivatives as a novel class of ß-cell protective agents against ER stress-induced dysfunction and death. A series of quinazoline derivatives were synthesized from dichloroquiazoline utilizing a sequence of nucleophilic reactions. Through SAR optimization, 2,4-diaminoquinazoline compound 9c markedly protects ß-cells against ER stress-induced dysfunction and death with 80% maximum rescue activity and an EC50 value of 0.56 µM. Importantly, 9c restores the ER stress-impaired glucose-stimulated insulin secretion response and survival in primary human islet ß-cells. We showed that 9c protects ß-cells by alleviating ER stress through the suppression of the induction of key genes of the unfolded protein response and apoptosis.

Identification of 1,2,3-triazole derivatives that protect pancreatic ß cells against endoplasmic reticulum stress-mediated dysfunction and death through the inhibition of C/EBP-homologous protein expression.

The C/EBP-homologous protein (CHOP) acts as a mediator of endoplasmic reticulum (ER) stress-induced pancreatic insulin-producing ß cell death, a key element in the pathogenesis of diabetes. Chemicals that inhibit the expression of CHOP might therefore protect ß cells from ER stress-induced apoptosis and prevent or ameliorate diabetes. Here, we used high-throughput screening to identify a series of 1,2,3-triazole amide derivatives that inhibit ER stress-induced CHOP-luciferase reporter activity. Our SAR studies indicate that compounds with an N,1-diphenyl-5-methyl-1H-1,2,3-triazole-4-carboxamide backbone potently protect ß cell against ER stress. Several representative compounds inhibit ER stress-induced up-regulation of CHOP mRNA and protein, without affecting the basal level of CHOP expression. We further show that a 1,2,3-triazole derivative 4e protects ß cell function and survival against ER stress in a CHOP-dependent fashion, as it is inactive in CHOP-deficient ß cells. Finally, we show that 4e significantly lowers blood glucose levels and increases concomitant ß cell survival and number in a streptozotocin-induced diabetic mouse model. Identification of small molecule inhibitors of CHOP expression that prevent ER stress-induced ß cell dysfunction and death may provide a new modality for the treatment of diabetes.

VAV2, a guanine nucleotide exchange factor for Rac1, regulates glucose-stimulated insulin secretion in pancreatic beta cells.

AIMS/HYPOTHESIS: Rho GTPases (Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division cycle 42 [Cdc42]) have been shown to regulate glucose-stimulated insulin secretion (GSIS) via cytoskeletal remodelling, trafficking and fusion of insulin-secretory granules with the plasma membrane. GTP loading of these G proteins, which is facilitated by GDP/GTP exchange factors, is a requisite step in the regulation of downstream effector proteins. Guanine nucleotide exchange factor VAV2 (VAV2), a member of the Dbl family of proteins, has been identified as one of the GDP/GTP exchange factors for Rac1. Despite recent evidence on the regulatory roles of VAV2 in different cell types, roles of this guanine nucleotide exchange factor in the signalling events leading to GSIS remain undefined. Using immunological, short interfering RNA (siRNA), pharmacological and microscopic approaches we investigated the role of VAV2 in GSIS from islet beta cells. METHODS: Co-localisation of Rac1 and VAV2 was determined by Triton X-114 phase partition and confocal microscopy. Glucose-induced actin remodelling was quantified by live cell imaging using the LifeAct-GFP fluorescent biosensor. Rac1 activation was determined by G protein linked immunosorbent assay (G-LISA). RESULTS: Western blotting indicated that VAV2 is expressed in INS-1 832/13 beta cells, normal rat islets and human islets. Vav2 siRNA markedly attenuated GSIS in INS-1 832/13 cells. Ehop-016, a newly discovered small molecule inhibitor of the VAV2-Rac1 interaction, or siRNA-mediated knockdown of VAV2 markedly attenuated glucose-induced Rac1 activation and GSIS in INS-1 832/13 cells. Pharmacological findings were recapitulated in primary rat islets. A high glucose concentration promoted co-localisation of Rac1 and VAV2. Real-time imaging in live cells indicated a significant inhibition of glucose-induced cortical actin remodelling by Ehop-016. CONCLUSIONS/INTERPRETATION: Our data provide the first evidence to implicate VAV2 in glucose-induced Rac1 activation, actin remodelling and GSIS in pancreatic beta cells.

Identification of small molecules that protect pancreatic ß cells against endoplasmic reticulum stress-induced cell death.

Endoplasmic reticulum (ER) stress plays an important role in the decline in pancreatic ß cell function and mass observed in type 2 diabetes. Here, we developed a novel ß cell-based high-throughput screening assay to identify small molecules that protect ß cells against ER stress-induced cell death. Mouse ßTC6 cells were treated with the ER stressor tunicamycin to induce ER stress, and cell death was measured as a reduction in cellular ATP. A collection of 17600 compounds was screened for molecules that promote ß cell survival. Of the approximately 80 positive hits, two selected compounds were able to increase the survival of human primary ß cells and rodent ß cell lines subjected to ER stressors including palmitate, a free fatty acid of pathological relevance to diabetes. These compounds also restored ER stress-impaired glucose-stimulated insulin secretion responses. We show that the compounds promote ß cell survival by reducing the expression of key genes of the unfolded protein response and apoptosis, thus alleviating ER stress. Identification of small molecules that prevent ER stress-induced ß cell dysfunction and death may provide a new modality for the treatment of diabetes.

High glucose exposure promotes activation of protein phosphatase 2A in rodent islets and INS-1 832/13 ß-cells by increasing the posttranslational carboxylmethylation of its catalytic subunit.

Existing evidence implicates regulatory roles for protein phosphatase 2A (PP2A) in a variety of cellular functions, including cytoskeletal remodeling, hormone secretion, and apoptosis. We report here activation of PP2A in normal rat islets and insulin-secreting INS-1 832/13 cells under the duress of hyperglycemic (HG) conditions. Small interfering RNA-mediated knockdown of the catalytic subunit of PP2A (PP2Ac) markedly attenuated glucose-induced activation of PP2A. HG, but not nonmetabolizable 3-O-methyl glucose or mannitol (osmotic control), significantly stimulated the methylation of PP2Ac at its C-terminal Leu-309, suggesting a novel role for this posttranslational modification in glucose-induced activation of PP2A. Moreover, knockdown of the cytosolic leucine carboxymethyl transferase 1 (LCMT1), which carboxymethylates PP2Ac, significantly attenuated PP2A activation under HG conditions. In addition, HG conditions, but not 3-O-methyl glucose or mannitol, markedly increased the expression of LCMT1. Furthermore, HG conditions significantly increased the expression of B55α, a regulatory subunit of PP2A, which has been implicated in islet dysfunction under conditions of oxidative stress and diabetes. Thapsigargin, a known inducer of endoplasmic reticulum stress, failed to exert any discernible effects on the carboxymethylation of PP2Ac, expression of LCMT1 and B55α, or PP2A activity, suggesting no clear role for endoplasmic reticulum stress in HG-induced activation of PP2A. Based on these findings, we conclude that exposure of the islet ß-cell to HG leads to accelerated PP2A signaling pathway, leading to loss in glucose-induced insulin secretion.

Glucotoxic conditions induce endoplasmic reticulum stress to cause caspase 3 mediated lamin B degradation in pancreatic ß-cells: protection by nifedipine.

Nuclear lamins form the lamina on the interior of the nuclear envelope, and are involved in the regulation of various cellular processes, including DNA replication and chromatin organization. Despite this evidence, little is known about potential alterations in nuclear metabolism, specifically lamin structure and integrity in isolated ß-cells subjected to stress conditions, including chronic exposure to hyperglycemia (i.e., glucotoxicity). Herein, we investigated effects of glucotoxic conditions on the catalytic activation of caspase 3 and the associated degradation of one of its substrate proteins, namely lamin-B. We report that incubation of insulin-secreting INS-1 832/13 cells, normal rat islets or human islets under glucotoxic conditions (20 mM; 12-48 h) results in the degradation of native lamin B leading to accumulation of the degraded products in non-relevant cellular compartments, including cytosol. Moreover, the effects of high glucose on caspase 3 activation and lamin B degradation were mimicked by thapsigargin, a known inducer of endoplasmic reticulum stress (ER stress). Nifedipine, a known blocker of calcium channel activation, inhibited high glucose-induced caspase 3 activation and lamin B degradation in these cells. 4-Phenyl butyric acid, a known inhibitor of ER stress, markedly attenuated glucose-induced CHOP expression (ER stress marker), caspase 3 activation and lamin B degradation. We conclude that glucotoxic conditions promote caspase 3 activation and lamin B degradation, which may, in part, be due to increased ER stress under these conditions. We also provide further evidence to support beneficial effects of calcium channel blockers against metabolic dysfunction of the islet ß-cell induced by hyperglycemic conditions.

Nifedipine prevents etoposide-induced caspase-3 activation, prenyl transferase degradation and loss in cell viability in pancreatic ß-cells.

Emerging evidence implicates novel roles for post-translational prenylation (i.e., farnesylation and geranylgeranylation) of various signaling proteins in a variety of cellular functions including hormone secretion, survival and apoptosis. In the context of cellular apoptosis, it has been shown previously that caspase-3 activation, a hallmark of mitochondrial dysregulation, promotes hydrolysis of several key cellular proteins. We report herein that exposure of insulin-secreting INS 832/13 cells or normal rat islets to etoposide leads to significant activation of caspase-3 and subsequent degradation of the common α-subunit of farnesyl/geranylgeranyl transferases (FTase/GGTase). Furthermore, the above stated signaling steps were prevented by Z-DEVD-FMK, a known inhibitor of caspase-3. In addition, treatment of cell lysates with recombinant caspase-3 also caused FTase/GGTase α-subunit degradation. Moreover, nifedipine, a calcium channel blocker, markedly attenuated etoposide-induced caspase-3 activation, FTase/GGTase α-subunit degradation in INS 832/13 cells and normal rat islets. Further, nifedipine significantly restored etoposide-induced loss in metabolic cell viability in INS 832/13 cells. Based on these findings, we conclude that etoposide induces loss in cell viability by inducing mitochondrial dysfunction, caspase-3 activation and degradation of FTase/GGTase α-subunit. Potential significance of these findings in the context of protein prenylation and ß-cell survival are discussed.

Rab-geranylgeranyl transferase regulates glucose-stimulated insulin secretion from pancreatic ß cells.

A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet ß-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and ß-subunits of RGGT in clonal INS 832/13 ß-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or ß-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.

Expression of sweet receptor components in equine small intestine: relevance to intestinal glucose transport.

The heteromeric sweet taste receptor T1R2-T1R3 is expressed on the luminal membrane of certain populations of enteroendocrine cells. Sensing of sugars and other sweet compounds by this receptor activates a pathway in enteroendocrine cells, resulting in secretion of a number of gut hormones, including glucagon-like peptide 2 (GLP-2). This subsequently leads to upregulation in the expression of intestinal Na(+)/glucose cotransporter, SGLT1, and increased intestinal glucose absorption. On the basis of the current information available on the horse genome sequence, it has been proposed that the gene for T1R2 (Tas1R2) is absent in the horse. We show here, however, that horses express both the mRNA and protein for T1R2. Equine T1R2 is most closely homologous to that in the pig and the cow. T1R2 protein, along with T1R3, α-gustducin, and GLP-2 proteins are coexpressed in equine intestinal endocrine cells. Intravenous administration of GLP-2, in rats and pigs, leads to an increase in the expression of SGLT1 in absorptive enterocytes and enhancement in blood glucose concentrations. GLP-2 receptor is expressed in enteric neurons, excluding the direct effect of GLP-2 on enterocytes. However, electric stimulation of enteric neurons generates a neural response leading to SGLT1 upregulation, suggesting that sugar in the intestine activates a reflex increase in the functional expression of SGLT1. Horses possess the ability to upregulate SGLT1 expression in response to increased dietary carbohydrates, and to enhance the capacity of the gut to absorb glucose. The gut sweet receptor provides an accessible target for manipulating the equine gut to absorb glucose (and water), allowing greater energy uptake and hydration for hard-working horses.

Expression of Na+/glucose co-transporter 1 (SGLT1) in the intestine of piglets weaned to different concentrations of dietary carbohydrate.

Na+/glucose co-transporter 1 (SGLT1) transports dietary sugars from the lumen of the intestine into enterocytes. Regulation of this protein is essential for the provision of glucose to the body and, thus, is important for maintenance of glucose homeostasis. We have assessed expression of SGLT1 at mRNA, protein and functional levels in the intestinal tissue of 28 d old piglets weaned onto isoenergetic diets with differing concentrations of digestible carbohydrate (CHO). We show that expression of SGLT1 remains constant when piglets are fed up to 40 % CHO-containing diets. However, there is a significant increase in SGLT1 expression when the CHO content of the diet is>50 %. Morphometric analyses indicate that the increased expression is not due to a trophic effect. It has been proposed that in rat intestine, in response to a high-CHO diet, GLUT2 (the classical basolateral membrane monosaccharide transporter) is translocated to the luminal membrane of enterocytes to absorb excess dietary glucose. We show, using immunohistochemistry and Western blotting with antibodies raised to amino acids in different epitopes of GLUT2, that under all dietary conditions, low to high CHO, GLUT2 is expressed on the basolateral membrane of pig enterocytes. Furthermore, functional studies indicate that there is no uptake of 2-deoxy-D-glucopyranoside, a specific substrate of Na+-independent glucose transporters into brush-border membrane vesicles isolated from the intestines of piglets either maintained on low- or high-CHO diets. Thus, SGLT1 is the major route for absorption of dietary sugars across the luminal membrane of swine enterocytes.

Expression of Na+/glucose co-transporter 1 (SGLT1) is enhanced by supplementation of the diet of weaning piglets with artificial sweeteners.

In an intensive livestock production, a shorter suckling period allows more piglets to be born. However, this practice leads to a number of disorders including nutrient malabsorption, resulting in diarrhoea, malnutrition and dehydration. A number of strategies have been proposed to overcome weaning problems. Artificial sweeteners, routinely included in piglets' diet, were thought to enhance feed palatability. However, it is shown in rodent models that when included in the diet, they enhance the expression of Na+/glucose co-transporter (SGLT1) and the capacity of the gut to absorb glucose. Here, we show that supplementation of piglets' feed with a combination of artificial sweeteners saccharin and neohesperidin dihydrochalcone enhances the expression of SGLT1 and intestinal glucose transport function. Artificial sweeteners are known to act on the intestinal sweet taste receptor T1R2/T1R3 and its partner G-protein, gustducin, to activate pathways leading to SGLT1 up-regulation. Here, we demonstrate that T1R2, T1R3 and gustducin are expressed together in the enteroendocrine cells of piglet intestine. Furthermore, gut hormones secreted by the endocrine cells in response to dietary carbohydrates, glucagon-like peptides (GLP)-1, GLP-2 and glucose-dependent insulinotrophic peptide (GIP), are co-expressed with type 1 G-protein-coupled receptors (T1R) and gustducin, indicating that L- and K-enteroendocrine cells express these taste elements. In a fewer endocrine cells, T1R are also co-expressed with serotonin. Lactisole, an inhibitor of human T1R3, had no inhibitory effect on sweetener-induced SGLT1 up-regulation in piglet intestine. A better understanding of the mechanism(s) involved in sweetener up-regulation of SGLT1 will allow the identification of nutritional targets with implications for the prevention of weaning-related malabsorption.

How do the satellite glia cells of the dorsal root ganglia respond to stressed neurons?--nitric oxide saga from embryonic development to axonal injury in adulthood.

Dorsal root ganglia (DRG) respond to peripheral nerve injury by up-regulating nitric oxide (NO) production by neurons and glia in addition to local fibroblasts, endothelium and macrophages. We hypothesise that NO produced from these cells has specific roles. We have shown that when neuronal NO synthase (nNOS) is blocked in axotomised DRG, neurons undergo degenerative changes (Thippeswamy et al., 2001, 2007a). Further, we demonstrated that increased neuronal NO production, in response to axotomy/growth factor-deprivation in vitro, signals glial cells to produce trophic factors to support neuronal survival (Thippeswamy et al., 2005a). Recently, we found that treating satellite glia-neuron co-cultures with nNOS inhibitor, 7-nitroindazole (7NI), decreases the number of nestin+ cells that show neuron-like morphology. Cultured/axotomised DRG also upregulate inducible NOS (iNOS) in non-neuronal cells. Therefore, it is plausible that degenerative changes following nNOS inhibition are also due to iNOS-mediated excessive NO production by non-neuronal cells, which indeed is cytotoxic. NG-nitro-l-arginine methylester (L-NAME), the pan NOS inhibitor did not significantly change nNOS+ neuron number in axotomised DRG compared to 7NI suggesting that iNOS-mediated NO contributes to the degenerative process. In this paper, these findings from our and others' past work on NO-mediated neuron-glia signalling in axotomised DRG are discussed.

Nitric oxide-NGF mediated PPTA/SP, ADNP, and VIP expression in the peripheral nervous system.

Nerve growth factor (NGF)-deprivation or axotomy of dorsal root ganglion (DRG) neurons causes stress, which they cope by triggering various mechanisms. Among several molecular changes, in the present study, we demonstrate preprotachykinin-A-substance P (PPTA-SP) and activity-dependent neuroprotective protein-vasoactive intestinal peptide (ADNP-VIP) expression pattern using DRG neurons-Schwann cells coculture and axotomy model. In the presence of NGF, DRG cultures showed high levels of PPTA and ADNP mRNA expression, which were significantly suppressed in the absence of NGF and/or nitric oxide synthase (NOS) inhibition by NG-nitro-L-arginine methyl ester (L-NAME), suggesting that both NGF and nitric oxide (NO) can regulate PPTA and ADNP expression. However, treating coculture with NO donor, diethylenetriamine nitric oxide (DETA-NO) did not increase PPTA and ADNP expression in the presence or absence of NGF, although there was a marginal increase in ADNP expression in the absence of NGF. NGF-deprivation increases endogenous NO; thus, DETA-NO had no further effect on PPTA and ADNP expression. Alternatively, NGF produced from NO-stimulated Schwann cells influence gene expression. In addition, interestingly, DETA-NO treatment of Schwann cells alone suppresses both PPTA and ADNP, suggesting differential response of DRG neurons-Schwann cells coculture to DETA-NO. SP and ADNP immunostaining of axotomized DRGs revealed significant reduction in SP and ADNP compared to intact DRG, which was partially recovered in neuronal NOS blocker, 7-nitroindazole (7-NI)-treated DRGs, particularly intense ADNP staining in satellite glia. As ADNP is VIP-responsive gene, we further explored VIP expression in DRGs. Axotomy increased VIP in DRG neurons, but 7-NI treatment caused intense VIP staining in satellite glia. These observations suggest a complex interaction of NO-NGF with PPTA/SP and ADNP-VIP in neuron-glial communication when neurons are stressed.

Evidence of postnatal neurogenesis in dorsal root ganglion: role of nitric oxide and neuronal restrictive silencer transcription factor.

The various mechanisms underlying postnatal neurogenesis from discrete CNS regions have emerged recently. However, little is known about postnatal neurogenesis in dorsal root ganglion (DRG). BrdU incorporation and subsequent immunostaining for BrdU, neural stem cell marker, nestin and neuronal marker, PGP 9.5 have provided evidence for postnatal neurogenesis in DRG. We further demonstrate, in vivo and in vitro, that nitric oxide (NO) regulates neural stem cells (nestin+) proliferation and, possibly, differentiation into neurons. Surprisingly, nerve growth factor (NGF) had no effect on nestin+ cells proliferation. Axotomy or NGF-deprivation of DRG neurons-satellite glia co-culture increases NO production by neurons and treating with a NO synthase (NOS) inhibitor, N G-nitro-L-arginine methylester (L-NAME) in vitro or 7-nitroindazole (7NI) in vivo, causes a significant increase in nestin+ cell numbers. However, a soluble guanylyl cyclase (sGC) blocker, 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (ODQ) treatment of NGF-deprived DRG neurons-satellite glia co-culture had no significant effect on nestin+ cell numbers. This implies NO regulates nestin+ cell proliferation independent of cGMP. We hypothesised that the neuronal-restrictive silencer transcription factor (NRSF, also termed REST), a master regulator of neuronal genes in non-neuronal cells, may be modulated by NO in satellite glia cultures. A NO donor, dimethyl-triamino-benzidine (DETA)-NO treatment of satellite glia cell cultures results in a significant increase in the NRSF/REST mRNA expression. The majority of cultured satellite glia cells express nestin, and also show increased levels of NOS, thus L-NAME treatment of these cultures causes a dramatic reduction in NRSF/REST mRNA. Overall these results suggest that NO inhibits neurogenesis in DRG and this is correlated with modulation of NRSF, a known modulator of differentiation.